Carbonic anhydrase 9 (CA9) expression in tumor cells enhances sensitivity to tirapazamine.
OBJECTIVE
Carbonic anhydrase 9 (CA9) is over-expressed in many human solid tumors under conditions of low oxygen concentration and can be associated with a low probability of survival. In this study, stable CA9-expressing cell lines were established using the CA9 gene-defective human C33a cell line and the HeLa cell line to investigate the role of CA9 in response to ionizing radiation and hypoxia-selective cytotoxin, Tirapazamine (TPZ).


METHODS
Human CA9 cDNA or an empty vector was transfected into the C33a and HeLa cell lines and C33a-vector, C33a-CA9, HeLa-vector, and HeLa-CA9 cell lines were produced accordingly. Sensitivity of the C33a-vector/C33a-CA9 cells to ionizing radiation and TPZ was measured using clonogenic assays. The alkaline comet assay was used to measure single strand DNA breaks caused by TPZ in the C33a-vector, C33a-CA9, HeLa-vector, and HeLa-CA9 cell lines.


RESULTS
Radiation sensitivity, as determined with clonogenic survival assays, of C33a-vector/C33a-CA9 cells did not differ under either normoxic or hypoxic conditions. However, increased clonogenic sensitivity to TPZ was observed in C33a-CA9 cells under the hypoxic condition by 26% (95% CI 14-39%, P = 0.02 in comparison to the C33a-vector cells). The comet assay showed significantly greater DNA damage in the C33a-CA9 cells compared with that of the C33a-vector cells with the same treatment under hypoxic conditions, supporting the results of the clonogenic survival data. Because this difference in the amount of DNA damage was not observed for the hypoxic HeLa-CA9/HeLa-vector cell lines, both of which have induced CA9 expression by hypoxia, the enhanced sensitivity of C33a-CA9 cells to TPZ is considered to be due to the specific condition of CA9 over-expression.


CONCLUSIONS
Our results suggest the possibility that CA9 over-expression in tumors might be exploited to increase the treatment effects of TPZ.
Shin
Hye-Jin
HJ
Radiation Oncology, Research Institute and Hospital, National Cancer Center, 809 Madu-dong, Ilsan-gu, Goyang-si, Gyeonggi-do 411-769, South Korea.
Kim
Joo-Young
JY
Yoo
Chong-Woo
CW
Roberts
Stephen A
SA
Lee
Sun
S
Choi
Soo-Jin
SJ
Lee
Hee-Young
HY
Lee
Doo-Hyun
DH
Kim
Tae Hyun
TH
Cho
Kwan Ho
KH
D000951:Antigens, Neoplasm / Q000235:genetics*; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics*; D018288:Carcinoma, Small Cell / Q000188:drug therapy / Q000201:enzymology; D015687:Cell Hypoxia; D004249:DNA Damage / Q000528:radiation effects; D004273:DNA, Neoplasm / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D000077704:Tirapazamine; D014162:Transfection; D014227:Triazines / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D004273:DNA, Neoplasm; D011838:Radiation-Sensitizing Agents; D014227:Triazines; D000077704:Tirapazamine; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases