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V Rotter

Publications and source records attributed to V Rotter.

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

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery.

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature-sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild-type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.

Animals↗
PubMed1999

Accentuated apoptosis in normally developing p53 knockout mouse embryos following genotoxic stress.

In order to identify the alternative pathways which may substitute for the p53 function during embryogenesis, we have focused our studies on p53 -/- normally developing mouse embryos that survived a genotoxic stress. We assumed that under these conditions p53-independent pathways, which physiologically control genomic stability, are enhanced. We found that while p53 +/+ mouse embryos elicited, as expected, a p53-dependent apoptosis, p53-/- normally developing mice exhibited an accentuated p53-independent apoptotic response. The p53-dependent apoptosis detected in p53+/+ embryos, was an immediate reaction mostly detected in the brain, whereas the p53-independent apoptosis was a delayed reaction with a prominent pattern observed in epithelial cells of most organs in the p53-deficient mice only. These results suggest that in the absence of p53-dependent apoptosis, which is a fast response to damaged DNA, p53-independent apoptotic pathways, with slower kinetics, are turned on to secure genome stability.

Animals↗
PubMed1999

Hydrogen peroxide induces nuclear translocation of p53 and apoptosis in cells of oligodendroglia origin.

The observation that apoptosis is an inherent pathway in oligodendrocytes development coupled with the notion that wild-type p53 is expressed in these cells, prompted us to investigate the interrelationship between the two phenomena. Using a permanent oligodendroglia-like cell line (OLN 93), we examined the role of p53 protein in apoptosis following a DNA insult induced by a brief exposure to H2O2. A marked translocation of p53 from the cytosolic to the nuclear compartment was notable by 20 min, following a 5 min treatment with 1 mM H2O2 as identified by cell immunostaining. By 48 h following H2O2 addition, nearly 60% of the cells exhibited p53 in the nuclei. At this time, a large proportion of the cells underwent apoptosis as identified by DAPI nuclear staining. The genotoxic-induced p53 relocalization appeared to be cell cycle phase specific; thus OLN 93 cultures enriched for cells in the G0/G1 stage by serum starvation, and abundant in nuclear-associated p53, were more susceptible to H2O2-induced apoptosis than their untreated counterparts and than double thymidine block, G1/S enriched, cultures. Analysis of the expression of p53 downstream genes indicated that p21 and mdm2 were upregulated following p53 nuclear translocation. From the kinetics of protein accumulation, it appears that mdm2 enhancement accelerated the exit of p53 from the nucleus to the cytosol. Our results suggest that following stress, oligodendroglia-like cells are induced to undergo p53-dependent apoptosis, an event that coincides with p53 nuclear translocation and is cell-cycle related.

Animals↗
PubMed1999

COOH-terminal domain of p53 modulates p53-mediated transcriptional transactivation, cell growth, and apoptosis.

The tumor suppressor protein p53 contributes to the control of cell cycle checkpoints and stress-induced apoptosis and is frequently mutated in many different types of human cancers. The COOH terminus of p53 modulates the transcriptional and apoptotic activities of the protein. Although COOH-terminal mutants of p53 are uncommon, we proposed that these p53 mutants nevertheless contributed to the selective clonal expansion of the cancer cells. Therefore, we analyzed the tumor-derived p53 COOH-terminal domain (CTD) mutants (352D/H, 356G/W, 342-stop, 360-del, and 387-del) functionally. The results have revealed that all mutants have impaired apoptotic activity when compared with wild-type p53. However, some of these mutants still transcriptionally transactivate p21Waf/Cip1 and inhibit cell growth. Interestingly, of the tumor-derived CTD mutants, oligomerization-defective mutant 342-stop was the only one that did not exhibit sequence-specific DNA binding or failed to transactivate p21Waf1/Cip1, Bax, and IGF-BP3 transcriptionally. The failure to inhibit cell growth by this tumor-derived CTD mutant supports the hypothesis that p53 sequence-specific transcriptional transactivity to p21Waf1/Cip1 is correlated with induction of cell cycle arrest and that the p53 transcriptional transactivity requires oligomerization of the p53 protein. These and other data indicate that the CTD of p53 is an important component of p53-mediated apoptosis and cell growth arrest and that inactivation of the apoptotic function, but not the inhibition of growth, is an important step during human tumorigenesis.

Apoptosis↗
PubMed1999

Introduction: p53--the first twenty years.

