STUDIES OF INHIBITOR COMBINATIONS ON ARCHITECTURE

Uppal Sonal

A Dissertation

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

December 2006

Committee:

Carol A. Heckman, Advisor

James H. Albert Graduate Faculty Representative

Carmen F. Fioravanti

Michael E. Geusz

Tami C. Steveson ii

ABSTRACT

Carol A. Heckman, Advisor

Previous work from this laboratory employed an assay for cell morphology, which is

based on computer-assisted microscopy and sophisticated classification methods. The assay

quantifies aspects of three-dimensional shape, based on values of 102 mathematical variables. To

evaluate a phenotype in an experiment, variable values for treated and control cells are compared

with a database of values for normal and oncogenically transformed cells.

In the current work, I evaluated the effect of inhibitor combinations on the cell features

defined by the above assay. The studies confirmed the previous finding that treating cells

simultaneously with the MT-polymerizing and MT-depolymerizing agent, ,

caused a reversal of the cancer cell phenotype. All combinations of paclitaxel with a MT- depolymerizing agent, including podophyllotoxin, nocodazole, or , had the same effect. Based on immunolocalization of tubulin, control cells exhibited numerous MTs arranged parallel to cell edge. Paclitaxel, in combination with any depolymerizing agent, caused MTS to

be arranged perpendicular to the cell edge. A review of clinical data showed greater reduction of

tumor size in patients exposed to simultaneous combination of MT-polymerizing and the MT-depolymerizing , than in patients exposed to either agent alone.

The effect of a coupled compound, Colchitaxel, on MT organization and MT plus ends,

was studied by immunolocalization. By laser confocal scanning microscopy, the EB1 was localized in sheaths around MT ends in control cells. Treated cells exhibited a more diffuse iii localization, which was restricted to the tips of the MTs. Cultures treated for 48 hours with the novel drug showed an elevated percentage of multinuclear cells. The level was even greater in cultures exposed to the paclitaxel and colchicine combination.

The conclusions of the studies are: 1) reversal of the morphometric phenotype is not restricted to the combination of paclitaxel with colchicine, 2) the MT arrangement that best correlates with reversal is one where the periphery is cleared of MTs and MTs are arranged perpendicular to the cell edge, and 3) as a cancer therapy, the highest efficacy is observed when the MT-polymerizing and MT-depolymerizing agents are administered together, not interspersed with single agents.

iv

ACKNOWLEDGEMENTS

I have learned over the years that there are many people that have a hand in our day to

day lives. I have saved writing the acknowledgements for the end of my thesis. However, I

could never have made it this far without the help of many wonderful people. I would like to

thank all those people who gave me support and encouragement on my way to completion of my

doctorate degree. My sincere and deepest thanks go to my graduate advisor, Dr. Carol Heckman, for her longtime patience, continuous guidance and spiritual encouragement in these years. I would also like to express my gratitude to my doctorate committee which includes Drs. Carmen

Fioravanti, Michael Geusz, and Tami Steveson for their helpful guidance, critical suggestions

and continuous encouragement in my research. Additionally, I would like to express my sincere

thanks to Dr. James Albert for his suggestions and serving as a graduate faculty representative on

my dissertation committee. I would like to thank BGSU Statistical Center for statistical

guidance. My deepest thanks to Dr. Marilyn Cayer, whose help and assistance in my research and above all, friendship, will always be remembered. Special thanks to Dr. Yingxin Li, John

DeMuth, Matt Franz, Chris Nowak, for their support and assistance. I would like to thank my lab members Surya Amarachintha, Santosh Malwade and Mita Varghese. I would like give my deepest appreciation to my family and Chandra for their support and encouragement.

I would like to acknowledge God for His guidance and wisdom. This work was partially supported by DAMD17-0101-0484 from US Army Medical Research and Material Command,

Department of Biological Sciences, and the Graduate College of Bowling State University

v

TABLE OF CONTENTS

Page

CHAPTER 1. INTRODUCTION ...... 1

Cytoskeleton and Its Types...... 1

MT Structure and Dynamic Behavior...... 2

MT Dynamicity...... 3

MTOC...... 7

Role of MAP in MT Dynamicity...... 9

Cancer and Anti-Cancer Therapy ...... 9

MT Depolymerizing Agents ...... 11

MT Polymerizing Agents...... 14

Cellular Level Effects of Combined MT Depolymerizing and Polymerizing Agents 16

Clinical Effects of Combined MT Depolymerizing and Polymerizing Agents...... 20

Cellular Level Effects of Combined MT Depolymerizing and Polymerizing Agents 21

Objectives of the Current Work...... 24

CHAPTER 2. MATERIALS AND METHODS...... 27

Cell Culture ...... 27

MT Inhibitors ...... 27

Analysis of In Vitro By Computerized Morphometry Method...... 27

Clinical Data Analysis ...... 31

Localization of MTs and Plus End Proteins by Immunofluorescence...... 31

Laser Confocal Scanning Microscopy (LCSM) and MT Visualization ...... 32

Effect of MT Inhibitors on ...... 33 vi

CHAPTER 3. RESULTS...... 34

In Vitro Analysis of the effects of the MT Inhibitor Combinations ...... 34

Factor Analysis...... 36

Cancer-Type Phenotype Classification...... 47

CHAPTER 4. EFFECTS ON MT ARRAY...... 55

CHAPTER 5. ANALYSIS OF PATIENTS IN CLINICAL TRIALS. 61

Analysis of Clinical Data...... 61

CHAPTER 6. EFFECT OF COLCHITAXEL...... 71

Effects of Colchitaxel on MTs...... 71

Effects of Colchitaxel on MT Plus End-Binding Proteins...... 74

Cell Cycle Effects ...... 80

CHAPTER 7. DISCUSSIONS ...... 86

Reversal of Shape Phenotype...... 87

Rearrangement of MT Arrays...... 92

Importance of In Vitro Models ...... 94

Basic Studies on the Clinical Combination ...... 95

Effects on EB1 ...... 97

Effects on Cell Cycle ...... 99

CHAPTER 8. REFERENCES ...... 103

vii

LIST OF FIGURES

Figure

1 MT structure showing the arrangement of α and β tubulin in a protofilament ...... 4

2 Assembly of MT from its building blocks of tubulin ...... 5

3 Assembly and disassembly of the MT from its building blocks of tubulin ...... 6

4 Schematic summary of positions of drug-binding sites on tubulin heterodimer...... 12

5 Structure of Colchicine ...... 13

6 Structure of Paclitaxel (Taxol)...... 15

7 Images of cells grown on anodic oxide interferometer...... 17

8 The anodic oxide interferometer, composed of tantalum metal and its oxide...... 29

9 Typical picture of a scanned cell with lowermost three contours...... 35

10 A typical transformed cell treated with MT inhibitor combination of paclitaxel

and colchicine...... 49

11 Data showing the distribution of cells (sample size =50) in different ranges of fractal

dimensions...... 52

12 Data showing the distribution of cells (sample size =50) in different ranges of fractal

dimensions...... 53

13 Data showing the distribution of cells (sample size =50) in different ranges of fractal

dimensions ...... 54

14 Micrographs of IAR20 PC1 cells showing the arrangement of MTs ...... 56

15 IARC20 PC1 cells exposed to 2 µM paclitaxel + 2 µM vinblastine...... 57

16 IARC20 PC1 cells treated with unequal molar combinations of MT inhibitors...... 58 viii

17 IARC20 PC1 cells treated with unequal combination 6 µM paclitaxel and 2 µM of

colchicine...... 60

18 Combined response of cancer patients against varying concentration of vinorelbine. 65

19 Combined response of cancer patients against length of time vinorelbine...... 66

20 Combined response of cancer patients against varying concentration of vinorelbine 67

21 Combined response of cancer patients against length of time vinorelbine ...... 68

22 MT arrangement visualized by indirect immunofluorescence localization of β-tubulin 73

23 Distribution of MT plus ends visualized by indirect immunofluorescence localization of

EB1 proteins ...... 75

24 Projections of a VRO from a cell treated with 6 μM Colchitaxel and then stained with

antibody against EB1 ...... 76

25 Projections of a VRO from a control cell stained with antibody against EB1 ...... 77

26 Appearance of nuclei in cells treated with MT Inhibitors compared with untreated

controls...... 85 ix

LIST OF TABLES

Table

1 Factors used to solve for transformed phenotype and their direction of change… ... 19

2 Solution of factor #4 with significance testing by Tukey’s multiple comparison ..... 38

3 Solution of factor #5 with significance testing by Tukey’s multiple comparison ..... 39

4 Solution of factor #7 with significance testing by Tukey’s multiple comparison ..... 40

5 Solution of factor #1 with significance testing by Tukey’s multiple comparison ..... 42

6 Solution of factor #8 with significance testing by Tukey’s multiple comparison ..... 43

7 Solution of factor #12 with significance testing by Tukey’s multiple comparison ... 44

8 Solution of factor #13 with significance testing by Tukey’s multiple comparison ... 46

9 Solution of phenotype classification with significance testing by Duncan’s multiple

comparison ...... 48

10 Solution of fractal analysis with significance by Two-sample t-test ...... 47

11 Combined responses (CR) with different schedules and therapeutic dosage protocols 62

12 Statistical analysis of responses to combined MT inhibitor therapy and combination

therapy alternating with a single agent...... 63

13 Odds of improved results with various variables using logistic regression analysis .... 70

14 Quantitative analysis of perinuclear EB1-positive caps in treated and control cells.... 79

15 Length and width of EB1-positive caps in Colchitaxel-treated and control cells .... 81

16 ED50 values on various endpoints for cell cycle aberrations ...... 82

1

CHAPTER 1. INTRODUCTION

Cell doctrine defines cells as basic elements, from which all plant and animal tissues are

constructed (1). This theory shapes the concepts of biological sciences. Various cellular

activities such as cell division, extracellular and intracellular signaling, transportation of

organelles are carried in the cells. The above activities are cytoskeleton dependent. Studies on

the plant microtubule (MT), one of the type of cytoskeleton filaments have stated that the spatial

distribution of chromosomes during cell division is controlled by MTs (2). MTs also provide directional and spatial routes for transport of organelles (3).

1.1 Cytoskeleton and Its Types

Cytoskeleton is literally the “cell skeleton”. Cytoskeletal protein polymers, such as the

MT and actin filaments, mediate many of the behavioral characteristics of cells. The

cytoskeleton also plays a crucial role in cell motility, cell growth, and intracellular trafficking of

organelles (3). The various functions of the cytoskeleton depend on the following three types of

protein filaments:

a) microfilaments, also known as actin filaments, make up the contractile substructure of the

cell;

b) intermediate filaments provide mechanical support and resistance against shear stress; and

c) MTs are required for direct intracellular trafficking and positioning of the proteins needed to

pull the chromosomes apart during (4).

2

Cytoskeleton acts as a force generator required to derive mechanical energy from

chemical energy (1). The two types of forces generated are primitive force based on the

polymerization of MT and actin and sophisticated force based on motors, such as myosin, kinesin and dynein, required for dragging cargoes along the MTs and actin filaments (4). The function of these filaments is dependent on the interactions with the nearby accessory proteins which bind to the filaments and control their assembly and disassembly (3, 4).

1.2 MT Structure and Dynamic Behavior

MTs are highly dynamic filamentous fibers made of tubulin subunits. Tubulin was originally identified through its colchicine-binding activity. Colchicine binds to polymerized MT in a biphasic manner; the first step is rapid but weak, with the second step slower but stronger.

The binding of colchicine inhibits the MT formation and disrupts MT at high concentration (5).

Tubulins are some of the most conserved proteins through evolution. Tubulin heterodimers are

made by the non-covalent bonding between globular protein subunits of α-tubulin and β-tubulin.

Tubulin is a heterodimeric protein of 110,000 Daltons (6). The tubulin dimers polymerize end to

end in protofilaments (Fig. 1). In the tubulin heterodimer, the α-tubulin subunit is always bound

to GTP (5). This site is non-exchangeable. The nucleotide associated with the β subunit can

exchange freely. The presence of non-hydrolysable GTP analogues maintains stability, because

the GTP cap at the MT end prevents unraveling of the MTs.

MTs are stiff hollow cylindrical structures, which are composed of 9 to at most 16

protofilaments. The wide range in the number of protofilaments provides flexibility in lateral

association within the MTs. All protofilaments are in parallel, which gives the MT a distinct 3

polarity. The diameter of the MT is 25nm and the thickness of the wall is 5nm. Protofilaments of

MT are formed by longitudinal and lateral interactions between tubulin heterodimers. Tubulin subunits assemble head-to tail in the same direction (4). Therefore, MTs show distinct polar plus and minus ends. The assembly of MTs is faster at the plus end (fast growing) than the slow- growing minus end (5). MT plus ends are more dynamic in nature than minus ends. The minus ends of MTs are located at the centrosome, which is also known as a microtubule organizing center (MTOC). The plus ends are located near the plasma membrane and are free in the cytoplasm (7). Some MTs are entirely free in cytoplasm rather than being anchored in the

MTOC. MTs undergo complex polymerization dynamics which are regulated by hydrolysis of

GTP (5).

1.3 MT Dynamicity

Formation of protofilaments is a process which relies upon formation of short polymers of

subunits which are elongated with the addition of more heterodimer subunits. This pathway is

defined as the nucleation-elongation pathway (5, 6). During the elongation phase, subunits are

added to the free ends of the existing MTs (Fig. 2). The concentration of free tubulin declines

until it reaches a plateau, where the polymerization and depolymerization rates are exactly

balanced, the critical concentration (Fig. 3). MTs known to be in equilibrium at the critical

subunit concentrations are called treadmilling (4), resulting in kinetically identical rates of

addition/removal of tubulin.

4

Plus End of MT

Minus End of MT

Exchangeable GTP β- tubulin

Non-Exchangeable GTP α- tubulin

Fig. 1: MT structure showing the arrangement of α and β tubulin in a protofilament

5

Plus End of MT

Fig. 2: Assembly of MT from its building blocks of tubulin

6

Fig. 3: Assembly and disassembly of the MT from its building blocks of tubulin

7

MT dynamic behavior is important during mitosis and cell division (5), and possibly

during other cell motility functions as well. One of the dynamic behaviors of the MT is dynamic

instability, where individual MTs can alternate between periods of slow growth and rapid

disassembly. Dynamic instability plays a major role in the positioning of MTs in the cells. The

diverse MT functions are achieved either by the post translational modification of tubulin or by

the binding of regulatory proteins, including MT associated proteins (MAPs), either to

unpolymerized tubulin or to MT ends (6). Dynamic behavior of MT is the key regulator in

spindle formation in the , when the centrosome is duplicated. The bipolar spindle is

formed by rapid shortening and elongation of MT originating from the centrosomes on the

opposite poles. This dynamic behavior enables MTs to be captured by the during

mitosis. The alignment of the spindle is an important step in separating the replicated chromosomes to form two nuclei, and it requires a process with rapid dynamicity (6). MAPs

also contribute to enhance the spindle formation and regulate the MTs. However, little is known about the way cell growth is regulated in a MT-dependent fashion except during M phase of the cell cycle.

1.4 MTOC

The MTOC, also known as the centrosome, plays a crucial role in the organization of the

cell’s cytoplasm (8). One of the major functions of the centrosome is the nucleation of

cytoplasmic MTs. Studies have localized MTs in the mitotic apparatus of every type of

eukaryotic cell. MTs are connected to each chromatid at the centromere, also known as the

. This connection is maintained firmly during and anaphase movements.

Under normal conditions the eukaryotic cell centrosome duplicates only once in its division 8 cycle. The centrosome is composed of two centrioles surrounded by pericentriolar material.

Centriole pairs in the centrosome consist of nine triplets of MTs (9). One of the proteins identified in centrosome, known as Cep55, shares a 39% similarity to the yeast spindle pole body

(SPB) protein, Spc110p, in Schizosaccharomyces pombe. Studies have demonstrated that the

SPB of S. pombe is a dynamic organelle, undergoing significant changes in morphology and cellular localization as cells progress through their growth and division cycle. Another of the major components of the centrosome is gamma-tubulin. A recent study shows that centrosome proteins are essential in processes like early mitosis and cell cleavage (10). Cep55 is found in the centrosome in the early stages of mitotic maturation. It is lost from the centrosome when the cell enters mitosis. In anaphase, Cep55 is relocalized in the midbody area between two cells which are about to separate (9). A juxtanuclear centrosomal MTOC is the main, but not the only, MT- nucleating site in animal cell types (10).

The centrosome functions to regulate the MT initiation rate, the number of MTs initiated, and their direction. The kinetically inactive minus end is anchored in the MTOC. In cells lacking centrioles, but containing a focus of MT growth, material similar to the antigenic determinants of the centrosome is found, suggesting that there are common structural components in all interphase MT organizing sites (5). Studies have shown that the centrosome is responsible for organizing the spindle and mitotic division (8, 11). Recent studies suggest that aberrant centrosome replication might be a key to tumor progression (11, 12). The number of centrosomes is an essential factor in determining the uniform distribution of chromosomes at mitosis and the preservation of euploidy (12). Supernumerary centrosomes are a distinguishing 9

feature of certain tumor cells. Numerous centrosomes contribute to formation of multipolar

spindles and, thus, to defective chromosome segregation.

1.5 Role of MAP in MT dynamicity

Some MAPs are known to stabilize MTs (13), whereas others encourage MT-

depolymerization within the cell. BIM1 (binding-to-) found in yeast, a homolog of

the end-binding protein (EB) family (14), has recently been shown to promote MT dynamics in the G1 phase of the cell cycle. BIM1 has been visualized both along cytoplasm of MTs (14) and

localized at their ends (13). BIM1 has also been linked to MTs ends in the yeast cell cortex, where it is thought to facilitate orientation of the spindle. The presence of EB1 at the tips is thought to promote the polymerization of the MTs (14). The mechanism of action of the EB1 protein in forming the MT protofilament is still poorly understood. Colchicine, which is a known MT depolymerizing agent, is known to prevent EB1 proteins becoming anchored to the plus ends (5).

1.6 Cancer and Anti-Cancer Therapy

Cancer is the second leading cause of death in the United States. Cancer cells can

impinge on surrounding tissues or organs, leading to abnormal body functions and in turn

causing death. The progression of cancer from one site to other distant organs or tissues is called

metastasis. The secondary tumor formed due to metastasis also disrupts bodily functions. The

drugs used to treat the primary tumor are sometimes toxic and frequently cause side effects

which can increase the death rate. In addition, resistance developed over time to the drugs, can

enhance the mortality rate in cancer patients. 10

Protocols to eradicate cancer include chemotherapy, immunotherapy, radiation therapy and cavitation, singly or in combination. Cancer cure rate by surgery and radiotherapy is 30-

50%. The choice of treatment is dependent on a number of factors such as the type and stage of the cancer, health status, age, and so forth. These two methods alone cannot be relied upon for curing cancer, because of secondary tumor formation (15). Cancers that have spread to secondary sites require systemic treatments by chemotherapy.

Introduction of chemo (toxico) therapy started after World War II with the discovery of {methyl-bis (chloroethyalmine)}, as an effective anti-cancer treatment (15).

Since there are relatively few biochemical differences between cancer cells and normal cells, the efficacy of many anti-cancer drugs is limited by their toxicity to normal, rapidly growing cells.

The unregulated control of cell growth and cell division serves an attractive and achievable objective for drug design. Since one of the key components required for cell division and cell growth is the MT, MT-targeted agents are exploited in cancer treatment (16, 17). Tubulin- binding drugs, including colchicine, vinca alkaloids, , and their analogues, are classified as MT inhibitors/poisons. These agents have various tubulin binding sites (Fig. 4) (5).

Anti-mitotic drugs are divided into two functional groups, MT-depolymerizing and MT- polymerizing agents. Since 1960, natural plant and animal products representing both groups have been used as chemotherapeutic agents.

11

1.7 MT Depolymerizing Agents

Compounds such as colchicine, estamustine, vinca alkaloids, halichondrins, and combretastatins

depolymerize MT at high doses (6). In addition, the natural hormone, estradiol, inhibits cell

growth, depolymerizes MT networks, and induces cell division arrest with higher drug

concentrations. In vitro studies have shown that estradiol binds to tubulin at a site different from

that of vinblastine and colchicine (18).

Colchicine is a major alkaloid found in the Colchicum autumnale plant (Fig. 5). It has

anti-inflammatory and anticancer properties and is used in the treatment of gout. C. autumnale

extract is still one of the most effective treatments for the intense pain associated with a gout attack. When colchicine binds with tubulin at the polymerizing end, it blocks the polymerization process and prevents the formation of MTs. The study of colchicine provided scientists with the

first understanding of anti-microtubular drug action (6). The binding of colchicine to tubulin

results in a conformational change in tubulin (19).

Although colchicine might be expected to push the tubulin subunits apart by binding in the cleft between them, it might actually pull them closer together, which makes the tubulin become disordered and more protease sensitive. Colchicine appears to reduce inflammatory symptoms by targeting both leukocyte division and locomotion (6). The compound nocodazole is thought to bind at the same site as colchicine. Nocodazole was first used as an effective agent against Ascaris lumbricoides. A. lumbricoides is the largest intestinal roundworm and is a common helminth infection of humans. Nocodazole inhibits the binding of colchicine to tubulin

(17, 18). 12

Vinblastine Binding Exchangeable site of GTP Paclitaxel Binding β- tubulin

Colchicine Binding Non exchangeable site of GTP

α- tubulin

Fig. 4: Schematic summary of positions of drug-binding sites on MT heterodimer.

13

OMe

MeO 3 2 4

4a 5 MeO 1 1a O 12 12a 6

11 7a 7 N Me H 8 10 9 MeO O

Fig. 5: Structure of Colchicine

14

Amongst the earliest MT-depolymerizing agents to be studied were Vinca alkaloids,

which are extracted from periwinkle leaves (Catharanthus roseus). Members of this group,

which include vinblastine and , were used in the treatment of leukemia. Since then,

semi-synthetic analogues, including , vinorelbine, and , have been found to

have clinical efficacy (6). Vinblastine and its semi-synthetic analogue, vinorelbine, bind to α and

β-subunits and block the ability of tubulin to polymerize into MTs (20). Vinca alkaloids depolymerize MTs and, at high concentrations, destroy the mitotic spindle. It has been shown that at low concentrations, there is no depolymerization of MTs, yet vinblastine powerfully blocks cell division. Studies have concluded that the cell division block is due to the suppression of MT dynamicity instead of MT depolymerization (21).

1.8 MT Polymerizing Agents

Paclitaxel, a product of the pacific yew, is another natural anti-cancer agent (Fig. 6).

Paclitaxel binds to the β-subunit of tubulin in MTs and disrupts the flexible nature of the MT.

Binding of paclitaxel decreases the magnitude of the dissociation constant for tubulin at both the

plus and minus ends of the MT (22) and promotes MT assembly (23, 24) leading to hyper-

stabilization of the MT. At high concentrations, paclitaxel causes assembly of all the free tubulin

into MTs in a cell. Paclitaxel ultimately arrests cell division and leads to (18).

Paclitaxel is also known to modify rheologic properties of MTs, leading to spiral curling.

Paclitaxel directly or indirectly displaces MAPs and thereby causes formation of stable bundles

(20).

15

O

O O 18 19 OH 10 9 O 12 7 11 8 6 3' 16 2' 13 15 3 5 HN 1' O 17 4 14 1 O 4' OH 2 HO O 20 O O O O

Fig. 6: Structure of Paclitaxel (Taxol)

16

Both colchicine and paclitaxel bind near the amino terminus of ß-tubulin (25-27). As

mentioned above, colchicine caps the MT and thereby prevents further subunit addition. Thus,

the two drugs have opposite effects on MT integrity at high concentrations. At substoichiometric

ratios to tubulin however, their effects are similar. Paclitaxel restricts both the periods of growth and shrinkage of the MT (28). The additive amounts of growth and shrinkage (dynamicity) were also suppressed by vinblastine (29). In combination, colchicine and paclitaxel exerted complex effects on the MT array in cells and created arrangements of MTs that were rarely found in control cells (16). Such combinations decreased the tendency of MTs to be anchored in the

MTOC (16, 30, 31). The combination of colchicine and paclitaxel also caused arrays of MTs to form from focal points in the cytoplasm. Inclusion of colchicine with paclitaxel partially suppressed the usual bundling of MTs induced by paclitaxel. It also allowed individual MTs to emerge perpendicular to the cell edge (16). It is difficult to determine which aspects of the MTs reorganization (if any) were related to the therapeutic effects of inhibitor combinations, however.

1.9 Cellular Level Effects of Combined MT-Depolymerizing and Polymerizing Agents

The current research is focused on studying the effects of the above mentioned MT inhibitors on shape features. Previous studies from our laboratory suggested that all drugs affecting MTs produced effects on cell shape (16). When cell shape dynamics were studied over time in long-term culture, time could be used as an independent variable to sort out features of cancerous cells from those of normal cells. To assay cell phenotypes, we sampled interference images in 3D (Fig. 7) and then analyzed the shape of the contours by calculating the values of

102 mathematical variables, of which only a small sunset was used in classifying the treated samples. 17

Fig. 7: Images of cells grown on an anodic oxide interferometer.

Resolution of the edge details was optimized by positioning the first dark fringe at the cell

margin. This interference color was dark due to the subtraction of certain wavelengths from the

spectrum of reflected light. The appearance of a topographic map was due to the alteration of dark and light colors within the interference order. Bar = 40 μm. 18

These variables provided a means of categorizing each cell according to the shape characteristics, so that we knew which agents mimicked the effects of transformation. In order to learn which defects were enhanced by each of the MT inhibitors alone or in combination, it was essential to make qualitative as well as quantitative distinctions. The development of a new method of analyzing the data allowed like changes among the variables to be grouped together and represented by a single number. The original contour shape data was broken down by principal components (latent factors). Table 1 defines features described by the various factors used in the classification equation for the transformed phenotype.

Latent factors could be used in the same way as primary shape variables to create a standard curve of morphogenetic changes. This curve was used to define a “signature” phenotype of cancer-type cells. Subsequently, the signature value could be solved for cells from experiments by comparing values of their shape variables to those of the standard. Shape changes that occurred in a short-term experiment but resembled those occurring during long-term maintenance of the cell line were considered promoter-like. In other words, they led to cells having more cancer-type properties. Such effects were observed in cells treated with several different agents, including colchicine (32). Experiments in which cells were exposed simultaneously to paclitaxel and colchicine revealed the opposite effect on the phenotype, leading the cells to adopt more normal cell features. Thus, this combination served as an anti- promoter (16, 33). This finding, along with other data on colchicine and docetaxel (31), gave clinicians a rationale to conduct tests of MT inhibitor combinations.

19

Table 1. Factors used to solve for transformed phenotype and their direction of change

Position Factor Value increases with Direction of in cell numbera change in

transformation

Edge 4 Number of sharp, tapering features at cell edge Decreases

Edge 5 Broad or long projections or deep invaginations Increases

Edge 7 Size of knobby projections at cell edge Increases

Upper 1 Coarse protrusions or invaginations in upper contour Decreasesb

Second 8 Presence of spiky structures in second contour Decreasesb

Second 12 Size of hollowed out regions in second contour Decreases

Global 3 Ellipsoidal shape of overall cell Increases

Global 13 Rounding-up of cell Increases aFactors used to classify the phenotypes of transformed and normal IAR20 PC1 cells. bFactor entered into the equation with a sign opposite to factor’s direction.

20

1.10 Clinical Effects of Combined MT-Depolymerizing and Polymerizing Agents.

The current research is focused on studying the effects of the simultaneous combination

of paclitaxel with various MT-depolymerizing agents on shape features of an epithelial cell line.

