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. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/24198 |
| Date | 09 1900 |
| Creators | Zacal, Natalie J. |
| Contributors | Rainbow, A.J., Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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