BCR/ABL Kinase Disrupts Formation of Mismatch Repair Complex To Induce Genomic Instability.

Abstract

Among different genotoxic agents, BCR/ABL cells are more resistant to N-methyl-N'-nitro-N-nitrosoguanine (MNNG), which methylates 06 position of guanine. 06 -MeG bases pair with either T or C during replication giving rise to mismatches. Mismatch repair (MMR) proteins are responsible for detecting and removing misincorporated nucleotides, which escaped proofreading activity of DNA polymerases. MMR proteins assembled on the mismatch can signal to repair or apoptosis. Defects in expression of MMR genes leads to drug resistance and mutator phenotype, observed in several different solid tumors. The role of MMR system in drug resistance and/or genomic instability of leukemic cells remains poorly understood. Parental cells and BCR/ABL expressing clones were incubated with MNNG for 4 weeks resulting in their MNNG-resistant derivatives, which may accumulate mutations in their genomic DNA resulting from methylating activity of the drug. To investigate the mutation rate and phenotype, ouabain-resistance test was employed. Cells become resistant to ouabain, a glycoside that inhibits ATP1A1 subunit of Na/K ATP-ase, when mutations arise in the gene fragment encoding this subunit. The clonogenic assay revealed over 5 times more ouabain resistant colonies in MNNG-resistant BCR/ABL-positive cells than in parental counterparts. To investigate the type of mutations, a fragment of Na/K ATP-ase gene encoding the ATP1A1 subunit was sequenced from MNNG-resistant BCR/ABL-positive and parental cells. The dominating mutation in BCR/ABL MNNG-resistant cells was C to T, while A to G mutations were prevalent in parental cells. In order to check the status of MMR proteins, Western blotting and immunofluorescence studies were performed. Expression of MMR proteins in BCR/ABL transformed cells was similar to parental cells, however immunofluorescence visualized dramatic changes after DNA damage in the nuclear co-localization of MMR proteins in BCR/ABL-transformed cells (CML patient cells and leukemic cell lines) in comparison to normal cells. Co-localization of MSH2 and MSH6 proteins, forming a heterodimer homologous to bacterial MutS, remained similar in parental and leukemia cells upon MNNG treatment. However, co-localization of MLH1 and PMS2 proteins, which form a heterodimer homologous to bacterial MutL, was detected in non-transformed cells, but not in BCR/ABL leukemia cells. In addition, MLH1 and MSH2 co-localized in normal, but not BCR/ABL-positive cells after MNNG treatment. The defects in interaction of MMR proteins in leukemia cells were reversed by inhibition of BCR/ABL kinase by STI571. Thus, the assembly of MMR proteins on mismatched bases and subsequent signaling to repair and/or apoptosis may be impaired in BCR/ABL leukemia cells. These results suggests a novel mechanism of regulation of DNA damage response proteins by oncogenic tyrosine kinase that can lead to genomic instability and drug resistance of leukemic cells.