90% of all clinically diagnosed neoplasms arise in the epithelia. Hence, the study was focused on epithelial model systems. Rather than using the original combination of colchicine and paclitaxel which was studied in the laboratory, I chose clinical reports which represented protocols developed in various European countries (see Chapter 5). Instead of using colchicine, the clinical studies employed a different depolymerizing agent, 5’-noranhydrovinblastine, also called vinorelbine, in combination with docetaxel, an analogue of paclitaxel (18, 34-38). The objective response, expressed in terms of tumor size reduction after therapy, was greater than if the individual MT inhibitors were used alone (39). Since the therapy was most effective when the agents were administered as nearly simultaneously as possible, the results suggested an additive or synergistic interaction of the MT inhibitor combination.

There are four reasons why a combination of MT inhibitors may be more effective than a single therapeutic drug. Combination therapy targets four objectives: (a) higher number of cells killed within the toxicity range of an individual drug tolerated by the patient; (b) a decreased likelihood of drug resistance to improve complete response rates; (c) the prevention of the formation of new resistant clones; (d) a positive interaction of two drugs at the cellular level

(18). In support of mechanism (d), studies have suggested that altered tubulins which are resistant to taxoids are increasingly sensitive to drugs such as vinorelbine (40). In support of mechanism (a), studies have shown that the combination of docetaxel and vinorelbine synergized in vitro, when vinorelbine was given before or both drugs were given simultaneously. In 21

contrast, an antagonistic effect was seen when taxanes were administered before vinorelbine

(18). In combination, the optimal dosage of docetaxel and vinorelbine by injection was 29.6 mg/kg and 12 mg/kg, respectively. This corresponded to 1.55 combination toxicity index. A combination toxicity index of 1 indicates that only 50% of each agent (or any other ratio, e.g.

70:30, 40:60, etc.) can be given in the combination. Therefore, nearly 100% of the optimal level of agent administered singly can be administered in the docetaxel-to-vinorelbine combination without increasing the toxicity to vital normal cells in mammary tumor models (41, 42).

1.11 Cellular Level Effects of MT-Depolymerizing and Polymerizing Agents

The dynamic nature of the MTs is key to carrying out normal mitotic division. Nearly all

MT inhibitors, including colchicine and paclitaxel, cause mitotic arrest. Paclitaxel and a number

of diverse natural cytotoxic substances, including , discodermolide and eleutherobin,

mimic the cytotoxic effects of binding to the binding site (43). In contrast, colchicine,

vinblastine, vincristine and nocodazole disrupt MT function by depolymerization. There is

substantial agreement that at least some of the MT inhibitors can arrest cells in mitosis by

mechanisms other than preventing spindle formation. Compared to the estimated 0.22 μM

paclitaxel delivered in a clinical setting, it was shown that the delivery of as little as 10 nM

paclitaxel suppressed growing and shortening of MTs and blocked mitosis in HeLa S3 cells.

Similar results were obtained with vinblastine, podophyllotoxin, colchicine, and nocodazole.

None of these agents exerted significant effects on the total mass of the MT polymer. Since the

spindles formed were aberrant, the workers concluded that the agents halted mitosis by inhibiting

spindle dynamicity. Nevertheless, at lowest effective concentrations, blocked spindles were

indistinguishable from the control (44). 22

The cytotoxic effects of MT inhibitors at the cellular level are controversial. Since

dynamic MTs are required for the correct assembly of spindle or for signaling the transition from

metaphase to anaphase, mitotic block by low doses of spindle poisons may involve kinetic

stabilization of the spindle MTs rather than alterations in MT structure. Some investigators

consider that subsequent failure of the cell to negotiate the spindle checkpoint triggers

programmed cell death (45, 46). In fact, until recently, the view that mitotic arrest was essential

for the cytotoxic effects of MT inhibitors was widely held.

Cell death occurs in cancer cells treated with MT polymerizing agents, and a question

arises as to the relationship between the mitotic block and apoptosis. Paclitaxel in a human cell

line and mouse embryo cells induced two forms of cell cycle arrest, which in turn induced two independent apoptotic pathways. Arrest in prophase induced rapid onset of a p53-independent pathway, whereas G1-block and the resulting slow (3-5 days) apoptotic pathway were p53 dependent (47). Even in the absence of mitotic arrest, however, abnormal cell products were

found in some studies. In lung carcinoma A549 human cells, it was shown that that a low

concentration of paclitaxel inhibited cell proliferation (48) without blocking mitosis. In vivo

studies in murine tumors indicated that baseline apoptosis and paclitaxel-induced apoptosis

strongly correlated with cell death and antitumor effect, but there was no correlation between

antitumor effects and mitotic arrest (49). This result suggested that mitotic arrest was irrelevant

to the mechanism of cell killing by paclitaxel. Since some studies failed to find evidence of

mitotic arrest, the results suggested that the earlier studies on apoptotic death may have failed to

identify the true mechanism.

23

The studies reviewed above were important, because they provided no rationale for

expecting the MT inhibitor combination to exert a different effect on cells than the components

individually. Moreover, there was some evidence that cell death pathways could be triggered in

the absence of mitotic arrest. Recent studies sought to determine whether there were synergistic

effects of paclitaxel and vinorelbine at relatively low concentrations. In human melanoma cells,

growth inhibition and metaphase block were induced without MT depolymerization and spindle disorganization. In prostatic carcinoma cells exposed to a combination of estramustine

(concentration 100-fold less than paclitaxel) and paclitaxel (1 nM), the two agents showed an additive effect on the inhibition of cell survival of both wild-type and estramustine-resistant

cells. Although the combination of estramustine and paclitaxel increased cells in of cell

cycle, increases in mitotic arrest or percentage of micronucleated cells were not found.

Inhibition of cell survival was not significant in combination of paclitaxel and vinblastine (50,

51). Such results suggested a possibility that cell death occurred in the absence of mitotic arrest.

A third experimental outcome has been described in recent reports. With exposure to

high levels of antimitotic agent, cells may pass through mitosis while avoiding apoptotic cell

death (52). Studies have shown that, when KB 3-1 and J82 lines were treated with high doses of

the combination, docetaxel and colchicine, the cells formed pseudo-asters of short MTs

surrounded by a depolymerized MT network. If the cells entered G1 phase without undergoing

chromosome segregation and , they may reconstitute more than one nucleus (53, 54).

As suppression of spindle dynamics by low concentrations of drugs may be responsible

for mitotic arrest induced by MT depolymerizing and polymerizing agents, I questioned if the 24

combination of MT polymerizing and depolymerizing agents would produce the same results as

the single agents alone. As MT polymerizing and depolymerizing agents bind to different sites

on the same target, namely tubulin, their combination could lead to synergistic, additive or

antagonistic effects, depending on the nature of interactions. One study has suggested that MT

inhibitors in combination have an additive effect on mitosis. At low concentrations, the number

of multinucleated KB 3-1 cells increased with the combination of docetaxel and colchicine, but

no short bundles of MTs or abnormal structures such as pseudo-asters were seen (31). Based on

the above studies, there is evidence for three mechanisms by which the cells are killed upon

exposure to the MT inhibitors. It remains to be determined whether there is a preferred

mechanism for cell killing in tumors.

1.12 Objectives of the Current Work

Heckman et al. found that cancerous cells treated with simultaneous combination of

colchicine and paclitaxel reverse their phenotype and appear more normal. Clinical trials were

carried out by other investigators, with a combination consisting of a docetaxel and vinorelbine.

The latter has effects similar to those of colchicine. The combination of agents proved to be

more effective than treatment with either drug alone, raising the frequency of complete

remissions from 3% to 13%. Therefore, results of clinical research suggested that a combination

of compounds had beneficial chemotherapeutic effects. The efficacy of the paclitaxel/vinorelbine therapy suggests that it involves a new therapeutic principle. Paclitaxel and colchicine (or vinorelbine) have the same target in cell cytoplasm, but invoke different mechanisms. One stabilizes microtubules, whereas the other prevents addition of new subunits and thereby causes the microtubule to unravel. Although the scientific basis of combination 25 therapy is under investigation, the therapeutic effects were predicted by results from the shape assay.

The current work has two approaches that offer new tools with which to address the question of MT inhibitor combination therapy. One is the assay for cancer-type phenotypes, which can be used to correlate the phenotype with the morphological effects on the MT array.

The hypothesis, that a MT inhibitor combination had a better effect on MT architecture and its behavior than the individual inhibitors administered alone, was investigated using this method.

Based on the original work by Heckman et al. where a combination of colchicine and paclitaxel reverse the phenotype of cancerous cell to normal features, the hypothesis that was explored in this experiment was that various depolymerizing agents were interchangeable for purposes of phenotype reversal. The compounds substituted for colchicine were vinblastine, podophyllotoxin, and nocodazole. Another primary aim was to determine whether the knowledge of phenotype reversal, presented in detail below (Chapters 3 and 4), corresponds to the clinical experience. The hypothesis underlying the clinical inquiry was that, if the therapeutic effect relied upon rearrangement of the MT array, then the objective response would not depend on the ratio of the combination.

A second approach consisted of synthesizing a portmanteau compound that could bind to either the colchicine site or the paclitaxel site and to determine whether it had effects on the MT array resembling those of the paclitaxel/colchicine combination. The new compound synthesized from the starting compounds is known as Colchitaxel.

26

Chapters are arranged by topic in the following order:

Chapter 3:

Objective 1a: Effect of MT inhibitor combinations on morphometric cell shape features.

Objective 1b: Use of fractal dimension to supplement shape analysis.

Chapter 4:

Objective 2: Effect of MT inhibitor combinations on MTs in cultured cells.

Chapter 5:

Objective 3: Statistical evaluation of results from clinical trials.

Chapter 6:

Objective 4a: Evaluate the effects of coupled compound, Colchitaxel, on MTs.

Objective 4b: Study the effect of the novel agent on the EB1 localization.

Objective 4c: Study the effect of the MT inhibitor combination and novel agent on cell division. 27

CHAPTER 2. MATERIALS AND METHODS

2.1 Cell Culture

The IAR20 (International Agency for Research on Cancer) PC1 line was derived from the liver of inbred BD-VI rats, as described elsewhere (55). It was cloned two months after being established. Liver cells were routinely grown in William’s E medium supplemented with penicillin, streptomycin (50units/mL each), and 10% fetal bovine serum (GIBCO Invitrogen,

Grand Harbor, NY). The line was selected because it gradually became tumorigenic while being maintained in culture over a period of 8 months (56, 57). This enabled the laboratory to identify shape changes that were correlated with transformation.

2.2 MT Inhibitors

Colchicine and vinblastine sulphate were obtained from Sigma-Aldrich Company (St.

Louis, MO) and were dissolved in ethanol and stored at -20°C (16). Paclitaxel (Taxol®) was a gift from Natural Products Branch of the Development Therapeutics Program, Division of

Cancer Treatment, National Cancer Institute. Later, paclitaxel was obtained from Dabur, Inc.,

India. The newly designed compound, Colchitaxel is a conjugate of two agents, paclitaxel and colchicine, connected with a glutarate linker. Its synthesis has been described elsewhere (58).

2.3 Analysis of in vitro by Computerized Morphometry Method

Heckman et al. developed an assay for cell shape phenotypes, which was based on extracting interference contours from cells subcultured onto solid substrate interferometers. The same technique was performed to analyze the effect of various MT inhibitor combinations on the 28

phenotypes of IAR20 PC1 cells. The methods followed are summarized briefly here, as they are

described in detail elsewhere (16). IARC20 PC1 cells were plated on anodized tantalum

substrates in replicate dishes, left overnight, and then exposed to experimental agents for 2 h.

The samples were collected by fixation in buffered glutaraldehyde (pH7.3). Glutaraldehyde was

used because it was a better cross-linker of proteins than formaldehyde. Samples were washed in

distilled water, and air-dried at room temperature. The samples were viewed by reflected light in

a Zeiss universal light microscope.

A precise pattern of destructive interference in certain wavelengths is established due to

the high refractive index of the oxide and the opportunity for light to pass through the oxide layer

(Fig. 8). Thus, at the zero-order contour, the cell edge was visible against the substrate, and two

additional contours were visible against contrasting interference (59). Edge coordinates derived from the image were input into a VAX 6620 computer via a Tektronix 4112 terminal.

Information about the shape features was extracted by a series of computer programs in C++

running on the anonymous FTP host, . The values of the 102 variables for the

contour shape were calculated from the digitized contours. Each experimental treatment was

represented by a total of 50 cells.

In previous work, the combination of optical and computing methods proved capable of

distinguishing a great variety of different phenotypes in cultured cell lines (56, 57). Heckman et

al. had also analyzed time-dependent changes in lines undergoing transformation in vitro using

equations based on the values of single “indicator” variables. For IAR20 PC1 cells, the

classification equation combined a number of variables that were normalized to actual 29

Fig. 8: The anodic oxide interferometer, composed of tantalum metal and its oxide.

The variety of wavelength represented in the white light create optical interactions representing complete destructive (A), partially destructive 9B), and constructive (C) interference effects (56). 30

dimensions of the cell with others that were normalized to dimensions of the ellipse of

concentration (57). Data from each 50 cell sample was submitted to multiple linear regression

analysis for classification alongside a group of cells representing normal and transformed

reference samples. Because the signature-type value was assigned relative to these samples, the

solutions were in units of predicted time in culture. After obtaining such a value for each cell line

in an experiment, the difference in mean predicted times for treated and control samples were

calculated as described by Heckman and coworkers (57). Classification of samples from

experiments in the current work employed multiple linear regression method based on factor scores (see below) rather than on contour shape variables.

The factors used in classification were selected by SAS (Statistical Analysis Software)

REG procedure. Factors which explain the covariance of the real variables were derived by solving for principal components by the SAS ‘PROC FACTOR’ procedure. The factor scores could then be calculated by SAS procedure, ‘SCORE‘. Predicted time-in- culture values were obtained by submitting the experimental cells to analysis, as described above for simple variables. The statistical significance of differences among sample means within each experiment was determined by the Duncan multiple comparison range test which is a modification of the t-test. The application of this method is such that experiment-wise error rate is controlled by the alpha value. To determine whether any factor’s values differed among treatment groups within an experiment, another multiple comparison method was incorporated which is known as Tukey’s test. GLM and MODEL discriminant analysis procedures of SAS were used. It was found that a few of them were entered into the equation by multiple linear regression selection but were not well correlated with transformation. Presumably, these were 31

entered because they counterbalanced fluctuations in the value of one or more factors that were

well correlated with transformation. Examples of this effect include factor #8, a factor for sharp

features in the second contour. This was included in the classification equation for IAR20 PC1

cells, although it showed no significant correlation with transformation in this line.

