Cell biological responses of prostatic tumour cell lines to irradiation and anticancer drugs

[摘要] ENGLISH ABSTRACT: The classic prostate cell lines, DU145, PC-3 and LNCaP, have served as a valuablecell biological model for research into prostate cancer. However, their relevance maybe limited because they derive from metastatic, and not from primary normal andtumour epithelium. The cell lines (1532T, 1535T, 1542T, 1542N and BPH-l) havebeen derived from primary benign and malignant human tumour prostate epitheliumand may be more representative. Using these cell lines I have examined the role ofbasic cell damage responses (repair, checkpoint activation, apoptosis and associatedsignalling proteins, and the influence of androgen status) in cell inactivation, and itsrelevance to treatment.Numerous studies have suggested that loss of p53 function leads to resistance tochemotherapeutic agents and irradiation. It is shown here that the p53-inactive celllines are, in fact, the most sensitive to chemotherapeutic agents such as etoposide,vinblastine and estramustine, whilst the p53 wild-type cell line, LNCaP, is the mostradiosensitive. Notwithstanding the effects of p53 degradation by the HPV -16 E6viral protein, the results on chemosensitivity raises the possibility that differentchemotherapeutic agents may have different p53-dependent effects in differenttumour cells.Androgen deprivation is demonstrated to sensitise prostate cancer cells tochemotherapeutic agents and it is shown that the hormone independent cell lines arethe most chemosensitive. The LNCaP cell line displayed an increased resistance toapoptosis induced by etoposide and gamma irradiation, suggesting that androgens arecapable of protection against both these DNA damaging agents.The major factors determining radiosensitivity in human tumour cell lines are knownto be DNA double-strand break (dsb) induction and repair. In the prostate cell lines Ifind that cellular radiosensitivity correlates with the number of DNA double-strandbreaks measured within 2 hours of irradiation, and that the more radioresistant celllines show better repair competence. Conclusions as to the influence of androgen dependence on radiosensitivity and repair are not possible at this stage since only theLNCaP cell line was androgen sensitive. The fact that the 2 hour repair period canseparate radiosensitive from radioresistant cells in 2 groups of human tumour celllines highlights the role of non-homologous end-joining repair. This has implicationsfor therapy, and is consistent with the clinical observation that prostate tumours canbe successfully controlled by low dose rate-brachytherapy.To evaluate the role of apoptosis, cells were exposed to TD50 concentrations ofchemotherapeutic drugs, and 60Co y-irradiation. Apoptosis was found to be low,overall, and ranged from 0.1% - 12.1%,3.0% - 6.0% and 0.1% - 8.5% for etoposide,estramustine and vinblastine, respectively. The percentage of cells undergoing druginducedapoptosis was, on average, higher in the tumour cell lines than in the normalcell lines. Gamma irradiation-induced apoptosis levels ranged from 1.3% - 7%. TheLNCaP cell line yielded the lowest percentage of apoptotic cells after exposure. Thel532T cell line yielded the highest percentage of apoptotic cells after exposure.Apoptotic propensity did not rank the cell lines according to their radiosensitivity.Immunoblotting demonstrated that the apoptosis-associated proteins, bax and bcl-2,are expressed at a basal level in all the cell lines tested, but no increase was detectedafter exposure to TD50 doses of etoposide, vinblastine and estramustine. The ratio ofbax and bcl-2 also was not altered by DNA damage.No evidence was found that a correlation may exist between reproductive cell deathand the expression of genes which control apoptosis. My results show that apoptosisis not a major mechanism of drug- or radiation-induced cell death in prostate celllines.In conclusion, loss of p53 function and loss of androgen dependence was not found tobe correlated with resistance of tumours to chemotherapeutic drugs. Cellularradiosensitivity was found to be correlated with the number of DNA double-strandbreaks remaining after 2 hours of repair. The more radioresistant cell lines showedbetter repair competence. Apoptosis and genes affecting apoptosis, such as p53 andmembers of the bcl-2 family, do not seem to contribute significantly to the sensitivityof prostate cancer cells to anticancer drugs and irradiation.

[发布机构] Stellenbosch University