Biomedical subjects

V Rotter

Publications and source records attributed to V Rotter.

At least 19 recordsLinked to original sources

PubMed2001

Activation of p53 protein by telomeric (TTAGGG)n repeats.

Genome instability is a primary factor leading to the activation of the p53 tumor suppressor protein. Telomeric repeat (TR) sequences are also responsible for genome integrity. By capping the termini of the chromosomes, TRs prevent them undergoing nucleolytic degradation, ligation or chromosome fusion. Interestingly, telomere shortening was suggested to activate p53, which in turn may cause primary cells to senesce. In order to elucidate the nature of a possible cross talk between the two, we introduced into cells TRs of defined length and investigated their effect on p53 activation and subsequent cellular response. We found that the introduction of a TR into cells leads to stabilization of the p53 protein. This stabilization was specific to TRs and was not observed in response to exposure of cells to plasmids containing non-TR sequences. p53 stabilization requires the presence of an intact p53 oligomerization domain. TR-activated p53 exhibited enhanced transcriptional activity. Eventually, TRs induced p53-dependent growth suppression, measured as a reduction in colony formation.

Base Sequence↗

PubMed2001

p53 is dispensable for apoptosis but controls neurogenesis of mouse dentate gyrus cells following gamma-irradiation.

Mammalian cells respond to DNA insults by activating cell-cycle checkpoints. This may result in a temporary cell growth arrest which allows DNA repair before proliferation or induces apoptosis. p53 is one of the main contributors in regulating these activities. To get a better insight on the molecular mechanism underlying these activities we studied the role of p53 in apoptosis and neurogenesis of brain cells from adult p53(+/+) or p53(-/-) mice exposed to gamma-irradiation. Apoptosis and neurogenesis were assessed up to 14 days following the injury. Five-ten hours following gamma-irradiation, cells with TUNEL positive nuclei were identified within the subgranular zone of dentate gyrus (DG) of both p53(+/+) and p53(-/-) mice. At the same time-points, pyknotic and shrinking nuclei were visualized by Hoechst 33258 staining. Furthermore, gamma-irradiation increased the number of proliferating cell nuclear antigen (PCNA) positive cells with a peak at 5-10 h in both animal groups. PCNA immunoreactivity was detected in cells exhibiting condensed nuclei as visualized by Hoechst 33258 staining. Neurogenesis, assessed by mitotic marker p34(cdc2) immunoreactivity, showed a biphasic response to gamma-irradiation both in p53(+/+) and p53(-/-) mice which was characterized by an early inhibition and a delayed stimulation. In p53(-/-) mice, the time required by DG granule cells to recover from the lesion and to stimulate proliferation was significantly shortened in comparison with wild-type mice thus resulting in an accelerated neurogenesis. Our data indicate that following gamma-radiation p53 plays a role in regulating cell-cycle progression rate but it is dispensable for promoting apoptosis of DG granule cells.

Animals↗

PubMed2001

The C-terminus of mutant p53 is necessary for its ability to interfere with growth arrest or apoptosis.

The ability to suppress wild type p53-independent apoptosis may play an important role in the oncogenicity of p53 mutant proteins. However, structural elements necessary for this activity are unknown. Furthermore, it is unclear whether this mutant p53 mediated inhibition is specific to the apoptotic pathway or a more general suppression of the cellular response to stress. We observed that an unmodified C-terminus was required for the suppression of apoptosis by the p53 135(Ala to Val) oncogenic p53 mutant. It was also required for the novel activity of G2 arrest suppression, the predominant response at low levels of genotoxic stress. These observations are consistent with a model whereby mutant p53 suppressive activity is not specific to the apoptotic pathway, but rather increases the threshold of genotoxic stress needed for a DNA damage response to occur. Furthermore, these observations indicate that it may be possible to selectively kill mutant p53 expressing cells based on the lower sensitivity of their growth arrest response.

Animals↗

PubMed2001

Integrity of the N-terminal transcription domain of p53 is required for mutant p53 interference with drug-induced apoptosis.

