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M F Lavin

Publications and source records attributed to M F Lavin.

At least 55 records · Page 3Linked to original sources
PubMed1998

An anti-sense construct of full-length ATM cDNA imposes a radiosensitive phenotype on normal cells.

The cloning of a full-length cDNA for the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) has been described recently. This cDNA, as well as a fragment representing a functional region from ATM, are capable of rescuing various aspects of the radiosensitive phenotype in A-T cells. We have subcloned full-length ATM cDNA in the opposite orientation in an EBV-based vector under the control of an inducible promoter to determine whether this anti-sense construct might sensitize control lymphoblastoid cells to ionizing radiation. The effectiveness of expression of this construct in control cells was monitored by loss of ATM protein which was evident over a period 6-12 h after induction. Under these conditions radiosensitivity was enhanced approximately threefold in control cells, approaching the degree of radiosensitivity observed in A-T cells. Expression of the anti-sense construct also increased the number of radiation-induced chromosomal breaks and led to the appearance of radioresistant DNA synthesis in these cells. Abrogation of the G1/S checkpoint was evident from the loss of the p53 response and that of its downstream effector, p21/WAF1, post-irradiation. The extent of accumulation of transfected cells in G2/M phase at 24 h post-irradiation was similar to that observed in A-T cells and the induction of stress-activated protein kinase by ionizing radiation was prevented by antisense ATM cDNA expression. These data demonstrate that full-length ATM anti-sense cDNA, by reducing the amount of ATM protein, is effective in imposing a series of known defects characteristic of the A-T phenotype. This inducible system provides an experimental model to further investigate mechanisms underlying radiosensitivity and cell cycle control.

Ataxia Telangiectasia↗
PubMed1998

Cloning and expression of the ataxia-telangiectasia gene in baculovirus.

The gene mutated in the human genetic disorder ataxia-telangiectasia, ATM, is implicated in the response to radiation-induced DNA damage and to a more widespread signalling defect. The ATM protein is predominantly a nuclear protein where it interacts with p53 and c-Abl as part of a radiation signal transduction pathway(s). We describe here the cloning of full-length ATM cDNA in a baculovirus vector to produce recombinant protein. Expression of ATM, as a soluble protein, was observed by 36 h post-infection using immunoblotting with anti-ATM antibody. The presence of a hexahistidine tag on ATM was used as the basis for purification of the protein by affinity chromatography. The protein yield was only 20 ng/100 ml of infected cells, presumably because of the size of the protein and adverse effects on cell growth when overexpressed. ATM was found to have autophosphorylation activity in immunoprecipitates with antibodies directed against the hexahistidine tag sequence. These results demonstrate that ATM can be expressed inefficiently in baculovirus infected insect cells and the data suggest that it phosphorylates itself.

Animals↗
PubMed1998

The radiosensitive cell line 180BR is not defective in the major DNA damage-sensing proteins.

The fibroblast culture 180BR, established from a patient showing an adverse response to radiotherapy, has been shown previously to be hypersensitive to ionizing radiation and to be defective in the repair of DNA double-strand breaks. We demonstrate here that the products of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and its regulatory subunits (Ku 70 and Ku 80) are present at normal levels and possess functional activity. The product of the gene mutated in the human genetic disorder ataxia-telangiectasia was also detected in these cells. Apoptosis was detected after high-dose ionizing radiation exposure, and this process was accompanied by specific degradation of DNA-PKcs, ATM, and poly(ADP-ribose) polymerase. Activation of CPP32, an interleukin 1beta converting enzyme-like protease implicated in apoptosis, was also observed in 180BR cells in response to radiation damage. The radiosensitivity observed in 180BR cells can be accounted for, at least in part, by radiation-induced apoptosis, and the defect in these cells is not a gross one in DNA-PKcs or ATM.

Ataxia Telangiectasia↗
PubMed1998

Radiosensitivity and oxidative signalling in ataxia telangiectasia: an update.

