Increased DNA-repair capacity and the modulation of 2 proteins in and metallothionein overexpressing Chinese hamster cell line

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.