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

1985 ◽  
Vol 5 (1) ◽  
pp. 127-132 ◽  
Author(s):  
D Wolf ◽  
N Harris ◽  
N Goldfinger ◽  
V Rotter
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cells ◽  
Cdna Clone ◽  
P53 Protein ◽  
P53 Gene ◽  
Full Length ◽  
Antigenic Determinants ◽  
Entire Group ◽  
P53 Expression ◽  
P53 Antibodies

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.

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

1985 ◽  
Vol 5 (1) ◽  
pp. 127-132
Author(s):  
D Wolf ◽  
N Harris ◽  
N Goldfinger ◽  
V Rotter
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cells ◽  
Cdna Clone ◽  
P53 Protein ◽  
P53 Gene ◽  
Full Length ◽  
Antigenic Determinants ◽  
Entire Group ◽  
P53 Expression ◽  
P53 Antibodies

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.

Immunologically distinct p53 molecules generated by alternative splicing

1986 ◽  
Vol 6 (9) ◽  
pp. 3232-3239
Author(s):  
N Arai ◽  
D Nomura ◽  
K Yokota ◽  
D Wolf ◽  
E Brill ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cdna Clone ◽  
P53 Protein ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Cdna Clones ◽  
P53 Expression ◽  
Mrna Species

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.

scholarly journals Immunologically distinct p53 molecules generated by alternative splicing.

1986 ◽  
Vol 6 (9) ◽  
pp. 3232-3239 ◽  
Author(s):  
N Arai ◽  
D Nomura ◽  
K Yokota ◽  
D Wolf ◽  
E Brill ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cdna Clone ◽  
P53 Protein ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Cdna Clones ◽  
P53 Expression ◽  
Mrna Species

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.

scholarly journals Tumor Eradication by Wild-type p53-specific Cytotoxic T Lymphocytes

1997 ◽  
Vol 186 (5) ◽  
pp. 695-704 ◽  
Author(s):  
Michel P.M. Vierboom ◽  
Hans W. Nijman ◽  
Rienk Offringa ◽  
Ellen I.H. van der Voort ◽  
Thorbald van Hall ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Tumor Cells ◽  
Cytotoxic T Lymphocytes ◽  
Normal Tissue ◽  
P53 Protein ◽  
P53 Gene ◽  
Wild Type ◽  
Wild Type P53 ◽  
Tumor Outgrowth ◽  
Tumor Eradication

The tumor suppressor protein p53 is overexpressed in close to 50% of all human malignancies. The p53 protein is therefore an attractive target for immunotherapy. Cytotoxic T lymphocytes (CTLs) recognizing a murine wild-type p53 peptide, presented by the major histocompatibility complex class I molecule H-2Kb, were generated by immunizing p53 gene deficient (p53 −/−) C57BL/6 mice with syngeneic p53-overexpressing tumor cells. Adoptive transfer of these CTLs into tumor-bearing p53 +/+ nude mice caused complete and permanent tumor eradication. Importantly, this occurred in the absence of any demonstrable damage to normal tissue. When transferred into p53 +/+ immunocompetent C57BL/6 mice, the CTLs persisted for weeks in the absence of immunopathology and were capable of preventing tumor outgrowth. Wild-type p53-specific CTLs can apparently discriminate between p53-overexpressing tumor cells and normal tissue, indicating that widely expressed autologous molecules such as p53 can serve as a target for CTL-mediated immunotherapy of tumors.

scholarly journals p53 Protein Detected By Immunohistochemical Staining is Not Always Mutant

1993 ◽  
Vol 11 (5-6) ◽  
pp. 239-250 ◽  
Author(s):  
Catriona Macgeoch ◽  
Diana M. Barnes ◽  
Julia A. Newton ◽  
Shehla Mohammed ◽  
Shirley V. Hodgson ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Direct Sequencing ◽  
P53 Protein ◽  
Pcr Amplification ◽  
P53 Gene ◽  
Tumour Suppressor Gene ◽  
Nuclear Staining ◽  
Breast Carcinomas ◽  
Direct Dna Sequencing ◽  
P53 Antibodies

The expression of the tumour suppressor gene p53 was analyzed in a variety of human solid tumours by immunohistochemistry and direct DNA sequencing. Positive nuclear staining using a panel of anti-p53 antibodies was used to select tumours for further genetic analysis. Using PCR amplification followed by immobilization onto magnetic beads and direct sequencing, we sequenced exons 5-9 of the p53 gene fro m 9 melanomas, 8 nasopharyngeal carcinomas, 16 sporadic breast carcinomas and 11 patients from familial breast cancer families. No sequence alterations of the p53 gene were detected in either the melanoma or nasopharyngeal tumours and only 19% of the primary breast carcinomas showed a variant band indicative of a mutation. Our results indicate firstly that p53 mutations are not generally involved in the tumour types studied and secondly the data emphasize the disparity encountered when attempting to correlate p53 immunohistochemical positivity with mutations within the p53 gene.

