Two modes of microsatellite instability in human cancer: differential connection of defective DNA mismatch repair to dinucleotide repeat instability.

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes of < or =6 bp. Type B changes are more drastic and involve modifications of > or =8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI+ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.

Monoallelic BUB1B mutations and defective mitotic-spindle checkpoint in seven families with premature chromatid separation (PCS) syndrome.

Cancer-prone syndrome of premature chromatid separation (PCS syndrome) with mosaic variegated aneuploidy (MVA) is a rare autosomal recessive disorder characterized by growth retardation, microcephaly, childhood cancer, premature chromatid separation of all chromosomes, and mosaicism for various trisomies and monosomies. Biallelic BUB1B mutations were recently reported in five of eight families with MVA syndrome (probably identical to the PCS syndrome). We here describe molecular analysis of BUB1B (encoding BubR1) in seven Japanese families with the PCS syndrome. Monoallelic BUB1B mutations were found in all seven families studied: a single-base deletion (1833delT) in four families; and a splice site mutation, a nonsense mutation, and a missense mutation in one family each. Transcripts derived from the patients with the 1833delT mutation and the splice site mutation were significantly reduced, probably due to nonsense-mediated mRNA decay. No mutation was found in the second alleles in the seven families studied, but RT-PCR of BUB1B and Western blot analysis of BubR1 indicated a modest decrease of their transcripts. BubR1 in the cells from two patients showed both reduced protein expression and diminished kinetochore localization. Their expression level of p55cdc, a specific activator of anaphase-promoting complex, was normal but its kinetochore association was abolished. Microcell-mediated transfer of chromosome 15 (containing BUB1B) into the cells restored normal BubR1 levels, kinetochore localization of p55cdc, and the normal responses to colcemid treatment. These findings indicate the involvement of BubR1 in p55cdc-mediated mitotic checkpoint signaling, and suggest that >50% decrease in expression (or activity) of BubR1 is involved in the PCS syndrome.

Induction of premature chromatid separation (PCS) in individuals with PCS trait and in normal controls.

Cultured peripheral blood lymphocytes from ten normal individuals, treated with 0.075 M KCl at 37 degrees C for 20 min, showed 0-2% cells in premature chromatid separation (PCS), a configuration with split centromeres and chromatids of most or all chromosomes. When treated for 30 min, they increased to 19% in the average, and at 45 min to 63%. Similar and significant effects of temperature and duration of hypotonic treatment on the frequencies of PCSs were found also in mitotic lymphocytes from patients with homozygous PCS trait, a cancer-prone disorder with >50% lymphocytes in PCS, mosaic variegated aneuploidy, and a variety of clinical manifestations; and from their heterozygous carrier parents. B lymphoblastoid cells from two infants with the homozygous PCS trait did not show PCSs when processed without hypotonic treatment. The frequencies of their PCSs increased with increasing temperature and duration of hypotonic treatment, attaining more than 65% after 20 min treatment and 90% after 45 min at 37 degrees C. PCS is thus likely to be induced largely by hypotonic treatment. Treatment at 37 degrees C for 20 min was found to be most suitable for the count of PCSs, in which the frequency of PCSs becomes almost zero in cells from normal individuals, and the difference in frequency of PCSs was most remarkable between the patients and heterozygous carriers, and between the heterozygous carriers and normal individuals. Chromosomes from the patients with the homozygous PCS trait tended to be long, and their PCSs tended to have a large number of widely separated sister chromatids. Chromosomes from normal individuals tended to be short, and the sister chromatids in their PCSs were set close to each other.

Alternative mechanisms of gene amplification in human cancers.

Gene amplification is a common phenomenon in cancer. Cytogenetic analyses have indicated that breakage-fusion-bridge (BFB) cycles drive intrachromosomal amplification of some oncogenes in a head-to-head manner in human cancers. However, the complex structures of an amplified sequence found in cancers are not always explained by the BFB model. At the 17q21 locus, which is not linked to common fragile sites, we discovered a recombination hot spot harboring amplicon repeats in tandem in a head-to-tail orientation, with the interamplicon junctions in each cancer cell being homogeneous. These findings clearly show the presence of alternative mechanisms other than BFB cycles in oncogene amplification.

Common cytological and cytogenetic features of Epstein-Barr virus (EBV)-positive natural killer (NK) cells and cell lines derived from patients with nasal T/NK-cell lymphomas, chronic active EBV infection and hydroa vacciniforme-like eruptions.

In this study, we describe the cytological and cytogenetic features of six Epstein-Barr virus (EBV)-infected natural killer (NK) cell clones. Three cell clones, SNK-1, -3 and -6, were derived from patients with nasal T/NK-cell lymphomas; two cell clones, SNK-5 and -10, were isolated from patients with chronic active EBV infection (CAEBV); and the other cell clone, SNK-11, was from a patient with hydroa vacciniforme (HV)-like eruptions. An analysis of the number of EBV-terminal repeats showed that the SNK cell clones had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patients, and SNK cells had the type II latency of EBV infection, suggesting that not only the cell clones isolated from nasal T/NK-cell lymphomas but also those isolated from CAEBV and HV-like eruptions had been transformed by EBV to a certain degree. Cytogenetic analysis detected deletions in chromosome 6q in five out of the six SNK cell clones, while 6q was not deleted in four control cell lines of T-cell lineage. This suggested that a 6q deletion is a characteristic feature of EBV-positive NK cells, which proliferated in the diseased individuals. The results showed that EBV-positive NK cells in malignant and non-malignant lymphoproliferative diseases shared common cytological and cytogenetic features.