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Chemotherapeutic Drug Cytotoxicity Enhancement in Human Cells in Culture

by Seamus Coyle B.Sc. M.Med.Sc

A thesis submitted for the degree of Ph.D.

Dublin City University

The research work described in this thesis was carried outunder the supervision of

Professor Martin Clynes

National Cell & Tissue Culture Centre School of Biological Sciences

August 1996

I hereby certify that this material, which I now submit for the assessment on the programme of study leading to the award of Ph.D. is entirely my own work and has not been taken from tiie work of others save and to the extent that such work has been cited and acknowledged within the text of my work.

Signed: ID. No.: 9 2

TABLE OF CONTENTS

Abstract 1

Acknowledgements 2

Abbreviations 3

1.0 Introduction 6

1.1 Introduction 71.2 Chemotherapy 81.2.1 Anthracyclines 81.2.2 V inca Alkaloids 101.2.3 Epipodophyllotoxins 131.2.4 Antipyrimidines 141.3 The Cell Cycle 151.3.1 Gi phase 171.3.2 S phase 191.3.3 G2/M phase 201.3.4 Cyclin Dependent Kinase Inhibitors 231.4 Chemotherapy and the Cell Cycle 241.4.1 G2 arrest 241.4.2 G2 andp34cdc2 251.5 Apoptosis 281.5.1 Bcl-2 291.5.2 ICE / Apoptotic proteases 331.6 Apoptosis and Chemotherapy 351.7 Cell Cycle and Apoptosis 371.8 Necrosis 381.9 Multidrug Resistance 401.9.1 P-glycoprotein 411.9.2 Multidrug Resistance Related Protein 441.9.3 Transporter-related with Antigen Presentation 451.9.4 Lung Resistance-related Protein 461.10 Alternative Mechanisms of Resistance 471.10.1 Topoisomerase II 471.10.2 Glutathione S-transferases 491.10.3 Bcl-2 / p53 501.10.4 Metallothioneins 511.10.5 DNA repair mechanisms 521.11 MDR circumvention 531.11.1 Pharmacological circumvention 531.11.2 Genetic circumvention 5 6

Section Title Page

1.12 Non-MDR chemosensitisers 581.13 Aims of this thesis 60

2.0 Materials and Methods 61

2.1 Water 622.2 Glassware 622.3 Sterilisation 622.4 Medium preparation 622.5 Cell lines 632.5.1 Sub-culture of cell lines 642.5.2 Cell counting 652.5.3 Cell freezing 662.5.4 Cell thawing 662.5.5 Sterility checks 672.6 Mycoplasma detection 672.6.1 Indirect staining procedure 672.6.2 Direct Staining 682.7 Toxicity Assay 692.7.1 Toxicity assays 702.7.2 Acid phosphatase assay 702.7.3 Circumvention assays 712.7.4 Chemotherapeutic drug pulse assays 722.7.5 IC50 calculation 722.7.6 Combination Index calculation 732.8 Flow cytometry 752.9 Time lapse videomicroscopy 762.10 Western blotting 772.10.1 Sample preparation 772.10.2 Protein quantitation 782.10.3 Gel electrophoresis 782.10.4 Western blotting 792.10.5 ECL detection 802.10.6 Autoradiograph film processing 812.11 Immunoprcipitation 812.12 Protein kinase assay 822.13 Plasmid DNA Isolation 832.13.1 Preparation of competent cells for transformation 832.13.2 Transformation of E. Coli 832.13.3 Plasmid Miniprep 842.14 Transfection procedures 852.14.1 Electroporation 852.14.2 Calcium phosphate co-precipitation 862.15 fi-Galactosidase activity assay 872.15.1 Preparation of cell extract 872.15.2 Assay 872.16 Selection of transfectants 88

2.16.1 Isolation of clonal transfectants 882.17 RNA extraction 892.18 Reverse transcriptase reaction 912.19 Polymerase chain reaction 912.20 Electrophoresis of PCR products 92

3.0 Results 94

3.1 Chemotherapeutic drug combinations 953.2 MDR circumvention 1103.3 MDR 1 ribozyme transfection 1173.4 MDR1 gene transfection 1253.5 Test Compound Screening 1333.6 Drug/Pulse / Scheduling Assays 1433.7 DLKP pHaMDRl/A #2 Scheduling 1503.8 Cell cycle distribution 1543.9 Time-Lapse Videomicroscopy 1793.10 Cyclin E/CDK2 kinase activity 1863.11 Cyclin E/CDK2 protein analysis 190

4.0 Discussion 197

4.1 Chemotherapeutic drug combination 1984.2 Circumvention 2004.3 MDR1 ribozyme transfection 2034.4 MDR1 transfection 2064.5 Test compound screening 2084.6 Scheduling 2114.7 DLKP pHaMDRl/A #2 Scheduling 2134.8 Cell cycle distribution 2154.9 Cell death kinetics 2174.10 Cell cycle proteins 220

5.0 Conclusions 224

6.0 References 230

Abstract

Chemotherapeutic Drug Cytotoxicity Enhancement in Human Cells in Culture

Seamus Coyle

This thesis examined the effect of combinations of different drugs on toxicity to cancer cells in vitro. Combinations of a known anticancer agent with one of a series of coded non-toxic compounds known to be safe for human use were assayed. Significant enhancement of toxicity of adriamycin and vincristine but not 5- fluorouracil, was observed with three compounds from the series. Studies in a wide range of cellular models showed that this combination effect was not observed in cells overexpressing P-glycoprotein and that the mechanism involved was not inhibition of P-glycoprotein.