2.4 Clinical Data Analysis

The use of two MT inhibitors, one which stabilizes and one which destabilizes MT

structure, has become a standard anti-cancer therapy. Seventeen different clinical studies were considered which collectively included data on 567 patients. I studied the parameters partial response, complete response, combined response, duration of treatment, length of treatment, and molar ratio of the drugs. Partial objective response is the partial reduction in tumor size after the treatment is administered in the cancer patient by a specific protocol. Complete response is

defined as the complete objective remission of the cancer disease, as judged by CAT scan, MRI,

or other objective measurement technique. All the studies followed a regimen of exposing the

patients to different dosages of combination treatments, in units of mg/m2, as mentioned in the

respective studies. The drug combination used Navelbine (vinorelbine) as the MT-

depolymerizing drugs and docetaxel as the MT-polymerizing agent. The drugs were

administered at different intervals and dosages, and for different numbers of treatment cycles,

which were set by the clinicians themselves (see Chapter 5).

2.5 Localization of MTs and Plus End Proteins by Immunofluorescence

For immunofluorescence localization studies, cells were subcultured onto glass coverslips, left in the incubator for 18-48 h to attach, and then treated with various compounds. 32

Treatments were carried out for 2 h unless otherwise specified. The coverslips were collected by immersion in methanol at -70˚ C. N357 monoclonal antibody against β-tubulin raised in

Balb/C57 mice (Amersham Biosciences, Piscataway, NJ) was diluted 1:600 for use in MT localizations. Mouse antibody against EB1, obtained from BD Transduction Laboratories (San

Jose, CA), was diluted 1:100 for use. As secondary antibodies, FITC-conjugated goat anti- mouse (U. S. Biochemical Corp., Cleveland, OH) or Alexa 488-conjugated goat anti-mouse immunoglobulin G (Molecular Probes, Eugene, OR) were used. Results with the two antibodies were indistinguishable. After having been stained with antibodies, the preparations were mounted on a slide in a solution of 1, 4-diazobicyclo[2.2.2]octane (Sigma) made up to 25mg/ml in glycerol (16).

Representative cells were photographed on a Zeiss Axiophot microscope equipped with a

63x Neofluar lens, using Kodak Ektachrome film. Later, images were acquired using a Roper

Scientific RTE/CCD camera and IBM PC running MetaMorph 4.6r5 software (Universal

Imaging Corp., Buckinghamshire, UK). In the case of MT plus-end images, various approaches to quantification were tried. The area around the nucleus was designated for each cell in the images, using transparent overlays placed over the image. EB1 positive comets in the areas overlying and surrounding the nucleus were counted using Student’s t-test.

2.6 Laser Confocal Scanning Microscopy (LCSM) and MT Visualization

LCSM is a method of acquiring many two-dimensional image planes in a microscope and reconstructing the three-dimensional (3D) volume of the subject. LCSM was used to visualize the EB1 antibody localization. LCSM was performed on a Zeiss Pascal confocal microscope 33

equipped with a 63x Neofluar lens. Software used for deconvolution included the Volocity

(Improvision, Coventry, UK) and Huygens (Advanced Volume Imaging, Hilversum,

Netherlands) packages. Virtual reality objects (VRO) were made in VRWorx 2.5 software

(VRTool box, Pittsburgh, PA). The 3D images were viewed in Quickview on Apple Macintosh

or Dell PC microcomputers. Such 3D images were rotated, in order to project the VRO views

and to measure length and width of the plus end cap protein distribution. To evaluate the statistical significance of the differences, the standard distribution of means was tested in

Student’s t-test.

2.7 Effect of MT Inhibitors on Cell Cycle

In order to understand the effect of MT inhibitors on cell cycle mechanisms, I wanted to

study cell populations at specific phases of the cell cycle. To study the frequency of

multinucleated cells, cells were subcultured as above into 60-mm plastic tissue culture dishes and

treated for 48 h with Colchitaxel or with colchicine (Sigma-Aldrich, St. Louis, MO) in the

presence or absence of paclitaxel. The cells were fixed in ethanol-to-glacial acetic acid (7:1),

stained with 10% Giemsa solution, mounted in ImmunoFluore and viewed on a Zeiss Axiophot

microscope (Thornwood, NY) equipped with a 40x Planapo lens. Mitotic figures were

categorized as normal or aberrant. The aberrant figures included divisions with multiple poles or

chromosomes far from the metaphase plate. Cells with single or multiple nuclei were also

counted. At least 200 cells were counted in each sample.

34

CHAPTER 3. RESULTS

3.1 In Vitro Analysis of the Effects of MT Inhibitor Combinations

The first objective of the experiments was to learn whether the effect of MT inhibitor combinations, composed of various depolymerizing agents in combination with paclitaxel, on shape reversal was restricted to the combination of paclitaxel and colchicine. The hypothesis that was evaluated in this experiment was that various depolymerizing agents were interchangeable for purposes of phenotype reversal. The compounds substituted for colchicine were vinblastine, podophyllotoxin, and nocodazole. Shape variables were measured from three contours (Fig. 9), yielding a total of 102 variables, as mentioned above. The results showed that reversal of the transformed shape phenotype also occurred after exposure to combinations of paclitaxel with MT-depolymerizing agents other than colchicine.

In addition to analyzing changes in the cancer-type phenotype which indicates the rounding up of the cells after treatment with various MT-depolymerizing agents in combination with paclitaxel, I analyzed effects on the individual factor values that were predictors of the transformed phenotype. These results would supply information about the type of features affected by MT inhibitors. Data on morphological features in cells treated with the MT inhibitor combinations are described, followed by results of the cancer-type phenotype analysis.

35

Fig. 9: Typical picture of a scanned cell with lowermost three contours which were used to extract the 102 variables. The outer contour is the cell edge contour, followed by the middle and the inner (upper) contour near the nucleus.

36

3.2 Factor Analysis

Based on Tukey’s multiple comparison statistical analysis a modification of t-test, factors

#1, 4, 5, 7, 8, 12, and 13 were significantly affected when various MT inhibitor combinations were administered to IARC20 PC1 cells. In previous experimentation with the combination of paclitaxel and colchicine, the only factor which had a significant difference from control was factor # 5. Its values were shifted down relative to control [Heckman, unpublished data]. The factor accounting for the greatest quantitative change in the shape of transformed cells was factor

#4, which represented the prevalence of filopodia. Factor #4 values were entirely computed from the lowermost (zero-order) interference contour (16). Although all of the treated groups showed a shift to values exceeding the control value, only the treatments with equimolar concentrations of paclitaxel and colchicine or 1:3 ratio of podophyllotoxin to paclitaxel significantly affected the #4 values (Table 2). This was opposite to the direction taken by #4 values in cell populations that became transformed, suggesting that the treatment enhanced the molecular arrangement of adhesion specializations that underpin the filopoium.

The values of factor #5, like those of factor #4, were based on variables of the lowest interference contour (cell edge). The values of factor #5 increased in transformed cells, but again, all combinations of taxanes with depolymerizing agents reduced the #5 values relative to control. This reflected a decline in broad expanses of cytoplasm or deeply eroded invaginations in the treated cells. Again, not all of the samples were significantly affected compared to the control group (Table 3). Samples exposed to the equimolar combinations of paclitaxel with any depolymerizing agent were generally significantly affected (except for the combination with 37 vinblastine). Exposure to 3:1 paclitaxel with vinblastine or nocodazole also significantly decreased factor #5 values (Table 3).

Factor #7 has been identified as a descriptor of P-21 Activated Kinase-dependent protrusions. The primary variables that contribute to factor #7 include both descriptors of the first and second contours. These descriptors, along with those representing three-dimensional aspects of cell shape, were unique features of the interferometric methods of analysis (16). Since experimental treatment with a combination of paclitaxel and any depolymerizing agent enhanced the values of this factor, the treated cells tended to resemble the cancer-type rather than the normal phenotype. In this respect, it differed from the other edge factors, in which all treatment induced a more normal phenotype. As with factor #4, the equimolar ratio of paclitaxel to colchicine and the unequal ratio of paclitaxel to podophyllotoxin differed significantly from the control (cf. Tables 2 and 4).

The most normal phenotypes in the analysis were provided by paclitaxel administered in equal ratio with podophyllotoxin or in unequal ratios with either nocodazole or colchicine, which gave results indistinguishable from control. Remarkably, in the case of factor # 4 the remaining treated samples all differed significantly from the control group (Table 2). Factor #1 represents coarse protrusions or deeply eroded features in the upper contours of the cells. These features are less prevalent in transformed cells than in normal cells, and they are not generally induced by treatment with MT inhibitor combinations. Values of #1, like those of factor #7, tend to shift only in the direction suggesting a more transformed phenotype

38

Table 2. Solution of factor #4 with significance testing by Tukey’s multiple comparison using

α =0.05

Mean (sample size=50) Combination of agents (ratio)

-1.18a colchicine + paclitaxel (1:1)

-1.23a podophyllotoxin + paclitaxel (1:3)

-1.28a,b vinblastine + paclitaxel (1:3)

-1.34a,b podophyllotoxin + paclitaxel (1:1)

-1.35a,b vinblastine + paclitaxel (1:1)

-1.39a,b nocodazole + paclitaxel (1:1)

-1.43a,b nocodazole + paclitaxel (1:3)

-1.48a,b colchicine + paclitaxel (1:3)

-1.60b control (solvent treated)

Means with the same superscript letter a,b are not significantly different.

39

Table 3. Solution of factor #5 with significance testing by Tukey’s multiple comparison using

α =0.05

Mean (sample size=50) Combination of agents (ratio)

-0.57a control (solvent treated)

-0.83a,b colchicine + paclitaxel (1:3)

-0.94a,b,c vinblastine + paclitaxel (1:1)

-1.00a,b,c podophyllotoxin + paclitaxel (1:3)

-1.06b,c colchicine + paclitaxel (1:1)

-1.10b,c podophyllotoxin + paclitaxel (1:1)

-1.14b,c vinblastine + paclitaxel (1:3)

-1.18b,c nocodazole + paclitaxel (1:1)

-1.31c nocodazole + paclitaxel (1:3)

Means with the same superscript letter a,b,c are not significantly different.

40

Table 4. Solution of factor #7 with significance testing by Tukey’s multiple comparison using

α =0.05

Mean (sample size=50) Combination of agents (ratio)

0.64a colchicine + paclitaxel (1:1)

0. 30a,b podophyllotoxin + paclitaxel (1:3)

0.21a,b,c nocodazole + paclitaxel (1:1)

0.14b,c vinblastine + paclitaxel (1:1)

0.056b,c vinblastine + paclitaxel (1:3)

-0.058b,c,d nocodazole + paclitaxel (1:3)

-0.067b,c,d podophyllotoxin + paclitaxel (1:1)

-0.20c,d colchicine + paclitaxel (1:3)

-0.46d control (solvent treated)

Means with the same superscript letter a,b,c,d are not significantly different.

41

Only in one sample, namely equimolar paclitaxel with colchicine, did the values

significantly differ from those found for the control (Table 5). Interestingly, the sample which

showed a significant change in factor #1 values also gave a high response in terms of factor # 7

values, adopting values expected of a more transformed cell population. Thus, cells treated with

equimolar concentrations of paclitaxel and colchicine, were particularly susceptible to exhibiting some transformed features. Factors #8 and #12 were among the other variables describing the upper contours. They represented different aspects of vesicle trafficking (see Introduction).

Although all of the treatments changed the factor #8 values in the direction expected for more normal cells, the changes were only significant in the case of one treatment. That was the equimolar ratio of nocodazole to paclitaxel (Table 6). Although the factor’s values declined in transformation, they were entered into the equation with the opposite sign, evidently because they compensated for variable values of another factor that was important in the regression.

For factor #12, the samples’ values again all increased the values of #12 in the direction contrary to that expected for transformed cells. All of the samples except paclitaxel in equimolar combination with podophyllotoxin differed significantly from control (Table 7). The results of analyzing factor # 8 and # 12 suggested that the changes induced by MT inhibitor combinations included some general effect on a vesicle trafficking process. The resulting phenotypes suggested a restoration to a more normal arrangement of trafficking organelles in the treated cells. Since the equimolar combination of paclitaxel and nocodazole reversed both of the vesicle trafficking factors, compared to the direction they were changed in transformed cells, this combination appeared particularly well-suited to further studies of the basis of this phenotype.

42

Table 5. Solution of factor #1 with significance testing by Tukey’s multiple comparison using

α =0.05

Mean (sample size =50) Combination of agents (ratio)

-0.28a podophyllotoxin + paclitaxel (1:1)

-0.38a,b vinblastine + paclitaxel (1:3)

-0.38a,b podophyllotoxin + paclitaxel (1:3)

-0.42a,b nocodazole + paclitaxel (1:3)

-0.50a,b control (solvent treated)

-0.52a,b colchicine + paclitaxel (1:3)

-0.57a,b vinblastine + paclitaxel (1:1)

-0.58b,c nocodazole + paclitaxel (1:1)

-0.91c colchicine + paclitaxel (1:1)

Means with the same superscript letter a,b,c are not significantly different.

43

Table 6. Solution of factor #8 with significance testing by Tukey’s multiple comparison using

α =0.05

Mean (sample size =50) Combination of agents (ratio)

2.71a nocodazole + paclitaxel (1:1)

2.14a,b colchicine + paclitaxel (1:1)

2.13a,b vinblastine + paclitaxel (1:3)

2.12a,b podophyllotoxin + paclitaxel (1:3)

1.91a,b nocodazole + paclitaxel (1:3)

1.87b podophyllotoxin + paclitaxel (1:1)

1.67b colchicine + paclitaxel (1:3)

1.51b vinblastine + paclitaxel (1:1)

1.47b control (solvent treated)

Means with the same superscript letter a,b are not significantly different.

44

Table 7. Solution of factor #12 with significance testing by Tukey’s multiple comparison using

α =0.05

Mean (sample size =50) Combination of agents (ratio)

10.31a nocodazole + paclitaxel (1:1)

10.15a,b vinblastine + paclitaxel (1:3)

10.15a,b colchicine + paclitaxel (1:1)

9.63a,b,c nocodazole + paclitaxel (1:3)

9.41b,c,d colchicine + paclitaxel (1:3)

9.07c,d vinblastine + paclitaxel (1:1)

8.91c,d podophyllotoxin + paclitaxel (1:3)

8.73d,e podophyllotoxin + paclitaxel (1:1)

7.95e control (solvent treated)

Means with the same superscript letter a,b,c,d,e are not significantly different.

45

There were several factors’ values that are computed from variables of all the contours

and are therefore called global variables. Those that were included in the equation for solving

for the cancer-type phenotypes included factors #3 and #13. The latter was a measure of the

slope of rise of the cell boundary from the substrate to the third contour. It was enhanced by at

least one of the combinations with all depolymerizing agents except for those containing

podophyllotoxin (Table 8). Whereas rounding-up was generally an index associated with transformation, the treated cells in this experiment were generally more rounded-up than the control cells. In the case of both combinations containing colchicine and nocodazole, and the 3:1 combination of paclitaxel with vinblastine, the results showed statistical significance compared to the untreated controls (Table 8). Although factor #3 values generally decreased in the treated samples compared to the control, none of the samples’ values showed a significant difference from control (data not shown).

The above results conclude that features represented by factors #1, # 4, #5, #7, #8, #12, and #13 can be influenced by various combinations of paclitaxel with MT-depolymerizing agents. However, in several cases, the direction of the changes was not that that would be expected of the more normal phenotype. Thus, some of the features (#7, #1, and #3) are changed so as to resemble more transformed cells. The implication of this is that the overall direction of change in the phenotype must be attained by overcoming some shift in selected features toward the transformed phenotype with other shifts toward normal. The results suggest that only factors

#4, #5, #8 and #12 “carry” the cells toward a more normal phenotype.

46

Table 8. Solution of factor #13 with significance testing by Tukey’s multiple comparison using

α =0.05

Mean (sample size =50) Combination of agents (ratio)

2.73a vinblastine + paclitaxel (1:3)

2.52a nocodazole + paclitaxel (1:1)

2.51a,b colchicine + paclitaxel (1:1)

2.48a,b nocodazole + paclitaxel (1:3)

2.33a,b colchicine + paclitaxel (1:3)

2.07b,c podophyllotoxin + paclitaxel (1:3)

1.86c control (solvent treated)

1.80c podophyllotoxin + paclitaxel (1:1)

1.71c vinblastine + paclitaxel (1:1)

Means with the same superscript letter a,b,c are not significantly different.

47

3.3 Cancer-Type Phenotype Classification

Previous studies showed that cells exposed to MT inhibitor combinations made up with

either cephalomannine or 7-deoxytaxol did not exhibit phenotype reversal (16). In current

studies, it was seen that all combinations composed of paclitaxel with any of the MT

depolymerizing agents induced cells to became more like normal cells. Table 9 shows the

phenotype classification analysis. The analysis showed that although the equimolar concentration

of colchicine to paclitaxel had the maximum effect, its effects on the cancer - type phenotype were indistinguishable from other equimolar combinations with the exception of the Paclitaxel- to- podophyllatoxin combination.

In addition to the computerized phenotype solved above, differences between the control and equimolar combination of colchicine and paclitaxel (1:1) were investigated using fractal geometry. We used fractal analysis as a novel quantitative approach with a single cumulative parameter, the fractal dimension. Not only did the fractal analysis shed light on an additional property of the cells, but it enabled a comparison on a contour by contour basis.

48

Table 9. Solution of phenotype classification with significance testing by Duncan’s multiple comparison using α =0.05

Mean (sample size =50) Combination of agents (ratio)

146.32a Control

124.38b podophyllotoxin + paclitaxel (1:3)

120.33b.c podophyllotoxin + paclitaxel (1:1)

116.62b,c colchicine + paclitaxel (1:3)

111.13b,c,d nocodazole + paclitaxel (1:3)

108.88b,c,d nocodazole + paclitaxel (1:1)

107.96b,c,d vinblastine + paclitaxel (1:3)

101.89d vinblastine + paclitaxel (1:1)

94.29d colchicine + paclitaxel (1:1)

Means with the same superscript letter a,b,c,d are not significantly different.

49

(a) (b)

Fig. 10: (a) A typical transformed cell treated with MT inhibitor combination of paclitaxel and colchicine (1:1) (b) control cell (solvent treatment)

50

Table 10. Solution of fractal analysis with significance by Two-sample t-test using α =0.05

Sample Outer Dimension Middle Dimension Inner Dimemsion

(No = 50) (Do) (Dm) (Di)

Control Treated Control Treated Control Treated

Mean 1.113 1.128 1.127 1.134 1.110 1.132

S.Da 0.040 0.024 0.033 0.024 0.043 0.027

P-valueb 0.022 0.256 0.003 a Standard deviation.

51

The treated cells were characterized by the following values of fractal dimensions:

dimension of upper (inner) contour (Di) = 1.132±0.027, dimension of middle contour (Dm) =

1.134±0.024 and dimension of outer (edge) contour (Do) = 1.128±0.024. In most cases, it was

possible to distinguish visually between cells belonging to different groups. Generally, treated

cells possessed a larger degree of fractality (Fig. 10). This can be illustrated by comparison with

the control population which had values of Di = 1.110±0.043, Dm = 1.127±0.033, and Do =

1.113±0.040 (Table 10). Results indicate that inner and outer contours undergo the most drastic changes in the distribution of fractal dimensions.

To evaluate global differences between the treated and control samples, I plotted histograms showing fractal dimension of cumulative probabilities as well as of attaining a given degree of self- similarity (Figs. 11-13).The histograms demonstrate that the degree of fractality is higher in the treated cells. Cumulative probabilities indicate the total number of cells with the values of fractal dimension less than Df as a function Df. These plots suggest that the degree of

roughness or fractality increases the treated cells. The edge (outer) contour is involved in extensive changes related to factors #4 and #5. Factor #4 is defined as a number of sharp, tapering features at the cell edge (filopodia). Factor #5 is related to broad protusions or enlarged

invaginations at the cell edge (mass displacement). Factors #1, #2 describe the upper (inner)

contour and are defined as coarse protrusions, bumpiness (size of protrusions). But factors # 8

and # 13 might be related because they are reflective of the size of protusions or spikes on the

contour. 52

S35,

15 inner

100 10

80 5 60 0 1.05 1.10 1.15 1.20 40

counts Control, 20 10 inner Cumulative Probability Cumulative 0

5 1.00 1.05 1.10 1.15 1.20 D i

0 1.05 1.10 1.15 1.20 D i

(a) (b)

Fig. 11: Data showing the distribution of cells (sample size =50) in different ranges of fractal dimensions. (a) Fractal histogram; (b) cumulative probability for the upper contour of the control

(dashed line) and treated (solid line) cell. 53

15 S35, middle 100 10 75 5 50 0 1.05 1.10 1.15 1.20 25

counts Control, Cumulative ProbabilityCumulative 10 middle 0

5 1.05 1.10 1.15 1.20 D m 0 1.05 1.10 1.15 1.20 D m (a) (b)

Fig. 12: Data showing the distribution of cells (sample size =50) in different ranges of fractal dimensions. (a) Fractal histogram; (b) cumulative probability for the middle contour of the control (dashed line) and treated (solid line) cell lines. 54

S35, 15 outer 100 10 80 5 60 0 1.05 1.10 1.15 1.20 40

counts 15 Control, 20 10 outer Cumulative Probability Cumulative

0 5 1.00 1.05 1.10 1.15 1.20 D 0 o 1.05 1.10 1.15 1.20 D o

(a) (b)

Fig. 13: Data showing the distribution of cells (sample size =50) in different ranges of fractal dimensions. (a) Fractal histogram; (b) cumulative probability for the edge contour of the control

(dashed line) and treated (solid line) cell lines.

55

CHAPTER 4. EFFECTS ON MT ARRAY

In the original shape reversal results as described in Chapter 2, combinations of 3:1 and

9:1 paclitaxel were used (16). These had equivalent effects on both the morphometric phenotype

and on the arrangement of MTs. In this chapter, I determine whether the effects exerted by

equimolar concentrations of these MT inhibitors, and combinations of paclitaxel with various

depolymerizing drugs, on the MT array resemble the results originally described. Morphological

correlates of IARC20 PC1 cell features were sought by immunofluroscence localization of MTs.

Control cells exhibited numerous MTs arranged parallel to the cell edge (Fig. 14A). Only

where cells made contact did MTs typically appear to be perpendicular to the cell edge. In cells

treated with the combination of equimolar paclitaxel and colchicine, MTs were rearranged so

that a part of the array extended straight out from the peripheral cytoplasm (Fig. 14B). The

patterns of MT organization in the cell edge and cytoplasm described previously by Heckman et

al. (16) were conserved. A small number of MTs radiated from points in cytoplasm, making an

image of criss-crossing MTs.

The cell in Fig. 15 was exposed to the equimolar combination of paclitaxel and

vinblastine. MT arrays were found in bundles at the periphery of the nuclear region, with some

MTs also found perpendicular to the cell edge.

56

(A) (B)

Fig. 14: Micrographs of IAR20 PC1 cells showing the arrangement of MTs. (A) Control with vehicle only. At the cell boundary (two way arrows), MTs are arranged parallel to the edge. (B)

Cells treated with combination of 2 µM paclitaxel and 2 µM colchicine. The straight arrow points to the perpendicularly arranged MTs. The criss-crossed arrangement of MTs is designated by the curved arrow (Bar = 10 µm).

57

Fig. 15: IARC20 PC1 cells exposed to 2 µM paclitaxel + 2 µM vinblastine. MTs are found in bundles at the periphery of the nuclear region (straight arrow), with some MTs perpendicular to the cell edge (curved arrow) (Bar = 10 µm) as compared to the control in Fig. 14A.

58

(A) (B)

Fig. 16: IAR20 PC1 cells treated with unequal molar combinations of MT inhibitors, as follows

(A) 6 µM paclitaxel and 2 µM vinblastine, are arranged in the form of an arc (B) 6 µM paclitaxel and 2 µM podophyllotoxin. Cells show MTs extending in a direction perpendicular to the substrate (arrow) (Bar = 10 µm).

59

The overall effect of exposure to paclitaxel and podophyllotoxin appeared somewhat different from the effects of other MT inhibitors. In cells treated with a combination of paclitaxel and podophyllotoxin, the MTs were short and often appeared discontinuous, indicating some erosion of the MT structure. In the podophyllotoxin-treated cells, the MTs appeared to be less numerous (Fig. 16B). Generally, all of the tubulin was recruited into MTs by paclitaxel treatment (see Discussion). However, the overall effect of exposure to paclitaxel and podophyllotoxin led to a portion of the MT array extending straight out from the periphery. In contrast, with the combination of 3:1 paclitaxel and colchicine, MTs appeared stabilized even at the periphery of the cells (cf. Fig. 16B and Fig. 17).

60

Fig. 17: IARC20 PC1 cells treated with unequal combinations of 6 µM paclitaxel and 2 µM of colchicine. Many of the MTs are arranged perpendicular to the cell edge (straight arrow). The bundling effect of the taxane treatment is seen both near the edges of the nuclear region and near the cell edge (curved arrows) (Bar = 10 µm) as compared to the control in Fig. 14A.

61

CHAPTER 5. ANALYSIS OF PATIENTS IN CLINICAL CHEMOTHERAPY TRIALS

The third section of results investigates the cumulative objective response of cancer patients who were treated with various dosages given based on the tumor size in the patients as mg/m2 of the MT inhibitors combination, vinorelbine and docetaxel. The primary aim was to

determine whether the knowledge of phenotype reversal, presented in detail above (Chapters 3

and 4), corresponds to the clinical experience. In the following section, the results are analyzed

by breaking out different parameters from clinical publications for investigation by statistical

methods. I was also interested in finding the best possible combination therapy schedule and

optimal dosages of the commonly-used MT inhibitors.

5.1 Analysis of Clinical Data

I analyzed eighteen clinical research reports to determine whether the administration of

the combination of one MT-depolymerizing agent with one MT-polymerizing agent is superior

to use of a single agent, based on combined objective response. A systematic overview of the

clinical data available from the clinical studies is shown in Table 11. The table describes the

dosages of docetaxel and vinorelbine given to patients in the form of injection as mg/m2 based on the tumor size in the respective patient. Since the majority of studies showing a high response rate, e.g. above 50%, had only dosed patients with the combination of the MT inhibitors, the analysis first addressed the question of whether these results differed from those in protocols that alternated combined treatment with a single agent. Based on statistical analysis, I found that

combination of docetaxel and vinorelbine alone is superior when the end-point, partial objective

response, was used. The result was similar when combined response was used as the endpoint. 62

Table 11. Combined responses (CR) with different schedules and therapeutic dosage protocols

CR D V D1 D3 D6 D8 D15 D22 D29 D36 Time

(%) (mg/m2) (mg/m2) (Days)

7.14 75 20 V X D+V X X X X X 42

10.53 25 20 D+V X X D+V D+V D+V D+V D+V 84

18.18 60 15 D+V X X V V X X X 63

21.43 75 20 D X V X V X X X 42

26.47 75 20 D+V X V X X X X X 63

36.59 100 25 V D X X X X X X 63

42.86 75 20 D+V X V X X X X X 42

45.45 60 25 D+V X X X X X X X 112

46.94 60 24 D+V X X X X X X X 126

48.28 80 20 D+V X X X X X X X 105

48.72 85 20 V X X D+V X X X X 126

51.85 90 15 D+V D X X X X X X 126

56.1 60 30 D+V X X X X X X X 168

58.06 80 30 D+V X X X V X X X 168

60 60 24 D+V X X X X X X X 126

60 80 20 D+V X X X X X X X 126

60 80 20 D+V X X X X X X X 105

61.4 30 30 D+V X X D D+V X X X 168

63

Table 12. Statistical analysis of responses to combined MT inhibitor therapy and combination therapy alternating with a single agent

Response Combination plus single agent Combination only p-value

Partial 0.331 0.408 0.029*

Complete 0.092 0.091 0.496

Cumulative 0.423 0.500 0.034*

* Samples differed at a 95% significance level 64

Statistical results indicated no significant difference when complete objective response

was used as an endpoint, however (Table 12). It can be stated that, generally speaking, the

alternation of the drug combination with a single drug has a less outcome than treating only with combination of drugs. In addition, I was interested in learning whether there was any change in the response depending on the dosage of either of the MT inhibitors. In the MT inhibitor combination alone group, it can be seen that there was no change in response rate with vinorelbine concentration (Fig. 18). Similar results were seen when the combined response was plotted against time (Fig. 19).

