Expression of the hMSH 6 mismatch-repair protein in colon cancer and HeLa cells

A phenotype characterised by widespread instability of microsatellite sequences in DNA has been reported to occur in 10–15% of human colon cancers [1, 2]. Microsatellites are stretches of repeated DNA sequences consisting of mono-, dior tri-nucleotide repeats. Monoand di-nucleotide microsatellites are either shorter or longer by a few repeat units in this subset of colon cancers, when compared to those in the DNA of normal tissues. Microsatellite instability (MSI) is caused by a defect of the Mismatch Repair (MMR) system, which has evolved to correct errors of DNA polymerases [3, 4]. These enzymes are error-prone and, during DNA replication, they can cause strand misalignments with the formation of loops of extrahelical bases in the newly synthesised or in the template strand. These intermediates (i.e., insertion/deletion loops), if not repaired, result in insertions or deletions of repeat units in the microsatellites of the progeny DNA. In addition, DNA polymerases incorporate non-complementary nucleotides into the newly synthesised DNA strand (i.e., base mismatches) with a frequency of approximately one error per 100 000 nucleotides synthesised. Also base misincorporations are substrates for the MMR system, which thus efficiently reduces the mutation rate from one error every 107 to one error every 1010 nucleotides. Base mismatches and insertion/deletion loops are recognised by a complex consisting of the hMSH2 and hMSH6 proteins [5] (figure 1). Another complex (hMSH2/hMSH3) (not shown in figure 1), which is less represented Principles: 10 to 15% of human colon cancers are associated with an inherited or somatic defect of the DNA Mismatch Repair (MMR) system, which has evolved to correct biosynthetic errors such as nucleotide mis-incorporations or misalignments arising during DNA replication in the S phase of the cell-cycle. Although expression of the MMR genes was expected to be cell-cycle dependent, we and others observed that the MMR proteins hMSH2 and hMLH1 are expressed constitutively in proliferating cells. Methods: In this study we extend our observations to another essential MMR protein, hMSH6. We used immunohistochemistry to evaluate the expression pattern of this protein in human colorectal mucosa and tumours, as well as in synchronised HeLa-S3 cells, in which we analysed its steady-state levels during the cell-cycle. Results: We show that the immunohistochemical pattern of expression of hMSH6 in normal colorectal crypts and in colon cancers differs significantly from that of the other MMR proteins, with a much lower percentage of replicating cells being hMSH6-positive. This implies that hMSH6 could be cell-cycle regulated. In order to test this hypothesis in a model system, we synchronised HeLa-S3 cells with mitotic shake-off and found that the hMSH6 protein was detectable throughout the cell-cycle, but that its steady-state level increased when cells progressed from G1 to S-phase. Discussion: The increase of hMSH6 steadystate level when cells enter S-phase was expected, since MMR acts during DNA replication. However, the overall low level of oscillations of hMSH6 during the cell-cycle in this cellular model apparently does not fit the immunohistochemical phenotype. We believe that this discrepancy is due to the fact that human cell lines proliferate at a much higher rate than normal and neoplastic colorectal cells in vivo.