Co-amplification of dhfr and a homologue of hmsh3 in a Chinese hamster methotrexate-resistant cell line correlates with resistance to a range of chemotherapeutic drugs.
OBJECTIVE
To characterize a methotrexate-resistant Chinese hamster cell line, designated as M5, which had previously been shown to be resistant to gamma radiation, at the cellular and molecular levels.


METHODS
Sensitivity towards a number of chemotherapeutic drugs was determined by colony-forming ability and compared with that of parental V79 cells. Expression of the hamster homologue of the human mismatch repair gene hmsh3 was also determined by RT-PCR.


RESULTS
Induced killing by chemotherapeutic agents cis-diamminedichloroplatinum II (cisplatin). the antimetabolite 6-thioguanine (6-TG), camptothecin, a topoisomerase I inhibitor, and 4-(9-acridinyl-amino)-methanesulfon-m-anisidide (mAMSA), an inhibitor of topoisomerase II, was less in M5 cells than in the parental V79 cells. The IC50 values, defined as the concentration of the drug that reduced the survival to 50% that of the untreated control, in V79 cells for mAMSA and camptothecin treatment were 0.35 +/- 0.02 microg/ml and 84.3 +/- 16.0 ng/ml, respectively. For M5 cells, equivalent values were 0.52 +/- 0.10 microg/ml and 186 +/- 40.8 ng/ml. Treatment with 30 microM cisplatin reduced the survival of V79 cells to 0.09 +/- 0.07, whereas the same treatment reduced the survival of M5 cells to 0.67 +/- 0.16. Treatment of M5 cells with 6-TG did not induce appreciable killing up to the concentrations studied. However, for V79 cells, 6-TG was very toxic. We further observed that the dihydrofolate reductase (dhfr) gene as well as the hamster homologue of the human mismatch repair gene hmsh3 was amplified in the methotrexate-resistant M5 cells.


CONCLUSIONS
Resistance to this group of chemotherapeutic drugs observed in M5 cells could be due to the amplification of the hamster homologue of hMSH3, which in turn possibly sequesters all the hMSH2 making M5 cells functionally deficient in the mismatch repair system.
Pandit
B
B
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Calcutta, India.
Roy
M
M
Dutta
J
J
Padhi
B K
BK
Bhoumik
G
G
Bhattacharyya
N P
NP
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D020137:Base Pair Mismatch; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004354:Drug Screening Assays, Antitumor; D005784:Gene Amplification; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D008727:Methotrexate; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D013762:Tetrahydrofolate Dehydrogenase; C078444:MSH3 protein, human; C421109:multidrug resistance-associated protein 1