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Acute myeloid leukemia is a neoplasm characterized by recurrent molecular aberrations traditionally demonstrated by cytogenetic analyses. We used high density genome-wide genotyping and gene expression profiling to reveal acquired cryptic abnormalities in acute myeloid leukemia. By genome-wide genotyping of 137 cases of primary acute myeloid leukemia, we disclosed a recurrent focal amplification on chromosome 14q32, which included the genes BCL11B, CCNK, C14orf177 and SETD3, in two cases. In the affected cases, the BCL11B gene showed consistently high mRNA expression, whereas the expression of the other genes was unperturbed. Fluo

The position of chromosomal neighborhoods in living cells was followed using three different methods for marking chromosomal domains occupying arbitrary locations in the nucleus; photobleaching of GFP-labeled histone H2B, local UV-marked DNA, and photobleaching of fluorescently labeled DNA. All methods revealed that global chromosomal organization can be reestablished through one cell division from mother to daughters. By simultaneously monitoring cell cycle stage in the cells in which relative chromosomal domain positions were tracked, we observed that chromosomal neighborhood organization is apparently lost in the early G1 phase of the cell cycle. However, the daughter cells eventually regain the general chromosomal organization pattern of their mothers, suggesting an active mechanism could be at play to reestablish chromosomal neighborhoods.

Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents.

Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (gamma-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced gamma-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, gamma-H2AX foci were also induced in Ercc1(-/-) cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1(-/-) cells, MMC-induced gamma-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1(-/-) and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.

PURPOSE: In uveal melanoma, specific chromosomal abnormalities are known to correlate with the risk of metastases; changes in chromosomes 3 and 8q correlate strongly with a decreased survival of the patient, whereas chromosome 6 abnormalities are associated with a better prognosis. Usuall

Recently, we and others reported a recurrent t(7;12)(q36;p13) found in myeloid malignancies in children < or =18 months of age and associated with a poor prognosis. Fluorescence in situ hybridization studies mapped the 12p13 breakpoint to the first intron of ETV6 and narrowed down the region of 7q36 involved. By using the sequences made public recently by the Human Genome Project, two candidate genes in 7q36 were identified: the homeobox gene HLXB9 and c7orf3, a gene with unknown function. Reverse transcription-PCR of two cases with t(7;12), using primers for c7orf3 and ETV6, was negative. However, reverse transcription-PCR for HLXB9-ETV6 demonstrated alternative splicing; the two major bands corresponded to fusion of exon 1 of HLXB9 to exons 2 and 3, respectively, of ETV6. The reciprocal ETV6-HLXB9 transcript was not detected. It remains to be elucidated if the leukemic phenotype is attributable to the formation of the HLXB9-ETV6 fusion protein, which includes the helix-loop-helix and E26 transformation-specific DNA binding domains of ETV6 or to the disruption of the normal ETV6 protein.

The majority of patients with DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS) and a minority of patients with non-syndromic conotruncal heart defects are hemizygous for a region of chromosome 22q11. The chromosomal region that is commonly deleted is larger than 2 Mb. It has not been possible to narrow the smallest region of overlap (SRO) of the deletions to less than ca 500 kb, which suggests that DGS/VCFS might be a contiguous gene syndrome. The saturation cloning of the SRO is being carried out, and one gene (TUPLE1) has been identified. By using a cosmid probe (M51) and fluorescence in situ hybridization, we show here that the anonymous DNA marker locus D22S183 is within the SRO, between TUPLE1 and D22S75 (probe N25). A second locus with weak homology to D22S183, recognized by cosmid M56, lies immediately outside the common SRO of the DGS and VCFS deletions, but inside the SRO of the DGS deletions. D22S183 sequences are strongly conserved in primates and weaker hybridizing signals are found in DNA of other mammalian species; no transcripts are however detected in polyA+ RNA from various adult human organs. Probe M51 allows fast reliable screening for 22q11 deletions using fluorescence in situ hybridization. A deletion was found in 11 out of 12 DGS patients and in 3 out of 7 VCFS patients. Two patients inherited the deletion from a parent with mild (atypical) symptoms.