In some studies, MT inhibitors were delivered in a more complex protocol, which interspersed the delivery of docetaxel and vinorelbine with a dose of a single MT inhibitor.

When such reports are broken out separately for analysis, the data illustrate that the pattern of the combined response against varying concentrations of vinorelbine and against time is scattered

(Fig. 20, 21).

65

80 60 40 20 0 010203040 Response (percentage) Vinorelbine (mg/m2)

Fig. 18: Combined response of cancer patients against varying concentration of vinorelbine.

Patients were treated only with the combination of vinorelbine and docetaxel injection in mg/m2

66

80 60 40 20 0 0 50 100 150 200 Response (percentage) Time (days)

Fig. 19: Combined response of cancer patients against length of time vinorelbine was administered. Cancer patients were treated only with the combination of vinorelbine and docetaxel. 67

70 60 50 40 30 20 10 0 Response (percentage) 0 10203040 Vinorelbine (mg/m2)

Fig. 20: Combined response of cancer patients against varying concentration of vinorelbine.

Cancer patients were treated with the combination of vinorelbine with docetaxel alternating with a single agent depending on the treatment schedule.

68

80 60 40 20 0 0 50 100 150 200 Response (percentage) Time (days)

Fig. 21: Combined response of cancer patients against length of time vinorelbine was administered. Cancer patients were treated with the combination of vinorelbine and docetaxel alternating with a single agent depending on the treatment schedule.

69

The complex protocols of drug delivery, therefore, show considerable variation in their therapeutic effects depending on the exact dosage and time over which the agents are administered. It was important to determine whether there was any change in response when other key factors, namely length of the treatment, number of cycles, ratio of MT inhibitors, etc., were considered. This was studied by using a logistic regression model to see whether the beneficial effects showed dependency on such variables. This is used when the dependent variable is categorical variable with two levels. The regression analysis failed to reveal any significant differences (results not shown). Variations in the individual studies may have obscured such differences, however. The results were also analyzed using the odds ratio values

(Table 13).

Based on the odds ratio, it was seen that with an increase in vinorelbine dosage, the odds of

“success” in the combined response decreases by 7% on average, per unit increase in vinorelbine. There was no change in combined response with the change in docetaxel dosage.

With an increase in length of cycle time and number of cycles, the odds of “success” increases by 22%, and 9% on average, per unit increase in ‘number of cycles’ and length of cycle respectively.

70

Table 13. Odds of improved results with various variables using logistic regression analysis

Variable Odds Ratio

Vinorelbine (mg/m2) 0.93

Docetaxel (mg/m2) 1.00

Number of cycles 1.22

Length of cycle 1.09

71

CHAPTER 6. EFFECTS OF COLCHITAXEL

The objectives were to evaluate the effects of the coupled compound on the MT and its

related proteins. Studies have stated that EB1 induce MT bundling. It was interesting to evaluate

the effects of Colchitaxel on EB1. Paclitaxel binds to the plus ends and displaces EB1 (6), so it

was decided to study the effect of the coupled compound on plus ends of MTs. The study aimed

to compare the potential effect of, Colchitaxel, to individual agents already used in clinical

settings. The result from chapter 3 and 4, showed that the combination of paclitaxel and

colchicine are better than the other MT inhibitor combinations studied. Thus, the result this

section describes the biological effect of the coupled compound, Colchitaxel on the MT

architecture and its associated proteins.

6.1 Effects of Colchitaxel on MTs

In order to determine whether Colchitaxel had an obvious effect on the structure or

distribution of MTs, we exposed cells to varying concentrations of the compound and then visualized the arrangement of the MTs by indirect immunofluorescence. Images were inspected to determine whether the treated cells showed details resembling those reported for cells exposed simultaneously to colchicine and paclitaxel. The integrity of MTs structure was conserved even when cells were treated with a high concentration of Colchitaxel. At an elevated concentration

(12 µM), there was evidence of a subtle difference in structure (Fig. 22). MTs were occasionally found to be oriented perpendicular to the cell edge, a pattern that was previously observed in cells exposed simultaneously to colchicine and paclitaxel. In control cells, the MTs typically

curved so that their distal ends ran parallel to the cell edge (60). The treated cells showed ‘x’ 72

shaped cytoplasmic foci from which MTs appeared to radiate, which resembled MTs arrays

observed in previous studies on the combination of inhibitors (16). Bundles of MTs, which had

characterized cells treated with the combination of starting compounds, were not formed in

Colchitaxel-treated cells (Fig. 22). The above results suggested that Colchitaxel had some but not

all of the same effects as the combination of starting compounds. Imaging the whole MTs array by immunofluorescence techniques had certain drawbacks. For example, it was difficult to gain information about the underlying biochemical defects from the appearance of the array.

Obtaining measurements of the comparative length, number, or arrangement of MTs in the cells

was also problematical. 73

Fig. 22: MTs arrangement visualized by indirect immunofluorescence localization of β-tubulin.

Cells were treated with: A) 2 μM Colchitaxel, B) 12 μM Colchitaxel, or C) solvent vehicle alone.

With the low concentration of compound, MTs occasionally appear to be arranged perpendicular

to the cell edge (arrows). With higher concentrations, cytoplasmic foci are observed with radiating MTs (curved arrows). In addition, the region around the centrosome (asterisks) is more evenly stained in treated cells. Control cells typically show a concentration of staining at one side of the nucleus. In control cells, the MTs usually bend to run parallel to the edge (arrowhead). A) bar = 25 μm, B) and C) bar = 10 μm.

74

6.2 Effect of Colchitaxel on MT plus end-binding proteins

Plus-end cap proteins such as EB1 form a comet-like track at one end of the MT, which

would lend itself better to quantitative assessment. Since MTs which are not growing but are

shrinking or in a stationary phase have lost the EB1 cap, a large fraction of cellular MTs do not

show up in the image. EB1 comets in untreated IAR20 PC1 cells showed a more compact shape and a higher density in the peripheral cytoplasm than in the center. These differences were less obvious and the comets more diffuse after Colchitaxel treatment (Fig. 23). In order to obtain more information about the distribution of comets in the cells, we imaged the same samples by a

3D imaging technique. In treated cells, the plus ends appeared as diffuse, fuzzy dots or short streaks when viewed from the substrate. Long, finger-like comets were relatively rare at this site

(Fig. 24).

When control cells were viewed in the same perspective, the plus ends were typically long and high in contrast (Fig. 25). In previous studies, it is mentioned that growth ends are straight with GTP-cap comprised of GDP alpha-Beta tubulin. Shrinking plus ends have outwardly curved protofilaments that peel off from MT wall, potentially due to presence of only

GDP beta-tubulin. When viewing the VRO displays from the underside of the cell, one could see that the ends descending toward the substrate were concentrated near the cell edge. Moreover, it was obvious that the longest comets found in the cell were at this site. These appeared as dot-like structures in 2D immunofluorescent images, because they were viewed in a perpendicular perspective.

75

Fig. 23: Distribution of MT plus ends visualized by indirect immunofluorescence localization of

EB1 proteins. Cells were treated with: A) solvent vehicle only or B) 6 μM Colchitaxel. Comet- shaped EB1-stained streaks are common throughout the cytoplasm, with shorter streaks or dots preferentially distributed at the cell edge. The ends appear shorter and more symmetrical in shape in the treated cells. Bar = 10 μm.

76

Fig. 24: Projections of a VRO from a cell treated with 6 μM Colchitaxel and then stained with antibody against EB1. A) When viewed from the underside, the cell shows short comets throughout which are particularly obvious at the edge (arrows). B) At higher magnification, the

EB1-positive ends appear compact in shape and diffuse in structure. Typical comets are designated by arrows. A) bar = 5 μm, B) length between parallel arrow tips is 3.5-4.5 μm. 77

Fig.25: Projections of a VRO from a control cell stained with antibody against EB1. A) When viewed from the underside, the cell shows finger-like structures directed downward toward the substratum (arrows) B) At higher magnification, the EB1-positive ends appear long and smooth.

Typical comets are designated by arrows. A) bar = 5 μm, B) length between parallel arrow tips is

6-7 μm.

78

The above data suggested that the novel compound particularly affected the elongated plus end of the MT near the cell periphery. Further information was gathered about the size and distribution of the comets. Since the comets’ distribution varied widely between treated and control cells, it was doubtful that uniform sampling of control and treated cells could be done by selecting random areas from the 2D images.

Therefore, counts of the comets’ number were made in an area where they were uniformly distributed, namely that overlying and surrounding the nucleus. A circular area around the nucleus was designated so as to include the centrosome. The density of comets was 15% less in Colchitaxel-treated cells than in controls. Moreover, the difference between control cells and those treated with 6 µM Colchitaxel was statistically significant (Table 14). This conclusion was supported by additional observations. In control samples, the highest number of comets found in the perinuclear region was 23, whereas after 12 µM Colchitaxel treatment, the highest number observed was 10. In order to determine whether the comets’ structure differed in control and treated cells, the samples were imaged by LCSM and the comets’ dimensions in 3D images were measured.

The results showed that the structures were significantly shorter after Colchitaxel treatment. The plus end caps were also smaller in diameter. Because of local variations in their width, it became increasingly difficult to measure the diameter in cells treated with high concentrations of the novel compound (Table 15). Thus, analysis of the plus end cap size and distribution in treated and untreated cells suggested that Colchitaxel reduced the amount of EB1 bound to the MT. 79

Table 14. Quantitative analysis of perinuclear EB1-positive caps in treated and control cells

Colchitaxel (μM) (No. cells sampled) No. ‘comets’ (No. images sampled)

0 (115) 5.58 ± 2.02 (32)a

6 (141) 4.53 ± 1.63 (35)a

12 (130) 4.62 ± 1.51 (24) aSamples differed at 95% significance level. 80

6.3 Cell Cycle Effects

Cells treated with spindle poisions typically arrest in mitosis and then enter an apoptotic pathway (61). Some fail to remain in arrest (62, 63), however, and proceed through mitosis by a process known as ‘mitotic slippage.’ They may fail cytokinesis as well and then undergo cell cycle arrest in the next G1 phase (64). We compared the frequency of aberrant mitotic figures and mitotic slippage in IAR20 PC1 cells. Mitotic slippage was analyzed by comparing the frequency of multinucleated cells in untreated cultures and those treated with various agents. In the case of colchicine, we found ED50 values of 3-4 nM for both aberrant mitoses and multinucleated cells.

The number of cells with multiple nuclei was counted for cells in control cultures and compared to those treated with paclitaxel. Similarly, abnormal versus normal mitotic figures were counted. With paclitaxel, the ED50 values were similar for the two endpoints but were about 15-fold higher than those found for colchicine (Table 16). Cells exposed to varied concentrations of paclitaxel in 3:1 molar proportion with colchicine, however, showed a different pattern. Surprisingly, the two endpoints showed a greater divergence than had been observed in cells treated with either compound singly. The ED50 for abnormal mitosis following paclitaxel in combination with colchicine, was reduced to about 3-4 nM. The ED50 for appearance of multinucleated cells was only half of this value (Table 16).

81

Table15. Length and width of EB1-positive caps in Colchitaxel-treated and control cells

Colchitaxel (μM) Mean Length (μm) ± S.E.M (No.) Mean Width (μm) ± S.E.M

0 2.24 ± 0.224 (4)a,b 0.44 ± 0.043

6 1.58 ± 0.086 (3)a,c 0.38 ± 0.037

12 1.28 ± 0.148 (6)b,c 0.38 aMeans differ significantly from one another by P ≤0.02 in Student t-test bMeans differ significantly from one another by P ≤0.005 in Student t-test cMeans differ significantly from one another by P ≤0.05 in Student t-test 82

Table 16. ED50 values on various endpoints for cell cycle aberrations

Compound tested ED50 (nM) for Biological Effect

Multinucleated cells Abnormal mitoses colchicine 3.8 3.6 paclitaxel 58 53 paclitaxel + colchicine 1.8 + 0.9 3.6 + 1.2 colchitaxel 6,600 ~9,900

83

The results suggested that paclitaxel and colchicine had a synergistic effect on formation of abnormal spindles and an even greater impact on multinucleation. When cells were treated with Colchitaxel, aberrant mitotic figures were observed with lower frequency than in cultures treated with the starting compounds. This made it more difficult to determine the ED50 for this endpoint. Likewise, fewer multinucleated cells were found under the same conditions as employed to study colchicine or paclitaxel alone. Exposure to Colchitaxel for a longer time or to a higher concentration than shown (Table 16) gave percentages of multinucleated cells of around

40% (data not shown). However, the same result would have been observed if more cells were directed into a death pathway at mitosis leaving fewer exiting by mitotic slippage, or if more were eliminated from the postmitotic multinucleated cell population.

