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V Rotter

Publications and source records attributed to V Rotter.

At least 109 records · Page 6Linked to original sources
PubMed1988

Meth A fibrosarcoma cells express two transforming mutant p53 species.

Expression plasmids directing the synthesis of various forms of the p53 cellular tumor antigen were compared with respect to their biological activities. All plasmids encoding wild type p53, derived from two different cDNA libraries, had absolutely no detectable activity when assayed for transformation of primary rat embryo fibroblasts in collaboration with Ha-ras. In contrast, p53 variants carrying point mutations in the protein coding region exhibited at least some transforming activity. Most notably, this was true for both types of mutant p53 cDNA clones isolated from Meth A cells. The data indicate that these cells, derived from a chemically-induced tumor, carry two independently mutated p53 alleles, each encoding a transformationally activated protein. This may imply that the mutations in the p53 gene played a role in the development of the Meth A tumor. Finally, cells overexpressing a transfected mutant p53 exhibit a physical complex between this exogenous p53 and its endogenous counterpart, possibly resulting in the stabilization of the latter.

Animals↗
PubMed1987

Inhibition of cell growth mediated by plasmids encoding p53 anti-sense.

p53 is an oncogene product which has been shown to be directly involved in malignant transformation. Furthermore, it has been proposed that this protein plays an essential role in the control of cell proliferation. In the present study, we investigated the involvement of p53 in growth regulation of cells by employing anti-sense RNA methodology to inhibit p53 expression. Transfection with p53-specific constructs spanning the entire mRNA molecule or the 5' region of the gene led to reduced p53 protein synthesis. Clones derived from such transfected cells exhibited a slower rate of DNA synthesis, as assayed by incorporation of [3H]thymidine. In most cases, transfection of plasmids encoding anti-sense RNA eventually brought about the complete cessation of cell proliferation. No such effects were observed in L12 cells, which do not synthesize p53 and therefore do not depend on p53 expression for their growth, thus excluding the possibility of a non-specific toxic effect exerted by the anti-sense p53 RNA. These findings support the notion that p53 is essential for continuous cell proliferation.

Animals↗
PubMed1986

Differential expression of metastasis-associated cell surface glycoproteins and mRNA in a murine large cell lymphoma.

A metastatic variant cell subline of the Abelson virus-transformed murine large lymphoma/lymphosarcoma RAW117 has been selected in vivo ten times for liver colonization. Highly metastatic subline RAW117-H10 forms greater than 200 times as many gross surface liver tumor nodules as the parental line RAW117-P. Analysis of cellular proteins and glycoproteins indicates reduced expression of murine Moloney leukemia virus-associated p15, p30, and gp70, and increased expression of a sialoglycoprotein, gp150, in the highly metastatic H10 cells. Northern analyses of oncogene expression suggested that mRNA of various oncogenes was expressed equally or not expressed in the RAW117 cells of differing metastatic potential. Differential gene expression was examined using a cDNA library of 17,600 clones established from poly A+ mRNA isolated from H10 cells. The cDNA library was screened by the colony hybridization technique using probes made from both RAW117-P and -H10 cells. Approximately 99.5% of these cDNA clones were expressed identically in P and H10 cells. Of the few differentially expressed cDNA clones (approx. 150/17,600), one-half of these were identified as Moloney leukemia virus sequences in a separate probing with a radiolabeled Moloney leukemia virus probe. The remainder of the differentially expressed mRNA detected by colony hybridization of the cDNA library were expressed at higher levels (approx. 1/6) or lower levels (approx. 1/3) in the highly metastatic H10 cells.

Animals↗
PubMed1986

Molecular basis for heterogeneity of the human p53 protein.