The p53 protein was discovered 20 years ago, as a cellular protein tightly bound to the large T oncoprotein of the SV40 DNA tumour virus. Since then, research on p53 has developed in many exciting and sometimes unexpected directions. p53 is now known to be the product of a major tumour suppressor gene that is the most common target for genetic alterations in human cancer. The nonmutated wild-type p53 protein (wtp53) is often found within cells in a latent state and is activated in response to various intracellular and extracellular signals. Activation involves an increase in overall p53 protein levels, as well as qualitative changes in the protein. Upon activation, wtp53 can induce a variety of cellular responses, most notable among which are cell cycle arrest and apoptosis. To a great extent, these effects are mediated by the ability of p53 to activate specific target genes. In addition, the p53 protein itself possesses biochemical functions which may facilitate DNA repair as well as apoptosis. The role of p53 in normal development and particularly in carcinogenesis has been elucidated in depth through the use of mouse model systems. The insights provided by p53 research over the years are now beginning to be utilized towards better diagnosis, prognosis and treatment of cancer.

Apoptosis↗
PubMed1999

Cellular events and the pattern of p53 protein expression following cyclophosphamide-initiated cell death in various organs of developing embryo.

This study was aimed at characterizing the temporal patterns of cell responses and p53 protein expression in the limbs, head, and liver of embryos responding to cyclophosphamide (CP)-induced teratogenic insult. ICR murine embryos were examined 24, 48, or 72 h after injection of 40 mg/kg CP on day 12 of pregnancy. The cellular events and temporal pattern of p53 protein expression were determined by FACS analysis and by TUNEL (apoptosis) in the head, limbs, and liver of the embryos. All tested organs showed apoptosis and a significantly decreased proportion of live cells after 24 h. Subsequent events were organ-dependent. In the liver, there were no dysmorphic events at any time and excessive cell death had been almost compensated for by 48 h. Compensation was preceded by G(1) arrest and accompanied by an increased level of p53 protein in surviving cells. Excessive cell death in the head and the limbs resulted in structural anomalies. In the head, there was an increased level of p53 protein and G(1) arrest after 24 h and the number of live cells at 48 h was equal to that seen in earlier samples, despite apoptosis. In the limbs, however, only isolated viable cells were seen by 48 h, but there was no increased level of p53 protein or G(1) arrest. Results of this study suggest that the differential sensitivity of tested organ systems to CP may be associated with differences in cellular events following CP-initiated cell death. They also suggest that the input of p53 in determining the response of these organ systems to CP-induced teratogenic insult may be different. Teratogenesis Carcinog. Mutagen. 19:353-367, 1999.

Animals↗
PubMed1999

p53 controls low DNA damage-dependent premeiotic checkpoint and facilitates DNA repair during spermatogenesis.

Previously, it was implicated that p53 plays a role in spermatogenesis. Here we report that p53 knockout mice exhibit significantly less mature motile spermatozoa than their p53(+/+) counterparts. To better understand the role of p53 in spermatogenesis, we analyzed the response of spermatogenic cells to DNA insult during prophase. It was found that although low-level gamma-irradiation activated a p53-dependent premeiotic delay, higher levels of gamma-irradiation induced a p53-independent apoptosis during meiosis. Furthermore, p53 knockout mice exhibited reduced in vivo levels of unscheduled DNA synthesis, indicative of compromised DNA repair. Thus, p53 provides another level of stringency in addition to other spermatogenic "quality control" mechanisms.

Animals↗
PubMed1999

p53 tumor suppressor gene expression in the mouse ovary during an artificially induced ovulatory cycle.

OBJECTIVE: To evaluate the expression of p53 in the mouse ovary during an artificially induced ovulatory cycle. STUDY DESIGN: Ovulation induction was performed using pregnant mares' serum gonadotropin/human chorionic gonadotropin (PMSG/hCG). First, a p53 promoter-chloramphenicol acetyl transferase (CAT) transgenic mouse model was used. Protein samples from ovaries of transgenic mice were assayed for CAT activity as evidence of p53 promoter activation. Next, RNA extracted from CD-1 mouse ovaries was used for reverse transcription/polymerase chain reaction (PCR) and northern blot analysis using a p53-specific probe. RESULTS: Increased CAT activity was noted in transgenic mice treated with PMSG/hCG as compared with controls. PCR studies on transgenic mice using primers for CAT and on CD-1 mice using primers for wild type p53 substantiated this observation. Furthermore, CAT assay and northern analysis, performed on samples obtained at serial time intervals from induction, indicated that maximal p53 expression occurs around the time of ovulation, beginning 48 hours after PMSG and peaking 6-12 hours after hCG administration. CONCLUSION: The temporal expression of p53 in the ovary during a PMSG/hCG artificially induced ovulatory cycle may indicate a role for p53 in processes of differentiation of granulosa cells into luteal cells.

