DIFFERENTIAL REGULATION OF MATRIX METALLOPROTEINASE3 (MMP-2) BY PHORBOL MYRISATE ACETATE, AND BY DGa-DIFLUOROMETHYLORNLTHINE,IN CELL LXNES OF VARYING TUMORlGENIC AND METASTATIC POTENTIAL

Oliver Yeung

A thesis submitted with the requirements for the degree of Masters of Science, Graduate Department of Laboratory of Medicine and Pathobiology University of Toronto

O COPYRIGHT BY OLIVER YEUNG 1999 National Library Bibliothèque nationale B*B m-da du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Sireet 395. rue Wellington Ottawa ON K1A 0N4 OctawaON KlAW Canada canada

The author has granted a non- L'auteur a accordé une licence non exclusive Licence allowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seU reproduire, prêter, distribuer ou copies of this thesis in microfom., vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/fiJm., de reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thése ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. DLFFERENTIAL REGULATLON OF MATRIX METALLOPROTEINASE-2 (MMP-2) BY PHORBOL MYRISATE ACETATE, AND BY DL-a-DIFLUOROMETHYLO-, IN CELL LINES OF VARYING TUMORIGENIC AND METASTATIC POTENTIAL

Oliver Yeung

A thesis submitted with the requirements for the de- of Masters of Science, Graduate Department of Laboratory of Medicine and Patûobiology University of Toronto

O COPYRIGHT BY OLIVER YEUNG 1999

The matrk metaiioproteinases (MM's) are thought to play key roles in tumor formation and malignant progression. The present study demonstrates novel alterations in the regulation of matrix metalloproteinase-2 (MMP-2) expression in celis of varying tumorigenic and metastatic potential. No change in MMP-2 gelatinolytic activity in response to PMA occurred in any of these cells. PMA did affect MMP-2 message expression in three of the four celi lines studied. Protein kinase C (PKC) mediated events were also found to play a role(s) in the regdation of MMP-2 message expression. An invoIvernent of cellular polyamine Levels in PMA modulated expression of MMP-2 was also observed. DFMO treatment altered PMA mediated regulation of MMP- 2 message expression, but not MMP-2 gelatinolytic activity. Interestingly, DFMO alone increased the basal expression of MMP-2 message in two of the cell Lines. Alterations in MMP-2 message expression. by PMA or by DFMO, involved both transcriptional and pst-transcriptional events. DEDECATION

For myfa- andfnenris.

For my fafher, wifhout whorn tkcn wovld be no MPrtcrpsdegree.

For AL,for always being ut my sidR 1 would like to sincerely thank my supervisor Dr. Robert Hurta for giving me the opportunity to pursue a Masters degree and who opened my eyes to the real worid. His advice and financial support over the course of my degree is greatly appreciated. His criticism was especidy helpful during the writing of this thesis. 1 would like to thank my committee members, Dr. Andrew Bognar, Dr. Linda Penn, and Dr. Eva Turley for theu helpfbl comments and suggestions.

1 would also like to thank the department faculty, especially Dr. Philip Conelly who generously provided aU the cornputer hardware and software needed to put this thesis together. 1 would like to express my gratitude to Graham Maguire, Zakaria Ahmed, Maureen Maguire, Yun Lam, Patrick Lai, and Saeid Babaei, their continuai technical assistance has been invaluable to the completion of my degree. A special thanks goes out to Janet Lee, Alice Yoon, Massimo Cimi, and Danial Voskas, my new fnends, who provided companionship and support in a lab situated in the quiet corner of St. Michael's Hospital.

Finally, 1 would like to thank my family and fkiends for their constant love and support. My deepest gratitude goes out to them. TABLE OF CONTENTS

ABSTRACT

DEDICATION

ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF FIGURES

LIST OF ABBREVIATIONS

LIST COMPANIES

CHAPTER 1: INTRODUCTION

1. Cancer

II. Polyamines Regdation of the Polyamine Pal hway Polyamines and Cancer

III. Matrix Metailoproteinases Regdation of the MMPs by TIMPs Activation of MMPs Activation of MMP-2 Transcriptional Regulation of the MMPs MMPs and Cancer

N. Rationale for the Study

CWTER2: MATERLALS AND METHODS

1. Cell Line Description II. Ce11 Culture DI. Assay for Gelatimlytic Activity IV. Total RNA Isolation V. cDNA Probes VI. RNA Blot Analysis VII. Northem Blot Analysis

CEAPTER 3: PHORBOL MYRISATE ACETATE DIF'FERENTXALLY REGULATES THE EXPRESSION OF MAT= METALLOPROTEINASE-2 IN NONTUMORIGEMC AND TUMOIUGEMC CELL LINES

1. The Addition of Phorbol Myrisate Acetate @MA) Alters MMP-2 Message but not MMP-2 Gelatinolytic Activity 25

II. The TranscnptionaI Process PIays a Role in the Regdation of MMP-2 Expression by PMA in Both 10T1/2 and NR3 Ceil Lines 33

III. Post-transcriptional Regulation of MMP-2 mRNA Expression Occurs In 1OT1/2 Cells and in NR3 Cells in Response to PMA 37

IV. De Novo Protein Synthesis is a Requirement for PMA Mediated Aiterations in MMP-2 Message Expression in 10T 1/2 Ceils but not in NR3 Cells 40

V. Protein Kinase C Mediated Events Are Involved in the Regulation of MMP-2 Message Expression in 10T112 and in NR3 Cells 44

CHAPTER 4: CELLULAR POLYAMINE DEPLETION CAN DtFFERENTIALLY REGULATE MATRIX METALLOPROTEINASE-2 EXPRESSION IN NONTUMORIGENIC AND IN TUMORIGEMC CELLS

1. Cellular Polyamine Depletion Induces MMP-2 Expression in IOTU2 Cells and in NR3 cells, and Alters PMA Mediated Regulation of MMP-2 in Cell Lines of DifSering Tumorigenic And Metastatic Potential 55

II. Differential Response to DFMO Treatment in 10T1/2 and NR3 Cells 56

In. The Transcriptional Process Plays a Role in DFMO Regulation of MMP-2 Expression in 10T 1/2 and in NR3 Cells 65

N. Stability of the MMP-2 Message is AEected by the Addition of DFMO in NR3 Cells but not in 10T1/2 Cells 70 v. Effect of de Novo Protein Synthesis Inhibition on MMP-2 Induction by DFMO in 10T1/2 Cells and NR3 Cells 75 CHAPTER 5: DISCUSSION

1, The Effect of Phorbol Myrisate Acetate (PMA) on MMP-S Gelatinolytic Activity

II. The Effect of PMA on MMP-2 Message Levels

III. The Effect of a-Difhoromethylomithine (DFMO) on MMP-2 Gelatinolytic Activity

IV. The Effect of DFMO on MMP-2 Message Levels

V. Proposed Mode1

VI. Summary / Future Direction

REFERENCES Figure Page #

The polyamine biosynthesis and interconversion pathway. 2

The effect of PMA on MMP-2 gelatinolytic activity in NM 3T3 cells. 27

The effect of PMA on MMP-2 gelatinolytic activity in v-fes-NM 3T3 cells. 28

The effect of PMA on MMP-2 gelatinolytic activity in 10T1i2 cells. 29

The effect of PMA on MMP-2 gelatinolytic activity in NR3 ceils. 30

The effect of PMA on MMP-2 message levels in NIH 3T3 cells. 31

The effect of PMA on MMP-2 message levels in v-fes-WH 3T3 cells. 32

The effect of PMA on MMP-2 message levels in 10T1/2 cells. 34

The effect of PMA on MMP-2 message levels in NR3 cells. 35

Cornparison of basal MMP-2 message expression in confluent 10T 112 cells and confluent NR3 cells. 36

Inhibition of the transcriptional process by actimmycin D and its effect on PMA mediated increases of MMP-2 message levels in 10T1/2 ceils. 38

Inhibition of the transcriptional process by actinomycin D and its effect on PMA mediated decreases of MMP-2 message levels in NR3 cells. 39

MMP-2 message half iife in lOTln cells and NR3 cells. 41

The effect of PMA on the haif Life of MMP-2 mRNA in 10T1/2 cells. 42

The effect of PMA on the haif life of MMP-2 mRNA in NR3 cells. 43

Inhibition of de novo protein synthesis by cycloheximide and its effect on PMA mediated increases of MMP-2 message levels in 10T1/2 cells. 45

Inhibition of de novo protein synthesis by cycloheximide and its effect on PMA mediated decreases of MMP-2 message levels in NR3 celis. 46

vii Down regulation of PKC by PMA in 10T1/2 cets and its effect on PMA mediated increases in MMP-2 message levels.

Down regulation of PKC by PMA in NR3 cells and its effect on PMA mediated decreases in MMP-2 message levels.

Down regulation of PKC by calphostin C in NR3 cells and its effect on MMP-2 gelatinolytic activity.

Down regulation of PKC by caiphostin C in NR3 cells and its effect on MMP-2 message levels.

Down regdation of PKC by cdphostin C in 10TI/2 cells and its effect on MMP-2 gelatinolytic activity.

Down regulation of PKC by calphostin C in 10T1/2 cells and its effect on MMP-2 message levels.

The effect of DFMO and PMA on MMP-2 gelatinolytic activity in NIH 3T3 cells.

The effect of DFMO and PMA on MMP-2 gelatinolytic activity in v-fes--NM. 3T3 cells.

The effect of DFMO and PMA on MMP-2 gelatinolytic activity in 10T112 cells.

The effect of DFMO and PMA on MMP-2 gelatinolytic activity in NR3 cells.

The effect of DFMO and PMA on MMP-2message levels in NIH 3T3 cells.

The effect of DFMO and PMA on MMP-2 message levels in v-fes-NIH 3T3 cells.

The effect of DFMO and PMA on MMP-2 message levels in 10T1/2 celis.

The effect of DFMO and PMA on MMP-2message levels in NR3 cells.

Time course investigating the effect of DFMO alone on MMP-2 gelatinolytic activity in 10T1/2ceils.

Time course investigating the effect of DFMO alone on MMP-2 gelatinolytic activity in NR3 cells.

viii Time course investigating the effect of DFMO alone on MMP-2 rnRNA levels in 10T1/2 cells.

Time course investigating the effect of DFMO alone on MMP-2 mRNA levels in NR3 cells.

Inhibition of the transcriptional process by actinomycin D and its effect on DFMO mediated increases of MMP-2 message levels in 10T1/2cells.

Inhibition of the transcriptional process by actinomycin D and its effect on DFMO mediated increases of MMP-2 message levels in NR3 cells.

The effect of DFMO on the half Me of MMP-2 mRNA in 10T1/2 cells.

The effect of DFMO on the half life of MMP-2 mRNA in NR3 cells,

Inhibition of de novo protein synthesis by cycloheximide and its effect on DFMO mediated increases of MMP-2 message levels in 10T1/2 ceus.

Inhibition of de novo protein synthesis by cycloheximide and its effect on DFMO mediated increases of MMP-2 message levels in MU ceiis.

Schematic diagram depicting the proposed model of PMA and DFMO signaling interaction-in the regulation of MMP-2 expression in 10T 112 ceils.

Schematic diagram depicting the proposed model of PMA and DFMO signaling interaction in the regulation of MMP-2 expression in NR3 cells. a-MEM a-Minimum Essential Medium bFGF basic fibroblast growth factor BM basement membrane Ca calcium cDNA complimentary deoxyribonucleic acid cpm counts per minute DAG diacylglycerol DEPC diethyl-pyrocarbonate DFMO DLa-Difluoromethylornithine ECM extracelluiar matrix EDTA ethylenediamine tetra-acetic acid EGF epidedgrowth factor FAD flavine adenine dinucleotide FBS fetal bovine semm GAPDH glyceraldehyde-3-phosphate dehydrogenase IL-1 interleukin-1 JNK c-Jun terminal kinase kDa kilodaltons Mg Magnesium Pl3 micrograms Pl microlitres mM miliimolars MMPs Matrix Metalloproteinases MMP- 1 collagenase- 1 MMP-2 gelatinase-A / 72kD type-N collagenase MMP-3 stromelysin- 1 MMP-7 matriiysin MMP-8 collagenase-2 MMP-9 gelatinase-B / 92kD type-N collagenase MMP-10 stromelysin-2 MMP-11 strometysin-3 MME'-12 metalloelastase MMP-13 collagenase-3 MOPS p-Morpholino]propanesulfonic acid mRNA messenger ribonucleic acid MT-MMP membrane-type MMP NaCl sodium chloride PBS phosphate buffered saline PKC protein kinase C PMA Phorbol 12-Myrisate 13-Acetate ODC Ornithine decarboxylase SAMDC S-adenosyimethionine decarboxy1ase SDS Sodium dodecylsulphate SSAT spemiidhe/spermine-~l-acetyltransferase TIMP tissue specific inhibitors of metalloproteinase TGF-P Wonning growth factor+ TNF-U twnor necrosis factor-cx TPA 12-O-tetradecanolyphorbol- 13-acetate PA tissue-type plasminogen activator TRE TPA response element uPA urokinase plasmhogen activator UTR mtranslated region Zn zinc LIST OF COMPANLES

Amersham Canada Ltd. Oakville, Ontario Amersham Phannacia Biotech Arlington Heights, Illinois BI0 101 Inc. Vista, California Bio/Can Scientific Mississauga, Ontario Bio Rad Laboratories Hercules, California Difco Laboratories Detroit, Michigan Eastman Kodak Company Rochester, New York Gibco BRL Life Technologies. Butlington, Ontario HycIone Logan, Utah ICN Biomedicals Inc. Cleveland, Ohio Ilex Oncology San Antonio, Texas Mandel Scientific Company Ltd. Guelph, Ontario Nalge Nunc International (division, Gibco BRL Life Technologies) Burlington, Ontario Pbarrnacia Biotech Baie d'Urfe, Quebec Qiagen Inc. Santa Clara, California Sigma-Aldrich Canada Ltd. Mississauga, Ontario Sarstedt Newton, North Caroiina 1, Cancer

Cancer metastasis is responsible for the majority of cancer related deaths. Therefore, a large nurnber of studies have been performed in order to achieve a better understanding of this disease, with a particular fôcus on the processes associated with malignant progression. It is now widely accepted that a relatively smali number of genes are responsible for carcinogenesis, and the various changes in biological properties associated with cancer (Wright et ai., 1990). These genes are referred to as oncogenes and tumor suppressor genes, which are normaliy essential for the regdation of diverse cellular fùnctions including proliferation, differentiation, cell to cet1 communication and motility. Some examples of these genes include ras, which encodes for p21, v-fes, which encodes for a tyrosine kinase, and the wild type pS3, which acts as a turnor suppressor. Recently, the gene encoding for ornithine decarboxylase has been classineci as a proto- oncogene (Auvinen et ai., 1992). Ornithine decarboxylase is the first and rate limiting enzyme in the biosynthesis of polyamines. These genes are also thought to play a key role in the progression of benign tumor cells towards the metastatic phenotype (Wright et ai., 1990; Chambers and Tuck, 1993). This concept is supported by the idea tbat there is a set of common changes that can lead to metastasis (Chambers and Tuck, 1993). These changes cmbe elicited by multiple signals, which are regulated by oncogenes or turnor suppressor genes. An alteration in maûix metalloproteinase expression is one of these 'common changes' thought to be important in the progression of the benign -or type to one that is metastatic (Chambers and Tuck, 1993). A criticd proteolytic event early in the metastatic cascade is the degradation of the basement membrane surroundhg tuinor cells (Ray and Stetler- Stevenson, 1994). There is abundant evidence irnpiicating the matrix metalloproteinases in the creation of this proteolytic defect (Ray and Stetler-Stevenson, 1994). II. The Polyamines

The polyamines putrescine, spermidine, and spermine are ubiquitous components of mammalian cells (Pegg and McCann, 1988; Janne et al., 199 1). These organic aliphatic cations remain fUy protonated at physiological pH. The biosynthesis and interconversion of polyamines is outliaed below.

SADENOSYLMETHIONME 0- ARGINME -ARCINASE S-ADENamtMETHIONINE ORNrmINE DECXRBOXYLASE DEClRdOXïLASE (SAMDC) (ODC)

DECARBOXYLATEDI I S-ADENOSYLMETEIIONINE

A~oPRoPyL SPERMIDINE N~~ACETYLSPERMIDINE

POLYMINE ACEI?US&

THIOADENOSME

b SPERMIDINE

POLYAMINE AC€ITUSE

Figure 1. The polyamine biosynthesis and interconversion pathway (Pegg and McCann, 1988).

The biosynthesis of the polyamines begins with the decarboxytation of ornithine by ornithine decarboxylase (ODC)to produce putrescine. Putrescine is then converteci to spermidine by spermidine synthase via the addition of an aminopropyl group. Spennine is formed by the Meraddition of an aminopropyl group to spermidine by spexmine synthase. Spermidine synhse and spehesynthase compte for the aminopropyl groups which are formed by the action of S-adenosylmethionine decarboxylase (SAMDC) which decarboxylates S-adenosyimethionine. Spermine and spennidine cm be recycled by the dual action of ~~e~dine/s~~e-~~-acetyltransferase(SSAT) and flavine adenine dinucleotide (FAD) - dependent polyamine oxidase ultimately leading to putrescine production. Spermine is acetylated by SSAT to produce ~bcetylspemiine.The polyamine oxidase then converts N'-acetylspermine to spennidine and 3-acetamidopmpanai. Spermidine can be acetylated by SSAT to give NI-acetylspermidine, which is then degraded by the Fmdependent polyamine oxidase to putrescine and 3-acetamidopropanal.

Regdation of the Polyamine Pathwav

The levels of polyamines in ceUs are regulated p-y by SSAT, ODC, and SAMDC (Pegg and McCann, 1988; Janne et al., 1991). The other enzymes involved in the biosynthesis and interconversion of polyamines (spermidine synthase, spennine synthase, and the FAD-dependent polyamine oxidase) are al1 expressed in large amounts and regulated by substraîe availability. On the other hand, SSAT, ODC, and SAMDC are not expressed in large arnounts in the ceii and it is thought the amounts of each protein determines the leveIs of polyamines (Pegg and McCann, 1988; Janne et al., 1991). It is possible to regulate polyamine levels in such a mamer because each of the enzymes has such a short half life. The halflife of these proteins is less than one hou. The presence of PEST sequences within the structure of these proteins is thought to have some part in mediating their short half life since tbis sequence is conserved in other proteins which also have a short haif Me. Since polyamine levels increase dramatically in response to growth factors and hormones, it is not surprishg that al1 thtee of these key enzymes are stimulated by the addition of these compounds (Pegg and McCann, 1988). In addition to stimulation by growth factors and hormones, SSAT is also induced by increased intracellular polyamine levels (Pegg, 1986). It is believed stimulation of SSAT production occurs in order to avoid the toxic effects associated with abnormaiiy high polyamine levels. Both an inçrease in the rate of its synthesis and a decrease in its rate of degradation mediate increased levels of SSAT. The importance of the FAD- dependent polyamine oxidase remaios unciear (Pegg and McCann, 1988). Experiments inhibithg FAD-dependent polyamine oxidase Ied to a buüd up of acetylated polyamines. Unlike high levels of unacetylated polyamines, this build up of acetylated polyamines had no toxic effect on the cells. It has been suggested that the FAD dependent polyamine oxidase serves as an important salvage pathway for putrescine. ODC and SAMDC are the two enzymes which are critical for the regulation of polyamine synthesis, and as such, have been the subject of intense scrutiny and study (Pegg and McCann, 1988; Janne et al., 1991). ODC, which is responsible for the synthesis of putrescine fiom ornithine, responds strongly to the addition of some growth factors and hormones (Pegg et al., 1994). Greaîer ODC activity is attributed to an increased rate of synthesis which is mediateci in part by an incfease in ODC mRNA levels. PoIyamine levels also strictly regulate the activity of ODC (Pegg et al., 1994). in general, when polyamine levels are low there is an increase in ODC activity, and when polyamine levels are high there is a decrease in ODC activity. Cellular polyamine levels regulate both the rate of synthesis and the rate of degradation of ODC. When polyamine levels are elevated, the increased rate of ODC degradation is facilitated by increased production of the ODC inhibitory protein, antizyme (Kanamoto et al., 1993). Antizyme binds to ODC, thereby inhibithg its activity, and at the same tirne facilitatuig its degradation. Polyamines regulate ODC synthesis primarily at the levels of translation and post-translation, with little, or no changes in the cellular content of ODC mRNA - (Shimogon et ai., 1996; Svensson and Persson, 1996). Hence, regulation of ODC synthesis occurs at multiple levels. The decarboxylation of S-adenosylmethionine by SAMDC is the other key enzyme in the biosynthesis of polyamines. Like ODC, cellular polyamine levels seictly regulate SAMDC. However, the control exerted by spermidine and spermine on SAMDC synthesis is quite dinerent fiom the control exerted by putrescine. Similar to the regulation of ODC, spermidiie and spemiiw act as negative feedback regdators of SAMDC synthesis. They control the activity of SAMDC by regulating SAMDC synthesis at the level of transcription, translation and pst-translation (Ruan et al., 1996; Shantz et ai., 1992). Spennine appears to be the more potent regdator of the two polyamines since spermine exhibits a greater influence on SAMDC synthesis than does spermidine (Shantz et al., 1992). Putrescine mediated regdation of SAMDC expression differs from htby spemidine and spermine in that high levels of putrescine, in general, enhance the activity of SAMDC (Wang et al., 1992). SAMDC is synthesized as a proenzyme and requires cleavage into two subunits in order for activation to occur. Putrescine increases the activity of SAMDC by increasing the conversion of the proenzyme to its active form (Pegg et al., 1988). In the absence of patrescine, SAMDC exists in greater amounts in the proform than when putrescine is present. Unlike spermine or spermidine, whether or not putrescine has any role in the synthesis of SAMDC remains to be elucidated (Wang et al., 1992).

