An anaplerotic approach to correct the mitochondrial dysfunction in ataxia-telangiectasia (A-T).

BACKGROUND: ATM, the protein defective in the human genetic disorder, ataxia-telangiectasia (A-T) plays a central role in response to DNA double-strand breaks (DSBs) and in protecting the cell against oxidative stress. We showed that A-T cells are hypersensitive to metabolic stress which can be accounted for by a failure to exhibit efficient endoplasmic reticulum (ER)-mitochondrial signalling and Ca2+ transfer in response to nutrient deprivation resulting in mitochondrial dysfunction. The objective of the current study is to use an anaplerotic approach using the fatty acid, heptanoate (C7), a metabolic product of the triglyceride, triheptanoin to correct the defect in ER-mitochondrial signalling and enhance cell survival of A-T cells in response to metabolic stress. METHODS: We treated control cells and A-T cells with the anaplerotic agent, heptanoate to determine their sensitivity to metabolic stress induced by inhibition of glycolysis with 2- deoxyglucose (2DG) using live-cell imaging to monitor cell survival for 72 h using the Incucyte system. We examined ER-mitochondrial signalling in A-T cells exposed to metabolic stress using a suite of techniques including immunofluorescence staining of Grp75, ER-mitochondrial Ca2+ channel, the VAPB-PTPIP51 ER-mitochondrial tether complexes as well as proximity ligation assays between Grp75-IP3R1 and VAPB1-PTPIP51 to establish a functional interaction between ER and mitochondria. Finally, we also performed metabolomic analysis using LC-MS/MS assay to determine altered levels of TCA intermediates A-T cells compared to healthy control cells. RESULTS: We demonstrate that heptanoate corrects all aspects of the defective ER-mitochondrial signalling observed in A-T cells. Heptanoate enhances ER-mitochondrial contacts; increases the flow of calcium from the ER to the mitochondrion; restores normal mitochondrial function and mitophagy and increases the resistance of ATM-deficient cells and cells from A-T patients to metabolic stress-induced killing. The defect in mitochondrial function in ATM-deficient cells was accompanied by more reliance on aerobic glycolysis as shown by increased lactate dehydrogenase A (LDHA), accumulation of lactate, and reduced levels of both acetyl CoA and ATP which are all restored by heptanoate. CONCLUSIONS: We conclude that heptanoate corrects metabolic stress in A-T cells by restoring ER-mitochondria signalling and mitochondrial function and suggest that the parent compound, triheptanoin, has immense potential as a novel therapeutic agent for patients with A-T.

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families: functional analysis in heterozygous individuals.

A protein-truncating variant of CHEK2, 1100delC, is associated with a moderate increase in breast cancer risk. We have determined the prevalence of this allele in index cases from 300 Australian multiple-case breast cancer families, 95% of which had been found to be negative for mutations in BRCA1 and BRCA2. Only two (0.6%) index cases heterozygous for the CHEK2 mutation were identified. All available relatives in these two families were genotyped, but there was no evidence of co-segregation between the CHEK2 variant and breast cancer. Lymphoblastoid cell lines established from a heterozygous carrier contained approximately 20% of the CHEK2 1100delC mRNA relative to wild-type CHEK2 transcript. However, no truncated CHK2 protein was detectable. Analyses of expression and phosphorylation of wild-type CHK2 suggest that the variant is likely to act by haploinsufficiency. Analysis of CDC25A degradation, a downstream target of CHK2, suggests that some compensation occurs to allow normal degradation of CDC25A. Such compensation of the 1100delC defect in CHEK2 might explain the rather low breast cancer risk associated with the CHEK2 variant, compared to that associated with truncating mutations in BRCA1 or BRCA2.

Ataxia-telangiectasia: chronic activation of damage-responsive functions is reduced by alpha-lipoic acid.

Cells from patients with the genetic disorder ataxia-telangiectasia (A-T) are hypersensitive to ionizing radiation and radiomimetic agents, both of which generate reactive oxygen species capable of causing oxidative damage to DNA and other macromolecules. We describe in A-T cells constitutive activation of pathways that normally respond to genotoxic stress. Basal levels of p53 and p21(WAF1/CIP1), phosphorylation on serine 15 of p53, and the Tyr15-phosphorylated form of cdc2 are chronically elevated in these cells. Treatment of A-T cells with the antioxidant alpha-lipoic acid significantly reduced the levels of these proteins, pointing to the involvement of reactive oxygen species in their chronic activation. These findings suggest that the absence of functional ATM results in a mild but continuous state of oxidative stress, which could account for several features of the pleiotropic phenotype of A-T.

