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Biomedical subjects

V Rotter

Publications and source records attributed to V Rotter.

At least 73 records · Page 4Linked to original sources
PubMed1995

Accumulation of wild-type p53 protein upon gamma-irradiation induces a G2 arrest-dependent immunoglobulin kappa light chain gene expression.

The exposure of cells to DNA-damaging agents leads to the accumulation of wild-type p53 protein. Furthermore, overexpression of the wild-type p53, mediated by transfection of p53-coding cDNA, induced cells to undergo apoptosis or cell differentiation. In this study we found that the gamma-irradiation that caused the accumulation of wild-type p53 in 70Z/3 pre-B cells induced, in addition to apoptosis, cell differentiation. This was manifested by the expression of the kappa light chain immunoglobulin gene that coincided with the accumulation of cells at the G2 phase. Overexpression of mutant p53 in 70Z/3 cells interferes with both differentiation and accumulation of cells at the G2 phase, as well as with apoptosis, which were induced by gamma-irradiation. Furthermore, the increment in the wild-type p53 protein level following gamma-irradiation was disrupted in the mutant p53 overproducer-derived cell lines. This suggests that mutant p53 may exert a dominant negative effect in all of these activities. Data presented here show that while p53-induced apoptosis is associated with the G1 checkpoint, p53-mediated differentiation, which may be an additional pathway to escape the fixation of genetic errors, may be associated with the G2 growth arrest phase.

Amino Acid Sequence↗
PubMed1995

The DNA binding activity of wild type p53 is modulated by blocking its various antigenic epitopes.

Interaction of wild type p53 with specific DNA target sequences, which is dictated by several structural domains, can be modified by blocking the different antigenic epitopes of the protein. Comparison of p53 protein expressed by recombinant bacteria (wtp53-Bac) to that produced in an eukaryotic system by a vaccinia expression vector (wtp53-Vac), indicated that only the later exhibited spontaneous DNA-binding activity. Furthermore, DNA-binding patterns of these wild type p53 proteins were affected differently by their interactions with monoclonal anti-p53 antibodies recognizing individual antigenic epitopes of the molecule. While the vaccinia derived p53 that spontaneously bound DNA is supershifted by the N'-terminal specific antibodies PAb-248, the bacterial derived p53 protein that retains this antigenic epitope but does not bind DNA spontaneously, is not affected. The C'-terminal specific PAb-421 antibodies accelerated binding of the bacterial p53 protein and modified the pattern of the interaction of the vaccinia derived p53 DNA. DNA-binding patterns generated by PAb-421 and PAb-248, suggest that either interaction of wild type p53 is dependent on modification of the p53 protein or that it interacts with cellular factors which their activity can be mimicked by PAb-421. Saturation of both types of wild type p53 with several anti-p53 monoclonal antibodies directed against the wild type p53 specific epitope that maps to the N'-terminal border of the DNA-binding region, blocked specific DNA-binding. 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. The wild type specific epitope which maps to the N'-terminal border of the DNA-binding region is neighboring the first beta-strand detected by the recent crystallographic analysis.

Animals↗
PubMed1995

Direct involvement of p53 in programmed cell death of oligodendrocytes.

A covalent dimer of interleukin (IL)-2, produced in vitro by the action of a nerve-derived transglutaminase, has been shown previously to be cytotoxic to mature rat brain oligodendrocytes. Here we report that this cytotoxic effect operates via programmed cell death (apoptosis) and that the p53 tumor suppressor gene is involved directly in the process. The apoptotic death of mature rat brain oligodendrocytes in culture following treatment with dimeric IL-2 was demonstrated by chromatin condensation and internucleosomal DNA fragmentation. The peak of apoptosis was observed 16-24 h after treatment, while the commitment to death was already observed after 3-4 h. An involvement of p53 in this process was indicated by the shift in location of constitutively expressed endogenous p53 from the cytoplasm to the nucleus, as early as 15 min after exposure to dimeric IL-2. Moreover, infection with a recombinant retrovirus encoding a C-terminal p53 miniprotein, shown previously to act as a dominant negative inhibitor of endogenous wild-type p53 activity, protected these cells from apoptosis.

Animals↗
PubMed1995

p53 mutations in matched primary and metastatic human tumors.