The present study examined whether the ability of mutant p53 to block apoptosis depended on its transcriptional activity. A core domain mutant p53 (143 Val to Ala), in which two N-terminal residues (22 and 23) essential for transactivation were also mutated (Leu to Glu and Trp to Ser, respectively), was examined. While p53 containing only the core mutation efficiently interfered with drug-induced apoptosis, further modification at the N-terminus abolished this blocking activity. Furthermore, expression of c-myc, a suggested target for core mutant p53 transactivation, was elevated in the core mutant p53-expressing cells, but was abolished in the presence of the transcription-deficient p53 core mutant. In addition, wild-type p53, mutated in the N-terminus (residues 22 and 23), was unable to induce apoptosis by itself. Nevertheless, it synergized with drugs in the induction of apoptosis. This suggests that the integrity of the N-terminus is essential for both the activity of wild-type p53 in apoptosis and for mutant p53-mediated block of drug-induced apoptosis. This supports the notion that core p53 mutants act via a gain of function mechanism.

Alanine↗

PubMed2001

Structural and functional involvement of p53 in BER in vitro and in vivo.

p53 is involved in several DNA repair pathways. Some of these require the specific transactivation of p53-dependent genes and others involve direct interactions between the p53 protein and DNA repair associated proteins. Previously, we have shown that p53 acts directly in Base Excision Repair (BER) when assayed under in vitro conditions. Our present data indicate that this involvement is independent of the transcriptional activity of the p53 molecule. We found that under both in vitro and in vivo conditions, a p53 transactivation-deficient molecule, p53-22-23 was more efficient in BER activity than was wild type p53. However, mutations in the core domain or C-terminal alterations strongly reduced p53-mediated BER activity. These results are consistent with the hypothesis that the involvement of p53 in BER activity, a housekeeping DNA repair pathway, is a prompt and immediate one that does not involve the activation of p53 transactivation-dependent mechanisms, but rather concerns with the p53 protein itself. In an endogenous DNA damage status p53 is active in BER pathways as a protein and not as a transcription factor.

Cell Line↗

PubMed2001

p53 modulates base excision repair activity in a cell cycle-specific manner after genotoxic stress.

To elucidate the nature of the cross-talk between the p53 protein and the DNA repair machinery, we have investigated the relationship between the two throughout the cell cycle. Base excision repair (BER) was analyzed in cell cycle phase-enriched populations of lymphoid cells expressing wild-type p53. Our study yielded the following novel findings: (a) BER exhibited two distinct peaks of activity, one associated with the G0-G1 checkpoint and the second with the G2-M checkpoint; (b) although the overall BER activity was reduced after exposure of cells to 400R, there was an augmentation of the G0-G1-associated BER activity and a reduction in the G2-M-associated BER activity; and (c) modulations in these patterns of BER after genotoxic stress were found to be p53 regulated. p53 protein levels induced after gamma-irradiation were distributed evenly in the various cell cycle populations (analyzed by the PAb-248 anti-p53 monoclonal antibody). However, both the dephosphorylation of serine 376 of p53 (contained in the PAb-421 epitope) and the specific DNA binding activity, as well as apoptosis, were enhanced toward the G2-M populations. Furthermore, inactivation of wild-type p53, mediated by mutant p53 expression, abolished the alterations in the BER pattern and showed no induction of a G2-M-associated apoptosis after gamma-irradiation. These results suggest that after genotoxic stress, stabilized p53 enhances the G0-G1-associated BER activity, whereas it predominantly reduces BER activity at the G2-M-enriched populations and instead induces apoptosis. After genotoxic stress, p53 functions as a modulator that determines the pattern of BER activity and apoptosis in a cell cycle-specific manner.

Animals↗

PubMed2001

Autoimmunity to the p53 protein is a feature of systemic lupus erythematosus (SLE) related to anti-DNA antibodies.

The induction of anti-DNA autoantibodies in systemic lupus erythematosus (SLE) patients is problematic because mammalian DNA is poorly immunogenic at best. Here we demonstrate a chain of connected antibodies in SLE patient sera that could account for the induction of anti-DNA antibody, and possibly for some of the pathogenic features of SLE. We now report that SLE patients, in addition to anti-DNA, produce antibodies to the carboxy-terminal domain of the tumour suppressor molecule p53; this p53 domain recognizes damaged DNA. Hence, these anti-p53 antibodies could mimic damaged DNA immunologically. Indeed, SLE sera do contain anti-idiotypic antibodies to a prototypic anti-p53 antibody. Moreover, SLE anti-DNA antibodies also recognize this type of anti-p53 antibody. Indeed, binding of affinity-purified anti-DNA both to DNA and to the anti-p53 antibody could be blocked by a p53 peptide derived from the DNA-binding domain. This mimicry of the p53 DNA-binding domain by the SLE anti-DNA antibodies is functional: activation of the p53 molecule could be inhibited by such anti-DNA antibodies. Thus, anti-DNA antibodies may arise in SLE patients by a chain of idiotypic autoimmunity centered around p53 autoimmunity. The SLE anti-DNA and anti-p53 antibodies can functionally block p53 activation, and so could affect apoptosis.