An assay was developed to analyse how synergy was affected when cells were exposed to the compounds at different times relative to chemotherapeutic drug pulse exposure. Activity of the active compounds was observed only during 24 hours subsequent to 2 hours drug exposure. Pre-treatment or treatment at later times was ineffective.

Flow cytometric analysis was used to determine the cell cycle distribution of these test compounds in DLKP-SQ, a human lung carcinoma clonal cell line. U-l (active in the combination assay) on its own was shown to induce a transient G /S arrest. There was no apparent effect on the cell cycle distribution using A-l (inactive) or N-l (active). A combination of adriamycin and U-l caused an increased delay in the G, phase and the S phase compared to adriamycin alone. Vincristine in combination with U-l displayed an increased G2/M arrest compared to vincristine treated cells alone. DLKP-SQ treated with 5-fluorouracil alone and in combination showed no effect on cell cycle distribution.

Time lapse videomicroscopic studies demonstrated that vincristine induced apoptosis within 24 hours and the rate of apoptosis increased in the combination. Cells underwent apoptosis 24 hours after treatment with adriamycin but surprisingly there was no significant difference in the rate of apoptosis between adriamycin alone and in combination.

To investigate if the combination effect was due to interference with proteins controlling cell cycle progress, the levels of cyclin E and CDK2 were determined using western blotting; Cyclin E/CDK2 kinase activity was also measured.

1

Abstract

Chemotherapeutic Drug Cytotoxicity Enhancement in Human Cells in Culture

Seamus Coyle

This thesis examined the effect of combinations of different drugs on toxicity to cancer cells in vitro. Combinations of a known anticancer agent with one of a series of coded non-toxic compounds known to be safe for human use were assayed. Significant enhancement of toxicity of adriamycin and vincristine but not 5- fluorouracil, was observed with three compounds from the series. Studies in a wide range of cellular models showed that this combination effect was not observed in cells overexpressing P-glycoprotein and that the mechanism involved was not inhibition of P-glycoprotein.

An assay was developed to analyse how synergy was affected when cells were exposed to the compounds at different times relative to chemotherapeutic drug pulse exposure. Activity of the active compounds was observed only during 24 hours subsequent to 2 hours drug exposure. Pre-treatment or treatment at later times was ineffective.

Flow cytometric analysis was used to determine the cell cycle distribution of these test compounds in DLKP-SQ, a human lung carcinoma clonal cell line. U-l (active in the combination assay) on its own was shown to induce a transient Gj/S arrest. There was no apparent effect on the cell cycle distribution using A-l (inactive) or N-l (active). A combination of adriamycin and U-l caused an increased delay in the Gj phase and the S phase compared to adriamycin alone. Vincristine in combination with U-l displayed an increased G2/M arrest compared to vincristine treated cells alone. DLKP-SQ treated with 5-fluorouracil alone and in combination showed no effect on cell cycle distribution.

Time lapse videomicroscopic studies demonstrated that vincristine induced apoptosis within 24 hours and the rate of apoptosis increased in the combination. Cells underwent apoptosis 24 hours after treatment with adriamycin but surprisingly there was no significant difference in the rate of apoptosis between adriamycin alone and in combination.

To investigate if the combination effect was due to interference with proteins controlling cell cycle progress, the levels of cyclin E and CDK2 were determined using western blotting; Cyclin E/CDK2 kinase activity was also measured.

1

Abbreviations

ABC ATP binding cassette

ActD Actinomycin D

Adr Adriaraycin

AMSA Amsacrine

ATCC American Tissue Culture Collection

ATP Adenosine tri-phosphate

bp Base pair

BSA Bovine serum albumin

BSO DL-Buthionine-[S ,R]-sulfoximine

CaCl2 Calcium chloride

CAK Cdc2-activating kinase

CaP04 Calcium phosphate

CDI Cyclin dependent kinase inhibitor

cDNA Complementary deoxyribonucleic acid

CDK Cyclin dependent kinase

CHO Chinese hampster ovary

CHX Cycloheximide

Cl Combination Index

CML Chronic myelocytic leukeamia

CsCl Cesium chloride

CTL Cytotoxic T-cell

CysA Cyclosporin A

Da Dalton

DEM Minimum essential medium eagles

DEPC Diethyl pyrocarbonate

DHFR Dihydrofolate reductase

DMEM Dulbeccos mimimum eagles medium

DMSO Dimethyl sulphoxide

DNA Deoxyribonucleic acid

3

DTT DL-Dithiothreitol

ECL Enhanced chemiluminescence

EDTA Ethylene glycol-bis(J3-aminoethylether)-N,N

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