Mutations in the p53 tumor suppressor gene have been found to be the most frequent genetic alterations in human malignancies. To further examine the idea that neoplastic progression is associated with mutations in the p53 gene, we analyzed matched primary and metastatic tumor samples. The samples included 15 pairs of breast cancer and metastases to lymph nodes, four pairs of gastrointestinal adenocarcinomas and metastases to liver, one colon adenocarcinoma and metastasis to a lymph node, and one lung carcinoma and metastasis in the pleura. Genomic DNA or cDNA from each tumor sample was amplified by the polymerase chain reaction and labeled by using one biotinylated primer. The DNA strands were separated with magnetic streptavidin beads and sequenced directly. p53 mutations were detected in 11 of 21 patients (52%) in either primary tumors, metastases, or both. In six of these patients the primary tumor and matched metastasis shared the same single mutation. In the other patients an additional mutation in the primary tumor only or a mutation in the metastasis only was observed. Our data suggest that tumor development and progression toward metastasis involves structural alterations in the p53 gene that occur early in carcinogenesis. In some cases, genetic changes in metastatic spreading may also include the appearance of a mutation in a metastasis derived from a primary tumor expressing wild-type p53, a selection of metastatic cells with a single mutation from a primary tumor expressing two different mutations, or loss of heterozygosity.

Adenocarcinoma↗
PubMed1995

A novel polymorphism in intron 6 of the human p53 gene: a possible association with cancer predisposition and susceptibility.

We present a novel polymorphic 8-bp sequence in intron 6 of the p53 gene that maps between bp 55 and 62 of the 3' end of exon 6. Of normal blood samples, 32% were heterozygotic for this polymorphism and display a NN' genotype, whereas 68% of the population is homozygotic for the N genotype. The rare homozygotic genotype N' was detected only in four blood samples of cancer patients. Peripheral blood of gastrointestinal (GI) and breast tumor patients demonstrated a higher incidence of heterozygosity (50%) than that of normal individuals. Analysis of the distribution of this polymorphism in tumor samples showed loss of heterozygosity (LOH). This LOH during tumor progression could exhibit preference to each one of the polymorphic alleles. The rare presentation of one allele and the increased incidence of heterozygosity in carcinoma patients may suggest an association between this polymorphism with cancer predisposition and susceptibility. The fact that genetic alterations occurring in noncoding regions may play a role in tumor development only further increases the extent of involvement of p53 in carcinogenesis.

Base Sequence↗
PubMed1995

Immunohistochemical detection of p53 protein expression in HPV-induced condyloma acuminatum.

Immunohistochemical peroxidase staining for p53 protein was performed on 22 condyloma acuminatum tissue samples from patients infected with human papillomavirus (HPV). The purpose of our study was to understand the benign character of this syndrome. The patients studied were infected by HPV type 6 and 11. Two monoclonal antibodies, PAbs DO-1 and 240, were used to detect the p53 protein. Overexpression of wild-type p53 was found in the nuclei of the basal cell layers. In healthy tissues and non-infected patients no p53 protein expression was detected. We would like to speculate that infection with HPVs and their viral protein E7, which is implicated in disruption of normal growth, may regulate the induction of wild-type p53 over-expression, as is known for DNA-damaging agents such as UV- or X-radiation.

Adolescent↗
PubMed1994

Histochemical studies of progressive p53 mutations during colonic carcinogenesis in Sprague-Dawley rats induced by N-methyl-N-nitro-nitrosoguanidine or azoxymethane.

We studied the increasing expression of the p53 tumor suppressor gene in Sprague-Dawley rats, chemically induced to develop colon cancer. p53 expression was evaluated histochemically at various stages of tumor progression (during a period of 40 weeks) that can be followed by colonic hyperproliferation labeled by 3H-thymidine incorporation. We found that high level nuclear expression of p53 protein correlates with progression of malignancy in carcinogen-induced animals, whereas cytoplasmic staining is related to the onset and early development of malignancy.

Adenocarcinoma↗
PubMed1994

Does wild-type p53 play a role in normal cell differentiation?

Inactivation of the p53 tumor suppressor gene plays a major role in malignant transformation. The central question in this issue is concerned with the understanding of the function of p53 in normal cells and its deregulation in cancer cells. Several in vitro and in vivo experimental models have indicated that induction of cells to undergo differentiation involve up-regulation in the expression of the p53. In the case of B cell differentiation, p53 was found to be involved in several steps of the differentiation pathway. The conclusion that p53 plays a role in normal development and differentiation in vivo is substantiated by the observation that p53 is expressed during embryonic development and is detected at low levels in a number of organs of adult mice. Accentuated levels of p53 in testes of adult mice, suggests that p53 plays a role in the meiotic process of spermatogenesis. B cell differentiation and spermatogenesis are biological pathways which normally involve DNA reshuffling and rearrangements. In accordance with the notion that p53 is associated with DNA repair it is tempting to speculate that at least in these physiological pathways p53 functions as a master gene that controls genome integrity.