Polvamines and Cancer

Though intensive research bas been done on polyamines, their exact fbction is stili under investigation* However, it is known that the&compouods play important roles in ce11 growth and differentiation. It has been demonstrated that increased leveb of polyamines are associated with ce11 division (Fredlund et al, 1995). inhibitor studies have also shown that depletion of polyamine pools induces cell cycle arrest (Ray et al., 1999; PateI and Wang, 1999). The effect of polyamine levels on dserentiation appears to be ce11 type specific. In some cells, increases in cellular polyamine pools induce differentiation. For exarnple, increased polyamine pools induce fibroblasts to differentiate into adipocytes (Pegg and McCann, 1988). While in other ce11 types, decreasing the polyamine levels induces differentiation. Decreasing the polyamine pools induces F9 teratocarcinorna stem cells to differentiate into a phenotype resembling the parietal endoderm ceiis of the early mouse embryo (Frostesjo et al., 1997). In addition to roles in ce11 growth and differentiation, there is growing evidence suggesting polyamines play a critical dein carcinogenesis (Auvinen et al., 1997). A number of studies have shown increased ODC activity and polyamine ievels in a variety of human cancers. Manni et al. (1 996) demonstrated increased ODC activity in primary tumors, and this increase predicted shorter disease fiee and ovdsurvival of breast cancer patients. ODC activity, as well as polyamine levels, were signifïcantly higher in hepatoma tissue than in noncancerous liver tissue (Tamori et al., 1994). As weii, ODC activity and the level of polyamines were increased in specimens of colorectal cancer when compared to the normal surrounding tissue (Linsalata et al., 1993). There is also abundant experimental evidence directly Linking aberrant polyamine metabolism to malignancy. Most of these experiments involve overexpression of ODC, the fkst key regdatory enzyme in the biosynthesis pathway of polyamines. Auvinen et ai. (1992) were the fhtto demonstrate that aberrant expression of ODC is not just a coincident, pleiotropic response to transformation, but a critical factor contributing to oncogenesis. They demonstrated that increased expression of ODC alone is sufiïcient in transforming NM 3T3 cells; and thaî the degree of transformation directly correlates with the level of ODC expression. Furthemore, Auvinen et al. (1992) demonstrated that blocking ODC activity prevents transformation of rat 2R cells by v-STC. Transformation by increased ODC expression has also been demonstrated in the 10T1/2 mouse fibroblast ce11 line (Kubota et al., 1997). These transformants exhibited anchorage independent growth and possessed an increased invasive phenotype. Anchorage independent growth has been shown to correlate with tumor aggressiveness (Manai et al., 1995). Kubota et al. (1 997) also showed theu 10T1/2ODC overexpressing clones exhibited increased MAP kinase activity demonstrating for the first time a connection between polyarnindODC and MAP kinase signaling pathways. They suggested the MAP kinase pathway may be involved in ODC induced ceii transformation and invasion. The effect of SAMDC overexpression and its role in carcinogenesis has also been investigated. Manni et al. (1 995) stably transfected MCF-7 breast cancer cells with SAMDC. These clones overexpressing SAMDC exhibited increased anchorage independent growth and an altered polyamine profile where spmiine levels were elevated 80% and spermidine and putrescine levels were reduced when compared to control cells. These MCF-7 clones overexpressing SAMDC dso had reduced ODC expression, indicating increased ODC expression is not always essentid to induce transformation mediated by aberrant polyamine rnetaboIism. Recently, it has been demonstrated that overexpressing ODC NIH 3T3 cells when injected into nude mice form highly vascularized, invasive tumors (Auvinen et al., 1997). These experiments add to the growing body of evidence indicating a roIe for polyamine metabolisrn in carcinogenesis.

III. The Matrix Metailoproteinases

It is now believed, dong with their putative role in metastasis, the matrix metalloproteinases (MMPs) also have a role in carcinogenesis (Chambers and Matrisian, 1997). The MMP family consists of fïfteen different proteases, most of them secret& as zymogens (Giambemardi et al, 1998; Matrisian, 1992). The MMPsare zinc2+dependent endopeptidases that are abIe to cleave one or more components in the extracellular ma& (ECM). Al1 MMPs have a catalytic domain containing the HEXGH motif responsible for ligating zinc. Zinc is essential for catalytic activity. Also, there exists a characteristic PRCGVPD sequence in the prodomain responsible for maintaining latency of the zyrnogens. MMPs dif5er in their structure by the addition or absence of other domains which have a variety of hctions including substrate specificity, inhibitor binding, ma& binding, and ce11 daceloçaljzation (Chambers and Matrisian, 1997). The MMPs are loosely assembled into four groups based on their structure, and to a certain extent, substrate specificity (Matrisian, 1992; Chambers and Matrisian, 1997). The htgroup consists of the interstitial collagenases which includes MMP-1 (collagenase-1), MMP-8 (collagenase-2), and MMP- 13 (collagenase-3). The interstitial collagenases are capable of cleaving fibrillar collagens (types 1, II, and III). The second group consists of MMP-2 (gelatinase-N72kD type-N collagenase) and MMP-9 (gelatiaase-B/ 92kD type-N collagenase) which degrade denatured collagen (gelatin), elastin, and types IV, V, W,and X collagem. They may also act synergistically with the interstitial collagenases in degrading fibrillar collagens. The largest group of MMPs is the stromelysins: which includes MMP-3 (stromelysin- 1), MMP-7(matrilysin), MMP- 1O (stromelysin-2), MMP-Il (stromelysin-3), and MMP-12 (metalloelastase). The stromelysins have broad substrate specificity degrading many components of the ECM including proteoglycans, laminin, elastin, and fibronectin. The finai group of MMPs are the membrane-type MMPs (MT-MMP),which includes MMP- 1 4, MMP- 1 5, MMP- 16, and MMP- 17. Unlike the rest of the MMPs which are secreted hmthe ceLi upon synthesis, the MT-MMPs are not secreted as they possess a membrane spanning sequence in the fourth plexin-like repeat of the C-tenninal domain. They also degrade a wide range of ECM proteùis. It has been suggested the MMPs play an important regulatory role in ECM turnover (Witty et al., 1995). This is supportai by the fact that the collagenases are the oniy known enzymes capable of degrading the fibrillar collagens which are found in the ECM (Mattrisian, 1992). Since MMPs play such a cntical role in the ECM, it is not surprishg that these enzyxnes are under tight regdation. Although the MMPs are very similar to each other in tenns of gene and protein structure, as well as in having overlapping substrate specincity, each MMP has its own distinct regdation profile (Marta et al., 1996; Crawford and Markisian 1996; Denhardt et ai., 1993). Regulation of MMP expression can be very intricate and complex and can occur at many levels including gene activation and transcription, mRNA stability, proenzyme activation, and inactivation by endogenous inhibitors.

Reaulation of the MMPs bv TIMPs

There exists a family of four specific endogenous inhibitors of MMPs activity known as the tissue specific inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) (Chambers and Matrisian, 1997). The balance of activated proteases and endogenous inhibitors is crucial for determining the extent of ECM turnover, since it is this baiance that determines the net MM'activity (Denhardt et al., 1993; Ray and Stetler- Stevenson, 1994). The TIMPs inhibit the activity of the MMPs in two ways (Denhardt et al., 1993; Ray and Stetler-Stevenson, 1994). They are able to bind to the latent proenzyme form of the MMP thereby preventing its activation, or TIMPs cm inûibit MMP activity by binding to its active site and thereby blocking substrate accessibility. These two potential binding sites for the TIMPs are distinct fiom one another (Kleiner et al., 1992). The binding is usually tight, and is the result of a non-covalent interaction. There does not seem to exist any signifïcant differences among the TIMPs ability towards inhibiting the active form of MMPs (Denhardt et al., 1993). However, the TIMPs do exhibit a certain degree of specificity when inhibithg the latent MMP fom. TIMP-1 is able to form a 1: 1 complex with the 92-LDa type IV procollagenase (MMP-9) (Goldberg et al., l992), whereas TIMP-2 selectively forms a complex with the 72-kDa type IV procoliagenase (MMP-2) (Stetler-Stevenson et al., 1989; Fridman R et al., 1993).

Activation of MMPs

The activation of the MMPs proenzyme form constitutes another critical step in the regdation of MMP activity. Once MMPs are synthesized, most of them are excreted as zymogens and require activation (Corcoran et al., 1996). MMPs are believed to be activated by the 'cysteine switch mechanism' (Van Wart and Birkedal-Hansen, 1994). It is referred to as the 'cysteine switch mechanism' because within the prodomain there exists an unpaired cysteine residue that coordinates with the zinc atom found in the active domain. It is this interaction that maintains MMPs latency. Once this interaction is disrupted, either by chernical or physicd means, a series of events are initiateci leading to conformational change of the MMPs- This change results in MMP activation and cleavage of the prodomain. investigation into the mechanism of in vivo activation of MMPs is ongoing. There are currently four mechanisms proposed for the in vivo activation of MMPs (Corcoran et al.,1996). The fïrst of these mechanisms is the activation of MMPs by proteases other than MMPs. The plasmin cascade was the nist mechanism identified responsible for the activation of certain MMPs (Corcoran et al., 19%). Plasminogen is cleaved by the tissue-type plasminogen activator (PA) or by the urokinase plasminogen activator (uPA) to yield the active serine protease plasmia. Once activated, plasmin can activate the coIlagenases, progelatinase B, and stromelysin-1 ami stromelysin-2 by cleaving part or al1 of their respective prodomain (He et al., 1989; Corcoran et al., 1996; Mazzieri et al., 1997). This cascade may occur lucally at the ce11 surface via the urokinase plasminogen activator receptor or at a site distant hmthat of enzyme secretion (He et al., 1989). The activation of progelatinase B by the plaunin cascade system only occurs at the celi sdace (MaPieri et al., 1997). Cathepsin G activation of pro-MMP -9 is another example of a pro-MMP activation cascade initiated by a protease that is not part of the MMP farnily (Okada et al., 1992). Interacting with a second active MMP can aiso activate pro-MMPs. Some examples of this type of regulation include the activation of pro-MMP-9 by MMP-3 (Okada et al., 1992) or by MMP-2 (F-ridman et al., 1995), and the activation of pro- MMP-7 by MMP-3 (Imai et al., 1995). For al1 of these interactions the conversion of the pro-MMP is inhibited when the activated MMP is bound by any of the TIMl? species or pro-MMP-9 is bound by TIMP-1. VonBredow et al. (1998) demonstrated the activation of pro-MMP-9 bound to TIMP-I by MMP-7. Incubation for 20 hours of the MMP- 9/ïlM?-1 complex with active MMP-7 in a 1:2 ratio resulted in complete conversion of the pro-MMP-9 (92-Ba) to the active species (estimated to be 78-kDa). The demonstration of MMP-7activation of pro-MMP-9/TIMP complex is the f'irst of its nature (VonBredow et al., 1998). Generaily, once pro-MMP-9 is bound to TïMP it can no longer be activated by another MMP, suggesting a noveI role for MMP-7in MMP-9 regulation. The third proposed mechanism of MMP activation involves the serine protease fUrin found in the golgi network (Corcoran et al., 1996). This mechanism is unique in that the MMP is activated prior to secretion. Furin recognizes proteins which contain a RXKR sequence. Only MMP- 11, as well as the MT-MMPmembers, contain this furin recognition sequence which is located between the propeptide and the catalytic domain (Cao et al., 1995). To date, oniy MMP-11 activation has been demonstrated by this mechanism (Pei and Weiss, 1995). However, it is specdated that the MT-MMPs are also activated by this mechanism (Corcoran et al., 1996).

Activation of MMP-2

Sato et al. (1994) first demonstrated the fourth mechanism of MMP activation proposed by Corcoran et al. (1996). Through a series of transfection experiments with MT-MMP, it was demonstrated that pro-MMP-2 can be activated by the MT-MMP family (Sato et OZ., 1994; Takino et al., 1995). This type of activation was specific as the MT-MMPs were unable to activate pro-MMP-9. TlMP-2 was fond to be very efficient at inhibithg this activation process. While TïMP-1, in these experiments, was inefficient or unable to inhibit the activation of pro-MMP-2 by MT-MMP(Foda et aï., 1996; Sato et al., 1994; Takino et ai., 1995). MT-MMPrequires its transmembrane domain in order to convert pro-MMP-2 to the active form (Cao et al., 1995). Once the transmembrane domain is removed, the MT-MMP was released fiom the ce11 surface, and MT-MMPlost its ability to activate MMP-2. This suggests MMP-2 mutcome in contact with the cell surface in order for its activation to occur. The discovery of a ceîi surface TIMP-2 receptor, coupled with the knowledge that newly synthesized MMP-2 is secreted hmthe cell bound to TIMP-2, has Ied researchers to suggest a mode1 for MMP-2 activation (Strongin et al., 1995). The secreted pro-MMP-UTIMP-2 complex attaches to the surface of the ce11 via the TIMP-2 ce11 surfàce receptor. Once on the dace, pro-MMP- UTIMP-2 then foms a complex with a MT-MMP allowing for the activation of MMP-2 and its release fiom the ce11 surface. It has also been suggested that the plasmin cascade plays a critical role in the activation of pro-MMP-2 (Ray and Stetler-Stevenson, 1994). However, the activation of pro-MMP-2 by the plasmin cascade is still controversial. Mazzieri et al. (1997) demonstrated the activation of pro-MMP-2 by plasminogen in HI' 1080 human fibrosarcoma cells, whereas, Lim et al. (1996) failed to activate pro-MMP-2 by plasmin in the same ce11 line. Based on these observations and othen (eg. Ray and Stetler- Stevenson, 1994) it is clear Merwork in this area must be done in order to determine the importance of the plasmin cascade in pro-MMP-2 activation.

Transcri~tionalRemlation of the MMPs

Transcriptional regdation of MMPs is dependent upon tbe ce11 type, MMP, and biochemical agent under investigation. However, induction of gene expression of most MMPs cm be achieved by a number of difEerent cytokines, growth factors, and turnor promoting agents. For example, interleukin- 1 (IL- 1 ) and tunior necrosis factor- (TNF- a)have been shown to induce MMP-1, MMP-3, and MMP-9 message levels in rabbit synovial fibroblast cells (Vincenti et al. , 1998; Nakano et al., 1995). Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have been shown to induce message expression of MMP-1, MMP-3, and MMP-9 in a series of glioma ceii lines and mouse fibroblast ceils (Aho et al-, 1997; Nakano et al., 1995). As well, the turnor promoter phorbol 12-myrisate 13-acetate (PMA) has ken shown to upregulate the message of MMP- 1, MMP-3, and MMP-9 in a nuniber of different fibroblast ceii lines and stroma1 cell hes(Rawdanowicz et al., 1994; Vincenti et al., 1998; Huhtala et al., 1991). It is believed induction of message by these agents occurs chiefly by the binding of AP- i to TRE sites found in the promoter region of the MMPs (Huhtala et al., 1991 ; Crawford and Matrisian, 1996; Aho et al., 1997). Decreases in MMP message have generaily ken seen with the addition of TGF-Pl - For exarnple, the addition of TGF-9i has ken shown to decrease MMP-I and MMP-3 message levels, and moderately reduce MMP-9 expression in a number of ce11 lines including fibroblast cell lines and glioma ce11 lines (Overali et al., 1989; Kerr et al., 1990; Huhtala et al., 199 1; Nakano et al., 1995). TGF-Bi is thought to block transcription of MMPs by binding to TEsites found in their promoter region (Kerr et al., 1990; Huhtala et al., 1991). In general, the changes seen at the message level in response to the addition of these biological agents are also seen at the protein level. It is important to note that these are just general trends, and as with any trend exceptions do exist. For exarnple, autocrine TGF-QI stimulation of MMP-9 has been demonstrated in ras-transfomied fibrosarcomas (Samuel et al., 1992). TGF-B 1 has also been shown to induce MMP-9 activity in a m.unmary adenocarcinorna ce11 line in which TGF-B induced collagenolytic activity conelated with increased invasion and metastasis (Welch et al., 1990). Regdation of MMP-2 is unique in that it dws not contain a TE site in its promoter region. Furthemore, the addition of TGF-Bi generaily induces MMP-2 mRNA levels (Overall et al., 1991). The exact mechanism by which TGF-9 1 induces the message is unknown. However, it is speculated the elevated levels of MMP-2 message are the result of increased message stability and not due to an increased rate of transcription (Overd et al., 1991). MMP-2 is also unique in its response to PMA. Although no TRE site has been found in the promoter region of MMP-2 (Huhtala et al., 1990), the addition of PM.has been shown to induce changes in MMP-2expression not only at the message level but at the protein level as well. How PMA regulates MMP-2 expression appears to be largely ce11 type specific. For exarnple, NaLano et al. (1995) demonstrated the addition of PMA increased MMP-2 message levels in some glioma celi lines while decreasing MMP-2 message levels in other giioma cell iines. Brown et al. (1990) demonstrated that PMA has Little to no effect on MMP-2 message levels in WI 38 cells, which are nontumorigenic fetal lung fibmblasts, while decreasing message levels in A2058 celis, which are human melanoma celis. Interestingly, ciifferences in PMA mediated regdation of MMP-2 expression have also been demonstrated within the same cell line. In some studies the addition of PMA to HT 1080 cells (a human fibrosarcoma ce11 line) resulted in a decrease in MW-2 message levels (HuhtaIa et al., 1991 ; Brown et al., 199û), whiie in other studies the addition of PMA had no affect on MMP-2 message levels in HT 1O80 cells (Lim et al., 19%). DifTerences were also seen in MMP-2 gelatinolytic when PMA was added to HT 1080 ceils. Both Huhtala et al. (1 99 1) and Brown et al. (1990) demonstrated a decrease in MMP-2 message, but only Brown et al. (1990) demonstrated a correspondhg decrease in MMP-2 gelatinolytic activity. No change in h4MP-2 gelatinolytic activity was noted by Huhtala et al. (1 99 1) even though a decrease in MMP-2 message was demonstrated. These observations underscore the complexity and the intricacies aswciated with MMP-2 expression and regdation. 'Ihe expression of TIMP-î, which binds selectively to pro-MMP-2, is not affected by the addition of PM.(Stetier-Stevenson et al., 1990). However, increased expression of MT-MMP-1 (the putative activator of pro-MMP-2) has been demonstrated in response to PMA (Foda et al., 1996; Lim et al., 1996). This increase in MT-MMP expression resulted in elevated levels of activated MMP-2. It bas been suggested, therefore, that the increased levels of activated MMP-2 due to elevated MT-MMP levels in response to PMA occurs through protein kinase C mediated events (Foda et al., 1996).

MMPs and Cancer

The MMPs have been shown to play key roles in normal biological processes such as those associated with the basement membrane (BM) and ECM turnover. Mme examples of these processes include menstruation (Rodgers et al., 1994), embryonic bone development (Vu et al., 1998), and wound healing (Stncklin et al., 1993). MMPs are also thought to play an important role in many pathologicai processes, including arthntis and metastasis. Metastasis is the process by which malignant cells lave their prhary site and spread to distant locations throughout the body (De,1996). The metastatic cascade consists of a nurnber of linked seguential steps. These steps are the ability of the tumor to grow and vascularize at the primary site, invade the surroundhg host tissue, disseminate throughout the body, arrest in the capiilary bed of distant organs, extravasate into the secondary organ, and grow at the secondary site (Dm, 1996). During metastasis, a number of naturai tissue barriers mut be degraded. In fact, this is one of the denning characteristicsseparating benign disorders hminvasive carcinomas (Ray and Stetler-Stevenson, 1994). Benign disorders aiways contain an intact BM surounding the tumor fiom the stroma, while invasive carcinomas are characterized by a discontinuous BM and zones of matrix loss. It has been suggested that the breakdown of the BM represents an early cntical event in the metastatic cascade since it is the fi& extracehlar banier to be crossed by the hvading cancer celis (Du£@, 1996). The proteolysis of the BM is also thought to be important in the subsequent steps of the metastatic cascade, in particular intravasation and extravasation fiom the blood Stream @*, 1996). Quantitatively, the most important protein found in the BM is type-N collagen (Du@, 1996). Since type N coiiagen is a major substrate of the MMPs, it is not surprishg that a variety of studies have indicated a role for the MMPs in tumor invasion and metastasis. MMP expression is associated with the malignant phenotype in a wide variety of tissues. Yamashita et al. (1998) showed enhanced production of MMP-7 in human gastric carcinoma tissues compared to control normal gastric mucosa Additionally, the proportion of immunoreactive carcinoma cells was markedly higher in the carcinomas which exhibited invasion and metastasis compared to those without invasion or metastasis. This fïnding suggested that the production and activation of MMP-7 was implicated in invasion and metastasis of human gastric carcinomas. Sugiura et al. (1998) found higher levels of MMP-9 in samples taken hmneuroblastoma tumors ciassified as stage N (hîghly metastatic and poor clinical outcome) compared to samples fiom neuroblastoma tumors classified as stage 1 and II (benign, localized turnor). Levy et al. (199 1) demonstrated increased levels of MMP-2 mRNA in primary human colon carcinomas compared to those levels found in the normal adjacent colonic mucosa Immunohistochemical studies have also shown increases in MMP-2 mRNA resulted in increased synthesis of enyme by the tumor celis, and that the increased enzyme synthesis correlated with metastatic potential (Levy et ai-, 1991). MMP expression has also been associated with the malignant phenotype in Iruig, prostate, neck, and breast cancers (Ray and Stetler-Stevenson, 1994). There is also abundant experimental evidence suggesting that the MMPs play a functional role in metastasis. Levels of a variety of MMPs correlate with metastatic potentiaf in model tumor systems. For example, there is increased gelatinolytic activiy in highly metastatic T-24 H-ras transformed NTH 3T3 cells compared to untransfected NIH 3T3 cells (Chambers and Tuck, 1993). Sreenath et ai. (1992) demonstrated that transfomed rat embryo ce11 Lines with high metastatic potentiaf produce high levels of MMP-3 and MMP-1 O, while the nonmetastatic ce11 lines produceci low or undetectable levels of these tw~MMPs. In vivo experimental evidence also directly Links MMPs to metastasis. DU445 celis are a prostate tumor ce11 line which is nometastatic. Matrilysin (MMP-7) overexpressing DU- 145 cells are sipnificantly more invasive than the nontransfected DU-145 cells (Powell et al., t 993). Bernhard et al, (1994) demonstrated tumongenic but nonmetastatic Ha-rer/EIA transfonned rat embryo cells became highly metastatic when transfected with MMP-9. Furthetmore, highly metastatic Ha-rudv-myc transformed rat embryo ceils lost their metastatic ability if MMP-9 expression was blocked (Hua and Muschel, 1996). Experiments involving TIMPs provide Merevidence for a functional role for MMPs in metastasis. hcreasing the levels of TIMPs, specific inhibitors of MMP activity, has been shown both by in vitro and in vivo experiments to block invasiveness and metastasis of tumor cells. When a recombinant fomi of TTMP-2 was added to 4R cells (a rat embryo ce11 line), these cells showed a significant decrease in invasive ability in an in vitro dynamic invasion model (DeClerck et al-,1 99 1). Stable TIMP-2 overexpressing 4R cells exhibited reduced tumor size and decreased invasive capacity compared to control4R cells, which fonned much larger tumors and were more invasive @eClerk et al., 1992). Similar studies with TIMP-I yielded comparable results (Chambers and Matrisian, 1996). N: Rationale for the Study

In order to potentially develop therapeutic strategies to address the problems associated with the development of cancer and its possible disease progression, it is very important to understand the differences that exist between the normal ceU and the ceii that is transformed. In this regard, previous studies, including desin our lab, have deterrnined and elucidated a number of ciifferences between normal cells and transformed cells. These differences include alterations in the expression and in the regulation of a number of genes associateci with cellular pwth, Ïncludhg dtered OM)expression and regulation, and altered polyamine biosynthesis (Hurta et al-,1993; Hurta and Wright 1995). TGF-P increased ODC expression in 1OTln cells transformed with H-rus, but did not alter ODC expression in nontransfomed lOTln celis. Furthermore, using cell lines expressing different levels of rus, it was demomtrated that basai levels of ODC and SAMDC expression correlated with the level of rar expression (unpublished results). Alterations in MMP expression and regulation in the nontransformed and the transformed cell have aiso been demonstrated in our laboratory (Samuel et al., 1992; Baruch and Hurta, 1996). MMP-2 and MMP-9 basal levels of expression was higher in H-ras transformed 10T112 cells versus nontransformeci 10T1/2 cells. Regdation of MMP expression by a number of ciifferent growth factors and cytokines also differed in the nontransformed and transformed ceIl (unpublished results). MMPs have bcen linked with the metastatic process. Additiody, there is a growing body of evidence suggesting that MMPs play an important deaiso in the fonnation of primary tumors (Chambers and Matrisian, 1997). In particular, MMP-2 has shown the greatest incidence among tumors of al1 types (Corcoran et al., 1996). There is also evidence that links aberrant polyamine metabolism and carcinogenesis (Auvinen, 1997). A putative role between aberrant polyamine metabolism and altered expression of MMP-2 has been proposed, but not yet proven. The objective of this thesis was to elucidate the possible link(s) between altered polyamine metabolism and MMP expression in normal and in transfonned mammalian cells. in order to focus the nature of this investigation, only the expression and the regulation of MMP-2 was investigated. The tumor promoter, phorbol 12-myrisate 13-acetate, has been shown to regdate a number of dflerent MMPs, including MMP-2, in a wide variety of ceil types. In order to dissect out possible alterations in the expression and regulation of MMP-2 whkh might exist between normal versus transfonned cells, the e&t(s) of PMA (phorbol 12- myrisate 13-acetate) a phorbol ester tumor promoter, was investigated. Additionally, the role of dtered cellular polyamine levels in the regdation of MMP-2 expression was investigated.