Multinucleated cells were created or retained far less frequently in cultures treated with the novel compound in comparison to treatments with single microtubule inhibitors. Thus, the

ED50 for formation of multinucleated cells with Colchitaxel was about 100-fold higher than that obtained with paclitaxel. An unexpected result was the divergence between ED50 values for the two endpoints in tests with the novel compound. It was possible that the apparent reduction in

ED50 for multinucleated cells, common to the novel compound and the combination of individual starting compounds, was due to a greater likelihood of cells forming a normal metaphase plate. Since the rate of multinucleated cell formation competes with the rates of entry into cell death pathways in treated cells before, at, and after mitosis, however, it is not clear how the sensitivity of the multinucleated endpoint was enhanced (see Discussion, Effects on Cell

Cycle).

84

Micronuclei were commonly found in paclitaxel-treated cells due to asymmetrical chromosome arrangements at the division plane (see Introduction). In order to determine whether this effect was also observed in Colchitaxel-treated cells, we compared the morphology of post- mitotic cells after exposure to various compounds. Cells exposed to colchicine were rounded-up with obvious morphological abnormalities. Although mitotic slippage was apparent, as some cells showed a large nucleus along with one or more sizeable, satellite nuclei, micronuclei were not usually observed in colchicine-treated cells. A sizeable fraction of the paclitaxel-treated cells showed them, however. Like colchicine-treated cells, cells treated with Colchitaxel rarely showed micronuclei. In the multinucleated cells, the nuclei could either be entirely separate or partially fused, so that the outline of the nucleus was irregular (Fig. 26). 85

Fig. 26: Appearance of nuclei in cells treated with MT inhibitors compared with untreated

controls. Cells were treated with: A) 500 nM colchicine, B) 600 nM paclitaxel, C) 16 μM

Colchitaxel, or D) solvent vehicle alone. Cells containing multiple, large nuclei are indicated by straight arrows. Paclitaxel-treated cells contain micronuclei (curved arrow). Some Colchitaxel

treated cells treated show multiple nuclei or enlarged, lobed nuclei suggestive of nuclear fusion.

These cells appear flatter and more adhesive than those treated with the other compounds. Bar

=25 μm.

86

CHAPTER 7. DISCUSSION

MTs are highly dynamic assemblies of protein tubulin, which polymerize and depolymerize in cells, and undergo two interesting kinds of dynamics called dynamic instability and treadmilling. MTs are also involved in maintaining the shape of interphase cells and forming the mitotic spindle. Given the success of tubulin as a cellular target for antitumor agents, a large and diverse group of natural tubulin-interacting compounds have attracted great attention as potential antimitotic agents and anticancer drugs. Paclitaxel is known to polymerize MTs and increase the bundling effect when given as a single agent. In contrast, colchicine depolymerizes

MTs by binding to tubulin dimers and preventing addition of new subunits to the MT.

The current research takes advantage of quantitative and qualitative methods of morphometric analysis, to determine if the reversal of the transformed phenotype by MT inhibitors is a general phenomenon. A signature-type effect was defined for cells maintained in culture for a prolonged period of time. Some of the theoretical variables, also call factors, related to the status of “cancer-type” cells, including: a) filopodia at the cell edge, b) aberrant vesicle trafficking, c) PAK-dependent protrusions, and d) degree of rounding up. The great array of shapes and features are evolved and maintained by the cell for specific physiological reasons.

The shape may mirror the effect of physical forces that operate on cell membranes (65).

Intracellular trafficking and cell adhesiveness are also key processes contributing to the shape of the cell. These determinants are implicated in the shape changes accompanying the development of tumorigenicity (16). In addition to the features of cells itemized above, changes of shape can be detected as changes in fractality of the cell and its contours. 87

7.1 Reversal of Shape Phenotype

Previous studies from our laboratory showed that a combination of colchicine and

paclitaxel restored the shape phenotype to normal-type (16). The data suggested that reversal of

the phenotype by this combination was restricted to IAR20 PC1 cells. The studies of Chapters 3

and 4 were designed to compare the effects of other combinations of paclitaxel delivered

simultaneously with various depolymerizing agents, in the IAR20 PC1 cell line. Because it was

found previously that replacing the paclitaxel with other taxanes (baccatin II, cephalomannine)

did not lead to phenotype reversal, paclitaxel was the only MT-stabilizing compound used.

I first investigated the hypothesis that each combination of paclitaxel with a

depolymerizing agent might have a unique mode of action and a unique effect on MT

organization. In this case, there might be no connection between the MT inhibitor of vinblastine and paclitaxel, which modeled the combination used in clinical practice, and the results previously obtained with colchicine and paclitaxel in the in vitro shape analysis assay. The

analysis indicated, however, that combinations of paclitaxel with various depolymerizing agents

all changed the shape phenotype of the IAR20 PC1 cells when compared to the control. There

was no one treatment that could be said to have effects reversing the phenotype more towards

normal as compared to the others. In addition, the MT inhibitor combination of vinblastine and

paclitaxel was indistinguishable from colchicine and paclitaxel, in its effects on MT arrangement

(see below).

Thus, any combination of MT depolymerizing agent with paclitaxel reversed the shape

phenotype as evaluated on a quantitative basis. The shape analysis method also enabled me to 88

define specific features that were changed in cells exposed to a combination of paclitaxel with

various MT depolymerizing agents. The features represented by factors #4, #5, #8, and #12

generally moved towards their normal phenotype. For other factors, especially #7 and #13, the means moved towards a more cancer-type value. In some treatments, the feature moved in the same overall direction designated but didn’t undergo a significant change. For example, the 3:1 combination of paclitaxel to podophyllotoxin (3P+1P) had significant effects on factors #1 and

#12, which shifted toward more normal values, but it had the opposite effect on factor #7. (All combinations tested shifted the #7 values towards more transformed phenotype.) Assuming that such factors might be deleterious, because they may reflect features related to invasion or metastasis, I assigned a penalty to any treatment that caused a shift toward more transformed values. In the case of 3P+1P, if a penalty value was assigned that was twice the value assigned to a reversal, the penalty score balanced out at zero.

In cells exposed to equimolar colchicine and paclitaxel, factors #1, #7, and #13 changed in the direction of more transformed phenotype. They were countered by changes in their properties represented by #4, #5, and #12 toward the more normal phenotype, so that the overall score was -3. The highest scores were awarded to the equimolar combination of paclitaxel to podophyllotoxin (+2) and the 3:1 paclitaxel to nocodazole (0). Equimolar combination of

Paclitaxel to nocodazole balanced out to be (-1). None of these scores are bad. This result suggested that nocodazole and especially podophyllotoxin might be good subjects for clinical investigation. Based on the results, it can be interpreted that other interesting combinations might be equimolar vinblastine with paclitaxel (-1) and the combination used in the original in vitro studies, 3:1 paclitaxel with colchicine (-1). These combinations might be suitable for future 89

preclinical investigation, as they have some effects maximizing the more normal phenotype.

Moreover, the results indicated that changes in shape phenotype were not dependent upon the

exact ratios of paclitaxel and depolymerizing agents, since equimolar ratios in many cases gave a

similar response to the 3:1 ratios.

Equimolar combinations of paclitaxel and colchicine or 3:1 paclitaxel and

podophyllotoxin increased the values of #4, the factor for prevalence of the filopodia. Because

#4 accounted for the greatest quantitative change in transformed cells, the regulation of these

features has been subjected to numerous studies. In other model systems, filopodia were induced

by the activation of the GTPase, Cdc42 (66, 67). Although the mechanisms of filopodia regulation are poorly understood, it has been suggested in studies of primary hepatocytes that paclitaxel induced a reorganization of the apical actin network and extension of filopodia (68).

A third concept of regulation involves endocytic and exocytic pathways, as it has been suggested that membrane trapping leads to a secondary reduction in the amount of membrane available for filopodium formation. The MT inhibitors might reduce the membrane stored in cells by reducing exocytosis and endocytosis, leading to an increased prevalence of filopodia.

In both unicellular and multicellular organisms, polarized cell growth is crucial for precise cell morphologies that carry out specialized functions. Polarization activity can be well explained in the model yeast S. cerevisiae which undergoes polarized cell growth in various stages of the life cycle (69). Polarized growth in yeast requires organization of actin cytoskeleton, polarity proteins, and regulation of signal transduction. The formation of the bud involves Ras family of G proteins, Bud1p, required for bud site selection, and Rho family small 90

G protein, Cdc42, which is important for the organization of the bud site (70). Formin found in

mammalian cell is the yeast homologue of Bni1p. Both are involved in the nucleation of new

actin filaments. In mammals, formin is involved downstream of Rho to form focal contacts and

stress fibers. Bni1p, which is a cortical protein, has been shown to interact with Cdc42 and induce actin cables in an Arp2/3-independent manner (71, 72). Bni1p is not only involved in the actin-dependent polarization but also in MT function in the formation of spindle formation during cell division (70, 73). Studies have indicated that bud formation is due to the complex

formed of Bni1p physically interacting with Rho small G proteins Cdc42, actin, and two actin-

binding proteins (profilin and Bud6p). The proline- rich FH1 domain of Bni1p binds profilin and this complex regulates actin polymerization at cortical sites. The actin structures involved in bipolar budding are also required for spindle orientation in cell division (74). Studies indicate that they are involved in the polarization of the MT array by stabilizing MT and their effectors at the cell cortex, which in turn modulates the activities of Rho family GTPases (75).

Pre-bud initiation in yeast serves as a model for cortical actin organization in a primitive

organism where Bni1p appears to designate the site of filament assembly. A second model

system has been studied, to further elucidate the mechanism of actin bundle formation. Two

intracellular pathogenic bacteria, Shigella flexneri and Salmonella typhimurium, show the ability to arrange the actin filaments in parallel bundles. In S. flexneri, the bacterial protein IpaC is sufficient to promote actin polymerization and the formation of filopodial extensions when introduced in the host cell cytosol (76, 77). IpaC interacts with beta-catenin and destabilizes the cadherin-mediated cell adhesion complex in order to exit the cell (75). IpaC-mediated actin 91

organization is Cdc42-dependent. The actin-dependent motility relies upon filament extension by

subunit additions.

All pathogens evaluated so far have been shown to utilize Arp2/3 complex. The

important difference between the other invasive pathogens and S. flexneri is that the latter shows

little cell binding ability (77). Actin polymerization and movement through the cytosol is mediated by bacterial protein IcsA/VirG. IcsA are the member of family involved in their own translocation across the cell membrane. Once the IcsA is exposed on the bacterial surface, it is targeted to and inserted into one pole of the bacterial body, where actin assembly is initiated.

IcsA is involved in binding to the host N-WASP (Wiskott-Aldrich syndrome protein) via the latter’s glycine-rich repeats and recruiting it to bacterial surface. N-WASP binds to Cdc42, via its

Cdc42/Rac interactive domain (CRIB), leading to Arp2/3 activation (77). This causes ARP2/3 activation at the bacterium surface by unfolding cytoskeletal proteins containing Ena/VASP homology domains. The above mechanism has been explained in Listeria where the Ena/VASP proteins interact with profilin-actin complex, leading to actin filament addition abutting the bacterial surface.

Much less is known about the remaining features that contribute to the transformed phenotype of IAR20 PC1 cells. The activation of PAK5 destabilizes the F-actin network and increases formation of PAK dependent protrusions (78), of which factor #7 is the descriptor. The factor reflecting the cell spreading was #13. Since the elevated levels of #13 suggested a membrane trapping phenotype, this could have accounted in part for the reduction of factor #4 in treated cells. The values of #4 were uniformly enhanced in treated cells, whereas those of #13 92

also generally increased. This effect suggested that the rounding up of cells treated with MT

inhibitors originated in adhesive or cytoskeletal changes. Previous studies have shown that factor #8 of the middle contour was depressed in cells treated with MT inhibitors.

7.2 Rearrangement of MT Arrays

As previously reported, cells treated with single agents individually showed complete

polymerization and depolymerization of the MT array after paclitaxel and colchicine exposure,

respectively (16). The conventional view of MT inhibitors as chemotherapeutic compounds is that they halt chromosome separation, and this causes the cells to enter a cell death pathway. I

postulated that each MT inhibitor combination would have a unique effect on the MT arrangement, which would correlate to effects in the phenotype assay. But results from the

current study indicated there were subtle differences between the combination of paclitaxel with

colchicine or vinblastine or podophyllotoxin. Extreme contraction of the MTs occurred after certain combinations, and left a MT free cell boundary.

This was seen in the combinations of paclitaxel with vinblastine or podophyllotoxin. In the combination of colchicine with paclitaxel, there were free MTs extending out either perpendicular or at an angle to the cell edge. Frequent bundling of MTs was among the effects seen when the MT pattern was analyzed. With the combination of paclitaxel and vinblastine, the

MTs frequently formed a completely parallel arrangement or a fountain-like spray pattern. The latter was occasionally associated with fragmentation of MT ends. Due to similar patterns of MT rearrangement upon exposure to various combinations, it might be concluded that the combinations might not differ substantially in their MT arrangement effects. 93

There might be a possibility that the combinations which do not have a very dramatic

effect on MT arrangements, might be affecting in the MT dynamicity, MTOC or at the

posttranslational level. At substoichiometric ratios to tubulin, both paclitaxel and MT-

depolymerizing agents restrict periods of growth and shrinkage of the MT (28, 29, 54). Thus,

synergy at the molecular level could be due to the two classes of agents affecting dynamicity

through different mechanisms. Even when two MT-polymerizing agents were used, however,

synergistic inhibition of MT dynamicity could be observed (79). Paclitaxel and discodermolide

also synergistically affected G2-M arrest, proliferation, and apoptosis. Some workers speculate

that such synergy might arise from the different binding affinities of the two molecules for

different tubulin isotypes present in the MT.

Another study of three MT polymerizing agents confirmed that paclitaxel and

discodermolide had complementary effects, whereas eleutherobin and B could

substitute for Paclitaxel (80). Considering the complexity of interrelationships among various

signaling and cell death pathways, however, it is easy to imagine that changes in dynamicity

affect other MT-mediated processes in addition to mitotic division. Inhibition of the extracellular

regulated kinase (ERK) blocks the cytotoxic effect of paclitaxel and the accumulation of sub-G1 cells (81), suggesting that the ERK pathway is essential to the mechanism of cell killing.

