HSV-1 amplicon vector-mediated expression of ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and radiation sensitivity. Previous research has shown that it is possible to correct the hereditary deficiency A-T by DNA transfection in cell culture, but the large size of the ATM cDNA (9 kb) limits the use of many vector types for gene replacement. HSV-1 amplicon vectors provide a means to deliver large genes to cells efficiently and without toxicity. In this study, the FLAG-tagged cDNA for human ATM was inserted into an HSV-1 amplicon under control of the CMV promoter (designated as HGC-ATM). FLAG-ATM expression was confirmed in 293T/17 cells and human A-T fibroblasts (GM9607) after transduction, by immunoprecipitation, Western analysis, and immunocytochemistry. Functional recovery was assessed by two independent assays. First, in vitro kinase assay showed that vector-derived ATM in GM9607 cells could successfully phosphorylate wt p53 using recombinant GST-p53(1-101). Second, in A-T cells infected with the HGC-ATM vector, the extent of accumulation in G2/M phase at 24 h postirradiation was similar to that observed in cells with wild-type endogenous ATM and lower than that observed in A-T cells infected with a control vector. Thus, these vectors provide a tool to test the feasibility of HSV-amplicons as gene therapy vectors for A-T.

Heat shock cognate 70 mutations in sporadic breast carcinoma.

Heat Shock Cognate 70 (HSC70) is a constitutively expressed molecular chaperone, functions of which regulate the structure, subcellular localization, and turnover of cell proteins. It is also a component of the centrosome facilitating rearrangements during mitotic/meiotic spindle formation and cytoplasmic microtubule organization. We localized HSC70 to 11q23.3, a region deleted in 40% of sporadic breast and other cancers. Sequencing demonstrated mutation in the NH2-terminal ATPase domain of HSC70 in 2 of 15 sporadic breast carcinomas examined. In both cases, mutation was coincident with allelic imbalance, suggesting that HSC70 is a target of somatic mutation and deletion in a fraction of breast carcinomas.

Chromosomes, 11Q and cancer: a review.

This review aims at providing a general understanding of how the multiple cytogenetic aberrations in cancer cells arise and exemplifies this by considering the specific role of chromosome 11q loci in carcinogenesis. Section I provides a theoretical molecular and structural framework for understanding the cytogenetic aberrations described in cancer. Given this background, Section II describes advances in the identification and localization of cancer susceptibility genes on chromosome 11q, highlighting ongoing areas of investigation.

11q23.1 and 11q25-qter YACs suppress tumour growth in vivo.

Frequent allelic deletion at chromosome 11q22-q23.1 has been described in breast cancer and a number of other malignancies, suggesting putative tumour suppressor gene(s) within the approximately 8 Mb deleted region. In addition, we recently described another locus, at the 11q25-qter region, frequently deleted in breast cancer, suggesting additional tumour suppressor gene(s) in this approximately 2 Mb deleted region. An 11q YAC contig was accessed and three YACs, one containing the candidate gene ATM at 11q23.1, and two contiguous YACs (overlapping for approximately 400-600 kb) overlying most of the 11q25 deleted region, were retrofitted with a G418 resistance marker and transfected into murine A9 fibrosarcoma cells. Selected A9 transfectant clones (and control untransfected and 'irrelevant' alphoid YAC transfectant A9 clones) were assayed for in vivo tumorigenicity in athymic female Balb c-nu/nu mice. All the 11q YAC transfectant clones demonstrated significant tumour suppression compared to the control untransfected and 'irrelevant' YAC transfected A9 cells. These results define two discrete tumour suppressor loci on chromosome 11q by functional complementation, one to a approximately 1.2 Mb region on 11q23.1 (containing the ATM locus) and another to a approximately 400-600 kb subterminal region on 11q25-qter.

Allelic deletion at 11q23.3-q25 is an early event in cervical neoplasia.

Twenty per cent of cervical intraepithelial neoplasias-III (CIN-III) progress to invasive cancer. Human papillomavirus (HPV) infection alone does not determine progression. CIN-III lesions were collected from 161 women. Each tissue was microdissected into a maximum of 32 contiguous units and assayed at multiple microsatellite loci on chromosome 11q, a region frequently deleted in invasive cervical and other cancers. Eight of 108 informative cases (7%) had 11q23.3-q25 deletions; focally intra-lesional in six (one with focal loss of alternate alleles), and pan-lesional in two cases. Hence, 11q deletion can occur early in cervical neoplasia, and possibly predisposes to invasion.