V. Hypothesis

MMPs expression and regulation differs between normal versus transformed ceiis, I hypothesize that (1) MMP-2 expression is differentially regulated in normal versus oncogene transfonned cells in response to the phorbol ester tumor promoter, PMA; and (2) that the alterations in MMP-2 expression due to the effects of PMA are mediatedllinked to cellular poiyamine levels; and (3) that the mechanisms contributhg to the altered expression/regulation of MMP-2are different between normal and transformed cells. MATERIALS AND M~I~NDDs

1. Ceii Line Description

The cell hes used were 10T1/2mouse fibroblasts and the NR.3 cell line which was derived fkom transfection of 10T 112 mouse fibroblast cells with the plasmid pAL8A which encodes for T-24 H-ras (Egan et al., 1986). 10T1/2ceiis do not express H-rar, and do not form tumors when injected into either syngeneic C3-N or irnmunodeficient BALB/c nulnu mice (Egan et al., 1986). NR3 cells express low levels of H-ras, and are tumorigenic but nonmetastatic when injected into C3WHeN mice (Egan et al., 1986). NIH 3î3 and v-fes transfected NIH 3T3 cell lines were also used (Egan et ai., 1987). The NM 3T3 ceils are nontumorigenic when injected into BALBlc nhurnice, while the v-fes transfected NIH-3T3 cells are tumorigenic and highly metastatic when injected into BALBk nulnu mice (Egan et al., 1987).

II. Ceii Culture

Cells were cultivated on 100 mm tissue culture plates (NUNC) with a-Minimum Essential Medium (a-MEM,GIBCO BRL) containhg 10% fetal bovine senun (FBS) (HYCLONE), 100 IU/d of penicillin G (SIGMA), and 68 pghl of streptomycih sulphate (SIGMA). The cultures were incubated at 37'~ in a humidified 5% CO2 atmosphere. Cells were grown to confiuence and then placed on a defined medium consisting of senun fiee a-MEMwithout the addition of 10Y0 FBS, and supplemented with 10 &ml of holo- transfemh (SIGMA) and 5 pghl of insirlin (SIGMA). AU experiments were conducted with cells on denned media For RNA extraction, cells were removed fiom tissue culture plates with approxirnately 2 ml of phosphate-buffered 5% bacto-trypsin (DIFCO) - 5 mM EDTA solution. Cellular polyamine depletion was achieved by treating cells with DL*- difluoromethylornithine @FMO)(ILEX ONCOLOGY DJC.) to a final concentration of 5mM, a concentration well documented to deplete cellular polyamine pools (Patel et al., 1998; Schaefer and Seidenf'eld, 1987). In stucfies involving the phorbol ester tumor promoter, phorbol 12-myrisate 13-acetate (PMA) (SIGMA), a nnal concentration of 0.1 pM was used. In experiments to assess the desof the transcriptional apparatus and message stability, actinomycin D (SIGMA) was used to block transcription. Actinomycin D, at a final concentration of 2.5 pg/ml was used. For experiments investigating the involvement of de novo protein synthesis, cycloheximide (SIGMA) was used at a nnal concentration of 10 pg/ml. In studies inhibithg the activity of protein kinase C (PKC)the drug calphostin C (SIGMA) was used at a nnal concentration of 0.5 PM

III. Assay for Gelatinolytic Activity

Gelatinoiytic activity was assayed essentially as described by Samuel et al. (1992). In experiments where gelatinolytic activity was examine4 cells were grown to confluence and then placed into defined media prior to initiation of the assay. The conditioned media was removed and assayed immediately or stored at -70'~ until Mer processed. Al1 samples were processed within two weeks. The conditioned media 4: 1 (v:v) with sample baer (2% SDS, 0.0625 M Tris -HCl (pH 6.8), 10% glycerol, and 0.025% bromophenol blue - without reducing agents) was then wanned at 37k for 5 minutes. 250 pI aliquots of each sample was loaded ont0 a 5% polyacrylarnide stacking gel dong with prestained molecular weight markers (GIBCO BRL) and resolved by electrophoresis on a 10% polyacrylamide resolving gel which containecl gelatin (SIGMA) as the substrate to a balconcentration of 1 mg/rnl. The Nnning baerwas 0.025 M Tris (- pH 8.3), 0.1% SDS, and 0.2 M glycine. Folloeg electmphoresis ovemight at a constant current of 10-20 mA, the gel was washed in a 0.05 M Tris-HCl @H 7.4) and 2% Triton X- 100 solution for 1 hou, and then rinsed in 0.05 M Tris-HCl (pH 7.4) for 0.5 hours. mer rinsing, the gel was incubated at 37'~for 24 hours in a substrate baer containhg 0.05 M Tris-HC1 (pH 7.4), 0.005 M CaC12, 1% Triton X-100, and 0.02% NaN3. Areas of gelatinolytic activity were visualized by staining the gel with 0.1% Coomassie Brilliant Blue R-250 (ICN) in acetic acid: methanol: water (10:40:50; v:v:v), followed by destaining in 40% methanol and 10% acetic acid. Gelatinolytic activity appears as zones of clearing due to deg-radation of gelatin. As a loading contrd identicaiiy loaded complementary Coomassie Brilliant Blue stained polyacrylamide gels without gelatin were used (Oetken et al., 1992). Staining of these gels produced a number of bands for each lane. The intensity of these bands was used to ensure an equal arnount of protein was added for each zymogram (data not shown).

IV. Total RNA Isolation

Cells were placed into sterile 15 ml conical screw cap tubes (SARSTEDT) and pelleted at 3,000 x g in a clinical centrifuge for 5 minutes. Celf pellets were resuspended in 2 ml of cold phosphate buffered saline (PBS) solution and aliquoted into two sterile 1.5 ml microfige tubes. Cells were then centrifuged at 6,000 x g for 5 minutes in the cold. Total RNA was then extracted using an RNA extraction kit which employed the Trizol reagent (GIBCO BRL). The procedure utilizes a single step RNA extraction modified fiom the procedure of Chomczynski and Sacchi (1987). In brief, pelleted cells were lysed by resuspending them in 500 pl of Trizol and allowed to sit at room temperature for 5 -10 minutes. 200 pl of chloroform was then added to each tube, followed by vigorous vortexing for 30 seconds. The tubes remained at room temperature for a Mer5 minutes and were then centrifuged at 12,000 x g for 20 minutes in the cold. AAer centrifugation the solution separated into a clear aqueous phase on top, a white interphase in the rniddle, and a lower red organic phase on the bottom. The aqueous phase was transferred into a clean sterile 1.5 ml microfige tube. RNA remains exclusively in this phase. Isopropanol(1 ml) was then added. The tubes were placed on ice for 20 minutes and then centrifuged for an additional 20 minutes at 12,000 x g. The supanatant was decanted and the pellet washed with 1 ml of 70% ethanol in 0.1% diethyl-pyrocarbnate (DEPC) buffer. The tubes were centrifuged for an additional 5 minutes at 7,500 x g and the supematant decanted. The tubes were air dried for 5 minutes and then resuspended in 20 - 30 pl of O. 1% DEPC treated water. The peiiets split eariier in the procedure were recombined to give a total volume of 40 - 60 11 of each sample. The samples were stored at -70'~until ready for Northern blot anaiysis.

V. cDNA Probes

The plasmid pMGe1PP wntaining the cDNA probe specific for murine MMPd was obtained fiom Dr. Lynn Matrisan (Department of Ce11 Biology, Vanderbilt University, Nashville, TN (Witty et al., 1995; Reponen et al., 1992). A cDNA probe specific for rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cloned into the plasmid pBssK' was a kind gift hmDr. Linda Penn (Department of Cellular and MoLecular Biology, Ontario Cancer Institute, Toronto, ON). Both plasmids were fmt transfonned into competent DHSa Escherichia di. Transfonned bacteria were grown ovemight in 100 ml LB media (ampicillin 50 pg/ml, bacto-tryptone @ECO) 10 g/L, bacto-yeast extract (DiFCO) 5 g/L, and NaCl 10 g/L) at 37 OC. Plasrnid isolation was prepared using a Maxi-Prep kit (QIAGEN) according to the manufacturer's instmctions. Bacteria were pelleted by centrifiiging in two 50 mi FALCON tubes at 3000 x g for 18 minutes and then resuspended in BeerPl in a total volume of 10 ml. 10 ml of Bmer P2 was then added and the entire mixture was incubated at roorn temperature for 5 minutes. After adding 10 ml of Bufîer P3, the solution was iced for 20 minutes, and aliquoted into eighteen 1.5 ml microfiige tubes. The tubes were spun at 12,000 x g for 30 minutes, supematants collected into fiesh microfbge tubes, and re-centrihiged for 20 minutes at 12,000 x g. The supernatant was then appiied to a QIAGEN tip and washed twice with 30 ml of Buffet QC. The DNA was eluted with 15 ml of Baer QF, collected into 16 microfiige tubes, each containing 700 pl of isopropanol, and centrifüged immediately at 12,OOC x g for 30 minutes. The supematant was decanted and the DNA pellet was washed with 75% ethanol, and then resuspended in a total volume of 300 pl of sterile water. The amount of DNA obtained was estimated by diluting 5 pl into 1.5 ml of sterile water and reading the absorbance at 260 nm. A 962 bp insert specific for murine MMP-2 was excised hmthe plasmid pMGelPP with EcoRI (GIBCO BRL) and Pst1 (GIBCO BRL). A 370 bp cDNA insert specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was excised hmthe plasmid pBssK' with BamHI (GIBCO BRL) and on1(GIBCO BRL). Digested pMGeLPP, dong with a 1 kB DNA ladder (GIBCO BRL), was loaded onto a 1% agarose gel and nui oveniight at 23 V in a 1 X TAE (0.04 M Tris-acetate, 0.001 M EDTA) buffer. Digested pBssK-, dong with a 1 kB DNA ladder (GIBCO BRL), was loaded onto a 1% agarose gel and nui for 3 hours at 60 V in a 1 X TAE (0.04 M Tris-acetate, 0.001 M EDTA) buffer. Under UV light, the insert was then excised hmthe gel and purified using the gel extraction kit Gene Clean II (BI0 101 INC.). The volume of Nd (6 M) added was three times the weight of the gel piece. The mixture was heated at 55'~for 5 -10 minutes (until the gel piece had completely dissolved). Glassmik was added, the solution vortexed, and placed on ice for 2 hours. The solution was centrifùged for 10 seconds and the supernatant discarded. The Glassmilk pellet was then washed 3 times with NEW (NaCl, ethanol, water) wash, and the DNA was eluted with a total of 250 pl of sterile water. The quantity of insert was estimated by diluting 15 pl into 1.5 ml of water and taking the absorbance at 260 m. The integrity of the cDNA insert isolated was checked by electrophoresis. Briefly, an aliquot of the isolated insert dong with a 1 kB ladder was Ioaded onto a 0.8% agarose gel and run for approximately 1 hour at 120V in a 1 X TAE buffer.

VI. RNA Blot Anaiysis

In brief, RNA samples were assayed by Northem Blot analysis essentially as described previously (Hurta et al., 1993). The amount of RNA present in each sample was detemillied by diluting 5 pl of the total volume into 1.5 ml O. 1% DEPC treated water and reading the absorbance at 260 nm and at 280 nm. Ratios at 260/280 were used as criteria to evaluate the purity of the RNA. Equal amounts of RNA were then resolved by electrophoresis. Prior to electrophoresis a 0.67 M 3-IN-MorpholinoJpmpanesulfonic acid (MOPS), 2.9 M formaldehyde, 65% formamide solution was added to each sample.

Samples were incubated between SOC - 60'~for 15 minutes. 5 pi of loading dye (0.25% Bromophenol Blue, 0.25% Xylene Cyanol, and 30% Glycerol) and 2 pl of ethidium brornide (10 rnghl) were added and the samples then hctionated on 0.8% agarose gels containhg 2.2M of formaldehyde at 30V overnight in a bSerof 1 X MOPS. After electrophoresis, ethidium bromide çtauied gels were visualized under UV light to ascertain qudity of RNA and as an indication of equal loading. RNA was transferred to nylon transfér membranes, Nytran Pius (MANDEL),using a capillary transfer technique; and the membrane was UV-cross linked using the auto cross link setting on an W Stratalinker 1800 (STRATAGENE).

VII. Northern Blot Analysis

The cDNA probes were labeled by Klenow (PHARMICIA BIOTECH) extension. 40-50 ng of cDNA insert was added to 10 pl of Reagent Mix, 1 pl of the Klenow fragment (both provided by the oligolabeIling kit), and boiled for approximately 5 minutes before adding 50 pCi of [32~]d~~~(1 0 pCi/pl, AMERSHAM). The reaction was incubated at room temperature for 1 hout. The probe was diluted fkom 50 pl to 250 pl with TE buffer pH 7.4, purified through a Sephadex G-50 spin columq and activity was determined in a liquid scintillation counter. The RNA-bound membranes were first prehybridized in the presence of 10 ml Rapid-Hyb baer (AMERSHAM LEE SCIENCE) for 2 hours at 65'~in a TEX STAR hybridization oven (BIO/CAN SCIENTWIC) and then hybridized in the same bder and at the same temperature in the presence of 106 CPM radiolabelled probe per ml of Rapid-Hyb baer. Mer hybridization was complete, the blots were washed appropriately. If the membranes were probed with MMP-2, the washing conditions used were two 20 minute washes with 5X SSC (LX SSC: 0.15M NaCl, 15 mM &C6&O7, pH 7.0), 0.1% SDS at room temperature, and then twice (3Ominutes/wash) with 0.2X SSC, 0.1% SDS at 59'~.For al1 mRNA stability experiments, the membranes were stripped of MMP-2(50% formamide, 0.1% SSC, at 65 OC for 1 hour) and reprobed with GAPDH- The washing conditions used for GAPDH were two 20 minute washes with 2X SSC, O. 1% SDS at room temperame, and then two 30 minute washes with 1X SSC, 0.1% SDS at 57%. Autoradiography was done using X-OMAT/AR (KODAK) x-ray film at -70'~. X-ray films were developed in a KODAK RP X-OMAT Rocessor (KODAK). Densitometric evaluation of autoradiognuns exposed in the liwar range for each set of samples was performed. Densitometric analysis of x-ray nIms (autoradiograms) was achieved using a GS 700 Imaging Densitometer (BI0 RAD) and the Molecular Analyst (BI0 RAD) software program. 1. The Addition of Phorbol Myrisate Acetate (PMA) AIters MMP-2 Message But Not MMP-2 Gelatinolytic Activity.

The tumor promoter, phorbol 12-myrisate 13-acetate (PMA)(also known as TPA, 12-O-tetradecanolyphorbol- 13-acetate), has a structure very similar to that of diacylglycerol and activates protein kinase C directly both in vivo and in vitro (Nishizuka, 1986). Treatment of cells with PMA has been shown to induce the expression of a fdy of AP-1 transactivator proteins that bind to the TPA response element (TRE) and initiate transcription (Ange1 et al., 1987). In general, PMA has shown the ability to increase the expression of the collagenases, stomelysins, and MMP-9 (Marta L et al.,1996). The addition of PMA to cells either has no effect, or can either increase or decrease the mRNA levels of MMP-2 (Marta L et al., 1996; Brown et al., 1990; Nakano et al., 1995). In ce11 lines of diBering turnorigenic and metastatic potential the effect of PMA on MMP-2 expression was determined. Experiments were performed with 10Tlj2, NR.3, NIH 3T3, and v-fes transfected MH 3T3 ce11 lines. Cells were grown to confluence and then placed on a dehed medium (semm fke a-MEM supplemented with 10 pg/ml of holo- transferrin and 5 pg/d of insulin) for 48 hours. PMA, to a £inalconcentration of 0.1 pM was then added to the cells for either 6 or 12 hours. This concentration of PMA that was used in this study was based on data in the literatute and previous work done in the laboratory (Huhtala et al., 1991 ; Hwta and Wright, 1995). Cells exposed to PMA for 12 hours were treated fïrst, 6 hours later PMA was added to ceUs designated for the 6 hou time point, and &er an additional 6 hours the conditioned media and cells from all the plates was collected. This manner of treatment was done to ensure the cells remained in defined media for the same amount of time, and that the only ciifference behiveen each time point was how long the ceils were exposed to PMA. The conditioned media was used to assay for gelatinolytic activity and the ceils were lysed and RNA isolated for determination of MMP-2 message levels (as described in Materials and Methods). Each experiment was performed, at least, in duplicate. All the ce11 lines produced and secreted MMP-2constitutively (Figures 2-5). Following gelatin gel electrophoresis (zymography) MMP-2 gelatinolytic activity was evident and identified by its characteristic size (M,62000-72000). The size was estimated by comparing the migration of the bands on the zymogram to prestained molecular weight markers. To ensure that the zones of clearing were due to the action of bonafide matrix metailoproteinases (MMPs) and not other protease(s), in control experiments 0.005 M EDTA was added to the substrate b&er (Rawdanowicz et al., 1994). EDTA inhibits the action of MMPs by chelating the ca2+required for MMP activity. Such complementary gels incubated in substrate bmer containhg EDTA showed no zones of clearing, indicating that the degradation of gelatin seen on the zymograms was indeed due to the action of a MMP (in this case MMP-2). The addition of PMA (O. 1PM) resuited in no readiiy apparent change in the gelatinolytic activity in any of the ce11 lines examine4 namely NIH 3T3 cells (Figure 2), v-fes-NIH 3T3 cells (Figure 3), 10T1/2 cells (Figure 4) aad TSR3 cells (Figure 5) respectively. As a loading control identically loaded complementary Coomassie Brilliant Blue stained polyacrylamide gels without gelatin were used (Oetken et al., 1992). Staining of these gels produced a number of bands for each lane. The intensiw of these bands was used to ensure an equal amount of protein was added for each zymograrn (data not shown). Northern blot analysis of total cellular RNA revealed there was a very srnall decrease in the message levels of MMP-2 in NIH 3T3 cells after PMA treatment for 6 hours, and a Merdecrease after 12 hours of PM.freatment (Figure 6). Interestingly, v-fes-NM 3T3 celis, which are highly metastatic, showed no change in the mRNA levels of MMP-2 aAer the addition of PMA either for 6 or 12 hours respectively (Figure 7). In 10T1/2 cells, which represents a normal celi, increased MMP-2 mRNA levels were noted in response to PM.treatment. This increase was evident as early as 6 hours and was - 1 I B O 6 12 PMA (Hours)

Figure 2. The phorbol ester tumor promoter, PM& does not alter MMP-2gelatinolytic activity in MH 3T3 cells. Conditioned media was prepared as described in Materials and Methods, and 200 pl aliquots were analyzed. A. Representative gelatin gel electrophoresis (ymography) of MMP-2 activity levels in NIH 3T3 cells is shown indicating MMP-2 gelatinolytic activity in (1) conaol cens, and in NIH 3T3 cells treaîed with PMA (O. 1 gM) for 6 hours (2) and 12 hours (3) respectively. Three zones of clearing were noted (approximately 62,68 and 72 kDa) comsponding to MMP-2 activity. As a loading control, complementary gels were anaiyzed as described in Materials and Methods (data not shown). B. Quantitative analysis as determineci by densitometry of relative MMP-2 gelatinolytic activity levels in NIH 3T3 cells in the absence and presence of PM..Similar observations were noted in duplicate 2 O 6 12 PMA (Hourr)

Figure 3. The phorbol ester tumor promoter, PMA,does not alter MMP-2 gelatinolytic activity in v-fes-NIH3T3 cells. Conditioned media was prepared as described in Materials and Methods, and 200 pl aliquots were analyzed. A. Representative gelatin gel electrophoresis (zymograpby) of MMP-2 activity levels in v-/e-NIH 3T3 ceils is shown indicating MMP-2 gelatinolytic activity in (1) control cells, and in v-fes-NIH 3T3 celis treated with PMA (O. I CIM) for 6 hours (2) and 12 hours (3) respectively. Three zones of clearïng were noted (approximately 62,68 and 72 kDa) correspondhg to MMP-2 activity. As a loading control, cornplementary gels were analyzed as described in Materials and Methods (data not shown). B. Quantitative iinalysis as detennined by densitometry of relative MM'-2 gelatinolytic activity levels in v-fes-NIH 3T3 cells in the absence and presence of PMA. Similar observations were noted in duplicate experirnents. Figure 4. The phorbol ester tumor promoter, PM& does not alter MMP-2 getatinolytic activity in 10T112 cells. Conditioned media was prepared as described in Materials and Methods, and 200 pl aliquots were analyzed. A Representative gelatin gel electrophoresis (zymography) of MMP-2 activity levels in lOTlC2 ceils is show indicaîing MMP-2 gelatinolytk activity in (1) control ceUs, and in lOTll2 cells treatd with PMA (0.1 FM)for 6 hours (2) and 12 hours (3) respectively. Two zones of clearing were noted (approximately 68 and 72 kDa) correspondhg to MMP-2 activity. As a Ioading control, complementary gels were analyzed as describecl in Materiais and Methods (data not show). B. Quantitative dysisas detemhed by densitometry of relative MMP-2 gelatinolytic activity levels in lOTlL2 ceils in the absence and presence of PMA. Similar obsewations were noted in duplicate experiments. PMA (Houn)

Figure 5. The phorbol ester tumor promoter, PM.,does not dter MMP-2 gelatinolytic activity in NR3 cells. Conditioned media was prepared as described in Materials and Methods, and 200 pl aliquots were analyzed. A Representative gelatin gel electrophoresis (zymography) of MMP-2 activity levels in NR3 ceiis is shown indicating MMP-2 gelatùiolytic activity in (1) control cells and in NR3 cells îreated with PMA (0.1 pM) for 6 hours (2) and 12 hours (3) respectively. Two zones of clearing were noted (approximately 68 and 72 kDa) corresponding to MMP-2 activity. As a loading control, complementary gels were analyzed as described in Materials and Methods (data not shown). B. Quantitative analysis as detemineci by densitometry of relative MMP-2 gelatinolytic activity levels in NR3 cells in the absence and presence of PM..Similar observations were noted in duplicate experiments. Figure 6. PMA decreases MMP-2 mRNA levels in NIH 3T3 ceus. Ao Northern blot analysis of MMP-2 mRNA Ievels in the absence of PMA (1) (wntrol), and in the presence of PMA (O. 1 CiM) for 6 hours (2) and12 hours (3) respectively. B. Eîhidium brornide staining of 28s rRNA is shown as a loading control. C. Quantitative analysis, as detennined by densitometry, of relative MMP-2 message Ievels in NIH 3T3 cells as shown in A.. Similar observations were noted in duplicate experiments. Fi yre 7. PMA does not affect MMP-2 mRNA levels in v-fes-NIH 3T3 cells. A Northem blot analysis of MMP-2 mRNA levels in the absence of PMA (1) (control), and in the presence of PMA (0.1 PM) for 6 hours (2) and 12 hours (3) respectively. B. Ethidium bromide staining of 28s rRNA is shown as a loading control. C. Quantitative analysis, as determhed by densitometry, of relative MMP-2 message levels in v-fes-NIH 3T3 cells as shown in A. Similar observations were noted in duplicate experiments. maintained after 12 hours of PMA treatment (Figure 8). In the tumorigenic low ras expressing NR3 cell line, PMA addition resulted in decreased levels of MMP-2 mRNA expression (Figure 9). The decrease was evident as early as 6 hours and was more pronounced aîter cells were exposed to PMA for 12 hours. These resuits suggest that the PMA mediated regdation of MMP-2 mRNA expression is dependent upon the cellular phenotype expressed, and differs between nontramformeci cells and îransformed ceils. When total cellular RNA was analyzed hmuntxeated celis grown to confluence, the basal levels of MMP-2 message expression also ciiffercd in the celi lines studied. Surprisingly, basal levels of MMP-2 message expression were higher in the parental 10T1/2 cell line compared to the NR3 ceil line (H-rus transformed 10T1/2 ce11 line) (Figure 10). Similar results were seen in the parental NIH 3T3 cell line and v-fes-NIH 3T3 ce11 lïne (v-fes transformed NIH 3T3 cell Liae). Basal MMP-2 message levels were higher in confluent NM 3T3 cells compared to confluent v-$es-NUI 3T3 ceiis (data not shown). These results indicate that basal MMP-2 message expression also differs between nontransformeci and transformed cells.

