Elevated DNA polymerase alpha, DNA polymerase beta, and DNA topoisomerase II in a melphalan-resistant rhabdomyosarcoma xenograft that is cross-resistant to nitrosoureas and topotecan.
Previous investigations have revealed that the human TE-671 MR human rhabdomyosarcoma xenograft selected in vivo for melphalan resistance (M. C. Rosenberg, et al., Cancer Res., 49: 6917-6922, 1989) is cross-resistant to a wide variety of alkylating agents and to bleomycin, but is collaterally sensitive to etoposide. Although glutathione levels were noted to be elevated in TE-671 MR compared to the melphalan-sensitive parental TE-671 xenograft, treatment with buthionine sulfoximine to deplete glutathione levels did not fully restore melphalan sensitivity in the TE-671 MR xenograft. The present studies were undertaken to search for additional mechanisms of resistance in the TE-671 MR xenograft. Drug sensitivity testing performed at the dose of agents that was lethal to 10% of the animals revealed that the TE-671 MR xenograft maintained resistance to the bifunctional cross-linking agent 1,3-bis(2-chloroethyl)-1-nitrosourea and was cross-resistant to the topoisomerase I poison topotecan. Treatment with buthionine sulfoximine did not sensitize the TE-671 MR xenograft to 1,3-bis(2-chloroethyl)-1-nitrosourea. Further, even though O6-alkylguanine-DNA alkyltransferase levels were high in both the TE-671 and TE-671 MR xenografts, depletion of O6-alkylguanine-DNA alkyltransferase activity by treatment with O6-benzylguanine substantially sensitized the TE-671 xenografts but not the TE-671 MR xenografts, suggesting an additional mechanism of resistance. Measurement of additional enzyme activities that might be involved in DNA repair revealed significant elevations in DNA polymerase alpha (46 +/- 8 (SD) units/mg protein in TE-671, 69 +/- 6 units/mg protein in TE-671 MR, P < 0.05) and DNA polymerase beta (0.43 +/- 0.01 units/mg protein in TE-671, 0.78 +/- 0.12 units/mg protein in TE-671 MR, P < 0.05) but not DNA polymerase delta or total DNA ligase. Examination of topoisomerases by activity assays and Western blotting revealed a 2-fold increase in topoisomerase II and a 2-fold decrease in topoisomerase I in the TE-671 MR xenograft compared to the parental xenograft, apparently explaining the collateral sensitivity to etoposide and cross-resistance to topotecan. These results suggest that TE-671 MR xenografts contain multiple changes in activities of DNA repair-related proteins and other nuclear proteins that could contribute to alkylating agent resistance.
Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710.
H S
HS
Friedman
M E
ME
Dolan
S H
SH
Kaufmann
O M
OM
Colvin
O W
OW
Griffith
R C
RC
Moschel
S C
SC
Schold
D D
DD
Bigner
F
F
Ali-Osman
D019883:Alkyl and Aryl Transferases; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002330:Carmustine / Q000494:pharmacology*; D004256:DNA Polymerase I / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004351:Drug Resistance; D004354:Drug Screening Assays, Antitumor; D005260:Female; D006801:Humans; D008297:Male; D008558:Melphalan / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D012208:Rhabdomyosarcoma / Q000188:drug therapy* / Q000378:metabolism*; D019772:Topotecan; D014166:Transferases / Q000378:metabolism; D014183:Transplantation, Heterologous
D000970:Antineoplastic Agents; D019772:Topotecan; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C050956:DNA alkyltransferase; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004250:DNA Topoisomerases, Type II; D008558:Melphalan; D002330:Carmustine; D002166:Camptothecin