Feyruz V. Rassool

Increased error-prone non homologous DNA end-joining: a proposed mechanism of chromosomal instability in Bloom's syndrome

BS is an inherited cancer predisposition disorder caused by inactivation of the RecQ family helicase, BLM. One of the defining features of cells from BS individuals is chromosomal instability, characterized by elevated sister chromatid exchanges (SCEs), as well as chromosomal breaks, deletions, and rearrangements. Although the basis for chromosomal instability is poorly understood, there is evidence that chromosomal abnormalities can arise through an alteration in the efficiency or fidelity of DNA double strand break (DSB) repair. Here, we show that BS cells demonstrate aberrant DSB repair mediated by the non-homologous end-joining (NHEJ) pathway for DNA repair, one of the two main pathways for the repair of DSBs in mammalian cells. Through a comparison of BS cell lines, and a derivative in which the BS phenotype has been reverted by expression of the BLM cDNA, we show that BS cells display aberrant end-joining of DSBs. Importantly, DNA end-joining in BS cells is highly error-prone and frequently results in DNA ligation at distant sites of microhomology, creating large DNA deletions. This aberrant repair is dependent upon the presence of the Ku70/86 heterodimer, a key component in the NHEJ pathway. We propose that aberrant NHEJ is a candidate mechanism for the generation of chromosomal instability in BS.

DNA double strand breaks (DSB) and non-homologous end joining (NHEJ) pathways in human leukemia

DNA double strand breaks (DSB) are considered the most lethal form of DNA damage for eukaryotic cells. DSB can either be properly repaired, restoring genomic integrity, or misrepaired resulting in drastic consequences, such as cell death, genomic instability, and cancer. It is well established that exposure to DSB-inducing agents is associated with chromosomal abnormalities and leukemogenesis. The non-homologous end joining (NHEJ) pathway is considered a major route for the repair DSB in mammalian cells. Although the mechanism(s) by which repair of DSB lead to leukemia are poorly understood, recent evidence is beginning to emerge that a poorly defined and error-prone branch of the NHEJ pathway plays a pivotal role in this process. This review discusses some of the ways in which error-prone NHEJ repair may be involved in the development of genomic instability and leukemia.

Constitutive DNA damage is linked to DNA replication abnormalities in Bloom's syndrome cells

Blooms syndrome (BS) is an autosomal recessive disorder associated with an elevated incidence of cancers. The gene mutated in BS, BLM, encodes a RecQ helicase family member. BS cells exhibit genomic instability, including excessive homologous recombination and chromosomal aberrations. We reported previously that BS cells also demonstrate increased error-prone nonhomologous endjoining, which could contribute to genomic instability in these cells. Here, we show that BS cells display an abnormality in the timing of replication of both early-replicating genes and late-replicating loci such as chromosomal fragile sites. This delayed replication is associated with a constitutively increased frequency of sites of DNA damage and repair, as determined by the presence of DNA repair factors such as RAD51 and Ku86. In addition, another RecQ family helicase, WRN, also localizes to these repair sites. The presence of these repair sites correlates with the temporal appearance of cyclin B1 expression, indicative of the cells having progressed beyond mid-S phase in the cell division cycle. Critically, these defects in BS cells are the direct result of loss of BLM function, because BS cells phenotypically ‘reverted’ following transfection with the BLM cDNA no longer show such defects. Thus, our data indicate that constitutive DNA damage is coupled to delayed DNA replication in BS cells.

Differentially expressed genes in adult familial myelodysplastic syndromes

The precise genetic events leading to myelodysplastic syndromes (MDSs) and leukemic transformation remain poorly defined. Even less is known about adult familial MDS. We report an adult MDS family in whom enriched tissue-specific transcripts were derived by subtractive hybridization of cDNA from the mononuclear and CD34+ cells of affected and unaffected family members. These expression libraries were then hybridized to Genome Discovery arrays containing 18 404 genes and expressed sequence tags, and several clusters of differentially expressed genes were identified. A group of 21 genes was underexpressed (>5-fold) in affected vs unaffected family members, and among these were transcription factors and genes involved in myeloid differentiation, such as ZNF140 and myeloid nuclear differentiation antigen (MNDA). Another group of 36 genes was overexpressed (>5-fold), and these encoded proteins belonging to signaling pathways, such as Ras- and Fos-related genes. The top two genes downregulated in this MDS family, ZNF140 and MNDA, were similarly altered in another MDS family, and in some cases of sporadic MDS. Our data suggest that we have identified genes differentially expressed in adult familial MDS, and that alteration of some of these genes may also be important for the evolution of different stages or severity of sporadic MDS.

Analysis of CHK2 in patients with myelodysplastic syndromes

The myelodysplastic syndromes (MDS) comprise a group of clonal hemopoietic stem cell disorders characterized by ineffective hematopoiesis with an increased propensity to myeloid leukemic (AML) transformation. The underlying molecular basis for MDS and its leukemic evolution is unclear. Except for patients with 17p syndrome, loss of function of the p53 tumor suppressor gene accounts for <10% of MDS and AML cases. Recently, mutations of the checkpoint gene, CHK2, the human homologue of the yeast CDS1 and RAD53 genes, have been reported in patients with Li-Fraumeni syndrome who also have normal p53. As p53 mutations are rare in MDS and AML, we investigated the status of the CHK2 gene by reverse transcriptase-polymerase chain reaction (RT-PCR) in patients with MDS (n=10) and patients in whom MDS had transformed into AML (n=3). In the MDS group, we found one patient with a conserved mutation (Lys-->Arg) in the forked head-associated (FHA) domain of the CHK2 coding sequence. We also found a deletion in the CHK2 transcript in one patient from the MDS-->AML group, resulting in a truncated protein lacking the kinase domain. We conclude that alterations of CHK2 and possible involvement in the pathogenesis of MDS may be a rare event.