Antibodies, Antinuclear↗

PubMed2001

The role of the C' terminus of murine p53 in the p53/mdm-2 regulatory loop.

Mdm-2 plays a central role in the regulation of p53 protein level and activity. Although the interaction of mdm-2 and p53 occurs through the N-terminus of the p53 protein, our present data suggest that the C' terminus plays an important role in the regulation of the p53/mdm-2 loop. Comparative analysis of the murine regularly spliced form of p53 (RSp53) and a physiological C-terminally modified p53 protein, which results from alternative splicing of the p53 mRNA (ASp53), indicated that the two isoforms behave differently in the p53/mdm-2 loop. We found that ASp53 can preferentially induce higher levels of the mdm-2 protein, compared with RSp53. Although the transactivation capacity of both forms is inhibited by mdm-2, only RSp53 is directed to proteolytic degradation by mdm-2, while ASp53 is relatively resistant. We present evidence that suggests that ASp53 protein levels determine the biological activities mediated by RSp53, such as the induction of apoptosis, through the mdm-2/p53 regulatory loop. We suggest, therefore, a new mechanism for the regulation of p53, and show that alteration of the p53 extreme C' terminus can significantly change the transcription activity and the resistance to degradation properties of the p53 protein.

Alternative Splicing↗

PubMed2001

Thiazolidinediones (PPARgamma agonists) but not PPARalpha agonists increase IRS-2 gene expression in 3T3-L1 and human adipocytes.

Thiazolidinediones (TZD) improve insulin sensitivity in human as well as in different animal models of insulin resistance and Type 2 diabetes. However, no clear link to the insulin signaling events has been identified. Using differentiated 3T3-L1 adipocytes, we found that TZD rapidly and markedly increased IRS-2 gene expression. This effect was specific for PPARgamma agonists and was not seen with PPARalpha agonists. It was rapidly induced (within 4 h) and maintained throughout the observation period of 48 h. It was also concentration dependent (EC50 approximately 50 nM) and not inhibited by cycloheximide, suggesting a direct effect on the IRS-2 promoter. There was no evidence that TZD altered IRS-2 mRNA stability, supporting that the increased mRNA levels were due to an increased gene transcription. IRS-2 protein expression was increased approximately 30% after 48 h and approximately 50% after 96 h. No effects of TZD were seen on IRS-1, PKB/Akt, or GLUT4 gene expression. TZD also increased IRS-2 mRNA levels in cultured human adipose tissue. These data show the first direct link between TZD and a critical molecule in insulin's signaling cascade in both 3T3-L1 and human adipocytes, and indicate a novel mode of action of these compounds.

3T3 Cells↗

PubMed2001

Change of the death pathway in senescent human fibroblasts in response to DNA damage is caused by an inability to stabilize p53.

The cellular function of p53 is complex. It is well known that p53 plays a key role in cellular response to DNA damage. Moreover, p53 was implicated in cellular senescence, and it was demonstrated that p53 undergoes modification in senescent cells. However, it is not known how these modifications affect the ability of senescent cells to respond to DNA damage. To address this question, we studied the responses of cultured young and old normal diploid human fibroblasts to a variety of genotoxic stresses. Young fibroblasts were able to undergo p53-dependent and p53-independent apoptosis. In contrast, senescent fibroblasts were unable to undergo p53-dependent apoptosis, whereas p53-independent apoptosis was only slightly reduced. Interestingly, instead of undergoing p53-dependent apoptosis, senescent fibroblasts underwent necrosis. Furthermore, we found that old cells were unable to stabilize p53 in response to DNA damage. Exogenous expression or stabilization of p53 with proteasome inhibitors in old fibroblasts restored their ability to undergo apoptosis. Our results suggest that stabilization of p53 in response to DNA damage is impaired in old fibroblasts, resulting in induction of necrosis. The role of this phenomenon in normal aging and anticancer therapy is discussed.

Acridine Orange↗

PubMed2000

Oncogenic mutations of the p53 tumor suppressor: the demons of the guardian of the genome.