Radiosensitivity is a major hallmark of the human genetic disorder ataxia telangiectasia. This hypersensitivity to ionizing radiation has been demonstrated in vivo after exposure of patients to therapeutic doses of radiation and in cells in culture. Clearly an understanding of the nature of the molecular defect in ataxia telangiectasia will be of considerable assistance in delineating additional pathways that determine cellular radiosensitivity/radioresistance. Furthermore, since patients with this syndrome are also predisposed to developing a number of leukaemias and lymphomas, the possible connection between radiosensitivity and cancer predisposition is of interest. Now that the gene (ATM) responsible for this genetic disease has been cloned and identified, progress is being made in determining the role of the ATM protein in mediating the effects of cellular exposure to ionizing radiation and other forms of redox stress. Proteins such as the product of the tumour suppressor gene p53 and the proto-oncogene c-Abl (a protein tyrosine kinase) have been shown to interact with ATM. Since several intermediate steps in both the p53 and c-Abl pathways, activated by ionizing radiation, are known it will be possible to map the position of ATM in these pathways and describe its mechanism of action. What are the clinical implications of understanding the molecular basis of the defect in ataxia telangiectasia (A-T)? As outlined above, since radiosensitivity is a universal characteristic of A-T, understanding the mechanism of action of ATM will provide additional information on radiation signalling in human cells. With this information it may be possible to sensitize tumour cells to radiation and thus increase the therapeutic benefit of radiotherapy. This might involve the use of small molecules that would interfere with the normal ATM-controlled pathways and thus sensitize cells to radiation or alternatively it might involve the efficient introduction of ATM anti-sense cDNA constructs into tumours to achieve the same end-point.

Animals↗
PubMed1998

Vaccine-induced cytotoxic T lymphocytes protect against retroviral challenge.

The development of prophylactic vaccines against retroviral diseases has been impeded by the lack of obvious immune correlates for protection. Cytotoxic T-lymphocyte (CTL), CD4-lymphocyteS, chemokine and/or antibody responses have all been associated with protection against HIV and AIDS; however, effective and safe vaccination strategies remain elusive. Here we show that vaccination with a minimal ovine CTL peptide epitope identified within gp51 of the retrovirus bovine leukemia virus (BLV), consistently induced peptide-specific CTLs. Only sheep whose CTLs were also capable of recognizing retrovirus-infected cells were fully protected when challenged with BLV. This retrovirus displays limited sequence variation; thus, in the relative absence of confounding CTL escape variants, virus-specific CTLs targeting a single epitope were able to prevent the establishment of a latent retroviral infection.

Animals↗
PubMed1998

ATM associates with and phosphorylates p53: mapping the region of interaction.

The human genetic disorder ataxia-telangiectasia (AT) is characterized by immunodeficiency, progressive cerebellar ataxia, radiosensitivity, cell cycle checkpoint defects and cancer predisposition. The gene mutated in this syndrome, ATM (for AT mutated), encodes a protein containing a phosphatidyl-inositol 3-kinase (PI-3 kinase)-like domain. ATM also contains a proline-rich region and a leucine zipper, both of which implicate this protein in signal transduction. The proline-rich region has been shown to bind to the SH3 domain of c-Abl, which facilitates its phosphorylation and activation by ATM. Previous results have demonstrated that AT cells are defective in the G1/S checkpoint activated after radiation damage and that this defect is attributable to a defective p53 signal transduction pathway. We report here direct interaction between ATM and p53 involving two regions in ATM, one at the amino terminus and the other at the carboxy terminus, corresponding to the PI-3 kinase domain. Recombinant ATM protein phosphorylates p53 on serine 15 near the N terminus. Furthermore, ectopic expression of ATM in AT cells restores normal ionizing radiation (IR)-induced phosphorylation of p53, whereas expression of ATM antisense RNA in control cells abrogates the rapid IR-induced phosphorylation of p53 on serine 15. These results demonstrate that ATM can bind p53 directly and is responsible for its serine 15 phosphorylation, thereby contributing to the activation and stabilization of p53 during the IR-induced DNA damage response.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1998

Calpain activation is upstream of caspases in radiation-induced apoptosis.

The molecular events involved in apoptosis induced by ionizing radiation remain unresolved. In this paper we show that the cleavage of fodrin to a 150 kDa fragment is an early proteolytic event in radiation-induced apoptosis in the Burkitts' Lymphoma cell line BL30A and requires 100 microM zVAD-fmk for inhibition. Caspases-1, -3, -6 and -7 were shown to cleave fodrin to the 150 kDa fragment in vitro and all were inhibited by 10 microM zVAD-fmk. We also show that the in vitro cleavage of fodrin by calpain is inhibited by 100 microM zVAD-fmk as was the calpain-mediated hydrolysis of casein. We demonstrate that calpain is activated within 15 min after radiation exposure, concomitant with the cleavage of fodrin to the 150 kDa fragment whereas caspase-3 is activated at 2 h correlating with the cleavage of fodrin to the 120 kDa fragment. These results support a role for calpain in the early phases of the radiation-induced apoptosis pathway, upstream of the caspases.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Evaluation of the potential of transferrin-adriamycin conjugates in the treatment of bladder cancer.