Another target where MT combinations may be affecting might be on the isoforms of

MTs. Also, it is been suggested that, combination may not have dramatic affect on the MT polymer mass, but might change the dynamicity at the MTOC level, which might suggest the mechanism of action especially at lower doses. Thus, it can be interpreted that combination 94 effects on MT pattern donot have a unique effects, but they definelty differ from the control which was treated by solvent. The phenotype induced by the MT inhibitor combination is qualitatively similar to that of promoter-treated cells but differs in direction. Therefore, reversal of cancer cell phenotype suggested that the same downstream mechanisms i.e. the activation or inhibition of protein kinase C-mediated networks, is implicated in both promotion and MT inhibitor combination effects. Like the apoptotic endpoint described above, effects in the shape assay were found after only a brief exposure to the agents. Although phenotype reversal could well rely upon inhibition of MT dynamicity, it could not be working at the level of MT integration with the kinetochore. Retardation of cell division could not be detected over so short a time as 2 h.

7.3 Importance of in Vitro Models

The importance of the in vitro experiments is that the amount of experimentation that can be done clinically, to elucidate mechanisms of the curative effect, is very limited. Patients cannot be exposed to a drug concentration beyond the optimal tolerated dose, because the side effects would be life-threatening. Due to this fact, clinical investigators cannot distinguish between the possibilities that drugs are additive or synergistic in their effects. Two drugs, for example, vinorelbine and docetaxel, can be delivered together at the optimal tolerated dose of each, due to their non-overlapping toxicities. In the in vitro assay, doubling the dosage of colchicine or vinblastine does not cause effects similar to those observed after administering the two agents together. Hence, the in vitro results on MT inhibitor combinations suggest that the cells exposed to such a combination have unique attributes. Thus, the possibility that the effects of such agents are synergistic can only be investigated in such an in vitro model. 95

The fractal dimension represented a new variable that could be used to study the

differences between control and treated cells. Many natural objects show similar levels of

complexity at different scales, a feature which can be quantified by fractal geometry. An

advantage of the fractal method is that it describes the irregular shapes of objects by assigning a

characteristic value of the fractal dimension. In contrast to factors, which integrate information

from 1, 2, or 3 contours, the fractal method provides a means of assessing a feature that is

specific to each level in the cell. In an analysis that was carried out by digitizing high fidelity paper images, I subjected data on the effects of an MT inhibitor combination on IAR20 PC1 cells to fractal analysis. The results indicated that the inner and outer contours undergo the most drastic changes in fractal dimension. The outer (cell edge) contour is involved in calculation of factors #4 and #5 (16, 82). I conclude that fractality is related to factors of IAR20 PC1 cells.

Fractal analysis reflects the complex effects of a combination of MT-interacting agents, and it may provide a practical index of complex morphological changes.

7.4 Basic Studies on the Clinical Combination

In addition to in vitro studies being used to predict the clinical efficacy of MT inhibitor

combinations, the results of clinical studies may shed light on fundamental effects of paclitaxel

and vinblastine that contribute to the mechanism of phenotype reversal. The hypothesis

underlying the clinical inquiry was that, if the therapeutic effect relied upon rearrangement of the

MT array, then the objective response would not depend on the ratio of the combination. But when results were analyzed it was found that with lower ratio of the combination, the response was slightly better than with the higher ratios. In clinical settings, the efficacy might be double 96

that found with either drug alone because both paclitaxel and vinblastine block mitosis and

inhibit cell proliferation by inhibiting the dynamics of spindle MTs, as described elsewhere.

Based on the frequency data derived from the clinical reports, it appeared that same day drug delivery, especially on Day 1 of the treatment cycle, ensured a higher cumulative response rate. Based on the analysis, it can also be concluded that the combination of docetaxel and vinorelbine (D+V) alone is better than the combination interspersed by vinblastine. This analysis of the clinical study was seen to correlate with the in vitro assay. Given that much more potent

MT-polymerizing and depolymerizing agents are now available, future work might be directed to determining whether their effects in combination are superior to those of paclitaxel and vinblastine.

Thus, an explanation of the synergy between MT inhibitors awaits the demonstration of a mechanism. In past studies, it has been difficult to determine which, if any, aspects of MT dynamicity or reorganization were related to the therapeutic efficacy of inhibitor combinations.

Although cell phenotype studies are suggestive of a synergy between MT inhibitors, it is more difficult to distinguish synergistic and additive effects in the clinical anticancer studies. The purpose of the present research was to make a single agent from paclitaxel and colchicine, which could be used to determine whether a single agent could have the same effect as the combination of agents. Indeed, the coupled agent retained some of the effects of the combination of starting agents but had fewer effects on MT structure. Future investigation of colchitaxel-treated cells by the shape assay will be useful in determining whether the MT rearrangements coincide with the reversal of the properties of cancer cells. 97

7.5 Effects on EB1

There has been great interest in EB1 because of its ability to bind to adematomous polyposis coli (APC) protein, which is mutated in a heritable form of familial colorectal cancer.

EB1 is localized on plus ends of microtubules along with APC, the p150glued subunit of dynactin, CLIP-170, and CLIP-115, and others, including subunits of MT motors. APC binds to the plus end in both EB1-dependent and -independent fashions (83). One of the roles of APC in cells is the formation of stable molecular linkages between plus end capping proteins, such as

EB1, and adhesive sites in the cortical cytoplasm (84-86). EB1 is distributed along the full length of the microtubule in mammalian cells and is concentrated at both its plus tip and at the centrosome (87, 88).

In the IAR20 PC1 cells used in the present research, however, EB1 was localized predominantly at the plus ends of the microtubule. The evidence suggested that substantial amounts of protein may exist free in cytoplasm but not specifically on the centrosome. The plus end binding protein, CLIP-115, showed the same distribution as the EB1 protein (data not shown). The results of the current research indicated that Colchitaxel displaced EB1 from plus ends. Moreover, the evidence suggested that it profoundly affected the stable, long EB1 comets at the cell edge, where the stabilizing cortical complexes are presumably formed. These comets were evident in reconstructions from the 3D images that were viewed in VRO form.

In the VRO, it was clear that MTs at the cell end appeared to attract a longer cap of EB1 than those elsewhere in the cell. Furthermore, these plus ends extended from near the upper surface to the substratum. In interphase cells, such structures move like comets in time-lapse 98 recordings, as plus end caps of microtubules emerge from the MTOC, proceed to the cell edge, and finally disassemble there (89). EB1-cortex interaction plays a poorly understood role in shape and motile specializations of mammalian cells, but it has been studied in more primitive and simpler cell types. The EB1 homologue in budding yeast, Bim1, interacts with a protein of the bud cortex, Kar9, to tether MTs from the spindle to the bud neck (90). Ablation of the Bim1 gene caused microtubules to undergo fewer transitions, grow less, and depolymerize more slowly than in wild-type cells. Thus, Bim1 promotes MT dynamicity in yeast (91). Consistent with these findings, EB1 was found to enhance the assembly or stability of the MT in vitro (92).

The current concept of EB1 is that it interacts with other proteins in a raft-like complex, which ultimately dictates how the plus end of the microtubules becomes stabilized at the cell edge or the kinetochore. At least in interphase, growth on the plus end is processive until the MT reaches the targeted area of cytoplasm. It is thought that episodes of growth and shrinkage then become an important mechanism for the MT to seek an anchoring point on the cell edge or kinetochore (83, 89, 91, 93, 94).

In vivo experiments on EB1 showed diverse effects upon MT dynamicity, which presumably depended upon what proteins were available in the cell to interact with it on the MT.

However, dynamicity was maximal in dividing cells (14) and EB1 depletion during mitosis or meiosis left the cells unable to form a functional aster. Although numerous studies have shown that EB1/Bim1 is an anti-pause, pro-rescue, and anti-catastrophe, its effects on microtubule growth and shortening rates were dependent on the cell cycle and model system studied. In

Xenopus, growth was enhanced by EB1 in interphase and shortening inhibited in meiotic 99

extracts. In Drosophila cells, EB1 had no effect on either rate (94). Since EB1 appeared to play a

role in stabilizing other proteins, for example APC and formins, mDia1 and mDia2, on the MT, it

is possible that its effects were modulated by the presence of other proteins on the surface. For

example, CLIP-170 and MAP2 acted as rescue factors (85, 95, 96). CLIP-170 and Tip1p, the

CLIP-170 homologue in fission yeast, also stabilize the microtubule at the cell end by

suppressing catastrophe (97, 98). In experiments where the mutant, truncated form of APC was

present, the dynamics of EB1 comets were compromised by an increased frequency of the pause

phase. Thus, rescue and catastrophe activities attributed to EB1 may be implemented by other

proteins that are bound to rafts in part by EB1. Only in the case of MAP2 has the exact role of

such proteins in MT dynamics been clarified (95).

7.5 Effects on Cell Cycle

Suppression of microtubule dynamics by enhancement of the stability of the plus end has

been associated with the enhancement of anti-proliferative activity of various antimitotic agents

(99). Mitotic arrest delays the onset of anaphase until all chromosomes have been connected to

the spindle and thus prevents chromosomes from becoming stranded at the metaphase plate at anaphase. Although investigators have assumed that, if cells are incapable of forming a

functional spindle, this would trigger the mitotic checkpoint and activate the mechanism of cell death implicated in the therapeutic effect, the relationship between the therapeutic effects of MT

inhibitors and mitotic arrest remains obscure. Some cells overcome mitotic arrest, escaping

apoptosis, decondensing the chromosomes, reconstituting the nuclear envelope and entering G1 phase (100). In some models, persistent block of mitosis is required in order to induce apoptosis

(101). Recent evidence suggests, however, that paclitaxel can induce DNA damage and inhibit 100

cell growth independent of arrest in the G2 or M phases (102,103). Thus, cells treated with MT-

stabilizing drugs might enter apoptosis long before they divide. In certain cells, there is evidence

that mitotic slippage induces an alternative death pathway involving nuclear fragmentation and

formation of a sub-G1 chromosomal complement. This is associated with a loss of viability, but

the mechanism of cell death is unknown. Cells in which the checkpoint protein BubR1 was

knocked down showed enhanced killing by paclitaxel (63). On the contrary, in leukemia cells,

the loss of checkpoint controls enhanced the cells’ resistance to paclitaxel (104). The existence

of a specific G1 checkpoint triggered by hyperploidy of postmitotic cells is controversial (100,

105, 106), and indeed, such a cell death pathway may be specific to certain cell lineages.

Workers have suggested enhancement of mitotic arrest after aberrant spindle formation as a

mechanism for cell killing, but it remains to be determined whether MT inhibitor combinations enhance the cell’s likelihood of taking a death pathway.

This view is supported by the highly synergistic effect of the inhibitor combination on

mitotic figure aberrations. If mitotic arrest were implicated, however, one would expect

colchicine (ED50 3-4 nM) to be far more effective as a chemotherapeutic agent than paclitaxel

(ED50 ~50 nM). Yet, paclitaxel is arguably the most potent anti-cancer agent ever discovered.

Many differences between the drugs’ effects at the cellular level were found. Micronuclei are

often formed after paclitaxel exposure, which is thought to be an indication that multiple spindles

were formed or that chromosomes failed to line up on a single division plane. Cells with

micronuclei were almost confined to cultures treated with paclitaxel. IAR20 PC1 cultures treated with the individual starting compounds developed a high percentage (>70%) of multinucleated cells. This represented the cells that entered mitosis but failed to undergo apoptosis after mitotic 101

arrest. Curiously, a lower percentage (<30%) was formed in cultures treated with Colchitaxel.

The simplest explanation for this finding was that the treatment stopped cells from cycling into

mitosis, or, alternatively, by conserving the integrity of the microtubule, the compound enhanced

formation of the spindle and thereby allowed normal segregation. Both possibilities are supported by results of the present research, since either halting of the cycle or cell killing at mitosis would limit the pool of cells subject to mitotic slippage and thereby reduce the frequency of multinucleated cells.

The comparable percentage of multinucleated cells formed with the combination of

starting compounds was high, and the endpoint suggested even more synergy between the

compounds that that of aberrant mitoses (Table 16). The data suggested that Colchitaxel retained

the comparatively large impact exerted by the inhibitor combination on formation of

multinucleated cells. Why are the ED50 values for the two endpoints dissimilar? At present, it is

hard to explain the therapeutic efficacy of MT inhibitor combinations in relation to a mechanism

of cell killing. There is little doubt that all the mechanisms involved some cell killing, since all

the levels of treatment above ED50 values led to obvious depletion of cells from the cultures

(data not shown).

Besides the cell cycle-dependent mechanisms of attrition mentioned above, one possible explanation was that the Colchitaxel and the inhibitor combination selectively affected a postmitotic mechanism of programmed cell death. Further work will be needed in order to understand the smaller impact of Colchitaxel on mitotic aberrations than on multinucleation. MT inhibitors and MT Array Concurrent exposure to the separate compounds was originally found to 102

affect MT organization in unique ways. The combination caused fringe-like arrays of MTs to emerge from the cell edge (16). Occasionally, MTs also appeared as if they were radiating from focal sites in the cytoplasm. These effects appeared common to cells treated with the combination of MTs or Colchitaxel. Since other effects of paclitaxel such as microtubule bundling were lacking, these MT rearrangements in Colchitaxel-treated cells may have represented subtle effects caused by displacement of the plus end raft proteins. Indeed, epithelial cells have recently been found to organize MT into focal structure in the basal cytoplasm. It is possible that the focal sites of MT initiation in cultured IAR20 PC1 cells are the equivalent of the acentrosomal MT arrays in intact tissue. Structural aspects of MT organization are fertile ground for further research on correlates of therapeutic efficacy.

Development of more effective agents and intervention earlier in the disease course are

two approaches that can be taken to improve chemotherapy. Indeed, a number of new MT

inhibitor such as estramustine (107) (108) and EM012 (99) have been described which may be

promising in this respect. The results of the current experiments suggest that MT inhibitors have

multiple and complex effects in vivo, and that further work will be required to find the

physiological features correlated with therapeutic effects of MT inhibitors.

103

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