The Effect of a Squamous Cell Carcinoma Associated
pi Integrin Mutation on Cell Behavior
Richard D. Evans
University College London
And
Cancer Research UK
Cancer Research UK Supervisor: Dr. Fiona M Watt
UCL Supervisor: Prof. Alan Hall
A thesis submitted in partial fulfillment of the requirements for the
degree of Doctor of Philosophy, University of London
September 2002 ProQuest Number: 10016079
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract
Integrins are the main receptors for extracellular matrix proteins and are responsible
for mediating attachment of most cell types to their surrounding matrix. In addition
integrins regulate growth and differentiation of many cell types, including epidermal
kératinocytes, through a variety of signalling pathways. Integrin loss or
overexpression contributes to the pathogenesis of benign epidermal disorders and
influences the incidence and prognosis of squamous cell carcinomas (SCC) and other
tumours. However, no integrin mutations have yet been reported in tumours of any
origin. In my thesis I have studied the SCC4 cell line isolated from a human oral
SCC. This cell line has normal integrin expression and is poorly differentiated. When
these cells are infected with a retrovirus encoding the wild-type chick (31 integrin
they regain the ability to differentiate. I found that these cells are heterozygous for a
(31 mutation, T188I, mapping to the A domain of the subunit. T188I results in
increased ligand binding, irrespective of the partner a subunit, both in solid phase
assays with recombinant protein and in living cells. The mutation promotes cell
attachment and spreading but not invasion or motility. It also alters integrin
signalling as shown by increased levels of MAPK phosphorylation. When introduced
into the SCC4 cell line the mutant (31 integrin fails to stimulate differentiation.
Activation of pi integrins in normal kératinocytes also suppresses differentiation.
To discover how frequently similar mutations occur in SCC I screened 124 skin tumours for mutations in exons surrounding T188I. A single alanine to valine substitution was found in a single case. My results establish mutation as a mechanism by which integrins can contribute to tumour development and provide new insights into how integrins regulate keratinocyte differentiation. Acknowledgments
I would like to thank Fiona, my supervisor, for all her help over the last four years. I would also like to thank Simon and Liz for all the times they have fed cells and helped out far beyond the call of duty. Also the various members of bay three over the last few years, John, Liz, Robin and Kristen for making it such as fun (and often surreal) place to work. The other integrin people in the lab (Ingo, Douglas, Sam and
Dave) have been of great help, as have all the other members of the lab.
The support staff at LIT have been of immense help, especially Cathy, Ayad, Gary and Derrick from the FACS lab, Colin, Debbie and Peter from microscopy and all the crew of the equipment park and cell production.
I would also like to acknowledge the help provided by Viv Perkins, Alistair Henry,
Paul Stephens and Martyn Robinson of CellTech PLC, Judith Jones of HMDS and
Claire Taylor of Cancer Research UK Leeds.
I would also like to thank my parents, for not complaining too much that they have barely seen me for the final months of this thesis. And Josie, for all her support (and cooking). Table of Contents
ABSTRACT...... 2
ACKNOWLEDGMENTS...... 3
TABLE OF CONTENTS...... 4
TABLE OF FIGURES...... 10
LIST OF TABLES...... 13
LIST OF ABBREVIATIONS...... 14
1 INTRODUCTION...... 15
Overview...... 15
1.1 Integrins ...... 15
1.1.1 Integrin families...... 17
1.2 Integrin Structure and Function ...... 19
1.2.1 a subunit...... 19
1.2.2 P subunit...... 23
1.2.3 Ligand binding and activation ...... 28
1.3 Integrin Interacting Proteins, Signalling and Focal Adhesion Assembly 34
1.3.1 Transmembrane and extracellular Integrin interacting proteins ...... 34
1.3.2 Integrin cytoplasmic domain binding proteins ...... 35
1.3.3 Focal Adhesions ...... 38
1.3.4 Focal adhesion assembly/disassembly ...... 41
1.3.5 Integrin Signalling...... 43
1.4 Structure and Function of the Skin...... 46 Table of Contents
1.4.1 The Dermis...... 46
1.4.2 The Epidermis ...... 47
1.4.2 The basal layer kératinocytes ...... 48
1.4.3 The upper epidermal layers ...... 49
1.4.4 Squamous Cell Carcinoma...... 51
1.5 Epidermal Integrins ...... 53
1.5.1 Functions o f Epidermal Integrins ...... 55
1.5.2 Epidermal Stem Cells ...... 57
1.5.3 Integrins and Squamous Cell Carcinoma...... 60
1.5.4 Regulation o f terminal differentiation ...... 61
1.6 Aim s...... 65
2 MATERIALS AND METHODS...... 66
2.1 Cell Biology ...... 66
2.1.1 General solutions ...... 66
2.1.2 J2-3T3 andJ2-3T3 Puro Cell Culture ...... 68
2.1.3 Culture o f human epidermal kératinocytes ...... 69
2.1.4 Squamous cell carcinoma cells...... 72
2.1.5 GD25 p i null mouse fibroblasts...... 72
2.1.6 Retroviral Producer Cells ...... 73
2.1.7 MOLT-4...... 75
2.2 Assays of proliferation and differentiation ...... 75
2.2.1 Proliferation Assay ...... 75
2.2.2 SCC4 Differentiation Assay...... 75
2.2.3 Suspension differentiation assay ...... 76
2.3 Adhesion Assays...... 77 5 Table of Contents
2.3.1 ECM proteins ...... 77
2.3.2 Cell Attachment Assay ...... 78
2.3.3 Recombinant Integrin Adhesion Assay ...... 79
2.4 Cell motility and spreading ...... 80
2.4.1 Time lapse microscsopy ...... 80
2.4.2 Determination o f rate o f spreading ...... 80
2.4.3 Cell motility towards serum ...... 80
2.5 Invasion Assay ...... 81
2.6 Immunological Methods ...... 81
2.6.1 General Solutions ...... 81
2.6.2 Antibodies ...... 83
2.6.3 Preparation o f cells for immunofluorescence staining ...... 84
2.6.4 Staining protocols ...... 84
2.7 Flow cytometry ...... 85
2.7.1 Staining protocol ...... 85
2.7.2 Sorting on basis o f extracellular epitope expression ...... 85
2.8 Biochemistry...... 86
2.8.1 Protein Extraction for MAPK assay ...... 86
2.8.2 Protein assay...... 86
2.9 Polyacrylamide Gel Electrophoresis (SDS Page) ...... 86
2.9.1 General Solutions ...... 86
2.9.2 SDS-PAGE...... 87
2.9.3 Western Blotting ...... 88
2.10 Molecular Biology ...... 89
2.10.1 Bacterial Culture General Solutions...... 89
6 Table of Contents
2.10.2 Transformation of bacteria ...... 90
2.11 DNA techniques...... 90
2.11.1 General solutions ...... 90
2.11.2 Isolation o f DNA from agarose gels ...... 91
2.11.3 Maxi/mini preps ...... 91
2.11.4 Genomic DNA preparation ...... 91
2.11.5 Restriction enzyme digestion ...... 92
2.12 PCR techniques...... 92
2.12.1 PCR...... 92
2.12.2 Site directed mutagenesis ...... 92
2.12.3 RT-PCR ...... 93
2.12.4 Sequencing...... 93
2.12.5 Primers...... 93
2.12.6 SNP analysis...... 95
2.13 List of Suppliers and Distributors ...... 97
3 ANALYSIS OF THE SCC4 PHENOTYPE...... 99
3.1 Introduction ...... 99
3.2 Results...... 100
3.2.1 Infection of SCC4 cells with chick pi subunits induces differentiation. 100
3.2.2 Expression o f endogenous integrin is normal when compared to
SCC12B2...... 102
3.2.3 SCC4 form focal adhesions when plated on ECM proteins ...... 104
3.2.4 Binding o f cj3l on anti-cj3l antibody coated surfaces causes clustering o f
the endogenous p i subunits...... 104
1 Table of Contents
3.2.5 Sequencing o f p i cDNA in SCC4...... 106
3.3 Discussion ...... 109
4 ANALYSIS OF THE EFFECT OF THE T188I MUTATION ON LIGAND
BINDING BY THE pi INTEGRIN SUBUNIT...... 115
4.1 Introduction ...... 115
4.2 Results...... 116
4.2.1 Analysis o f the effect o f the T188I mutation on integrin affinity in cell free
assays...... 116
4.2.2 Construction o f chick pi T 1881 equivalent retroviral construct. 119
4.2.3 Expression of chick T188I in pi null fibroblasts ...... 120
4.2.4 Adhesion of GD25 cells expressing WTand T188I integrin subunits.... 122
4.2.5 Adhesion ofSCC4 cells to ECM proteins ...... 125
4.2.6 Homology modelling o f the p i integrin A domain ...... 128
4.3 Discussion ...... 132
5 THE EFFECT OF INTEGRIN ACTIVATION ON CELL BEHAVIOUR.. 137
5.1 Introduction ...... 137
5.2 Results...... 138
5.2.1 Analysis o f the effect o f the T 1881 mutation on spreading, migration and
invasion ...... 138
5.2.2 The effect o f integrin activation on cell signalling ...... 148
5.2.3 Effect o f integrin activation on SCC and normal kératinocytes ...... 150
5.2.4 The activating anti- p i antibody TS2/16 can effect normal keratinocyte
differentiation ...... 156
5.2.5 Discussion ...... 160
8 Table of Contents
6 SCREENING FOR FURTHER INTEGRIN MUTATIONS ...... 167
6.1 Introduction ...... 167
6.2 Results...... 168
6.3 Discussion ...... 172
7 GENERAL DISCUSSION...... 174
7.1 The T188I mutation activates the pi subunit ...... 174
7.2 Integrin activation affects different aspects of pi null fibroblast behaviour. 175
7.3 Integrin activation by expression of the T188I subunit or treatment with
TS2/16 influences keratinocyte differentiation ...... 176
7.4 Mutation of the A domain of the pi subunit is not a common event in human
SCC...... 177
7.5 General Discussion ...... 178
APPENDIX 1: DETAILS OF pi MUTATION SCREEN...... 181
REFERENCES...... 185 Table of Figures
F ig 1.1: S t r u c t u r e o f t h e a v s u b u n it ...... 22
F ig 1.2: S t r u c t u r e o f t h e p3 s u b u n it extracellular d o m a in ...... 27
F ig 1.3; C onformational c h a n g e s in t h e I d o m a in o f t h e o 2 s u b u n it ...... 32
F i g 1.4: KERATINOCYTES EXPRESSING ENDOGENOUS W T HUMAN INTEGRIN (RED) AND Y PRF MUTANT
CHICK INTEGRIN (GREEN) SPREAD ON COLLAGEN ...... 38
F ig 1.5 : S c h e m a t ic s t r u c t u r e a n d c o m p o n e n t s o f (A ) f o c a l c o n t a c t s a n d (B ) f ib r il l a r
ADHESIONS...... 39
F ig 1.6 (A ) FA K DEPENDENT AND INDEPENDENT ACTIVATION OF E R K BY LIGATION OF INTEGRINS. (B)
In t e g r in s ig n a l l in g t o A k t t h r o u g h F A K /P I3 K ...... 45
F ig 1.7: S c h e m a t ic representation o f t h e s t r u c t u r e o f h u m a n s k in ...... 47
F ig 1.8: S c h e m a t ic representation o f t h e s t r u c t u r e o f t h e e p id e r m is ...... 48
F ig 1.9 H is t o l o g y o f (A ) N o r m a l h u m a n s k in a n d (B ,C ) s q u a m o u s c e l l c a r c in o m a ...... 52
F ig 1.10 (A) p i HIGH EXPRESSING CELLS ARE FOUND IN CLUSTERS WITHIN EPIDERMIS(B) THE FATE OF
EPIDERMAL STEM CELLS...... 59
F ig 1.11 S u p p r e s s io n o f s u s p e n s io n in d u c e d differentiation b y p i in t e g r in l ig a t io n ...... 62
F ig 1.12 T h e 8 cytoplasmic d o m a in a n d D 1 5 4 A extracellular d o m a in c p l c o n s t r u c t s u s e d
TO EXAMINE THE ROLE OF THE P l SUBUNIT IN REGULATION OF DIFFERENTIATION (LEVY ETAL,
2 0 0 0 )...... 64
F ig 2.1 E l u t io n o f V n f r o m h e p a r in s e p h a r o s e c o l u m n ...... 78
F i g 3.1 : E x p r e s s io n o f t h e c p l s u b u n it in t h e S C C 4 c e l l s ...... 100
F ig 3.2: (A) SCC4 CELLS INFECTED WITH C pl W T UPREGULATE THE KERATINOCYTE DIFFERENTIATION
MARKER INVOLUCRIN...... 101
F ig 3.3 : T h e S C C 4 c e l l l in e e x p r e s s e s e q u iv a l e n t l e v e l s o f p l in t e g r in t o S C C 1 2 B 2 ...... 103
F i g 3.4 SCC4 IS CAPABLE OF FORMING FOCAL ADHESIONS ON COLLAGEN COATED SURFACES ...... 103
F ig 3 .5 C l u s t e r in g o f e n d o g e n o u s p l o c c u r s w h e n S C C c o n t a in in g e it h e r W T o r Y P R F c p i
CONSTRUCTS ARE PLATED ON JG 22 (ANTI-CPl ANTIBODY)...... 105
F ig 3 .6 F r a g m e n t s o f t h e p i c D N A g e n e r a t e d f o r s e q u e n c in g s h o w in g p r im e r s u s e d ...... 106 Table of Figures
F ig 3.7C l u s t a l W alignm ent of the A domain of a ll eight known p s u b u n i t s 114
F ig 4.1 : T h e T 1 881 MUTANT p i s u b u n it e x h ib it s a g r e a t e r a f f in it y f o r fibronectin c o a t e d
SURFACES THAN THE WT SUBUNIT...... 118
F ig 4.2: PBABE-PURO retro v iral vector showing position of c p l i n t e g r i n CDNA AND THE
POSITION OF THE BASES CORRESPONDING TO MUTATION T1881...... 119
F ig4 .3 T h e T1 881 m u t a n t s u b u n it f o r m s is e x p r e s s e d o n t h e s u r f a c e o f t h e c e l l a n d is
CAPABLE OF FORMING FOCAL ADHESIONS...... 121
F ig 4.4: Adhesion of GD25 cells expressing WT (GD25WT), T188I c p l (GD25 T188I) OR
EMPTY VECTOR (GD25) TO ECM PROTEINS...... 124
F ig 4.5: SCC4 SHOWS GREATER ADHESION TO (A) FN AND (B) COL THAN SCC13, A WT/WT
UNDIFFERENTIATED CELL LINE. (C) THE HUTS21 Pl INTEGRIN ACTIVATION REPORTER EPITOPE
IS EXPRESSED ON S C C 4 IN THE PRESENCE OF M n 2+. IT IS ALSO PRESENT ON THE POSITIVE
CONTROL LINE M0LT4 BUT NOT ON NORMAL KERATINOCYTES...... 127
F ig 4.6 T h e C-C loop (red) bearing T1 881 (white) lies adjacent to the ligand binding site
(LIGHT BLUE) ON THE SURFACE OF THE Pl A DOMAIN...... 130
F ig 4.7: (A) Theoretical model of a v p i p p r o p e l l e r a n d p s u b u n i t A domain bound to an
RGD PEPTIDE (PRODUCED BY SWISS-MODEL BASED ON 1L5GB)...... 131
F ig 5.1: Expression of T1 881 c p i causes an increase in the rate of spreading of GD25 c e l l s . .
...... 141
F ig 5.2: T h e e f f e c t o f W T o r T 1 881 c p i o n t h e m o v e m e n t o f p i n u l l fibroblasts ...... 142
F ig 5.3: The effect of WT o r T1 881 c p l expression on GD25 movement through transw ells
IN THE PRESENCE OR ABSENCE OF MATRIGEL...... 147
F ig 5.4: ERK PHOSPHORYLATION IN Pl WT AND T1881 EXPRESSING GD25 CELLS...... 149
F ig 5.5(A) Expression of the T1881 c p i does not inhibit grow th more than the WT ...... 150
F ig 5.6: INVOLUCRIN expression in SCC4 CELLS RETROVIRALLY INFECTED WITH THE WT, T1881 CPl
SUBUNITS OR EMPTY VECTOR...... 152
F ig 5.7:CORNIFIN EXPRESSION IN SCC 4 CELLS RETROVIRALLY INFECTED WITH THE WT OR T1881 C Pl
SUBUNITS OR EMPTY VECTOR...... 153
11 Table of Figures
Fig 5.8: Expression of the WT o r T188I cpl constructs does not induce differentiation in
EITHER UP OR SCC12B2...... 155
Fig 5.9 : The activating mAb TS2/16 caused a decrease in the num ber of involucrin positive
KERATINOCYTES IN ADHERENT CULTURES AFTER INCUBATION FOR 48 HR ...... 158
Fig 5.10 Incubation of keratincoytes in suspension w ith TS2/16 reduces suspension induced
INVOLUCRIN e x p r e s s io n BY THE SAME DEGREE AS FN ...... 159
Fig 6.1: (A) Example of SSCP traces. Upper trace is sample 51 carrying the 776 O T A239V.
M utation. Lower trace is WT c o n t r o l . (B) Sequencing results of the same samples.
...... 170
Fig 6.2: Staining of pi subunit in SCC samples w ith the (A) 776 C>T A239V, (B) 471 O T
AND (C) IVS2-36 O A m utations. (D) HUTS-21 STAINING OF THE SCC CONTAINING THE 776
C>T A239V MUTATION...... 171
Fig 7.1 The inverse relationship between adhesion and differentiation observed in
KERATINOCYTES...... 180
12 List of Tables
T a b l e 1.1: Human integrin dimers divided by subunit composition showing typical ligands
AND CELLS ON WHICH THEY ARE EXPRESSED...... 18
T a b l e 1.2 : Epiderm al Phenotypes of integrin transgenic mice (W att, 2 0 0 2 ) ...... 56
T a b l e 2.1: Antibodies used in this thesis ...... 83
T a b l e 2.2: SDS-P AGE g e l r e c i p e ...... 88
T a b l e 2 .3 Prim ers used in this thesis...... 95
T a b l e 3.1 : Sequence changes found in the SCC4 p i cDNA f r a g m e n t s ...... 107
T a b l e 5.1 Antibodies screened for an ability to influence differentiation in cultured
KERATINOCYTES...... 156
T a b l e 6.1 Summary of sequence changes found ...... 168 List of Abbreviations
Abbreviation Definition
AM12 Gag, pol +env AM 12 packaging cells BSA Bovine serum albumin cPl Chick pi integrin CD Cluster of differentiation antigen Col Collagen DCS Donor calf serum DMEM Dulbecco’s modification of Eagle’s medium DMSO Dimethyl sulphoxide ECM Extracellular Matrix EOF Epidermal growth factor FACS Fluorescence activated cell sorter FAD FI 2 +adenine +DMEM FCS Foetal calf serum Fn Fibronectin hpl Human pl integrin fflCE Hydrox y cortisone, insulin, cholera enterotoxin and EOF HRP Horseradish peroxidase IVS Intron Variable Sequence kDa Kilo dalton Ln Laminin mAb Monoclonal antibody MAPK Mitogen activated protein kinase MT Mutant pAb Polyclonal antibody PBS Phosphate buffered saline S.D. Standard deviation TBS Tris buffered saline Vn Vitronectin VWF Von Willebrand Factor WT Wild-type Chapter 1
Introduction
Overview
The importance of the pi family of integrin extracellular matrix receptors in regulating the terminal differentiation of human kératinocytes has been known for many years. In my thesis I describe the effects of an integrin mutation on the behaviour of a cell line derived from a human squamous cell carcinoma. This introduction will begin with an overview of integrins and then proceed to discuss their role in the skin and how they regulate keratinocyte behaviour.
1.1 Integrins
Integrins are a family of cation dependent extra cellular matrix (ECM) receptors. An integrin consists of a heterodimer of a single a and p subunit (Hynes, 1992). Each subunit is a large (>100kDa) integral membrane glycoprotein (Tamkun et al, 1986).
Integrins have been implicated in a diverse range of processes including platelet aggregation, phagocyte adhesiveness, leucocyte extravasation and angiogenesis
(Hynes, 1987; Hynes, 1992). In all of these cases the essential property of the integrin is its ability to bind extracellular proteins and mediate the adhesion of the cell. The term integrin was coined denote its role as an integral membrane complex involved in the transmembrane association between the extracellular matrix and the cytoskeleton'' (Tamkun et al, 1986).
15 Chapter 1 Introduction
Integrins are amongst the most widespread family of proteins known with every nucleated human cell type expressing at least one family member. Integrin like proteins are found in many organisms. Drosophila has 4 a (aPSl, aPS2, aPS3 and av) and 2 P (PPS and Pv) subunits (Gotwals et al, 1994). These show changing patterns of expression during development. Their functional importance is shown by the effects of mutations of pPS {lethal myospheroid), aPSl {multiple edematous wings) and aPS2 {inflated) which result in embryonic lethality due to defects in muscle junctions (Gotwals et al, 1994). Two integrins homologous to a5 and av have been found in C. elegans (Sulston et al., 1992). Even primitive organisms such as the sponge have genes that have high degrees of similarity to mammalian integrins
(Wimmer et a l, 1999). The importance of integrins, particularly the pl family is further illustrated by the pi knockout phenotype which is lethal prior to implantation of the embryo (Fassler and Meyer, 1995; Stephens et a l, 1995).
Integrins function by binding to defined motifs in their ligands (such as the RGD or
REDV motifs in fibronectin, (Pierschbacher and Ruoslahti, 1984; Humphries et al,
1986a; Sonnenberg, 1993)) and clustering into complexes such as focal adhesions, focal contacts or hemidesmosomes where they associated with either the actin or keratin cytoskeleton. Signalling molecules can be recruited to their cytoplasmic tails and lead to cytoskeletal rearrangement and signalling (outside in signalling)
(Giancotti and Ruoslahti, 1999; Schwartz, 2001; Schwartz and Ginsberg, 2002).
Integrins can also modulate their affinity for ligands in response to signals from the cell (inside out signalling) by cytoplasmic proteins binding the integrin tails and propagating conformational changes up the structure to the ligand binding domain
16 Chapter 1 Introduction and by clustering (Yauch et al, 1997; van Kooyk and Figdor, 2000; Hogg and
Leitinger, 2001).
1.1.1 Integrin families
Integrins can be loosely divided into families depending on their subunit composition
(Hynes, 1992) (Tablet. 1). While this is a convenient division it does not show the complexity of the tissue distribution and ligand specificity of the different dimers, nor does it necessarily equate with their function.
Alternate Ligands Cell types(examples) Nomenclature a l CD49a/CD29 Collagen Fibroblasts, Activated T&B VLA-1 lymphocytes a2 CD49b/CD29 Collagen Fibroblasts, Platelets, VLA-2 Endothelial and Epithelial cells. Monocytes a3 CD49c/CD29 Laminin Fibroblasts, Epithelial cells. VLA-3 a4 CD49d/CD29 Fibronectin, VCAM-1 M elanocytes, Lymphocytes, VLA-4 monocytes pl a5 CD49e/CD29 Fibronectin Fibroblasts, Endothelial and VLA-5 epithelial cells, monocytes a6 CD49f/CD29 Laminin Monocytes, T lymphocytes, VLA-6 platelets a l Laminin Muscle a8 Fibronectin, Tenascin Neurons, Epithelial cells a9 Tenascin Muscle, hepatocytes av CD51/CD29 Fibronectin, Fibroblasts vitronectin The pl integrin family, ligands and typical cells on which they are expressed
17 Chapter 1 Introduction
Alternate Ligands Cell types(examples) Nomenclature p i CD51/CD29 Fibronectin, Vitronectin Fibroblasts P3 CD51/CD61 Fibrinogen, vWFA, Fibroblasts, Platelets, Thrombospondin, Endothelial and Epithelial cells. av Vitronectin, Monocytes P5 Vitronectin Epithelial cells. Fibroblasts P6 Fibronectin, Epithelial cells P8 Fibronectin, Laminin Sensory neurones The av integrin family, ligands and typical cells on which they are expressed.
Alternate Ligands Cell types(examples) Nomenclature aL CDlla/CD18, ICAM-1,2,3 All leucocytes LFA-1 aM GDI lb/CD 18, ICAM-1, Fibrinogen, Monocytes, Granulocytes, Mac-1, CR3, IC3b, Heparin CD8+ve T cells. NK cells Mol P2 aX CD11C/CD18, Fibrinogen, IC3b, EPS Monocytes, Granulocytes, CR4, LeuM5 activated B lymphocytes, NK cells aD ICAM-3 Monocytes, Granulocytes, most lymphocytes The P2 integrin family
Alternate Ligands Cell types(examples) Nomenclature P7 a4 LPAM-1 Fibronectin, VCAM-1, T & B lymphocyte Made AM-1 subpopulations P3 Fibrinogen, Platelets alip GpIIb/IIIa Fibronectin, vWFA a6 P4 Laminin Epidermal Kératinocytes aE P7 HML-1 E-Cadherin Intraepithélial lymphocytes The “orphan” integrins
Table 1.1: Human integrin dimers divided by subunit composition showing typical ligands and cells on which they are expressed.
18 Chapter 1 Introduction
1.2 Integrin Structure and Function
At its simplest an integrin dimer resembles a globular head and pair of stalks leading to the plasma membrane. This structure was first seen by electron microscopy
(Nermut et a l, 1988). The recent solution of the avp3 extracellular domain crystal structure has given a clearer picture of the structure of an integrin (Xiong et a l,
2001). The receptor is a large multi-domain structure capable of undergoing radical conformational changes which allows it to react both to signals from the cell and from the surrounding matrix (Xiong et al, 2002).
1.2.1 a subunit
The a integrin subunit consists of multiple domains (Fig 1.1). At the extracellular terminus there is a large P propeller structure made up of seven blades of four P sheets (Springer, 1997). Two of these blades (2 and 3) are involved in forming part of the ligand binding site in the majority of integrins (Mould et a l, 2000). Four of these blades (4-7) also contain calcium-binding motifs. These were once thought to be involved directly in ligand binding however it has now been shown that these lie on the underside of the propeller, well away from the regions interacting with either the P chain or the ligand (Xiong et al, 2002).
In a subset of integrins, mostly collagen receptors ( a l, a2) or leucocyte integrins
(aL, aM, aX, aE) the a subunit has an extra 2.00 amino acid domain inserted above the p propeller, between loops 2 and 3 (Zang et a l, 2000). This I domain (for
“inserted”) is a von Wilebrand Factor A domain (Lee et a l, 1995b) and appears to be the sole site of ligand binding in these integrins. The structure of this domain has been extensively studied, indeed it was the first part of the structure to be resolved at \9 Chapter 1 Introduction the atomic level (Lee et al, 1995b). The domain has a MIDAS site (Metal Ion
Dependent Adhesion Site) which mediates ligand binding.
The stalk region of the a subunit comprises a series of 3 P sandwich domains known as the thigh and the calf 1 and 2 domains (Xiong et al, 2001) (Fig 1.1). A single transmembrane region connects the extracellular domains to the cytoplasmic tail of the subunit. In a subset of a subunits the extracellular region is cleaved at a point in the calf 2 domain just before the transmembrane region and the subunit is then held intact by a disulphide bond (a 3,5,6,7,8). a4 also exists in a cleaved form («4 80/70) however this cleavage is further out at the base of the propeller domain (Rubio et a l,
1992; Teixido et a l, 1992). A truncated form of a6, a6p, has also been reported which is synthesised from a normal, full length, a6 mRNA (Davis et a l, 2001). This smaller form of a6 lacks the first 13 exons including the ligand binding domains.
