Biomedical subjects

D Pim

Publications and source records attributed to D Pim.

27 records · Page 2Linked to original sources

PubMed1991

Loss of HPV-16 E7 dependence in cells transformed by HPV-16 E7 plus EJ-ras correlates with increased c-myc expression.

We have previously shown that continued expression of the HPV-16 E7 gene was required to maintain the transformed phenotype of primary baby rat kidney cells transformed by HPV-16 E7 plus EJ-ras. In this study we aimed to investigate the possible mechanisms by which cells could overcome this continued requirement for E7 expression. Using an inducible system for expression of the E7 gene we were able to generate cell lines no longer dependent on continued E7 expression. All such lines were still fully transformed, maintained inducible E7 expression and showed increased proliferative activity in the presence of HPV-16 E7 gene product. Further analysis has demonstrated a marked increase in the presence of c-myc protein in these cell lines when compared with the parental cells, suggesting a possible mechanism by which E7 dependence can be overcome.

Animals↗

PubMed1988

Comparison of the in vitro transforming activities of human papillomavirus types.

The association of certain human papillomavirus (HPV) types with the majority of human cervical carcinomas suggests a role for the virus in the development of this type of cancer. In this paper, we have examined the transforming properties of several HPV types where the early region genes of the virus are under the control of a strong heterologous promoter and show that major differences exist between the HPV types in their ability to transform primary rat kidney epithelial cells in conjunction with an activated ras oncogene. Those HPV types most commonly found in carcinomas--types 16, 18, 31 and 33--are capable of co-operating with ras to transform primary cells, but those types most commonly found in benign lesions--types 6 and 11--are not. We further demonstrate that the E7 gene of HPV16 by itself is sufficient to co-operate with activated ras to produce transformed cells which are tumorigenic in immunocompetent animals.

Adenocarcinoma↗

PubMed1987

Expression of human papillomavirus type 6 and type 16 capsid proteins in bacteria and their antigenic characterization.

The L1 and L2 capsid proteins encoded by human papillomavirus types 6 and 16 (HPV-6 and HPV-16) have been synthesized in bacteria. Antisera were raised against the HPV-6 L1- and L2-beta-galactosidase fusion proteins and against an HPV-16 L1 C-terminal peptide which was 14 amino acids long. The HPV-16 L1 peptide antibodies have been shown to be highly reactive with the HPV-16 L1-beta-galactosidase protein but not against the equivalent HPV-6 L1-beta-galactosidase fusion protein. The effectiveness of these antibodies was compared with commercially available antibovine papillomavirus type 1 (BPV-1) antibodies and the results demonstrated that the anti-BPV-1 antibodies reacted well against HPV-6 L1-beta-galactosidase but not against HPV-16 L1-beta-galactosidase. In addition, the L2 portion of the HPV-6 L2-beta-galactosidase fusion protein appeared particularly immunogenic, since antibodies raised against this fusion protein were predominantly reactive with the L2 moiety. The HPV-16 L1 peptide antibodies described here will be preferred reagents for the specific detection of HPV-16 capsid antigens, which may be particularly important in early diagnosis of HPV-16 infection.

Antibodies, Viral↗

PubMed1987

Primary structure polymorphism at amino acid residue 72 of human p53.

We analyzed p53 cDNA and genomic clones from a variety of normal and transformed cells. Sequence analysis of these clones revealed that amino acid residue 72 can be an arginine, proline, or cysteine. This single codon difference results in electrophoretically distinct forms of human p53 seen in normal and transformed cells.

Amino Acid Sequence↗

PubMed1987

Alternative splicing of human p53 transcripts.

The human p53 gene is expressed in both normal and transformed human cells as a family of polypeptides. One type of heterogeneity, which results in variation in electrophoretic mobility, is found at amino acid residue 72 (Matlashewski et al, 1987). A second type of heterogeneity is described here which involves an alteration in the sequence of the open reading frame at the 5' end of the p53 mRNA. This heterogeneity is generated by alternative splicing at the acceptor site in intron 2 resulting in an altered 5' end coding region. This study represents the first description of an altered human p53 transcript arising by differential splicing.

