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M Montenarh

Publications and source records attributed to M Montenarh.

At least 55 records · Page 3Linked to original sources
PubMed1994

The emerging picture of p53.

1. The cellular phosphoprotein p53 is a negative regulator of the cell growth. A great majority of human malignancies expresses a mutated p53 that represents an oncogenic version of the protein. 2. However, in the meantime many tumors were identified containing a p53 protein without any mutation. Here also other events than genomic alterations of p53 might be implicated in the process of cell transformation. 3. The expression of wild-type or mutant conformation is not exclusively defined by the p53 DNA sequence but also influenced by the subcellular environment and the interaction of cellular proteins with p53. 4. In particular, the mdm-2 gene product appears to be an important partner of p53 somehow involved in these complex regulatory processes. 5. Recent findings supported a role for p53 in transcriptional regulation, perhaps by reducing the expression of genes that are needed for ongoing cell proliferation. 6. This property may be based upon the ability of p53 to bind DNA as well as different proteins from viral or cellular origin. 7. Especially transcription factors or further cellular proteins connected in any way with the regulation of cell proliferation are possible candidates. 8. Thus, it is not surprising that p53 is implicated in the regulation of the cell cycle and in the decision of a cell to replicate DNA or to go into apoptosis.

Animals↗
PubMed1994

Isolation and characterization of two monoclonal antibodies raised against tms1 protein of fission yeast.

Monoclonal antibodies were produced against recombinant tms1 protein of fission yeast. The antibodies of IgG3 subclass were isolated from serum-free cell culture medium and purified by affinity chromatography on protein A-Sepharose. The antibodies can be used to detect specifically the tms1 protein on immunoblots of total yeast lysates. In addition, native tms1 protein is specifically precipitated by these antibodies from yeast lysates.

Animals↗
PubMed1994

Overexpression of wild-type p53 interferes with normal development in Xenopus laevis embryos.

We have cloned and sequenced a Xenopus p53 homologue which differs by one amino acid deletion from a previously published Xenopus sequence (Soussi et al., 1987). Transcription analysis revealed that this gene is activated during early oogenesis and that zygotic transcription initiates after midblastula transition. Transcripts are also present in all tested tissues of adult animals. Whole mount in situ hybridization with Xenopus oocytes and embryos revealed, that transcripts are ubiquitously distributed although some accumulation is observed in certain tissues. Microinjection of p53 mRNA into early cleavage stages effectively resulted in overexpression of p53 protein and interfered with normal development. Lethal defects until and during gastrulation and aberrant phenotypes of surviving embryos were probably caused by cleavage arrest or cleavage delay of injected cells with subsequent distortions of cell movements, induction processes and tissue differentiations.

Amino Acid Sequence↗
PubMed1992

Protein-protein interactions in high molecular weight forms of the transformation-related phosphoprotein p53.

The transformation-related cellular phosphoprotein p53 interacts with a variety of viral and cellular proteins and with itself to form high molecular weight complexes. The formation of high molecular weight complexes correlates with the transformed morphology of the cells whereas in non-transformed cells low molecular weight forms are predominant. Thus, aggregation seems to be involved in the regulation of biological functions of p53. Analyzing wild-type and mutant p53 in the same cellular environment i.e. after an in vitro transcription/translation reaction in rabbit reticulocytes we found high molecular weight forms for wild-type and mutant p53. The sedimentation profile resembled the profile obtained for mutant p53 from transformed cells. As shown by dilution experiments, aggregation of p53 was not due to high p53 protein concentrations. Although p53 is known to bind RNA, treatment with RNAse did not change the aggregation state of p53 suggesting that RNA may not contribute to the quaternary structure of p53. High molecular weight aggregates of p53 were resistant to treatment with 1 M NaCl and also stable in weak acidic conditions. Alkaline pH as well as treatment with 3.5 M NaCl led to a disaggregation of high molecular weight complexes of p53. This treatment resulted in low molecular weight forms consisting probably of dimers to tetramers whereas monomers of p53 are hardly detectable. A nearly complete disaggregation was obtained only with the ionic denaturing detergent sodium dodecyl sulfate. Therefore, one has to assume different types of protein-protein interactions leading to the various quaternary structures of p53.

