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P R Walker

Publications and source records attributed to P R Walker.

At least 37 records · Page 2Linked to original sources
PubMed1999

Apoptosis-related functional features of the DNaseI-like family of nucleases.

Rat DNaseYb and its human homolog DHP2 are members of a new family of DNaseI-like endonucleases. They contain all the conserved amino acid residues to engage a DNaseI-like catalytic activity and the same molecular mechanisms of DNA hydrolysis. The sequence similarity can be extended to other families of nucleases, such as FEN-1, DNA polymerases, RNaseH and exonuclease III, involved in the ion-dependent hydrolysis of nucleic acids. Their unique features include the NLS signals that place them in the nuclei and a high content of positively charged amino acid residues that results in their high affinity for the substrate. Their properties are consistent with a role in the early stage DNA degradation during apoptosis. The caspase-DFF45/CIDE-CPAN pathway is most likely involved in the second stage of internucleosomal DNA degradation. However, cells express constitutively multiple transcripts encoding DNA degrading enzymes and related molecules, hence they have the molecular diversity to engage the self-destructive pathway appropriate to a given trigger.

Amino Acid Sequence↗
PubMed1998

DNaseY: a rat DNaseI-like gene coding for a constitutively expressed chromatin-bound endonuclease.

A rat gene, designated DNaseY, encoding a 36 kDa endonuclease was identified and cloned. Sequence analysis of the cDNA showed it to be the rat homologue of human DNAS1L3. The DNaseY gene product had 42% identity to DNaseI, including conserved critical active site residues, the essential disulfide bridge, the calcium binding domain, and a signal peptide, as well as 2 of the 3 signature boxes. Significantly, DNaseY had 2 nuclear localization signals and was more basic (pI 9.5) than DNaseI (pI 4.8). The DNaseY gene contained a number of exons similar to that of DNaseI, separated by much larger introns, resulting in a gene of >17 kb compared to <4 kb gene of DNaseI. The 36 kDa DNaseY gene product was catalytically inactive but was converted to an active 33 kDa endonuclease following processing of the hydrophobic signal peptide. Antibody generated against peptides representing the predicted amino acid sequence of DNaseY cross-reacted with a 33 kDa nuclear protein which possessed endonucleolytic activity. The enzyme was active over a broad pH range (optimum pH 7-8), was Ca2+/Mg2+-dependent, was inhibited by Zn2+, and was capable of both single- and double-stranded DNA cleavage, producing DNA fragments with 3'-OH ends. Furthermore, the DNaseY gene was expressed constitutively in all cells and tissues tested, but it was not transcriptionally up-regulated in apoptotic cells. All these features were consistent with a role in the early stages of apoptotic DNA fragmentation.

Amino Acid Sequence↗
PubMed1998

Tumor expression of Fas ligand (CD95L) and the consequences.

Tumors of diverse origin have recently been shown to express CD95 ligand and to induce apoptosis in CD95-expressing targets in vitro; however, in vivo, enhanced tumor growth and rejection have both been observed as a consequence of either immunosuppressive or proinflammatory functions of CD95 ligand. The final in vivo outcome of CD95 ligand expression will depend upon a complex balance of interactions relevant for each tumor in its particular microenvironment.

Animals↗
PubMed1998

Splenic T lymphocytes die preferentially during heat-induced apoptosis: NuMA reorganization as a marker.