OBJECTIVE: To assess the ability of a transferrin-adriamycin conjugate (Tf-ADR) to target transferrin receptor (TfR)-positive cancer cells selectively and to overcome drug resistance in bladder cancer cell lines. MATERIALS AND METHODS: Two paired sets of cell lines were used: the first was Chinese hamster ovary (CHO) cells (TfR-negative TRVb cells, as a model for normal resting cells, and TRVb-1 cells which were transfected with human TfR), and the second was a pair of bladder cancer cell lines (ADR-sensitive MGH-U1 cells and ADR-resistant MGH-U1R cells). Cell survival curves were determined after treatment with ADR, Tf and Tf-ADR. RESULTS: MGH-U1, TRVb and TRVb-1 cells required similar concentrations of ADR and Tf-ADR for 50% inhibition of growth; MGH-U1R cells were resistant to both ADR and TF-ADR. CONCLUSION: Tf-ADR did not prevent toxicity to the TfR-negative cells nor did it overcome the resistance of the ADR-resistant cells. These results imply that Tf-ADR does not provide a better cytotoxic drug delivery system for the treatment of bladder cancer.

Animals↗
PubMed1998

Upregulation of ATM in sclerosing adenosis of the breast.

The gene mutated in ataxia telangiectasia (ATM) has an established tumour suppressor role in breast cancer. ATM appears to be expressed in most normal cells, including breast epithelium, where it has been postulated to have a nuclear role in cell cycle regulation following DNA damage. However, ATM is not upregulated after DNA damage. In this study, we demonstrate an absence of immunohistologically detectable levels of ATM in the normally quiescent myoepithelial cells that line normal breast ducts. This contrasts dramatically with the significant expression of ATM in the proliferative myoepithelium of sclerosing adenosis (n = 7). This upregulation of ATM suggests that ATM expression is coupled to the proliferative status of the myoepithelium. Our results also indicate that there are factors other than ATM gene mutations that can dramatically influence ATM expression in the breast and that these factors should be considered for their possible implications in carcinogenesis.

Actins↗
PubMed1998

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata.

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H. curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

Animals↗
PubMed1997

Resistance to radiation-induced apoptosis in Burkitt's lymphoma cells is associated with defective ceramide signaling.

Increased sensitivity to ionizing radiation has been shown to be due to defects in double-strand break repair and mutations in the proteins that detect DNA damage. However, it is now recognized that the cellular radiation response is complex and that radioresistance/radiosensitivity may also be regulated at different levels in the radiation signal transduction pathway. Here, we describe a direct relationship between resistance to radiation-induced apoptosis and defective ceramide signaling. Radiation sensitivity in human tumor cells correlated with the immediate accumulation of the second messenger ceramide. In the BL30A Burkitt's lymphoma line, ceramide increased 4-fold by 10 min postirradiation (10 Gy), and in the moderately sensitive HL-60 leukemia cells, ceramide accumulated 2.5-fold above basal levels. In contrast, in all radioresistant tumor cells examined, including several Burkitt's lymphoma lines (BL30K, BL29, and BL36) and the MO59K glioma cell line, ceramide did not accumulate postirradiation. The ability to abrogate ceramide production by pretreatment with the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, conferred resistance to radiation-induced apoptosis in the sensitive BL30A cells. An isogenic subline of BL30A, BL30K, was resistant to both C8-ceramide (20 microM) and ionizing radiation-induced apoptosis. Bypassing the block in radiation-induced ceramide production by the addition of exogenous ceramide was not sufficient to induce apoptosis; this suggests the existence of a second ceramide-associated signaling defect in these radioresistant cells that confers resistance to ceramide-induced apoptosis. Thus, these results provide compelling evidence that ceramide is an essential mediator of radiation-induced apoptosis and that defective ceramide signaling confers an apoptosis-resistant phenotype in tumor cells.

Apoptosis↗
PubMed1997

Isolation of full-length ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype.