Animals↗
PubMed1992

Cooperation of p53 and polyoma virus middle T antigen in the transformation of primary rat embryo fibroblasts.

Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation.

Animals↗
PubMed1992

Antisense effect of oligodeoxynucleotides with inverted terminal internucleotidic linkages: a minimal modification protecting against nucleolytic degradation.

The synthesis of a new class of antisense oligonucleotide compounds with 3'-3' and 5'-5' end inversion (INV-oligonucleotides) is described. Besides the advantage of simplicity of synthesis, physico-chemical studies show that these compounds do not disturb Watson-Crick base-pairing. INV-oligonucleotides have a half-life of 30 h in human serum. We show that they are capable of inhibiting SV40 large T-antigen expression in COS-1 cells, both in vitro and in vivo, and by modulation of the expression of cellular oncoprotein p53 in vitro.

Animals↗
PubMed1992

Biochemical properties of the growth suppressor/oncoprotein p53.

The cellular p53 protein is so called because of its molecular weight as determined by SDS-polyacrylamide gel electrophoresis. It was originally classified as a nuclear oncogene product when it was shown by DNA transfection experiments that p53 is able to extend the lifespan of primary rodent cell cultures and to cooperate with an activated ras oncogene to achieve complete transformation of primary cells. However, there is now conclusive evidence that loss of normal p53 expression may be an important step in cell transformation and tumorigenesis. Furthermore, it has been shown that mutant p53 was used for the experiments demonstrating the immortalizing and transforming capacity of p53. Wild-type p53 seems to negatively regulate cell growth and division. So far, the basic function of p53 is not known. Biochemical variability seems to be a key feature of p53 and an understanding of biochemical variations in the p53 protein may contribute to an understanding of how p53 is regulated or how p53 may regulate cell proliferation. Thus, the present review will focus on the biochemical properties of p53.

Animals↗
PubMed1992

Biochemical, immunological, and functional aspects of the growth-suppressor/oncoprotein p53.

The cellular phosphoprotein p53 was initially discovered in a variety of in vitro transformed and tumor-derived cell lines. Later on, p53 was also found in normal, nontransformed cells albeit at very low levels. Inhibition of p53 functions by microinjection of anti-p53 antibodies prevented quiescent cells from reentering the cell cycle after serum stimulation, indicating that p53 might somehow be involved in the regulation of cell proliferation. After detection of p53-specific mRNAs, the gene was discovered in diverse species ranging from fish and frog to man. Knowing the p53 DNA sequence, it became clear that mutant forms of p53 had immortalizing and, in cooperation with other oncogenes, transforming activities. On the other hand, wild-type p53 could suppress oncogene-mediated transformation. It seems clear now that wild-type p53 is a tumor suppressor. Moreover, analysis of p53 from many types of human tumors indicates that the p53 gene is a very frequent target for mutational alterations. Because the biochemical consequences of these alterations are not yet clear, it is not understood yet on the molecular level how p53 can act as an oncogenic protein, on one hand, and as a growth-suppressor protein on the other hand. Therefore, the present review aims to summarize the biochemical and immunological properties of p53 and to address some biological activities of p53 in order to allow more insight into how p53 might be regulated within the cell or how p53 might regulate cell proliferation.

Amino Acid Sequence↗
PubMed1991

Correlation of metabolic stability and altered quaternary structure of oncoprotein p53 with cell transformation.

The phosphoprotein p53 seems to be implicated in various processes connected with cell transformation and in particular with the regulation of cell cycle and probably DNA replication. In the present paper we have analyzed two sets of closely related cell lines expressing the same p53 which exhibited either a nontransformed or a transformed phenotype. These cell lines were used to study biochemical properties of the p53 protein which might be correlated with cell transformation. We found a positive correlation among an elevated stability of p53, the formation of high-molecular-weight forms of p53, and the transformed phenotype of the corresponding cell lines. Furthermore, these data indicate that self-aggregation prevents p53 from rapid degradation. By a comparative analysis of the stability and oligomerization properties of mutant p53 and wild-type p53, we could demonstrate that elevated stability and self-aggregation of p53 are correlated with the transformed phenotype of the cells and independent of a particular mutation in the p53 gene.