We are investigating nuclear events during apoptosis in mouse splenic lymphocytes cultured immediately after isolation (controls) or after heat treatment (42 degreesC, 30 minutes), and have found that hyperthermia increased the level of apoptosis to double that of spontaneous apoptosis in controls within 6 hours. Immunolabelling for Nuclear Mitotic Apparatus Protein (NuMA) suggested that splenocytes were responding heterogeneously to the heat treatment. Whereas all nuclei in controls and about half of nuclei in heat-treated samples showed the usual diffuse nucleoplasmic labelling, 40-60% of nuclei in heated samples also contained numerous bright spots. We then examined whether the heterogeneity in NuMA organization might be an indication of a differential response of B and T lymphocytes to hyperthermia, and whether the presence of NuMA spots is related to the apoptotic process. NuMA labelling of heated fractionated splenocyte populations showed that 90% of nuclei in T-enriched cultures (less than or equal to 4% IgG+ cells), but only 25% of nuclei in B-enriched samples (less than or equal to 80% IgG+ cells), contained spots. As well, 2 hours after heat treatment of unfractionated cultures, greater than or equal to 90% of nuclei that were accumulating DNA strand breaks, as detected by TUNEL, exhibited NuMA spots. These data indicate that cells with NuMA spots are targetted for, or have initiated, the death program. Since most T cells, but few or no B cells, were spotty after heating, we conclude further that hyperthermia induces apoptosis preferentially in splenic T lymphocytes. The observation that the proportion of T cells was, on average, threefold greater in control than in heated samples after 24 hours in culture reinforces this conclusion.

Animals↗
PubMed1997

Role of Fas ligand (CD95L) in immune escape: the tumor cell strikes back.

Several different tumor types have recently been shown to express functional Fas ligand (FasL), which may represent an additional mechanism of tumor escape. However, the in vivo significance of these findings in the context of potent adversaries such as activated CTL or NK cells remains to be fully established. The potential interactions between tumor cells and the host immune response are thus more complex than previously thought; their elucidation will benefit the rational design of cancer immunotherapy strategies.

Apoptosis↗
PubMed1997

Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain?

Astrocytomas are among the most common brain tumors that are usually fatal in their malignant form. They appear to progress without significant impedance from the immune system, despite the presence of intratumoral T cell infiltration. To date, this has been thought to be the result of T cell immunosuppression induced by astrocytoma-derived cytokines. Here, we propose that cell contact-mediated events also play a role, since we demonstrate the in vivo expression of Fas ligand (FasL/CD95L) by human astrocytoma and the efficient killing of Fas-bearing cells by astrocytoma lines in vitro and by tumor cells ex vivo. Functional FasL is expressed by human, mouse, and rat astrocytoma and hence may be a general feature of this nonlymphoid tumor. In the brain, astrocytoma cells can potentially deliver a death signal to Fas+ cells which include infiltrating leukocytes and, paradoxically, astrocytoma cells themselves. The expression of FasL by astrocytoma cells may extend the processes that are postulated to occur in normal brain to maintain immune privilege, since we also show FasL expression by neurons. Overall, our findings suggest that FasL-induced apoptosis by astrocytoma cells may play a significant role in both immunosuppression and the regulation of tumor growth within the central nervous system.

Animals↗
PubMed1997

Jun and JNK kinase are activated in thymocytes in response to VM26 and radiation but not glucocorticoids.

In order to establish what role members of the activating protein-1 (AP-1) gene families, i.e., c-fos, c-jun, junB, and junD, play in thymic apoptosis, we have analyzed changes in their expression in response to three different agents: a glucocorticoid analog dexamethasone, an inhibitor of topoisomerase II teniposide VM26, and gamma radiation. All three agents induced thymic death at a similar rate and with the same morphological and biochemical features. There was a rapid and transient increase in the steady-state mRNA level of junB and c-fos genes in all treatments, including control cultures, reminiscent rather of cellular stress response to the environmental changes than to the apoptotic stimuli. On the other hand, treatments with the DNA-damaging agents, VM26 and gamma radiation, resulted in superinduction of the c-jun mRNA and in the activation of the stress response signaling pathway of c-Jun N-terminal kinase. Gene transcription ceased completely in cells with fragmented DNA and the down-regulation of genes such as junD and tubulin was reflective of the thymocytes' commitment to apoptosis. The DNA-binding activities of the serum response factors, cyclic AMP response element binding proteins, and AP-1 factors, indicative of their transcriptional competence, were compromised shortly after induction of apoptosis regardless of the agent employed, consistent with previously reported enhancement in cellular proteolysis which is an essential component of the apoptotic cell death.

Animals↗
PubMed1997

Identification of a novel 97 kDa endonuclease capable of internucleosomal DNA cleavage.