A gene mutated in the human genetic disorder ataxia-telangiectasia (A-T), ATM, was recently identified by positional cloning. ATM is a member of the phosphatidylinositol-3-kinase superfamily, some of which are protein kinases and appear to have important roles in cell cycle control and radiation signal transduction. We describe herein, to our knowledge, for the first time, the cloning of a full-length cDNA for ATM and correction of multiple aspects of the radio-sensitive phenotype of A-T cells by transfection with this cDNA. Overexpression of ATM cDNA in A-T cells enhanced the survival of these cells in response to radiation exposure, decreased radiation-induced chromosome aberrations, reduced radio-resistant DNA synthesis, and partially corrected defective cell cycle checkpoints and induction of stress-activated protein kinase. This correction of the defects in A-T cells provides further evidence of the multiplicity of effector functions of the ATM protein and suggests possible approaches to gene therapy.

Ataxia Telangiectasia↗
PubMed1997

Interaction between ATM protein and c-Abl in response to DNA damage.

The gene mutated in the autosomal recessive disorder ataxia telangiectasia (AT), designated ATM (for 'AT mutated'), is a member of a family of phosphatidylinositol-3-kinase-like enzymes that are involved in cell-cycle control, meiotic recombination, telomere length monitoring and DNA-damage response. Previous results have demonstrated that AT cells are hypersensitive to ionizing radiation and are defective at the G1/S checkpoint after radiation damage. Because cells lacking the protein tyrosine kinase c-Abl are also defective in radiation-induced G1 arrest, we investigated the possibility that ATM might interact with c-Abl in response to radiation damage. Here we show that ATM binds c-Abl constitutively in control cells but not in AT cells. Our results demonstrate that the SH3 domain of c-Abl interacts with a DPAPNPPHFP motif (residues 1,373-1,382) of ATM. The results also reveal that radiation-induction of c-Abl tyrosine kinase activity is diminished in AT cells. These findings indicate that ATM is involved in the activation of c-Abl by DNA damage and this interaction may in part mediate radiation-induced G1 arrest.

Ataxia Telangiectasia↗
PubMed1997

Defective signaling through the B cell antigen receptor in Epstein-Barr virus-transformed ataxia-telangiectasia cells.

A characteristic series of immunological abnormalities are observed in the human genetic disorder ataxia-telangiectasia (A-T). The recent cloning of a gene mutated in this syndrome provides additional evidence for a defect in intracellular signaling in A-T. We have investigated the possibility that signaling through the B cell antigen receptor is one manifestation of the A-T defect. In response to cross-linking of the B cell receptor, several A-T cell lines were defective in their mitogenic response; in addition Ca2+ mobilization from internal stores was either absent or considerably reduced in these cell lines in response to cross-linking. The defect in signaling was not due to difference in expression of surface immunoglobulin. The defective response in A-T cells was also evident in several arms of the intracellular cascade activated by B cell cross-linking. Tyrosine phosphorylation of phospholipase Cgamma1, a key step in activation of the enzyme, was reduced or negligible in some A-T cell lines. This defect in signaling was also seen at the level of Lyn tyrosine kinase activation and its association with and activation of phosphatidylinositol 3-kinase. Our results provide evidence for a role for the ATM gene product in intracellular signaling which may account at least in part for the abnormalities in B cell function in A-T.

Ataxia Telangiectasia↗
PubMed1997

Resistance of actin to cleavage during apoptosis.

A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1beta-converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.

Actins↗
PubMed1997

Novel gene containing multiple epidermal growth factor-like motifs transiently expressed in the papillae of the ascidian tadpole larvae.

We have investigated molecular mechanisms of the embryonic development of an ascidian, a primitive chordate which shares features of both invertebrates and vertebrates, with a view to identifying genes involved in development and metamorphosis. We isolated 12 partial cDNA sequences which were expressed in a stage-specific manner using differential display. We report here the isolation of a full-length cDNA sequence for one of these genes which was specifically expressed during the tailbud and larval stages of ascidian development. This cDNA, 1213 bp in length, is predicted to encode a protein of 337 amino acids containing four epidermal growth factor (EGF)-like repeats and three novel cysteine-rich repeats. Characterization of its spatial expression pattern by in situ hybridisation in late tailbud and larval embryos demonstrated strong expression localised throughout the papillae and anteriormost trunk and weaker expression in the epidermis of the remainder of the embryo. As recent evidence indicates that the signal for metamorphosis originates in the anterior trunk region, these results suggest that this gene may have a role in signalling the initiation of metamorphosis.

Amino Acid Sequence↗
PubMed1997

Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: cloning and characterization.

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydia which are involved in the immunogenic response. Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an XbaI fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneumoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli.

Amino Acid Sequence↗