Animals↗
PubMed1991

Expression of p53 during mouse embryogenesis.

By in situ hybridisation we have examined the expression of p53 during mouse embryogenesis from day 8.5 to day 18.5 post coitum (p.c.). High levels of p53 mRNA were detected in all cells of the day 8.5 p.c. and 10.5 p.c. mouse embryo. However, at later stages of development, expression became more pronounced during differentiation of specific tissues e.g. of the brain, liver, lung, thymus, intestine, salivary gland and kidney. In cells undergoing terminal differentiation, the level of p53 mRNA declined strongly. In the brain, hybridisation signals were also observed in postmitotic but not yet terminally differentiated cells. Therefore, gene expression of p53 does not appear to be linked with cellular proliferation in this organ. A proposed role for p53 in cellular differentiation is discussed.

Animals↗
PubMed1991

Synthetic oligonucleotides for biomedical applications.

New developments of oligonucleotides for biomedical applications are surveyed. Diagnostic probes were conveniently labelled by enzymatic immunogenic tailing with 5-bromo-deoxyuridine triphosphate. "Antisense" oligonucleotides of potential therapeutic value were stabilized against nucleolytic decay by inversion of terminal internucleotidic linkages. Introduction of 2'-deoxy-2'-fluoro-nucleotide units enhances duplex stability and conveys resistance to ribonucleases.

Base Sequence↗
PubMed1991

A new DNA binding assay to study subclasses of SV40 large T antigen binding to individual binding sites on the SV40 DNA.

SV40 large T antigen interacts specifically with three different sequences, termed sites I, II or III, within the control region of the SV40 DNA to regulate transcription as well as the initiation and progress of SV40 DNA replication. We have biotinylated three different DNAs containing either site I, II or III and immobilized these constructs on a streptavidin agarose matrix. All three immobilized DNAs were shown to bind T antigen from a total cell extract of SV40 infected monkey cells although with different affinities. Pulse chase experiments revealed that newly synthesized T antigen bound efficiently to all three binding sites whereas mature T antigen bound only to site I and site III DNA. The analysis of non-binding and DNA-bound T antigen on sucrose density gradients showed that only low molecular weight forms of T antigen were bound to all three immobilized DNAs. However, incubation of T antigen in the total cell extract with site I DNA resulted in high molecular weight forms of T antigen, indicating that the presence of site I DNA influences the quaternary structure of T antigen which might result in a detachment from the DNA.

Animals↗
PubMed1991

Association of casein kinase II with immunopurified p53.

Viral and cellular oncogene products sometimes activate protein kinases, are protein kinases themselves, or share phosphorylation sequence motifs for different protein kinases. We have recently shown that a protein kinase activity is tightly associated with immunopurified p53. We have now expressed p53 in a baculovirus expression system and characterized this protein kinase activity in more detail. We found that casein could compete with p53 in the kinase reaction. Heparin efficiently inhibited the p53 associated protein kinase whereas the polyamine spermidine stimulated enzymatic activity. A synthetic peptide which was shown to be specifically phosphorylated by casein kinase II blocked the in vitro phosphorylation of p53, whereas a synthetic peptide with a potential phosphorylation site on human p53 at ser 315 was ineffective in blocking the phosphorylation of p53. GTP as well as ATP can be used as a phosphate donor in the in vitro kinase reaction. An antibody directed against casein kinase II coprecipitated p53 from insect cells as well as from mammalian cells. These data strongly indicate that casein kinase II is associated with immunopurified p53 and contributes to the phosphorylation of p53. A mutant p53 with a ser 389 to ala exchange was not phosphorylated in vitro by the p53 associated protein kinase.

Amino Acid Sequence↗
PubMed1990

Expression of p53 in human leukemic cell lines.

The cell-encoded p53 antigen seems to be tightly associated with various human malignancies. We have analyzed biochemical properties of p53 in two different cell lines derived from patients with ALL or ANLL. p53 was found in elevated levels in both leukemic cell lines compared to unstimulated or stimulated normal lymphocytes. High levels of p53 in these cell lines are due to an extended stability of p53 protein rather than to different rates of synthesis. p53 from both cell lines formed low- and high-molecular weight oligomers which revealed that p53 exists in a heterogenous population in these tumor cells. The presence of immunologically different subsets of p53 was demonstrated by sequential immunoprecipitation experiments with different p53 specific monoclonal antibodies. Our results showed structural and immunological variabilities of p53 in cell lines derived from human tumors and may thus provide an insight into the role p53 may play in human malignancies.