The two steps of DNA digestion seen in apoptotic cells were recreated in nuclei isolated from 5123tc rat hepatoma cells. The initial DNA cleavage, into high molecular weight fragments (300-50 kb), was stimulated by magnesium ions alone, whereas the second step required both calcium and magnesium ions and produced the ladder of oligonucleosomes. Endonucleolytic activities involved in both steps of DNA cleavage could be separated under appropriate conditions since the magnesium-modulated activity was tightly bound to the chromatin whereas the calcium/magnesium-dependent internucleosomal cleaving activity was easily extractable with a low ionic strength buffer. This calcium/ magnesium-dependent activity was attributed to a novel 97 kDa endonuclease which was also activated by manganese and cobalt and inhibited by millimolar concentrations of zinc, consistent with the properties ascribed to the apoptotic endonuclease. Furthermore, this activity became resistant to extraction with a low salt buffer in nuclei of apoptotic cells. Isoelectrofocusing revealed that the p97 protein existed in multiple forms of different isoelectric points (pI range 4.6-5.0), indicative of its postranslational modification. The p97 enzyme was present constitutively in a variety of cultured cells and rat tissues. It was active over a broad range of pH (6-9), but it was inactivated by reducing agents. In vitro, it displayed both endo- and exonucleolytic activities, and it was capable of both single- and double-stranded DNA cleavage. Rabbit polyclonal anti-p97 antibodies were generated and used to further distinguish this protein from other known cellular nucleases, namely, DNases I and II.

Animals↗
PubMed1997

Induction of apoptosis in neoplastic cells by depletion of vitamin B12.

Methionine synthase, a critical enzyme in deoxyribonucleotide biosynthesis for DNA replication, requires vitamin B12 as a cofactor. We have tested the hypothesis that depletion of cells of vitamin B12 would block growth of neoplastic cells and divert them into apoptosis and could form the basis of a new therapeutic strategy for cancer treatment. Using nitrous oxide to inactivate vitamin B12 we show that, in a variety of cell lines in vitro, methionine synthase is rapidly inhibited, the cells cease proliferation and undergo apoptosis. The kinetics of cell death, once started, are similar to those observed following methotrexate treatment or serum withdrawal. This is the first observation of apoptosis being induced following depletion of an essential metabolite as opposed to the more conventional strategy of adding a toxic drug to damage cells thereby triggering apoptosis. Moreover, vitamin B12 depletion has no effect on the nonproliferating cell population.

Journal Article↗
PubMed1997

Evidence that DNA fragmentation in apoptosis is initiated and propagated by single-strand breaks.

Apoptosis is characterised by the degradation of DNA into a specific pattern of high and low molecular weight fragments seen on agarose gels as a distribution of sizes between 50-300 kb and sometimes, but not always, a ladder of smaller oligonucleosomal fragments. Using a 2D pulsed field-conventional agarose gel electrophoresis technique, where the second dimension is run under either normal or denaturing conditions, we show that single-strand breaks are introduced into DNA at the initial stages of fragmentation. Using single-strand specific nuclease probes we further show that the complete fragmentation pattern, including release of small oligonucleosomal fragments can also be generated by a single-strand endonuclease. Three classes of sites where single-strand breaks accumulate were identified. The initial breaks produce a distribution of fragment sizes (50 kb to >1 Mb) similar to those generated by Topoisomerase II inhibitors suggesting that cleavage may commence at sites of attachment of DNA to the nuclear matrix. A second class of rare sites is also cut further reducing the size distribution of the fragments to 50-300 kb. Thirdly, single-strand breaks accumulate at the linker region between nucleosomes eventually causing double-strand scissions which release oligonucleosomes. These observations further define the properties of the endonuclease responsible for DNA fragmentation in apoptosis.

Journal Article↗
PubMed1997

DNase I primary transcript is alternatively spliced in both normal and apoptotic cells: no evidence of up-regulation in apoptosis.

