Doctoral Degrees (Radiation Oncology)

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    Cell biological responses of prostatic tumour cell lines to irradiation and anticancer drugs
    (Stellenbosch : Stellenbosch University, 2003-12) Serafin, Antonio Mendes; Bohm, E. L. J. F.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Radiation Oncology.
    ENGLISH ABSTRACT: The "classic" prostate cell lines, DU145, PC-3 and LNCaP, have served as a valuable cell biological model for research into prostate cancer. However, their relevance may be limited because they derive from metastatic, and not from primary normal and tumour epithelium. The cell lines (1532T, 1535T, 1542T, 1542N and BPH-l) have been derived from primary benign and malignant human tumour prostate epithelium and may be more representative. Using these cell lines I have examined the role of basic cell damage responses (repair, checkpoint activation, apoptosis and associated signalling proteins, and the influence of androgen status) in cell inactivation, and its relevance to treatment. Numerous studies have suggested that loss of p53 function leads to resistance to chemotherapeutic agents and irradiation. It is shown here that the p53-inactive cell lines 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 most radiosensitive. Notwithstanding the effects of p53 degradation by the HPV -16 E6 viral protein, the results on chemosensitivity raises the possibility that different chemotherapeutic agents may have different p53-dependent effects in different tumour cells. Androgen deprivation is demonstrated to sensitise prostate cancer cells to chemotherapeutic agents and it is shown that the hormone independent cell lines are the most chemosensitive. The LNCaP cell line displayed an increased resistance to apoptosis induced by etoposide and gamma irradiation, suggesting that androgens are capable of protection against both these DNA damaging agents. The major factors determining radiosensitivity in human tumour cell lines are known to be DNA double-strand break (dsb) induction and repair. In the prostate cell lines I find that cellular radiosensitivity correlates with the number of DNA double-strand breaks measured within 2 hours of irradiation, and that the more radioresistant cell lines show better repair competence. Conclusions as to the influence of androgen dependence on radiosensitivity and repair are not possible at this stage since only the LNCaP cell line was androgen sensitive. The fact that the 2 hour repair period can separate radiosensitive from radioresistant cells in 2 groups of human tumour cell lines highlights the role of non-homologous end-joining repair. This has implications for therapy, and is consistent with the clinical observation that prostate tumours can be successfully controlled by low dose rate-brachytherapy. To evaluate the role of apoptosis, cells were exposed to TD50 concentrations of chemotherapeutic 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 druginduced apoptosis was, on average, higher in the tumour cell lines than in the normal cell lines. Gamma irradiation-induced apoptosis levels ranged from 1.3% - 7%. The LNCaP cell line yielded the lowest percentage of apoptotic cells after exposure. The l532T 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 detected after exposure to TD50 doses of etoposide, vinblastine and estramustine. The ratio of bax and bcl-2 also was not altered by DNA damage. No evidence was found that a correlation may exist between reproductive cell death and the expression of genes which control apoptosis. My results show that apoptosis is not a major mechanism of drug- or radiation-induced cell death in prostate cell lines. In conclusion, loss of p53 function and loss of androgen dependence was not found to be correlated with resistance of tumours to chemotherapeutic drugs. Cellular radiosensitivity was found to be correlated with the number of DNA double-strand breaks remaining after 2 hours of repair. The more radioresistant cell lines showed better repair competence. Apoptosis and genes affecting apoptosis, such as p53 and members of the bcl-2 family, do not seem to contribute significantly to the sensitivity of prostate cancer cells to anticancer drugs and irradiation.
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    The influence of pentoxifylline on damage responses in tumour cells
    (Stellenbosch : Stellenbosch University, 2000-04) Theron, Catherina S; Bohm, E. L. J. F.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medical Imaging and Clinical Oncology. Radiation Oncology.