II. The Transcriptional Procas Plays A Role In The Regdation Of MMP- 2 Expression By PMA In Both lOTl/2 And NR3 CeU Liiies.

Since the most striking response in MMP-2 expression to PMA occdin lOTlf2 cells (normal) and in NR3 celis (low ras, capable of benign tumor formation) these ce11 lines became the focus of this investigation. Actinomycin D, a well recognized transcriptional inhibitor, was used in the following experiment to investigate if the transcriptional process was involved in regulating MMP-2 message expression in response to PMA in both the lOTl/2 and NR3 ceil lines. Cells were grown to confluence and placed on denned media for 48 hours. Cells were either mtreated, treated with PMA for 6 hours, treated with the transcriptionai inhibitor actinomycin D for 1 hou, or actinomycin D for 1 hour and then PMA for 6 hours. Treatment occurred in such a way as to ensure al1 the cells remained on defineci media for the same arnount of time. The concentration for actinomycin D (2.5 pg/ml) was established by previous work done in the lab (Hurta and Wright, 1995). As judged by Figure 8. PMA Uiaeases MMP-2 mRNA levels in lOTl/î ceiis. A Northern blot aaalysis of MMP-2 mRNA levels in the absence of PMA (1) (control), and in the presence of PMA (0.1 CiM) for 6 hours (2) and12 hours (3) respectively. B. Ethidium bromide stainllig of 28s rRNA is shown as a loading control. CmQuantitative adysis, as determined by densitometry, of relative MMP-2 message levels in lOTlQ cells as shown in A.. Similar observations were noted in duplicate experiments. Figwe 9. PMA decreases MMP-2 mRNA levels in NR3 cells. Decreased MMP-2 mRNA levels were noted within 6 hours of PMA treatment. A. Northem blot analysis of MW-2 mRNA IeveIs in the absence of PM.(1) (control), and in the presence of PMA (0.1 FM) for 6 ho- (2) and12 hours (3) respectively B. Ethidium brornide staining of 28s rRNA is shown as a loading control. C. Quantitative analysis, as determineci by densitometry, of relative MMP-2 message levels in NR3 ceiis as shown in A. Similar observations were noted in duplicate experiments. Figure 10. Basal levels of MMP-2 message are higher in lOTln cells versus NR3 ceiis. A. Northern blot analysis of MMP-2 mRNA levels in 10TlL2 cells grown to confluence and in the absence of any treatment (l), and in NR3 celis grown to confluence and in the absence of any treatment (2) respectively. B. Ethidium bromide staining of 28s rRNA is show as a loading control. Similar observations were noted in duplicate experiments. ce11 morphology adceii number, the dose of actinomycin D was not toxic over the the interval studied. AAer treatment, the cells were coiiected and the RNA was extracted for Northern blot analyses (as described in Matenals and Methods). Figures 1 1 and 12 indicate that addition of actinomycin D prior to PMA treatment abrogated any changes in the level of MMP-2 message in response to PMA treatment in both the 10T1/2and NR3 celi lines. When actinomyciin D was added to 10T112 cells prior to PMA addition there was no induction of MMP-2message. in NR3 cells, the addition of actinomycin D blocked the PMA mediated decreases in MMP-2mRNA. This suggests that for bthcell lines the transcriptional ptocess, at least in part, ptays a role in PMA regdation of MMP- 2 mRNA levels. For both ce11 Lines, the addition of actinomycin D did not affect the basal levels of MMP-2 -A.

m. Post-trnnscriptiond Reguiation Of MMP-2 =RNA Expression Occurs In 10Tl/t Ceüs And In NR3 Ceb In Response To PMA.

The rate of decay for mature message may also alter mRNA levels. In order to determine if PMA affects the message stability of MMP-2 in 10Tt/2 and MU cells the half life of MW-2 in the presence and in the absence of PMA was detennined in 10T1/2 cells and in NR3 celis, respectively. h brief, cells were gmwn to confluence and then placed on defined media for 48 hours. Cells were then treated first with PMA for 6 hours and then with actinomycin D for 6 and 18 hours respectively. To establish the normal half life of MMP-2 in both cells lines, an identical set of celis was treated only with actinomycin D for 6, and 18 hours after they were grown to confluence and placed on defined media for 48 hours. Again, treatment occurred in an inverse rnanner with actinomycin D added tkst to the 18 hour thepoint in order that al1 the cells remained on defined media for the same amount of time. As judged by ce11 morphology and celi number, the dose of actinomycin D was not toxic over the time intervals studied (data not show). Cells were lyseci, and RNA extracted for Northem blot dysis(as described in Materials and Methods). The blots were probed nrst for MMP-2 (as described in Figure 11. The transcriptional process plays a role in the regdation of MMP-2 gene expression by PMA in lOTll2 cells. Northern blot analysis of MMP-2 mRNA levels in 10T1/2cells. A. MMP-2 mRNA levels in control cells (1) in the absence of either actinomycin D (2.5 pg/rnl) or PMA (O. 1 pM), in cells treated with PMA for 6 hours (2), in cells treated with only actinornycin D for 1 hour (3), in cells treated first with actinomycin D for 1 hour and then with PMA for 6 hours (4) respectively. B. Ethidium bromide stained 28s rRNA is shown as a loading control. C. Quantitative analysis detennined by densitometry of relative MMP-2 message levels in control cells (1) in the absence of either actinomycin D (2.5 pgd) or PMA (O. 1 CIM), in cells treated with PMA for 6 hours (21, in cells treated with only actinomycin D for 1 hour (3), in celis treated fust with actinomycin D for 1 hou and then with PMA for 6 hours (4) respectively. Similar observations were noted in duplicate experiments. Figure 12. The transcriptional process plays a role in the regdation of MMP-2gene expression by PMA in NR3 cells. Northem blot anaiysis of MMP-2 mRNA levels in NR3 cells. A. MMP-2 mRNA levels in control cells (1) in the absence of either actinomycin D (2.5 pg/ml) or PM.(0.1 pM), in cells treated with PMA for 6 hours (2), in cells treated with only actinornycin D for 1 hour (3), in cells treated first with actinomycin D for 1 hour and then with PMA for 6 hours (4) respectively. B. Ethidiun bromide stained 28s rRNA is show as a loading control. C. Quantitative dysisdetermined by densitomeûy of relative MMP-2 message levels in control cells (1) in the absence of either actinomycin D (2.5 ~g/ml)or PMA (0.1 PM), in cells treated with PMA for 6 hours (2), in cells treated with only actinomycin D for 1 hou (3), in cells treated first with actinomycin D for 1 hour and thea with PMA for 6 hours (4) respectively. Similar observations were noted in duplicate experiments. Material and Methods) and a senes of exposures were taken to ensure densitometric evduation of autoradiograms exposed in the linear range occurred. The blots were stripped, and reprobed for GAPDH (as descnbed in Materials and Methods). A series of exposures were taken to enmte densitometric evaluation of autoradiograms exposed in the Linear range occured. Values obtained Erom densitometric analysis of MMP-2 message levels were normalized to the vaiues obtained for GAPDH. MMP-2 mRNA levels relative to cells untreated with actinomycin D were then calculated and plotted in a semilogarithmic manner. The half Life of the MMP-2 message was longer in NR3 cells than in 10T1Q cells. The haif Sie of MMP-2 was detemineci by extrapolation of semilogarithmic plots to be approximately 44 hours in IOT1/2 ceils and to be approximately 90 hours in NR3 cells respectively (Figure 13). How PMA affected the message stabiiity of MMP-2 also differed between these two ce11 lines. In 10T1/2the addition of PMA stabilized the message and moderately increased the half life 2 fold, approximately 90 hours (Figure 14). However, in NR3 cells the addition of PMA increased the rate of MMP-2 MA decay indicating a change in message stability (Figure 15). MMP-2 message in NR3 cells exposed to PMA had a reduced half life of approximately 13 hours. This represents a 6 fold reduction in MMP-2 mRNA half life in NR3 ceUs in response to PMA treatment. Therefore, in addition to an involvement of the transcriptional apparatus, PMA is able to reguiate MMP-2 message Ievels in lOT1/2 cells and in NR3 cells at the post- transcriptional level also.

IV. De Novo Protein Synthesis 1s A Requirement For PM.Mediateà Alterations In MMP-2 Message Expression In 10T1/2 Cells But Not In NR3 Cells

Regulation of MMP message by chemokines and cytokines has been shown to require de novo protein synthesis (Overall et al., 199 1; Huhtala et al-, 1991). Cyclohexùnide, an inhibitor of eukaryotic protein synthesis (Philips and Crowthers, 1986; Le et al., 1992). was used in the foliowing experiment to determine if PMA also Figure 13. MMP-2 mRNA half life is increased in H-rar transformed NR3 cells. Stability of MMP-2 mRNA in untreated IOTA2 cells and untreated NR3 cells. lOTl/2 cells (0)and NR 3 cells ( ) were placed on defined media for 48 hours and then treated with actinomycin D (2.5 )ig/ml). Total cellular RNA was isolated at 6 and 18 hours fier treatment with actinomycin D, and subjected to Northem blot analysis as described in Materials and Methods, The relative levels of MMP-2 mRNA were determined by densitometry and normalked to GAPDH. On the y-axis, ( d designates the relative value at 50%. The resdts presented are fiom duplicate experiments. Figure 14. PMA increases the haLfüfe of MMP-2message in lOTln cells. 10T112 cells in the absence of PMA (control ceus) ( O ), or exposed to PMA (O. 1 PM) for 6 hours (Cl ) were subsequentiy treated with actinomycin D (2.5 pg/ml). Total cellular RNA was isolated at 6 and 18 hours after treatment with actinomycin D, and subjected to Northem blot analysis as described in Materials and Methods. The relative levels of MMP-2 mRNA were determined by densitometry and nomalized to GAPDH. On the y-axis, ( A ) designates the relative value at 50%. The resdts presented are from duplicate experiments. Figure 15. PMA decreases the half life of MMP-2 message in NR3 cells. NR3 cells in the absence of PMA (control cells) ( O ), or exposed to PMA (0.1 CiM) for 6 hours (a ) were subsequently treated with actinomycin D (2.5 pg/ml). Totai cellular RNA was isolated at 6 and 18 hours after treatment with actinomycin D, and subjected to Northern blot analysis as described in Materials and Methods. nerelative levels of MMP-2 mRNA were determined by densitometry and normalized to GAPDH. On the y- axis, (A ) designates the relative value at 50%. The results presented are fiom duplicate experiments. requires de novo protein synthesis to regulate MMP-2 message levels in lOTlR and in NR3 cells. Briefly, ceils were grown to confluence and placed on defined media for 48 hours. Cells were then either left untreated, treated with PMA for 6 hours, cyclohexi&de for 1 hour, or cycloheximïde for 1 hour, then PMA for 6 hours. Treatment occurred in such a manner as that al1 the cells, regardless of treatment, could be collected at the same time for RNA extraction and subsequently anaiyzed by Northem blot analysis (as described in Material and Methods). The concentration of cycloheximide used (1 Opg/ml) was established by previous work done in the laboratory (Hurta and Wright, 1995). The addition of cycloheximide prior to that of PMA prevented any induction of MMP-2 message in 10T1/2 celis indicating PMA regdation of MMP-2 mRNA levels is dependent on de novo protein synthesis (Figure 16). In NR3 cells, the PMA mediated decrease in MMP-2 mRNA levels was not biocked by the addition of cycloheximide indicating PMA does not rquire de novo protein synthesis in its regdation of the MMP-2 message in NR3 cells (Figure 17). For both the 10T1/2 and NR3 ceil lines the addition of cycloheximide alone did not affect the basai levels of MMP-2 mRNA (Figures 16 and 17).

V. Protein Kinase C Mediated Events Are Invoived In The Regulation Of MMP-2 Message Expression In 10T1/2 And In NR3 Ceh

Since one of the effects of adding PMA to cells is to induce protein kinase C (PKC) activity (Nishizuka, 1986), the role of PKC mediated events in the PMA mediated alterations in MMP-2 expression was investigated. Prolonged exposure to PMA effectively down regulates PKC activity (Young et al., 1987). in effect, ceils treated with PMA for a long period of tirne will down regulate PKC by increasing its rate of degradation (Young et ai., 1987). In brief, cells were grown to confluence and then placed on either defined media for 48 hours, or defked media supplemented with PMA (0.1 ph4) for 48 hours. Cells that were in defined media only for 48 hours were either lefi untreated, or treated with PMA for 6 hours. Cells exposed to PMA for 48 hours were then re-exposed to PMA (O. 1pM) for 6 hours or to vehicle (ethanol) for 6 hours. RNA Figure 16. A role for de novo protein synthesis in the regdation of MMP-2 gene expression by PMA in 10T112 cells. Northem blot analysis in 10T1/2 cells. A. MMP-2 mRNA levels in control cells (1) in the absence of eitlîer cycloheximide (10 pg/ml) or PMA (O. 1 PM), in cells treated with PMA for 6 hours (2), in cells treated with only cycloheximide for 1 hour (3), in ceiis treated fmt with cycloheximide for 1 hour and then with PMA for 6 hours (4) respectively. B. Ethidium bromide stained 28s rRNA is shown as a loading control. C. Quantitative analysis determined by densitometry of relative MMP-2 message levels in control ceiIs (1) in the absence of either cycloheximide (10 pg/ml) or PMA (O. 1 CiM), in cells treated with PMA for 6 hours (2), in cells treated with only cycloheximide for 1 hour (3), in cells treated fim with cycloheximide for 1 hou and then with PMA for 6 hours (4) respectively. Similar observations were noted in duplicate experiments. Figure 17. No role for de novo protein synthesis in the regdation of MMP-2 gene expression by PMA in NR3 cells. Northem blot analysis of MMP-2 mlWA levels in NR3 cells. A. MMP-2 mRNA levels in control cells (1) in the absence of either cycloheximide (10 pg/ml) or PMA (O. 1 FM), in cells treated with PMA for 6 hours (2), in cells treated with only cycloheximide for 1 hout (3), in cells treated 6rst with cycloheximide for 1 hour and then with PMA for 6 hours (4) respectively. B. Ethidium bromide stained 28s rRNA is shown as a loading control. C. Quantitative analysis determined by densitometry of relative MMP-2 message levels in control cells (1) in the absence of either cycloheximide (10 pg/ml) or PMA (0.1 PM), in cells treated with PMA for 6 hours (2), in cells treated with only cycloheximide for 1 hour (3), in cells treated fhtwith cycloheximide for 1 hour and then with PMA for 6 hours (4) respectively. Similar observations were noted in duplicate experiments. Figure 18. A role for PKC mediated events in the regdation of MMP-2 gene expression by PMA in 1OT1/2 ceils. Northern blot analysis of MMP-2 message expression in 10T112 cells. A. MMP-2 mRNA levels in cells treated with PMA (0.1 CrM) for 6 hours (l),in control cells absent of PMA (2), in celis pretreated with PMA for 48 hours (3), in ceiis pretreated with PMA for 48 hours and then with fiesh PMA for an additionai 6 hours (4) respectively. B. Ethidium bromide stained 28s rRNA is shown as a loading control. C. Quantitative analysis determined by densitometry of relative MMP-2message levels in cells treated with PM.(0.1 PM)for 6 hours (l),in control cells absent of PMA (2), in cells pretreated with PMA for 48 hours (3), in cells pretreated with PMfor 48 hours and then with fiesh PMA for an additional 6 hours (4) respectively. Similar observations were noted in duplicate experhents. Figure 19. A role for PKC mediated events in the regulation of MMP-2 gene expression in NR3 cells. Northem blot analysis of MMP-2 expression in NR3 cells. Quantitative analysis, as determhed by densitometry, of relative MM'-2 message levels in cells in the absence of PMA (1) (control), in ceils in the presence of PMA (O.IpM) for 6 hours (2), in ceils pretreated with PMA for 48 hours (3), and in cells pretreated with PMA for 48 hours and then with fresh PMA for an additional 6 hours (4) respectively. Ethidium bromide staiwd 28s rRNA was used as a loading control. The histogram is representaîive of sMar observations in duplicate experiments. was extracted and analyzed by Northern blot analysis (as described in Materiais and Methods). Previously, the addition of PMA to 10T1/2 celis for 6 hom resulted in an increase in MMP-2 mRNA levels. Prolonged exposure to PMA (wbich resuited in PKC down regulation) prevented the PM.mediated induction of the MMP-2 message in 10T1/2 celis (Figure 18). This suggests a possible role for PKC mediated events in the PM4 mediated regulation of MMP-2 in 10T1/2 cells. For NR3 cells, the addition of PMA for 6 hours resulted in a decrease in MMP-2 message levels. When PKC was down reguIated in NR3 cells by treating them with PMA for 48 hours there was a decrease in the basal levels of MMP-2 mRNA (Figure 19). This suggested the possibility that the normal expression of MMP-2 requires PKC activity in NR3 cells. PMA added for an additionai 6 hours after the NR3 ceils were subjecced to PM.treatment for 48 hours, resulted in no Merdetectable changes in MMP-2 message levels (Figure 19). To confïrm the above results, the effect of calphostin C on MMP-2 expression in NR3 celis and in 10T1/2 ceils was evaluated. Calphostin C, is a specinc inhibitor of PKC. Calphostin C, at an effective concentration of OSpM (Kobayashi et ai., 1989) inhibits PKC activity 100%. To confirm that PKC has a role in reguladng basal levels of MMP-2 message expression in NR3 cells and not in 10T1/2 cells, both ce11 lines were grown to confiuence and then placed on defhed media for 48 hours. Cells were then left untreated, or treated with calphostin C, for 1 and 6 hours respectively. Foiiowing this, MMP-2 expression was evaluated by zymography and by Northern blot analysis (as described in Materials and Methods). As judged by ce11 morphology and ceii number, the dose of calphostin C was not toxic over the time interval studied. In NR3 cels down regulation of PKC foilowing treatment with calphostin C resulted in decreased MMP-2 message levels. This decrease occurred as early as 1 hour after calphostin C treatment. No effect on MMP-2 gelatinolytic activity was noted (Figures 20 and 21). This indicates PKC activity is essentid for normal basal levels of MMP-2 message expression in NR3 cells. When calphostin C was added to IOT1/2 cells no change in MMP-2 gelatinolytic activity was noted (Figure 22). 10T1/2MMP-2 message levels were unchanged in response to calphostin C treatment indicating fùnctional PKC is not essential for basal levels of MMP-2 message expression (Figure 23). I I O 6 12 PMA (Houn)

Figure 20. Inhibition of protein kinase C does not alter MMP-2 gelatinolytic activity in NR3 cells. Conditioned media was prepared as described in Materials and Methods, and 200 pl aiiquots were analyzed. A. Representative gelatin gel eiectrophoresis (zyrnography) of MMP-2 activity levels in NR3 ceils is shown indicating MMP-2 gelatinolytic activity in (1) control cells, and in MU cells treated with caiphostin C (0.5 PM) for 1 hour (2) and 6 hours (3) respectively. Equal Loading was determined by analysis of complementary gels, as described in Materials and Methods (data not shown). B. Quantitative analysis as determined by densitometry of relative MMP-2 gelatinolytic activity levels in NR3 cells as shown in A.. Similar observations were noted in duplicate experiments. Figure 21. A role for PKC mediated events in the regdation of basal levels of MMP-2 gene expression in NR3 cells. Northem blot analysis of MMP-2 message expression in NR3 cells. A. MMP-2 mRNA levels in control cells (1) in the absence of calphosth C (0.5 FM), in ceils treated with calphostin C for 1 hour (2), or 6 hours (3) respectively. B. Ethidium bromide stained 28s rRNA is shown as a loading contml. C. Quantitative analysis determineci by densitome- of relative MMP-2 message levels in control ceiis (1) in the absence of calphostin C (0.5 pM), in cells treated with calphostin C for 1 hour (2), or 6 hours (3) respectively. Similar observations were noted in duplicate experiments. LL -- r O 6 12 PMA (Houn)