The authors suggest that it is formed either by post-transcriptional modification of the full length a6 or that it results from translation of the mRNA starting from a second internal translation start site. The role of this form is not well understood but it appears to be upregulated in differentiating kératinocytes and it has been suggested to be a dominant negative form of the subunit involved in down regulating cell adhesion (Davis et al, 2001).
The cytoplasmic domains of the a subunits are not highly conserved although all start with a KXGFFKR motif. The tails range from the 67 amino acid a l to the 15 amino acid a l. The variation in a subunit tail sequence is thought to be indicative of the presence of a subunit specific binding proteins that allow the different effects seen upon ligation of different dimers that contain the same p subunit. No direct 2Ô" Chapter 1 Introduction interactions between a subunit tails and cytoplasmic components are known except for the high affinity interaction of a4 with paxillin (Liu et al., 1999) and an association with the chaperone calnexin prior to heterodimer assembly (Lenter and
Vestweber, 1994). Sequences in the a subunit tail are thought to negatively regulate integrin activation as experiments have shown that truncation of the a subunit tail can cause activation of the ligand binding activity of the dimer (Hibbs et al, 1991;
Chan et a l, 1992; Bauer et al, 1993; Rabb et al, 1993).
21 Chapter 1 Introduction
p propeller thigh
hinge - r # 'h calf1
calf2
Fig 1.1: Structure of the av subunit, a-helixes in red, p-sheets in yellow, random coil in grey. Based on 1JV2 (Xiong et al, 2001).
22 Chapter 1 Introduction
1.2.2 p subunit
All p subunits have a highly conserved complex structure (Fig 1.2). The subunit
consists of several domains, a ligand binding domain, an Ig fold, several cysteine
rich domains that comprise the “stalk”, a short transmembrane domain and a
cytoplasmic domain (Xiong et al, 2001).
The ligand binding domain is at the amino terminus and is in the form of a von
Willebrand factor A fold (Tuckwell and Humphries, 1997) and will be referred to as
the PA domain. It is also known as the I-like domain due to its similarity to the I
domain found in some a subunits although it contains two insertions that are not
found in the a subunit. The PA domain has been shown to be the domain responsible
for recognising the RGD sequence (Puzon-McLaughlin and Takada, 1996; Tozer et
al, 1996). The domain contains three cation-binding sites: the MIDAS site, which is
the primary site of ligand interaction (Tozer et al, 1996), the ADMIDAS (ADjacent
to MIDAS) site recently shown in the crystal structure (Xiong et al, 2001) and a
third cation, LIMBS, that is acquired upon ligand binding (Xiong et al, 2002). The
MIDAS site is thought to be normally occupied by Mg2+ but can also accept Mn2+
and Ca2+ but these cause changes in conformation leading to changes in ligand binding consistent with changes in activation of the receptor. Mutagenesis of the
side-chains that co-ordinate the cation cause loss of function of the integrin dimer
(Tozer et al, 1996). It is thought that the ADMIDAS site plays a role in stabilising the upper surface of the domain upon ligand binding. The third cation site known as
LIMBS (Ligand-associated Metal Binding Site) is not present in the unliganded integrin but is generated by the conformational changes caused by ligand binding. It
23" Chapter 1 Introduction is then thought to stabilise the ligand bound state (Xiong et al, 2002). Another important area of the ligand binding domain is the specificity loop, a small cysteine bridged loop not found in other vWFA domains which has been shown to control the specificity of both the ligand binding and signalling performed by the receptor. This loop also cross-links to integrin peptide ligands indicating it forms part of the ligand binding surface (Bitan et al, 2000; Yahalom et ai, 2002). Substitution of this loop in pi for the corresponding sequence from p3 has been shown to alter ligand specificity
(Takagi et al, 1997), signalling (Miao et al, 2002) and to influence folding and dimérisation (Takagi et al, 2002). Another region, the 3io helix, is also not found in a subunit 1 domains. An Wkbicme sidechain from this loop fits into the cavity at the centre of the P-propeller of the a subunit and is crucial for subunit dimérisation
(Xiong et al, 2001).
The region that lies below the pA domain is a hybrid Ig fold consisting partly of the
N terminus of the protein and partly from the region that lies C terminal to the PA domain (Xiong et al, 2001). This domain makes extensive contacts across the lower surface of the PA domain. It is thought that there is little movement between these two structures. The extreme N terminus of the protein forms a PSI (Plexu\
Semaphorin and Integrin) domain and this connects the N terminus back onto itself to the EGF-1 domain in the stalk below the Hybrid domain. To date the atomic level structure of this domain has not been determined.
The stalk of the p subunit comprises four EGF like units which together are often referred to as the cysteine rich region as each unit contains multiple internal disulphide bonds (_Shih,«zTd |qs3 ^Calvete et al, 1991). The four units appear to
ÏÂ Chapter 1 Introduction
divide into 2 tandem repeats (1 and 2, 3 and 4). The junction between the two units
inside the tandem repeat appears inflexible and includes a disulphide bridge as well
as H-bonding whereas the interface between units 2 and 3 appears more flexible
(Xiong et al, 2001). In addition to the EGF-like repeats there is a final extracellular
domain known as PTD (Xiong et al, 2001). This comprises a four stranded P sheet and a N terminal helix and has little homology to any other known structure. The
stalk region contains the epitopes for many of the affinity reporter antibodies
(Bazzoni et al, 1995; Luque et al, 1996). These antibodies function by binding to an epitope that only becomes exposed once the integrin undergoes structural rearrangement following ligand binding.
The cytoplasmic domain of P subunits is usually short, around 30-50 amino acids.
Apart from P4 and p8 the cytoplasmic domains of the remaining six known p
subunits are highly conserved, probably reflecting the importance of their role as
ECM receptors. The p4 subunit has a large cytoplasmic domain consisting of an additional thousand amino acids which reflects its role in hemidesmosome formation and connection to the intermediate filament network (Spinardi et a l, 1993). Other p
subunit cytoplasmic domains have 3 groups of conserved residues, normally designated cyto 1, 2 and 3 (Reszka et al, 1992). The region immediately adjacent to the membrane, before the cyto-1 region, has been implicated as a negative regulator
of integrin activation (Hughes et al, 1992). Cyto 1 is a region of 10 amino acids near the membrane. This has been shown to be required for binding of the focal adhesion
components FAK, filamin and a-actinin (Otey et a l, 1993; Schaller and Parsons,
1994; Lewis and Schwartz, 1995; Sharmaet a l, 1995). Cyto 2 and 3 are NPXY motifs. These have been shown to be sites of phosphorylation of the tail and 25 Chapter 1 Introduction disruption of these sites can modulated integrin function, preventing recruitment to focal adhesions (Bodeau et al, 2001; Mulrooney et a l, 2001). All 3 domains are vital for proper function of the integrin, deletion/disruption of any causes the integrin to lose the ability to mediate cell attachment although certain mutations can result in an increased affinity for ligand (Hughes et al, 1992),
26 Chapter 1 Introduction
Hybrid Ig domain
f % A domain (R ■V' S' ,
Cysteine
rich stalk
Specificty Loop
3io helix
Fig 1.2: Structure of the P3 subunit extracellular domain, a-helixes in red, P-sheets in yellow, random coil in grey. Based on 1JV2 (Xiong et al, 2001). The PSI domain is not shown as the atomic structure has not been resolved. Inset is an alternative view of the A domain in the ligand bound form (based on 1L5G (Xiong et al, 2002)) showing the specificity loop, the 3io helix and the LIMBS, MIDAS and ADMIDAS cation sites (blue spheres, left to right)
27 Chapter 1 Introduction
1.2.3 Ligand binding and activation
The heterodimeric structure of integrins is more complex than many other types of
cell adhesion molecule and this increased complexity allows modulation of integrin
function in a more subtle manner than simple up or down regulation at the protein
level. One of the requirements of integrin function in many situations is the necessity
to perform their adhesive functions in a very rapid and specific manner, often in
limited time frames and locations. Integrins achieve this regulation of their activity
through two main methods, receptor clustering and changes in receptor conformation
(Hynes, 1992; van Kooyk and Figdor, 2000; Hogg and Leitinger, 2001).
Integrin clustering is often associated with integrin ligation and this clustering of receptors leads to an increased avidity of the cell for its ligand. As an example
showing the importance of clustering, deletion of the a4 cytoplasmic domain prevents the clustering of a4pl into focal contacts and hence prevents cell
attachment despite not preventing the binding of the truncated integrin to VC AM-1
(Yauch et al, 1997). In this case the clustering of the receptor appears to be the important step in regulating the interaction between the cell and the ECM. In other cases structural rearrangements in the receptor itself lead to changes in affinity for ligand. The platelet integrin allbp3 exists in a low affinity form on unactivated platelets. In this state it is capable of binding to immobilised fibrinogen but not to any of its soluble ligands. This means the platelet can only bind to pre-existing sites of clotting. Exposure of the platelet to activating stimuli result in the aIIbP3 receptor switching into a high affinity state capable of binding to soluble ligands (Kieffer and
Phillips, 1990; Phillipset al, 1991). Similar activation events have been observed in
28" Chapter 1 Introduction the p2 integrins (Arnaout, 1990; Larson and Springer, 1990). The multiple
conformational states can be measured by the use of reporter antibodies that
specifically recognise one of the affinity states (Luque et al, 1996) and other
antibodies have been produced that are capable of inducing or stabilising a high affinity conformation (Kovach et al, 1992; Petruzzelli et al, 1995). Different
activating antibodies have also been reported to promote different active states recognising different ligands (Ortlepp et al, 1995)
There is no current consensus of whether the conformational shift or receptor clustering is the initial event in integrin activation. It is possible that ligand binding to activated, high affinity integrins causes recruitment of further receptors resulting in clustering. It is also conceivable that the initial interactions are between low affinity integrins and ligand and that clustering of these receptors results in changes in conformation leading to strengthening of the interaction due to increases in both avidity and affinity. The two mechanisms of integrin adhesion regulation are likely to have a synergistic effect with conformational changes increasing individual integrin-ligand interactions and integrin clustering increasing the avidity of the interaction and supporting cell attachment. It is also likely that whether integrin clustering or changes in conformation is the dominant factor in activation varies depending on the integrin dimer in question.
The crystal structures of individual integrin domains have helped to reveal the changes in conformation that occur on activation and ligand binding. Comparisons of the crystals ofa subunit I domains in the presence of Mg2+ and Mn2+ (Lee et a l,
1995a; Qu and Leahy, 1996) and collagen ligands (Emsley et al, 2000) as well as
1 3 Chapter 1 Introduction mutants that lock the domain in either the low or high affinity conformation
(Shimaoka et al, 2000; Shimaoka et al, 2001) have provided a mechanism for regulation of I domains. The crystals of the extracellular portion of the avP3 in the presence of an RGD peptide have shed light on how changes are transmitted throughout the structure (Xiong et al, 2002).
Information on the mechanisms of regulation of integrin activation has also been
obtained through studies using antibody epitopes that either cross the boundary between the a and P chains (Zang et al, 2000), block function (Greve and Gottlieb,
1982; Mould et al, 1997; Shihet al, 1997; Mould et al, 2000), report affinity
(Bazzoni et al, 1995; Lu et al, 2001a; Mould et al, 2002) or activate the integrin
(Faull et al, 1996). Other experiments involved the use of ligand mimetic peptides to map the critical residues in the ligand binding domain (Humphries et al, 2000;
Mould et al, 2000). These results led to a number of models of different regions of the integrin structure being proposed (Huang and Springer, 1997; Tuckwell and
Humphries, 1997; Huang et al, 2000a; You et al, 2002).
The quaternary structure of the dimer has recently been resolved in both the ligand bound and an unbound form for the avp3 dimer (Xiong et al, 2001; Xiong et al,
2002). The interaction of the p subunit A domain and the p propeller domain closely resembles the structure of the G-protein a/p subunit interface with the 3io helix of the pA domain located in the gap in the axis of the P-propeller domain (Xiong et al,
2001). The two avP3 crystal structures only included the extracellular domains of avp3. Because of this the effect that the ligand induced conformational change has on the cytoplasmic domains is still not well understood. The role of the cytoplasmic 3Ô" Chapter 1 Introduction domains in conducting signals both into the cell and out to the ligand binding domains is crucial in understanding integrin function (Takagi et al, 2001).
In integrins where an a subunit I domain is the principal site of ligand binding the I domain undergoes some very specific changes upon activation (Fig 1.3). In the al I domain a single turn of helix is transferred from helix C to helix a6 which allows the opening of the ligand binding domain (Emsley et al, 2000). The MIDAS cation moves, its co-ordination sphere changes and it interacts directly with the ligand.
Most notably small changes in other regions of the structure result in a large movement of the C terminal al helix which drops downwards by 10 Â in the liganded structure (Emsley et al, 2000). Mutated domains where this helix has been restrained in either the up or down position show that this movement is capable of activating or inactivating the entire I-domain (Lu et al, 2001b; Shimaoka et al,
2001). The movement of the al helix is thought to allow the conformational transition to be passed down the structure. Recent work has shown that a sequence bottom of this domain acts as a ligand for the P subunit A domain and that the interplay between these two domains is vital for function of the integrin (Alonso et al, 2002).
31 Chapter 1 Introduction
Fig 1.3: Conformational changes in the I domain of the a2 subunit (after (Emsley et al, 2000)). On ligand binding the cation moves towards helix a5, the aC helix unwinds and transfers a loop to a6 and the a l helix is displaced downwards and away from a l. a-helix in red, P sheets in yellow and random coil in grey. Cations shown as blue spheres. Prepared using SWISSPdB viewer ft-om structures lAOX (left) and IDZI (right).
32 Chapter 1 Introduction
In subunits lacking an a subunit I domain the principal site of ligand binding is the p subunit A domain. This domain is similar in structure to the a I domain, however activation appears to occur in a different manner. In the p A domain the major events are rearrangement of the cation binding sites with the MIDAS site becoming occupied and co-ordinating with the ligand (Xiong et al, 2002). The LIMBS cation binding site forms and becomes occupied to stabilise the conformation and the region containing the specificity loop moves towards the MIDAS site (Xiong et al, 2002).
It has also been suggested that the a l helix may become displaced in a similar fashion to the a l movement seen in a subunit I domains (Mould et al, 2002).
Sequences in the second and the third repeats of the a subunit P-propeller domain play a role in defining ligand specificity by interacting with “synergy sequences” in integrin ligands that surround the RGD motif (Mould et al, 1997; Humphries et al,
2000; Mould et al, 2000)..
In addition to the local changes around the ligand binding sites the integrin undergoes large scale structural rearrangement. The inactive form of the integrin seen in the avP3 crystal is bent back on itself at two hinge regions, one in each subunit (Xiong et al, 2001). On activation the integrin is thought to straighten into the classic “globular head with stalks” conformation seen in electron-micrographs
(Nermut et al, 1988; Beglova et al, 2002). This switch exposes a large region of the stalk, which is hidden in the closed conformation, and many activation reporter antibodies bind to epitopes in this region (Luque et al, 1996). It is likely that this conformational transition is passed through the membranes and leads to relative changes in the position of the cytoplasmic domains and thus influences integrin signalling. 33" Chapter 1 Introduction
1.3 Integrin Interacting Proteins, Signalling and Focal
Adhesion Assembly
Integrins are capable of assembling large complexes of proteins through interactions mediated by their cytoplasmic, transmembrane and extracellular domains. These complexes function not only as structural junctions between the cell and ECM but also control complex signalling systems allowing the cell to respond to changes in its environment.
1.3.1 Transmembrane and extracellular Integrin interacting proteins
Integrins mediate several processes through cis-interactions with other membrane proteins. Caveolin has been shown to interact with a subset of a subunits (al,a5,ocv) via the integrin transmembrane domains. Its ability to bind Src family kinases, such as Fyn, allows a signalling pathway to ERKl/2 to be activated (Wary et al, 1998).
This pathway is associated with caveolin’s ability to bind to cholesterol and glycosphingolipids and organise membrane rafts that are rich in the Src family members which are both myristolated and palmitoylated such as Fyn, Yes and Lck
(Harder and Simons, 1997). Integrins which are in the active conformation have been shown to interact with these lipid rafts in certain cell types (Leitinger and Hogg,
2002).
lAP (CD47) is a large 5 transmembrane Ig family protein which is widely expressed.
It has been shown to act as a receptor for thrombospondin. It interacts with a2pl
(Wang and Frazier, 1998) and avP3 (Lindberg et al, 1996) and appears to act to regulate cell migration by signalling through pathways involving FAK and heterotrimeric G proteins. 3? Chapter 1 Introduction
Many a integrin subunits form highly stable interactions with members of the tetraspanin or transmembrane-4 superfamily (TM4SF). These interactions are mediated through the one of the extracellular loops of the tetraspanin and the extracellular domain of the a subunit although the exact point of interaction seems to differ in different tetraspanin/integrin complexes (Hemler et al, 1996; Yauchet al,
2000). The cytoplasmic domains of the tetraspanins are associated with signalling pathways involving PI-4-K and PKC and have been implicated in processes including cell spreading, migration, MMP induction and differentiation in different cell types (Hemler, 1998; Sugiura and Berditchevski, 1999; Stipp and Hemler, 2000;
Berditchevski, 2001),
1.3.2 Integrin cytoplasmic domain binding proteins.
The interactions between integrin cytoplasmic domains and their associated adapter proteins has been extensively studied. However because of the complex nature of these interactions, coupled with the fact that many of them appear to be transient, rely on phosphorylation by other proteins, or require multiple proteins to be present to be stable, they are not fully understood. One of the best characterised is that of paxillin with the a4 cytoplasmic domain (Liu et al, 1999; Liu and Ginsberg, 2000).
In this case the interaction is high affinity, allowing the two proteins to be co- immuno-precipitated. Paxillin is also thought to be capable of forming a weaker interaction with some |3 subunits and interacts with a large number of proteins present in the large complex which assembles around the P subunit tail (Turner,
2000). a-actinin, filamin, tensin and talin are all structural components of the focal
35 Chapter 1 Introduction adhesions which interact with the p subunit tail (reviewed in (Petit and Thiery,
2000)).
Other proteins that interact directly with the p cytoplasmic domain have been identified by yeast two hybrid screens; ICAP, RACK-1, caspase-8 and ILK. ICAP has been shown to interact with the region between the cyto-2 and 3 motifs of pi (Chang et al, 1997; Zhang and Hemler, 1999). Overexpression of ICAP inhibits cell spreading but this is reversed by overexpression of consitutively active Cdc42.
ICAP interacts with both cdc42 and Rac as an inhibitor of GDP dissociation (Degani et al, 2002).
RACK-1 interacts with an area just before the cyto-1 motif and has been shown to co-immuno-precipitate with p subunits under certain conditions (Liliental and
Chang, 1998). RACK-1 is an adapter protein for the active form of PKC and recruits activated PKCaand e (Liliental and Chang, 1998; Besson et al, 2002). PKC isoforms are involved in processes governing transport of p subunits to the membrane (Ng et al, 1999). PKCa may interact directly with some p subunits through the NPXY motifs in the pi tail (Parsons et al, 2002).
Caspase-8 is reported to bind directly to the membrane proximal region of unligated pi and p5 subunits (Stupack et al, 2001).
ILK (Integrin Linked Kinase) was initially identified as a kinase that binds directly to the pi tail (Hannigan et al, 1996). ILK has been implicated in processes including anchorage dependent growth (Attwell et a l, 2000), suppression of apoptosis (Persad
36 " Chapter 1 Introduction et al, 2001) and tumor^genicity (Marotta el al, 2001). Over expression of ILK induces an epithelial/mesenchymal transition in mammary epithelial cells (Somasiri et al, 2001). There is also evidence that ILK can promote P-catenin signalling (Tan et al, 2001). The kinase activity of ILK does not appear to be essential for its function in integrin signalling (Zervas and Brown, 2002) and it is now thought to act primarily as an adapter molecule (Tu et al, 1999; Guo and Wu, 2002).
The kinase FAK (Focal Adhesion Kinase) has also been reported to bind to the pi tail (Lewis and Schwartz, 1995; Hannigan et al, 1996) and has a role in focal adhesion assembly/disassembly and in integrin mediated signalling .
37 Chapter 1 Introduction
1.3.3 Focal Adhesions
Focal adhesions were first identified in the 1970s as regions of the cell that were in close contact with the substratum (Abercrombie et al, 1971; Couchman and Rees,
1979). These are large protein structures that assemble around integrin tails to mediate adhesion and motility. Focal adhesions can be divided into the two types: focal contacts and fibrillar adhesions (Zamir el al, 1999). Focal contacts are the
“classical” focal adhesion, enriched in scaffolding proteins such as talin and paxillin and are highly tyrosine phosphorylated. Fibrillar adhesions are enriched for tensin, another scaffold protein, and are less highly phosphorylated.
Fig 1.4: Kératinocytes expressing endogenous WT human integrin (Red) and YPRF mutant chick integrin (Green) spread on collagen. WT integrin forms large focal adhesions. The YPRF disrupts the cyto-2 motif in the pi tail and this prevents recruitment of the chick subunit to these focal adhesions.
38 Chapter 1 Introduction
A Integrin binding to rigid matrix
Focal contact forms
under generation of
tension from actin
cytoskeleton
Integrin binding to deformable
matrix
Fibrillar adhesion forms
as matrix deforms
Structural Kinases/Adapter
Src/Fyn O Tensin Paxillin o
a-actinin F-actin
Vinculin
Fig 1.5 : Schematic structure and components of (A) focal contacts and (B) fibrillar adhesions.
39 Chapter 1 Introduction
Focal contacts
The structure of the focal contact (Fig 1.5A) is provided by a series of scaffolding proteins such as talin, vinculin, filamin and a-actinin upon which is built a series of signalling systems. Talin is present as a dimer and may associate directly with the P subunit tail. Cells expressing integrins that lack the binding sites for a-actinin and talin are deficient in migration and adhesion (Bodeau et ai, 2001). In culture focal contacts appear to form at the periphery of the cell and can be promoted by plating the cells onto rigid matrixes such as glass bound vitronectin. These contacts apply significant amounts of force onto the matrix generated by myosin II driven cytoskeleton contraction (Zamir et al, 1999).
Fibrillar adhesions
Fibrillar adhesions are more elongated structures (Hynes and Destree, 1978) containing tensin, a protein with an actin filament capping function (Lo et al, 1994;
Chuang et al, 1995) (Fig 1.5B). These adhesions also appear to be simpler, and not to involve as many other proteins as the talin containing focal contacts (Katz et a l,
2000; Zamir et al, 2000). They form at sites of low matrix rigidity and tend to migrate towards the centre of the cell under the contractile force of the cytoskeleton.
As they move the carry the matrix components bound to the integrins with them. It is thought that this is responsible for generation of the elongated fibrils of fibronectin seen under cells in culture (Katz et al, 2000).
40 Chapter 1 Introduction
1.3.4 Focal adhesion assembly/disassembly
As previously described focal adhesions are large complexes of proteins, the primary role of which is to anchor the cell to the basement membrane. However this strong adhesion has the effect of severely limiting the migration of the cell. In order for effective cell movement the stability of focal adhesions has to be tightly regulated
(Rodriguez Fernandez et al, 1992; Rodriguez Fernandez et al, 1993), allowing the cell to attach to the matrix, apply sufficient force across the adhesion to move the cell body over it then detach from the matrix at the rear of the cell and recycle components to the leading edge.
The initial step of focal adhesion formation is clustering of integrin subunits. This process is highly dependent on the p cytoplasmic tail and is mediated by adapters such as talin binding to the cyto2/3 motifs (Horwitz et al, 1986). Subunits in which these motifs are disrupted are incapable of being recruited to integrin clusters and are present diffusely across the whole membrane surface (Solowska et al, 1989; Hayashi et al, 1990). FAK, talin, tensin, a-actinin and vinculin are recruited to early adhesion structures followed by their associated signalling molecules. Once the focal adhesion has begun to assemble around the clustered integrins the complex associates with actin fibres. This process is regulated by the Rho GTPase and its interactions with pl40Dia, PI-5-Kinase and Rho Kinase (ROCK) ((Ridley and Hall,
1992), reviewed in (Petit and Thiery, 2000; Geiger et al, 2001)). ROCK has numerous targets including myosin phosphatase and myosin light chain.
Phosphorylation of these proteins allows myosin II to interact with F-actin causing contraction of stress fibres. The generation of tension through the stress fibre matrix allows the cell to generate contractile force and propel itself over the matrix. This
4 T Chapter 1 Introduction tension is also essential for the proper formation of the focal adhesion. Experiments have shown that the type of adhesion that forms, either fibrillar adhesion or focal contact, is directly dependent on the amount of contractile force that the matrix can support. If the matrix is non-rigid little tension is exerted on the focal adhesion as the cell deformations the matrix. If the matrix is rigid the force across the focal adhesion increases and this leads to recruitment of proteins such as talin and vinculin which stabilise the structure (Katz et al, 2000; Zamir et al, 2000; Riveline et al, 2001).
The degree of phosphorylation seen in the structure also appears to be dependent on the degree of tension (Jockusch et a l, 1995; Zamir et al, 1999).
At the rear of the cell many focal adhesions appear to physically break off from the cell and remain in contact with the matrix. However most focal adhesions are disassembled at the rear of the cell and their components recycled. Deactivation of
Rho has been shovm to lead to focal adhesion disassembly. This can be triggered by phosphorylation of Rho by PKA (Lang et al, 1996). Activation of Cdc42/Rac effectors such as N-WASP and PAK (p21 activated kinase) also induce stress fibre and focal adhesion disassembly by inactivating myosin light chain kinase.
Activation of tyrosine kinases has also been linked to focal adhesion disassembly. V-
Src transformed cells show increased phosphorylation of focal adhesion proteins but show a rounded morphology and disorganised focal contacts (Petit and Thiery,
2000). Phosphorylation of Src targets such as FAK and paxillin can also lead to their degradation suggesting that Src is important both in focal adhesion disassembly as well as signalling (Petit and Thiery, 2000).
42 Chapter 1 Introduction
1.3.5 Integrin Signalling
Unlike many other cell surface receptors integrins have no intrinsic kinase activity.
For many years it was thought that, because of this, they had no signalling function beyond their role in cell adhesion and focal adhesion formation. It is now understood that, by recruiting a range of adapter proteins and kinases through interactions with both extracellular and cytoplasmic domains, integrins are involved in many different signalling pathways (reviewed in Giancotti and Ruoslahti, 1999; Schwartz, 2001;
Schwartz and Ginsberg, 2002).
One of the major kinases involved in integrin mediated signalling is the Focal
Adhesion Kinase (FAK). As its name suggests FAK plays an important role both in focal adhesion formation and disassembly but it is also involved in several signalling cascades leading from the integrin. One of the best characterised roles for FAK is in
FAK mediated MAPK activation (Fig 1.6A). FAK is initially recruited to focal adhesions through its interactions with talin and vinculin (Chen et al, 1995) and possibly the p subunit tail (Lewis and Schwartz, 1995). Auto phosphorylation of
Y397 activates FAK as a tyrosine kinase. This allows binding of the membrane bound kinase Src via an SH2 domain interaction with Y397 (Schaller et al, 1994;
Schlaepfer et al, 1994). Further phosphorylation events lead to the recruitment of the adapter proteins such as CAS and CRK leading to activation of the JNK pathway.