    ENGLISH ABSTRACT: Pentoxifylline enhances the toxicity of radiation and has emerged as an effective modulator of the radiation response of tumour cells. The molecular mechanisms involved in the enhancement of radiotoxicity by pentoxifylline have not yet been elucidated. Cell cycle blocks, DNA repair and programmed cell death (apoptosis) are all pert of the cellular response to DNA damage and as such must be considered as targets of the drug. In this study, the influence of pentoxifylline on radiosensitisation, G2 block abrogation, DNA repair inhibition and the induction of apoptosis have been investigated in 8e11 and MeWo melanoma and 4197 and 4451 squamous cell carcinoma (SCC) cell lines. The influence of pentoxifylline on radiation-induced apoptosis in Jurkat J5 T-lymphocytic leukemia cells has also been assessed. Hela cervical carcinoma cells were used to investigate the molecular events involved in the abrogation of the G2 block by pentoxifylline. It is shown that pentoxifylline preferentially sensitises the TP53 mutant MeWo and 4451 cell lines and enhances radiotoxicity by factors of up to 14.5. In the MeWo melanoma, but not in the 4451 SCC cell line, radiosensitisation is accompanied by inhibition of DNA repair. No significant enhancement of radiation-induced apoptosis was observed in MeWo melanoma and 4451 SCC cells. However, Jurkat J5 cells showed an increase in apoptosis after irradiation in the presence of the drug. In irradiated Hela cervical carcinoma cells, pentoxifylline affects the expression of the two components of the mitosis promoting factor (MPF), namely cyclin 81 and p34cdC2, and rapidly restores cyclin 81/p34cdC2 ratios to control levels. Analysis of cyclin 81 expression in whole cells and isolated nuclei furthermore reveals an influence of the drug on the subcellular translocation of the MPF. It is concluded that G2 block abrogation is not the only mechanism involved in the radiosensitisation of tumour cells by pentoxifylline, but that DNA repair inhibition plays a role in certain cell types. Although pentoxifylline induces apoptosis in Jurkat J5 thymocytes, radiation-induced apoptosis plays no role in the radiosensitisation of the two TP53 mutant melanoma and sec cell lines. Abrogation of the G2 block by pentoxifylline, which sensitises tumour cells to a second irradiation or chemotherapeutic challenge, involves a modulation of the levels of cyclin 81 and p34cdC2, and the subcellular location of the MPF. These results are of utmost importance for the clinical potential of pentoxifylline as a dose modifier in cancer therapy.
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    Examination of irradiated neuroblastoma and neuroepithelial cell lines for the interrelationship between cell survival, micronucleation, apoptosis and DNA repair
    (Stellenbosch : Stellenbosch University, 2000-12) Akudugu, John Mbabuni; Bohm, E. L. J. F.; Slabbert, J. P.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medical Imaging and Clinical Oncology. Radiation Oncology.
    ENGLISH ABSTRACT: Predictive assays are of key importance in clinical radiotherapy, chemotherapy and toxicology. Prior to exposing malignant tissues to irradiation or drugs in the clinic, a good understanding of the damage response to the cytotoxic agent is required. Such information is necessary for effective planning and treatment. Regrettably however the methods which detect DNA damage, namely micronucleus, apoptosis and DNA repair assays do not rank cells according to their intrinsic survival response to cytotoxic agents. The application of predictive assays based on micronuclei and apoptosis in the clinic therefore remains unreliable. Using a panel of 7 neuroblastoma and 6 neuroepithelial cell lines, it is shown that damage assays also do not rank cell lines according to cell survival. However, radiosensitivity can be reconstructed from micronuclei formation and apoptosis, and a new parameter, cell death due to small deletions, chromosome aberrations and misrepair. The interrelationships between radiation-induced micronuclei, apoptosis and repair is complex and varies between cell lines. Micronuclei formation and apoptosis are exponentially interrelated. This suggests that these cell inactivation pathways are strongly correlated. Evidence exists to show that the expression of apoptosis and micronuclei is influenced by the extent of DNA double-strand break repair within the first 2 hours after irradiation. Cell lines which repair more damage in the first 2 hours express more micronuclei and less apoptosis. Micronuclei formation and apoptosis and are not significantly correlated with the 20 hours slow repair component. There is however a strong correlation between 20 hours of repair and radiosensitivity, with the more radioresistant cell lines being more repair proficient. This suggests that the 2 hours (fast) DNA repair component is more error prone, and that cells lines repairing more damage late after irradiation tend to show better survival. In conclusion, micronuclei formation, apoptosis and DNA repair are strictly cell type specific and are not suitable for predicting radiosensitivity in terms of cell survival. However, these assays are very useful for studies on the influences of dose modifying agents i.e. oxygen tension, radiation modality, pH, cytotoxic sensitisers and radiation protectors which alter cellular responses and provide insight into damage mechanisms.