Figure 22. Inhibition of protein kinase C does not alter MMP-2 gelatinolytic activity in 10T 1/2 cells. Conditioned media was prepared as described in Materiais and Methods, and 200 pl aliquots were aualyzed. A. Representative gelatin gel electrophoresis (zymography) of MMP-2 activity levels in 10T1/2 cells is shown indi~thgMMP-2 gelatinolytic activity in (1) controi cells, and in lOT1/2 cells treated with calphosth C (0.5 PM) for 1 hour (2) and 6 hours (3) respectively. Equai loading was detennined by analysis of complementary gels, as described in Materials and Methods (data not shown). B. Quantitative analysis as determined by densitometry of relative MMP-2 gelatinolytic activity levels in 10T1/2 cells as shown in A.. Similar observations were noted in duplicate experiments. Figure 23. No role for PKC mediated events in the regdation of basal levels of MMP-2 gene expression in I OT1/2-11s. Northem blot analysis of MMP-2 message in 10T1/2 cells. A. MMP-2 mRNA levels in control cells (1) in the absence of calphostin C (0.5 FM), and in cells treated with calphostin C for 6 hours (2) respectively. B. Ethidium bromide stained 28s rRNA is shown as a loading control. C. Quantitative analysis detemiined by densitometry of relative h4MP-2 message levels in control cells (1) in the absence of calphostin C (0.5 PM), and in cells treated with calphostùi C for 6 hours (2) respectively. Similar observations were noted in duplicate experiments. CHAPTER4 CELLULARPOLYAMLNE DEPLETION Cm DIFFERENTIALLYREGULATE MATRIX METALLOPROTEINASE-~ EXPRESSIONIN NONTUMORIGENICAND IN TUMORIGENKCELLS

1. Cellular Polyamine Depletion Induces MMP-2 Expression In lOTl/2 Celh And In NR3 CeUs And Altm PMA Meâiateà Regdation Of MMP-2 in CeU Lines Of Diffe~gTumorigenic And Metastatic Potential

The dmg a-difluoromethylomithine {DFMO) is an ineversible inhibitor of ODC (Pegg and McCann, 1988; Pegg, 1986). DFMO binds covalently to ODC, rendering ODC inactive (Pegg and McCann, 1988). This inhibition of ODC causes a depletion of polyamine pools. Putrescine and spermidine levels are depleted greater than 90%, whereas spermine levels are decreased by 50% in cells treated with DFMO (Heby et al., 1996; Wallon et al., 1994; HurÉa, unpublished results). To examine if DFMO may alter regulation by PMA of MMP-2 at the message and the activity level in celis of varying tumorigenic and metastatic potential the foiiowing experiment was performed. 10T1/2, NR3, NIH 3T3, and v-fes-transfected NIH 3T3 cells were grown to confluence and placed on defined datreated with DFMO (5mM) for 48 hours. This concentration of DFMO has ken established in the literatwe and in our laboratory to be effective in inhibiting ODC activity and depleting the polyamine pools (Wang et al., 1992; Patel et al., 1998; Schaefer and Seidenfeld, 1987; Hurta, unpublished results). Mer 48 hours in DFMO, cells were treated with PMA (O.1p.M) fot either 6 or 12 hours. Cells were then removed, and MMP-2 message and activity was analyzed by Northem blot analysis and zymography respectively (as described in Materials and Methods). As with cells treated only with PMA, when cells were pretreated with DFMO for 48 hours and then with PMA for 6 or 12 hours there was no change in the gelatinolytic activity of MMP-2 in any of the cell lines examined, namely NM 3T3 cells (Figure 24), v-fes-NIH 3T3 cells (Figure 25), 10T1/2celis (Figure 26) and NR3 cells (Figure 27) respectively. As weU, there was no change in MMP-2 gelatinoIytic activity of celis exposed to DFMO when compared to cells receiving no DFMO treatment. This suggests DFMO treatment has no effect on the gelatinolytic activity in these cell lines. Northem blot analysis of MMP-2 expression revealed pretreatment with DFMO altered the effects of PMA in NM 3T3 and v-fes-transfected NIH 3T3 celis. In the DFMO treated NïH 3T3 celis there was an increase in MMP-2 message expression &er PMA addition for 6 hours Figure 28). This induction was mainfained up to 12 hours of PMA treatment. In DFMO treated v-fes-transfected NiH 3T3 ceils (metastatic ceUs) the addition of PMA for 6 hours also tesuiteci in increased MMP-2 message levels (Figure 29). This induction was also maintained up to 12 hours of PMA treatment. Therefore, while DFMO does not alter the regulation of MMP-2 gelatinolytic activity by fMA, DFMO alters PMA regulation of the message in MH 3T3 cells (nontransformeci cells) and v-fes-transfected NIH 3T3 cells (metastatic celis). Pretreatment with DFMO also altered PMA regulation of MMP-2 message levels in 10T112 cells, but not in NR3 cells (Figures 30 and 3 1). When 10T1/2celis were pretreated with DFMO for 48 hours, and then with PMA for 6 hours, the induction of MMP-2 message was abrogated. When NR3 celis were pretreated with DFMO aud then treated with PMA ,there was dlapproximately a 2 fold decrease in MMP-2message ievels. There was also an uicrease in MMP-2 message seen in both cell lines when the basal levels were compared to cells treated only with DFMO (Figures 30 and 3 1). This could indicate that DFMO treatment alone can afkct the expression of MMf-2 message levels in the 10T 112 and in the NR3 cell lines.

II. Differential Response to DFMO Treatment In 10T1/2 And NR3 Ceb

The observation that DFMO affects the expression of MMP-2 message levels in lOTlR and in NR3 cells, prompted a Merinvestigation into how this occurs. When both 10T112 and NR3 cells were exposed to DFMO for 48 hours there was an increase in Figure 24. Pretreatment of NIH 3T3 cells with DFMO (5m.M) does not alter the basal level of MMP-2 gelatinolytic activity, nor does pretreatment with DFMO alter the effect of PMA on MMP-2 gelatinolytic activity in NM 313 cells. Conditioned media was prepared as described in Matends and Methods, and 200 pl alipuots were analyzed. A Representative gelatin gel electrophoresis (zymography) of MMP-2activity levels in NTH 3T3 cells is shown indicating MMP-2 gelatinolytic activity in (1) control cells, in NM 3T3 cells treated with PM.(0.1 pM) for 6 hours (2) and 12 hom (J), in NJH 3T3 cells pretreated with DFMO only for 48 hours (4), and in NTH 3T3 cells pretreated with DFMO for 48 hours and then treated with PMA for 6 ho= (5) and 12 hours (Q, respectively. Three zones of clearing were noted (approximately 62,68, and 72 kDa) comesponding to MMP-2 activity. As a loading contml, complementary gels were used as descnbed in Materials and Methods (data not shown). B. Quantitative anaiysis as detemiined by densitometry of relative MMP-2 gelatinolytic activity Levels as shown in A. Simila.observations were noted in duplicate experiments. Figure 25. Pretreatment of v-fes-NIH 3T3 cells with DFMO (5mM) does not alter the basai level of MMP-2 gelatinolytic activity, nor does pretreatment with DFMO alter the effect of PMA on MMP-2 gelatinolytic activity in v-fes-NIH 3T3 cells. Conditioned media was prepared as described in Materials and Methods, and 200 pl aliquots were analyzed. A. Representative gelatin gel electrophoresis (zyxnography) of MMP-2 activity leveis in v-fes-NM 3T3 cells is shown indicating MMP-2 gelatinolytic activity in (1) control cells, in v-fes-NM. 3T3 cells treated with PMA (O. 1 pM) for 6 hours (2) and 12 hours (3), in v-fes-NM 3T3 celis pretreated with DFMO only for 48 hours (4), and in v- fes-NIH 3T3 cells pretreated with DFMO for 48 hours and then treated with PMA for 6 hours (5) and 12 hours (6), respectively. Three zones of clearing were noted (approximately 62,68, and 72 kDa) correspondhg to MMP-2activity. As a loading control, complementary gels were used as described in Materials and Methods (data not shown). B. Quantitative analysis as determined by densitometry of relative MMP-2 gelatinolytic activity levels as shown in A. Similar observations were noted in duplicate experiments. Figure 26. Pretreatment of 10Tll2 cells with DFMO (5mM) does not alter the basal Ievel of MMP-2 gelatinolytic activity, nor does pretreatment with DFMO alter the effect of PMA on MMP-2 gelatinolytic activity in 10T1/2 cells. Conditioned media was prepared as described in Materiais and Methods, and 200 pl aliquots were analyzed. A. Representative gelatin gel electrophoresis (zymography) of MW-2 activity levels in 10T112 cells is shown indicating MMP-2 gelatinolytic activity in (1) control cells, in 10T 112 ceils treated with PMA (0.1 pM) for 6 hows (2) and 12 hours (3), in 10T1/2 ceUs pretreated with DFMO only for 48 hours (4), and in 10T1/2 ceils pretreated with DFMO for 48 hours and then treated with PMA for 6 hours (5) and 12 hours (6), te~pectively. Two zones of clearing were noted (approximately 68 and 72 kDa) corresponding to MMP-2 activity. As a loading control, complementary gels were used as described in Materials and Methods (data not shown). B. Quantitative analysis as determineci by densitometry of relative MMP-2 gelatinolytic activity levels as shown in A. Simüar observations were noted in duplicate experiments. . Figure 27. Pretreatment of NR3 cells with DFMO (5mM) does not alter the basal level of MMP-2 gelatinolytic activity, nor does pretreatment with DFMO alter the effect of PMA on MMP-2 gelatinolytic activity ui NR3 ceils. Conditioned media was prepared as described in Materials and Methods, and 200 pl aliquots were analyzed. A* Representative gelatin gel electrophoresis (zymography) of MMP-2 activity levels in NR3 cells is shown indicaring MMP-2 gelatinolytic activity in (1) control cells, in NR3 ceIIs treated with PMA (0.1 pM) for 6 hours (2) and 12 hours (3), in NR3 cells pretreated with DFMO only for 48 hours (4), and in NR3 cells pretreated with DFMO for 48 hours and then treated with PM.for 6 hous (5) and 12 hours (6), respectively. Two zones of clearing were noted (approximately 68 and 72 kDa) corresponding to MMP-2activity. As a loading control, complementary gels were used as described in Materials and Methods (data not shown). B. Quantitative analysis as determined by densitometry of relative MMP-2 gelatinolytic activity levels as shown in A. Similar observations were noted in duplicate experiments. Figure 28. DFMO alters PMA regdation of MMP-2 message in NIH 3T3 cells. PM& done, decreases MMP-2 mRNA levels in NIH 3T3 cells. However, DFMO and PMA together, increase MMP-2 message levels in NIH 3T3 cells. Northern blot analysis of MMP-2 mRNA in NM. 3T3 cells. A. MMP-2 mRNA levels in the absence of PMA and DFMO (1) (control), in the presence of PMA (0.1 FM)for 6 hours (2) and 12 hours (3), in the presence of DFMO (5mM) for 48 hours (4), and in the presence of DFMO for 48 hours with the addition of PMA for 6 hours (S) and 12 hours (6) respectively. B. Ethidium bromide staining of 28s rRNA is shown as a loading control. C. Quantitative analysis, as determined by deasitometry, of relative MMP-2 message levels in NIH 3T3 cells as shown in A.. Similar results were seen in duplicate experiments. Figure 29. DFMO alters PMA regulation of MMP-2 message in v-fes-NIH 3T3 cells. PMA, alone, does not aect MMP-2 mRNA levels in v-fis-MH 3T3 cells. However, DFMO and PMA together, increase MMP-2message levels in v-fes-NIH 3T3 cells. Northem blot anaiysis of MMP-2 mRNA in v-fes-NM 3T3 celis. A. MMP-2 mRNA levels in the absence of PMA and DFMO (1) (control), in the presence of PMA (0.1 PM) for 6 hom (2) and 12 how (3), in the presence of DFMO (5mM) for 48 hours (4), and in the presence of DFMO for 48 hours with the addition of PMA for 6 hours (3and 12 hours (6) respectively. B. Ethidium bromide staining of 28s rRNA is show as a loading control. C. Quantitative analysis, as determined by densitometry, of relative MMP-2 message levels in NIH 3T3 cells as show in A.. Similar results were seen in duplicate experiments. Figure 30. Interaction between DFMO and PMA in the regulation of MMP-2 message expression in 10T1/2cells. DFMO abrogates PMA induction of MMP-2 message levels in 10T1/2 ceils. Furthemore, alone, DFMO increases MMP-2 mRNA levels in 10T1/2 ceiis. Quantitative analysis, as determined by densitometry, of relative MMP-2 message levels in the absence of PMA and DFMO (1) (control), in the presence of PMA (O.lCrM) for 6 hours (2), in the presence of DFMO (5mM) for 48 hours (3), and in the presence of DFMO for 48 hours with the addition of PMA for 6 hours (4) respectively. Ethidium brornide stained 28s rRNA was used as a loadimg controi. The histogram is representative of similar observations noted in duplicate experïments. Figure 31. Interaction between DFMO and PMA in the regulation of MMP-2 message expression in NR3 cells. DFMO does not affect PWmediated decreases of MMP-2 message levels in NR3 cells. Furthemore, alone, DFMO increases MMP-2 mRNA levels in NR3 cells. Quantitative analysis, as determined by densitometry, of relative MMP-2 message levels in the absence of PMA and DFMO (1) (control), in the presence of PMA (0.1pM) for 6 hours (2), in the presence of DFMO (5mM) for 48 hours (3), and in the presence of DFMO for 48 hours with the addition of PMA for 6 hours (4) respectively. Ethidium brornide stained 28s rRNA was used as a loading control. The histogram is representative of similar observations noted in duplicate experiments. MMP-2 message levels. To determine if responsiveness to DFMO treatment was the same for both lines or distinct for both ceIl lines, the following time course was performed. Cells were grown to confiuence and placed on dehed media only for 24 hours. The cells were then treated with DFMO for 0,3,6,24, and 48 hours. Mer completion of the thecourse MMP-2 mRNA and activity was measured by Northern blot andysis and zymogram analysis, respectively (as descnbed in Materials and Methods). There was no change in the gelatinolytic activity when DFMO was added to 10T112 cells or NR3 celis up to 48 hours (Figures 32 and 33). However, merences in the response of 1OTln cells and NR3 ceils to DFMO mediated basesin MMP-2 message levels were depicted by Northern blot analysis (Figures 34 and 35). tu 10T1/2 cells the MMP-2 message was increased fier 24 hours of DFMO treatment and was maintained up to 48 hours. In NR3 cells, DFMO mediated increases in MMP-2 message as early as 6 hours, and was maintained up to 48 hours after DFMO treatment. These results show that even though MMP-2 message is increased in both the nodceus (1 OT1/2 cells) and transformed ceUs (NR3 ceik), their responsiveness to DFMO is di fferent.

III. The Transcriptional Process Plays A Role In DFMO Regdation Of MMP-2 Expression In lOTl/2 And In NR3 CeUs

The involvement of the transcriptional process was investigated to Mer determine how DFMO may regulate MMP-2 expression at the message level in 10T1/2 and NR3 cells. Both ce11 lines were grown to confluence and placed on defined media for 24 hours. The 10T112 cells were then left untreated or were treated with actinornycin D for 1 hour, DFMO for 24 hours, or actinomycin D for 1 hour and then DFMO for 24 hours. NR3 cells were left untreated or were treated either with actinomycin D for 1 hour, DFMO for 6 hours, or actinomycin D for 1 hour and then DFMO for 6 hom. Cells were treated in such a manner as to ailow for their removal fiom the plates to occur at the same the. Figure 32. DFMO does not alter MMP-2 gelatinolytic activity in 10T1/2cells. Conditioned media was prepared as described in Materials and Methods, and 200 pl aiiquots were anaiyzed. A. Representative gelatin gel electrophoresis (zymography) of MMP-2 activity levels in 10T1/2cells is shown indicating MMP-2 gelatinolytic activity in (1) control cells, and in 10T112 cells treated with DFMO (5mM) for 3 hours (2), 6 hours (3), 24 hours (4), and 48 hours (3respectively. As a loading control, complementary gels were wdas described in Materials and Methods (data not shown). B. Quantitative analysis as determined by densitometry of relative MMP-2 gelatinolytic activity levels in 10T 112 cells as shown in A.. Similar observations were noted in duplicate experiments. Figure 33. DFMO does not alter MMP-2 gelatinolytic activity in NR3 cells. Conditioned media was prepared as descnbed in Materials and Methods, and 200 pi aliquots were analyzed. A Representative gelatin gel electrophoresis (ymography) of MMP-2 activity levels in NR3 cells is shown indicating MMP-2 gelatinolytic activity in (1) control cells, and in NR3 cells treated with DFMO (5mM) for 3 hours (2), 6 hours (3), 24 hours (4), and 48 hours (5) respectively. As a loading control, complementary gels were used as described in Materials and Methods (data not shown). B. Quantitative dysisas determined by densitometry of relative MMP-2 gelatinolytic activity levels in NR3 celis as shown in A.. Similar observations were noted in duplicate experiments. Figure 34. DFMO treatment of 10T1/2 ceiis increases MMP-2 mRNA expression. Quantitative analysis, as detemllned by densitometry, of relative MMP-2 message levels in the absence of DFMO (1) (control cells), and in cells treated with DFMO (5mM)for 3 hours (2), 6 hours (3), 24 hours (4), and 48 hours (5) respectively. Ethidium bromide stained 28s rRNA was used as a loading controi. The histogram is representative of similar observations noted in duplicate experiments. Figure 35. Early induction of MMP-2 mRNA in NR3 ceiis in response to DFMO treatment. Quantitative analysis, as deter-ed by densitometry, of relative MMP-2 message levels in the absence of DFMO (1) (control cells), and in ceils treated with DFMO (5mM) for 3 hours (2), 6 hours (3), 24 hours (4), and 48 hours (5) respectively. Ethidium brornide stained 28s rRNA was used as a loading control. The histogmm is representative of similar observations noted in duplicate experiments. In 10T1/2 ceUs the addition of actinomycin D prior to the addition of DFMO for 24 hours prevented the induction of MMP-2 message indicatîng the transcriptional process plays a role in DFMO regulation of MW-2 (Figure 36). It should be noted 10T1/2 cells treated with actinomycin D for 1 hour and then DFMO for 24 hours were beginning to die, possibly due to inhibition of global gene expression. In NR3 celis the addition of actinomycin D pnor to the addition of DFMO resulted in blocking the induction of MMP-2 message (Figure 37). Actinomycin D for 1 hout did not alter the basai levels of expression in 10T1/2 and NR3 cens. For both ce11 hes this indicates the induction of MMP-2 by DFMO, at least in part, requUes the transcriptional process.

IV. Stability Of The MMP-2 Message 1s Affêcted By The Addition Of DFMO In NR3 Celh But Not In lOTlI2 Celis

Since increases in message can also occur by alterations in the decay rate of mature message, the stabiiity of MMP-2 was investigated in response to DFMO treatment. Cells were grown to confluence and then placed in defked media for 48 hours in the presence of DFMO. Actinomycin D was then added to the plates for 6 and 18 hours. Exposure to actinomycin D for 18 hours was non-toxic to both 10T1/2 and NR3 cells as judged by ce1 rnorphology and celi number. CeUs were removed and the RNA was extracted as described in Materiais and Methods. The haif life of MMP-2 message was calculated as described earlier on page 37. Treatment of 10T1/2 ceils with DFMO did not alter the half life of MMP-2 message Figure 38). Surprisingly, for NR3 celis the addition of DFMO increases the rate of decay of MMP-2 message (Figures 39). When NR3 cells were exposed to DFMO, the half life of MMP-2 message was estimated by extrapolation of a semilogarithmic plot to be 26 hours. In the absence of DFMO, the half life for the MMP-2 message in NR3 cells was estimated by extrapolation of a semilogarithmic plot to be 90 hours. Figure 36. The transcriptional process plays a role in the regulation of MMP-2 gene expression by DFMO in 10T112 cells. Northem blot anaiysis of MMP-2 message expression in 10T1/2cells. A. MMP-2 mRNA levels in control cells (1) in the absence of either actinomycin D (2.5 pglml) or DFMO (SmM), in cells treated with DFMO for 24 hours (2), in cells treated with only actinomycin D for 1 hou (3), in cells treated fht with actinomycin D for 1 hour and then with DFMO for 24 hours (4) respectively. B. Ethidium bromide stained 28s rRNA is shown as a loading control. C. Quantitative analysis determined by densitometry of relative MMP-2 message levels in control cells (1) in the absence of either actinomycin D (2.5 pg/ml) or DFMO (SmM), in celis treated with DFMO for 24 hours (2), in cells treated with only actinomycin D for 1 hour (3), in cells treated fmt with actinomycin D for 1 hour and then with DFMO for 24 hours (4) respectively. Sllnilar observations were noted in duplicate experiments. Figure 37. The transcriptional process plays a role in the regulation of MMP-2 gene expression by DFMO in NR3 cells. Northem blot analysis of MMP-2message expression in NR3 cells A. MMP-2 mRNA levels in control cells (1) in the absence of either actinomycin D (2.5 &ml) or DFMO (SmM), in celis mated with DFMO for 6 hours (2), in cells treated with only actinomycin D for 1 hour (3), in cells treated fint with actinomycin D for 1 hour and then with DFMO for 6 hours (4) respectively. B. Ethidium bromide stained 28s rRNA is shown as a loading control. C. Quantitative analysis detennined by densitometry of relative MMP-2 message levels in control cells (1) in the absence of either actinomycin D (2.5 or DFMO (SmM), in cells treated with DFMO for 6 hours (2), in cells treated with only actinomycin D for 1 hour (3), in cells treated fVst with actinomycin D for 1 hour and then with DFMO for 6 hours (4) respectively. Similar observations were noted in duplicate experiments. cantral

DFMO

Figure 38. DFMO does not affect the half life of MMP-2 message in 10T112 cells. 10T1/2cells in the absence of DFMO (control ceiis) ( O ), or exposed to DFMO (0.5 mM) for 48 hours (0) were subsequently treated with actinomycin D (2.5 pglml). Total cellular RNA was isolated at 6 and 18 hours &er treamient with actinomycin D, and subjected to Northem blot analysis as descnbed in Materials and Methods. The relative levels of MMP-2 mRNA were detennined by densitometry and normalized to GAPDH. On the y-axis, ( A ) designates the relative value at 50%. The resuits presented are fiom duplicate experiments. contml 1iOFMO

Figure 39. DFMO decreases the haif life of MMP-2 message in NR3 cells. NR3 cells in the absence of DFMO (control cells) ( O ), or exposed to DFMO (0.5 mM) for 48 hours ( O ) were subsequently treated with actinomycin D (2.5 pglml). Total cellular RNA was isolated at 6 and 18 hours fier treatment with actinomycin D, and subjected to Northem blot analysis as described in Materials and Methods. The relative levels of MMP-2 mRNA were detennined by densitometry and normalized to GAPDH. On the y-axis, ( A ) designates the relative value at 50%. The resuits presented are nom duplicate experiments. V. Effect Of de Now Protein Synthesis Inhibition On MMP-2 Induction By DFMO In lOTlrZ Cells And NR3 Ceüs