Recruitment of the Grb2/S0S complex leads to activation of the ERKl/2 MAP
Kinase via Ras and Raf (reviewed in Giancotti and Ruoslahti, 1999).
Integrin mediated ERKl/2 activation can also occur independently of FAK activation
(Wary et al, 1998) (Fig 1.6A). In this pathway She binds to the Fyn SH3 domain
43" Chapter 1 Introduction leading to phosphorylation of She and recruitment of the Grb2/S0S complex. This allows activation of Ras and triggers the MAPK cascade .The two pathways leading to Ras are thought to act in tandem, She initially triggering the high levels of MAPK activation seen shortly after adhesion and FAK causing the sustained activation following it (reviewed in Giancotti and Ruoslahti, 1999).
Integrin ligation can influence signalling to MAPK stimulated by ligation of the EGF receptor. Studies have shown that in the absence of integrin ligation EGF induced signalling is largely inhibited (Miyamoto et al., 1996), in contrast integrin derived signalling to MAPK can occur in the absence of EGF, albeit at lower levels than when growth factors are present. Other work has suggested an association between the EGF receptor and various integrins (Miyamoto et al, 1996). It has been shown that co-clustering of the EGF receptor and the a2pl integrin at sites of cell-cell contact induce tyrosine phosphorylation and activation of the EGF receptor in the absence of its ligand (Moro et al, 1998; Yu et al, 2000). EGF receptor signalling uses many of the same components found in integrin mediated signalling pathways, such as She, Grb and SOS so a synergistic role for these two pathways is likely. In addition the PDGF (Schneller et al, 1997; Woodard et al, 1998) and VEGF (Soldi et al, 1999) receptors have also been reported to form complexes with integrins.
PI-3-Kinase plays a role in integrin signalling downstream of FAK (Fig 1.6B). This lipid kinase is an important regulator of adhesion and migration and activates the anti-apoptotic protein kinase Akt (Chen et al, 1996). PTEN, a tumour suppressor lipid/protein phosphatase acts as a negative effector on these pathways, acting to reduce phosphorylation in the absence of integrin activation (Parekh et a l, 2000).
4? Chapter 1 Introduction
A
SRC phosphorylates CAS Caveolin
> Rac Grb/SOS
Fyn phosphorylates She FAK Auto-P Y397 and binds SRC and Grb/SOS JNK
RAS B
ERK
PTEN FAK
PI3K
Fig 1.6 (A) FAK dependent and independent activation of ERK by ligation of integrins. (after (Giancotti and Ruoslahti, 1999)) (B) Integrin signalling to Akt through FAK/PI3K. PTEN is thought to act as a repressor on this pathway. 45 Chapter 1 Introduction
1.4 Structure and Function of the Skin
Human skin consists of several layers (Fig 1.7). The outermost layer is the epidermis.
Below the epidermis lies a basement membrane that separates the epidermis from the underlying dermis. The epidermis and the dermis interdigitate, forming the rete ridge and dermal papillae (Fig 1.7).
1.4.1 The Dermis
The most common cells in the dermis are fibroblasts although there are also numerous blood vessels and nerve endings. The dermis can be divided into two layers. The upper layer, the papillary dermis, is a loosely arranged matrix of type I and III collagen fibres (Goldsmith, 1991). Below this lies the reticular dermis, a layer of densely packed collagen I fibres along with filamentous type V and VI collagens.
Elastin is also present in this layer and this provides the elastic strength of the skin
(Goldsmith, 1991). The dermis also contains large amounts of mucopolysaccharides, primarily hyaluronates and dermatan sulphates. These occupy the space between the collagen fibres (Nasemann et al, 1983). Below the dermis lies a layer of subcutaneous fat that separates the skin from the underlying muscles.
46 Chapter 1 Introduction
Hair shaft
Epidermis Dermal 'papilla
Basement membrane
Blood Vessel Rete Ridge Sebaceous gland Smooth Dermis muscle
Apocrine Hair bulb Eccrine gland gland
Nerve Subcutaneous Fat
Fig 1.7: Schematic representation of the structure of human skin.
1.4.2 The Epidermis
The epidermis is the main barrier between the body and the surrounding environment
(Fig 1.8). It is predominantly made up of kératinocytes although smaller numbers of
melanocytes (pigment cells)(Jimbow c/ ai, 1991), Merkel cells (sensory cells) and
Langerhans cells (a type of antigen presenting cell) (Hauser et al, 1991) are also
present. The outer layers of the epidermis consist of tough squames that provide the
barrier function of the skin. Below these are layers of living cells that arise from the
population of dividing kératinocytes in the basal layer. Within the epidermis and
dermis are also found hair follicles, apocrine and eccrine sweat glands and sebaceous
glands (Odland, 1991). These are all derived from the epidermis.
47 Chapter 1 Introduction
— ^ —PT-r—-1 Cornified czzzzzCr--- —--- IT"------, ——!r3L__ 1 Shedding o o o o 3 Granular f \ r N
# # # # V Jk VV Jk------Jk------V Terminal ~s f % r differentiation Cytoplasmic # keratohyaiin # # # # Spinous granules k Jk Jk______/ \ ------/
Basal Proliferation
Basement membrane
Fig 1.8: Schematic representation of the structure of the epidermis.
1.4.2 The basal layer kératinocytes
The cells of the basal layer (Fig 1.8) are in contact with an underlying membrane
comprised of a range of ECM proteins. The most abundant are collagens, especially
type IV collagen (Timpl, 1989; Odland, 1991). In addition there are large amounts of
laminins and other proteoglycans (Burgeson and Christiano, 1997). Fibronectin is not
normally present except in wounded epidermis (Mosher, 1989; Fine, 1994). The
basement membrane components are synthesised both by kératinocytes and dermal
fibroblasts (Marinkovieh et al., 1993). The basal kératinocytes adhere to the basal
membrane through (34 mediated hemidesmosomes and (31 mediated focal adhesions
(Carter et ai, 1990a; Carter et ai, 1990b; Stepp et ai, 1990).
48 Chapter 1 Introduction
1.4.3 The upper epidermal layers
The various layers of the skin can be distinguished by the range of proteins they express.
The spinous layer comprises the first 4-8 cell layers above the basal layer and is characterised by an increase in cell size and an increase in desmosomal junctions between the kératinocytes (Holbrook, 1994). Most suprabasal layers express involucrin (Rice and Green, 1979), a protein synthesised early in the differentiation pathway, which is part of the cornified envelope of a mature squame.
The granular layer is identified by the synthesis of electron dense keratohyaiin granules of loricrin and profilaggrin (Holbrook, 1994). Loricrin is a component of the cornified layer (Mehrel et al, 1990) and profilaggrin is the precursor to filaggrin, a protein involved in the aggregation of keratin filaments (Rothnagel et al, 1987;
Rothnagel and Steinert, 1990).
The final layer of the skin, the cornified layer, consists of flattened squames.
Cornified squames are extremely flat, being approximately 40 p.m in diameter but only 0.5 pm in thickness. These cells have lost their organelles and their nucleus and function as a barrier to desiccation and mechanical damage (Nemes and Steinert,
1999). The contents of a squame are largely keratins surrounded by a 12 nm thick insoluble cornified envelope that is deposited beneath the remains of the plasma membrane (Holbrook, 1994). These squames are continually being shed from the surface of the skin.
49 Chapter 1 Introduction
The cells of all layers adhere to each other in a calcium dependent manner through adherens junctions and desmosomes (Cowin, 1994). Adherens junctions, also known as zonula adherens are found as a belt around the cell and appear as electron dense regions where the membranes of adjacent cells run parallel about 20 nm apart
(Geiger and Ginsberg, 1991). This type of cell-cell junction is found in many tissues including the epithelium (Geiger et al, 1987).
The intracellular section of adherens junctions consists of dimers of cadherin family proteins (Bussemakers et al, 1993; Kemler, 1993). These are transmembrane proteins that form calcium dependent head-to-tail dimers through their extracellular domain with corresponding proteins from adjacent cells. Kératinocytes of all epidermal layers express E-cadherin while P-cadherin is expressed only in the basal layer (Hirai et al, 1989). Adherens junctions link to the actin cytoskeleton through vinculin, a-actinin and catenin family proteins (Kemler, 1993).
Desmosomes link adjacent cells through desmoglein and desmocollin, members of the cadherin superfamily (Schwarzet al, 1990; Kowalczyk et al, 1999). In vivo desmosomes cover a large proportion of the membrane surface of kératinocytes
(Cowin, 1994). Desmosomes are attached to the keratin cytoskeleton through a group of proteins termed plakins. This junction effectively interconnects the keratin cytoskeletons of surrounding kératinocytes into one mesh, increasing the ability of the skin to resist damage.
50 Chapter 1 Introduction
Gap junctions are also present in the kératinocytes. These are small transmembrane channels that permit the transfer of small molecules between adjacent cells (Evans,
1988). These are found in all layers of the epidermis
1.4.4 Squamous Cell Carcinoma
Squamous cell carcinomas (SCC) are common tumours of the skin and epithelia (Fig
1.9). The incidence of SCC is so high and the mortality rate so low that, along with basal cell carcinomas of the skin, they are excluded from the national statistical analysis of cancer cases. The combined incidence of these two types of non melanoma skin cancer was over 40,000 in England and Wales in 1997 and around
20% of these were recorded as SCC. Less than 500 mortalities per year are recorded as being directly due non-melanoma skin cancer (Source: National Office of
Statistics).
SCC most frequently arise from other skin lesions such as actinic keratosis or benign papilloma although they can also form due to exposure to chemical carcinogens or radiation (Nasemann et al, 1983). Most SCC do not show great metastatic potential with less than 3% of patients showing any secondary tumours. The exceptions to this are carcinoma of the oral cavity and especially the lower lip of which 11% are metastatic. SCC induced by chemical or radiation exposure also frequently metastasise (Lever and Elder, 1997).
51 Chapter 1 Introduction mmi%
Fig 1.9 Histology of (A) Normal human skin and (B) squamous cell carcinoma. (C) SCC showing keratin pearls (arrow heads).
52 Chapter 1 Introduction
Treatment is normally by excision followed by chemo- or radiotherapy and survival rates are normally 90% over 5 years if treated early. If the condition is not treated promptly large scale tissue destruction, can occur, especially in oral tumours
(Nasemann et al, 1983). SCC show disruption of the normal skin architecture with islands of kératinocytes present within the dermis and loss or disruption of the basement membrane (Wheater, 1991). The keratinocyte differentiation program is also perturbed. Well-differentiated SCC produce large amounts of keratin and frequently large masses of protein known as keratin pearls can be seen (Fig 1.9C).
Poorly differentiated tumours show reduced keratin expression (Wheater, 1991).
Spindle like cells may be present in more severe undifferentiated tumours (McGee et al, 1992)
1.5 Epidermal Integrins
Kératinocytes express a range of integrin subunits. In vivo a2, a3, a6, a9 and av are expressed along with the pi, P4 and pS subunits (Watt and Hertle, 1994; Watt,
2002). These make up the a2pi collagen receptor, the a3pi laminin receptor, a6P4 laminin receptor, «9pi tenascin receptor and the avPS vitronectin receptor.
Upregulation of aSpi and avp6 is seen in wounded or hyperproliferative epidermis as well as in culture (Adams and Watt, 1991; Watt and Hertle, 1994). avps is also reported to be present (Stepp, 1999). Despite expressing both the a6,av and pi subunits neither a6pi or avpi is expressed in kératinocytes (Adams and Watt,
1991).
In normal, undamaged epidermis integrin expression is restricted to the basal layer of kératinocytes in the epidermis and outer root sheath of the hair follicle. The
53" Chapter 1 Introduction exception to this is avpS which is expressed only in the suprabasal layers of the epidermis (Stepp, 1999). a6p4 is concentrated on the basal membrane of kératinocytes where it forms hemidesmosomes, anchoring the cell to the basement membrane (Watt and Hertle, 1994). Wholemount labelling of basal kératinocytes shows some focal adhesion like clusters of pi integrins interspersed with the hemidesmosomes on the basal surface of the cell but most pi integrins are present in a ring around the cell periphery (Jensen et a l, 1999). Although pi integrins appear to be concentrated at cell-cell borders this is likely to be a result of the heavily interdigitated membranes at these sites rather than due to any role in cell-cell adhesion (Braga et al, 1998). In wounded or hyperproliferative skin integrin expression may be seen in the suprabasal layers of the epidermis (Watt and Hertle,
1994).
Alternative splicing events occur in the integrin subunits expressed by kératinocytes.
The most common pi expressed is PlA although kératinocytes are also one of the few cell types to express piB. This subunit has a unique 12 amino acid sequence that replaces the last 21 amino acids of piA. piB is expressed on the cell surface but does not mediate cell adhesion and acts in a dominant negative manner against pi A function in transfection experiments (Balzac et a l, 1994). The importance of piB is questionable as it is expressed at very low levels in human kératinocytes (Kee et a l,
2000) and is not conserved in the mouse genome (de Melker and Sonnenberg, 1999).
a 6 A is the normal « 6 form expressed in kératinocytes (Hogervorst et a l, 1993).
However the a 6 B subunit is found in some keratinocyte cell lines derived from mouse tumours (Tennenbaum et a l, 1995). p4A is the most common variant 54 Chapter 1 Introduction expressed in kératinocytes although trace amounts of (34B (Hogervorst et al, 1993) and P4E are present. (34E has a shorter (232 amino acid) tail and lacks the structures required for hemidesmosome formation (van Leusden et a l, 1997; de Melker and
Sonnenberg, 1999).
1.5.1 Functions of Epidermal Integrins
The primary function of integrins in the epidermis is to secure the cells of the basal layer to the basement membrane through their interactions with the actin and keratin cytoskeletons. Integrins also play a vital role in mediating keratinocyte migration over ECM (Watt and Hertle, 1994; Watt, 2002). a3pl is required at the leading edge of the cell for keratinocyte migration over laminin (DiPersio et al, 1997; Goldfinger et a l, 1999). In migrating cells a6p4 is present at the leading of edge of the cell where it can be associated with membrane protrusions and show a reduced association with hemidesmosomes on the basal surface (Nguyen et a l, 2000;
Mercurio et al, 2001).
Mutation of specific integrin subunits has confirmed the role of the different subunits in mediating adhesion and migration in vivo (Table 1.2). Deletion of either the a6 or P4 subunits in mice result in a loss of hemidesmosomes, severe blistering of the skin and other epithelia and death die shortly after birth (Dowling et al, 1996;
Georges-Labouesse et a l, 1996; van der Neut et a l, 1996). This closely mimics the human condition junctional epidermolysis bullosa where mutations in the a6p4 integrin result in severe blistering of the skin.
55 Chapter 1 Introduction
Subunit Phenotype Reference a3 Disorganised basement membrane, (DiPersio et al, 1997) occasional blistering. (36 Juvenile hair loss (Huang e ta l, 1996) a9 No defects (Huang et a l, 2000c) (35 Reduced migration of kératinocytes (Huang et a l, 2000b) (31 Floxed p i X K5Cre (Grose et a l , 2002) Abnormal hair follicles, hair loss, blistering, reduced proliferation and abnormal differentiation, disruption of basement membrane, impaired wound healing
Floxed p i X K14Cre (Brakebusch et a l, 2000) Blistering, basement membrane disruption, reduced hemidesmosomes, thin epidermis, reduced hair follicles, reduced a6P4 a 6 Severe blistering (Georges-Labouesse et al, 1996) P4 Severe blistering (Dowling et al, 1996; van der Neut et al, 1996) a3+a6 Blistering (DiPersio et a l, 2000) a2 No defects (Holtkotter et a l, 2002)
Table 1.2 : Epidermal Phenotypes of integrin transgenic mice (Watt, 2002)
Loss of a3 by targeted deletion in mice also causes a disruption of the basement membrane and occasional epidermal blistering on the feet and legs (DiPersio et al,
1997). The double knockout of «3pi and a6P4 does not result in a more severe phenotype than either alone (DiPersio et al, 2000). (x2, a9 and pS knockouts do not result in skin phenotypes (Huang et al, 2000b; Huang et al, 2000c; Holtkotter et al,
2002) although loss of p5 does result in severely impaired migration of kératinocytes
(Huang et al, 2000b).
The pi knockout is early embryonic lethal (Fassler and Meyer, 1995; Stephens eta l,
1995). Mice expressing floxed pi alleles have been generated and crossed with mice
56 ^ Chapter 1 Introduction expressing Cre under the control of either the keratin 5 or 14 promoter. This results in Cre activity in the basal layer of the epidermis (Brakebuschet al, 2000; Raghavan et a l, 2000). These mice suffer from epidermal blistering but not to the degree reported in the a6p4 null mice. Wound healing studies performed on these mice confirm that |3l is essential for keratinocyte migration in vivo (Grose et al, 2002).
Mouse kératinocytes lacking pi due to K5-Cre expression show an increase in the proportion of cells expressing differentiation markers, the number of cells expressing involucrin increased from 1% to 20-40% (Grose et al, 2002). Other than the increase in the number of differentiated cells the differentiation program itself appears to proceed normally. The mice also show a general reduction in proliferative cells in their hair follicles and by 7 weeks lack both hair follicles and sebaceous glands
(Brakebusch et al, 2000). No change in the differentiation program was seen in K14-
Cre mice, however the proportion of differentiated cells was not measured
(Raghavan et al, 2000).
1.5.2 Epidermal Stem Cells.
Epidermal stem cells are a sub-population of kératinocytes that are responsible for renewing the epidermis. The cells of the interfollicular epidermis, the hair follicle and the sebaceous glands all arise from the same population of stem cells (Watt,
2001). Human epidermal stem cells can be identified in culture and in the epidermis by their high expression of the pi integrin subunit (Jones and Watt, 1993; Jensen et al, 1999). High pi expressing cells appear to be clustered into groups at the tip of the dermal papillae and are surrounded by areas of lower expressing cells (Jensen et al, 1999) (Fig l.lOA). In hair follicles a region of high pi expression, called the
i?" Chapter 1 Introduction bulge region, is also thought to contain stem cells (Jones et a l, 1995; Akiyamaet al,
2000). In the mouse various locations for stem cells have been suggested including the bulge region of the hair follicle (Cotsarelis et a l, 1990), the inter-follicular epidermis (Allen and Potten, 1974) and the outer root sheath (Lenoir et al, 1988).
The cells surrounding the areas of high Pl expression are the offspring of stem cells,
-known as~transit ampli^dng-ceUs. These cells have left the stem cell compartment and divide a limited number of times before withdrawing from the cell cycle and differentiating (Watt, 2001)(Fig 1.1 OB). These cells are known as transit amplifying cells. These cells have lower pi integrin levels and are less adhesive to ECM than stem cells (Bickenbach and Chism, 1998).
The high level of pl integrin in human epidermal stem cells has two significant roles in maintaining the stem cell compartment. Expression of a chimera of the CD 8 extracellular domains and the pi integrin cytoplasmic domain has a dominant negative effect on the endogenous pi integrin. Kératinocytes expressing this construct show reduced levels of endogenous pl, reduced pi mediated adhesion and reduced colony forming efficiency in culture (Zhu et a l, 1999). This effect appears to be due to changes in integrin signalling to MAPK, making the cells behave as transit amplifying cells. Expression of a constitutively active MAPK or over expression of WT pi integrin restores normal function in these cells (Zhu et al,
1999). The high level of pi also makes stem cells less migratory than their offspring and helps ensure the segregation of the stem and transit cell compartments (Jensen et al, 1999).
58 Chapter 1 Introduction
B
Basal layer Supra Basal
unlimited <5
Epidermal Transit Commited Terminal
Stem Cell Amplifying Cell Cell Differentiation
integrin expression
Fig 1.10 (A) pi high expressing cells are found in clusters within epidermal whole mounts with the highest expression at the centre of the cluster (inset). Scale bar 100 pm. (Jensen et ai, 1999) (B) The fate of epidermal stem cells. (Zhu et ai, 1999)
59 Chapter 1 Introduction
1.5.3 Integrins and Squamous Cell Carcinoma
SCC, in human and mouse, often show considerable variation in integrin expression both between tumours and between regions of the same tumour. Normal expression, over expression and total or focal loss of the normal keratinocyte integrin subunits has been observed as well as de novo expression of integrins such as avp 6 . (Peltonen et al, 1989; Jones et al, 1993; Jones et al, 1997; Bagutti et a l, 1998).
Changes in expression of a6p4 have been implicated in epithelial carcinogenesis.
Over expression of this integrin in the layers of the tumour that are not adjacent to the stroma is associated with poor prognosis in human disease (Van Waes et a l,
1991) and a high risk of malignant conversion in mouse carcinogenesis
(Tennenbaum et al, 1995). Loss of a6(34 in the layers of tumour adjacent to the stroma is associated with a loss of the basement membrane (Downer et a l, 1993). In many tumours #6^4 expression is maintained but becomes associated with the actin cytoskeleton to promote invasion and migration (Mercurio et al, 2001).
Changes in integrin expression, especially the induction of avp 6 , are often linked to direct changes in cell motility through induction of matrix metalloproteinases
(Thomas et al, 2001).
The expression of integrins in suprabasal kératinocytes also appears to result in changes in tumorigenesis. By expressing integrin subunits under the control of the involucrin promoter mice can be generated that express various integrins in the suprabasal layers of the skin. Expression of integrins in suprabasal kératinocytes
60 Chapter 1 Introduction does not lead to spontaneous tumours. However, after treatment with DMBA to initiate Ras mutations followed by TP A to promote clonal expansion effects of integrin expression on tumour development can be seen. In normal mice benign papilloma appear first, some of these progress to SCC but others spontaneously regress. In mice expressing suprabasal a3|3l the rate of conversion from papilloma to
SCC is reduced. Expression of suprabasal a6p4 increases the formation of both types of tumour. Suprabasal a2pi has no apparent effect on SCC formation (Owens and
Watt, 2001). Although skin tumours are derived from the proliferative cells in the basal layer this shows that the cells surrounding the tumour are capable of influencing the tumours development.
Integrin expression is also linked to differentiation in SCC cell lines. Transfection of av into a cell line lacking this subunit has been shown to restore the ability of the cell to undergo terminal differentiation and inhibits anchorage dependent growth
(Jones et a l, 1996).
1.5.4 Regulation of terminal differentiation
Cultured kératinocytes that are placed in suspension withdraw from the cell cycle and undergo terminal differentiation (Green, 1977; Adams and Watt, 1989). After 24 hours up to 80% of cells will express involucrin, compared to 5-20% in adherent cultures. This process can be partially inhibited by ligation of the a5pl integrin, either by fibronectin or anti-pl antibodies (Adams and Watt, 1989; Watt et a l, 1993)
(Fig 1.11). Monovalent antibody Fab fragments and ROD peptides also cause suppression of suspension induced differentiation showing that receptor ligation
6T Chapter 1 Introduction
rather than clustering is the essential factor (Adams and Watt, 1989). The
suppression of differentiation does not involve polymerisation of the actin
cytoskeleton as cytochalasin D treatment does not prevent the effect (Adams and
Watt, 1989; Watt and Jones, 1993). Addition of integrin ligands can only prevent
differentiation if added within a few hours of the start of suspension culture. After 5
hours the cells have committed to differentiation and cannot then be rescued (Adams
and Watt, 1989).
+ pi ligands or antibodies
O 5 hr Commitment + pl ligands or antibodies
24hr Differentiation
Fig 1.11 Suppression of suspension induced differentiation by pl integrin ligation.
Function of the pl integrin is regulated at two levels during keratinocyte
differentiation. In the initial stages of suspension induced differentiation
kératinocytes become less adhesive due to inactivation of their pi integrin (Hotchin
et al., 1993). This inactivation can be reversed by the use of activating antibodies to
the pl subunit. However, the down regulation of integrin function occurs after the 6 Î Chapter 1 Introduction cell has committed to differentiation and the use of activating antibodies to reverse this does not reverse commitment (Hotchin et ah, 1993). After 24 hours in suspension the |3l mRNA is down regulated leading to loss of the subunit (Hotchin and Watt, 1992; Hotchin et al, 1995).
While there are clearly parallels between suspension induced differentiation and anoikis the two are distinct processes and primary human kératinocytes do not undergo apoptosis in suspension (Gandarillas et a l, 1999). Deletion of pl does not induce apoptosis in the skin (Brakebusch et al, 2000). While there are reports that loss of P4 causes apoptosis (Dowling et al, 1996) a6 null epidermis does not show an increase in apoptotic cells (DiPersio et al, 2000). Although there is evidence that unligated integrins can recruit caspase -8 to the plasma membrane and thereby stimulate apoptosis in adherent cells (Stupack et al, 2001) this is not seen in mice where a variety of integrins are expressed suprabasally in the absence of ligand
(Carroll et al, 1995; Romero et al, 1999; Owens and Watt, 2001).
The role of integrins in the regulation of keratinocyte differentiation has been further investigated by the use of chick pi integrin (cpl) constructs retrovirally expressed in human kératinocytes. The cpl subunit is highly homologous to the human pi subunit, dimerises with the endogenous a subunits and functions identically to the endogenous subunit when retrovirally expressed in kératinocytes (Levy et al, 1998).
Experiments with mutant constructs of the cpl have helped to reveal the specific regions of the pi subunit that are involved in regulation of differentiation (Fig 1.12)
(Levy et al, 2000).
63 Chapter 1 Introduction
DIFFERENTIATION
FOCAL ADHESION e CM SUSPENSION- SCC4 TARGETTING ADHESION INDUCED 3
WT KLLMIIHf^RREFAKFEKEjKMNAKWDTGEfvIPI’ijKSAVTTW^PKY^GK _|_ + 4. +
757 803
YPRF KLLMIIHDRREFAKFEKEKMNAKWDTGEYPRFKSAVTTWNPKYEGK _ —. 4 4 4 4
YTRF KLLMIIHDRREFAKFEKEKMNAKWDTGEYTRFKSAVTTWNPKYEGK — — + + + +
N797I KLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTWIPKYEGK 4 4 * * II + + + KLLMIIHDRREFAKFEKEKMNAKWDTGEIPIYKSAVTTWIPKYEGK _ __ + + + Y788A/ KLLMIIHDRREFAKFEKEKMNAKWDTGENPIAKSAVTTWIPKYEGK — — 4 4 4 N7971 ’ ' '
A759-771 KL ------EKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK _ — — —
A771-79Ü KLLMIIHDRREFAKL AVTTWNPKYEGK _ __ _ _
DI54A ______+ 757 803
Fig 1.12 The 8 cytoplasmic domain and D154A extracellular domain cPl constructs used to examine the role of the pi subunit in regulation of differentiation (Levy et 6//., 2000).
These experiments show that disruption of the cyto-2 motif in the integrin
cytoplasmic domain removes the ability of the integrin to be recruited to focal
adhesions and to mediate cell attachment to ECM. However this does not remove the
ability of the integrin to suppress suspension induced differentiation. Deletion of
either amino acids 759-771 or 771-790 makes the integrin non-functional in any of
the assays. Disruption of the MIDAS motif in the PA domain by the D154A
substitution prevents the integrin from binding ligand. This prevents the integrin
supporting cell attachment but because the focal adhesion targeting motif in cyto-2 is
intact it is still recruited to existing focal adhesions. This mutant subunit has lost the
ability to suppress suspension induced differentiation. These results reiterate that the
important event in regulation of terminal differentiation is ligand binding and that
sequences involved in receptor clustering are not required (Levy et al, 2000).