The human p53 tumor antigen comprises several physically distinct proteins. Two p53 proteins, separable by polyacrylamide gel electrophoresis, are expressed by the human transformed cell line SV-80. The individual cDNAs which code for these proteins were isolated and constructed into the SP6 transcription vector. The proteins encoded by these clones were identified by in vitro transcription with the SP6 vector and translation in a cell-free system. p53-H-1 and p53-H-19 cDNA clones code for the faster- and slower-migrating p53 protein species, respectively, of SV-80. The in vitro-expressed proteins of p53-H-1 and p53-H-19 had the same antigenic determinants and were structurally indistinguishable from their in vivo counterparts. By expressing defined restricted cDNA fragments in vitro, the region of heterogeneity between the respective cDNAs was located at the 5' end of the cDNAs. Exchanging the 5' fragments of interest and expressing the chimeric clones in vitro confirmed that the DNA heterogeneity was responsible for the difference in the electrophoretic mobility of these proteins. The sequences of the two cDNAs revealed a single base pair difference (G versus C) in the coding region of the clones. This sequence difference resulted in an arginine being coded for in clone p53-H-1 and a proline being coded for at the equivalent position in clone p53-H-19. This variation accounted for the change in the electrophoretic mobility of the individual p53 protein species.

Amino Acid Sequence↗
PubMed1986

Immunologically distinct p53 molecules generated by alternative splicing.

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.

Abelson murine leukemia virus↗
PubMed1986

Expression of p53 in human leukemia and lymphoma.

Analysis of fresh human tumors have indicated that patients with B type lymphoproliferative diseases and the majority of patients with acute lymphoblastic leukemia (ALL) express elevated levels of p53 production. It is suggested that in these human malignancies, p53 may provide a novel tool for monitoring cancer activity. Conversely, p53 is not expressed in acute myeloid leukemias, myeloproliferative diseases, or myeloid leukemic cell lines. Analysis of the p53 gene structure indicated the existence of similar patterns of p53 restriction fragments in producer and nonproducer cells, which suggests that the p53 gene is not altered in the latter. However, in one case of acute promyelocytic leukemia (APL), we have observed a rearrangement in the p53 gene. Karyotype analysis has indicated that these APL cells do not contain the typical 15;17 translocation. In other APL patients who exhibit a 15;17 translocation, we found no genomic changes of the p53, suggesting that the p53 gene, which was recently mapped to the short arm of chromosome 17 in the human, is not structurally related to the typical chromosomal break point found in the long arm of chromosome 17 of APL patients.

Antibodies, Monoclonal↗
PubMed1985

Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells.

The tumor antigen p53 is overproduced in transformed cells of various species, including man. HL-60 is an exceptional human tumor cell line that does not express this protein. Hybridization of polyadenylylated mRNA of these cells with a human p53 cDNA probe (p53-H14), which we cloned, had indicated a total absence of the mature-size (3.0 kilobases) or any aberrant p53 mRNA species. Analysis of the genomic HL-60 DNA indicated that the p53 gene in these cells was significantly altered. Most of the gene was deleted, and the residual p53 sequences of these cells, which show weak homology, mapped to the corresponding 5' region of the p53 gene. In agreement with previously documented results, we found that HL-60 cells have an amplified c-myc gene. We suggest that the deficiency of the p53 protein in HL-60 cells could have been overcome by using an alternative metabolic pathway. The c-myc product is a candidate for such an alternative protein.

Animals↗
PubMed1985

Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.

Animals↗
PubMed1985

In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons.

Cell Line↗
PubMed1984

Reconstitution of p53 expression in a nonproducer Ab-MuLV-transformed cell line by transfection of a functional p53 gene.

L12 are Ab-MuLV-transformed cells that express the abl p120 oncogene product but lack the cellularly encoded p53. The functional p53 gene in these cells has been inactivated by the insertion of Moloney virus-like sequences into the first p53 intron. Transfection of L12 cells with a functional p53 gene, contained in a 16 kb Eco RI genomic cloned fragment gave rise to L12-derived cell lines with novel p53 sequences of various sizes and copy number. A high percentage of L12-derived clones efficiently transcribed p53 mRNA and synthesized the p53 protein. Whereas injection of L12 parental cells into syngeneic mice caused the development of local tumors that later regressed, L12-derived clones that expressed p53 caused lethal tumors in syngeneic mice, thus behaving similarly to other Ab-MuLV-transformed p53-producer cell lines. These results suggest that the expression of p53 is essential for tumor cells to exhibit a fully transformed phenotype, manifested in lethal tumors in syngeneic mice.