To determine whether the DFMO mediated elevations in MW-2 message required de novo protein synthesis, NR3 cells were t%st grown to confluence and then placed on defined media for 24 hours. Cells were then left either untreated, treated with DFMO for 6 hours, treated with cycloheximide for I hour, or treated with cycloheximide for 1 hour and then DFMO for 6 hours. Cycloheximide, an inhibitor of eukaryotic protein synthesis (Philips and Crowthers, 1986; Le et al., 1992), was used at a concentration (10 pglmi) established by previous work in the lab (Hurta and Wright, 1995). Figure 40. shows cycloheximide treatment enhanced DFMO mediated inmeases of MMP-2 mRNA in NR3 cells. This suggests de novo protein synthesis alters DFMO rnediated increases in MMP-2 message in NR3 cells. Cycloheximide alone had no affect on the basal levels of MMP-2 mRNA in NR3 cells. Since 10T112 cells do not exhibit an elevation in MMP-2 mRNA levels until after 24 hours of DFMO exposure, the above experimental design would require cells to be exposed to cycloheximide for 25 hours. However, when 10T112 cells were exposed to cycloheximide for this length of the it resulted in considerable ce11 death. In order to circumvent this problem cells were first treated with DFMO for 44 hours after reaching confluence and then treated with cycloheximide for 4 hours before removing the cells and extracthg the RNA. As controis, plates were either lefi untreated, treated with cycloheximide for 1 hou, treated with DFMO for 24 hours, or treated with DFMO for 48 hours. Previous results showed DFMO mediated elevations in MMP-2 mRNA is maintained between 24 and 48 hours of treatment. If blocking protein synthesis afker cells have ken treated with DFMO for 44 hours decreases the levels of MMP-2 mRNA seen at 48 hours when compared to cells exposed to DFMO for 24 hours we felt this would suggest elevations in MMP-2 mRNA by DFMO requires de novo protein synthesis. As shown in Figure 41., 10T1/2 ceUs exposed to DFMO for 44 hours and then treated with cycloheximide for 4 hours enhaaced the effect of DFMO mediated elevations in MMP-2 mRNA. This suggests de novo protein synthesis, as with NR3 cells, &ers DFMO mediated increases in MMP-2 message Levels in 10T112 cells. - Figure 40. De novo protein synthesis plays a role in the regdation of MMP-2 gene expression by DFMO in NR3 cells. Northern blot analysis of MMP-2message expression in NR3 cells. A. MMP-2 mRNA leveis in control cells (1) in the absence of either cycioheximide (10 pg/ml) or DFMO (SmM), in ceils treated with DFMO for 6 hours (2), in cells treated with only cycloheximide for 1 hour (3), in cells treated first with cycloheximide for 1 hour and then with DFMO for 6 hours (4) respectively. B. Ethidium bromide stained 28s rRNA is shown as a loading control. C. Quantitative analysis deterrnined by densitornetry of relative MMP-2 message levels in control cells (1) in the absence of either cycloheximide (10 pg/ml) or DFMO (SmM), in cells treated with DFMO for 6 hours (t),in cells treated with only cycloheximide for 1 hour (3)- in celis treated first with cycloheximide for 1 hour and then with DFMO for 6 hours (4) respectively. Similar observations were noted in duplicate experiments. Figure 41. De novo protein synthesis plays a role in the regdation of MMP-2 gene expression by DFMO in 10T1/2 cells. Northern blot dysisof MW-2 message in 10T1/2cells. A. MMP-2 mRNA levels in control celis (1) in the absence of either cycioheximide (10 pgM) or DFMO (SmM), in cells treated with DFMO for either 24 hours (2) or 48 hom (3), in cells treated with oniy cycloheximide for 1 hour (4), in celis treated first with DFMO for 44 hours and then cycloheximide for 4 hours (5) respectively. B. Ethidium bromide stained 28s rRNA is shown as a loading control. C. Quantitative analysis determined by densitomeüy of relative MMP-2 message levels in control cells (1) in the absence of either cycloheximide (10 pg/ml) or DFMO (SmM), in cells treated with DFMO for either 24 hows (2) or 48 hours (3), in cells treated with only cycloheximide for 1 hour (4), in cells treated first with DFMO for 44 hours and then cycloheximide for 4 hours (5) respectively. Similar observations were noted in duplkate experiments. 1. The Effect of Phorbol Myrisate Acetate(PMA) on MMP-2 GelatinoIytic Activity.

There are a nmber of biologicd ptoperties thaî ciiffer between nontransfonned, ''normal" cells and cells which possess a transformed phenotype (Egan et al., 1986; Denhardt et al., 1987). Some of these ciifferences have been elucidated in our laboratory; and indude alterations in the expression and the regulation of a number of genes associated with cellular growth, including altered ODC expression/ regulation and altered polyamine biosynthesis (Hurta et al-, 1993; Hurta and Wright, 1995) and alterations in MMPs expression and regulation (Samuel et al., 1992; Baruch and Hurta, 1996). These alterations in gene expression fregulation constitute a part of the aitered growth regulatory program inherent to transfonned ceiis. In this regard, the present study explored another aspect of this altered growth regulatory program. Specificdy, this study focused on the expression and the regulation of MMP-2. This study has demonstrated that matrix metalloproteinase-2 (MMP-2) is regulated differentially in the transformed ce11 compared to the nontransfonned celI. The matrix metalloproteinases (MMPs), including MMP-2, play important roles in the metastatic process, and are also thought to be involved in primary tumor formation (Chambers and Matrisian, 1997). The regulation of MMPs is very intricate and cornplex, and can occur at transcriptional, post- transcriptional, and translational levels of regulation. In order to examine possible altered regulatory rnechanisms affecthg MMP-2 expression in normal versus transformed cells, the phorbol ester tumor promoter, PMA, was used as a "probe" to dissect out potentially interesthg reçponses. MMP-2 gelatinolytic activity was assayed by polyacrylamide gelatin gel electrophoresis (zymography). Zymography offers one of the most sensitive techniques for detecting MMP-2 gelatinolytic activity (Quesada et al.,1 997). As well, ymography offers detection not only of the MMP activated form, but also the MMP proenzyme form (Zucker et al., 1994). Furthemiore, it has been demoastrated that gelatinolytic activity can correlate with protein levels (Kleiner and Stetler-Stevenson, 1994). This has led ti the suggestion that zymography is a usefbl technique to determine total enzyme activity, and hence total enzyme protein (Kieiner and Stetler-Stevenson, 1994). The effect of PMA on MMP-2 gelatinolytic activity was investigated in a number of ce11 lines. No change in MMP-2 gelatinolytic activity was noted in any of the ce11 Iines studied. No change in MMP-2 message expression was also observed in vfes-NIH 3T3 cells (metastatic cells) following PMA treatment. Interestingly, PMA treatment decreased MMP-2 message levels in the parental NIH 3T3 ceiis. PMA also affected MMP-2 message levels in 1OTlD cells and NR3 cells (NR3 cek express low levels of tas and form benign himors). PMA increased MMP-2 message levels in lOTln cells, and decreased MMP-2 message levels in NR3 cells. The effect of PMA on MMP-2 expression in NR3 cells is very simüar to that found in HT 1080 cells (a human fibrosarcoma ce11 Iine) (Huhtala et al., 199 1). HT 1080 cells, when treated with PM& exhibited decreased MMP-2 message levels but no change in MMP-2 geIatinolytic activity (Huhtala et al., 199 1). Interestingly, others have shown the addition of PMA actually induces activation of the proenzyme form to its activated fom in HT 1080 cells (Brown et al., 1990; Lim et al., 1996). This activation was mediated by the membrane type matrix metalloproteinsise (MT-MMP), a known activator of proMMP-2 (Lim,et al., 1996). PMA mediated the activation of proMMP-2 by increasing the amount of MT-MMP. One possible explmation for the differences in MMP-2 gelatinolytic activity observed by Huhtala et al. (1 991) versus those by Lim et al. (1996) and Brown et al. (1 990) is the length of time the cells were exposed to PMA. Huhtala et al. (1 99 1) ody exposed their cells to PMA for 12 hou before investigating MMP-2 gelatinolytic activity, while the othet two groups treated their cells for at least 16 hours with PMA. Consistent with tfiis idea, Lim et al., (1996) did not observe activation of proMMP-2 until 16 hours dertreatment with PMA. There was no activation of proMMP-2 observed for earlier time points. Importantly, Lim et al. (1996) did not observe any changes in total MMP-2 gelatinolytic activity (i.e. there appeared to be no decrease in the level of MMP-2 protein). Brown et ai. (1990) did notice a deccease in total MMP-2 activity, but ody when cells were exposed to PM.for 48 hours. These observations underscore the cornplexities associated with the regulation of MMP-2 expression in cells. Nevertheless, these findings raise the possibility of the existence of a lag thne between changes in MMP-2 message levels and co~espondingchanges in the gelatinolytic activity in response to PMA treatment. This is consistent with rny observation that changes in MMP-2 message levels did not result in any changes in the level of total MMP-2 gelatinolytic activity. The level of MMP-2 gelatinolytic activity was oniy observed up to 12 hours after PMA treatment, Whether or not alterations in the levei of MMP-2 gelatinolytic activity, in response to PM& murdue to prolonged exposure to PMA (greater than 12 hours) remains to be determined. Future studies codd be done to address this question.

II. The Effect of PMA on MMP-2 Message Levels

Despite the fact that MMP-2 does not contain any known PMA response eiement within its promoter region (Huhtala et al., 199 1; Corcoran et al., 1W6), a number of studies exist which clearly demonstrate that PMA cm alter the expression of MMP-2 message levels. The responses to PMA appear to be celi type dependent. For example, the addition of PMA has been shown to increase the message levels in GB-1 and U-373 MG ce11 lines @iman glioma ceIl lines) (Nakano et al., 1995). Alternativeiy, exposure to PMA decreased MMP-2 message levels in HT 1080 cells (human fibrosarcoma cells) and A2058 cells (Brown et al., 1990; Huhtala et al., 199 1). WI 38 cells (human embryonic lung fibroblasts) and HT 144 cells (human melanoma cells) are examples of two ceil lines which show no change in MMP-2 message levels in response to PMA (Brown et ai., 1990). Differential regulation of MMP-2 message levels, in response to PMA,was also observed in the ce11 lines investigated in this study. A small decrease in MMP-2 message levels was evident in NIH 3T3 cells in response to PMA, but relatively no change in v- fes-NIH 3T3 cells (the transformed cells, metastatic cells). A differential response to PMA was also noted in lOTlM cells and its ras transformed counterpart, NR3 cells. NR3 cells showed a decrease in MMP-2message levels in response to PMA, whiie 10T1/2 cells exhibited increased MMP-2 message levels in response to PMA. The response to PMA in 10T 1/2 cells (mouse fibroblasts) ciiffers fiom the response reporteci by Brown et al. (1990), which demonstrated that PMA did not change MMP-2 message expression in WI 38 cells (nontumorigenic, human embyonic lung fibroblasts). This ciifference may reflect distinct responses to PMA in fibroblasts denved hmdifferent species. in fact, variations even within celi types of the same species have shown a differential response in MMP-2 message levels to PMA (Brown et al., 1990; Nakano et al., 1995). Nakano et al. (1 995) investigated 5 different human glioma ceIl lines. GB-1 and U-373 MG showed an increase in MMP-2message, while the A-172, T-98G, and U-87 MG ce11 lines al1 showed a decrease in MMP-2message levels in response to Pm. Brown et al. (1990) demonstrated a decrease in MMP-2message levels in the A 2058 human meianoma ce11 line and no change in MMP-2 message levels in the HT 144 human melanoma ce11 line. The length of time cells were exposed to PMA rnay provide another explaaation to why 10T1 /2 ceils and W13 8 cells differentiaily respond to PMA. Brown et al. (1990) did not investigate MMP-2 message levels until24 hours &et PMA addition. On the other hand, MMP-2 message ievels were investigated as early as 6 hours &er PMA treatment in 10T1/2 cells. The induction of MMP-2 message by PMA in 10T1/2 ceils probably represents a transient response. This is supported by the fact that: (1) the increase in MMP-2 message after 12 hows of PMA treatment is not as hi& as that observed after 6 hours, and (2) after 48 hours of PMA treatment there is no increase in MMP-2 message levels in 1OT112 cells (Figure 18). For Figure 18., MMP-2 message levels expressed (at 48 hours post PMA exposure) represent baseline level of MMP-2 expression, in this regard, MMP-2 and other MMPs have shown transient induction of their messages in response to a number of stimuli. For example, MMP- 13 (collagenase- 3) has shown an increase in message levels in response to interleukin-1 at 4 and 8 hours, followed by a decline at 12 hours (Vincenti et al., 1998). MMP-2 message levels were induced 48 hours following exposure to interleukùi-6 (IL-6), and subsequently retumed to basal levels 5 days following IL-6 treatment (Kusano et al., 1998). PMA decreases MMP-2 message expression in NR3 cells. Decreased MMP-2 message levels in response to PM& has also been demonstrated in the HT 1080 cell, a human fibrosarcoma ce11 line (Brown et a', 1990; Huhtala et al., 1991). Brown et al. (1990) Merdemonstrated this decrease in MMP-2 message in HT 1080 celis was accompanied by a similar reduction in gelatinolytic activity 48 hours after PMA addition. Fridman et al. (1990) have also demonstrated a decrease in type- N collagenolytic activity in HT 1080 cells in response to PMA. The type-N collagenase assay is specificaliy designed to investigate MW-2 and MMP-9 activity. Unfortunately, it is unable to distinguish between the two MMPs. in HT 1080 cels, MMP-9 expression has been shown to be induced by the addition of PMA. These PMA mediated alterations in MMP-9 expression were regdated at the Ievel of transcription, coupled with increases in MMP-9 gelatinolytic activity (Corcoran et al., 1996; Huhtaia et d , 1991 ; Lim et al., 1996). Therefore, the reported decrease in typeN collagenolytic activity observed by Fridman et aL (1 990) was due primarily to decreases in MMP-2 synthesis. Furthermore, Fridman et al. (1 990) suggested the decreased type-N collagenolytic activity seen in HT 1080 cells in response to PMA was due to dom regdation of protein kinase C (PKC). High levels of diacylglycerol @AG) may down regulate PKC activity (Nishïzuka, 1984). Malignant HT 1O80 cells have an activated N- ras gene, accompanied by elevated levels of DAG production (compared to noamalignant fibroblast cells). These elevated DAG levels result in decreased PKC activity. This elevation in DAG production and subsequent lowering of PKC activity has been demonstrated in other ce11 lines transformed with the ras gene (Weyman et al., 1988). Prolonged exposure to PMA has been shown to down regulate PKC activity primarily due to accelerated rate of degradation of PKC (Young et al., 1987). Fridman et al. (1990) have suggested that it is this decrease in PKC activity mediated by both elevated levels of DAG and the addition of PMA that results in the decrease in collagenase-IV activity in HTl O80 cells. In fact, addition of PMA for 24 hours did result in the disappearance of PKC protein (Fridman et al., 1990). A significant decrease in PKC protein levels as early as 5 hours after exposure to PMA also occurred in HT 1O80 ceiis (Fridman et al., 1990). The above mode1 may be pertinent to and provide a possible explanation to account for the PMA mediated decreased MMP-2 message levels in NR3 cells. Transformation of the 10T1/2 ce11 line with H-ras (the same gene used to produce NR3 cells) has resulted in lower levels of PKC activity compared to parental 10T1/2 ceiis (Weyrnan et al., 1988). When hWcdls were exposed to PMA for 48 hours an almost complete disappearance of the MMP-2 message occurred. Furthemore, when calphostin C, a specific inhibitor of PKC, was added to NR3 ceils, a decrease in MMP-2 message levels was evident. These findiags suggest that PKC mediated events may play a role in regulating MMP-2 expression in these cells as well. Contrary to the result seen in NR3 cells, prolonged exposure to PM.did not Sectthe expression of basal MMP-2 message levels in 10T112 celis (Figure 18). The addition of calphostin C also did not alter MMP-2 message levels in lOTlR cells. These observations are consistent with the hdings of Fridrnan et aL (1990) which did not observe any decrease in collagenolytic activity in Wi 38 ceils (nontumorigenic fibroblast cell line) after prolonged exposure to PMA. This suggests PKC mediated events probably do not play a role in regulating MMP-2 basal level of expression in 10T1/2 cells. The apparent dependency which exists between the maintenance of basal levels of MMP-2 message expression, and fiuictional PKC mediated events may be a novel property associated with rus mediated cellular transformation. Further study is warranted to test this possibility. While there was no effect on basal MMP-2 message levels, down regulation of PKC @y prolonged exposure to PMA) did abrogate PMA mediated MMP-2 message level increases in 10T1/2 cells. This suggests PKC mediated events play a role in PMA regulation of MMP-2 message expression in 10T1/2 cells. Whether this is also true for the NR3 cell line remaüls to be elucidated. The effect of down regulating PKC on basal MMP-2 message levels in NR3 ceils makes it difficult to determine if PMA mediated decreases in MMP-2 message expression is dependent on PKC. Down regulating PKC by prolonged exposure to PMA, and domregulatbg PKC by prolonged exposure to PMA and then re-exposing the cells to PMA yielded comparable, very low, MMP-2 message levels. If PMA regulation of MMP-2 expression was not dependent on PKC mediated events then one would expect an additive or synergistic effect on MMP-2 message levels by down regulating PKC and the addition of PMA. This was difficult to ascertain in this study, since the decreases in MMP-2 message levels were so low for both instances. It is possible an addititive or synergistic effect was present, but uadetected, since the decrease in MMP-2 message levels by down regulation of PKC @y prolonged exposure to PMA) was so great that any eerdecrease by re-exposure to PMA would be small in cornparison. Future studies investigating the effect of PMA on PKC in NR3 cells, coupled with specific inhibitors of PKC (eg. calphosth C) rnay provide insight into whether PMA regulation of MMP-2 message in NR3 cells is distinct fiom that of PKC effects. A difference in the basal levels of MMP-2message expression was observed in confluent 10T112 cells and confluent NR3 cells. MMP-2 message expression was higher in the parental 10T112 ceil hecompared to the NR3 ceU line (H-ras transformed 10T112 celi line). These findings are consistent for the role that has been proposed for PKC. Basai level of MMP-2 message expression was found to be dependent on PKC activity in NR3 cells, but not in LOT112 cells. The expression of an oncogenic ras gene has ken shown to correlate with lower ievels of PKC expression (Weyman et al-, 1988; Fridman er al., 1990). It is possible, therefore, that the reason why MMP-2 message levels are lower in NR3 cells than in 10T1/2 ceUs may be due to lower levels of PKC and its effects on MMP-2 message expression. In support of this possibility, Brown et ai. (1990) observed lower basal levels of MMP-2 message in HT 1080 cells (fibrosarcoma ce11 line) compared to WI 38 cells (nontumorigenic fibroblast ce11 Lie). Previous work in our labotatory showed MMP-2 message expression and MMP-2 gelatinolytic activity correlated with H-ras expression, and that NR3 cells expressed higher levels of MMP-2 message and activity levels when compared with 10T112 cells (Baruch and Hurta, 1996). Baruch and Hurta (1996) investigated MMP-2 expression in logarithmically growing cells, while this present study investigated MMP-2 expression in cells grown to confluence. This suggests the growth state of the cells may also play a role in regulating MMP-2 message expression. Consistent with this idea, Wallon et al. (1 994) demonstrated that in SW 11 16 cells (human adenocarcinorna ceil line) MMP-7 message expression was higher in cells in the exponential phase cornpared to cells in the stationary phase. Further evaluation of MMP-2 message levels with respect to the growth state of the cells is required. Other difTerences exist between 10T1/2 cells and NR3 cells in PMA mediated regulation of MMP-2 expression. The decrease in MMP-2 message levels by PMA in NR.3 cells does not require de novo protein synthesis. This is in agreement with fïndings in HT 1080 ceUs which showed the addition of the translational inhibitor cycloheximide had no affect in blocking PMA mediated decreases in MMP-2 message levels (Huhtala et al., 1991). These fïndings also suggest the possibility that PMA mediated MMP-2 expression in NR3 cells is regulated in a similar manner to that found in HT 1080 cells. In contrast, the addition of cycloheximide blocked the PM.mediated increases of MMP- 2 message levels in 10T112 cells. This would indicate PMA regulation of the MMP-2 message does require de novo protein synthesis in 10T1/2 celk PMA mediated elevations in MMP-2 message levels in 10T112 celis occurred bot&ttanscriptionally and post-transcriptionally. This may suggest the synthesis of new proteins is requued to increase either the rate of transcription, or to stabilize the message, or both. Future studies are required in order to chri@ the role of "new proteins" in PMA mediated regulation of MMP-2 message in the 10T1/2 and the NR3 ce11 lines. Transcriptional and pst-transcriptional events were found to play a role in the PMA regulation of MMP-2 message in both ce11 lines. The effect of PMA on message stability differed in the 10T1/2 ce11 line fiom the NR3 ceii line. An increase in the half life of MMP-2 message occurred following PMA treatment in IOTU2 celis. This indicates increased levels of MMP-2 message following PMA treatment in 10T1/2 ceils was in part due to a mechanism of pst-transcriptional stabibtion. In NR3 cells, a reduction in the Wlifeof MMP-2 message occurred following exposure to PMA, indicating that PMA is capable of regulating MMP-2 mRNA levels through a mechanism of post-transcriptional desbbilization. The stability of MMP-2 message in 10T1/2 cells may be explained by the presence of an AUUUA sequence found in the 3' untranslated region (UTR)of MM.-2riRNA (Overall et al., 1991). This sequence has been -implicated in the stabilization of lymphokine and oncogene mRNAs after phorbol ester induction (Overall et al., 199 1). Why PMA does not stabilize MMP-2 mRNA in NR3 cells may result fiom changes caused by expression of H-ras. Further studies investigating the potentiai role of the MMP-2 3' UTRshould help elucidate the mechanism by which PMA affects MMP-2 message stability in 10T112 cells and in NR3 cells. LU. The Effect of a-Dïfluoromethylornithine (DFMO) on MMP-2 Gelatinoiytk Activity

There is increasing evidence in the literature indicating a role for aberrant polyamine metabolism in the role of carcinogenesis (Auvinen, 1997). Depleting the polyamine pools has also been shown to have an ad-metastatic effect on some ce11 types (Sunkara and Rosenberger, 1987). To establish ifthere was a link between MMP-2 expression and polyamine levels in our ce11 lines, depletion of the polyamine pools was achieved by exposing the celis to DL-aitiauoromethyIornithhe @FMO) an irreversible inhibitor of ODC for 48 hours. The concentration of DFMO (5mM) and the length of tirne we exposed our cells to DFMO has been well established in the literature and in ou. laboratory to signinicantiy deplete the levels of polyamines (Heby et al., 1996; Wallon et al., 1994; Hurta, personal communication and unpublished results). Putrescine and spennidine levels are depleted greater than 90%, whereas spermine levels are decreased by 50% in cells treated with DFMO for 2-3 days (Heby et al., 1996; Waiion et al., 1994). The depletion of the polyamine pools did not affect MMP-2 gelatinolytic activity in any of the ceil lines studied. Also, the depletion of the polyamine pools did not alter the effect of PMA on MMP-2 gelatinolytic activity up to 12 hours of treatment. As with PMA alone, DFMO induced changes in MMP-2message levels without accompanying changes in MMP-2 gelatinolytic activity. Again, there is the possibility of the existence of a lag tirne between changes in MMP-2 message levels and corresponding changes in MMP-2 gelatinolytic activity. Marti et al. (1994) demonstrated increases in MMP-2 gelatinolytic activity 2 days followiag TGF-B mediated increases in MMP-2 message levels. The effect of polyamine pool depletion on protein synthesis may provide another possible expianation why no corresponding increases in MMP-2 gelatinolytic activity were observed. Along with growth and differentiation, polyamines are thought to play an important role in protein synthesis. Recent studies have demonstrated a large proportion of polyamines is closely associated with roua ribosomes (Fujiwara et al., 1998). As well, decreasing the polyamine pools have been shown to interfere with the translational process. Specifically, polyamines are believed to interfere with the tRNA and mRNA interaction (Corella et al., 1998). Finally, depletion of polyamine pools has been demonstrated to cfecrease the half Me of some proteins (Corella et al., 1998).