Animals↗
PubMed1998

Immunity to p53 induced by an idiotypic network of anti-p53 antibodies: generation of sequence-specific anti-DNA antibodies and protection from tumor metastasis.

The general overexpression of p53 by different types of tumor cells suggests that p53 immunity might be generally useful for tumor immunotherapy. We describe here the induction of immunity to p53 and resistance to tumor metastasis using an idiotypic network. Mice were immunized with domain-specific anti-p53 monoclonal antibodies (Ab1): PAb-248 directed to the N-terminus; PAb-246 directed to the specific DNA-binding region; or PAb-240 directed to a mutant p53 that does not bind specific DNA. Immunized mice responded by making anti-idiotypic antibodies (Ab2) specific for the Ab1 inducer. Ab1 PAb-246 induced Ab2 that, like p53 itself, could bind the specific DNA oligonucleotide sequence of the p53 responsive element. Mice immunized with Ab1 PAb-240 or PAb-246 spontaneously made Ab3 anti-p53 antibodies that reflected the specificity of their Ab1 inducers: Ab1 PAb-246 induced Ab3 specific for wild-type p53; PAb-240 induced Ab3 specific for mutant p53. Ab1 PAb-248 induced only Ab2. The spontaneously arising Ab3 were of T cell-dependent IgG isotypes. Peptides from the complementarity determining regions of the Ab1 antibodies PAb-240 and PAb-246 could also induce Ab3 anti-p53. Finally, mice that produced Ab3 anti-p53 acquired resistance to tumor metastases. Therefore, an anti-idiotypic network built around certain domains of p53 seems to be programmed within the immune system, specific Ab2 antibodies can mimic the DNA binding domain of p53, and Ab3 network immunity to p53 can be associated with resistance to tumor cells.

Amino Acid Sequence↗
PubMed1998

Mutant p53 protein expression interferes with p53-independent apoptotic pathways.

Loss of normal p53 function was found frequently to interfere with response of cancer cells to conventional anticancer therapies. Since more than half of all human cancers possess p53 mutations, we decided to explore the involvement of mutant p53 in drug induced apoptosis. To further evaluate the relationship between the p53-dependent and p53-independent apoptotic pathways, and to elucidate the function of mutant p53 in modulating these processes, we investigated the role of a p53 temperature-sensitive (ts) mutant in a number of apoptotic pathways induced by chemotherapeutic drugs that are currently used in cancer therapy. To that end, we studied the M1/2, myeloid p53 non-producer cells, and M1/2-derived temperature-sensitive mutant p53 expressing clones. Apoptosis caused by DNA damage induced with gamma-irradiation, doxorubicin or cisplatin, was enhanced in cells expressing wild type p53 as compared to that seen in parental p53 non-producer cells; mutant p53 expressing clones were found to be more resistant to apoptosis induced by these factors. Actinomycin D, a potent inhibitor of transcription, as well as a DNA damaging agent, abrogated the restraint apoptosis mediated by mutant p53. These observations suggest that while loss of wild type p53 function clearly reduces the rate of apoptosis, p53 mutations may result in a gain of function which significantly interferes with chemotherapy induced apoptosis. Therefore, to achieve a successful cancer therapy, it is critical to consider the specific relationship between a given mutation in p53 and the chemotherapy selected.

Animals↗
PubMed1998

Pooled analysis of p53 mutations in hematological malignancies.

A computerized database is described that contains information about 507 mutations in the p53 gene of hematologic tumors and corresponding cell lines. Analysis of these mutations indicated the following findings: First, mutational spectrum analysis in these tumors was found to be similar to the pattern found for other solid tumors. However, when the patterns of base substitutions were examined separately according to the types of hematologic malignancies, followed by subgroup analysis, notable differences (in some cases of statistical significance) emerged. Second, mutational pattern analysis indicates that about 48% of base substitutions in hematologic tumors are suspected to be associated with carcinogen exposure. Third, deletions and insertions are localized mainly to exons 5-8 and repeated DNA sequences. However, the unusual profile of variations in frequency within each type of tumor suggests that, in addition to endogenous damage to template DNA, there is the factor of exposure to environmental physical and chemical carcinogens/mutagens. Fourth, p53 protein alterations analysis indicate that most of the changes in the amino acids are "semiconservative," presumably in order to avoid disrupting the structure of the p53 monomer. Consistent with this notion, structural mutations are more conservative than the binding mutations. Finally, molecular mechanisms that lead to p53 mutations, etiological factors that play a role in their formation, and the pathophysiological significance of consequent p53 protein alterations are discussed.

