Implication of Protein Kinase C in the Regulation of DNA Mismatch Repair Protein Expression and Function

Abstract

The DNA mismatch repair (MMR) proteins are essential for the maintenance of genomic stability of human cells. Compared with hereditary or even sporadic carcinomas, MMR gene mutations are very uncommon in leukemia. However, genetic instability, attested by either loss of heterozygosity or microsatellite instability, has been extensively documented in chronic or acute malignant myeloid disorders. This observation suggests that in leukemia some internal or external signals may interfere with MMR protein expression and/or function. We investigated the effects of protein kinase C (PKC) stimulation by 12-O-tetradecanoylphorbol-13-acetate (TPA) on MMR protein expression and activity in human myeloid leukemia cell lines. First, we show here that unstimulated U937 cells displayed low level of PKC activity as well as MMR protein expression and activity compared with a panel of myeloid cell lines. Second, treatment of U937 cells with TPA significantly increased (3-5-fold) hMSH2 expression and, to a lesser extent, hMSH6 and hPMS2 expression, correlated to a restoration of MMR function. In addition, diacylglycerol, a physiological PKC agonist, induced a significant increase in hMSH2 expression, whereas chelerythrine or calphostin C, two PKC inhibitors, significantly decreased TPA-induced hMSH2 expression. Reciprocally, treatment of HEL and KG1a cells that exhibited a high level of PKC expression, with chelerythrine significantly decreased hMSH2 and hMSH6 expression. Moreover, the alteration of MMR protein expression paralleled the difference in microsatellite instability and cell sensitivity to 6-thioguanine. Our results suggest that PKC could play a role in regulating MMR protein expression and function in some myeloid leukemia cells.

Structure and function of the components of the human DNA mismatch repair system

“…The same group found that hMSH2 and hMSH6 are phosphorylated in vitro and in vivo, and that phosphorylation is required for nuclear translocation and G-T binding. 35,36 Phosphorylation of hMSH2 can be accomplished by PKC-f and protects hMutSa from degradation by the ubiquitin/proteasome pathway. 37 Cadmium, a known carcinogen and an environmental concern from improperly disposed rechargeable batteries, was recently shown to inhibit MMR, leading to an increase in mutation rates up to 2,000-fold.…”

G2/M checkpoint stringency is a key parameter in the sensitivity of AML cells to genotoxic stress

“…The results presented here are representative of three independent experiments. (Humbert et al, 2002). In the same experiment, we also examined the G2/M checkpoint recovery efficiency by monitoring histone-gH2AX labeling in mitotic cells, as it has been shown that the level of its expression correlates with the amount of double-strand breaks (DSBs) in the DNA (McManus and Hendzel, 2005;Bouquet et al, 2006).…”

The Ubiquitin-Proteasome System and DNA Repair

“…Ubiquitination and degradation rates of hMSH2 and hMSH6 appear quite similar, suggesting that UPS-mediated proteolysis may maintain a constant ratio of these two proteins (Hernandez-Pigeon et al, 2004). While no strong correlation between total proteasomal activity and the degradation rate of hMutS has been observed in vitro, low hMutS expression in cells is a limiting factor for MMR and indicative of proteolytic activity of the UPS in MMR regulation (Ciechanover, 1994;Humbert et al, 2002;Hernandez-Pigeon et al, 2004). This process is regulated also by an atypical protein kinase C (PKC ); this kinase increases hMutS protein levels and the binding of hMutS to G•T mismatches (Hernandez-Pigeon et al, 2005).…”

“…The UPS is involved in post-transcriptional regulation of hMutS protein expression (Humbert et al, 2002;Hernandez-Pigeon et al, 2004). Ubiquitination and degradation rates of hMSH2 and hMSH6 appear quite similar, suggesting that UPS-mediated proteolysis may maintain a constant ratio of these two proteins (Hernandez-Pigeon et al, 2004).…”