IV. The Effkct Of DFMO On MMP-2 Message Levels

Increased levels of TGF-B message in IEC-6 cells (intestinal epithelial ce11 line) exposed to DFMO was demonstrated by Patel et ai- (1 998). This increase was mediated by stabilization of the TFG-f3 message. In the sarne ceIl line, DFMO increased the message levels of jun D (Patel and Wang, 1999). As well, MMP-7 message Levels were increased in SW 11 16 cells (human colon adenocarcinoma cell he) exposed to DFMO when compared to untreated SW 11 16 cells (Wallon et al., 1994). These studies iilustrate that DFMO, bg domregulating intracelMar polyamine pools, cmaffect the expression of many genes at the message level. It was demonstrated in this study that DFMO can affect MW-2 message expression, either by interacting with PMA,or on its own. The addition of DFMO clearly altered the effects of PMA on MMP-2 message levels in al1 the ce11 lines studied indicating a role for polyamine metabolism in the regulation of MMP-2 message by PMA. PMA alone, mediated a decrease in MMP-2 message expression for NIH 3T3 cells, but did not affect MMP-2 message expression in v-Ses-NIH 3T3 cells. Yet depIetion of polyamine pools folbwed by PM.treatment caused an increase in MMP-2 message in NIH 3T3 cells and v-fes-NIH 3T3 cells. This suggests that PMA induction of MMP-2 message is linked to attenuation of cellular polyamine levels in a positive marner in NIH 3T3 cells and v-fes-NIH 3T3 cells. To the best of our knowiedge, this interaction between polyamine pools and PM.regulation of MMP-2 message is a novel observation, and the mechanism involved in regulating MMP-2 message by both PMA and DFMO is not known. Perhaps by decreasing the polyamine levels potential MMP-2 transcriptional activators or stabilizers of MMP-2 message can then be induced by PMA. Future experiments investigating transcriptional and pst-traouriptional events, after MH 3T3 cells and v-fes-NIH 3T3 cells have been treated with both DFMO and PMA, may provide clues into the mechanism behind DFMO and PM.mediated increases in MMP-2 message. An interaction between PMA and DFMO in regulating MMP-2 message expression was also demonstrateci in 10T1/2 cells. DFMO prevented the same induction of MMP-2 message in 1OTln cells by PMA when compared to lOTl/2 ceiis treated &th PMA only. This suggests that in 10T1/2 cells, induction of MMP-2 message by PMA in some way interacts with cellular polyamine levels, and is probably a cellular polyamine dependent event. In NR3 cells, PMA mediated inhibition of MMP-2 appears to be independent of cellular polyamine levels. The decreases in MMP-2 message expression mediated by PMA in NR3 celis pretreated with DFMO were comparable to decreases observed in NR3 celis tteated with PMA dy. These results demonstrate, once again, differential regulation of MMP-2 expression in 10T1/2 cells, and in MU cells (tramsformed ce11 line). DFMO increased MMP-2 message levels, without the addition of PMA, in 10T 112 cells. PMA aione also increased MMP-2 message levels in 10T1/2 cells. However, the effect of DFMO and PMA on MMP-2 message was neither synergistic nor additive. The addition of DFMO and PM.increased MMP-2message levels in 10T1/2 cells was comparable to increases mediated by DFMO alone, and was substantially less than that observed by treatment of 10T1/2 cells only with PMA. Perhaps the depletion of polyamines by DFMO blocks the effect of PMA on MMP-2 message. This could account for why the increase in MMP-2 message leveis in 10T1/2 cells pretreated with DFMO and then with PMA resembled the increases observed in IOTA2 cells exposed to DFMO alone, and did not resembie increases observed in 10T112 cells exposed only to PMA. In NR3 cells, DFMO alone also increased MMP-2 message levels. However, since in the presence of DFMO, PMA still mediated a decrease in MMP-2 message levels in NR3 cells comparable to that observed in NR3 cells exposed only to PM& suggests the effects of DFMO, and that of PM& on MMP-2 are distinct. Furthennore, it also suggests PMA mediated events can counteract (suppress) the eEects of DFMO on MMP- 2 message. This proposai diEers fiom that made for lOT1/2 cells where it is proposed DFMO blocks the effect of PMA mediated events on MMP-2 message levels. Further studies investigating the interaction between PMA and DFMO,and the mechanisms by which this interaction regulates MMP-2message levels in 10T112 celis, and in NR3 cells is required. 10T1/2 and NR3 celis exposed to DFMO for 48 hours exhibited increased MMP- 2 message levels. Upon closer examination, DFMO mediated an increase in MMP-2 message levels as eady as 6 hours in NR3 cells. DFMO did not mediate an increase in MMP-2 message Ievels util24 hours after treatment in 10T112 cells, indicating a differential responsiveness to DFMO. Though many studies report depletion of polyamine pools only der2-3 days of exposure to DFMO (Patel et al., 1998; Frostesjo et al., 1997), alterations in the polyamine pools begins ahnost immediately following DFMO treatment. Putrescine levels have been shown to drop quickly following exposure to DFMO, and decreasing as much as 3 fold after exposure to DFMO for 6 hours (Ashida et al., 1992). Furthemore, a signincant decrease in putrescine and spermidine is observed as early as 24 hours after exposure to DFMO (Frostesjo et al., 1997). DFMO h=rs also been shown to affect other biologicai processes. Afker 24 hours of exposure to DFMO, regulation of protein synthesis by various growth factors has been altered (Blachowski et al., 1994). Also, phosphorylation of proteins has been altered as early as 3 hours afler the addition of DFMO, and JNK (c-Jun N-terminal kinase) activity as early as 5 hours after DFMO treatment (Oetken et al., 1992; Ray et al., 1999). Though polyamine levels are not completely depleted, even alte~gthe polyamine pools can induce rapid cellular responses. This is consistent with the observation that DFMO altered MMP-2 message expression in 10T112 cells after 24 hours of treatment, or as early as 6 hours, as is the case with NR3 cells. The effect of DFMO on MMP-2 message stability also differed between 10T112 cells and NR3 cells. For 10T1/2 cells, DFMO did not alter MMP-2 message stability. Interestingly, for NR3 cells DFMO decreased the half life of the MMP-2 message. This was not expected since DFMO increased MMP-2 message expression, and decreasing the half life would actually decrease MMP-2 message levels. This apparent paradox may reflect changes induced by transformation and underscores the complexity of responses in transformed cells and warrants Merstudy. The transcriptional process was also required in order for DFMO mediated changes in MMP-2message expression to occur in both 10T112 cells and NR3 cells. The addition of cycloheximide, an inhibitor of protein synthesis, prior to DFMO exposure for NR3 ceils or, during DFMO exposure for 10T112 cells, enhanced the effects of DFMO on MMP-2 message levels in both cell lines- Perhaps cyclohexllnide blocks the synthesis of "inhibitors" or, promotes the action of "activators" which can influence the effects of DFMO on MMP-2 message in the 10T112 and NR3 ce11 lhes. These "inhibitors" or "activators" may affect transcriptional andlor post-transcriptional events involved in DFMO mediated increases of MMP-2 message. Further study is warranted in this area. As stated before, the addition of DFMO bas been shown to inhibit the metastatic activity of some ceU types (Sunkara and Rosenberger, 1987). Therefore, it is of some surprise that decreasing the polyamine pools induced increased levels of MMP-2 mRNA expression. However, similar results with MMP-7 have been desctibed previously (Wallon et al., 1 994). Wallon et al. (1 994) demonstrated that DFMO treated SW 1 1 1 6 cells (human colon adenocarcinorna derived cell line) exhibited decreased expression of MMP-7 at the protein level, but an increase in MMP-7 message ievels. Furthexmore, this decrease in MMP-7 enzyme did not occur until7 days after DFMO treatment. It is quite possible then that even though in our ce11 lines MMP-2 message levels were increased, this induction may actually correspond to a decrease in protein levels in time. Evaiuation of MMP-2 gelatinolytic activity and MMP-2 protein levels fiom cells exposed to DFMO for longer penods of time would clariQ this possibility. It is important to note MMP-2 regdation has been previously shown to deviate fiom the expected. For example, TGF-P has ben shown to induce comective tissue formation. This is accomplished by increasing the synthesis of comective tissue matrix components and, at the same tirne, decreasing the rate of synthesis of proteases involved in matrix breakdown (Overall et al., 199 1). For example, MMP-9,MMP-1, and MW-3 have al1 been shown to have lower message levels in response to TGF-P (Corcoran et al., 19%). In contrast, TGF-P increases the synthesis of MMP-2 (Overall et al., 199 1; Corcoran et al., 1996). This response by MMP-2 appears to oppose the net formative effects of TGF-P on promoting comective tissue formation (Overail et al., 1991). Therefore, the MMP-2response to polyamine pool depletion may be unique among the proteases and it would be interesthg to investigate the response of other MMPs to polyamine pool depletion in these ce11 lines, such as MMP-7 and MMP-9. Recently, Ray et al. (1999) has shown depietion of the polyamine pools by

DFMO induces the celi cycle inhibitors p2 1WWC~PI , p27Kip1, and p53 in IEC-6cells. This is of interest because Bian and Sun (1997) have demonstrated that within the MMP-2 promoter there exists a p53 transcriptional activation site. By transcription assays they demonstrated this site binds p53 resulting in activation of promoter activity. Furthemore, the addition of etoposide (a common p53 inducer) induced p53 DNA binding and transactivation activities in a tirnedependent manner; and that induction of MMP-2 message levels followed the p53 activation pattern. These findings strongly suggest that the MMP-2 gene could be a p53 target gene and that its expression is subject to p53 regdation. It is of interest to speculate that the increase in MMP-2 message levels in these cells upon addition with DFMO is mediated by an increase in p53 expression.

V. Proposed Mode1

A schematic diagram depicting the interaction of DFMO and PMA in the regulation of MMP-2 message levels for 10T112 and NR3 cells is presented in Figure 42 and Figure 43, respectively. in 10T112 cells DFMO, via the MAPK pathway, induces p53 production (Ray et al. 1999). Increased levels of p53 then transcriptionally activates MMP-2 (Bian and Sun, 1997), thereby, increasing MMP-2 mRNA levels- DFMO, by inducing TGF-P (Patel et al. 1998), may also increase MMP-2 message levels (Overall et al. 199 1). When 10T112 celb are treated with PMA for 6 hours, MMP-2 message levels are increased. PMA regulation of MMP-2 requires PKC mediated events in lOT112 cells. Pretreatrnent of 10T112 celis with DFMO for 48 hours effectively blocks PMA mediated regulation of MMP-2 mRNA levels, Uidicating PMA regulation of MMP-2 message is dependent on polyamine levels. The addition of PMA does not affect DFMO regulation of MMP-2 message levels in 10T112 cells. Similar to the 10T112 ce11 line, DFMO regulates MMP-2 message levels in NR.3 cells via the MAPK pathway, by inducing p53 (Ray et al. 1999), which then transcnptionally activates MMP-2 (Bian and Sun, 1997). Since MAPK is dowflstream of ras, NR3 cells may have higher MAPK activity compared to 10T1/2 cells due to H-ras expression. This may explain why an increase in MMP-2 message level is detected in NR.3 cells after exposure to DFMO at 6 hours, whereas changes in MMP-2 message levels are not detected in 10T1/2 cells until24 hours &et treatrnent with DFMO. As with IOT112 cells, TGF-j3 may dso mediate an increase in MMP-2 message by DFMO (Patel et al. 1998; Overall et al. 199 1). PMA decreases MMP-2message levels. Further investigation is required to detemine whether or not PKC mediated events are hvolved in the regulation of MMP-2by PMA in NR3 cells. Regdation of MMP-2 message levels in NR3 cells by PMA is not dependent on the polyamine pools. However, the effect of DFMO on the MMP-2 message level is blocked with the addition of PMA. This contrasts the modei proposed for the t OT1R ceU he, and underscores the difference in DFMO and PMA regulation of MMP-2 in the nontransfomed ce11 (10T112 celi line) and the ûmsformed ce11 (NR3 ce11 line).

VI. Summary/Future Direction

The resdts of this study demonstrate differential MMP-2 expression and regulation in the normal cell versus the transformed cell. Basal levels of MMP-2 message differed in the parental ce11 lines compared to the transformed ce11 Lines (1 0T 1/2 versus NR3, NIH 3T3 versus v-fis-NIH 3T3). The addition of PM.did not affect MMP- 2 message expression in v-fes-MH 3T3 cells (NIH 3T3 v-fes transfomed celi line, malignant), but did mediate a decrease in MMP-2 message expression in NIH 3T3 celis (mouse fibroblasts). PMA mediated increases in MMP-2 message expression in 10T112 cells (mouse fibroblasts), and decreases in MMP-2 message expression in NR3 cells (1 0T1/2H-ras transfonned ce11 line, benign). However, no corresponding change in MMP-2 gelatinolytic activity was observed in any of the ce11 lines studied- The changes obsewed in MMP-2 message without changes in gelatinolytic activity illustrate the complexity associated with the regulation of MMP-2. Brown et al. (1990) noted a decrease in MMP-2 gelatinolytic activity 24 hours after PMA mediated decreases in MMP-2 message expression in HT 1080 cells. While Marti et al. (1994) demonstrated increases in MMP-2 gelatinolytic activity 2 days following TGF-f3 mediated increases in MMP-2 message levels in glomerular cells. DFMO

PMA / \ 6 Hours 48 Hours

DFMO PMA

PKC4

Figure 42. Schematic diagram of the proposed mode1 depicting PMA and DFMO signaling interaction in the regulation of MMP-2expression in lOTl/2 cells. A. DFMO activates MAPK, which then induces p53 expression resulting in transcriptional activation of MMP-2. DFMO also increases TGF-P, which may increase MMP-2 expression in concert with p53 or independently of p53. B. PMA through PKC increases MMP-2 expression. Down regdation of PKC does not aect basal levels of MMP-2 expression. C. The effect of PMA on MMP-2 expression in lOTll2 cells is blacked when cells are pretreated with DFMO for 48 hours indicating PM.regulation of MMP-2 is dependent on the polyamine levels. The effect of DFMO on MMP-2regulation is not afYected by the addition of PMA. DFMO

PMA Ar' Ar' h 6 Hours 48 Hours 4 & PKC (?) & PKC

DFMO PMA

Figure 43. Schematic diagram depicting the proposed mode1 of PMA and DFMO signaling interaction in the regulation of MMP-2 expression in NR3 cells. A. DFMO activates WK, which then induces p53 expression resulting in transcriptional activation of MMP-2. MAPK levels are higher in NR3 cells than 10T112 cells. This rnay explain why increases in MMP-2 mRNA are detected earlier in MU cells versus 1OTlR cells. DFMO also induces TGF-P, which may inctease MMP-2 expression in concert with p53 or independently of p53. B. PMA decreases MW-2 expression. PKC mediated evenrs may or may not be involved in the regulation of MMP-2 message levels by PMA. Down regulation of PKC decreases basal levels of MMP-2 message expression. C. PMA blocks the effect of DFMO on MMP-2 expression. Regdation of MMP-2 expression by PM.is not dependent on the polyamine levels. These observations raise the possibility of the existence of a lag time between changes in MMP-2 message levels and correspondhg changes in the gelatinolytic activity. Since our experiments only investigated gelatinolytic activity up to 12 hours following PMA treatment, friture experiments investigating MMP-2 gelatinolytic activity at later tirne points would clarify if the changes observed at the message level also translated to changes in gelatinolytic activity in our ceIl iines. Furiher investigation revealed the mechanism by which PMA mediated the changes in MM.-2 message expression also differed between 10T112 cells (nontransformed celIs) and NR3 ceils (transfomeci celis). Furthemore, other studies have demonstrated that regulation of MMP-2 expression by PMA in HT 1080 cells is very similar to NR3 cells (Fridman et al., 1990; Brown et al., 1990; Huhtala et al-, 1991). HT 1080 cells express an activated N-ras gene, while NR3 cells contain an activated H- ras gene (Fridman et al,, 1990; Egan er al., 1986). It is interesthg to speculate that these alterations in the regulation of MMP-2 expression are a common phenornenon of ras transformation. Evaluation of the mechanisms by which PMA regulates MMP-2 expression in other H-ras transfomed 10T112 ce11 lines (such as NRQ, C 1, C2, and C3 which express increasing malignant potential) would provide insight as to whether a cornmon set of changes in MMP-2 regulation associated with ras transformation does exist. The maintenance of basal levels of MMP-2 message expression, and functional PKC mediated events appeared to be linked in NR3 cells. This dependency was not apparent in LOT 112 cells. These fmdings coupled with similar observations for HT 1O80 cells (Fridman et al., 1990) suggest this relationship between MMP-2 expression and functional PKC mediated events may be a novel property associated with ras mediated cellular transformation. Studies involving other celi lines transformed with H-ras, and celi lines transfomed with other oncogenes may help elucidate if this is in fact a novel property of ras mediated cellular transformation. This study demonstrated for the first time a direct link between MMP-2 message expression and DFMO treatment (alterations in cellular polyamine ïevels). DFMO dtered PMA regulation of MMP-2 message expression in 10T112 cells, NIH 3T3 cells, and v-fes-MH 3T3 cells, and also increased basal levels of MMP-2 message expression in !OT I/2 and NR3 cells. Previously, it has been demonstrated that decreasing the polyamine levels has an anti-metastatic effect (Sunkara and Rosenberger, 1987). However, studies linking depletion of polyamine pools with increased p53 expression (Ray et al., 1999), coupled with the recent discovery of a hction for p53 in the regulation of MMP-2 expression (Bian and Sun, 1997) provides a possible explanation for our results. It is possible the increases in MMP-2expression in response to DFMO are mediated by p53. Further experiments involviog antibodies agabst p53, ceU hes which contain no endogenous p53 gene (for example, Saos-2 cells, a human osteogenic sarcoma ceil line), and inducers of p53 (for example etopiside) would help dari@ the role of p53 in DFMO mediated increases of MMP-2 message in these cells. It is important to note that MMP-2 is just one member of the MMP family. Other MMPs have been implicated to play key roles in the metastatic proçess. Though many MMPs are regulated in a similar manner by growth factors, cytokines, and tumor promoters, MMP-2 often responds differently to these agents (Corcoran et al., 1996). Therefore, it would be interesthg to investigate if the other MMPs also exbibit the same type of response to PMA and polyamine depletion as MMP-;?, or if MMP-2 is unique in its response. In particda., investigation of MMP-7, MMP-9, and the MT-MMPs is warranted. The focus of friture investigations may be directed towards MMP-7 because of the previous links demonstrated between MMP-7 expression and DFMO treatment in SW 1 1 16 cells (Wallon et al., 1994). MMP-9 is of interest since preliminary results in our laboratory have aIso demonstrated links between MMP-9 expression and altered cellular polyamine levels. Furthemore, MMP-9 was regulated differentially fkom MMP- 2 in response to DFMO, and the regulation of MMP-9 by DFMO also ciiffered in the 10T 1/2 and NR3 ce11 lines (unpublished observations). MT-MMP expression, dong with the expression of tissue specinc inhibitor of metalloproteuiases (TIMPs) (especialIy TIMP-2), in response to DFMO, or PMA,should also be investigated in the IOTlQ and M3 ceil lines. MT-MMP is a specific activator of pro-MMP-2. Whereas, TIMP-2 binds specifidly to pro-MMP-2, thereby, inhibithg its activity. It would be of interest to determine if the changes mediated by PM& or DFMO in MMP-2 message levels result in correspmding changes in TIMP-2 expression andior MT-MMP expression. Though PMA is a usefiil tool in exploring the ciifferences in regulation of MMP expression in nontransformed versus the transformed cell, it is a synthetic agent not normally found in the ECM. It wouId be interesthg to investigate the affect of growth factors such as PDGF, or cytokines such as TNF-a on MMP expression. These growth factors and cytokines are found in the ECM and producd by cells surroundhg the ECM. Investigation of the growth factors and cytokines would not only provide insight into the diEerences associated with the regulation of MMPs in the nontransformed versus transformed cells, but could also serve as a mode1 for what may be occurring in vivo. To this end, studies investigating the effect of PDGF,and TNF-a on MMP expression in cell lines of varying tumorigenic and metastatic potential have begun in our laboratory. Results to date fkom these studies support the observations of this thesis. PDGF and TNF-a differentially regdate MMP-2 and MMP-9 in nontransformed versus transformed cells. Studies investigating the effect of PDGF and TNF-a, as well as a number of other growth factors and cytokines on MMP expression, in cell lines of varying tumorigenic and metastatic potential, is on going. In summary, this study underscores the intricate and complex nature of MMP-2 regulation in general. Furthemore, it was demonstrated that the expression and regulation of MMP-2 by phorbol ester tumor promoters @MA) is dtered in the transformed ceil versus the normal (nontransformed) cell. PKC regulation of basal levels of MMP-2 message ody in the ras-transformed ce11 Iine (NR3 ce11 line) suggests that this may represent a novel characteristic of ras transformed cells. This study was also the first to demonstrate a link between MMP-2 message expression and DFMO treatment, and suggests that cellular polyamine levels have a role in regulating MMP-2 expression. In conclusion, this investigation has contributed to and enhanced out understanding of the growth regulatory programs in nontransformed ceils and in transformed cells. This study has also elucidated novel aspects of these regulatory programs, and has Merelaborated many possible areas of investigation, which could fom the basis of future investigations. Aho S, Rouda S, Kennedy SH, Qin H, and Tan EML (1997). Regdation of human interstitial collagenase (ma& metailoproteinase-1) promoter activity by fibmblast growth factor. European Journal of Biochemishy. 247,503-5 10.

Angel P., Imagawa M, Chiu R, Stein B, Imbra RI, Rahmsdorf HJ, Jonat C, Herrfich P, and Karin M (1 987). Phorbal ester-inducible genes contain a comon cis elernent recognized by a TPA-modulated trans-acting factor. Cell. 49,729-739.

Ashida Y, Kido J, Kinoshita F, Nishino M, Shinkai K, Akedo H, and houe H (1992). Putcescine-dependent invasive capacity of rat ascites hepatoma ceHs. Cancer Resecuch. 52, 53 13-53 16.