Animals↗
PubMed1993

Mice with reduced levels of p53 protein exhibit the testicular giant-cell degenerative syndrome.

Transgenic mice which carry hybrid p53 promoter-chloramphenicol acetyltransferase (CAT) transgenes were found to express CAT enzymatic activity predominantly in the testes. Endogenous levels of p53 mRNA and protein were lower than in the nontransgenic control mice. The various p53 promoter-CAT transgenic mice exhibited in their testes multinucleated giant cells, a degenerative syndrome resulting presumably from the inability of the tetraploid primary spermatocytes to complete meiotic division. The giant-cell degenerative syndrome was also observed in some genetic strains of homozygous p53 null mice. In view of the hypothesis that p53 plays a role in DNA repair mechanisms, it is tempting to speculate that the physiological function of p53 that is specifically expressed in the meiotic pachytene phase of spermatogenesis is to allow adequate time for the DNA reshuffling and repair events which occur at this phase to be properly completed. Primary spermatocytes which have reduced p53 levels are probably impaired with respect to DNA repair, thus leading to the development of genetically defective giant cells that do not mature.

Animals↗
PubMed1993

The helix-loop-helix containing transcription factor USF binds to and transactivates the promoter of the p53 tumor suppressor gene.

Expression of the wild-type p53 tumor suppressor gene has been found to play an important role in the regulation of cellular proliferation and differentiation. In addition, in many transformed cells and primary tumors, the gene has undergone allelic deletions and mutant forms of the p53 gene are expressed at elevated levels. In defining transcriptional regulatory regions of the p53 gene, we have previously shown that both the human and murine p53 promoters contain a conserved consensus recognition sequence for the basic-helix-loop-helix (bHLH) containing family of DNA-binding proteins. In the murine p53 promoter this element is required for full promoter activity and contains the sequence CACGTG, a sequence identical to the recognition site for the bHLH containing transcription factors c-Myc, USF and TFE3. Here we examine the ability of one of these factors, USF, to bind to the p53 promoter. By assaying the binding activity of in vitro translated USF as well as factors present in nuclear extracts, we conclude that the transcription factor USF binds in a site-specific manner to a CACGTG motif within the murine p53 promoter and represents the major DNA-binding activity observed in nuclear extracts. Elevated levels of USF, generated upon transfection of a vector expressing USF, lead to enhanced activity of the p53 promoter. These findings indicate that USF may play a central role in regulating p53 expression.

3T3 Cells↗
PubMed1993

Testicular tissue-specific expression of the p53 suppressor gene.

The hybrid transgene approach was adapted to study the physiological pathway(s) in which the p53 suppressor gene is involved. p53 promoter-CAT transgenic mice were found to express enzymatic CAT activity predominantly in the testes. In situ hybridization indicated that expression of the transgene as well as the endogenous p53 agreed with the typical wave and cycle patterns of spermatogenesis. p53 promoter-CAT transgenic mice expressed in the testes reduced levels of endogenous p53 mRNA that correlated with the copy number of the mouse or human transgene. The spatial and cyclical expression of the p53 gene which is confined to the primary spermatocytes in the seminiferous tubuli suggested that p53 may play a role in the meiotic process of spermatogenesis in vivo.

Animals↗
PubMed1993

Isolation and characterization of DNA sequences that are specifically bound by wild-type p53 protein.

Wild-type p53 was shown to function as a transcription factor. The N-terminal region of the protein contains the transcription activation domain, while the C terminus is responsible for DNA binding. Localization of the DNA-binding domain of the p53 protein to the highly conserved carboxy-terminal region suggests that the interaction of p53 with DNA is important for its function. We have developed a strategy for studying the DNA sequence specificity of p53-DNA binding that is based on random sequence selection. We report here on the isolation of murine genomic DNA clones that are specifically bound by the wild-type p53 protein but are not bound by mutant p53 protein forms. The isolated p53 target gene contains the unique DNA-binding sequence GACACTGGTCACACTTGGCTGCTTAGGAAT. This fragment exhibits promoter activity as measured by its capacity to activate transcription of the chloramphenicol acetyltransferase reporter gene. Our results suggest that p53 directly binds DNA and functions as a typical transcription factor.

Animals↗
PubMed1993

Wild type p53 functions as a control protein in the differentiation pathway of the B-cell lineage.