Amino Acid Substitution↗
PubMed1998

Molecular alterations in the TP53 gene of peripheral blood cells of patients with chronic myeloid leukemia.

The TP53 gene has been extensively studied in patients with chronic myeloid leukemia (CML), both in chronic phase and in blast crisis. Mutations in the gene were found in up to 30% of the patients, especially among those in blast crisis. We report the results of an analysis of 29 blood samples from CML patients: 8 samples from chronic phase patients, 8 from patients in the accelerated phase, and 13 from patients in blast crisis. By using genomic DNA, we sequenced PCR products of the coding exons and most introns of the TP53 gene, finding genetic changes in 30% of the blast crisis samples and 12% in chronic phase. All mutations were found in introns and were previously unreported. Immunocytochemical studies revealed accumulation of TP53 in blood cells of samples both from chronic phase and blast crisis patients. Since these samples had no TP53 mutations, we believe that wild type TP53 accumulates in blood cells of CML patients. Our results, therefore, indicate that molecular changes in coding regions of the TP53 gene are rare. The significance of the abundance of intronic changes should be investigated further. Accumulation of wild type TP53 in CML cells may indicate an additional mechanism involving this gene in the pathogenesis of this disease.

Adult↗
PubMed1998

p53-dependent cell cycle control: response to genotoxic stress.

p53 protein is involved in key responses to genotoxic stress. These functions underlie the role of p53 as the 'guardian of the genome'. In a simplified manner, upon low or repairable levels of DNA damage, p53 mediates the delay or arrest at checkpoints preceding cell replication (the G1/S checkpoint), and is involved in delaying damaged cells prior premitotic chromosome condensation (the G2 and pre-meiotic check-points) and actual chromosome partition (the spindle check-point). During these delays, an opportunity is given to repair the DNA damage, before its fixation and propagation, that may lead to carcinogenesis. Upon high or irreparable DNA damage, p53 promotes the cells towards apoptosis. Here we review the known molecular pathways by which p53 controls the cell cycle, with a specific focus on the significance of p53-mediated checkpoint response for its 'tumor suppressor' function. The data reviewed is concerned with the in vivo mouse models including p53 knockout mice, transgenic mice harboring various mutant forms of p53 and mice knocked out for cell-cycle- and apoptosis-associated genes situated upstream or downstream from p53, that have been elaborated upon over the last few years.

Animals↗
PubMed1998

Introduction.

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Journal Article↗
PubMed1998

Idiotypic immunization induces immunity to mutated p53 and tumor rejection.

The p53 molecule might serve as a common tumor-associated antigen, as the tumor suppressor gene p53 is mutated and the p53 protein is often over-expressed in tumor cells. We report that effective immunity to p53 can be induced through an idiotypic network by immunization of mice with a monoclonal antibody (PAb-240) specific for mutated p53, or with a peptide derived from the complementarity determining region (CDR) 3 of the variable domain of the light chain (VL) of this antibody. The immunized mice produced IgG antibodies to p53 and mounted a cytotoxic reaction to a tumor line bearing mutated p53. The idiotypically immunized mice were resistant to challenge with the tumor cells. Thus antibodies to p53 might serve as immunogens for activating resistance to some tumors. At the basic level, these findings indicate that a network of p53 immunity may be organized naturally within the immune system.

Animals↗
PubMed1998

DNA-binding activity of wild-type p53 protein is mediated by the central part of the molecule and controlled by its C terminus.

The DNA binding activity of wild type p53 is central to its activity. The "central" part of the molecule, where most mutations appear in primary human tumors, is the actual DNA binding domain. The C-terminal part was shown to exert a negative effect on the DNA binding activity. In the present study we show that while anti-p53 antibodies recognizing the C terminus of the wild type p53 facilitate DNA binding activity, blocking of the wild type specific epitope by specific anti-p53 antibodies, inhibited the DNA binding activity of the wild type p53 protein. An alternatively spliced p53 protein exhibits an augmented DNA binding activity. The fact that most p53 mutants have lost the wild type p53 conformation specific epitope, coupled with the observation that blocking of this site by binding specific antibodies, prevents the interaction of wild type p53 with DNA, suggests that maintaining the correct structural conformation of this site is central for DNA binding activity. Still, the internal structure of the p53 target and particularly the length of the sequence between the two tandem inverted repeats, is critical for protein-DNA interaction behavior.

Antibodies, Monoclonal↗