Auvinen M (1997). Ce11 transformation, invasion, and angiogenesis: a regdatory role for ornithine decarboxylase and polyamines? Journal of the National Cancer Inrtitute. 89 (8): 533-537.

Auvinen M, Paasinen A, Andersson L, and Holtta E (1 992). Ornithine decarboxylase is critical for ce11 transformation. Nature. 360,355-358.

Auvinen M,Laine A, Pasinen-Sohm A, Kangas A, Kangas L, Saksela O, Andersson LC, and Holtta E (1 997). Hunan ornithine decarboxylase-overpoducing NIH 3T3 cells induce rapidly growing, highly vascularked tumors in nude mice. Cancer Researck 57, 3016-3025.

Baruch RR, and Hurta RAR (1996). Matrix metalloproteinase expression in transformed cells. (Abstract) American Society of Ce12 Biology, San Francisco, CA. Bernhard EJ, Gruber SB, and Muschel RJ (1994). Direct evidence linking expression of matrk metalloproteinase 9 (92-kDa gelatinase/coflagenase) to the metastatic phenotype in transformed rat embryo cells. Proceedings of the National Academy of Science OSA. 91,4293-4297.

Blachowski S, Motyl T, Grzelkowska K, Kasterka M, Orzechowski A, and Interewicz B (1 993). invohement of polyamines in epidermal growth factor (EGF),transforming growth factor (TGF)-a and -pl action on culture of L6 and fetal bovine myoblasts. International Journal of Biochemistry. 26 (7),89 1-897-

Brown PD, Levy AT, Margulies IMK, Liotta LA, and Stetler-Stevenson WG (1990). Independent expression and cellular processing of M, 72,000 type iV collagenase and interstitial collagenase in human tumorigenic ce11 Iines. Cancer Reseurch. 50,6184- 6191,

Cao J, Sato H, Takino T, and Seiki M (1995). The C-terminal region of membrane type matrix metalloproteinase is a fiuictional transmembrane domain required for pro- gelatinase A activation. Journal of Biological Chernistry. 270 (2), 80 1-806.

Chambers AF, and Matrisian LM (1997). Changing views of the role of matrix metalloproteinases in metastasis. Journal of the National Cancer Imtiture. 89 (17), 1260-1270.

-ChambersAF, and Tuck AB (1 993). Ras-responsive genes and tumor metastasis. Crifical Reviews in Oncogenesis. 4 (2), 95-1 14.

Chomczynski P, and Sacchi N (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry. 162, 156- 159. Corcoran ML, Hewitt RE, Kleiner DE, and Stetier-Stevenson WG (1996). MMP-2: expression, activation and inhibition. Enzyme Protein. 49,7019.

Corella D, Guillen M, Hemandez J, and Hemandez-Yago J (1 998). Effixts of polyamine levels on the degradation of short-lived and long-lived proteins in cultured L-132 human Iuag ceils. Biochemistry Jownal. 334,367-375.

Crawford HC, and Matrisian LM (1996). Mechanisms controlling the transcription of matrk metalloproteinase genes in normal and neoplastic cells. . Enzyme Protein. 49,20- 37.

DeClerck YA, Yean TD, Chan D, Shimada H, and Langley KE (1991). Inhibition of tumor invasion of smooth muscte celi layers by recombinant human metalloproteinase inhibitor. Cancer Research. 51,2 15 1-2 157.

DeClerck YA, Perez N, Shimada H, Boone TC, Langley KE, and Taylor SM (1 992). Inhibition of invasion and metastasis in celk transfected with an inhibitor of metalloproteinases. Cancer Research. 52,70 1-708,

Denhardt DT, Greenberg AH, Egan SE, Hamilton RT, and Wright JA (1987). Cysteine proteinase cathepsin L expression closely correlates with the metastatic potential of H- ras-transfonned murine fibrobiasts. Oncogene, 2-55-59.

Denhardt DT, Feng B, Edwards DR, Cocuzzi ET, and Malyankar UM (1993). Tissue inhibitor of metalloproteinases (TIMP, aka EPA): structure, contml of expression, and biological functions. Phurmacology Therapeutics. 59,329-34 1.

Duffy M (1996). The biochemistry of metastasis. Advonces in Clinical Chemis~y.32, 135-165. Egan SE, McClarty GA, Jarolim L, Wright IA, Spiro 1, Hager G, and Greenberg AH (1 986). Expression of H-ras correlates with metastatic potentid: evidence for direct regdation of the metastatic phenotype in 10T1/2 and NIH 3T3 cells. MolecuZar and CeIIuIar BioIogy. 7 (2), 830-83 7.

Egan SE, Wright JA, Jarolim L, Yanagihara K, Bassin RH, and Greenberg AH (1987). Transformation by oncogenes encoding protein kinases induces the metastatic phenotype. Science. 238,202-205.

Foda HD, George S, Corner C, Drews M, Tompkins D, and Zucker S (1996). Activation of human urnbilical vein endothelid ce11 progelatinase A by phorbal myristate acetate: A protein kinase C-dependent mec hanism involving a membrane-type matrix metalloproteinase. Laboratory Investigation. 74 (t),538-545.

Fredlund JO, Johansson MC, Dahlberg E, and Oredsson SM (1 995). Ornithine decarboxylase and S-adenosyimethionine decarboxylase expression during the cell cycle of chinese hamster ovary cells. Werimental Ce11 Research. 2 16,86-92.

Fridman R, Lacal J, Reich R, Bod3 Dyand Ahn C (1990). Differential effects of phorbof ester on the in vitro invasiveness of malignant and non-malignant human fibroblast cells. Journal of Cellular Physiology. 142,55-60.

Friciman R, Bird RE, Hoyhtya M, Oelkuet M, Komarek D, Liang CM, Berman ML, Liotta LA, Stetler-Steevenson WG, and Fuerst TR (1993). Expression of human recombinant 72 kDa gelatinase and tissue inhibitor of metalloprotei~se-2(TTMP-2): characterization of complex and fiee enzyme. Biochemical Journal. 289 (Pt 2), 41 1- 416.

Fridman R, Toth M, Pena D, and Mobashery S (1 995). Activation of progelatinase B (MMP-9) by gelatinase A (MMP-2). Cancer Research. 55,2548-2555. Frostesjo L, Holm 1, Grahn B, Page AW, Bestor TH, and Heby O (1997). Interference with DNA methyftransfrase activity and genome methylation during F9 teratocarcinorna stem ce11 differentiation induced by polyamine depletion. The Journal of BioIogicd - Chernistry. 272 (1),43594366.

Fujiwara K, Bai G, Kitagawa T, and Tsuru D (1998). Immuooelectron microscopic study for polyamines. Journal of Histohemisrry and Cytochemishy. 46 (Il), 132 1- 1328.

Giarnbemardi TA, Grant GM, TayIor GP,Hay RJ, Maher VM, McCormick JJ, and Klebe EU ( 1998). Overview of matrix metailoproteinase expression in cultured human cells. Muîrir BioIogy- 16,483-496.

Goldberg GI, Strongin A, Collier IE, Genrich LT, and Marmer BL (1992). Interaction of 92-kDa Type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromolysin. Journal of Biological Chernis~.267,4583 -459 1.

He C, Wilhelrn SM, Pentland AP, Marmer BL, Grant GA, Eisen AZ, and Goldberg GI (1 989). Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. Proceedings of the National Academy of Science USA. 89,2632-2636.

Hua J, and Muschel RJ (1996). Inhibition of rnatrix metalloproteinase 9 expression by a rîbozyme blocks metastasis in a rat sarcoma mode1 system. Cancer Research. 56,5279- 5284.

Huhtala P, Tuuttila A, Chow LT, Lohi J, Keski-Oja 3, and Tryggvason K (1 99 1). Complete Structure of the human gene for 92-kDa type-iV colagenase. Journal of Biological Chemistry. 16485- 16490. Huta RAR,Greenberg AN,and Wright JA (1993). Transforming pwthfactor Pi selectively regulates ornithine decarboxylase gene expression in malignant H-rus transformed fibrosarcorna ce11 Lines. Jownal of Cellular Physiology. 156,272-279.

Hurta RAR and Wright JA (1995). Malignant transformation by K-rm results in aberrant regdation of ribonucleotide reductase gene expression by transforming growth factor+ [. Journal of Cellular Biochemishy. 57,543-556. hai K, Yokohama Y, Nakanishi 1, Ohuchi E, Fujii Y, Nakai N, and Okada Y (1995). Matrix metalloproteinase 7 (matrilysin) fiom human rectal carcinoma cells. Journal of BioIogical Chemisny. 270 (12), 669 1-6697.

Janne J, Alhonen L, and Leinonen P (199 1). Polyamines: hmmolecdar biology to clinical applications. Annals of Medicine. 23,24 1-25 1.

Kerr LD, Miller DB, and Matrisian LM (1 990). TGF-B inhibition of transin/stomeIysin gene expression is mediated through a fos binding sequence. Cell. 61,267-278.

Kleiner DE, Unsworth EJ, htzsch HC, and Stetler-Stevenson WG (1992). Higher order complex formation between the 72-Kilodalton Type N collagenase and tissue metalIoproteuiase-2. Biochenzisfry. 31: 1665-1672.

Kleiner DE, and Stetler-S tevenson WG ( 1994). Quantitative zymography: detection of picogram quantities of gelatinases. Analytical Biochemistry. 2 18 (2), 325-329.

Kobayashi E, Nakano H, Monmoto MTand Tamaoki T (1989). Calphostin C (UCN- 1028C), a novel microbial compound, is a highly potent and specinc inhibitor of protein kinase C. Biochenticul und Biophyscal Research Communicatiom. 159 (f), 548-553. Kubota S, Kiyosawa H, Nomura Y, Yamada T, and Seyama Y (1997). Ornithine decarboxylase overexopression in mouse 10T1/2 fibroblasts: cellular transformation and invasion. Journal of me National Cancer Imtitute. 89 (81,567-571.

Kusano K, Miyaura C, Inada M, Tarnwa T, Ito A, Nagase H, Kamoi K, and Su& A (1 998). Regdation of matrix metaNoproteinases (MMP-2,-3 ,-9,and -1 3) b y interieukin- 1 and interleukin-6 in mouse calvaria: association of MMP induction with bone resorption. EndocrinoZogy. 139 (3), 1338-1 345.

Le A, Ferrell GA, Dishon DS, Le QQ-A, Sifers RN (1 992). Soluble aggregates of the human PiZd-antitrypsin variant are degraded within the endoplasrnic reticulum by a mechanism sensitive to inhibitors of protein synthesis. Joudof Biological Chemistry. 267,1072-1OSO.

Levy AT, Cioce V, Sobel ME, Garbisa S, Grigioni WF, Liotta LA, and Stetler-Stevenson WG (199 1). Increased expression of the Mr 72,000 type IV collagenase in human colonic adenocarcinorna Cancer Research. 51,43944.

Lim Y, Sugiura Y, Laug WE, Su.B, Garcia A, and DeClerck YA (1996). Independent regdation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells. Journul of Cellular PhysioZogy. 167,333-340.

Linsaiata M, Russo F, Cavallini A, Berloco P, and DiLeo A (1993). Polyamines, diamine oxidase, and ornithine decarboxyiase activity in colorectal cancer and in normal surrounding rnucosa. Diseases of the Colon and Rechim. 36,662-667.

Manni A, Badger B, Grove R, Kunselman S, and Demers L (1995). Isolation and charactenzation of human breast cancer cells overexpressing S-adenosylmethionine decarboxy lase. Cancer Letters. 95,23-28. Manni A, Mauger D, Gimotty P, and Badger B (1996). Propostic influence on survival of Uicreased ornithine decarboxylase activity in human breast cancer. Chical Cancer Research- 2,19014904.

Marti H, Lee L, Kashgarian M, and Lovett D (1994). Transforming growth factor+ 1 stimulates glomular mesaagial ce11 synthesis of the 72-kd type-IV collagenase. American Journal of Parhology. 144 (l), 82-94.

Matrisian LM (1992). The matrix degrading metalIoproteinases. Bioessays. 14,455- 463.

Mazzieri R, Masiero L, Zanetta L, Monea S, Onisto M, Garbisa S, and Mignatti P (1997). Control of type IV collagenase activity by components of the urokinase-plasmin system: a regdatory mechanism with cefi-bound reactants. The EMûO Journal. 16 (9), 23 1 9- 2332.

Nakano A, Tani E, Miyazaki K, Yamamoto Y, and Furuyama J (1995). Maûix metalloproteinases and tissue inhibiton of metalloproteinases in human gliomas. Journal of Neurosurgery. 83,298-307.

Nishizuka Y (1986). Studies and perspectives of protein kinase C. Science. 233,305- 3 10.

Oetken C, Pessa-Morikawa T, Autero My Andersson LC, and Mustelin T (1992). Reduced tyrosine phosphorylation in polyamine-starved cells. Erperimenfd Cell Research. 202,370-375.

Okada Y, Gonoji Y, Naka K, Tomita K, Nakanishi 1, Iwata K, Yarnashita K, and Hayakawa T (1992). Matrix metalloproteinase 9 (92-kDa gelathaseltype IV coliagenase) fiom HT 1080 human fibrosarcoma cells. Journal of Biologieal Chemisfry. 267 (30), 21712-21719. Overall CM,Wrana J, and Sodek J (1989). Transforming growth factor-p regulation of collagenase, 72-kDa-progelatinase, TIMP, and PM-1 expression in rat bone ce11 populations and human fibroblasts. Connecrive Tissue Research. 20,289-294.

Overall CM, Wrana JL, and Sodek J (1991). Transcriptional and pst-transcriptional regulation of 72-kDa gelatinasdtype IV collagenase by traasforming growth factor-f3 1 in human fibroblasts. Jownal of BioIogz*culChemisby. 266 (21), 14064-1 407 1.

Patel AR, Li J, Bass BL, and Wang JY (1 998). Expression of the transforming growth factor-beta gene during growth inhibition following polyamine depletion. American Journal of Physiology. 275 (2 Ptl), CS90-C598.

Patei AR, and Wang JY (1999). Polyamine depletion is associated with an increase in JunD/AP- 1 activity on small intestinal crypt cells. American Journal of Physiology. 276, G44 1-G4SO.

Pegg AE (1986). Recent advances in the biochemistry of polyamines in eukaryotes. Biochemical Journal. 234,249-262.

Pegg AE, and McCann PP (1988). Polyamine metabolism and function in mammalian cells. ISI Atlas of Science: Biochemisw. 11-1 8.

.Pegg AE, Kamej i T, Shirahata A, Stanley B, Madhubala R, and Pajunen A (1 988). Regulation of mammalian S-adenosy lmethionine decarboxylase. Advances in Enzyme Regdarion. 27,43-55.

Pegg AE, Shantz LM, and Coleman CS (1994). Ornithine decarboxylase: structure, fimc tion, and translation regulation. Biochemical Society Transactions. 22,846-852. Phillips DR, and Crowthers DM (1986). Kinetics and sequence specincity of dmg-DNA interactions: An in vitro transcription assay. Biochemistry. 25,73 55-73 62.

Powell WC, Knox JD, Navre M, Grogan TM, Kittleson J, Nagle RB, and Bowden TG (1 993). Expression of the metallopmteioase matrilysin in DU445 cells increases their invasive potential in severe combined immunodeficient rnice. Cancer Resemch 8, 417-422.

Quesada AR, Barbacid MM, Mira E, Femandez-Resa P, Marquez G, and Aracil M (1 997). Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelathases. CIinincal and Eiperimental Metastasis. 15 (l),26-32.

Rawdanowicz TJ, Hampton AL, Nagase H, Woolley DE, and Salamonsen LA (1994). Matrix metalloproteinase production by cultured human endometrial stroma1 cells: identification of interstitiai collagenase, gelatinase A, gelatinase B, and stromefysin- 1 and their differential regdation by interleukin-la and -or necrosis factor-. Journal of CZinical Endocrinology and Metabolism. 79 (2), 5 30-536.

Ray JM,and Stetler-Stevenson WG (1994). The role of matrix metailoproteinases and their inhibitors in tumour invasion, metastasis and angiogenesis. European Respiratory Journal. 7,2062-2072.

Ray RM, Zimmerman BJ, McCormack SA, Patel TB, and Johnson LR (1 999). Polyamine depletion arrests ce11 cycle and induces inhibitors p2 1Wafl JCip 1, p27"P', and p53 in IEC-6 cells. Arnerican Jownal of Physiology. 276, C684-C69 1.

Reponen P, Sahlberg C, Huhtala P, Hurskainen T, Thesieff 1, and Tryggvason K (1 992). Molecular cloning of muririe 72-kDa type IV collagenase and its expression during mouse development. nte Journal of BiologicaI Chemistry. 267 (11), 7856-7862. Rogers WH, Matrisian LM, Giudice LC, Dsupin B, Cannon P, Svitek C, Gorstein F, and Osteen KG (1994). Patîerns of ma& metailoprotek expression in cychg endometrium irnply Werentiai fimctions and regulation by steroid hormones. Jownol of Clinical Investigation 94,946-953.

Ruan H, Shantz LM,Pegg AE, and Moms DR (1996). The upstream open reading frame of the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive translational control element. The Journi of BiologicaZ Chernistry. 271 (47), 295 76- 29582.

Samuel SK, Hurta RA, Kondaiah P, Khalil N, Turley EA, Wright JA, and Greenberg AH (1 992). Autocrine induction of tumor protease production and invasion by a metallothionein-regulated TGF-P 1 (Ser223,225). EmOJournul. 11 (4), 1599-1605.

Sato H, Takino T, Oka& Y, Cao J, Shinagawa A, Yamamoto, and Seiki M (1994). A matrut metalloproteinase expressed on the surface of invasive tumor cells. Nature. 370 (7),6 1-65.

Schaefer EL, and Seidenfeld J (1987). Effects of polyamine depletion on semm stimulation of quiescent 3T3 murine fibroblast cells. Journal of Cellular Physiology. 133, 546-552.

Shantz LM, Holm 1, Jame OA, and Pegg AE (1992). Regdation of S- adeno sylmethionine decarboxylase activity by aiterations in the intracelluiar poIy amine content. Biochemical Journal. 288,s 1 1-5 18.

Shimogori T, Kashiwagi K, and Igarashi K (1996). Spermidine regulation of protein synthesis at the level of initiation complex formation of met-tRNAi, rnRNA, and ribosomes. Biochemical and Biophysicui Research Communications. 223,544-548. Sreenath T, Matrisian LM, Stetler-Stevenson W, ûattoni-Celli S, and Popatti RO (1 992). Expression of matrut metailoproteinases genes in transformed rat ceIl lines of high and low metastatic potential. Cancer Resemch. 52,494204947.

Stetler-Stevenson WG, Krutzsch HC, and Liotta LA (1989). Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metaîioproteinase inhibitor family. Journal of Biological Chentistry. 264 (29), 17374-17378.

Stricklin GP, Li L, Jancic V, Wenczak BA, and Nanney LB (1993). LocaIization of mRNAs representing collagenase and TIMP in sections of healing human burn wounds. American Journal of Pathology. 143, 1657-1666.

Sugiura Y, Shimada H, Seeger RC, Laug WE, and DeClerck Y (1998). Matrix metalloproteinase-2 and -9 are expressed in human neuroblastoma: contribution of stroma1 cells to their production and correlation with metastasis. Cancer Research 58, 2209-22 16.

Sunkara P and Rosenberger A (1 987). Antirnetastatic activity of DL*- difluoromethylornitbine, an inhibitor of polyamine biosynthesis, in mice. Cancer Research- 47,933-935.

Svensson F, and Persson L (1996). Regdation of ornithine decarboxylase and s- adenosy lmethionine decarboxylase in a polyamine auxotrophic ce11 line. Molecular and Cellular Biochemisby. 162, 1 13- 1 19.

Takino T, Sato H, Shinagawa A, and Seiki M (1995). Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene fkom a human placenta cDNA library . Journai of BiofogicaI Chemishy. 270 (39), 23 0 13-23020- Tamori A, Nishiguchi S, Kuroki T, Seki S, Kobayashi K, Kinoshita H, and Otani S (1994). Relatioaship of ornithine decarboxylase activïty and histological hdings in human hepatocellular carcinoma HepaoIogv. 20, 1 1 79- 11 86.

Van-Wart HE and Birkedal-Hansen H (1990). The cysteine switch: A principle of regulation of metalloproteinase activity with potentiai applicability to the entire matrix metailoprotienase gene family. Proceedings of the National Academy of Science USA. 87,5578-5582.

Vincenti MP, Coon CI, Mengshol JA, Yocum S, Mitchell P, and Brinckerhoff CE (1 998). Cloning of the gene for interstitial collagenase-3 (matrix metalloproteinase- 13) fÏom rab bit synovial fibroblasts: differential expression with col! agenase- 1 (matrix metalloproteinase-1). Biochemishy JournalL 331,341-346.

Vodredow DC, Cress AE, Howard EW, Bowden GT, and Nagle RB. Activation of gelatinase-tissue-inhibitors-of-metalloprotee complexes by matrilysin (1 998). Biochemistry Journal. 331,965-972.

Vu TH, Shipley M, Bergers G, Berger JE, Hehs JA, Hanahan D, Shapiro SD, Senior RM, and Werb Z (1998). MMP-9/gelatinase B is a key reguiator of growth plate angiogenesis and apoptosis of hypertrophie chondrocytes. Cell. 93,411-422.

Wallon U, Shassetz L, Cress A, Bowden T, and Gemer E (1994). Polyamine-dependent expression of the matrix metalloproteinase matrilysin in a human colon cancerderived ce11 line. Molecular Carcinogenesis. f 1, 138-144.

Wang J, Viar MJ, McCormack SA, and Johnson LR (1992). Effect of putrescine on S- adenosylmethionine decarboxylase in a srnaIl intestinai crypt cell line. Arnerican Journal of Physiologly. GG494-GSO 1. Weyman C, Taparowsky E, Wolfson M, and Ashendel C (1988). Partial down regdation of protein kinase C in C3H 10T1/2 mouse fibroblasts transfected with the human Ha-ras oncogene. Cancer Research. 48,6535-6541.

Witty JP, Wright JH, and Matrisian LM (1995). Matrix metaIloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin- 1 in transgenic mice induces unscheduled dveolar development. Molecular Biology of the Cell. 16, 1287-1303.

Wright JA, Egan SE, and Greenberg AH (1990). Genetic regdation of metastatic progression. Anficancer Reseurch. 10, 1247- 1256.

Yamashi ta K, Azurnano 1, Mai M, and Okada Y (1998). Expression and tissue localization of matrix metalloproteinase 7 (matrilysin) in human gastric carcinomas. Impiications for vesse1 invasion and metastasis. International Journal of Cancer. 79, 187- 194.

Young S, Parker PJ, Ullrich A, and Stabel S (1987). Down-regulation of protein kinase C is due to an increased rate of degradation. Biochemical Journal. 244,775-779.

Zucker S, Mancuso P, DiMssimo B, Lysik R, Conner C, and Wu C (1997). Cornparison of techniques for the measurement of gelatinace/type IV collagenases: enzyme-linked immunoassays versus substratte degradation assays. Chical and lQperimentai Metasfasis. 12 (l), 13-23.