W Chapter 1 Introduction
SCC4, a keratinocyte cell line derived from a human oral squamous cell carcinoma, normally differentiates at a low rate in culture (< 1%) but does not differentiate further in suspension culture (Rheinwald and Beckett, 1981; Levy et al, 2000).
When this cell line is infected with WT c|3l integrin 10-15% of the cells begin to differentiate. In this case the restoration of differentiation in not due to replacement of a lost integrin subunit as the SCC4 line has normal p 1 expression (Sugiyama et a l, 1993). When these cells are infected with the same panel of mutant cpl subunits all the constructs that are capable of suppressing suspension induced differentiation in normal kératinocytes are capable of inducing differentiation in SCC4 in adherent culture (Fig 1.12). All constructs that were unable to affect differentiation in kératinocytes failed to induce differentiation in SCC4 (Levy et al, 2000).
1.6 Aims
The pi subunit is an important regulator of keratinocyte differentiation (Adams and
Watt, 1989; Adams and Watt, 1993) and the SCC4 cell line is a useful tool to study this process (Levy et al, 2000). The aim of this project was to investigate the SCC4 differentiation phenotype. Initial observations suggested either a defect in the signalling pathway leading from the endogenous pi integrin or a defect in the integrin itself. To investigate these ideas I decided to investigate the signalling hypothesis by plating SCC4 expressing the cpl WT integrin on surfaces coated with anti-cPl or anti-human pi to investigate the signalling events triggered by the two subunits. To look for a defect in the endogenous pi I generated a cDNA of the pi gene expressed in the SCC4, sequenced it and looked for mutations that could affect function.
65 Chapter 2
Materials and Methods
2.1 Cell Biology
2.1.1 General solutions
The central cell services of Cancer Research UK fund provided sterile distilled, de ionised water and solutions that are indicated by “CR-UK”. All reagents used were tissue culture grade and sterile.
Phosphate buffered saline (PBS, CR-UK)
8 g NaCl, 0.35 g KCl, 1.43 g Na 2HP04 and 0.25 g KH 2PO4 were dissolved in 1 1 dH 2Û, adjusted to pH 7.2 and autoclaved. PBSabc was PBS supplemented with 1 mM CaCl2 and 1 mM MgCh
Tris Buffered Saline (TBS) lOx Stock solution was prepared by dissolving 24.2g Trizma base and SOg NaCl in
IL dH 2 0 . The pH was adjusted to 7.6 and the solution was sterile filtered.
EDTA solution (versene, CR-UK)
8g NaCl, 0,2g KCl, 1.15g Na 2HP0 4 , 0.2g KH 2PO4 and 0.2g ethyldiaminotetraacetic acid disodium salt (EDTA) and 1.5ml 1% (w/v) phenol red solution were dissolved in IL dH 2 0 . The pH was adjusted to 7.2 and the solution was autoclaved.
66 Chapter 2 Materials and Methods
Trypsin solution (CR-UK)
8 g NaCl, 0.1 g Na2HP04, 1 g D-glucose, 3 g Trizma base, 2 ml 19% (w/v) KCl solution and 1.5 ml of 1% phenol red solution were dissolved in 200 ml dHiO. The pH was adjusted to 7.7 and 0.06 g penicillin and 0.1 g streptomycin (Gibco BRL) were added. 2.5 ml pig trypsin (Difco 1:250) was dissolved in 250 ml dH20. Air was bubbled through the solution until the trypsin dissolved. The trypsin solution was then added to the tris buffered saline, made up to 1 1 with dHiO. The solution was sterilised through filtration through a 0.22 pm filter and stored at - 2 0 °C
Mitomycin C stock solution
Mitomycin C is an inhibitor of DNA synthesis and nuclear division. It is used to metabolically inactivate J2-3T3 cells to form a feeder layer for the culture of primary kératinocytes. A 1 OOx stock solution was prepared by dissolving 4 mg of mitomycin
C powder (Sigma) in 10 ml PBS. The final concentration used was 4 ug/ml.
Puromycin Stock solution
100 mg puromycin powder was dissolved in 100 ml PBS, sterilised using a 0.22 pm filter and stored at -20°C.
Polybrene Stock solution lOOOx stock was prepared at 5 mg/ml in sterile conditions and stored in frozen aliquots
67 Chapter 2 Materials and Methods
2.1.2 J2-3T3 and J2-3T3 Puro Cell Culture
J2-3T3 feeder cells (J2F) are a 3T3 derived clone selected for its ability to support keratinocyte growth (Rheinwald and Green, 1975). 75 cm^ flasks of J2 cells were washed with versene, incubated with versene/trypsin to detach cells then passaged
1:10 and grown to confluence. J2-3T3-puro (J2P) cells are J2F cells infected with the pBabe-puro retrovirus and were used to culture kératinocytes in medium containing puromycin. Cultures of feeders could be maintained for 2-3 months before transformed cells appeared which made them unsuitable for keratincyte culture.
J2-3T3 andJ2-3T3 Puro culture medium (E4+DCS)
J2-3T3 cells were cultured in Dulbecco’s modification of Eagle’s medium (DMEM)
(E4, CR-UK) suplimented with 10% (v/v) donor calf serum (DCS, Gibco BRL). For
J2-3T3 Puro cells (J2-3T3 transfected with the puromycin resistance gene) the medium was supplemented with 2.5 pg/ml puromycin. Medium was stored at 4°C until use.
Freezing and thawing o f J2-3T3 cells
Cells were harvested as above then resuspended in DCS containing 10% (v/v) sterile dimethyl sulphoxide (Gibco BRL). 1ml aliqouts were then frozen in each cryovial overnight at -70°C in insulated containers. One 75 cm^ flask was aliquoted into 3-5 cryovials. The vials were then transferred to liquid nitrogen storage. Thawing of the cells was performed by transfering the tubes to a 37°C water bath. As soon as the cell suspension was thawed it was added to 10ml of medium and centrifuged at 1,000 rpm for 5 minutes. The cells were then resuspended in medium and plated onto 25 cm^ flasks.
68 Chapter 2 Materials and Methods
2.1.3 Culture of human epidermal kératinocytes
Isolation o f primary human kératinocytes
Neonatal foreskins were kindly provided by Dr Cohen of the Fitzroy Clinic, London.
Isolation of primary kératinocytes was carried out as soon as possible after circumcision (Levy et al, 1998). Under sterile conditions, using a pair of forceps and curved scissors, a piece of foreskin was trimmed of dermal and fatty tissues. The foreskin was cut into pieces of about 5 mm^ and transferred into a Wheaton Cellstir
(Jencons) containing 5 ml trypsin and 5 ml versene and stirred over a magnetic stirrer at 37°C. Dissociated cells were collected every 30 minutes and added to 5 ml keratinocyte culture medium. The number of cells obtained was estimated using a haemocytometer. Dissociation of cells from the tissue was continued with addition of fresh versene and trypsin solution. This procedure was repeated 2 to 3 times before the number of cells obtained started to decrease. The yield from a neonatal foreskin was usually between 1-5x10^ cells. Feeder cells had been plated onto 25 cm^ flasks in readiness. Isolated cells were pooled, pelleted and plated at a density of
10^ cells per 25 cm^ flask. Cells were cultured until just confluent. One flask of cells was tested for mycoplasma infection by the CR-UK Cell Production Unit, while the remaining cells were harvested and frozen at 10^ cells/ml as for J2-3T3 cells.
Kératinocytes from 3 isolations were used; km, kp and kc.
69 Chapter 2 Materials and Methods
Keratinocyte culture medium (FAD+FCS+HICE)
FAD powder (Imperial Labs.) consisting of 1 part Ham’s F12 and 3 parts
DMEM+180 |iM adenine (final conc.) was supplemented with 3.07 g/1 NaHCOs, 100 lU/L penicillin and 100 pg/1 streptomycin. FAD medium (CR-UK) was bubbled with CO2 until acidic in pH before sterilising by filtration through a 0.22 \xm filter.
Medium was stored at 4°C until use.
Stock solutions of additives were prepared. 10'^ M cholera enterotoxin (ICN) was stored at -20 °C. 100 ug/ml recombinant human epidermal growth factor (Sigma) was prepared by first dissolving in 1/10 volume O.IM acetic acid (BDH) before adding to FAD medium containing 10% (v/v) batch tested fetal calf serum (FCS,
Imperial Labs) and stored at -20°C. The additives were combined into a lOOOx cocktail (HCE): 1ml hydrocortisone, 100 \i\ cholera enterotoxin and 1 ml epidermal growth factor stock solutions were added to 7.9 ml FAD medium with 10 % FCS and stored at -20°C. The final concentrations in the medium were 10'^® M cholera enterotoxin, 0.5 g/ml hydrocortisone and 10 ng/ml epidermal growth factor. lOOOx insulin stock solution (5 mg/ml in 5 mM HCl, Sigma) was stored at -20°C. The final concentration in the medium was 5 |ig/ml insulin.
Complete keratinocyte medium (FAD+FCS+HICE) was prepared by adding 10%
(v/v) FCS, cocktail and insulin solutions to the FAD medium prior to use. For kératinocytes infected with retrovirus puromycin was added to the medium at 1 pg/ml. Complete medium was stored at 4°C for up to 14 days.
70 Chapter 2 Materials and Methods
Preparation ofJ2-3T3 cells as feeder cells
The culture of human kératinocytes requires co-cultivation with mitotically inactive
J2-3T3 cells which are referred to as feeder cells (Rheinwald and Green, 1975).
Feeder cells were incubated with 4 |ig/ml mitomycin C for 2-3 hrs at 37“C. Cells were then harvested and plated onto flasks as described earlier. Cells from a confluent 75 cm^ flask were played onto between 3x 75 cm^ flasks or 9x 25 cm^ flasks. J2-puro cells were used as feeder cells when kératinocytes were infected with retroviruses.
Serial culture o f human kératinocytes
Frozen kératinocytes (passage 1-4) were thawed as described for J2-3T3 cells. The strains used were named km, kq, and kn. Each strain corresponds to kératinocytes isolated from a single individual. The usual number of cells seeded in Ix 75 cm^ was
2x10^ actively growing cells; 5x10^ cells from a frozen cryotube were plated in a
75cm^ flask to allow for loss of viability resulting from freezing and thawing. Fresh medium was applied every 2 days. A day prior to any experimental manipulation, kératinocytes were fed with fresh medium.
Kératinocytes were passaged just before they reached confluence. The cultures were rinsed once with versene and then incubated with versene for 5-10 minutes at 37°C.
This treatment caused any remaining feeder cells to detach. Kératinocytes would round up but would not detach from the flask. The versene solution was discarded and the remaining kératinocytes were incubated in 5ml trypsin/versene solution (1 part trypsin and 4 parts versene) at 37°C for about 10 minutes, until all kératinocytes had detached from the flask. 5ml medium was added to the suspension and the
tT Chapter 2 Materials and Methods number of cells was counted using a haemocytometer. The cells were pelleted and resuspended in medium as described and plated onto flasks with feeder cells; 10^ cells were added to a 25 cm^ flask or 2x10^ cells were added to a 75 cm^ flask.
2.1.4 Squamous cell carcinoma cells
SCC Cell Culture
The SCC cell lines used in this work were originally derived from patients with advanced oral squamous cell carcinomas (Rheinwald and Beckett, 1981). These lines were cultured in the same manner as normal primary kératinocytes.
2.1.5 GD25 pi null mouse fibroblasts
GD25 culture medium (E4+DCS)
GD25 cells were cultured in Dulbecco’s modification of Eagle’s medium (DMEM)
(E4, CR-UK) suplemented with 10% (v/v) donor calf serum (DCS, Gibco BRL). For
GD25 cells infected with puromycin resistant constructs the medium was supplemented with 2.5 pg/ml puromycin.
GD25 cell culture.
GD25 cells (Wennerberg et a/., 1996) are a fibroblast line derived from pi integrin null ES cells. When GD25 cells approached confluence they were harvested by washing in versene then incubating in 1:4 trypsin/versene at 37°C from 5 minutes.
The cells were pelleted at 1,000 rpm for 5 minutes then resuspended in medium.
Cells were subcultured at dilutions of 1:5 or 1:10.
72 Chapter 2 Materials and Methods
2.1.6 Retroviral Producer Cells
Retroviral producer cell culture
Helper free ecotropic packaging cells GP + ^«vAM12 (abbreviated as AMI2), were obtained from Dr P. Patel of the Institute of Cancer Research, London. These cells were designed in conjunction with the pBabe puro vector to reduce the risk of generation of wild type Mo MuLVirus via homologous recombination (Markowitz et a l, 1988a; Markowitz et a l, 1988b). The packaging cells were cultured in E4 medium supplemented with 10% (v/v) FCS (Sigma). For infected retroviral producer cells the culture medium was supplemented with 2.5 pg/ml puromycin. Phoenix ecotropic packaging cells were obtained from the American tissue culture collection.
Phoenix is a second-generation retrovirus producer lines derived from the human embryonic kidney 293 cell line for the generation of helper free ecotropic and amphotropic retroviruses. The lines had been created by placing into 293T cells constructs capable of producing gag-pol, and envelope protein for ecotropic and amphotropic viruses. For both the gag-pol and envelope constructs non-moloney promoters were used to minimize recombination potential. Different promoters for gag-pol and envelope were used to reduce their inter-recombination potential. Gag- pol was introduced with hygromycin as the co-selectable marker and the envelope proteins were introduced with diptheria resistance as the co-selectable marker.
Transfection o f phoenix cells.
Ecotropic virus producer cells lines were generated by transfecting the phoenix packaging cells with retroviral vector, and then using the virus containing supernatant to infect GD25 or AM 12. This two step protocol gives higher viral titres than from normally transfected producers (Sally Lowell, personal communication)
ÏÏ Chapter 2 Materials and Methods and allows production of amphotrophic virus for infection of human cells. The transfection process used was a calcium phosphate precipitation infection protocol
(Nolan protocol). 8x10^ phoenix cells were seeded onto a 100 mm dish the day before transfection. 5 minutes prior to transfection, chloroquine was added to each plate at 25 pM. Chloroquine increases the transfection efficiency by neutralizing vesicle pH and thereby inhibiting lysosomal DNAse. 10 pg DNA. 438 pl dH20, 61 pl 2 M CaCh were mixed and brought to a 500 ul total volume with dHzO. 0.5mL
2xHBS was added, mixed by bubbling air through the mixture and the HBS/DNA was added dropwise into 1 ml medium on the cells.
24 hours post-transfection the medium was changed. Retroviral supernatants were collected at 65- to 90 hours after infection, filtered though a 0.45 pm filter to remove cells and added to GD25 or AM 12 cells.
Infection o f AMI 2 and GD25 cells
Producer lines that are generated by retroviral infection have higher viral titres than those generated by transfection (Morgenstern and Land, 1990b; Morgenstern and
Land, 1990a), hence virus released into the culture medium by confluent phoenix cells was used to produce stable high titre AM 12 producer cells. AM 12 or GD25 cells were seeded on 100 mm dishes at 1-2x10^ density the day before infection. 2.5 ml infection medium (virus-containing medium collected from the phoenix cells, supplemented with 5 pg/ml polybrene; Sigma) was added to the cells. After 12h infection at 37°C the infection medium was replaced with fresh culture medium (E4
+ FCS). The selection medium containing 2.5 pg/ml puromycin was applied 48h later and changed every 2 days until cells reached confluence.
74 Chapter 2 Materials and Methods
Where required the lines produced by this process where FACsorted to give populations with high surface expression of the transferred construct.
2.1.7 MOLT-4
Molt-4 is a lymphocyte cell line derived from a patient with acute lymphobastic leukaemia. These cells were provided by ICRF cell production. These cells were used as a control in HUTS21 epitope expression experiments.
2.2 Assays of proliferation and differentiation
2.2.1 Proliferation Assay
Cultures of SCC4 cells expressing integrin constructs were set up with 1x10^ cells in each well of a 24 well tissue culture dish. At intervals over the next 17 days triplicate wells were harvested in small volumes of liquid and the number of cells determined by counting on a haemocytometer.
2.2.2 SCC4 Differentiation Assay.
1x10^ SCC4 cells expressing integrin constructs were plated into a 25 cm^ flask with
J2 feeders and cultured until just preconfluent. The culture wash then harvested as normal by ty psin/versene, washed in PBS several times and resuspended at 1x10^ cells/ml in PBS. Small amounts of each culture were then air dried onto duplicate wells of an 8 well slide. Multiple slides were prepared. The slides were then fixed in
4% formaldehyde for 10 minutes and stored in PBS. Just prior to staining the cells were permeabilised in cold methanol for 10 minutes. The slides were then stained using DH-1 or SY5 (human involucrin) or SQ13C (human comifm). Multiple fields for each condition were counted directly. Fields totalling three hundred cells were counted in each case. ______75 Chapter 2 Materials and Methods
2.2.3 Suspension differentiation assay
PolyHEMA coating o f dishes
To minimise attachemnt of cells to dishes bacteriological grade plastic petri dishes were coated with polyHEMA (type NCC, cell culture grade, Hydro Medical). A 10% polyHEMA (w/v) solution in 95% ethanol was prepared by muxing end over end overnight at room temperature. To coat dishes stock solution was diluted to 0.4% in ethanol/acetone (1:1). 5 ml of solution was added to a 100 mm dish and swirled repeatedly to coat the base of the dish and then removed immediately. This was repeated once. Coated dished were left under sterile conditions to dry before use.
Methyl cellulose medium
3.5 g methyl cellulose (Sigma) was autoclaved in a 400 ml centrifuge tube. 180 mM
FAD was heated to 60 °C and added to the tube. The contents were stired for 30 minutes at 30 °C and then overnight at 4°C (Green, 1977). After addition of 20 ml
PCS the medium was centrifuged at 9,500 rpm at 4 °C. The medium was then aliquotted and strored at -20°C until use. Immediately before use the medium was supplimented with HICE as for keratinocyte medium.
Suspension culture
Newly confluent cultures of keratinccytes were harvested and resuspended at 10^ cells/ml in complete FAD medium. The cells were then added slowly to the methyl cellulose medium simulateously swirling the medium to ensure homgenous distribtion of the cells. The final concentration of cells was 10^ cells/ml. 50 ml of cell suspension was poured into polyHEMA coated dishes and cultured overnight at 37
°C. Cells were recoverd by diluting the methyl cellulose with 10 volumes of versene
16 Chapter 2 Materials and Methods and centrifuged in a 250 ml conical bottom Beckman bottle at 2,000 rpm for 10 minutes at 4 °C. The recovered cells were then dried onto coverslips and stained for differentiation markers in the same manner as SCC4 cells.
2.3 Adhesion Assays
2.3.1 ECM proteins
Bovine plasma fibronectin was purchased from Sigma-Aldrich. LaminTwas also sourced from Sigma-Aldrich. Collagen was supplied by Vitrogen Corp. Vitronectin was purified from human plasma by the method of Cheresh e/ al. Briefly 100ml plasma was clotted by addition of 2 ml of 1 M CaCh. The clot was removed and the serum filtered. 0.5 ml of 0.2 M PMSF and 2.5 ml of 0.2 M EDTA was added and the plasma run over a sepharose 4B column. The flow through was then run on a heparin-sepharose column (equilibrated in 10 mM Na 2? 0 4 , 0.13 M NaCl, 5 mM
EDTA pH 7.7) to remove fibronectin and other heparin binding proteins. The plasma was then treated with 8M urea incubated at room temperature for 2 hr and recirculated over a second heparin column, equilibrated in (eqnlhbr3ted4n^l OmM
Na2P0 4 , 8 M urea, 5 mM EDTA pH 7.7' for 12 hours to isolate the vitronectin. The column was then washed in 100ml of the equilibration buffer, then 100ml lOmM
Na2P0 4 , 8 M urea, 5 mM EDTA, 0.13 M NaCl pH 7.7. 1 column volume (5ml) of 10 mM Na2P0 4 , 8 M urea, 5 mM EDTA, 0.13 M NaCl, 10 mM p-mercaptoethanol was run onto the column and the column incubated overnight at room temperature. The column was eluted with 10 mM Na 2P0 4 , 8 M urea, 5 mM EDTA, 0.13 M NaCl.
Fractions of eluted protein were pooled and analysed by SDS-PAGE to determine purity and A280 to meausre concentration. Over 10 mg of vitronectin was isolated from 100 ml plasma.______77 Chapter 2 Materials and Methods
Elution curve for Vitronectin
3,0 T
2.0 --
E oc o 2 LL
0.5 -- A
‘A - A — A— A - 0.0 0 5 10 15 20 25 30 mg / ml
Fig 2.1 Elution of Vn from heparin sepharose column. Fraction 6 is not plotted as A280 was > 3.
2.3.2 Cell Attachment Assay
96 well plates were coated overnight at 4°C with ECM proteins in PBS at appropriate
concentrations. 100 pi of the ECM solution was applied to each well. The plate was
then blocked by incubation with 5% heat denatured BSA in PBS for 1 hr at 37°C.
The plate was then washed three times in PBS and used immediately.
Precontluent Basks of cells were treated with 5 pM of CellTraker dye (Molecular
Probes) in serum free medium for 20 minutes at 37T. The cells were then washed
twice and placed in full medium for a further 20 minutes. The cells were then
harvested using trypsin/versene, washed in complete medium and then in TBS twice.
78" Chapter 2 Materials and Methods
The cells were finally resuspended at 5x10^ cell/ml in TBS. Mg2+, Mn2+, Ca2+ or
EDTA (1 mM final concentration) was added as appropriate and the 100 pi of the cell suspension was added to each well. The plate was incubated at 37°C for an appropriate length of time and then washed twice with TBS. The fluorescence from each well was measured on a fluorescent plate reader and then quantitated by comparison with a standard curve prepared from the same cell suspension.
2.3.3 Recombinant Integrin Adhesion Assay
These methods were used by Drs A Henry and V Perkins at CellTech, Slough to screen for affinity changes in mutant integrins.
Integrin cDNAs were cloned into the vector pEE12.2h which contains the human yl
Fc domain as a SalMEcoRX genomic fragment. Separate vectors encoding the extracellular regions of the human a5, av, wild type pi and T188I pi integrin subunits were constructed, essentially as described previously (Stephens et al., 2000).
The appropriate a and P integrin vectors were transiently co- expressed in
CHOL761h cells. Solid phase binding to fibronectin coated plates was performed.
Breifly, equal concentrations of WT and MT integrin supernatent were incubated in fibronectin coated 96 well plates, for 2 hr at room temperature. The plates were then washed and the bound integrin quantitated using a fluorescently labelled anti-Fc antibody.
79 Chapter 2 Materials and Methods
2.4 Cell motility and spreading
2.4.1 Time lapse microscsopy
In order to quantitate cell motility subconfluent cultures were harvested and plated onto dishes coated in fibronectin, at a density that allowed tracking of individual cells. Frames were taken every 2 minutes for 24 hours using microscopes fitted with a digital cameras. Motility was measured using Kinetic image analysis software and speed was calculated using a program written in Mathematica (Wolfram Research) by Dr. D. Zicha (CR-UK).
2.4.2 Determination of rate of spreading
Cells were plated onto fibronectin, allowed to attach and filmed as above while they spread over 3-4 hours. Every third frame was analysed and the number of small spherical unspread cells and the total number of cells was counted. From this the percentage of cells that had begun to spread was determined and plotted against time.
Any cells that failed to spread at all during the assay were discounted as non-viable.
2.4.3 Cell motility towards serum
GD25 cells expressing the WT or MT integrin or empty vector were placed into the upper section of duplicate 6 well plate cell culture insert with 8 pm pores in serum free medium. The lower part of the well contained medium supplemented with 10 % serum. The cells were left to migrate for 6 hr after which the cell culture insert membranes were fixed in methanol and then either the upper or lower side of the membrane was wiped clean of cells. The membranes were then stained in crystal violet solution, washed twice in PBS and air dried. Three random fields were photographed using a digital camera and the number of pixels of each field stained 80 Chapter 2 Materials and Methods by the crystal violet measuresd using the NIH-Image analysis program. The percentage of cells that had migrated through the membrane was determined by dividing the number of coloured pixels on the bottom side of the membrane by the total number of positive pixels on the top and bottom (For an example see Fig 5.3).
2.5 Invasion Assay
Invasion assays were performed basically as described (Thomas et al.,, 2001). GD25 cells expressing WT or T188I MT integrin or empty vector in serum free medium were mixed with matrigel to give 1:2 ratio of matrigel to medium. 100 pi of matrigel mixture containing 30,000 cells were then placed in the top part of a 24 well cell culture insert with 8 pm pores. 150 pi of serum free medium was placed on top and
750 pi serum containing medium in the lower half of the well. The cells were allowed to migrate for 2 days then the inserts were removed, the matrigel cleaned from the top part and the membranes fixed in methanol. The cells were then stained with crystal violet and the centre of each membrane photographed at 2.5x with a digital camera. The number of pixels occupied by the crystal violet stained cells was then quantitated using NIH-Image. This value was converted into cells/mm^ (the average size of a single cell and the scale of the photograph having previously been determined).
2.6 Immunological Methods
2.6.1 General Solutions
Gelvatol mounting solution
The Gelvatol mounting solution was prepared as described by Harlow and Lane
(Harlow and Lane, 1988). 2.4 g Gelvatol (Monsanto Chemicals) was mixed with 6 g sT Chapter 2 Materials and Methods glycerol (Sigma) and vortexed; 6ml dHzO was added, mixed and left to stand for 90 minutes at room temperature. 12.5 ml of 200 mM Tris-HCl, pH 8.5 was added and the solution was vortexed, heated to 50 °C and vortexed again. Heating and vortexing were repeated three times and the solution placed on an end over end mixer overnight at room temperature. The solution was then centrifuged at 2000 rpm for 10 minutes at room temperature and stored in aliquots at 4 °C.
82 Chapter 2 Materials and Methods
2.6.2 Antibodies
Antibody Specificity Species Reference/Source
JG22 Adhesion blocking cpi integrin Mouse (Greve and Gottlieb, 1982) V2E9 Anti pi integrin Mouse (Hayashiet al, 1990)
P5D2 Adhesion blocking hpi integrin Mouse (Dittel et al, 1993)
HUTS21 Active hpi integrin Mouse (Luque et al, 1996) Pharmingen TS2/16 Activating anti-hpi integrin Mouse (van de Wiel-van Kemenade et a l, 1992) A2B2 Adhesion blocking Anti-hpi Rat (Bohnsack et a l, integrin 1990) 9EG7 Activating/Activating anti-pl Mouse (Lenter et a l, 1993) Pharmingen Phospho-ERKl/2 Rabbit NEB
ERK 1/2 Mouse Santa Cruz
SY-5 Human involucrin Mouse (Hudson et al, 1992) DH-1 Human involucrin Rabbit (Dover and Watt, 1987) SQ37C Human comifin Rabbit
Alexa488 Anti Rabbit Alexa 488 conjugate Goat Molecular Probes
Alexa488 Anti Mouse Alexa 488 conjugate Goat Molecular Probes
Alexa594 Anti Rabbit Alexa 594 conjugate Goat Molecular Probes
Alexa594 Anti Mouse Alexa 594 conjugate Goat Molecular Probes aMo RPE Anti Mouse R-PE conjugate Goat DAKO
Anti Mouse HRP Sheep Amersham
Anti Rabbit HRP Donkey Amersham
Table 2.1: Antibodies used in this thesis
83 Chapter 2 Materials and Methods
2.6.3 Preparation of cells for Immunofluorescence staining
Cells were cultured on tissue culture plastic microscope slides (Nunc) or on glass coverslips (Chance Propper Ltd.). Coverslips were first boiled in 7x detergent (ICN) for 30 minutes to remove silicone coating. They were rinsed thoroughly first in tap water and then in dHzO. Washed coverslips were rinsed briefly in absolute ethanol and spread out on filter paper to dry completely before autoclaving. Alternatively the washed coverslips were coated with ECM proteins overnight at 4°C. Cells were then plated onto the coverslips and allowed to spread before fixation.