Burkitt Lymphoma↗
PubMed1990

Protein kinase activity associated with immunopurified p53 protein.

Enhanced protein phosphorylation seems to be characteristic for cell transformation. Viral or cellular oncogene products which are functionally implicated in cell transformation sometimes activate protein kinases, or they are protein kinases themselves. In the present paper we have shown that a protein kinase activity is tightly associated with immunopurified oncoprotein p53, either uncomplexed or in complex with SV40 large T antigen. Furthermore, we could demonstrate that the protein kinase associated with immunopurified p53 was independent of SV40 large T antigen. p53 in the immunocomplexes served as a substrate for this protein kinase. Phosphoamino acid analysis of in vitro phosphorylated p53 revealed a phosphorylation predominantly on serine residues similar to p53 phosphorylated in vivo. The use of different monoclonal antibodies did not reveal a total inhibition of the protein kinase activity. However, p53 precipitated with monoclonal antibodies which recognize a C-terminal domain, was phosphorylated in vitro to a lesser extent than p53 which was precipitated with monoclonal antibodies that recognize an N-terminal epitope. All subclasses of immunopurified p53 separable by sucrose density gradients or by sequential immunoprecipitation exhibited a protein kinase activity and served as substrates for this protein kinase. Moreover, a protein kinase activity was found to be associated with baculovirus expressed p53 which allows us to attribute this enzymatic activity more directly to p53.

Amino Acids↗
PubMed1990

Regulation of the level of the oncoprotein p53 in non-transformed and transformed cells.

In order to contribute to the understanding of the activation of the oncoprotein p53 we determined the metabolic stability of p53 in a variety of non-transformed, immortalized and SV40- and non-SV40-transformed cell lines. In addition, we analyzed the metabolic stability of the SV40 large T antigen in SV40 transformed cell lines. Pulse-chase experiments revealed a low stability (t1/2 = 20 min) of p53 in non-transformed cells and in cells immortalized by the p53 construct pLTRp53cG9. In cells transformed by an activated ras oncogene and pLTRp53cG9 and in methylcholanthrene induced mouse sarcoma cells p53 proved to be progressively more stable with half-lives ranging from 5.5 h to 7 h. Sequential immunoprecipitation with p53- or T antigen specific monoclonal antibodies allowed us to separate T-p53 complexes, uncomplexed p53 and free T antigen in cell extracts from cells transformed by SV40 and pLTRp53cG9. In these transformed cells uncomplexed p53 showed an increased stability (t1/2 = 2.8 h) when compared to p53 from non-transformed cells. Complex formation with T antigen resulted in an additional stabilization of p53 (t1/2 = 13.3 h). Furthermore, T-p53 complex formation also seems to increase the stability of T antigen nearly sixfold. In transformed cells two immunological variants of p53, a PAb246 precipitable and a non-precipitable form showed distinctly different stabilities, indicating a correlation between the ability of p53 subclasses to bind hsc70 protein and their metabolic stability. Moreover, binding to hsc70 correlated with the stabilization of T antigen in CTM cells also where the mutant T antigen is localized exclusively in the cytoplasm. In abortively infected cells p53, even in complex with T antigen, exhibited a relatively low stability (t1/2 = 87 min) indicating that complex formation per se is not sufficient for fully stabilizing p53.

Animals↗
PubMed1990

[Thrombocyte growth factor].

PDGF is one of the principal mitogens for cultured cells of mesenchymal origin. Apart from aggregated platelets PDGF can be secreted by a number of cells. There is a striking homology between the amino-acid sequence of PDGF and that of a transforming protein of simian-sarcoma virus. PDGF is an important chemotactic protein for cells implicated in wound healing. Furthermore, recent publications suggest a possible role in the pathogenesis of several diseases. This article summarizes studies and recent findings on the structure, biology, and functional role of PDGF.

Animals↗