The reverse transcriptase polymerase chain reaction (RT-PCR) was used to elucidate the expression of DNase I and its possible involvement in apoptotic genome degradation. Multiple PCR products were obtained from cDNAs of different rat and human cells. The subsequent cloning and sequence analysis of seven PCR products revealed that only one of them had the expected size (639 bp) and sequence identity to that of rat DNase I cDNA. The other six PCR products were characterized by either sequence insertions or deletions. To establish the origin of this molecular diversity, a genomic fragment of the rat DNase I gene was also isolated, subcloned, and sequenced. Sequence comparison of six cDNAs with the rat genomic DNA revealed that they, indeed, resulted from inclusion of introns or deletion of exons. Southern hybridizations of the RT-PCR products from a variety of mammalian cell lines, using the major DNase I cDNA fragment as a probe, showed that in some cells as many as 20 alternatively spliced transcripts could be detected. This complexity of splice variants was widespread, and cell-specific profiles differed by the relative concentration of each transcript. None of these spliced transcripts maintained an open reading frame containing an intact catalytic site, suggesting that they do not encode any functional proteins. These complicated alternative splicing events might, however, significantly contribute to the regulation of DNase I expression. There was no apparent increase of the major DNase I transcript after induction of apoptosis in the cell lines studied. Apoptotic cells appeared to have similar normal/alternative transcript ratios as the control cells, suggesting that DNase I may not be the endonuclease involved in DNA degradation during apoptosis.

3T3 Cells↗
PubMed1997

TCR analysis reveals significant repertoire selection during in vitro lymphocyte culture.

The in vitro stimulation of T lymphocytes is frequently used as a technique to expand specific cells present at low precursor frequency in vivo. However, cells analysed after such procedures may no longer reflect those originally present in vivo because of the variable efficiency of outgrowth of different T cell subpopulations. To systematically assess this and to complement functional assays, we have analysed the TCR repertoire using a new high resolution RT-PCR method to determine TCR beta chain CDR3 transcript length. In the ex vivo analysis of tumor infiltrating lymphocytes (TIL) of renal cell carcinoma and glioblastoma patients, we observed and quantified oligoclonally expanded populations of T cells that were very susceptible to repertoire modification upon subsequent in vitro culture with autologous tumor cells. This in vitro repertoire skewing occurred preferentially with TIL rather than peripheral blood lymphocytes and we noted that tumor cells rather than normal cells of the same tissue type were the most potent inducers of the effect. It was striking that this selection was sometimes negative: certain prominent T cell populations that were highly represented in vivo disappeared after in vitro re-stimulation. This suggests that the presentation of tumor associated antigens during culture may eliminate rather than enrich for in vivo primed T cells. It is clear that in vitro functional tests cannot adequately describe all T cells with tumor specificity. Approaches that allow the assessment of potentially antigen-reactive T cell populations ex vivo are thus an important advance in the global appraisal of anti-tumor T cell immune responses.

Amino Acid Sequence↗
PubMed1997

New aspects of the mechanism of DNA fragmentation in apoptosis.

In this article we review the current status of research into the mechanism of DNA fragmentation in apoptosis. There is accumulating evidence that there is no single conserved endonuclease that is responsible for DNA fragmentation in all cells. Not only are the two stages of high molecular weight fragment and DNA ladder formation likely to be catalysed by different activities, but multiple activities appear to be involved in internucleosomal DNA fragmentation in various cells. In addition to any one of a number of Ca2+/Mg2+-dependent endonucleases, acidic enzymes (i.e., endonucleases that are active at acid pH) also appear to be involved. Moreover, some enzymes currently being identified are active over a broad range of acidic and neutral pH values and have complex cation requirements that are a function of pH. DNA fragmentation in apoptosis appears to be controlled by either Ca2+ ions or a decrease in pH, depending on the cell type. DNA degradation to oligonucleosomes is not essential for apoptosis, but all cells must undergo DNA degradation to produce the large fragments. This is believed to occur in regions of DNA attached to the nuclear matrix, but the activity responsible has not yet been identified. Changes in both Ca2+ ions and pH have been recorded in a variety of cells, indicating that both mechanisms of regulation of endonuclease activation are involved in apoptosis.

Animals↗
PubMed1997

Unique behaviour of NuMA during heat-induced apoptosis of lymphocytes.