Abelson murine leukemia virus↗
PubMed1984

Chromosomal assignment of the murine gene encoding the transformation-related protein p53.

p53 is a transformation-related protein that is encoded by the cellular genome and is synthesized at elevated levels in a wide range of different cell line types and in primary tumors of various species. By using several independently established anti-p53 monoclonal antibodies, it was possible to distinguish between p53 of mouse origin and p53 of Chinese hamster origin. By analysis of a series of mouse X Chinese hamster hybrid cell lines containing various mouse chromosomes, we mapped the p53 gene product to mouse chromosome 11.

Animals↗
PubMed1984

Abelson murine leukemia virus-transformed cells that lack p53 protein synthesis express aberrant p53 mRNA species.

Cells of the Abelson murine leukemia virus-transformed line L12 that lack the p53 protein also lack polyadenylated mRNA capable of directing the synthesis of p53 in a cell-free system. Direct analysis of stable polyadenylated mRNA from a variety of cell lines shows that all p53 producers shared a common mRNA species (2.0 kilobases) which hybridized with a p53-specific cDNA probe. This species, which appears to be the mature, normal-sized p53 mRNA, was totally undetectable in L12 cells, which did not produce p53 in vivo. However, L12 cells contained two major p53-specific mRNA species of a substantially larger size (3.5 and 6.5 kilobases) than the p53-specific mRNA in the p53-producing cells. Genomic DNA analysis uncovered an apparent alteration in the 5' proximal part of only one p53 gene, which is unique to the L12 cell line. It is thus possible that the nonproducer phenotype of L12 cells is due at least in part to an alteration within a p53-specific DNA sequence. These findings define a system in which production of p53 appears to be efficiently regulated at the level of stable mRNA and which can be used to study the mechanisms controlling p53 expression in Abelson murine leukemia virus-transformed cells.

Abelson murine leukemia virus↗
PubMed1984

Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences.

Analysis of Abelson murine leukemia virus-transformed L12 cells which lack the p53 cellular encoded tumor antigen revealed alterations in the p53-specific genomic DNA sequences. The active p53 gene, usually contained in a 16-kilobase EcoRI DNA fragment of p53 producer cells, went through major alterations leading to the appearance of a substantially larger 28.0-kilobase p53-specific EcoRI fragment. Detailed restriction enzyme analysis, with genomic probes spanning throughout the whole active p53 gene, indicated that the L12 p53 altered gene contains all the exons and principal introns of the normal p53 16.0-kilobase gene. However, its structure was interrupted by the integration of a novel DNA segment into the noncoding intervening sequences of the first p53 intron. Analysis of the inserted sequences revealed close homology to Moloney murine leukemia virus. This Moloney leukemia murine virus-like particle resides in a 5' to 3' transcriptional orientation, similar to the p53 gene, permitting the transcription of aberrant fused mRNA molecules detected in these cells.

Animals↗
PubMed1983

The presence of p53 transformation-related protein in Ab-MuLV transformed cells is required for their development into lethal tumors in mice.

p53, a cellular-encoded protein, is synthesized at elevated levels in a wide range of tumor cells. Ab-MuLV-transformed cells expressing both the viral-encoded p120 oncogene and the cellular-encoded p53 display a lethal tumor phenotype in syngeneic mice. L12 is an exceptional Ab-MuLV-transformed cell line that expresses the p120 oncogene and lacks the p53 cellular protein. Injection of L12 cells into syngeneic mice is followed by the development of local tumors that are subsequently rejected. Prolonged treatment of L12 cells with TPA, a tumor cell promoter, gave rise to L12T cells that synthesize the p53 protein and exhibit a lethal tumor phenotype. Comparison of one-dimensional proteolytic partial peptide map of p53 obtained from L12T to that obtained from other Ab-MuLV-transformed cell lines confirmed their identity. These results suggest a correlation between the cellular expression of p53 in Ab-MuLV-transformed cells and their capacity to develop into lethal tumors in syngeneic mice.

