Investigation of the molecular basis of cisplatin sensitivity in testicular germ cell tumours

Over 80% of metastatic testicular germ cell tumours (TGCT) are cured using cisplatin- based chemotherapy. Five studies were undertaken to investigate the molecular basis for the hypersensitivity of testicular tumour cells to cisplatin. Firstly, to determine the number of genes involved in the hypersensitivity of testicular tumour cells to cisplatin, four cell lines were fused with each other and complementation analysis was performed. Hybrids between testicular tumour cell lines were sensitive to cisplatin, indicating that the sensitivity might be controlled by a common mechanism and possibly by one gene. Secondly, to try to identify the gene, a cDNA library carrying a G418-resistant marker from a cisplatin resistant human cell line was transfected into a cisplatin-sensitive mouse embryonal carcinoma cell line. G418 and cisplatin-resistant primary transfectants were selected and G418-resistant secondary transfectants were isolated. However, the secondary transfectants failed to show resistance to cisplatin indicating that the primary cisplatin and G418-resistant transfectants were non-specific. Thirdly, to characterize a cisplatin resistant secondary transfectant, which was isolated by transfection of a cDNA library into a human testicular tumour cell line, the plasmid carrying the DNA insert was amplified and transfected into 5 tumour cell lines. However, it failed to confer resistance to cisplatin suggesting that the cisplatin resistance in the primary transfectant was non-specific. Fourthly, to identify human chromosomes carrying genes controlling cisplatin sensitivity, a mouse embryonal carcinoma cell line was fused with a human bladder tumour cell line. However, in contrast to most of the human-mouse hybrids, the hybrids isolated in this study showed little loss of human chromosomes. Therefore, it was not possible to identify which individual human chromosomes are responsible for causing changes in the sensitivity to cisplatin in the hybrid cells. Lastly, to compare the gene expression of testicular and bladder tumour cell lines before and after exposure to cisplatin, differential display RT-PCR was performed. Ten differentially displayed bands were characterized and sequenced. A DNA fragment located on chromosome 5q31 was found to be upregulated by cisplatin in testicular tumour cells