Transactivation-deficient p73alpha (p73Deltaexon2) inhibits apoptosis and competes with p53.

p73 has recently been identified as a structural and functional homolog of the tumor suppressor protein p53. Overexpression of p53 activates transcription of p53 effector genes, causes growth inhibition and induced apoptosis. We describe here the effects of a tumor-derived truncated transcript of p73alpha (p73Deltaexon2) on p53 function and on cell death. This transcript, which lacks the acidic N-terminus corresponding to the transactivation domain of p53, was initially detected in a neuroblastoma cell line. Overexpression of p73Deltaexon2 partially protects lymphoblastoid cells against apoptosis induced by anti-Fas antibody or cisplatin. By cotransfecting p73Deltaexon2 with wild-type p53 in the p53 null line Saos 2, we found that this truncated transcript reduces the ability of wild-type p53 to promote apoptosis. This anti-apoptotic effect was also observed when p73Deltaexon2 was co-transfected with full-length p73 (p73alpha). This was further substantiated by suppression of p53 transactivation of the effector gene p21/Waf1 in p73Deltaexon2 transfected cells and by inhibition of expression of a reporter gene under the control of the p53 promoter. Thus, this truncated form of p73 can act as a dominant-negative agent towards transactivation by p53 and p73alpha, highlighting the potential implications of these findings for p53 signaling pathway. Furthermore, we demonstrate the existence of a p73Deltaexon2 transcript in a very significant proportion (46%) of breast cancer cell lines. However, a large spectrum of normal and malignant tissues need to be surveyed to determine whether this transdominant p73 variant occurs in a tumor-specific manner.

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites. In vivo assessment using phospho-specific antibodies.

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1.Mre11.Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after UV) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.

Role for ATM in DNA damage-induced phosphorylation of BRCA1.

The human genetic disorder ataxia-telangiectasia is characterized by immunodeficiency, progressive cerebellar ataxia, radiosensitivity, cell cycle checkpoint defects, and cancer predisposition. The gene product [ataxia-telangiectasia mutation (ATM)] mutated in this syndrome is a component of the DNA damage detection pathway. Loss of ATM function in human and mouse cells causes defects in DNA repair and cell cycle checkpoint control and, not surprisingly, humans and mice with compromised ATM function are prone to cancers. An excess of breast cancer in the relatives of ataxia-telangiectasia patients has also been reported by epidemiological studies. Predisposition to breast and ovarian cancers is also observed in women with germline mutations in BRCA1, a tumor suppressor gene. BRCA1 is a nuclear protein with a cell cycle-regulated expression pattern and is hyperphosphorylated in response to DNA-damaging agents. Here we show that rapid ionizing radiation-induced in vivo phosphorylation of BRCA1 requires the presence of functional ATM protein. Furthermore, we show that ATM interacts with BRCA1, and this association is enhanced by radiation. We also demonstrate that BRCA1 is a substrate of ATM kinase in vitro and in vivo. Using phospho-specific antibodies against serines 1387, 1423, and 1457 of BRCA1, we demonstrate radiation-induced, ATM-dependent phosphorylation of BRCA1 at these sites. These findings show that BRCA1 is regulated by an ATM-dependent mechanism as a part of the cellular response to DNA damage. This interaction between ATM and BRCA1 argues in favor of the involvement of particular aspects of ATM function in breast cancer predisposition.

ATM-dependent phosphorylation of nibrin in response to radiation exposure.

Mutations in the gene ATM are responsible for the genetic disorder ataxia-telangiectasia (A-T), which is characterized by cerebellar dysfunction, radiosensitivity, chromosomal instability and cancer predisposition. Both the A-T phenotype and the similarity of the ATM protein to other DNA-damage sensors suggests a role for ATM in biochemical pathways involved in the recognition, signalling and repair of DNA double-strand breaks (DSBs). There are strong parallels between the pattern of radiosensitivity, chromosomal instability and cancer predisposition in A-T patients and that in patients with Nijmegen breakage syndrome (NBS). The protein defective in NBS, nibrin (encoded by NBS1), forms a complex with MRE11 and RAD50 (refs 1,2). This complex localizes to DSBs within 30 minutes after cellular exposure to ionizing radiation (IR) and is observed in brightly staining nuclear foci after a longer period of time. The overlap between clinical and cellular phenotypes in A-T and NBS suggests that ATM and nibrin may function in the same biochemical pathway. Here we demonstrate that nibrin is phosphorylated within one hour of treatment of cells with IR. This response is abrogated in A-T cells that either do not express ATM protein or express near full-length mutant protein. We also show that ATM physically interacts with and phosphorylates nibrin on serine 343 both in vivo and in vitro. Phosphorylation of this site appears to be functionally important because mutated nibrin (S343A) does not completely complement radiosensitivity in NBS cells. ATM phosphorylation of nibrin does not affect nibrin-MRE11-RAD50 association as revealed by radiation-induced foci formation. Our data provide a biochemical explanation for the similarity in phenotype between A-T and NBS.

