Search PubMed⌕ Search

Biomedical subjects

V Rotter

Publications and source records attributed to V Rotter.

At least 91 records · Page 5Linked to original sources
PubMed1992

Expression of wild-type and mutant p53 proteins by recombinant vaccinia viruses.

To facilitate the purification of wild type p53 protein, we established a recombinant p53 vaccinia viral expression system. Using this efficient eukaryotic expression vector, we found that the expressed p53 proteins retained their specific structural characteristics. A comparison between wild type and mutant p53 proteins showed the conservation of the typical subcellular localization and the expression of specific antigenic determinants. Furthermore, wild type p53 exhibited a typical binding with large T antigen, whereas no binding was detected with mutant p53. Both wild type and mutant p53 proteins were highly stable and constituted 5-7% of total protein expressed in the infected cells. These expression recombinant viruses offer a simple, valuable system for the purification of wild type and mutant p53 proteins that are expressed abundantly in eukaryotic cells.

Antigens, Viral, Tumor↗
PubMed1991

Involvement of wild-type p53 in pre-B-cell differentiation in vitro.

Wild-type p53 protein is a growth modulator whose inactivation has been found to be a key event in malignant transformation. Reconstitution of wild-type p53 in the p53-nonproducer, Abelson murine leukemia virus-transformed pre-B-cell line L12 gave rise to stably growing clones. Wild-type p53-producer derived cell lines exhibit an altered cell cycle, however. More cells with an extended G0/G1 phase were found than in the p53-nonproducer parental cell line. Furthermore, when injected into syngeneic mice, these cells induced a lower incidence of tumors and these tumors were less aggressive. Analysis of immunoglobulin expression revealed that wild-type p53 induced the expression of cytoplasmic immunoglobulin mu heavy chain. In addition, these derived cells lines exhibited increased levels of a B-cell-specific surface marker, B220. These results suggest that wild-type p53 may function as a cell differentiation factor that can induce development of pre-B cells into a more advanced stage in the pathway of B-cell maturation. In these pre-B cells, wild-type p53 may induce cell differentiation without terminal growth arrest of the cell population.

Abelson murine leukemia virus↗
PubMed1991

A DNA binding domain is contained in the C-terminus of wild type p53 protein.

In the present study we evaluated the DNA binding activity of wild type and mutant p53 proteins that were isolated from bacterial expression vectors. A comparison of the binding activities of the various purified p53 proteins, assessed by their ability to bind DNA cellulose columns, indicated that wild type p53 has a higher affinity to DNA than have mutant p53 forms. Furthermore, only wild type p53 was able to bind genomic DNA upon electrophoretic protein blotting. As specific deletion of the C-terminal region of wild type p53 totally abolished binding to genomic DNA, it was concluded that the 47 C-terminal amino acids contain the DNA binding region. The fact that the N-terminus contains a transcription activation region whereas the C-terminus contains a DNA binding domain places p53 in the family of typical transcription factors. Our experiments show that the topographical positioning of these domains plays an important role in the activity of wild type p53.

Amino Acid Sequence↗
PubMed1991

Alterations in tumor development in vivo mediated by expression of wild type or mutant p53 proteins.

To study the mechanism of p53 involvement in malignant transformation, we compared the tumor development patterns induced by a parental p53 nonproducer pre-B cell line with those by cell lines generated from this parental cell line following transfection of either wild type or mutant p53. It was found that whereas mutant p53 facilitated tumor development, expression of wild type p53 restrained tumor development. Cell lines expressing the wild type p53 induced the development of faster regressing tumors than the parental cell line. The parental p53 nonproducer and the wild type p53 producer regressor tumors underwent in vivo cell differentiation, manifested as IgG production. Mutant p53, producer cell lines, on the other hand, failed to show any immunoglobulin synthesis and gave rise to highly proliferative lethal tumors. Our results support the conclusion that these pre-B cells develop regressor tumors because they have undergone differentiation. Whereas the wild type p53 facilitates this differentiation, mutant p53 cells block it. We suggest that, in addition to inactivating the growth-suppressive activity of wild type p53, the expression of mutant p53 facilitates malignant transformation.

Animals↗
PubMed1991

Expression from the murine p53 promoter is mediated by factor binding to a downstream helix-loop-helix recognition motif.

Expression of the p53 gene plays an important role in the regulation of cellular proliferation and malignant transformation. Overexpression of mutant forms of p53 is in fact a common feature of many transformed cells. Studies dealing with the transcriptional regulatory regions of the p53 gene indicate that, unlike most promoters transcribed by RNA polymerase II, the p53 promoter contains no TATA-like sequence upstream of the transcription start site. Here we demonstrate that the murine p53 promoter contains a cis-acting element that maps downstream to the transcription initiation site. The integrity of this element is required for high-level expression from the promoter in transformed cells. By DNase I protection and mobility-shift analysis, we show that a nuclear factor binds to this downstream element through the consensus recognition sequence for the helix-loop-helix (HLH)-containing proteins of the myc/MyoD family of transcriptional regulators. We propose that the activity of one or more members of this family of transcription factors is an important determinant in the expression of p53 and that at least one level of p53 overexpression in transformed cells may thus be due to aberrant expression of the relevant factor(s). Furthermore, the possibility that the regulation of expression of p53 occurs, in part, by means of a potential HLH-containing factor provides a possible mechanism for the suppression of proliferation by the MyoD family of transcriptional regulators.