The p53 guardian of the genome is inactivated in the majority of cancers, mostly through missense mutations that cause single residue changes in the DNA binding core domain of the protein. Not only do such mutations result in the abrogation of wild-type p53 activity, but the expressed p53 mutant proteins also tend to gain oncogenic functions, such as interference with wild-type p53-independent apoptosis. Because p53 mutants are highly expressed in cancer cells and not in normal cells, their reactivation to wild-type p53 function may eliminate the cancer by apoptosis or another p53-dependent mechanism. Several studies that embarked on this quest for reactivation have succeeded in restoring wildtype p53 activity to several p53 mutants. However, mutants with more extensive structural changes in the DNA binding core domain may be refractory to reactivation to the wild-type p53 phenotype. Therefore, understanding the structure and functions of oncogenic p53 mutants may lead to more potent reactivation modalities or to the ability to eliminate mutant p53 gain of function.

Animals↗

PubMed2000

Differential sensitivity of MCF-7 and LCC2 cells, to multiple growth inhibitory agents: possible relation to high bcl-2/bax ratio?

Comparison of LCC2, the E(2)-independent, tamoxifen-resistant subline of the MCF-7 human breast cancer cell line with its parent line, disclosed that it is more resistant to growth inhibition and apoptosis induction by a variety of agents acting by diverse mechanisms. Thus, LCC2 cells can serve as a useful in-vitro model for the study of the molecular mechanisms of this resistance. It was found that bcl-2 protein and mRNA were elevated and that bax protein and mRNA were reduced in LCC2 compared with MCF-7 cells. Incubation of both lines in the presence of bcl-2 antisense caused growth inhibition and reduced bcl-2 protein levels only in MCF-7 cells, suggesting the involvement of bcl-2 in the regulation of normal growth of breast cancer cells. Increased bcl-2 expression in breast cancer cells may correlate with their resistance to growth inhibitory agents. Bcl-2 is a useful target for enhancing the effects of growth inhibitory agents.

Antineoplastic Agents↗

PubMed2000

A fast signal-induced activation of Poly(ADP-ribose) polymerase: a novel downstream target of phospholipase c.

We present the first evidence for a fast activation of the nuclear protein poly(ADP-ribose) polymerase (PARP) by signals evoked in the cell membrane, constituting a novel mode of signaling to the cell nucleus. PARP, an abundant, highly conserved, chromatin-bound protein found only in eukaryotes, exclusively catalyzes polyADP-ribosylation of DNA-binding proteins, thereby modulating their activity. Activation of PARP, reportedly induced by formation of DNA breaks, is involved in DNA transcription, replication, and repair. Our findings demonstrate an alternative mechanism: a fast activation of PARP, evoked by inositol 1,4,5,-trisphosphate-Ca(2+) mobilization, that does not involve DNA breaks. These findings identify PARP as a novel downstream target of phospholipase C, and unveil a novel fast signal-induced modification of DNA-binding proteins by polyADP-ribosylation.

Animals↗

PubMed2000

p53-dependent apoptosis is regulated by a C-terminally alternatively spliced form of murine p53.

It is now well accepted that the p53 C-terminus plays a central role in controlling the activity of the wild-type molecule. In our previous studies, we observed that a C-terminally altered p53 protein (p53AS), generated by an alternative spliced p53 mRNA, induces an attenuated p53-dependent apoptosis, compared to that induced by the regularly spliced form (p53RS). In the present study we analysed the interrelationships between these two physiological variants of wild-type p53, and found that in cells co-expressing both forms, in contrast to the expected additive effect on the induction of apoptosis, p53AS inhibits apoptosis induced by p53RS. This inhibitory effect is specific for p53-dependent apoptosis and was not evident in a p53-independent apoptotic pathway induced by growth factor deprivation. Furthermore, the expression of p53AS in transiently transfected cells caused both inhibition of apoptosis and inhibition of the p53RS-dependent transactivation of a number of p53 target genes. These results suggest that expression of an alternatively spliced p53 form may serve as an additional level in controlling the complexity of p53 function by the C-terminal domain.

Alternative Splicing↗

PubMed2000

Systemic lupus erythematosus in mice, spontaneous and induced, is associated with autoimmunity to the C-terminal domain of p53 that recognizes damaged DNA.