Induction of myocardial heat shock protein 70 during cardiac surgery.

Animal experiments have shown that members of the heat shock protein (HSP) family have cytoprotective properties against ischaemia. In experimentally induced cardiac ischaemia, the induction of HSP70s correlates with reduced infarct size and enhanced myocardial function and endothelial recovery. Direct evidence that increased myocardial HSP70 expression result in cytoprotection during ischaemia has also been obtained using transgenic mice overexpressing either rat or human HSP72. This study examined the induction and expression of myocardial HSP70s after an obligatory period of ischaemia in patients during cardiac surgery. The level of HSP72/HSC73 protein in Tru-cut biopsies of the myocardium, taken before and after an acute ischaemic insult, was examined using a polyclonal antibody. The amount of HSP72 mRNA in the biopsies was also determined by reverse transcriptase polymerase chain reaction (RT-PCR) and correlated HSP72/HSC73 protein expression. In four patients subjected to brief alternating periods of normothermic ischaemia and reperfusion, the amount of myocardial HSP72/HSC73 protein was increased several fold after ischaemic insult. This was accompanied by increased expression of HSP72 mRNA. In contrast, the amounts of myocardial HSP72/HSC73 protein and HSP72 mRNA were unchanged in a patient subjected to a single prolonged period of hypothermic ischaemia. Given the proven myocardial protective properties of HSP72 in experimental models, it is postulated that the observed induction of HSP72 may have a similar function in man.

Allelic deletions at chromosome 11q22-q23.1 and 11q25-qterm are frequent in sporadic breast but not colorectal cancers.

We identified the chromosome 11q23 region as containing a putative tumour suppressor gene(s) frequently deleted in nonfamilial breast and other cancers. To define this region(s) further, we performed a systematic genetic analysis at chromosome 11q14-qterm in sporadic breast and colorectal cancer. Tumour and constitutional DNA from a panel of 81 cases (51 breast and 30 colorectal cancers) were analysed with multiple microsatellite markers distal to 11q13. Of 51 breast cancers, 31 of 49 informative cases (63%) showed LOH at the 11q22-q23.1 region (approximately 8 Mb). Furthermore, 23 of 45 informative cases (51%) had a deletion at 11q25 (approximately 2 Mb). Overall, LOH on 11q occurred in 37 of 51 breast cancers (72%). Colorectal cancers had LOH at 11q22 in two of 18 informative cases (11%), LOH at 11q23.3 in two of 17 informative cases (12%) and LOH at 11q25 in three of 20 informative cases (15%). Overall, LOH at 11q occurred in five of 30 colorectal cancers (16%). This data shows that chromosome 11q contains at least two independent regions (one novel) frequently deleted in breast cancers. Contrary to previous reports, LOH at distal 11q is not frequent in colorectal cancer. Chromosome 11q22-q23.1 and 11q25-qterm contain putative tumour suppressor genes with a significant role in breast but not colorectal carcinogenesis.

The 50-kDa primase subunit of Drosophila melanogaster DNA polymerase alpha. Molecular characterization of the gene and functional analysis of the overexpressed protein.

The gene encoding the 50-kDa subunit of Drosophila melanogaster DNA polymerase alpha has been cloned. A comparison of the predicted polypeptide sequence of the Drosophila protein with the equivalent subunits from mouse and yeast suggests that they are closely related and defines three conserved regions which are likely to be important for enzyme activity. The expression patterns of both the 50-kDa protein and its transcript (a single RNA message of 1.6 kilobases) throughout development are consistent with a role of the protein in DNA replication. When overexpressed and purified the 50-kDa subunit displays DNA primase activity. The products of the reaction, mainly oligoribonucleotides 12-14 nucleotides in length, plus dimers and some trimers, are similar to those synthesized by either the intact DNA polymerase alpha, or the biochemically isolated primase heterodimer. The isolated primase also shows similar sensitivity to antibodies, magnesium and monovalent cations, and the same nucleotide requirements as complexed forms of the primase. The isolated subunit, however, is more thermally labile, suggesting a role for the additional subunits in DNA polymerase alpha in stabilizing the primase activity of the 50-kDa primase subunit.