Fixation o f cells
Culture medium was discarded from cells and they were rinsed in PBS before fixing in either 4% paraformaldehyde solution or 4% formaldehyde solution for 15 minutes at room temperature. The specimens were rinsed three times in PBS, and then incubated in PBS abc containing 2% PCS to reduce binding to non-specific proteins, for at least 30 minutes at room temperature. To stain cells for focal adhesion components, cells were fixed for 10 minutes with 4% formaledehyde in PBS containing 0.1% Triton X-100
2.6.4 Staining protocols
Tissue sections or cultured cells were incubated in blocking solution for 30 minutes at room temperature. Primary antibodies were diluted in the blocking solution and applied to cells for 45 minutes at room temperature. The cells were rinsed three times in blocking solution and then incubated with the secondary antibody diluted in blocking for 45 minutes at room temperature. The cells were then rinsed three times
84 Chapter 2 Materials and Methods in blocking solution, once in PBS, and once in dH20, before mounting in Gelvatol solution.
In co-localisation experiments using a double immunofluorescence method, cells were stained with two primary antibodies. Both primary antibodies were of distinct species and these were subsequently probed with species-specific secondary antibodies conjugated to different fluorophore. The antibodies were applied in the following sequence: first primary; first secondary; second primary; second secondary, with thorough washes in blocking solution between each antibody application.
2.7 Flow cytometry
2.7.1 Staining protocol
Single cell suspensions of cells isolated directly from cultures harvested with trypsin/
EDTA solution were were incubated in primary antibodies diluted in 2% PCS PBS at
4°C for 20 min, washed, and incubated on ice for 15 minutes with an AlexaFluor488 conjugated secondary antibodies. Cells were washed 4 times and then resuspended in
PBSabc. Samples were analysed on a Becton-Dickinson FACScan.
2.7.2 Sorting on basis of extraceliuiar epitope expression
Cells were prepared for sorting by the above staining procedure but in sterile conditions. Cells were sorted using a pre-sterilised Becton-Dickinson F ACS Vantage cell sorter and collected in chilled complete medium. The cells were then replated in fresh pre-warmed medium.
85 Chapter 2 Materials and Methods
2.8 Biochemistry
2.8.1 Protein Extraction for MARK assay
Adherent cells were washed twice in ice cold PBS then lysed in RIPA buffer (150 mM NaCl, 50 mM Tris pH 8, 1% NP-40, 0.1% SDS, 0.5% Deoxycholate, 5 mM
EDTA, 1% Triton X-100) supplemented with a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail (Sigma). Lysates were then sonicated briefly and assayed for protein content using the EGA protein assay (Pierce).
2.8.2 Protein assay
To determine the amount of protein present in cell extract the EGA assay kit (Pierce) was used as per the manufacturers instructions.
2.9 Polyacrylamide Gel Electrophoresis (SDS Page)
2.9.1 General Solutions
Laemmli Sample Buffer
2x laemmli sample buffer (non reducing) comprised 125mM Tris-HGl, pH6.8, 2%
SDS, 20% glycerol and 0.02% bromophenol blue. 2x reducing sample buffer had an additional 10% (v/v) p-mercaptoethanol
SDS-PAGE running buffer
5OmM Trizma, 384mM Glycine and 0.1% SDS.
86 Chapter 2 Materials and Methods
2.9.2 SDS-PAGE
Vertical gel electrophoresis apparatus (Atto Corp. or Hoefer scientific instruments) were used. 1.5 mm gels were prepared between glass plates using the method of
Laemmli (Laemmli, 1970). The recipe for the gel is presented in table 2.2. After pouring the resolving gel a layer of 1ml dH 2 0 was applied to ensure a flat surface.
After the gel had polymerised the layer of water was poured away and the stacking gel was applied. A 1.5 mm comb was used to form the sample wells and the gel allowed to set. The comb was then removed and the wells flushed with SDS-Page running buffer. Samples were applied to the wells. 10 |il rainbow markers
(Amersham) were added to one well. The gel was then electrophorsed at 100-150V until the dye front had just left the bottom of the resolving gel. The gel was then removed and prepared for Western blotting.
Stock solutions
A 30% acrylamode / 0.8% bisacrylamide (Millipore)
B 3M Tris-HCl, pHS.S (BDH)
C 10% SDS
D 2M Tris-HCl, pH6.8 (BDH
AP 10% ammonium persulphate (Bio-Rad) in dHiO
TEMED N,N,N’,N’-tetramethylethylenediamine (Bio-Rad)
AB C dHzO TEMEDAP
7.5% 7.5 ml 3.75 ml 0.3 ml 18.45 ml 30^1 300 \i\
10% 10 ml 3.75 ml 0.3 ml 15.95 ml 30 ^il 300 ^il
12.5% 12.5 ml 3.75 ml 0.3 ml 13.45 ml 30 |al 300 ^il
87 Chapter 2 Materials and Methods
A C D dHzO TEMED AP
Stacking 1.5 ml 100 pi 0.625 ml 7.775 ml 10 pi 100 pi
Table 2.2: SDS-PAGE gel recipe
2.9.3 Western Blotting
Semi-dry transfer buffer lOx stock solution comprised 0.2 M Tris-HCl, pH 7.5 and 1.5 M Glycine. The solution was filtered through a 0.22 pm filter and stored at room temperatute. The semi-dry transfer solution was made by diluting the stock 10 fold in 0.1 % SDS and
20% (v/v) methanol.
Protocol
After SDS-PAGE electrophorese proteins were transferred ontp a a Millipore
Immobilon PVDF (polyvinylfiuoride) membrane (pre-wetted in methanol) using a semi-dry transfer unit (Trans-Blot-SD, Bio-Rad) for 1 hr at 200 mA. After transfer the membrane was breifly washed in TBS containing 0.05% Tween (TBST) then blocked in TBST containing 5% milk powder (Premier Brands UK Ltd) for 2 hr at room temperature to block non-specific binding of antibodies.
Blotting with antibodies and ECL
Membranes were incubated with primary antibodies diluted in 5% milk/TBST overnight at 4°C with agitation. The membrane was then washed with 5 changes of
TBST. The membrane was then incubated with a horse-radish peroxidase (HRP) conjugated secondary antibody diluted in TBST for 1 hr. The membrane was then washed 5 times. Detection was performed using the ECL chemiluminescence reagent is" Chapter 2 Materials and Methods
(Amersham) as per the manufacturer’s instructions. If a membrane was to be reprobed they were incubated in 200 mM glycine pH2.5, 0.2% SDS in dHzO for 20 minutes at room temperature then washed in TBST and reblocked in TBST/5% milk.
2.10 Molecular Biology
2.10.1 Bacterial Culture General Solutions
L-broth (ICRF)
L-broth comprised 1% Bacto-Tryptone (Difco), 0.5% yeast extract (Difco) and
170mM NaCl and was sterilised by autoclaving.
L-agar (ICRF)
L-agar comprised 1.5% bacto-agar (Difco, w/v) in L-broth. The agar was dissolved by heating in a microwave oven and allowed to cool to 50°C before adding the selection antibiotic. The solution was then poured into 100 mm bacteriological petri dishes and left to set on a level platform. Agar dishes were stored at 4°C, agar side up.
Ampicillin stock solution
Ampicillin (Sigma, stock 100 mg/ml in dH20) was used as a selection antibiotic and was added to LB or L-agar to a final concentration of lOOpg/ml.
Kanamycin stock solution
Kanamycin (Sigma, lOOOx at 25 mg/ml) was used as a selection antibiotic and was added to LB or L-agar to a final concentration of lOOpg/ml.
89 Chapter 2 Materials and Methods
2.10.2 Transformation of bacteria
Heat shock transformation o f TOP 10 cells
50 of competent bacteria were thawed at room temperature and then incubated on ice for 10 minutes. DNA (0.1 pg for ligations or 1 ng for plasmids) was added, mixed and left on ice for 30 minutes. The bacteria were then heat-shocked at 42°C for 45 seconds and transferred onto ice for 2 minutes. 4 volumes of SOC medium
(CR-UK) were added and the tube was incubated at 37°C for 1 hour. 100 pi culture was spread out onto plates containing L-agar and either ampicillin or kanamycin as appropriate and incubated overnight at 37°C. Single colonies were picked with a sterile loop to inoculate LB media.
2.11 DNA techniques
2.11.1 General solutions
All reagents used were of molecular biology grade. When possible, solutions were autoclaved after preparation to destroy DNases.
Tris/EDTA buffer (TE)
TE was used as a general storage buffer for DNA and comprised 10 mM Tris-HCl and 1 mM EDTA, pH8.0.
Tris-acetate-EDTA buffer (TAE)
A 5Ox stock solution was prepared by dissolving 242g Trizma base and 57.1 ml glacial acetic acid (BDH) in dHiO. 100 ml 0.5M EDTA, pH 8.0 were added and the final volume was made up to 1 1.
90 Chapter 2 Materials and Methods
Agarose/TAE gel
This was used in the electrophoresis of DNA. 0.8-2% (w/v) ultra pure agarose
(Gibco BRL) was melted in a microwave oven in Ix TAE buffer. Ethidium bromide was added at 0.05 pg/ml to agarose solution before casting in gel mould. Typically,
DNA was electrophoresed at constant voltage of 80-100 V in Ix TAE buffer.
DNA loading buffer
6x DNA gel loading buffer comprised 0.25% bromophenol blue (Sigma), 0.25% xylene cyano (Sigma) and 15% Ficcol (Sigma) in dHzO.
2.11.2 Isolation of DNA from agarose gels
DNA bands were isolated from agarose gels using the Qiaquick gel extraction kit
(Qiagen) as per the manufacturer’s instructions.
2.11.3 Maxi/mini preps
Plasmid preparation at the Maxi (500 mg) and Mini (5 mg) scale were performed using the appropriate Qiagen plasmid isolation kit as per the manufacturer’s instructions.
2.11.4 Genomic DNA preparation
Three 10 |xm sections of frozen tissue were placed in a 1.5 ml eppendorf tube on dry ice. 200 |xl of DNA extraction buffer (50 mM Tris pH8, 100 mM EDTA, 0.5% SDS) containing 5 mg/ml Proteinase K (Sigma) was added and the tube centrifuged breifly to pellet the tissue in the buffer. The Tube was then incubated at 55 °C for 4 hours.
200 pi of phenol/chloroform/iso-propyl alcohol (25:24:1 by volume, Amresco) was added and the tube vortexed. The tube was then centrifuged and the upper, aqueous 91 Chapter 2 Materials and Methods phase transferred to a fresh tube without disturbing the interface layer. Sodium acetate (pH 5.2) was then added to the aqueous layer to 0.3 M. 1 ml of ethanol was added and the tube placed at -20 °C to precipitate DNA. The tube was then centrifuged at 14,000 rpm, the pellet washed with 70 % ethanol, air dried and resuspended in 30 p-l TE.
2.11.5 Restriction enzyme digestion
All restriction enzymes were purchased from NEB and used as per manufactures instructions.
2.12 PCR techniques
2.12.1 PCR
PWO (Roche), an error checking polymerase was used as per the manufacturers instructions for producing blunt ended fragments. 2x PCR Master Mix (Promega) was used for producing small PCR fragments for sequencing.
2.12.2 Site directed mutagenesis
Site directed mutagenesis was performed using the QuickChange protocol (Sigma-
Aldrich). Briefly, complementary primers containing the mutated sequence were used to generate single strands covering the entire template plasmid by PCR. These strands anneal to each other to form complete, nicked plasmids which were used to transform comp<^.tent cells. Colonies were then picked, DNA prepared and analysed for the presence of the mutation by restriction analysis. Clones containing the
n Chapter 2 Materials and Methods mutation were then sequenced. For production of the cpl T188I plasmid pBabe-puro cpi WT was used as template, the T188I- and T188I+ primers for amplification and a EcoNl restriction site was removed by mutagenesis.
2.12.3 RT-PCR
RT-PCR was performed using the SuperScriptll polymerase (Promega). Random hexamer primers were used to generate a first strand cDNA. Specific sections of
DNA were then generated using gene specific primers.
2.12.4 Sequencing
Sequencing reactions were performed using BigDyeTerminator mix and analysed by the staff of the equipment park at 44 Lincoln’s Inn Fields.
2.12.5 Primers
Primer Sequence Specificity Use
IL CCGGACGCCGCGCGGA Human pi Sequencing and PCR AAAGATG
B1.2 TTGGCTGGAGGAATGT Human pi Sequencing and PCR TACACGGC
B1.3 CAGTTTCTGGACAAGG Human pi Sequencing and PCR TGAGCAAT
B1.5 GCAGATCTGTCCGTTGC Human pi Sequencing and PCR TGGCTTC
B1.4 ATCCAATGGCTTAATTT Human pi Sequencing and PCR GTGGAGG
B1.6 TAAAGCTACCTAACTGT Human pi Sequencing and PCR GACTCTG
93 Chapter 2 Materials and Methods
B1.9 GACTGTCATGCCTTACA Human P 1 / Sequencing and PCR TTGAGCACAAC genomic DNA Bl.lO TCCAGATATGCGCTGTT Human p i / Sequencing and PCR TTCCAACAAG genomic DNA T188I+ GAGAAATCCTTGTCTTG Chick pi Sequencing and PCR GTGACCAGAACTG
T188I- CAGTTCTGGTCACCAAT Chick pi Sequencing and PCR ACAAGATTTCTC
CBl GTGGAGAAAACTGTGA Chick pi Sequencing and PCR TGCC
CB2 CATCAAATCCACCTTCA Chick pi Sequencing and PCR GGAG
CB3 ATGGCCGAGACTAATT Chick pi Sequencing and PCR TAAC
CB4 AGTTTGGCTGGTGTTGT Chick pi Sequencing and PCR AC
CB5 CGTGAAGTTACACAAC Chick pi Sequencing and PCR GAGT
CB6 TTGGAATTTGGGATCCG Chick pi Sequencing and PCR TGC
CB7 CAATAGGAGATGAGGT Chick pi Sequencing and PCR TAGG
M13F GTTTTCCCAGTCACGAC Plasmid Sequencing sequence
M13R CAGGAAACAGCTATGA Plasmid Sequencing C sequence
3F ATGTAAAAAGGTTTGA Hum an pi Sequencing, PCR and SSCP CCATTAC genomic DNA 3R CGCAGGTATTCACAGA Human pi Sequencing, PCR and SSCP GTTG genomic DNA 4F ATTTGGTTTTTCTCGTT Hum an pi Sequencing, PCR and SSCP TGC genomic DNA
94 Chapter 2 Materials and Methods
4R AAACAACATTTGAGAA Human pi Sequencing, PCR and SSCP TTGCC genomic DNA 5F CCATCCATTCATTCATT Human pi Sequencing, PCR and SSCP TATG genomic DNA 5R ATATCCCCTGATAGGA Human pi Sequencing, PCR and SSCP AATG genomic DNA 6F AGGATTTTTGTTGAATT Human pi Sequencing, PCR and SSCP ACTGTG genomic DNA 6R AAAATCCAACATAAAC Human pi Sequencing, PCR and SSCP ACATTC genomic DNA 7F GAGTTATGTGTAATCTT Human pi Sequencing, PCR and SSCP GCATG genomic DNA 7R TGCCTCTAACAATCAA Human pi Sequencing, PCR and SSCP AAATG genomic DNA
Table 2.3 Primers used in this thesis
2.12.6 SNP analysis
These methods were used to screen for single point mutations by Dr Claire Taylor,
CR-UK Leeds
PCR
PCR amplification of genomic DNA was carried out using Amplitaq Gold polymerase (Applied Biosystems) in the buffer supplied by the manufacturer; 2.5-3.0 mM MgCli; 200 pM each dNTP and 0.5 pmol/pl each primer. For DNA sequencing
PCR primers were unlabelled; for FSSCP both primers were 5’ labelled with F AM,
HEX or NED. PCR was checked for yield and specificity by agarose gel electrophoresis as described in Sambrook et al., 1989. (Sambrook et a l, 1989)
95 Chapter 2 Materials and Methods
Fluorescent single strand conformation polymorphism (FSSCP) analysis.
Fluorescent PCR products were diluted with water by a factor of between 1 in 10 and
1 in 40, depending on the yield of the reaction. 1-2 pi of the diluted product was mixed with 0.5 pi of ROX-500 size standards (Applied Biosystems) and 10.5 pi of
HiDi Formamide (Applied Biosystems). The samples were denatured at 95°C for two minutes and snap cooled on ice. FSSCP analysis was carried out on a 3100
Genetic Analyser (Applied Biosystems) at 18, and 30°C using 5% Genescan polymer containing 10% glycerol and IxTBE. Data was analysed using Genescan 3.7.1 and
Genotyper 2.5 software (Applied Biosystems). Mutation detection was by visual inspection of electropherogram traces which were examined independently by two observers.
DNA sequencing
Unincorporated primers and deoxynucleotides were removed from PCR products using shrimp alkaline phosphatase and exonuclease I (AmershamPharmacia). PCR primers were used for all sequencing reactions. Sequencing reactions were carried out using ABI PRISM BigDye Terminator Cycle Sequencing Kit versions 2 and 3
(Applied Biosystems) with electrophoresis of the products on 310 and 3100 Genetic
Analysers (Applied Biosystems). Data analysis was carried out using Sequence
Analysis 3.0 (Applied Biosystems) and SeqMan (DNAStar) software and by visual inspection of electropherograms.
96 Chapter 2 Materials and Methods
2.13 List of Suppliers and Distributors
Aldrich Chemical Company Ltd. Dorset, UK. Amresco, Solon, Ohio, USA. Amersham International, Amersham, Buckinghamshire, UK. BDH Laboratory Supplies Inc., Hemel Hempstead, Hertfordshire, UK. Beckman Instruments, Palo Alto, California, USA. Becton-Dickinson, Lincoln Park, New Jersey, USA. Bio 101 Inc. La Jolla, California, USA. Bio-Rad Laboratories Inc. Hemel Hempstead, Hertfordshire, UK. Boehringer Mannheim UK Ltd. Lewes, East Sussex, UK. Calbiochem -Novabiochem (UK) Ltd. Nottingham, UK. Carl Zeiss Ltd. Welwyn Garden City, Hertfordshire, UK. Chance Propper Ltd., Swethwick, Warley, UK. Clontech, Palo Alto, UK. Coulter Electronics Ltd. Harpenden, Herts, UK. DAKO A/S, Denmark. Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa, USA. Difco Laboratories, Manston, Wisconsin, USA. Eastman Kodak Co. is distributed by Sigma Chemical Co. EOS Electronic, South Glamorgan, Wales, UK. European Collection of Animal Cell Cultures (ECACC), Salisbury, UK. Flow Laboratories Ltd., Aryshire, Scotland, UK. Genetics Research Instrumentation Ltd. Dunmow, Essex, UK. Gibco BRL/Life Technologies Ltd. Paisley, Renfrewshire, UK Hoefer Scientific Instruments is distributed by Biotech Instruments Ltd., Beds, UK. Hydro Medical Sciences Division, Brunswick, New Jersey, USA. ICN Pharmaceuticals Ltd. Thame, Oxon, UK. Imperial Laboratories (Europe) Ltd. Andover, Hampshire, UK. Integrated Separation Systems, Natick, Maryland, USA. Jackson Immunoresearch Laboratories, Luton, Bedfordshire, UK Jencons, Leighton Buzzard, Beds, UK. Chapter 2 Materials and Methods
Monsanto Chemicals. Springfield, Massachusetts, USA. Millipore, Harrow, Middlesex, UK Molecular Probes, Leiden, Netherlands. New England Biolabs (NEB). New York, USA. Nunc A/S, Roskilde, Denmark. Perkin-Elmer Co. Foster City, Carlifomia, USA. Pharmacia Biotech. Uppsala, Sweden. Pharmingen, San Diego, California, USA. Pierce, Rockford, Illinois, USA. Premier Brands UK Ltd. Knighton, Stafford, UK. Promega UK Ltd. Southampton, UK. Qiagen Ltd. Crawley, UK. RND Systems, Abingdon, Oxford, UK. Santa Cruz Biotech. Inc. Santa Cruz, Carlifomia, USA. Seikagaku Corp. Tokyo, Japan. Serotec Ltd. Kidlington, Oxford, UK. Scotlab Ltd. Coatbridge, Strathclyde, Scotland, UK. Sigma Chemical Co. Poole, Dorset, UK. Transduction Laboratories, Lexington, Kentucky, USA. US Biochemical Corp. Cleveland, Ohio, USA. Vector Laboratories, Burlingame, Carlifomia, USA. Whatman Intemational Ltd. Maidstone, Kent, UK Zeneca Pharmaceuticals, Macclesfield, UK
98 Chapter 3
Analysis of the SCC4 phenotype
3.1 Introduction
In this chapter I confirm that the expression of a WT chick pi subunit in the SCC4 cell line stimulates terminal differentiation. I also show that this cell line is heterozygous for a mutation in the A domain of the |3l integrin subunit.
99 Chapter 3 Analysis of the SCC4 phenotype
3.2 Results
3.2.1 Infection of SCC4 cells with chick Pl subunits induces differentiation.
SCC4 kératinocytes were induced to express the wild-type chick pi integrin subunit or an empty pBabe-puro retrovirus by co-culturing with AM 12 retroviral producer cells for 48 hours followed by puromycin selection for 1 week. The expression levels of the subunit in the SCC4 were then checked by flow cytometry (Fig 3.1). The number of cells expressing the terminal differentiation markers involucrin and cornifin was then determined. SCC4 cells in adherent culture exhibit extremely low levels of differentiated cells as detected by involucrin staining (less than 1% are positive for involucrin). Upon infection with the chick WT integrin subunit there is an increase in involucrin positive cells to 8-15% (Fig 3.2A). Induction of cornifin expression was also observed (Fig 3.2B). The empty vector had no effect on either differentiation marker.
Fig 3.1: Expression of the cPl subunit in the SCC4 cells (grey trace negative control, black JG22-anti-cpl )
100 Chapter 3 Analysis of the SCC4 phenotype
A SCC4 pBp
SCC4 c|i1 WT 15 2 + c 10 gÜ 5 Ç
1 ^
SCC4 SCC4 pBp SCC4 cp1 W T
B 15 !» 8 I '
SCC4 SCC4 pBp SCC4 cp1 WT
Fig 3.2: (A) SCC4 cells infected with c|31 WT upregulate the keratinocyte differentiation marker involucrin. Disaggregated colonies of SCC4, SCC4 infected with pBabe-puro or SCC4 infected with pBabe-puro c(31 WT stained with SY-5, an antibody to human involucrin. Data representative of 3 experiments. (B) Similar results were obtained when stained for cornifin
101 Chapter 3 Analysis of the SCC4 phenotype
3.2.2 Expression of endogenous integrin is normal when compared to SCC12B2.
In previous studies restoration of differentiation has been linked to replacement of a missing integrin subunit (Jones et al, 1996). In order to confirm that the induction of involucrin by WT c|3l in SCC4 was not due to replacement of a missing pi integrin subunit SCC4 were disaggregated, stained with P5D2 (anti-human pi). The levels of surface pi was measured by flow cytometry and compared to the levels found in
SCC12B2 another SCC line that does not respond to infection with the cpl WT subunit. Both lines expressed similar levels of pi subunit (Fig 3.3).
It has been previously shown that infection with the cpi does not cause changes in the normal a subunit profile of kératinocytes or SCC4 cells (Levy et al, 2000).
102 Chapter 3 Analysis of the SCC4 phenotype
SCC4 8 SCC12B2
S S 0 5
Fig 3.3 : The SCC4 cell line expresses equivalent levels of pi integrin to the SCC12B2 line, a similar SCC derived culture that does not differentiate on infection with the WT subunit.
Fig 3.4 SCC4 is capable of forming focal adhesions on collagen coated surfaces. Stained with P5D2 (anti-human pi). Bar 20 pm.
103 Chapter 3 Analysis of the SCC4 phenotype
3.2.3 SCC4 form focal adhesions when plated on ECM proteins.
To confirm that the endogenous integrin in the SCC4 cells forms focal adhesions cells were plated on 20 pg/ml collagen and allowed to spread. Cells where then simultaneously fixed and permeabilised and stained for human |3l integrin. The
SCC4 cells spread and formed large focal adhesions in a similar manner to normal kératinocytes. (Fig 3.4, Fig 1.4).
3.2.4 Binding of c^l on anti-cPl antibody coated surfaces causes clustering of the endogenous Pl subunits
One of the initial hypotheses of the project was that the SCC4 cells carried a defect in integrin signalling. One approach to investigate this is to plate SCC4 cells expressing the WT cpl or one of a range of mutant integrins onto surfaces coated anti cPl antibody (JG22) or anti hpi to specifically cluster either subunit. Cell lysates could then be prepared to analyse the signalling properties of the subunit.
However rather than selectively clustering the cpl the endogenous pi also formed clusters that resembled focal adhesions (Fig 3.5). This occurred even if YPRP cpl integrin subunits were used which are themselves incapable of being recruited to focal adhesions. If uninfected cells were used the cells did not adhere to the plate.
104 Chapter 3 Analysis of the SCC4 phenotype
YPRF
Fig 3.5 Clustering of endogenous pi integrin occurs when SCC containing either WT or YPRF cpi constructs are plated on JG22 (anti-cpl antibody). Stained with A2B2 (anti-hpi). Scale Bar 40 pm
105 Chapter 3 Analysis of the SCC4 phenotype
3.2.5 Sequencing of Pl cDNA in SCC4.
To determine whether the induction of involucrin in the SCC4 line was due a defect
in the endogenous pi subunit the pl cDNA was sequenced. mRNA was isolated
from cultured SCC4 cells and a first strand cDNA synthesised using random primers.
Three approximately 1 kb overlapping fragments covering the length of the 2.5 kb
coding region of the pl gene message were then synthesised using PWO, an error
checking polymerase (Fig 3.6). The resulting fragments were cloned into the pCR-
Blunt vector and used to transform competent bacteria. Clones from these were then
sequenced using primers against the flanking regions of the vector. In addition the
same fragments were generated from a culture of the kératinocytes.
B1.2 B1.5
r - ^ <4-1
1L B1.3 B1.4 B1.6 r > r <4-1
1 1 1 1
500 1000 1500 2000 2500 1 1
Coding Region
Fig 3.6 Fragments of the pl cDNA generated for sequencing showing primers used
It was found that the clones isolated from the SCC4 line differed from the published
sequence at four points (Table 3.1).
106 Chapter 3 Analysis of the SCC4 phenotype
Change in codon Amino Acid Effect % of SCC4 clones Conclusion CAT->ACT 92 His->Thr 100% (6/6) Error in published sequence/Common Polymorphism TAT -> TAC 133 Tyr->Tyr 100% (6/6) Silent polymorphism ACA ->ATA 188 Thr -> He 66% (7/10) Heterozygous mutation ACC -> AGC 195 Thr-> Ser 100% (6/6) Error in published sequence
Table 3.1 : Sequence changes found in the SCC4 pi cDNA fragments
Of the four changes found in the SCC4 cDNA the H92T substitution was deemed to be either a common polymorphism or an error in the original sequence (Argraves et a l, 1987) as both the SCC4 and the normal kq4 cells were found to be homozygous for this change. The T133T substitution causes no change in the translated protein and as such was discarded as irrelevant in this context. The T195S substitution is most likely to be an error in the published sequence as every clone sequenced in all
SCC, keratinocyte and normal DNAs that covered this point were found to have a serine in this position. In addition the pi integrin gene for every other species sequenced contains a serine at this point.
