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Title: | An Examination of Sensitivity of Photodynamic Therapy-Resistant HT29 Cells to Ultraviolet Radiation and Cisplatin |
Authors: | Zacal, Natalie J. |
Advisor: | Rainbow, A.J. |
Department: | Biology |
Keywords: | photodynamic therapy-resistant HT29 cells;ultraviolet radiation;cisplatin;sensitivity |
Publication Date: | Sep-2003 |
Abstract: | Photodynamic therapy (PDT) is a form of cancer treatment involving light, a photosensitizer and oxygen, whereby the photosensitizer is preferentially taken up by tumour cells, excited when exposed to light of the appropriate wavelength, and generates cytotoxic excited singlet oxygen that damages and destroys cells. Photofrin is the only approved photosensitizer for clinical use in treating esophageal and early and late lung cancers in the U.S., Canada and several other countries. Despite its effectiveness in treating some tumour types, Photofrin use has some limitations and thus photosensitizers are continuously being studied to find more efficient ways of killing tumour cells. Previous reports have described the isolation of photodynamic therapy resistant human colon carcinoma HT29 cells. HT29/P14, HT29/All and HT29/N8 were isolated by repeated in vitro PDT treatment to the 1-10% survival level followed by regrowth of single surviving colonies using the photosensitizers Photofrin, Aluminium Phthalocyanine Tetrasulphonate (AlPcS4) and Nile Blue A respectively. These PDT resistant HT29 variants all display increased levels of BNip3, Bcl-2 and the heat shock protein 27 (Hsp 27), but decreased levels of Bax and the mutant HT29 p53 protein. Since mutant p53 and increased expression of Hsp27 and Bcl-2 and have been associated with resistance to various chemotherapeutic agents in some tumour cells, whereas Bax and BNip3 are potent inducers of apoptosis, it was considered of interest to examine the sensitivity of these PDT resistant HT29 variants to other cytotoxic agents. Cell sensitivity to ultraviolet (UV) A radiation (UV A), a mixture of UV A and UVB (UV AlB), UVC, or cisplatin was determined by a comparison of the D37 values for clonogenic survival in the variants compared to that in parental HT29 cells. The HT29 PDT resistant variants were not cross-resistant to cisplatin or UVC. In contrast, HT29/P14, HT29/All and HT29/N8 all showed a significant increase in cisplatin sensitivity, while HT29/All cells also showed a significant increase in UVC sensitivity. HT29/N8, and HT29/P14 both showed a significant increase in UVA resistance compared to HT29 cells whereas HT29/All did not. HT29/P14 was the only POT-resistant cell line significantly cross-resistant to UVA/B relative to HT29. While HT29/P14 and HT29/All both showed a slight increase in resistance to Photofrinmediated PDT compared to HT29/Parental, this increase was only significant for HT29/All. However, HT29/N8 was significantly more sensitive to Photofrin-mediated PDT than HT29/Parental. To complicate matters, clonogenic variability was observed amongst the two HT29 sources examined, since one of the original HT29 cell lines showed a significantly higher resistance to Photofrin-mediated PDT compared to the other parental HT29 cells that were used to derive the PDT -resistant cell lines. To examine if the differences in sensitivity of the PDT-resistant cell lines compared to parental HT29 cells in response to cisplatin and UV radiation were due to differences in DNA repair, host cell reactivation (HCR) experiments were performed with a UVC damaged B-galactosidase reporter gene from the adenovirus Ad5HCMVSp1LacZ. HCR ofthe UV-damaged reporter gene was reduced in HT29/All (the cell line most sensitive to UVC) compared to the parental HT29 cells at high multiplicities of infection of the virus. This suggests the possibility of a decreased DNA repair capacity for HT29/ A 11 cells. However, due to differences in cellular morphology between HT29 and HT29/All cells, as well as possible differences in expression of the reporter gene, it was inconclusive that the difference in HCR reflects a true difference in DNA repair between HT29 and HT29/All cells. Hsp27 over expression alone was not responsible for the increased cisplatin sensitivity of the HT29 PDT resistant variants since there was no correlation of Hsp27 protein expression levels to l/D37 (used as a measure of sensitivity), for the cisplatin colony survival assays. In addition, Hsp27 protein expression levels did not correlate with UVC, cisplatin or UV A sensitivity suggesting that Hsp27 may be uniquely involved in making cells more resistant to PDT. p53 but not BNip3 protein levels correlated with sensitivity of cells to UV A, whereas no correlation was observed between p53 or Hsp27 protein expression levels and UVC sensitivity. p53 and p21 protein levels were not altered in either parental HT29 or the HT29/P14 POT-resistant variant following UVC and cisplatin exposure, respectively. In addition, introduction of wild-type p53 (using infection of a replication deficient adenovirus vector encoding the wild-type p53 gene), into parental HT29 or the PDT -resistant HT29/P 14 variant, had no effect on cisplatin sensitivity compared to cells infected with a control adenovirus vector expressing the LacZ gene. Taken together, these results suggest that the increased sensitivity of the PDT resistant variants to cisplatin did not result from differences in p53-dependent cisplatininduced cell cycle arrest. A strong correlation of cellular cisplatin sensitivity to the ratio of BNip3 to p53 protein levels, suggests that alterations in the expression of several different genes, including a reduced expression of the mutant HT29 p53 protein and an increased expression of BNip3, contribute to the increased cisplatin sensitivity of the HT29 PDT resistant variants. It has been reported previously that apoptosis induced by BNip3 is significantly inhibited by both wild type and mutated p53. Since pro-apoptotic BNip3 is over expressed in all three PDT-resistant HT29 cell lines, and BNip3/p53 protein expression levels were correlated to cisplatin sensitivity, this suggests that cisplatin kills HT29 cells through a BNip3-mediated apoptotic pathway. |
URI: | http://hdl.handle.net/11375/24198 |
Appears in Collections: | Digitized Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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zacal_natalie_j_2003sep_masters.pdf | 11.25 MB | Adobe PDF | View/Open |
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