Localization of a portion of extranuclear ATM to peroxisomes.

The gene mutated in the human genetic disorder ataxia-telangiectasia codes for a protein, ATM, the known functions of which include response to DNA damage, cell cycle control, and meiotic recombination. Consistent with these functions, ATM is predominantly present in the nucleus of proliferating cells; however, a significant proportion of the protein has also been detected outside the nucleus in cytoplasmic vesicles. To understand the possible role of extra-nuclear ATM, we initially investigated the nature of these vesicles. In this report we demonstrate that a portion of ATM co-localizes with catalase, that ATM is present in purified mouse peroxisomes, and that there are reduced levels of ATM in the post-mitochondrial membrane fraction of cells from a patient with a peroxisome biogenesis disorder. Furthermore the use of the yeast two-hybrid system demonstrated that ATM interacts directly with a protein involved in the import of proteins into the peroxisome matrix. Because peroxisomes are major sites of oxidative metabolism, we investigated catalase activity and lipid hydroperoxide levels in normal and A-T fibroblasts. Significantly decreased catalase activity and increased lipid peroxidation was observed in several A-T cell lines. The localization of ATM to peroxisomes may contribute to the pleiotropic nature of A-T.

ATM is upregulated during the mitogenic response in peripheral blood mononuclear cells.

Patients with the human genetic disorder ataxia-telangiectasia (A-T) are characterized by immunodeficiency and a predisposition to develop lymphoid malignancies. The gene mutated in A-T patients, ATM, codes for a high molecular weight protein that is implicated in DNA damage recognition and cell cycle control. The ATM protein does not change in amount or cellular distribution throughout the cell cycle or in response to DNA damaging agents. Because peripheral blood mononuclear cells (PBMCs) are largely in a state of quiescence and can be readily stimulated to enter a proliferative phase and because A-T cells exhibit growth abnormalities and senescence, indicative of a general intracellular defect in signalling, we chose PBMCs to examine the relationship of ATM to the proliferative status of the cell. We show here that ATM protein is present at low levels in freshly isolated PBMCs and increases approximately 6-fold to 10-fold in response to a mitogenic stimulus, reaching a maximum after 3 to 4 days. A similar, but delayed response, was evident in the presence of serum only. This increase in ATM protein was accompanied by an increase in ATM kinase activity. While expression of ATM protein increased during proliferation, ATM mRNA expression was unchanged in stimulated and unstimulated cells and there was no evidence for increased ATM protein stability in the phytohemagglutinin (PHA)-treated cells. In keeping with the reduced levels of ATM in quiescent cells, the extent of radiation-induction of the p53 pathway was significantly lower than in mitogen-stimulated cells. Basal levels of p21 were elevated in quiescent cells, and the response to radiation was negligible or reduced compared with proliferating cells over a 2-hour period. Overall, the data suggest that the increase in ATM protein in proliferating cells is due to posttranscriptional regulation and points to a role for ATM in more general signalling.

Ataxia telangiectasia-mutated gene product inhibits DNA damage-induced apoptosis via ceramide synthase.

DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2'deoxyuridine, which produces DNA dsb, signaled de novo ceramide synthesis by post-translational activation of ceramide synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.

Chk1 complements the G2/M checkpoint defect and radiosensitivity of ataxia-telangiectasia cells.

Cells from patients with the human genetic disorder ataxia-telangiectasia (A-T) are defective in the activation of cell cycle checkpoints in response to ionizing radiation damage. In order to understand the role of ATM in checkpoint control we investigated whether Schizosaccaromyces pombe chk1, a protein kinase implicated in controlling the G2 DNA damage checkpoint, might alter the radiosensitive phenotype in A-T cells. The fission yeast chkl gene was cloned into an EBV-based vector under the control of a metallothionein promoter and transfected into A-T lymphoblastoid cells. Induction of chk1 enhanced the survival of an A-T cell line in response to radiation exposure as determined by cell viability and reduction of radiation-induced chromosome aberrations. This can be accounted for at least in part by the restoration of the G2 checkpoint to chk1 expressing cells. There was no evidence that chk1 expression corrected either the G1/S checkpoint or radioresistant DNA synthesis in S phase in these cells. These results suggest that chk1 when overexpressed acts downstream from ATM to restore the G2 checkpoint in these cells and correct the radiosensitive phenotype. These data allow us to dissociate individual checkpoint events and relate them to the radiosensitive phenotype in A-T cells.