Both WT and T188I containing clones were found in the SCC4 cDNA at approximately 50% proportions. I therefore concluded that the SCC4 was heterozygous for this mutation. Smaller fragments comprising of amino acids 673 to
832 were generated from the SCC12B2, SCC12F2 and SCC 13 cell lines and a panel of fifteen human genomic DNAs using primers B1.9 and Bl.lO. The T188I substitution was not found in any of the other cell lines or individuals examined. All
107 Chapter 3 Analysis of the SCC4 phenotype of the sequence changes found map to the A domain of the P subunit. No changes were found in the cytoplasmic, transmembrane or stalk domains.
In an attempt to determine whether the T188I mutation occurred in the tumour or was carried by the patient DNA I isolated DNA from fibroblast like cells (provided by J.Rheinwald) grown out of an early (passage 2) frozen stock of SCC4 cells in fibroblast selective medium. I hoped that these represented dermal fibroblasts that were present in the original tumour sample and were representative of the genotype of the patient. When sequenced DNA isolated from these cells was found to be mix of WT and T188I clones, but the T188I clones were at a lower frequency than those found in the pure SCC4 DNA (1/6 WT). No other material was available for further analysis
108 Chapter 3 Analysis of the SCC4 phenotype
3.3 Discussion
The SCC4 line appears to be unique in that it responds to infection with WT^by
differentiating. Normal kératinocytes do not differentiate upon infection with WT |3l
although expression of high levels of a CDS extracellular domain, |3l cytoplasmic
domain chimera can increase the rate of differentiation due to a decrease in MAPK
signalling (Zhu et al, 1999). Previous work in the lab (Levy et al, 2000) had shown
that to induce differentiation the infected pl integrin construct had to have a
functional ligand binding site, but that the ability of the integrin to be cluster in focal A adhesions was not required.
There were two initial possibilities to explain the induction of differentiation by the cpl WT integrin. Firstly, the lack of differentiation could be due to a fault in the
integrin signalling pathway controlling differentiation that was overcome by the presence of the cpl WT integrin. Alternatively there may be a fault in the
endogenous pi integrin itself preventing correct signalling. I planned to investigate the first possibility by clustering the endogenous human integrin or the introduced
chick subunits and comparing the pathways stimulated. However similar experiments with primary keratinocyte lines failed to show differences between signalling from endogenous human and introduced chick pi subunits even when functionally dead chick integrin constructs were used (personal communication, H. Abedi).
Immunofluorescence staining of cells expressing chick integrins clustered on blocked anti-chick integrin antibody coated plates showed that the endogenous pl integrin tended to co-cluster with the chick construct explaining why the “dead” integrin
109 Chapter 3 Analysis of the SCC4 phenotype constructs appeared to signal. This co-clustering made it difficult to draw conclusions from the clustering assays and so I turned to the second hypothesis.
I have found three amino acid changes from the published human pl sequence in the
SCC4 cell line that could cause a change in the function of the subunit. The presence
of multiple changes from the published sequence of pl is interesting although of the three changes only one is likely to have any function. H92T may be a common polymorphism rather than an error in the database but T195S is almost certainly an error in the original submission. We have found no evidence of a serine at this point in any sample sequenced and in over 100 tumours (Chapter 6) T195S was found in every sequence. Furthermore the pi sequence from every other species that is in the
NCBI database contains a serine at this point. This shows that a database “WT”
sequences can not always be taken as absolute.
To try and establish if the T188I mutation arose as a result of genetic instability on the tumour or was present in the patient I tried to obtain non-tumour material. The only available source was a fibroblast like cell type that was grown out of an early passage of SCC4 under selective conditions. The lower frequency of T188I clones in these fibroblasts does not support any conclusions as to the origin of the tumour. It is possible that this lower frequency represents WT/WT fibroblasts contaminated with
WT/T188I SCC4 cells; however with the material available it is not possible to make a statistically safe statement either way. It is also possible that the cells are not dermal fibroblasts as suspected but are, in fact, a keratinocyte clone from the original tumour that is capable of growing in fibroblast selective medium. Further non
110 Chapter 3 Analysis of the SCC4 phenotype tumour tissue from the patient is not available so it is impossible to be certain whether the T188I substitution is germline or appeared in the tumour.
The position of the T188I mutation makes it a good candidate to have functional significance. The importance of the small C-C loop upon which T188I lies in integrin function has been shown many times and is part of a larger structure referred to as the specificity loop (Takagi et al., 1997). It has been shown that both the ligand and the signalling properties (Miao et al, 2002) of the av(33 integrin could be changed by substitution of this loop. The region is also important for folding of the P subunit
(Takagi et ai, 2002).
The C-C loop is on one of only four areas of the ligand binding domain where there is not a high degree of homology when the P1 is compared to the other known human P subunits (Fig 3.7). When these sites are mapped onto the surface of the
ROD bound p3 crystal (Xiong et a!., 2002) three of the sites map to the top surface of the protein, two of which lie adjacent to the residues that are close (<4 Â) to the
ROD ligand itself. These three sites lie in a line across the top of the domain and while the orientation of the ROD makes it unlikely that they contact that section of the ligand it is possible that they may come into contact with sections of ligand either side of the ROD site. The fourth site lies on the base of the domain away from the ligand binding site and has been identified as the binding site of the TS2/16 antibody, a mAb that can cause activation of pi integrins (van de Wiel-van Kemenade et al.,
1992).
Ill Chapter 3 Analysis of the SCC4 phenotype
Integrin mutations have been found to be the cause of several diseases (reviewed in
(Hogg and Bates, 2000)). Mutations in the a6p4 integrin can cause a skin blistering disease known as junctional epidermolysis bullosa (EB) (Pulkkinen et a l, 1998a;
Pulkkinen et a l, 1998b; Pulkkinen et a l, 1998c), due to a loss of hemidesmosome function resulting in poor adhesion between the basal kératinocytes and the basement membrane.
Leucocyte adhesion deficiency type 1 (LADl). Is characterised by a lack of leucocyte function due to loss of adhesion through the P2 family of integrins. LADl is usually caused by mutations causing loss of expression (Lipnick et a l, 1996; Kijas et a l, 1999), although mutations causing expressed but poorly functioning subunits have also been characterised (Wardlaw et al, 1990; Hogg et a l, 1999; Mathewet al,
2000; Shaw et al., 2001). The severity of the condition varies depending on the alleles present and range from a sub-clinical condition due to a single partly functioning mutation to a severe life-threatening condition where both alleles are non-functional (Kishimoto et a l, 1989; Mathew et al, 2000).
Glanzmann’s thrombasthenia (GT) is characterised by a failure of blood clotting due to a lack of platelet crosslinking and is caused by defects in either the allb or p3 genes. Again the severity of the disease depends on the number and type of mutations present. Most GT mutations are loss of function (Dong et a l, 2000;
Tadokoro et al, 2002; Watkins et al, 2002) although two cases have been identified where the integrin was locked in the high affinity state (Fullard et al, 2001; Ruiz et a/., 2001).
112 Chapter 3 Analysis of the SCC4 phenotype
Integrin mutations have also been found to contribute to other diseases including heart disease (avP3)(Undas et al, 2001; Douglas et al, 2002), isolated giant platelet disorder (aIIbP3)(Kunishima et a l, 2001) and certain forms of muscular dystrophy
(a? (Mayer et a l, 1997; Pegoraro et a l, 2002).
The presence of a mutation in the pl gene of the SCC4 provides a possible mechanism by which infection with WT cpi could restore differentiation. In most cell types the P1 integrin subunit is present in excess, while the levels of a subunits are limiting and control the amount of p l expressed on the surface. It has been previously shown that infection with the WT cpl does not influence the levels of a subunits expressed by the cell (Levy et al, 2000). If the mutant pi subunit is responsible for the lack of differentiation in the SCC4 line then infection with the
WT cpl will cause a dilution of the mutant integrin in the cytoplasmic pool of available pi subunits, thereby reducing the number of mutant integrins on the surface of the cell.
113 Q LV L R L E jS L^F K R A |E D Y P I DLTYLMDLS • MKDDLE nvk 8 L G T D LlM N E M T g D lF B I Ar’ S P Q KVT L 0 L S T v f r F R R A K g|y P I DLYYLMDL8 8M 0D D lJr]n V K K L G GDRAL f f j S P Q R I A L R L S P I V R Q V[E D Y P V 1>1T]y Y L M D L S 8 M K ]> D LjV S| I Q N L G TK L A T Q M g P Q G L R V R L S P LfflvfrE P l | e | s1p V D L Y [i] l M D 0 8 8 mi@D D L D H L K K M G Q N L A R V L g P V D BB T P Q E I A V 0 L S P EDYPVDLYYLMDLt K D D L D N I R 8 L 6 T K L A E E M B6 A P Q 8 L I L K L S P V H V R EDYPVDLYYLMDLS 8H ^D D L[F T I K E L 6 8 G L S KE H n BF A P O R V R V 0 L K P e JgIy PVDLYYLNPL8 8 M K P P L E r I v R Q L G H A l { ^ V RL TIH S V B BS T p Jg E V 8 I
G G VB H L L V F S T D A G FH FA G D G E - L G I V L PHDGQCHLE N N Y tJ|M i] H Y Y D Y P 8 i]Ajir L ELSEHHI P G VB H L L V F A T D D G FH FA G D G E - - L G a ] I L TP H D G R C H L d D |N y [k“R m N E F D Y P 8 V G ^ L K L A E H H I ffj D E K G VB H^D L L V F T T D A K TH I A L D G R - - L AG I V Q P H D G Q C H vfG S D Y 8 A s T T M D Y P 8 M ELS q[F1h I B4 T R D G V b [F|d s L L V F S TE 8 AFH Y E AD G U N V L AG I M 8 D g R C H L Y T Q Y R T Q D Y P 8 P T L g L a[kJH H ij K IÉ K I G V b |£ |d A L L V F T T D D V PH I Â)L D G E - - L G G L V P H D G Q C H Y T A « H q M D Y P 8 A L L E L A E H H I > K E K I G V B H D S L E V F 0 S D A D 8 H FG M d [s ] e - - L AG I V I PHD G 0C H L*D Y S M m T V L EYPT I G Q L E l [v]q H H V| 3 SF q [e Q I G V B H -VSR L L V F T S D D TFH TA G D G E - - L G G ifFM p [s] d G H C H L D Y 8 R m T E F D Y P 8 V G Q V ^ L s J a ^ H I SS E s [h I G V B [ F [Ë~Â L L L |V M|T D Q T8 H L A L d IFIe - - L AG I ^ V P H D G[n 1c H LflT n [n JT"V Yfv K m T T M e[h 1p 8 I G Q L E l I i d [ h H I % % B/ T I F A V T £ L I P K 8 A V G T L S A N jiTlTv" I Q L I I D A Y HlS L 8 m E V I L EJ O BB P I F A V T S R M V K I I P K 8 A V G EL S E D 8 8 H V v [ g L i ( F ^ a Y L 8 m R V FL g BS L IF A V T E L I P G T T V G V L S M D 8 8 H V L Q L I V D A Y[g K I R m K V E L g cr B 4 PIF A V T N Y f Ip V 8 8 L G V L q E D 8 8 H I V E L L [F E A F H R I R m N LD I [r fD BB L I F A V T K N H Y M L I P G T T V[Ê“ IL D G D 8 H I I Q L I I A Y H|S IR s K V E L[g C/) BB L I F A V T
Analysis of the Effect of the T188I Mutation on
ligand binding by the Integrin Subunit
4.1 Introduction
The majority of integrin mutations that have been characterised in human disease cause inactivation or loss of function of the affected subunit (reviewed in (Hogg and
Bates, 2000)). In this chapter I set out to discover if the T188I mutation affected integrin function in a similar way. Initially I used a cell free integrin assay system to measure the binding of the T188I subunit to its ligand (Stephens et al, 2000). This system uses the extra cellular domains of the integrin subunits fused to the Fc regions of IgG, combined with a “knob and hole” approach to ensure a/p dimer formation.
This allows ELISA style assays of integrin/ligand interactions to be performed in a cell free environment, removing the avidity issues seen in cell based systems. The assay is commonly used to screen libraries of small molecule inhibitors of integrin function.
To confirm the results obtained with recombinant integrins I expressed both the
T188I and WT cpl in pi null fibroblasts and performed adhesion assays in a variety of conditions.
115 Chapter 4 Analysis of T188I Integrin Function
4.2 Results
4.2.1 Analysis of the effect of the T188I mutation on integrin affinity in cell free assays.
To investigate the effect of the T188I mutation on individual a/p integrin heterodimers the T188I mutation and WT pl subunits were produced as secreted Fc fusion proteins and used to perform ELISA style assays to measure the binding of the av and a5pl dimers to 10 pg/ml Fn coated surfaces. Titration curves were performed for WT av and aSal and the single concentration points selected for comparison with the mutant corresponded to the concentration of half maximal binding. Changes in observed binding at this point are therefore indicative of changes in affinity. This was performed by Drs A. Henry and V. Perkins at CellTech
Chiroscience PLC, Slough UK.
Binding of a5~Fc/ pl -Fc fusion proteins to fibronectin.
It was found that the a5pl T188I subunit combination bound fibronectin with an affinity 20% higher than the WT in the presence of Mg2+ (Fig 4.1 A). Addition of either Mn2+ or TS2/16 as activating factors (van de Wiel-van Kemenade et al,
1992)caused the binding of the WT to increase 20 % to that of T188I. There was no observed increase in the binding of T188I upon activation. This suggested that for aSpi the T 1881 mutation causes a shift to the activated conformation as promoted by
Mn2+ even in the absence of the activating cation.
116 Chapter 4 Analysis of T188I Integrin Function
Binding o f ocv-Fc/pi-Fc fusion proteins to fibronectin. av-Fc/pl-Fc T188I dimers bound Fn with an affinity 80 % higher than that of the
WT in the presence of Mg2+ (Fig 4. IB). On addition of TS2/16 the affinity of both the WT and T188I increased considerably (over 6 fold). In this condition the T188I was still more adhesive than the WT, exhibiting 7.8 times the affinity of the WT non activated protein compared to 6.3 times for the activated WT.
117 Chapter 4 Analysis of T188I Integrin Function
a 3 .0 - WT WT T188I T188I 0 .5 2.5
1 4 1.0 < 12 0.5 0.1 0.0 0.0 1 10 100 1000 1 10 100 1000 [aSpI] ng/ml [avp1] ng/ml 2001 150 +.100 1001 Mq2*+TS2/16 Mn2" Mg2»+7S2/l6 Fig 4.1 : The T188I mutant pl subunit exhibits a greater affinity for fibronectin coated surfaces than the WT subunit when expressed as a Fc fusion protein with either thea5 (A,C) or av (BJD) subunit. The degree of binding was influenced by the presence of the cations Mg2+ and Mn2+ as well as the activating anti-P 1 antibody TS2/16. Mean of triplicate wells±S.D. Data representative of three experiments. (White Bars=WT, Black bars=T188I) 118 Chapter 4 Analysis of T188I Integrin Function 4.2.2 Construction of chick Pl T188I equivalent retroviral construct In order to express the T188I mutation in human cells and be able to distinguish it from the endogenous protein the equivalent chick mutant subunit was made. The cpl subunit is 80 % homologous to the human subunit and shows complete conservation of the cytoplasmic domain. WT cpl cDNA pBabe-puro retrovirus vector was used as a template for site-directed-mutagenesis by the quickchange protocol (Fig 4.2). Primers T1881+ and T1881- were used. T188I Fig 4.2: pBabe-puro retroviral vector showing position of cPl integrin cDNA and the position of the bases corresponding to mutation T1881. 119 Chapter 4 Analysis of T188I Integrin Function 4.2.3 Expression of chick T188I in Pl null fibroblasts To allow analysis of the effect of the T188I mutation on pi integrin function in the absence of any WT pi subunits I used a cell line that lacks pl integrin, the GD25 cell line (Wennerberg et al, 1996). GD25 cells are immortalised fibroblasts derived from pl null mouse embryonic stem cells. When the pl subunit is reintroduced the cells express a3pl, a5Pl and a6pi on their surface (Wennerberg et al, 1996). GD25 were induced to express either empty vector pBabe-puro, cpi WT or cPl T188I by retroviral infection using Phoenix ecotrophic packaging lines. The infected cells were then FAC Sorted to produce pure populations with high levels of surface expression (Fig 4.3A). T1881 integrin is recruited to focal adhesions. GD25 cells infected with either the WT or T188I pi integrin subunit were grown on coverslips overnight and the stained for cpi integrin using the simultaneous fix and permeabilization method. It was found that the infected GD25 cells form focal adhesions containing the introduced chick subunits (Fig 4.3B). There was no obvious difference in size or distribution between the focal adhesions formed from WT or T188I cPl. 120 Chapter 4 Analysis of T188I Integrin Function A WT T188I T188 B Fig 4.3 The T188I mutant subunit forms is expressed on the surface of the cell and is capable of forming focal adhesions. (A) GD25 cells were infected with either the T188I or WT cpi subunit and sorted to give a pure population with equal high expression. Cells stained with JG22 (anti c^l, Black profile). Grey profile - uninfected cells. (B) The introduced subunits were both able to form focal adhesions when plated on 10 pg/ml Fn. Green: V2E9 - anti cpi mAb, Red - Phalloidin, Blue - Hoescht 121 Chapter 4 Analysis of T188I Integrin Function 4.2.4 Adhesion of GD25 cells expressing WT and T188I integrin subunits T188I causes increased adhesion of GD25 cells to fibronectin and laminin but not vitronectin relative to WT controls. In order to confirm the results of the cell free integrin binding experiments the adhesion of empty vector, chick pi WT and chick p l T1881 infected GD25 cells to surfaces coated with various ECM proteins was assayed. Initial assays using serum free E4 as a buffer were performed to determine the optimum concentrations of ligand to use. 5 pg/ml FN represented the point at which 50% of the WT cells attached during the duration of the assay. Cells were then plated on 5 pg/ml Fn in TBS in the presence of 1 mM and 10 mM concentrations of Mg2+, Mn2+ or Ca2+ and were allowed to adhere for 15 minutes at 37 °C. When the cells were plated in TBS in the presence 1 mM of Mg2+ all three cell types adhered poorly (Fig 4.3A). In higher concentrations of Mg2+ (10 mM) and in 1 mM Mn2+ the cells expressing the mutant subunit were found to bind in higher numbers (approximately 50% more in Mg2+ and approximately double in Mn2+) than those expressing the WT. A higher 10 mM concentration of Mn2+ appeared to be toxic to the cells and was not used for further experiments. An activating effect was also observed in the presence of 1 mM Ca2+ but fewer cells adhered than in Mn2+. 10 mM Ca2+ did not support binding as effectively as 1 mM and was not used for further experiments. The empty vector cells adhered poorly in all cations and concentrations. 122 Chapter 4 Analysis of T188I Integrin Function The adhesion of T188I expressing GD25 cells for 10 |xg/ml Ln coated surfaces was measured as for Fn. It was found that T188I caused an increase in adhesion in the presence of Mn2+ of around 40-50 % but no increase was detected in the presence of Mg2+ (Fig 4.4B). The empty vector cells showed no adhesion to Ln. This is consistent with a lack of either a3 or a6 on the surface of uninfected cells. Fligher concentrations of Ln were not found to improve the numbers of cells binding. When assayed for adhesion to Vn neither the T188I nor the WT expressing cells showed any increase in binding over the levels shown by the empty vector cells (Fig 4.4C). This is consistent with the published observations that GD25 do not express an avpl dimer after infection with the pi subunit (Wennerberg et al, 1996). Adhesions assays were not performed on collagen as the GD25 cells lack collagen receptors and do adhere to this ECM protein. 123 Chapter 4 Analysis of T188I Integrin Function 80% A 60% c 2 ^ 40% 1 ■o CD 0% G D 25 GD25 WT GD25T188I B 50% 40% - c 2 30% ^ (D I x■a : CD 20% . 10% j 0% : GD25G D 25 W T GD25 71881 80% 60% c 2 IZCD 40% ■O CD 20 % - 0% XX GD25 GD25WT GD25T188I » TBS o +Mg lOmMr +Mn ImMw +Ca 1mM Fig 4.4: Adhesion of GD25 cells expressing WT (GD25WT), T188I c(3l (GD25 T i881) or empty vector (GD25) to ECM proteins. (A) Cells expressing the T188I subunit show a greater affinity for Fn in the presence of Mg2+, Mn2+ and Ca2+. (B) When adhering to Ln an increase in adhesion is only seen in the presence of Mn2+ and is smaller than that seen on Fn. (C) Neither (31 subunit increases adhesion to Vn. *: p<0.05 that T1881=WT. Error bars are S.D. of triplicate wells. Data representative of four experiments on Fn, three on Ln, two on Vn 124 Chapter 4 Analysis of T188I Integrin Function 4.2.5 Adhesion of SCC4 cells to ECM proteins. SCC4 cells shows increased adhesion to fibronectin and collagen coated surfaces when compared to a similarly non-differentiated SCC WT/WT cell line. To have an effect on the behaviour of the SCC4 cells the T188I mutation must be able to influence integrin function when present as a heterozygous allele. The binding of SCC4 cells to surfaces coated in either 10 pg/ml Fn or Col was measured as for GD25 cells and compared to that of SCC13, a SCC line with a similar degree of differentiation (Rheinwald and Beckett, 1981) known to be WT/WT for the |3l subunit. It was found that the SCC4 cells exhibited greater adhesion to both matrix proteins than the SCC 13 cells in all conditions assayed, although the difference between the cell types was smaller than that seen in the GD25 WT/T188I comparison presumably due to the heterozygous nature of the mutation (Fig 4.5A,B). In the presence of 10 mM Mg2+ the SCC4 showed^approximately 30 % increase in cells adhering to both Fn than SCC13. In the presence of 1 mM Mn2+ the SCC4 cells showed an increase in adhesion of around 50-60 %. The pattern of adhesion was similar on Col, with a 50-75 % increase in adhesion in either cation. SCC4 can be induced to express the HUTS-21 epitope but normal kératinocytes cannot. The activation state of integrin dimers can be assayed by the binding of reporter antibodies. These bind to sites on the dimer that are only accessible when the integrin is in its high affinity or ligand bound state (Bazzoni et al, 1995; Tuque et al, 1996). 125 Chapter 4 Analysis of T188I Integrin Function Normal kératinocytes and SCC4 cells were harvested, resuspended in either TBS+Mg2+ (10 mM) or TBS+Mn2+ (1 mM) and then stained with the activated 131 integrin reporter antibody HUTS-21. No HUTS-21 binding was detected on normal kératinocytes in either cation, consistent with previous work suggesting that keratinocyte integrins are normally in a low affinity state unless ligand bound (Bishop et a l, 1998). The SCC4 cells showed the same lack of HUTS-21 binding in TBS+Mg2+ as normal kératinocytes but when in the presence of Mn2+ the mean fluorescence increased twofold indicating that some of the integrins present on the SCC4 cells were now in an active conformation (Fig 4.5C). 126 Chapter 4 Analysis of T188I Integrin Function 60% 1 A 50% 40% - % adherent 30% J 20 % - 10% - 0% « SCC13 SCC4 60% B * 50% * T 40% - % adherent 30% j 20% 4 10% 0% SCC13 SCC4 m TBS o +Mg 10ml\/- +Mn ImM 8 8 MOLT-4 SCC4 8 8 8 8 8 c I? ? FL2-II FL2-H FL2-H Fig 4.5: SCC4 shows greater adhesion to (A) FN and (B) Col than SCC13, a WT/WT undifferentiated eel! line. Means of triplieate wells±S.D. Data representative of three experiments (C) The HUTS21 (31 integrin activation reporter epitope is expressed on SCC4 in the presence of Mn2+. It is also present on the positive control line M0LT4 but not on normal keratinoeytes. Grey trace HUTS21 staining in presence of Mg2+, Black trace HUTS21 in presence of Mn2+. .c 127 Chapter 4 Analysis of T188I Integrin Function 4.2.6 Homology modelling of the Pl integrin A domain In order to better understand the T188I mutation, a model of the pi A domain was devised. This was possible due to the publication of the crystal structure of the avp3 integrin (Xiong et al, 2001). The residues corresponding to the A domain of the pi integrin were found by alignment of the p3 A domain sequence in Mac Vector sequence analysis software. This sequence and the corresponding T188I sequence were then fitted to the crystal structure of the ligand bound (1L5GB,(Xiong et al, 2001)) and the ligand unbound avP3 (lJV2B(Xiong et al, 2002)) by the SWISS- Model automated structure server (Guex and Peitsch, 1997). The resulting file was then adjusted in SwissPDB viewer to improve the fit and the file optimised by the SWISS-Model server to produce the final models (Fig 4.6, 4.7A). The pi A domain has a high degree of homology to corresponding P3 domain and the resulting models are all similar to those of the p3 crystals. It was found that the T1881 residue projected upwards from the C-C loop which itself projects from the upper surface of the main body of the domain. The presence of the isoleucine caused a change in the position of the C-C loop in both models however change in the body of the structure was seen. While there are fewer major structural changes in the P A domain upon activation than in the a 1 domain it is likely that this lack of change is due to the method used to prepare the model which always attempts to fit the modelled sequence as closely as possible to equivalent region of the template. This will tend to minimise the effects of changes in sequence that are not immediately proximal to the mutated region. 128 Chapter 4 Analysis of T188I Integrin Function When compared to the pi A domain models of You et a/.(You et al, 2002) and Rava Devi and Taj ne the model presented here differs in the presence of the disulphide bridge in the C-C loop (Fig 4.7 B-D). You et al state that to include the C-C bond forces the loop into unnatural kink. While the loop is in a tight hairpin SWISS- MODEL does not remove the C-C loop when the structure is optimised and energy minimised. 129 Chapter 4 Analysis of T188I Integrin Function I? 1 ^ 1 WT T188I ♦ Fig 4.6 The C-C loop (red) bearing T188I (white) lies adjacent to the ligand binding site (light blue) on the surface of the pi A domain. Model based on the crystal structure of avps in presence of ROD peptide (shown as trace (Xiong et al., 2002). MIDAS and ADMIDAS cations shown in orange. Light blue surface denotes residues within 4 Â of ligand. The presence of the mutation causes a change in the position of the C-C loop in the model. The isoleucine occupies a greater area on the surface of the protein (T188I) than the threonine (WT). Model produced using SWISS-MODEL and rendered using SWISS-PDBviewer and winPOV. 130 Chapter 4 Analysis of T188I Integrin Function Fig 4.7: (A) Theoretical model of avpi p propeller and p subunit A domain bound to an RGD peptide (produced by SWISS-MODEL based on 1L5GB). Comparison between the model of the pi A domain produced by SWISS-MODEL based on avp3 in absence of ligand (1JV2B) (B,D) and the theoretical model of S. Rama Devi and S. Tajne (ILHA, PDB) (C,E) Helixes shown in red, p-sheets in yellow and random coil in grey. RGD peptide in green. The C-C loop is highlighted in blue and its cysteines in yellow. MIDAS and ADMIDAS cations in blue. 131 Chapter 4 Analysis of T188I Integrin Function 4.3 Discussion It is clear from the results presented in this chapter that the T188I mutation has an effect on the function of the (31 subunit and that this is to cause an increased affinity for ligand. Prior to the solution of the av|33 crystal structure (Xiong et al, 2001) a mechanism for this activation would have been hard to envisage. With the benefit of the structure it is apparent that the equivalent residue in p3 lies on the top of the P3 A domain, close to the MIDAS site and plays some role in the structural rearrangements caused by ligand binding. The cell free adhesion assays show that the unstimulated conformation of the T188I subunit has a similar affinity to that of the stimulated conformation of the WT. In the case of a5 p i the T188I Mg2+ condition shows the same level of FN binding as the WT Mn2+ state although this represents only a small shift in the affinity of dimer. This suggests that the T188I is permanently in either the high affinity Mn2+ induced state or a similar activated conformation. The cell based assays show increases in adhesion of the T188I expressing cells in both the Mg2+ and the Mn2+ bound state suggesting a conformational change in the structure exposing the ligand-binding site more in both the low and high affinity conditions. The cell based assays have the disadvantage of measuring the adhesion of the cell to a ligand coated surface rather than the binding of an individual receptor to its ligand. Because of this the assay really measures the avidity of the cell, as the cell’s adhesion is affected by the rate at which the many thousands of individual 132 Chapter 4 Analysis of T188I Integrin Function integrins attach and detach from the surface. The ceil based assays have the advantage of working with intact full length integrins. The comparison between the cell based and cell free systems shows some interesting differences in activation patterns. The WT is activated by around 20% by Mn2+ or TS2/16 in the cell free system. This is also seen in the cell based system. The T188I subunit has the same affinity in Mg2+ as the WT in Mn2+ in both assays systems. However in the case of the a5pl cell free assay no further activation is seen in the T188I subunit after treatment with Mn2+ or TS2/16. In the cell based assays a large increase in binding can be seen when the T188I subunit is placed in Mn2+. This suggests that the replacement of the integrin cytoplasmic domains with the Fc domain in the cell free system may be somehow preventing maximal activation being achieved. While the a5pl/Fn cell based assay showed that the mutation caused an increase in both the Mg2+ bound and Mn2+ bound state, the a3/a6pi/Ln assays showed no difference in the low affinity Mg2+ bound state but did show a difference in the Mn2+ state. The cell free assays also showed differences in patterns of activation depending on the a subunit present. This shows that integrin activation is not purely a P subunit event and that the a subunit plays some role. While the structure does not suggest that the C-C loop or T188 itself interacts with the a subunit perhaps there is some difference in the tertiary changes upon ligand binding in the Pl A domain structure depending on the a subunit it is paired with. Alternatively as the recognition sequence in Ln differs from the RGD motif found in Fn perhaps there are 133 Chapter 4 Analysis of T188I Integrin Function different conformational changes on ligand binding. It is clear that the T188I mutation is capable of activating a range of different a subunits. The increased adhesion of the SCC4 cells to collagen shows that the conformational change can be transmitted from the p A domain to the a subunit I domain and influence the affinity of that ligand binding site. This is consistent with the mechanism recently proposed by Alonso et a/.