Nuclear collapse is a key feature of apoptosis, reflecting the DNA and protein fragmentation observed biochemically. We have compared nuclear events during spontaneous and heat-induced (42 degrees C for 30 min) apoptosis at the level of individual cells, monitoring overall chromatin organization by staining with 4,6'-diamidino-2-phenylindole (DAPI), DNA cleavage by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL), and the nuclear antigens NuMA, PI2, and lamin B by immunofluorescence microscopy. The data were correlated with analyses at the population level by flow cytometry and immunoblotting. We show that heat treatment doubles the level of apoptotic splenocytes with respect to the spontaneous apoptosis in controls within 6 honThe organization and disassembly of nuclear envelope antigens is identical during spontaneous and heat-induced apoptosis and proceeds in a temporally and spatially ordered manner relative to DNA fragmentation and chromatin collapse. In contrast, NuMA organization is affected by heat treatment, with about half of the cells containing many bright spots in addition to the usual diffuse labelling. The spots were still visible in many fully collapsed apoptotic nuclei. Further, this novel reorganization of NuMA and the hyperinduction of apoptosis by heat are correlated, suggesting that lymphocytes with reorganized NuMA are destined to undergo apoptosis. The data also indicate that DNA fragmentation precedes extensive remodelling of the nucleoplasm in these cells, and that chromatin begins to collapse before disassembly of nuclear envelope components.

Animals↗
PubMed1996

Single-cell PCR analysis of TCR repertoires selected by antigen in vivo: a high magnitude CD8 response is comprised of very few clones.

Taking advantage of a potent MHC class I-restricted response that allows the identification of antigen-selected CD8 T cells directly ex vivo, we characterized the antigen-specific T cell repertoires that develop in individual mice by single-cell PCR analysis. Each of the immune mice displayed distinct yet structurally similar TCR repertoires. The overall repertoire size was estimated to be in the range of 15-20 for most mice. No major differences were observed between primary and secondary responses. Moreover, for a hyperimmunized mouse the antigen-specific TCR repertoire expressed 8 months after the initial immunization was very similar to that found at the peak of the primary response. Our results demonstrate that a high magnitude immune response may be composed of very few clones, and that at least in the system analyzed, the memory response largely reflects the repertoire selected by the peak of the primary response.

Amino Acid Sequence↗
PubMed1996

Alpha interferon-2b, leucovorin, and 5-fluorouracil (ALF) in non-small cell lung cancer.

Eighteen patients with non-small cell lung cancer (NSCLC) who had previously received no systemic chemotherapy, were treated on this phase II study with interferon-alfa-2b 8 MU tiw, leucovorin 500 mg/m2 IVPB over 2 hours and 5-Fluorouracil 500 mg/m2 i.v. for 6 weeks followed by a 2-week rest. There were was no rest period for interferon. Median age of patients on this study was 63 years. Fatigue, nausea, and diarrhea were the most common toxicities. There were no grade IV toxicities and no treatment-related deaths. Seven partial responses (39%) with median duration of 4.5 months were seen. An additional patient has had stable disease/minor response for 12+ months. Median survival is 10 months. ALF has activity in NSCLC.

Adult↗
PubMed1996

Memory TCR repertoires analyzed long-term reflect those selected during the primary response.

Normal T cell repertoire selection and evolution in antigen-specific responses is poorly understood. We have recently described an MHC class I-restricted response characterized by an overwhelming expansion of CD8 cells expressing a Vbeta10 TCR, thus allowing the identification of antigen-selected cells directly ex vivo. Our present strategy to follow the overall TCR repertoire selection was to monitor the expression of a particular TCR alpha chain (Valpha8) on antigen-selected Vbeta10(+) cells by four-color flow cytometry. We demonstrate that while there is substantial variation among the responder mice in Valpha8 usage, the repertoires of individual animals remain relatively stable over long periods of time (>1 year), with or without repeated antigenic challenge. Thus if any evolution of this response occurs upon re-exposure to antigen, it would appear not to skew the TCR repertoire established during the primary response.

Animals↗