Animals↗
PubMed1991

Nuclear localization is essential for the activity of p53 protein.

p53 appears to be a growth regulator, the perturbation of which induces changes in normal cell proliferation. Wild-type p53 protein is thought to function as a growth arrest gene, whereas mutant p53, which accumulates in transformed cells, has been shown to enhance malignant transformation. Both wild-type and mutant p53 migrate into the cell nucleus by means of identical nuclear localization signals (NLS) inherent in their primary sequences. Results presented here show that the suppressive activity of wild-type p53 measured as the reduction of transformation of primary rat fibroblasts induced by co-transfection with ras and either E1A or mutant p53, as well as the transformation enhancement of mutant p53 estimated by cooperation with ras in transformation of primary rat fibroblasts, is dependent upon nuclear localization signals in p53 protein. While transfection of unmodified wild-type p53 significantly reduces the number of rat embryonic fibroblast-transformed foci induced by E1A and ras or mutant p53 and ras, the wild-type p53 protein without NLS has completely lost this suppressive activity. Partially defective NLS wild-type p53, with a reduced nuclear accumulation ability, still exhibits some suppressive activity. In addition, we found that plasmids coding for intact mutant p53 protein efficiently cooperate with the ras oncogene, whereas the corresponding plasmids without NLS are totally inert. On this basis we conclude that nuclear localization of both wild-type and mutant p53 is a fundamental feature for manifesting the activities of these proteins. Both the suppressor activity mediated by the wild-type p53 and enhancement of transformation mediated by the mutant p53 require nuclear localization of the proteins to function.

Amino Acid Sequence↗
PubMed1991

Involvement of wild-type p53 protein in the cell cycle requires nuclear localization.

Transfection of wild-type p53 into a pre-B, p53 nonproducer cell line yielded the generation of stable clones. Although constitutively expressing the growth-suppressor wild-type p53 protein, these cells proliferate continuously in vitro. However, expression of wild-type p53 in these cells altered their cell cycle pattern and reduced their growth in vivo. When the same parental cells were transfected with a plasmid coding for a wild-type p53 lacking nuclear localization signals, a wild-type cytoplasmic p53 protein was expressed. Expression of this cytoplasmic p53 product did not exert any changes in the growth of the parental cells, suggesting that wild-type p53 affects the cell cycle only when localized in the nuclear cell compartment.

Animals↗
PubMed1991

Mutant p53 proteins bind DNA abnormally in vitro.

The p53 gene encodes a phosphoprotein which binds DNA. Many types of tumors contain mutant p53 genes, but the effects of these mutations on the intrinsic properties of p53 are largely unknown. In the present study, we tested the effect of p53 mutations on DNA-binding. Each of 15 different mutant p53 gene products derived from human tumors or mouse transformants bound calf thymus DNA more weakly than did wild-type products. A significant subset of mutant proteins were also found to be underphosphorylated compared to the wild-type protein when produced in a reticulocyte lysate system, but this did not appear to explain the pattern of alterations of DNA-binding. The tested mutations were dispersed over several regions of the p53 gene and included representatives of all four of the evolutionarily conserved domains that are the known 'hot spots' for p53 mutation. The results suggest common pathways by which these various mutations affect the normal function of p53.

DNA↗
PubMed1990

Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis.

The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.

Amino Acid Sequence↗
PubMed1990

Subcellular distribution of the p53 protein during the cell cycle of Balb/c 3T3 cells.

The expression of p53, a transformation associated protein, has been found to be regulated during the cell cycle. We show here that the subcellular localization of p53 varies throughout the cell cycle. In growth stimulated Balb/c 3T3 cells, p53 is produced at elevated levels and the newly synthesized protein accumulates in the cytoplasm during the G1 phase. Around the beginning of the S phase, p53 accumulates in the cell nucleus, where it stays for about 3 h. Following this initial step of DNA synthesis, p53 is no longer found in the nuclear compartment, but rather accumulates in the cytoplasm. This modulation in the subcellular localization of p53 suggests that the protein is spatially regulated during cell cycle.

Animals↗
PubMed1990

Chromosomal mapping of the murine c-abl proto-oncogene by in situ hybridization.

Deletion and rearrangement of chromosome 2 were shown to be major cytogenetic characteristics of radiation-induced murine myeloid leukemias. Analysis of the localization of the murine protooncogene c-abl, previously assigned by Goff et al. to chromosome 2, was done using the in situ hybridization method. The c-abl was located close to the centromere, within bands 2A-2B. This site does not correspond to the common characteristic deleted segments (2C-2D) predominantly observed in radiation induced murine myeloid leukemias.