(Alonso et al, 2002). The pi integrin found on keratinoeytes is usually in a low affinity state, except at points where the integrin is bound to ligand. In culture this is observed in focal adhesions or the “o” ring of integrin seen around the periphery of the cell. In vivo the high affinity form is seen at the basement membrane (Bishop et al, 1998). Active integrins are not found in cell-cell junctions (Kim and Yamada, 1997).The increased exposure of the HUTS21 epitope indicates that at least some of the integrin on the SCC4 is in the active, extended form. The lower signal from the SCC4 compared to the M0LT4 line may be indicative of the heterozygous nature of the mutation. The HUTS21 epitope has been mapped to a region on the stalk of the p l subunit (Luque et al, 1996). That this epitope is exposed in SCC4 cells in suspension in the absence of ligand shows that the T188I mutation can activate not only the P A domain, but can also trigger the large scale rearrangements typical of integrin activation. The lack of any inducible epitope on normal keratinoeytes is also interesting as in many other cell lines, such as M0LT4, it is possible to induce activation of the pi integrin using Mn2+ treatment. It is possible that there is an inhibitory inside-out signalling pathway present in keratinoeytes and that the mutation, acting locally on the p subunit A domain, overrides this effect. Ï34" Chapter 4 Analysis of T188I Integrin Function Activation of integrins by mutation is a rare occurrence. To date only two naturally occurring activating mutations have been characterised, both of which are in the |33 subunit (Fullard et al, 2001; Ruiz et al, 2001). One of these, C560R maps to the stalk of the subunit, the other, V193M, the A domain. V193M lies close to the position of T188I, however it is outside the C-C loop although it is within the larger area known as the specificity loop. In addition several mutated subunits have been designed in the laboratory with high affinity conformations. Deletion of the I domain from the aL Subunit of aLp2 cause the subunit to adopt an active conformation as detected by activation reporter antibodies (Leitinger and Hogg, 2000). a subunit I domains have been locked into the active conformation by locking the a l helix into its active position (See Fig 1.6) (Shimaoka et al, 2001). Swapping the C-C loop of p2 to the equivalent from P3 has also been shown to activate the integrin aLP2 (Kamata et a l, 2002). Mutation of the threonine to isoleucine may cause the activation of the integrin in several ways. Movement of the specificity loop towards the cation at the MIDAS site appears to be a significant part of the conformational change in integrin activation (Xiong et al, 2002). The activating effect of the T188I mutation is likely to be due to some conformational change in the structure around this region. T188 may form a bond to another residue in the area that stabilises the low affinity form, loss of this bond leading to a constitutive high affinity state. Alternatively the presence of the large hydrophobic isoleucine side chain may cause a change in the position of the C- C loop causing the integrin to remain in a fully or partially active state rather than a low affinity state. This shift could be caused by the increased size of the side chain _ Chapter 4 Analysis of T188I Integrin Function forcing some rearrangement of the neighbouring amino acids or, as T188 is partially exposed to solvent, caused by the hydrophobic isoleucine sidechain moving away from the surface of the protein. When the pl A domain is modelled based on the P3 structure the T188 residue faces upwards from the C-C loop and lies along the top of the main body of the domain towards the ligand. The isoleucine side chain would lie along the surface in the direction in which the C-C loop is thought to move. This may interfere in some way with the correct movement of the loop. The position of the C-C loop does not seem to allow it to be in contact with the a subunit or be part of the RGD binding site (Fig 4.6A). This is similar to the original avp3 crystals (Xiong et al., 2001; Xiong et al, 2002) where there is no contact between the subunits or the ligand at this point. However as the C-C loop is positioned just outside the ligand binding site it is probable that the loop is involved in the conformational change upon ligand binding in a similar manner to that seen in the avp3 ligand bound crystal. In the avP3-RGD crystal the ligand is represented only by a small peptide and it is possible that another part of a larger ligand, such as intact Fn, may come into contact with the C-C loop. This interpretation is supported by photo-cross-linking experiments (Bitan et al, 2000; Yahalom et al, 2002). These studies showed that an RGD peptide with a C- terminal photo-cross-linking group would become covalently attached to the integrin. Subsequent proteolytic sectioning of the integrin showed the RGD peptide bound to a fragment that included the C-C loop. 136 Chapter 5 The Effect of Integrin Activation on Cell Behaviour 5.1 Introduction The finding that T188I causes an increase in the affinity of the pl subunit for its ligand led me to investigate the affect of integrin activation on cell behaviour. I investigated this using GD25 pl null fibroblasts, the SCC4 cell line and normal keratinoeytes. To cause activation of the integrin in these cell types I used two methods, retroviral infection with the T188I chick pl integrin or incubation with an activating anti human pl integrin mAh TS2/16. 137 Chapter 5 The Effects of Integrin Activation 5.2 Results 5.2.1 Analysis of the effect of the T188I mutation on spreading, migration and invasion. GD25 spread more quickly on FN when infected with the T188I cf8l than WT c ^l integrin subunit. GD25 cells expressing either empty vector pBabe-puro, the WT c(3l or T188I cpi constructs were plated onto coverslip bottomed cell culture dishes coated with 10 pg/ml fibronectin and allowed to adhere for 5 min. Fields containing 35-40 cells were filmed for 24 hr. The number of spread cells was then counted and plotted against time to determine the rate at which the cells spread. A spread cell was defined as any cell that was no longer round and refractile. Any cell that did not spread within the length of the recording was not included in the analysis on the assumption that it was non-viable. It was found that the WT expressing cells spread at a rate approximately half that of the T188I expressing cells. Half the WT cells were spread in about 30 minutes compared to approximately 10 minutes for the T188I expressing cells. The empty vector cells spread poorly and took in excess of 90 minutes for 50 % to spread (Fig 5.1). Once the cells had been plated for 5 hours and had fully spread state their morphology was compared. The WT and T188I infected cells were spread to o greater extent than the parental cells. No differences between the WT and T188I cells 138 Chapter 5 The Effects of Integrin Activation were observed that would indicate differences in Rac/Rho/Cdc42 signalling and there was no difference in the final degree of spreading observed (Fig 5.1 B). The movement of cells expressing the WT and T188I cPl subunits was tracked for 24 hr and quantitated. 40 cells were analysed in each experiment. It was found that the average speed of the uninfected GD25 was 3.55±0.045 pm/25min compared to 9.84±0.09 pm/25 minutes for the T188I cells and 7.91± 0.08 pm/25 minutes for the WT. The speeds of cells expressing the WT and T188I pl are not statistically different at the p<0.05 level (Fig 5.2). 139 Chapter 5 The Effects of Integrin Activation 1 2 0 -1 A 100 - AAAA AAA AAA AA o 55 40 - AA 0 1 0 20 3040 50 min O G D 2 5 n G D a S cp1 a GD 25 cp1 T 1 881 B GD25 GD25WT GD25T188I 20min « « 40min 5 hr 140 Chapter 5 The Effects of Integrin Activation Fig 5.1: Expression of T188I cpl causes an increase in the rate of spreading of GD25 cells. (A) GD25 cells expressing the T188I subunit (GD25 T188I) spread more quickly on 10 pg/ml Fn than those infected with the WT subunit (GD25 WT) or empty vector (GD25). At each time point the number of cells that had begun to spread was counted. Cells which did not spread over the course of the whole film (20 hrs) were discounted as non-viable. This represented fewer than 5 cells in any sample. (B) GD25 cells expressing the WT or T188I cPl or empty vector cells 20,40 or 300 minutes after plating. Bar= 100p.m. Data representative of three experiments. 141 Chapter 5 The Effects of Integrin Activation A 17.5 D25 T188I GD25 WT, 12.5 7.5 tHlHHt'millVt'tll,! 2.5 200 400 600 800 1000 1200 200 12Q[ 0 200 400 600 800 1000 1200 time [mm] time [mm] time [mm] B 10.0 7.5 I 5.0 E 2.5 0.0 GD25 T188I WT Fig 5.2: The effect of WT or T188I cfl on the movement of (31 null fibroblasts. The migration of GD25 cells expressing empty vector (GD25) , WT (GD25 WT) or T188I (GD25 T188I) chick pi subunits on 10 |Lig/ml Fn was measured over 20 hr. (A) Data plotted is the average speed of the cells±S.D. at each time point. (B) The average speed of each type of cell±standard error over the whole 20 hr. 40 cells tracked for each cell type, data representative of 3 experiments. 142 Chapter 5 The Effects of Integrin Activation GD25 cells expressing WT p l integrin invade through Matrigel at a higher rate than those expressing T1881 or empty vector. Invasion through the surrounding extracellular matrix is an important factor in tumorigenesis (Boyd, 1996). In order to determine whether the T1881 pl subunit would promote invasion, Matrigel assays were performed. In this assay the cells are placed above a gel made from a mixture of ECM proteins above a porous membrane. A chemoattractant is placed in the compartment below the gel and over several days the cells invade through the gel and the membrane to reach the lower (Thomas et al, 2001). The rate of invasion is controlled by several factors including the ability of the invading cells to produce proteases to break down the gel (Del Rosso et al., 2002), their responsiveness to the chemoattractant and their integrin expression (Thomas et al, 2001). Initial experiments where cells were allowed to invade through a gel formed from 1:1 Matrigel:medium were found to give poor results as GD25 cells did not spread well on this concentration of gel. This resulted in clumps of cells forming above the gel and when cells did migrate through the gel they tended to move as clumps (Fig 5.3A). In addition, the 3 day incubation used allowed these cells to start to divide on the lower side of the membrane and produce monolayers. To rectify these problems a 1:2 Matrigel:medium gel was used and the incubation period reduced to 48 hours. This improved the spreading of the cells on the surface of the gel and caused them to migrate individually (Fig 5.3A). The reduced time period also reduced the problem of growth on the lower side of the membrane. 143 Chapter 5 The Effects of Integrin Activation After the cells had been allowed to invade the membranes were fixed in methanol, stained with crystal violet and photographed at 2.5x using a microscope equipped with a digital camera (Fig 5.3B). The resulting photos were then converted to a black and white image and the number of black pixels (representing cells) measured by image analysis software (Fig 5.3C). This number was then converted to cells/mm^ by comparison with the average number of pixels occupied by single cells. It was found that cells transduced with the empty vector migrated slowly and few were found on the lower side of the membrane. Few T188I expressing cells were found to have invaded although there was a statistically significant increase in the number of the T188I cells present below the membrane when compared to the empty vector. The WT cells migrated significantly more quickly with at least three times as many cells present on the bottom of the membrane than the T188I expressing cells (Fig 5.3D). I concluded the T188I subunit does not promote invasion. GD25 expressing T188I migrate toward serum more slowly when infected with WT p i subunit. As cells expressing the T188I cpl subunit were less invasive than those expressing the WT subunit (Fig 5.3D) but there was no difference in the rate of random migration (Fig 5.2) it was important to determine whether there was any change in the attraction of these cells to serum. This assay was performed by measuring the number of cells that moved through a transwell filter from a low serum to a high serum environment. It was found that the number of T188I expressing cells that migrated through the membrane was lower than that of the WT. Approximately 15% of the T188I cells compared to 22% of the WT cells migrated through the membrane in 6 hr (Fig 5.3E). This result mirrors the difference in the rate of invasion through — Chapter 5 The Effects of Integrin Activation matrigel in that expression of the WT cpi promoted movement towards serum more efficiently than the mutant. This suggested that the lack of invasion through Matrigel of the T188I expressing cells may be partly due to a lack of attraction towards the serum in the lower chamber rather than simply an inability to penetrate the Matrigel layer. 145 Chapter 5 The Effects of Integrin Activation A W* m B c ; Î • . ^ • • • Vf^ . * * t • • > • - / • • .• . • . .f -, '■ 2 D 50 40 g 25% Z 20% "g 30 S) 15% 1 20 = 10% 10 0 GD25 GD25{ilT188I GD25 pi WT GD25 GD25P1T188I GD25 pl WT 146 Chapter 5 The Effects of Integrin Activation Fig 5.3: The effect of WT or T188I cPl expression on GD25 movement through transwells in the presence or absence of matrigel. (A) The lower surface of a transwell stained with crystal violet showing clumps of cells when a 1:1 Matrigel:medium gel was used with a 72 hr incubation period (left) or cells more evenly distributed across the membrane with a 1:2 gel and 48 hr incubation. (B) Lower surface of a transwell membrane imaged. Bar=300 pm. (C) Black and white image of B as used for quantitation of cell number. (D) Infection with the WT cpi promotes invasion more effectively than T18I cP l. Mean number of cells/mm^ on the lower surface of the transwell after 48 hours migration±S.D. of triplicate wells. (E) Expression of either the T188I and WT supports migration through transwells in the absence of Matrigel but fewer cells expressing the T188I construct are found on the lower side of the transwell. % of cells on the lower surface of the transwell±S.D. of triplicate wells after 6 hours migration. 147 Chapter 5 The Effects of Integrin Activation 5.2.2 The effect of integrin activation on cell signalling GD25 expressing T1881 show increased phosphorylation of ERKl/2 in response to adhesion to fibronectin when compared to cells expressing WT cPl Integrin signalling, particularly the MAPK pathway (Zhu et al,, 1999), plays an important role in the regulation of terminal differentiation. To investigate whether integrin activation could alter integrin signalling I used the GD25 cells to assay MAPK activation upon spreading on ECM. GD25 cells expressing either the WT or the T188I construct were trypsinised, washed in serum containing medium then twice in serum free medium and plated onto 10 p.g/ml Fn. Lysates were made at various time points. When the levels of ERKl/2 phosphorylation in the absence of EGF was quantified by western blotting both WT and T188I expressing cells showed an increase in activation for the first 7 minutes which then fell to very low levels of activation until at least 60 minutes after plating (Zhu et al, 1999). However the T188I expressing cells showed a greater activation than the WT with phosphorylated p44 ERK being easily detectable 7 minutes after plating (Fig 5.4). When the experiment was repeated in the presence of 10 ng/ml EGF the levels of ERKl/2 phosphorylation were found to be much higher and with little difference between the WT and T188I at the 7 minutes time point. However the high level of phosphorylation was found to be maintained for a longer period in the T188I expressing cells with high levels of phospho-ERK still detectable at 40 minutes. It was also observed that there was a slight increase in the level of phospho-ERK in the presence of EGF in T188I expressing cells held in suspension for 60 minutes when compared to the WT (Fig 5.4). These result are interesting as the duration of ERK — Chapter 5 The Effects of Integrin Activation activation in the WT expressing cells closely matches the rate of spreading, whereas in the T188I expressing cells MAPK activation continues long after the cells are spread (see Fig 5.1 ) +EGF -EGF 7 15 25 40 60 90 S 7 15 25 40 60 S Time (min) P-ERK1/2 W Tcpi total ERK P-ERK1/2 T188I Gp1 total ERK Fig 5.4 : ERK phosphorylation in pi WT and T188I expressing GD25 cells. Cells were plated on 10 pg/ml Fn in the presence or absence of 10 ng/ml EGF. ERK phosphorylation is maintained longer in the cells expressing T188IcPl than those expressing WT integrin, S = 60 minutes in suspension. 149 Chapter 5 The Effects of Integrin Activation 5.2.3 Effect of integrin activation on SCC and normal kératinocytes. Infection ofSCC4 with T1881 does not impede growth relative to the WT As increasing the adhesion of cells can, in some cases, lead to a reduction in growth the growth rate of cultures of SCC4 infected with empty vector, WT or T188I chick integrin was determined. 1x10^ cells were plated into each well of 24 well tissue culture dishes and over the space of 17 days wells were harvested and the number of cells counted. It was found that infection with the WT reduced the growth rate of the kératinocytes, the growth curve shifting slightly to the right (Fig 5.5). The presence of the activated integrin did not cause any further reduction. For most of the time- course the growth curves of the WT and the T188I cells were less than one standard deviation apart. A B WT 1,000,000 I 8 100,000 T188I g 10,000 0 2 4 6 8 10 12 16 1814 D ays — OpBp DWT ---AT188I Fig 5.5 (A) Expression of the T188I c(3l does not inhibit growth more than the WT. 1x10^ SCC4 cells expressing the WT cpl, T188I subunit or the empty vector were plated in 24 well plate wells on day 0 and harvested at intervals over the next 18 days. The number of cells per well was counted. Points are the mean number of cells in triplicate wells±S.D. of triplicate. Data representative of three experiments. (B) Expression of the WT and T188I constructs was equivelant. (grey trace - non specific control. Black - JG22, anti cpd) 150 Chapter 5 The Effects of Integrin Activation Infection of SCC4 with T188I cPl subunit does not induce differentiation whereas infection with WT does. In order to determine whether the T188I mutation was responsible for the lack of differentiation in the SCC4 line SCC4 cells transduced with the empty vector, WT cpi or T1881 cpi subunits were co-cultured with J2F feeder cells and allowed to grow to 75% confluence. The cultures were then dissaggregated and stained for the the differentiation markers involucrin and comifm. The presence of the WT integrin subunit induced both involucrin (Fig 5.6) and comifm (Fig 5.7), to a similar extent as seen before (15-20% for each marker)(Levy et al, 2000). Neither the empty vector or the T1881 subunit caused induction of either differentiation marker (<4% positive for either marker). 151 Chapter 5 The Effects of Integrin Activation SCC4 SCC4 pBp SCC4 cpi WT SCC4 cp1 T188I if 0 25% B ? 15% SCC4 SCC4 pBp SCC4 cpl T188I SCC4 cpl WT Fig 5.6: Involucrin expression in SCC4 cells retrovirally infected with the WT, T188I cpl subunits or empty vector. (A) Dissaggregated cultures of SCC4 cells infected with either the WT cpl, T1881 cpi or the empty vector were dried onto coverslips and stained with SY-5 (mAh to human involucrin). (B) Quantitation of involucrin staining. Triplicate slides were counted for each cell type and the meaniS.D. plotted. At least 300 cells per slide were counted. Data representative of four experiments. 152 Chapter 5 The Effects of Integrin Activation SCC4 pBp # SCC4 c(31 WT SCC4 cpi T188I 20% B 0I 15% 1g. 10% S « Ü 5% 'o 0% SCC4 SCC4 pBp SCC4 cBl mt SCC4 cBl wt Fig 5.7: Comifm expression in SCC4 cells retrovirally infected with the WT or T188I cpl subunits or empty vector. (A) Dissaggregated cultures of SCC4 cells infected with either the WT cpi, T188I cpi or the empty vector were dried onto coverslips and stained with SQ37C (pAb to human comifm). (B) Quantitation of comifm staining. Triplicate slides were counted for each cell type and the mean±S.D. plotted. At least 300 cells per slide were counted. Data representative of five experiments. 153 Chapter 5 The Effects of Integrin Activation Infection ofSCC12B2 or up with T 1881 does not effect differentiation. In order to examine whether the T188I mutation suppressed differentiation, failed to induce or was simply inactive in influencing differentiation 1 expressed the T1881 and WT integrin in two keratinocyte derived cell lines with WT endogenous pi subunits. SCC12B2 is a cell line similar to the SCC4 (Rheinwald and Beckett, 1981). up is a HPV transformed keratinocyte cell line (Hodivala et a l, 1994). These were chosen as they are both transformed keratinocyte lines that behave in a similar manner to the SCC4 but express higher levels of differentiation markers. In both cases high levels of cPl expression were obtained but in each case the levels of differentiation in the culture was too low to detect any reduction in the presence of the T1881 subunit. The WT integrin did not induce differentiation in either case (Fig 5.8). Therefore 1 could not conclude whether the T1881 mutation was able to actively suppress differentiation. Several attempts to retrovirally express the T1881 cpi in normal, untransformed kératinocytes did not produce any cells expressing the construct. 154 Chapter 5 The Effects of Integrin Activation A T188I B Cell Type / Construct % expressing involucrin Standard Deviation up pBp 223 1.66 up WT 0.20 0.44 upTlSSI T38 1.44 SCC12B2pBp 5.49 1.19 SCC12B2cPl WT 6.57 1.31 SCC12B2cPl T188I 6.70 1.03 Fig 5.8: Expression of the WT or T188I cPl constructs does not induce differentiation in either up or SCC12B2. (A) Involucrin staining (SY-5) of dissaggregated cultures of up cells expressing pBp (Up), cpl WT (WT) or cPl T188I (T188I). (B) Quantitation of involucrin positive cells in cultures of Up and SCC12B2. 155 Chapter 5 The Effects of Integrin Activation 5.2.4 The activating anti-Pl antibody TS2/16 can effect normal keratinocyte differentiation Incubation of normal adherent kératinocytes with TS2/16 causes a suppression of differentiation. To confirm that activation of the pi integrin subunit is capable of affecting the differentiation of primary kératinocytes, normal kératinocytes were cultured to confluence in the presence of various activating and non-activating antibodies for 48 hr. To avoid affecting plating efficiency by the use of the activating antibodies, the cultures were set up 48 hr before the antibodies were added. Four different monoclonal antibodies to the hpl subunit were used, each at 20 p,g/ml. In addition, a culture with no antibody and a culture with an antibody to the cpi subunit was used as a negative control. After 48hrs the cultures were washed in versene to remove the feeder cells, disaggregated and stained for differentiation markers. mAh Specificity Function P5D2 human pi Blocks adhesion A2B2 human pi Blocks adhesion 9EG7 human/mouse pi Recognises ligand bound conformation TS2/16 human pi Induces active conformation WIBIO chick pi Irrelevant antibody control Table 5.1 Antibodies screened for an ability to influence differentiation in cultured kératinocytes. 156 Chapter 5 The Effects of Integrin Activation Incubation of kératinocytes with 20 |xg/ml of the non-activating antibodies was found to have no effect on the kératinocytes expression of involucrin or comifin. However, the cultures incubated with the TS2/16 were found to contain 40 % fewer involucrin positive cells than the others. The percentage of cells expressing the differentiation markers was found to vary between experiments with the passage number of the parental cultures (approximately 10 % involucrin positive at passage 3 to 25 % at passage 6) but in all cases treatment with TS2/16 reduced this by around 40% (Fig 5.9). None of the antibodies caused any affect on cell spreading under these assay conditions. 157 Chapter 5 The Effects of Integrin Activation 140% 1 120% # 100% - ve W1B10 P5D2 A2B2 9EG7 TS 2/16 B Km5 +A2B2 +TS2/16 Fig 5.9 : The activating mAh TS2/16 caused a decrease in the number of involucrin positive kératinocytes in adherent cultures after incubation for 48 hr. (A) No change in the number of involucrin positive cells was found when kératinocytes were treated with an irrelevent antibody control (WlBlO), two other non activating mAbs (A2B2, P5D2) or an activation reporter (9EG7). Incubation with the activating antibody TS2/16 supressed differentiation by approximately 40%. (B) Triplicate dissaggregated cultures of kératinocytes incubated with A2B2, TS2/16 or untreated. Stained with DH-1, a polyclonal antibody to human Triplicate wells were analysed±S.D.. Data representative of three experiments. is F Chapter 5 The Effects of Integrin Activation TS2/16 is capable of suppressing keratinocyte differentiation in suspension It has been previously shown that suspension induced differentiation of kératinocytes can be suppressed by incubation with fibronectin or by certain adhesion blocking anti-integrin antibodies (Adams and Watt, 1989, Watt et ai, 1993). It has also been shown that restoration of integrin adhesion by using the activating anti (31 antibody 8A2 does not prevent differentiation in kera tinocytes previously placed in suspension (Hotchin et al.,, 1993). In order to examine whether the activating antibody TS2/16 could enhance the suppressing effect of fibronectin suspension experiments were performed with kératinocytes in the presence of fibronectin (25 pg/ml) TS2/16 (20 pg/ml) or both. It was found that fibronectin produced a suppression of differentiation of 20-30%. TS2/16 produced a similar inhibition (Fig 5.10). The combination of both treatments was found to be no more effective than either component alone. 100% o 75% - m 50% - 25% - TS2/16 TS2/16+FN Fig 5.10 Incubation of keratincoytes in suspension with TS2/16 reduces suspension induced involucrin expression by the same degree as Fn. The combination of both treatments was no more effective than either alone. Cells placed in suspension overnight with 25 jag/ml Fn and/or 20 pg/ml TS2/16. 159 Chapter 5 The Effects of Integrin Activation 5.2.5 Discussion The results of the previous chapter showed that the T188I mutation had an activating effect on the function of the pi integrin. In this chapter I have extended my investigation by testing additional aspects of cell haviour. I have shown that the activation of the integrin causes effects on the behaviour of the cell carrying the mutation. I found that expression of the XI881 integrin caused various changes in behaviour of the GD25 line when compared to the WT infected cells. This included an increase in the rate of spreading and changes in ERK activation but a lack of promotion of invasion through Matrigel and migration towards serum. Kératinocytes also show changes in behaviour on integrin activation. SCC4 was found not to differentiate in response to infection with the mutant subunit while the WT caused differentiation as before. Incubation of normal kératinocytes with TS2/16 was found to reduce the number of differentiated cells in culture and suppressed differentiation in suspension. The increased rate of spreading in GD25 cells expressing the T188I cPl subunit is likely to be a direct result of the increased integrin affinity. The lack of spreading observed in the cells infected with the empty vector is likewise probably due to the lack of integrin on the cell surface. Spreading of cells is linked to various events such as MAPK phosphorylation and differentiation in kératinocytes (Watt et a l, 1988; Zhu et al,, 1999). Increased adhesion has been linked to an inability to migrate (Kuijpers et al, 1993). It is clear that in the case of GD25 cells infected with the T188I cPl subunit the Ï6Ô" Chapter 5 The Effects of Integrin Activation increase in adhesion is not of an order to preclude cell movement as the T188I promoted migration to the same extent as the WT. It is possible that on very high or very low concentrations of Fn a greater difference in migration may have been observed as high affinity for ligand tends to reduce migration on high densities of ligand (Palecek et al, 1997). It can also be seen that a lack of adhesion due to a lack of integrin precludes cell motility as the cells infected with the empty vector showed little ability to migrate. These results are consistent with the results of Rose et a/.