Robson T, Grindley H, Hall A, Vormoor J, Lohrer H
Cancer Research Campaign Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex, UK.
Mutat Res. 1994 Mar;314(2):143-57. doi: 10.1016/0921-8777(94)90078-7.
Elevated intracellular levels of metallothionein have been associated with resistance to the cytotoxic effects of some alkylating agents. In order to study the mechanisms responsible for this resistance, we used a pair of CHO cell lines consisting of normal K1-2 cells and their derivative K1-2MT, which overexpresses the human metallothionein II-A gene (Lohrer et al., 1989). K1-2MT cells were found to be resistant to cadmium chloride and the alkylating agents N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), but resistance did not extend to the alkylating agent, 1,3-bis(2-chloroethyl)- 1-nitrosourea, nor to adriamycin, an inhibitor of DNA synthesis. The DNA damage caused by MNNG, was only marginally less in resistant cells compared with the parental cell line, thus excluding drug scavenging as a possible mechanism for resistance. Also, glutathione S-transferases (GSTs) were present at equal levels in both cell lines (acidic and basic type GST) or slightly reduced in drug resistant K1-2MT cells (neutral type GST), thereby ruling out metabolic inactivation of the alkylating agents. However, the drug resistant phenotype was accompanied by a more efficient block of DNA synthesis after MNNG treatment and by a 3-h delay in the G2 phase of the cell cycle. Using two-dimensional gel electrophoresis of total protein extracts, we identified a 24-kDa protein (MIP1), which is only present in the resistant K1-2MT cells, and a 23.5-kDa protein (MIP2) which is 2-3 times over-synthesized in K1-2MT cells. MNNG treatment reduced the level of both proteins MIP1 and MIP2. These results suggest that the proteins MIP1 and possibly MIP2 may be responsible for the alkylating agent resistant phenotype and are probably modulated by the human metallothionein II-A protein.
细胞内金属硫蛋白水平升高与对某些烷化剂细胞毒性作用的抗性有关。为了研究这种抗性的机制，我们使用了一对CHO细胞系，由正常的K1-2细胞及其衍生物K1-2MT组成，后者过表达人金属硫蛋白II-A基因（Lohrer等人，1989年）。发现K1-2MT细胞对氯化镉和烷化剂N-甲基-N'-硝基-N-亚硝基胍（MNNG）具有抗性，但这种抗性并不延伸至烷化剂1,3-双（2-氯乙基）-1-亚硝基脲，也不延伸至阿霉素（一种DNA合成抑制剂）。与亲代细胞系相比，MNNG引起的DNA损伤在抗性细胞中仅略微减少，因此排除了药物清除作为抗性可能机制的可能性。此外，谷胱甘肽S-转移酶（GST）在两种细胞系中的水平相等（酸性和碱性类型的GST），或者在耐药的K1-2MT细胞中略有降低（中性类型的GST），从而排除了烷化剂的代谢失活。然而，耐药表型伴随着MNNG处理后DNA合成更有效的阻滞以及细胞周期G2期3小时的延迟。通过对总蛋白提取物进行二维凝胶电泳，我们鉴定出一种24 kDa的蛋白质（MIP1），其仅存在于耐药的K1-2MT细胞中，以及一种23.5 kDa的蛋白质（MIP2），其在K1-2MT细胞中的合成量是正常细胞的2 - 3倍。MNNG处理降低了MIP1和MIP2两种蛋白质的水平。这些结果表明，蛋白质MIP1以及可能的MIP2可能是烷化剂耐药表型的原因，并且可能受到人金属硫蛋白II-A蛋白的调节。