Animals↗
PubMed1989

Rearrangements in the p53 gene in Philadelphia chromosome positive chronic myelogenous leukemia.

Molecular structural analysis of the p53 gene in patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) indicates a significant incidence of gene rearrangements in patients at either accelerated phase or blastic crisis. Southern blot analysis of genomic DNA hybridizing with either genomic or cDNA p53 specific probes indicated that 30% of the CML patients at blastic crisis phase exhibited rearrangements, mostly mapping downstream to the first non-coding exon. This is compatible with the observation that the progression of CML from the chronic to the acute phase involves frequent aberrations in chromosome 17, to which the p53 oncogene has been mapped. Therefore, we suggest that one of the pathways of development of CML to the acute phase is associated with aberrations in the p53 nuclear oncogene.

Blast Crisis↗
PubMed1989

Induced expression from the Moloney murine leukemia virus long terminal repeat during differentiation of human myeloid cells is mediated through its transcriptional enhancer.

Transcription from the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) is inhibited in murine stem cells and induced during maturation of these cells. We have investigated whether alterations in the activity of this viral regulatory element also occur during differentiation of human myeloid leukemia cells. The Mo-MuLV LTR and the simian virus 40 (SV40) early promoter were introduced into HL-60 promyelocytes on Epstein-Barr virus-derived chloramphenicol acetyltransferase expression vectors. When these cells were induced to terminally differentiate, transcription from the Mo-MuLV LTR was induced approximately 10-fold. Expression from the SV40 promoter remained constant during differentiation of these cells. Replacing the SV40 transcriptional enhancer with the Mo-MuLV LTR transcriptional enhancer rendered the SV40 promoter inducible during differentiation. We conclude that sequences within the transcriptional enhancer of the Mo-MuLV LTR contain cis-acting elements responsible for induction of gene expression during differentiation of human myeloid cells.

Cell Differentiation↗
PubMed1989

Two promoters that map to 5'-sequences of the human p53 gene are differentially regulated during terminal differentiation of human myeloid leukemic cells.

p53 is overexpressed in many transformed cells and expression of the gene is known to alter during terminal differentiation of cells in culture. Through analysis of recombinant vectors expressing the chloramphenicol acetyl transferase (CAT) gene we found that two promoters map to the 5'-portion of the human p53 oncogene. One promoter, p53p1, maps upstream of the non-coding first exon and the second, p53p2, maps within the first intron. By primer extension analysis of cellular RNA from a number of human cell lines, we found that p53p2 is a functional promoter in vivo. In order to test whether differential regulation of these promoters may be correlated with the control of expression of the p53 gene during differentiation, we have measured the activity of the two promoters by their ability to direct expression of the CAT gene during terminal differentiation of the human promyelocytic leukemia cell line HL-60. HL-60 cells stably harboring Epstein-Barr virus-derived recombinant plasmids that express the CAT gene from either p53p1 or p53p2 were induced to undergo terminal differentiation by a variety of chemical inducers to either granulocytes or monocytes and expression of the CAT gene was measured. The results indicate that while expression of p53p1 remained constant, expression from p53p2 was induced 5- to 10-fold during differentiation of these cells to either granulocytes or monocytes. Similarly, the endogenous p53p2 was found to be induced in HL-60 cells undergoing differentiation. Although the product of the p53p2 initiated transcript has not yet been characterized these results indicate that altered regulation of these two promoters may be important in modulating the expression of mRNA from this gene during terminal differentiation.

Cell Cycle↗
PubMed1988

Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1.

To gain insight into how transcription of the human p53 oncogene is controlled, we characterized the regulatory regions of the gene. A 3.8-kilobase-pair (kbp) EcoRI restriction fragment encompassing the 5' end of the human p53 gene, as well as subfragments generated by restriction digests, was cloned upstream of the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and CAT activity was assayed in extracts of transfected cells. Two types of CAT vectors were used: Epstein-Barr virus oriP-derived constructs that were stably introduced into the human cell lines K562, Raji, and HL-60, and pSV0-CAT-derived constructs that were transiently introduced into the monkey cell line COS. By this approach we have identified two promoters for the human p53 gene. One promoter, p53P1, is located 100-250 bp upstream of the 218-bp noncoding first exon; a second, stronger promoter, p53P2, maps within the first intron. CAT activity and expression of CAT RNA indicate that p53P2 functions up to 50-fold more efficiently than p53P1. We conclude that the expression of the human p53 gene may be controlled by two promoters and that differential regulation of these promoters may play an important role in the altered expression of the gene in both normal and transformed cells.

Acetyltransferases↗
PubMed1988

p53 increases experimental metastatic capacity of murine carcinoma cells.

Transfection of a cloned p53 gene into a murine bladder carcinoma cell with a low metastatic capacity led to elevated levels of p53 protein in clonal transfectants. After intravenous inoculation into syngeneic mice, p53-transfected clones showed significantly increased metastatic potential in comparison with control transfectants. The observed change did not seem to be due to a change in growth potential per se since the cell lines showed similar growth properties in vitro.

Animals↗