scholarly journals Mutation of the p53 gene in human acute myelogenous leukemia

Blood ◽  
1991 ◽  
Vol 77 (7) ◽  
pp. 1500-1507 ◽  
Author(s):  
JM Slingerland ◽  
MD Minden ◽  
S Benchimol
Keyword(s):  
Acute Myelogenous Leukemia ◽  
P53 Protein ◽  
Point Mutations ◽  
P53 Gene ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
P53 Expression ◽  
Coding Sequence ◽  
Acute Myelogenous ◽  
Blast Cells

Abstract Heterogeneity of p53 protein expression is seen in blast cells of patients with acute myelogenous leukemia (AML). p53 protein is detected in the blasts of certain AML patients but not in others. We have identified p53 protein variants with abnormal mobility on gel electrophoresis and/or prolonged half-life (t 1/2). We have sequenced the p53 coding sequence from primary blast cells of five AML patients and from the AML cell line (OCIM2). In OCIM2, a point mutation in codon 274 was identified that changes a valine residue to aspartic acid. A wild type p53 allele was not detected in these cells. Two point mutations (codon 135, cysteine to serine; codon 246, methionine to valine) were identified in cDNA from blasts of one AML patient. Both mutations were present in blast colonies grown from single blast progenitor cells, indicating that individual leukemia cells had sustained mutation of both p53 alleles. The cDNAs sequenced from blast samples of four other patients, including one with prolonged p53 protein t 1/2 and one with no detectable p53 protein, were fully wild type. Thus, the heterogeneity of p53 expression cannot be explained in all cases by genetic change in the p53 coding sequence. The prolonged t 1/2 of p53 protein seen in some AML blasts may therefore reflect changes not inherent to p53. A model is proposed in which mutational inactivation of p53, although not required for the evolution of neoplasia, would confer a selective advantage, favoring clonal outgrowth during disease progression.

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

1985 ◽  
Vol 5 (8) ◽  
pp. 1887-1893 ◽  
Author(s):  
D Wolf ◽  
Z Laver-Rudich ◽  
V Rotter
Keyword(s):  
Cdna Clone ◽  
P53 Protein ◽  
Cdna Probe ◽  
P53 Gene ◽  
Heteroduplex Analysis ◽  
Cdna Clones ◽  
Free System ◽  
Human Cdna ◽  
Mrna Molecule

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.

Effects of Bcl-2, Bax and P53 Gene Protein Expressions in Gastric Cancer Tissue on the Apoptosis of Cancer Cells

2013 ◽  
Vol 423-426 ◽  
pp. 358-361
Author(s):  
Lei Zhang ◽  
Da Peng Li ◽  
Guan Nan Lu
Keyword(s):  
Gastric Cancer ◽  
P53 Protein ◽  
P53 Gene ◽  
Gastric Cancer Tissue ◽  
Normal Lung ◽  
Cancer Tissue ◽  
P53 Expression ◽  
Bax Protein ◽  
Significant Difference ◽  
Positive Rate

Objective: To investigate the expression of p53 gene in gastric cancer tissue and its correlation with bcl-2 and bax protein expression in relationship and discuss the significance of their correlation in clinic. Methods: Pathological specimens from 100 gastric cancer patients with complete medical data and 24 normal lung tissue specimens were selected. Immunohistochemical streptavidin-biotin-peroxidase assay was used to detect the expression of bcl-2, bax and p53 protein. Results: p53 expression was positively correlated with bcl-2 expression (Pearson's R =0.491, P <0.05). The positive expression of p53 was no significantly correlated with bax expression (P> 0.05). The positive rate of p53 expression in the well-differentiated gastric cancer tissues was 17.6%, the positive rate in the differentiated tissues was 90.9%, the positive rate in the poorly differentiated tissues was 72.7%, and There was a significant difference in the expression of p53 in the three tissues (P <0.05). The expression of p53 in the TNM staging showed that the positive rate in stage Iand IIwas 27.3%, the positive rate in stage III and stage IVwas 89.3%, the difference between them was significant (P <0.05). Conclusions: The expression of p53 gene has an obvious clinical significance for the assessment of degree of malignancy and the prognosis of gastric cancer. Bcl-2 and p53 gene expression could jointly promote the development of gastric cancer in a synergistic way by acting on different stages of apoptosis, or bcl-2s participating in p53-induced mitochondria-mediated apoptosis signaling pathways.

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies

1984 ◽  
Vol 4 (2) ◽  
pp. 276-281
Author(s):  
W E Mercer ◽  
C Avignolo ◽  
R Baserga
Keyword(s):  
Cell Proliferation ◽  
Monoclonal Antibodies ◽  
Mammalian Cells ◽  
P53 Protein ◽  
S Phase ◽  
Human Cells ◽  
Antigenic Determinants ◽  
3T3 Cells ◽  
Species Specific

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.

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

1985 ◽  
Vol 5 (8) ◽  
pp. 1887-1893
Author(s):  
D Wolf ◽  
Z Laver-Rudich ◽  
V Rotter
Keyword(s):  
Cdna Clone ◽  
P53 Protein ◽  
Cdna Probe ◽  
P53 Gene ◽  
Heteroduplex Analysis ◽  
Cdna Clones ◽  
Free System ◽  
Human Cdna ◽  
Mrna Molecule

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.

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