Amino Acid Sequence↗

PubMed1986

Analysis of human p53 proteins and mRNA levels in normal and transformed cells.

p53 mRNA and proteins were examined in a variety of human transformed cells and in normal human foreskin fibroblast cells. Both the steady-state and translatable levels of p53 mRNA were the same in normal and transformed human cells. In vitro synthesized p53, programmed by mRNA from normal and transformed human cells, revealed that there was heterogeneity in the primary structure of p53 from these cells. Pulse labeling of cells and immunoprecipitation analysis with a panel of human reactive anti-p53 antibodies demonstrated that the types of p53 synthesized in vitro corresponded to the types made in vivo from SV80 and COLO 320 cells. No p53 was detectable by similar pulse-labeling analysis of HeLa and normal foreskin fibroblast cells. Since it was necessary to use anti-p53 sera from cancer patients to carry out much of the immunoprecipitation analysis in this study we therefore further characterised these sera to determine if they reacted with one or more than one epitope. p53-beta-galactosidase fusion proteins were synthesized in Escherichia coli and used to analyse the anti-p53 antibodies produced by cancer patients. We demonstrate that the antisera contain antibodies directed against epitopes in both the N-terminal and C-terminal regions of the p53 molecule.

Antibody Specificity↗

PubMed1986

The expression of human papillomavirus type 18 E6 protein in bacteria and the production of anti-E6 antibodies.

Human papillomavirus type 18 (HPV-18) has recently been closely linked with human malignant cervical lesions. The early region of the genome of the related bovine papillomavirus (BPV) has been shown to be important for the production of the transformed phenotype. BPV E6 has been shown to be a transforming protein. We report the primary structure of the HPV-18 E6 open reading frame and its predicted amino acid sequence. Both E6 protein and E6-beta-galactosidase fusion protein were synthesized in bacteria. Antisera were raised against the E6-beta-galactosidase fusion protein and against an E6 N-terminal peptide which was 14 amino acids long. We show that these antisera reacted on Western blots against E6 synthesized in bacteria. The HPV E6 antigen and antibodies described here will be useful in understanding HPV expression and its association with human malignancies and may also be diagnostically useful.

Amino Acid Sequence↗

PubMed1984

Isolation and characterization of a human p53 cDNA clone: expression of the human p53 gene.

A cDNA clone for human p53 cellular tumor antigen has been isolated and characterized. This clone contains the complete 3'-untranslated region and most of the open reading frame for the protein. Nucleotide sequence analysis revealed that p53 mRNA contains an Alu repeat in the 3'-untranslated region. Hybridization selection experiments showed this clone was capable of selectively binding p53 mRNA. In vitro translation of SV80 mRNA resulted in the synthesis of two immunoreactive p53 polypeptide species. Northern blot analysis showed that human p53 mRNA was 2.8 kb in length and was present in cell lines containing high and low levels of p53 protein. There appears to be only a single p53 gene in human cells and Southern blot analysis demonstrated no major genomic rearrangements or amplification of the p53 gene in the transformed cell lines examined.

Antigens, Neoplasm↗

PubMed1982

Radioimmunoassay of the cellular protein p53 in mouse and human cell lines.

We have developed quantitative radioimmunological solid phase assays for the host protein p53 from mouse cells and from human cells. The first assay, for mouse p53, depends on having two monoclonal antibodies reacting with different determinants on the p53 molecule. With this assay we have shown that SV40-transformed cells have approximately 100-fold more p53 than untransformed mouse cells and that other transformed cells have intermediate levels. Embryonal carcinoma cell lines have approximately 50-fold less p53 than SV40-transformed cells. This is in contrast to the high levels of incorporation of [35S]methionine into p53 in these cells and indicates that metabolic labelling is not a valid approach for measuring p53 levels. The second assay, for human p53, required a different approach and made use of the anti-p53 antibodies detected in the sera of some breast cancer patients. Human tumour cell lines contained amounts of p53 varying from the high level seen in SV40-transformed human fibroblasts down to less than one hundredth of this amount. Normal human cells showed low levels of p53. The data confirm that many, but not all, human tumour cell lines contain more p53 than normal cells.

Animals↗