Friedman H S, Dolan M E, Kaufmann S H, Colvin O M, Griffith O W, Moschel R C, Schold S C, Bigner D D, Ali-Osman F
Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710.
Cancer Res. 1994 Jul 1;54(13):3487-93.
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.
先前的研究表明，在体内筛选出的对美法仑耐药的人TE-671 MR人横纹肌肉瘤异种移植瘤（M. C. 罗森伯格等人，《癌症研究》，49: 6917 - 6922, 1989）对多种烷化剂和博来霉素具有交叉耐药性，但对依托泊苷具有侧链敏感性。尽管与对美法仑敏感的亲本TE-671异种移植瘤相比，TE-671 MR中的谷胱甘肽水平有所升高，但用丁硫氨酸亚砜胺处理以降低谷胱甘肽水平并不能完全恢复TE-671 MR异种移植瘤对美法仑的敏感性。本研究旨在寻找TE-671 MR异种移植瘤中其他的耐药机制。在对10%的动物致死剂量下进行的药敏试验表明，TE-671 MR异种移植瘤对双功能交联剂l,3 - 双(2 - 氯乙基)-l - 亚硝基脲保持耐药性，并且对拓扑异构酶I抑制剂拓扑替康具有交叉耐药性。用丁硫氨酸亚砜胺处理并没有使TE-671 MR异种移植瘤对l,3 - 双(2 - 氯乙基)-l - 亚硝基脲敏感。此外，尽管TE-671和TE-671 MR异种移植瘤中的O6 - 烷基鸟嘌呤 - DNA烷基转移酶水平都很高，但用O6 - 苄基鸟嘌呤处理以耗尽O6 - 烷基鸟嘌呤 - DNA烷基转移酶活性，显著使TE-67l异种移植瘤敏感，但对TE-671 MR异种移植瘤却没有作用，这表明存在另一种耐药机制。对可能参与DNA修复的其他酶活性的测量显示，DNA聚合酶α（TE-671中为46±8（标准差）单位/毫克蛋白，TE-671 MR中为69±6单位/毫克蛋白，P < 0.05）和DNA聚合酶β（TE-671中为0.43±0.01单位/毫克蛋白, TE-671 MR中为0.78±0.12单位/毫克蛋白，P < 0.05）显著升高，但DNA聚合酶δ或总DNA连接酶没有升高。通过活性测定和蛋白质印迹法对拓扑异构酶的检测显示，与亲本异种移植瘤相比，TE-671 MR异种移植瘤中的拓扑异构酶II增加了2倍，拓扑异构酶I减少了2倍，这显然解释了其对依托泊苷的侧链敏感性和对拓扑替康的交叉耐药性。这些结果表明，TE-671 MR异种移植瘤中DNA修复相关蛋白和其他核蛋白的活性存在多种变化，这可能导致对烷化剂的耐药性。