The tumor suppressor molecule p53 features a regulatory domain at the C terminus that recognizes damaged DNA. Since damaged DNA might be involved in activating anti-DNA autoantibodies, we tested whether autoimmunity to the C terminus of p53 might mark murine systemic lupus erythematosus (SLE). We now report that MRL / MpJ-Fas(lpr) mice, which spontaneously develop SLE, produce antibodies both to the C terminus of p53 and to a monoclonal antibody (PAb-421) that binds the p53 C terminus. Anti-idiotypic antibodies to PAb-421 (sampled as monoclonal antibodies) could also bind DNA. Thus, the PAb-421 antibody mimics DNA, and the anti-idiotypic antibody to PAb-421 mimics the p53 DNA-binding site. This mimicry was functional; immunization of BALB / c mice to PAb-421 induced anti-DNA antibodies and antibodies to the C terminus of p53, and most of the mice developed an SLE-like disease. Immunization of C57BL / 6 mice to PAb-421 induced antibodies to p53, but not to its C-terminal domain. The C57BL / 6 mice also did not develop anti-DNA antibodies or the SLE-like disease. Thus, network autoimmunity to the domain of p53 that recognizes damaged DNA can be a pathogenic feature in SLE in genetically susceptible strains of mice.

Animals↗

PubMed1999

An intragenic deletion of nuclear localization signal-1 of p53 tumor suppressor gene results in loss of apoptosis in murine fibroblasts.

We established mouse lines containing either full-length wild-type p53 or nuclear localization signal-I (NLS-I) deleted p53 to study the role of NLS-I in p53 translocation and function. Induction of apoptosis in response to DNA damage, a primary function of p53, was tested in these cell lines. After exposure to gamma-ionizing radiation or hydrogen peroxide, DNA ladders and labeling of nucelosomal fragments were detected in cells with wild-type p53 gene, but not in p53 null cells or NLS-I deleted cells, suggesting that the NLS-I of p53 protein is necessary for apoptosis. Analysis of p53 protein from subcellular fractions indicated that NLS-I deprived p53 remained in the cytoplasmic fraction, which may explain why NLS-I deleted p53 failed to induce apoptosis.

Animals↗

PubMed1999

Epithelial cells of different organs exhibit distinct patterns of p53-dependent and p53-independent apoptosis following DNA insult.

The present study shows that DNA damage induces different patterns of p53-dependent and p53-independent apoptosis in epithelial cells of various organs of adult mice. Genotoxic stress induced a biphasic apoptotic response in the small intestine and tongue. While the first immediate apoptotic wave was p53-dependent, the second was slower in rate and was p53-independent. Under the same experimental conditions a single rapid, but a more extended, p53-independent response was evident in the skin of the tail. Indeed, exposure of p53+/+ mice to 400 R induced in epithelium of the small intestine and tongue an immediate rapid response that was followed by a second delayed p53-independent apoptotic wave. p53-/- mice exhibited in these organs the second wave only. However, epithelium of the tail derived from the same mice showed a single rapid apoptotic response that lasted much longer than the p53-dependent response and was similar in the p53-/- and the p53+/+ mice. Variations in apoptotic patterns observed in epithelial cells derived of the different tissues may point to differences in the physiological pathways expressed.

Animals↗

PubMed1999

Tissue-specific p53 expression in the nervous system.

P53 is a transcription factor that has been found to be expressed in association with cell proliferation and apoptosis. Previously, bacterial chloramphenicol acetyl transferase (CAT) enzymatic expression was predominantly found in the testes of p53 promoter driven-CAT transgenic mice. In the current study, we extended this study to survey p53 expression across both the central and peripheral nervous systems of the same strain of transgenic mice as well as their parental strain. High levels of p53 promoter driven-CAT activity was observed in the cerebellum, hippocampus, hypothalamus, pons, thalamus and upper cerebral spine. Furthermore, we consistently found unexpectedly high levels of p53 promoter-driven CAT expression in the eyes. These observations were reinforced by p53 protein analysis using a p53 pan ELISA assay. Immunohistochemical studies confirmed and further defined p53 expression in several regions of the nervous system. Significantly, p53 promoter-driven CAT expression was visualized in the Ammon horn of the hippocampus, in the Purkinje cells of the cerebellum and in the cornea as well as in the retina of the eye. Furthermore, strong p53 protein expression was found in the cornea of the parental mouse strain. p53 ELISA demonstrated a profile of p53 protein concentration, which correlate well with the high p53 promoter-driven CAT activities observed in the cerebellum, hindbrain, hypothalamus, thalamus, hippocampus, whole eyes as well as with the low CAT activities observed in the cortex and spinal cord. In both of these assays considerable p53 promoter activity and p53 protein levels were found in post-mitotic non-dividing cells.

Animals↗