Abelson murine leukemia virus↗
PubMed1983

P53 transformation-related protein accumulates in the nucleus of transformed fibroblasts in association with the chromatin and is found in the cytoplasm of non-transformed fibroblasts.

The subcellular localization of the p53 molecule was studied in transformed and non-transformed fibroblasts. A newly established transformed cell line obtained by treating primary embryonic mouse cells in vitro with the chemical carcinogen methylcholanthrene was compared with the embryonic parent fibroblasts. The transformed cells lost the spindle shape characteristic of the parent fibroblasts, acquired an accelerated growth rate, developed into tumors when injected into syngeneic mice and expressed high levels of p53 synthesis estimated by immunoprecipitation of [35S]methionine-labeled cell extracts. The cellular localization of the p53 molecule was studied by immunofluorescent staining of fixed cells with monoclonal antibodies and by immunoprecipitation of [35S]-methionine-labeled p53 from various subcellular fractions. p53 was mainly found in the nucleus of the transformed fibroblast, while in the parent non-transformed primary embryonic cells, p53 was detected in the cytoplasm in a Triton X-100 soluble fraction, and associated with the cytoskeleton. The modulated distribution of p53 was also confirmed by analyzing a wide range of independently established transformed and non-transformed fibroblastic cell lines growing in vitro. The switch from the cytoplasmic localization of p53 in the non-transformed fibroblasts to a chromatin-associated accumulation in the transformed cells suggests a possible mechanism by which this protein may function in the transformed fibroblasts.

Animals↗
PubMed1983

p53, a transformation-related cellular-encoded protein, can be used as a biochemical marker for the detection of primary mouse tumor cells.

p53, a transformation-related cellular-encoded protein, was found to accumulate at high concentration in transformed cell lines. The results presented here show that p53 biosynthesis is also increased in most induced and spontaneous mouse tumors. Judged by the identity in antigenic determinants (estimated by binding to monoclonal antibodies), size, and partial peptide mapping, I conclude that the p53 molecule found in primary tumors is indistinguishable from that in established cell lines. The fact that p53 is found in heterogeneous populations of primary tumors makes it a convenient biochemical diagnostic marker for the detection of primary tumors in mice. It is found in primary tumors as a phosphoprotein, just as it was found previously in established cell lines. On the other hand, the p53 found at low concentration in normal thymocytes is labeled with [35S]methionine but cannot be found in its phosphorylated form.

Animals↗
PubMed1983

Variation in antigenic determinants of p53 transformation-related protein obtained from various species.

p53 is a cellular-encoded transformation-related protein. It is synthesized at elevated levels in tumor cells but has also been detected at low concentrations in several types of nontransformed cells. The p53 of tumor cells is immunogenic and elicits specific antibody production. The antigenic determinants of the p53 protein were studied by specific binding to anti-p53 monoclonal antibodies obtained from the RA3-2C2, PAb122, and PAb421 established hybridoma cell lines, and their conservation was followed in various animal species. We found that whereas mouse p53 efficiently immunoprecipitated with all three anti-p53 monoclonal antibodies, human and rat p53 bound PAb122 and PAb421 but lacked a determinant binding RA3-2C2. The hamster p53 molecule represented a third category, which immunoprecipitated with polyclonal anti-p53 antibodies but failed to bind all three monoclonal antibodies analyzed here. Using these monoclonal antibodies, we detected no variations between p53 found in transformed and p53 found in nontransformed cells, within a given species. The results also showed that RA3-2C2, which recognizes a mouse-specific determinant, binds a site located at a proteolytic digestion fragment of the p53 molecule that differs from that containing PAb122 and PAb421 recognition site(s). p53 is a single protein that can be immunoprecipitated through different antigenic determinants that vary between species.

Animals↗