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.

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.

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.

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.

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.

Cellular localisation of the ataxia-telangiectasia (ATM) gene product and discrimination between mutated and normal forms.

The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted protein sequence detected a approximately 350 kDa protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM protein is present in the nucleus and cytoplasmic vesicles. This distribution did not change after irradiation. We also provide evidence that ATM protein binds to p53 and this association is defective in A-T cells compatible with the defective p53 response in these cells. These results provide further support for a role for the ATM protein as a sensor of DNA damage and in a more general role in cell signalling, compatible with the broader phenotype of the syndrome.

Genetic complementation of radiation response by 3' untranslated regions (UTR) of RNA.

The molecular basis of radiosensitivity was studied using a cDNA complementation approach to correct radiosensitivity in cells. Four cDNAs of sizes 1.6, 2.0, 2.2 and 2.5 kb were isolated that corrected several aspects of the phenotype of cells from patients with the human genetic disorder ataxia-telangiectasia, characterized by hypersensitivity to ionizing radiation. The criteria used to assess correction included cell viability, induced chromosome aberrations, G2 phase delay and induction of p53 after exposure to radiation. One cDNA (2.5 kb) was identified as the complete sequence of the RNA helicase p68, which was capable of correcting radiosensitivity based on two of the above four criteria, with p53 induction post irradiation being partially corrected. The 2.2 kb cDNA was shown to correspond to the complete sequence of arginyl tRNA synthetase and the other two cDNAs were identical to the 3' untranslated regions (UTR) of the transcription factor TFIIS (1.6 kb) and phospholipase A2 (2.0 kb) respectively. Additional transfections with the 3'UTR (198 nucleotides) of p68 RNA helicase and its inverse sequence revealed that the 3'UTR had the same complementation capacity as the full-length cDNA, whereas the inverse construct failed to complement radiosensitivity. These data provide additional support for a novel role for 3'UTRs in the regulation of gene expression.

Recombinant viral vaccines for enzootic bovine leucosis.

Recently published studies on the development and use of recombinant vaccinia virus (VV) vaccines incorporating either the complete envelope (env) gene or only a fragment of the env gene consisting of the coding sequence for the env glycoprotein 51 (gp51) and part of gp30 of the bovine leukaemia virus (BLV) are described. It has been reported that vaccination of sheep with recombinant VV vaccines containing the complete env gene appears to protect sheep against challenge infection with BLV. The evidence for this protection is based on the lack of persistence of high titres of anti-gp51 antibodies compared with unvaccinated BLV infected controls, on the enhanced CD4 proliferative responses to specific BLV gp51 synthetic peptides in the vaccinated sheep, and on the inability to detect BLV pro-virus by polymerase chain reaction in the vaccinated sheep after 4 months following challenge infection compared with continual detection in unvaccinated sheep over a 16 month trial period. It has been suggested that cell-mediated immune responses may be an important aspect of protective immunity against BLV infection and it has been reported that large tracts of amino acid sequences within the env and pol genes are highly conserved in different isolates from different countries which is of importance in designing peptide derived vaccines.

T-cell responses to highly conserved CD4 and CD8 epitopes on the outer membrane protein of bovine leukemia virus: relevance to vaccine development.

Bovine leukemia virus (BLV) is a retrovirus that infects cattle and sheep and may provide a model for studying human leukemia. Cell-mediated immune mechanisms may play a major role in protection against BLV infection. We describe here for the first time the identification of proliferative (CD4) and cytotoxic T-lymphocyte (CD8) epitopes of the gp51 envelope (env) protein of BLV. This protein and a recombinant form expressed by a vaccinia virus construct have been shown to be potential vaccine candidates. A complete series of overlapping peptides, 20 amino acids in length, was prepared to identify epitopes from gp51. These peptides were tested for the ability to elicit peripheral blood lymphocyte proliferation and cytotoxic T-lymphocyte responses in infected and uninfected cattle and sheep. Peptides 51-70 and 61-80 produced a proliferative response in lymphocytes from only uninfected animals (both sheep and cattle), and this was shown by T-cell subset deletion to be a CD4-mediated response. Seven BLV-infected sheep did not show a response to either peptide. Cytotoxic T-lymphocyte activity, however, was associated only with peptides 121-140 and 131-150. In this case, the response was demonstrated to be CD8 dependent and was found only in BLV-infected animals (sheep). Knowledge of the location of these T-cell recognition domains will complement data available on B-cell epitopes in gp51 and may be useful in the design of a subunit vaccine.