(Rose et a l, 2001) in which cells with an inability to effectively activate their integrin had a reduced ability to migrate. The invasion assay system used here, comprising a transwell chamber with a layer of semi-permeable ECM gel and a serum gradient, is a crude attempt to mimic the in vivo process of a tumour cell migrating away from the primary tumour, through the surrounding matrix and towards the vasculature. In order to move into the lower portion of the chamber the cells must be able to migrate effectively, produce appropriate MMPs to penetrate through the gel and also be attracted to the serum in the lower chamber. The link between integrin signalling and MMP production has been shown in numerous cases and involves signalling via proteins including PKC (Niu et a l, 1998) and tetraspanins (Sugiura and Berditchevski, 1999). Integrin subunits implicated in these systems include (x2 (Dumin et a l, 2001 ), a3 (Sugiura and Berditchevski, 1999), av|33 (Brooks et a l, 1996) and av(36 (Thomas et al, 2001), (Niu et al, 1998). In several cases ligation of the integrin not only induces expression of the MMP but also allows the MMP to directly associate with the integrin to form a ternary adhesion/ECM degradation complex (Dumin et al, 2001). In the case of the GD25 cells the lack of invasion of the uninfected cells may well be îô T Chapter 5 The Effects of Integrin Activation due to a lack of MMP production caused by a lack of integrin signalling. Alternatively without the presence of integrin a subunits on the cell surface the MMP may not be able to localise to the correct part of the membrane to assist migration (Brooks et al., 1996; Dumin et al, 2001). The increase in invasion on infection with the WT cPl could be explained by a possible induction of MMPs by the a3 subunit now present on the surface. However the lack of invasion by GD25 cells expressing the T188I cPl subunit is hard to explain in terms of MMP production. Palecek et al propose that the optimum ligand concentration for cell migration is inversely proportional to the affinity of the cell for its ligand (Palecek et al, 1997). As the matrigel presents the cell with a very high effective concentration of ligand it is possible that this is causing inhibition of migration through the gel. In an attempt to answer this question we performed similar assay in the absence of a matrigel layer. In these assays the GD25 cells failed to migrate. This is likely to be due to the absence of integrin on their surface as was seen in the random migration assays. The number of WT cells that migrated through the membrane was higher than the number of MT cells, although the difference was not as marked as for the matrigel assays. This suggests that the T188I subunit fails to promote directional movement towards serum as effectively as the WT expressing cells and this may be a contributory factor in the lack of invasion of the T188I cells. The interplay between integrin signalling and growth factor receptor signalling is well known (Moro et al, 1998). It is possible that over activation of one pathway can support signalling in both when the second stimulus is at a sub optimal level. In this situation increased signalling from the integrin may be compensating for the low growth factor signalling in the low serum ïô T Chapter 5 The Effects of Integrin Activation environment above the membrane resulting in reduced migration into the high serum medium below the membrane. The ERK 1/2 pathway has been shown to be capable of influencing kératinocytes behaviour, in particular the ability of the cell to differentiate (Zhu et al, 1999). Two pathways that can lead to ERK are signalling from the EGF receptor (reviewed in Yarden and Sliwkowski, 2001) and from integrin ligation to ECM (Chen et al, 1994). The signalling pathways from these two sources share many common components and for maximal activation of ERK both signals are required. The strength of ERK signalling from pi integrin ligation has been shown to be proportional to the number of ligated integrin receptors (Asthagiri et a l, 1999) and a correlation between the strength of adhesion and the strength of ERK signalling has been observed (Ishida et a l, 1996). An increase in ERK signalling due to a increase in the number of ligated integrin subunits due to the increased affinity of T1881 is likely to explain the increased signalling at the initial time points in the absence of EGF and the increased duration of ERK signalling in the presence of EGF. The signal from the integrin is often described as being “permissive”, for the more important EGF signalling event (Miyamoto et al,, 1996). In both the WT and T188I expressing cells this can be seen as ERK activation drops to low levels in the absence of integrin ligation even in the presence of EGF. This interplay between ERK activation and integrin ligation is linked to the initiation of cell spreading due to changes in the cytoskeleton (Asthagiri et al,, 1999). This is seen in the WT cpl expressing GD25 cells where ERK activation drops back to basal levels once the cells are spread at 40 to 60 min. However the T188I cpl expressing GD25 show ïô T Chapter 5 The Effects of Integrin Activation strong activation of ERK at 40 minutes, at a point where most of the cells have spread suggesting the mutation can affect integrin signalling independently of its effects on spreading. The absence of any induction of differentiation in the SCC4 cells expressing the T188I subunit suggests that the mutant integrin is, in some way, involved in the differentiation phenotype seen in the SCC4 cell line. I have not been able to express this construct in normal kératinocytes or any keratinocyte line that differentiates at levels high enough to see any suppresive effect so I cannot determine whether the construct simply fails to induce differentiation or actively suppresses it. The results of the experiments using TS2/16 to activate the endogenous integrin on normal keratincoytes suggest that raising the affinity of the pi integrin does, in fact, actively supress differentiation. TS2/16 and T188I both causes integrins to switch into a higher affinity state, increasing the affinity of the cell for extracellular matrix and promoting ligand binding. It is probable that this increase in ligand binding causes the decrease in the number of differentiated cells. This is consistent with previous work (Adams and Watt, 1989; Levy et al.,, 2000) where it was shown that pi integrin binding to ligand sends a "do not differentiate" signal. This would also explain the restoration of differentiation seen after infection with the WT cpi subunit. The WT subunit competes out the effect of the T188I by diluting the number of T188I integrins, reducing the strength of the "do not differentiate" signal and restoring differentiation. The effect of TS2/16 in suspension is harder to reconcile with the previous study using a different activating mAh, 8A2 (Hotchin et al,, 1993). 8A2 was not found to — Chapter 5 The Effects of Integrin Activation suppress suspension induced differentiation either as whole antibody or as Fab fragments. It is possible that the whole antibodies used in the TS2/16 experiments were simply crosslinking the pi subunits and thereby activating signalling. However if this was the case the whole 8A2 antibodies would have been likely to be equally effective. It is clear from the results from Chapter 4 that there are a range of ways of stimulating a high affinity state (cations, antibodies, mutation) and that they do not necessarily have identical effects on adhesion (Ortlepp et al, 1995). It has also been shown that different functional forms of integrins are often found on cells (Chan and Hemler, 1993) and that different conformations of the extracellular domains can cause different signalling events (Miao et al, 2002). The TS2/16 epitope lies close to the site of the T188I mutation in the A domain of the p subunit. The epitope of 8A2 is not known. It is possible that the TS2/16 activation occurs in a way that is sufficiently different to that of 8A2 that the signalling that results differs. This would also explain why 9EG7, a anti hpl antibody that has been reported to activate integrins in certain situations also did not affect differentiation (Tenter et al,, 1993; Bazzoni et al,, 1995). Modulation of integrin affinity has been shown to influence cell fate in other cell systems. Stem Cell Factor has been shown to cause a transient increase in integrin mediated cell adhesion in M07E cells and that this correlated with mobilisation of haemopoetic cells from the bone marrow in vivo (Kovach et al, 1995). Myoblast development is also influenced by integrin signalling. Cell cycle withdrawal is suppressed by integrin signalling through FAK and ERK, and perturbation of integrin function results in faults in muscle differentiation (Sastry et a l, 1999). Activation of a5pl has been shown to disrupt myotube morphogensis although not lëT Chapter 5 The Effects of Integrin Activation to prevent differentiation (Boettiger et al, 1995). My results show that modulation of integrin affinity can similarly affect the fate of kératinocytes. 166 Chapter 6 Screening for Further Integrin Mutations 6.1 Introduction There have been many previous studies published comparing integrin expression patterns on different types of tumour, relating this to tumour grade or prognosis (Peltonen et a l, 1989; Jones et al, 1993; Jones et al, 1997; Bagutti et a l, 1998, Van Waes et a l, 1991). To date there has been no previous report of integrin mutations having an effect in tumour development and no published studies of Pl integrin mutations or polymorphisms in tumours exist. In order to investigate the frequency of pi mutations we screened other oral SCCs from the tumour collection of St Thomas’s Hospital (London) for other integrin mutations. 167 Chapter 6 Screening for Further Mutations 6.2 Results Frozen sections of 124 human SCCs were provided by Dr J. Jones, UMDS (London) from the histopathology collection of St. Thomas’s hospital, London. For each of these tumour blocks a matched specimen of normal tissue or blood was available. Genomic DNA was extracted from the tumour samples by proteinase K digestion, phenol/chloroform extraction and ethanol/sodium acetate precipitation. Sequencing af-"the all the exons of the pi was not feasible due to limits on access to the equipment. Therefore I examined exons 3-7, the region surrounding the location of the T188I mutation. Each exon was analysed by SSCP analysis and where changes from the WT trace were found the presence of a mutation was confirmed by sequencing (example traces shown in Fig 6.1). SSCP and sequencing were carried out at the Cancer Research UK mutation detection facility (Leeds) by Dr Claire Taylor. Sequence Change Amino Acid effect Num ber of Frequency tumours. IVS2-36 OA N/A 43 34.9% 459 O T No change 29 23.5% 471 OT No change 2 1.6% 776 O T A239V 1 0.8 % 783 T>C No change 30 243 94 IVS6+8 O T N/A 2 1.6% Table 6.1 Summary of sequence changes found. (I VS - Intron Variable Sequence) Sections of tumour positive for the 471 C>T, 776 C>T A239V and IVS2-36 C>A variants were stained with antibodies to the pi subunit. All tumours showed expression of the pi subunit. In addition the 776 C>T A239V was stained with the HUTS21 activation reporter antibody (Fig 6.2). This was performed by Dr J.Jones, 168 Chapter 6 Screening for Further Mutations UMDS (London). Strong staining for the pi subunit was found in tumours carrying each mutation indicating that they did not affect the expression of the subunit. HUTS21 staining, indicating high affinity or ligand bound Pl, in the 776 C>T A239V was only seen in the layers of tumour adjacent to a basement membrane (Bishop et al, 1998) Normal tissue was analysed from patients expressing the 776 C>T A239V, IVS2-36 C>A and 471 C>T variants. All three sequence changes were found in normal tissue and were therefore not tumour acquired. 169 Chapter 6 Screening for Further Mutations A 5 Green ITGB1 ex 5 7 Green ITGB1 ex 5 B C AAGF TGCAGTTT G T 210 C A A G T GCAGTTTGT 80 190 Fig 6.1; (A) Example of SSCP traces. Upper trace is sample 51 carrying the 776 C>T A239V. Mutation. Lower trace is WT control. The extra peak (arrowhead) shows the presence of a sequence change, which was then determined by sequencing. (B) Sequencing results of the same samples. Upper trace shows 776 C>T A239V heterozygous mutation, lower trace is WT 170 Chapter 6 Screening for Further Mutations W -M * i-ienwa Fig 6.2: Staining of pi subunit in SCC samples with the (A) 776 C>T A239V, (B) 471 C>T and (C) IVS2-36 C>A mutations, pi expression is not perturbed by any of these sequence changes. (D) HUTS-21 staining of the SCC containing the 776 C>T A239V mutation. Bar =500 pm. 171 Chapter 6 Screening for Further Mutations 6.3 Discussion The results of this chapter suggest that, although a second mutation causing a change in amino acid sequence was found, amino acid mutations in the pi integrin subunit in human SCC are not a common occurrence. The single amino acid change found, A239V, is in a highly conserved region of the A domain (see Fig 3.7). Based on the model presented in Chapter four (fig 4.7A) it lies in the a3 helix facing into the centre of the domain. While an alanine to valine change is a significant change in side chain character (a small -CH3 group to a larger hydrophobic -CH(CH 3)2 ) there appears to be sufficient space to accept the larger side chain without any clashes with the surrounding amino acids. The surrounding residues are already mostly hydrophobic so this change would not appear to alter the surrounding environment. As A239V was also found in normal tissue from this patient and is heterozygous in both tumour and patient I conclude that this mutation is not tumour derived and is unlikely to ^^ect integrin function or the characteristics of the tumour. Several changes in sequence that do not change the amino acid sequence were also found. Two of these were found at a high frequency (almost 1 in 4 tumours). While these will not affect integrin function this does show that the database WT sequence does not represent the only “normal” pi subunit sequence. Two sequence changes were found in the non-coding intron sequences between exons. Mutations in certain areas of the intron can lead to aberrant splicing of mRNA. However the IVS6+8 C>T mutation is not in a region thought to influence splicing. IVS2-36 C>A is in an area that could be part of a branch site consensus — Chapter 6 Screening for Further Mutations sequence although this could only be confirmed experimentally ((Hertel et a l, 1997) and personal communication C. Taylor). However tumours containing this mutation have normal pi integrin staining making a change in splicing of the subunit seem unlikely. Three inherited human diseases are thought to be caused by mutations in branch sites. Familial hypercholesterolaemia is caused by an intron mutation 30 bp upstream of the start of exon 10 in the low density lipoprotein receptor gene (Webb et a l, 1996). Fish eye disease, a condition involving clouding of the cornea and atherosclerosis, is caused by a mutation 22 bp upstream of exon 4 of the lecithimcholesterol acyltransferase gene (Kuivenhoven et a l, 1996; Li et a l, 1998). Glycogenosis type II is an inherited disorder caused by mutations in intron 1 of the acid maltose gene (Raben et al, 1996). In this work I examined only exons 3 to 7 as the primary aim was to find mutations that acted on the A domain in a similar manner to the T188I. It may be of use to screen other parts of the gene. Naturally occurring mutations in the stalk region have been shown to activate aIIbP3 in OT (Fullard et al,, 2001; Ruiz et al,, 2001). The cytoplasmic domains have been shown play vital roles in the regulation of integrin activation so both of these areas may prove interesting. In summary while the results from the previous chapter show alterations in integrin affinity can modulate tumour and keratinocyte function in vitro, this may be a rare event in SCC development. 173 Chapter 7 General Discussion In this chapter I will summarise the main findings of this thesis and suggest directions for further research 7.1 The T188I mutation activates the Pl subunit. The T188I mutant integrin subunit causes an increased rate of binding to integrin ligands, both in cell free and cell based systems (Chapter 4). The degree of activation observed varies according to the type of assay, the partner a subunit and the ligand used. The T188I mutation lies on a small cysteine bridged structure previously shown to be important in regulation of integrin function. In this thesis I have compared the binding of the WT integrin to the T188I mutant. In order to discover more about the importance of the loop on which T188I lies further A domain mutants could be made. Mutation of the threonine to other residues may show whether it is the presence of the isoleucine or absence of the threonine that is the cause of the activation. It would also be very interesting to look at the effect of complete substitution of the C-C loop for the equivalent region of other subunits, as in the work of Takagi. This is a substitution that has been shown to cause alterations in both signalling and ligand binding properties and thus has the potential to alter keratinocyte behaviour (Takagi et al, 1997). Ultimately deeper understanding of the process of activation of the A domain and the P subunit as a whole will most likely require the solution of further crystal structures. — General Discussion Specifically, solution of a (31 subunit containing integrin both in a ligand bound and ligand unbound state would provide a great deal of information as to the mechanics of integrin function. Solution of avpi would also allow comparison of a dimer containing pi with p3 and give more information on how structures are conserved between the various P subunits. Two of the constructs in Levy et al. (Levy et a l, 2000), containing deletions of regions of the cytoplasmic domain and were judged to be effectively non-functional as they lacked the ability to regulate differentiation or cell attachment. These subunits have intact ligand binding domains and there is some evidence that in CD8/pi cytoplasmic domain chimera these deletions cause gain of function (personal communication D. Campbell). It may be worth expressing the full length cpl constructs in GD25 cells to determine if, although they cannot support cell attachment, their ligand binding domains are constitutively active. 7.2 Integrin activation affects different aspects of Pl null fibroblast behaviour. Integrin activation causes changes in the behaviour of GD25 cells. Integrin activation due to T188I promotes attachment to cdl-attaohmcnt matrix coated surfaces and subsequent spreading (Fig 4.4, 5.1). T188I expression appears to have no effect on random cell migration across matrix coated surfaces when compared to WT (Fig 5.2) but T188I does not promote invasion of GD25 cells through Matrigel as effectively as the WT (Fig 5.3). The discrepancy between the observed effects on random migration and invasion may be resolvable with further experimentation. Palecek et V75 General Discussion al. (Palecek et al, 1997) suggest that the optimum concentration for efficient cell migration is related to the affinity of the cell for the matrix. Therefore it is likely That that the lack of observed difference between the migration rate of the T188I and WT expressing cells is due to the concentration of the matrix protein used in these experiments. It is possible that higher concentrations would show a reduction in the rate of movement of the T188I relative to the WT while lower concentrations may promote migration of the activated mutant. The lack of invasion of T188I expressing cells through Matrigel may be due to differences in MM? expression. This could be investigated by gelatin gel zymography to screen for changes in MM? activity induced by expression of the T1881 or WT integrin subunits. 7.3 Integrin activation by expression of the T188I subunit or treatment with TS2/16 influences keratinocyte differentiation. Infection with the T188I subunit does not induce differentiation of the SCC4 cell line, whereas infection with the WT does (Fig 5.6, 5.7). In a similar manner activation of the endogenous pi subunit on normal kératinocytes reduced the number of differentiated cells (Fig 5.9, 5.10). There are several further experiments that could be performed. Expression of the T188I subunit in normal kératinocytes should lead to a decrease in differentiated cells. This experiment has been attempted several times but no normal kératinocytes expressing T188I have been generated in four attempts. The retroviral producer cells Ï76" General Discussion are capable of infecting other cell lines as GD25 and SCC lines were generated using these producers. Concentration of the T188I virus did not result in successful infection. The WT pl subunit can be infected easily into normal kératinocytes. Other P subunit cytoplasmic domain mutations have been shown to cause activation of the subunit. Expression of the equivalent pi mutation in kératinocytes may provide more evidence of the effect of activation of the subunit. One of these, D764A, is thought to function by removal of a salt bridge between the a and p cytoplasmic tails (Hughes et a l, 1996). I have attempted to express this subunit in human kératinocytes without success, as have other members of the laboratory. It would also be interesting to look at the behaviour of SCC4 cells expressing the WT and T188I subunits in tumour formation experiments in vivo, for example by injection into nude mice. Blockade of integrins by peptide ligands has been shown to reduce the formation of tumours in the lungs of mice injected with mouse malignant melanoma (Humphries et al, 1986b). It would be interesting to see if an increase in integrin function could promote this process. 7.4 Mutation of the A domain of the subunit is not a common event in human SCC. I have found little evidence that mutations of the pi subunit are common in human SCC. Including the T188I mutation only two mutations that result in amino acid mutations have been found in 125 tumours. While I have found only one other sequence change in the area surrounding T188I it is possible that if the entire gene was sequenced in the each patient other sequence changes may be found. — General Discussion It may be informative to compare the frequency of the sequence changes found with their frequency in the general population as this may show whether any of the polymorphisms result in a predisposition to cancer. It may also be worth examining other types of tumour. It would also be interesting to look for other events that could lead to changes in integrin activity. PKCa signalling has been implicated in integrin function (Ng et al, 1999). It may be useful to look for changes in this pathway that may influence integrin affinity. 7.5 General Discussion The initial idea that a mutation that causes an increase in cell adhesion can affect a tumour in such a way as to make it less differentiated seems to be counter-intuitive as transformation of cell lines is normally associated with a loss of adhesion and a down regulation of ECM proteins (e.g. fibronectin) (Hynes et a l, 1978; Hynes et a l, 1979). However, the T188I mutation is not acting as a transforming mutation in the manner of an oncogene, rather it is acting on an already tumorigenic cell line to cause changes in the cells behaviour. In fact there may be a place for increased adhesion in the tumorigenic process. While fibronectin is initially downregulated in most tumours there is evidence that in cells isolated from métastasés fibronectin and other ECM genes are upregulated (Clark et al, 2000). This production of matrix hy the tumour cells allows the metastatic cells to ensure that they receive survival _ General Discussion signais from the matrix surrounding them. It is possible that activating integrin mutations could function in a similar manner, promoting the survival signals provided by the ECM present around the tumour. My work shows that it is not just the up or down regulation of integrin subunits that is relevant to tumour behaviour, mutation of integrins can also play an important role. The existing model of regulation of differentiation in cultured human kératinocytes revolves around the ligation of the pl integrin subunit being the prime source of a suppression of differentiation signal ((Adams and Watt, 1989),(Watt et al, 1993), (Levy et al, 2000)). The induction and commitment to terminal differentiation is triggered by the down regulation of the affinity of the pi subunit and subsequent withdrawal from the surface (Adams and Watt, 1990) as this removes the "do not differentiate" signal. Proliferative potential is observed to correlate with integrin expression in the epidermis. Cells with the highest levels of pi exhibit stem like behaviour and the lowest levels of differentiation (Jensen et al, 1999). Other results showing the importance of adhesion in regulating differentiation were provided by Zhu et al (Zhuet al, 1999). Reduction of cell/matrix adhesion by expression of a dominant negative CD8pl chimera caused a reduction in the levels of MAPK signalling and resulted in premature differentiation. This affinity/differentiation correlation is seen again in cells taken from the KSpi null mouse (Grose et al, 2002). In this situation mouse kératinocytes lacking pi show greatly reduced adhesion to ECM components and show massively increased differentiation. When added to the data presented in this thesis it is clear that the strength of matrix/cell binding is an important factor in the fate of the cell as regards to _ General Discussion differentiation (Fig 7.1). Whether this relationship is linear or whether a certain level of loss integrin ligation is required to trigger differentiation is a question that remains to be determined. c o TO c 2 ► Adhesion ài a ài a à i Cells in Infection Stem suspension with cells CD8P1 pl Transit T188I KO cells /TS2/16 Fig 7.1 The inverse relationship between adhesion and differentiation observed in kératinocytes. As adhesiveness and ligand binding increases differentiation is seen to decrease. Whether the relationship is linear (A, solid line) or whether a critical level of ligand binding is required for suppression of differentiaton (B, dotted) is unknown. 180 Appendix 1: Details of pi mutation screen Exon Patient Sample 3 5 Number -ve 1 El 2 459 O T 783 T>C F6 3 J2 4 459 O T 783 T>C J2 5 IV82-36 O A 459 O T 783 T>C homozygote D4 6 R6 7 P5 S 783 T>C P4 10 P7 11 MS 12 P2 13 IV82-36 OA 459 O T R6 14 El 15 459 O T 783 T>C L6 IS 459 O T 783 T>C A4 19 IV82-36 OA 459 O T MS 24 Y1 27 Y1 28 AlO 29 783 T>C L4 30 783 T>C Pl 31 T3 33 459 O T A2 34 IV82-36 O A LS 35 783 T>C 86 36 81 37 459 O T homozygote N4 38 783 T>C W2 39 K2 40 IV82-36 O A F5 41 IV82-36 O A 783 T>C AS 42 82 43 A3 45 A6 48 D2 49 J5 50 J3 51 776 O T Ala>Val LI 52 783 T>C 181 Appendix 1 Exon Patient Sample 3 4 5 6 7 Number A13 53 459 O T , 471 783 T>C OT C3 54 459 O T E2 56 D1 57 C2 58 783 T>C J6 59 N2 60 Ml 61 E4 62 S3 63 02 64 IVS2-36 O A 459 O T B1 65 M9 67 F3 69 M6 71 783 T>C J1 74 IVS2-36 O A 783 T>C M7 75 C3 76 IVS2-36 O A M3 77 IVS2-36 O A G3 79 IVS2-36 O A 783 T>C B2 80 IVS2-36 O A 783 T>C P3 81 L8 82 IVS2-36 O A 783 T>C G2 84 R4 85 K5 86 IVS2-36 O A 459 O T L7 87 IVS2-36 O A 459 O T Cl 88 M4 90 El 91 G4 92 G1 93 El 94 K1 96 E3 97 F4 98 N1 99 F2 100 G5 101 IVS2-36 O A 459 O T 783 T>C homozygote S4 102 IVS2-36 O A 459 O T homozygote homozygote A ll 103 A12 104 182 Appendix 1 Exon Patient Sample 3 4 5 6 7 Number M5 105 IVS2-36 O A 459 O T PS 106 IVS2-36 O A P4 108 J7 109 IVS2-36 O A 783 T>C A9 110 R5 111 IVS2-36 O A K6 112 IVS2-36 O A 783 T>C Y1 113 A7 114 IVS2-36 O A 459 O T homozygote L3 115 R3 116 IVS2-36 O A 459 O T IVS6+8 O T homozygote E5 117 IVS2-36 O A 783 T>C A7 119 IVS2-36 O A 459 O T homozygote FI 120 IVS2-36 O A 459 O T homozygote E6 121 IVS2-36 O A 783 T>C E7 122 IVS2-36 O A 459 O T L5 123 IVS2-36 O A 471 O T 783 T>C D5 124 IVS2-36 O A 783 T>C N3 125 J5 126 S5 127 K4 128 A5 129 HI 130 IVS2-36 O A K3 131 R2 132 IVS2-36 O A 459 O T P6 133 IVS2-36 O A 459 O T W1 135 T2 136 IVS2-36 O A 459 O T 783 T>C homozygote M2 137 LIO 138 IVS2-36 O A 459 O T C4 139 IVS2-36 O A 459 O T 783 T>C A1 140 IVS6+8 O T D6 141 L9 142 IVS2-36 O A 459 O T L2 143 IVS2-36 O A 783 T>C 01 144 IVS2-36 O A 459 O T homozygote P9 145 IVS2-36 O A J4 146 IVS2-36 O A 183 Appendix 1 Exon Patient Sample 3 4 5 Number T1 147 D3 148 IVS2-36 O A 783 T>C R1 149 W3 152 Summary Number of discrete samples (after duplicates/failed samples excluded) - 124 Mutation Number of Samples IVS2-36 C>A 4 3 459 C>T 29 471 C>T 2 783 T>C 3 0 776 C>T Ala>Val 1 IVS6+8 C>T 2 184 References Abercrombie, M., Heaysman, J. 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