An analysis of cell lines representing different stages of the B-cell differentiation pathway indicated that about 50% of the cell lines examined expressed exclusively wild type p53 protein. These lines therefore offer a convenient system to study the involvement of p53 in cell differentiation. When 70Z/3, a pre-B cell line which expresses wild type p53, was treated with the differentiation inducer lipopolysaccharide (LPS), it was seen that increased levels of p53 mRNA preceded specific changes in kappa (kappa) immunoglobulin expression. This increased expression of kappa specific mRNA, which was evaluated by specific PCR analysis, was blocked following transfection with mutant p53 coding plasmids. Treatment of 13A60, another cell line which endogenously expresses wild type p53, with LPS caused a secretion of IgA antibodies, also accompanied by increased p53 mRNA expression. The conclusion was that induction of B-cell differentiation involves the transcription of the p53 gene. This was further substantiated by experiments showing that differentiation of stable clones derived from the 70Z/3 cell line, harboring a p53-promoter-CAT plasmid, induced increased CAT activity. Furthermore, wild type p53 transactivated the promoter control sequences of the kappa light chain gene. Taken together, these results suggest that p53 is involved in B-cell differentiation, a pathway which involves DNA rearrangements that may be accompanied by generation of faulty DNA. The fact that wild type p53 was shown to function as a transcriptional factor, coupled with the notion that it is associated with DNA repair systems, may designate p53 as a control protein in the B-cell differentiation pathway.

Animals↗
PubMed1993

Wild-type but not mutant p53 can repress transcription initiation in vitro by interfering with the binding of basal transcription factors to the TATA motif.

It has previously been shown that excess wild type (wt) p53 can repress the transcriptional activity of a variety of promoters in intact cells. To determine whether this transcriptional repression represented a direct effect of p53, wt and mutant p53 were prepared from E. coli-produced p53 and from insect cells infected with a recombinant baculovirus. When added into an in vitro transcription system, wt p53, but not mutant p53 reduced markedly transcription from the c-myc promoter, as well as from an array of other promoters, with the exception of an MHC class I gene promoter. The presence of wt p53 seemed to affect specifically the formation of the transcription preinitiation complex because preformed initiation complexes were completely refractory to wt p53, as was also the process of transcript elongation. Wild-type but not mutant p53 interfered with the stable binding of TBP and TFIIA to the TATA motif, although both wt and mutant p53 could associate in vitro with purified TBP. We propose that upon binding to TBP, wt but not mutant p53 specifically blocks the ability of TBP to engage in interactions required for efficient transcriptional initiation. This may account, at least in part, for the ability of excess wt p53 to inhibit cell proliferation and to interfere with neoplastic processes.

Base Sequence↗
PubMed1993

c-Myc trans-activates the p53 promoter through a required downstream CACGTG motif.

c-Myc and wild-type p53 have been shown to play important roles in the regulation of cellular proliferation and oncogenic transformation. We have previously shown that the p53 promoter contains a conserved consensus recognition sequence for the basic-helix-loop-helix-containing proteins, identical to the specific binding site for c-Myc/Max heterodimers. Here, we demonstrate that this element, which is required for full promoter activity, is bound by in vitro translated c-Myc/Max heterodimers. Furthermore, we found that in cotransfection assays, c-Myc trans-activates the p53 promoter as well as a hybrid herpes simplex virus-thymidine kinase promoter containing multiple copies of a synthetic p53-derived c-Myc binding site. The p53 promoter deleted of the basic-helix-loop-helix consensus recognition sequence is not trans-activated by c-Myc, thus suggesting that c-Myc trans-activates the p53 promoter through the basic-helix-loop-helix recognition motif. These findings raise the possibility that the p53 gene may be a potential target for trans-activation by c-Myc in vivo.

Animals↗
PubMed1993

Expression of p53 protein in spermatogenesis is confined to the tetraploid pachytene primary spermatocytes.

The various steps of differentiation and maturation in spermatogenesis are well characterized and offer a convenient system to explore the possibility that p53 plays a role in cell differentiation in vivo. In situ hybridization experiments indicate that the p53 gene is expressed in tetraploid primary spermatocytes at the meiotic pachytene stage of the first round of spermatogenesis in young mice. An analysis of spermatogenic cells treated with anti-p53 antibodies reveals that the p53 protein is expressed in a discrete tetraploid cell population, with size and cellular structure parameters characteristic of midpachytene spermatocytes. The specific kinetics of p53 expression in the first round of spermatogenesis and its localization in adult testicular sections, coupled with the fact that the protein is expressed in the largest cells with lower DNA density, suggest that p53 expression is confined to the tetraploid primary spermatocytes of the meiotic pachytene phase. These observations identify p53 protein as a potential member of the meiosis control protein family.

Animals↗