Correlation between HPV positivity and state of the p53 gene in cervical carcinoma cell lines.

Human papillomavirus (HPV) infection is clearly associated with cervical carcinomas, yet it is also true that there are cervical carcinomas in which HPV DNA is absent. We examined eight established cell lines derived from cervical carcinomas for the presence of mutations of the p53 antioncogene in relation to the presence of HPV DNA sequences. Of these eight cell lines, seven were positive for HPV DNA and the remaining one was negative for HPV DNA. Single-strand conformation polymorphism analyses revealed a point mutation of the p53 gene in the cell line in which HPV DNA was absent. Sequencing analysis revealed a single-base mutation at codon 273 from CGT to CAT(Arg-->His) and immunocytochemical studies provided evidence that the p53 protein was overexpressed in this cell line. Our observations suggest that the loss of normal p53 gene function may be linked to the oncogenesis of cervical carcinoma.

Altered structure and expression of the p53 gene in human neuroepithelial tumors.

The p53 gene, located on chromosome 17p13.1, may be important in the pathogenesis of human neuroepithelial tumors, because it is a tumor suppressor gene and genetic alteration is essential for certain human cells to acquire the neoplastic phenotype. The structure and expression of the p53 gene were investigated in cultured human glioma cells and biopsied specimens of neuroepithelial tumors. Immunocytochemical examination of p53 gene expression revealed positive nuclear staining in six of seven glioma cell lines tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis demonstrated unequivocal heterogeneity of migration rate in p53 bands. Pulse-chase analysis clearly showed an increased half-life of p53 in cultured human glioma cells. These abnormalities are presumably due to genetic alterations in the p53 gene. Nucleotide substitutions in exon 5, 7, or 8 of the p53 gene could be detected by polymerase chain reaction-single strand conformational polymorphic analysis in four of seven (57%) human glioma cell lines, and nine of 29 (31%) biopsied specimens of neuroepithelial tumors examined. The present results indicate that genetic alterations in the p53 gene are responsible for the tumorigenesis of at least some human neuroepithelial tumors.

Detection of human papillomavirus genome and analysis of expression of c-myc and Ha-ras oncogenes in invasive cervical carcinomas.

Invasive carcinomas of the uterine cervix of 38 patients were examined for the presence of human papillomavirus (HPV) genomes and for the state of the c-myc and Ha-ras oncogenes. A combination of Southern blot hybridization and polymerase chain reaction revealed the presence of the genome of HPV type 16 in 17 tumors (45%), that of HPV type 18 in 3 tumors (8%), and that of unknown types in 16 others (42%), while no viral DNA sequences were detected in 2 tumors. Of the 38 tumors, c-myc amplification was found in only 1 tumor, while there was no Ha-ras amplification. Overexpression of the c-myc gene was observed in 15 (44%) of the 34 tumors analyzed, while there was no overexpression of Ha-ras. Of the 23 squamous cell carcinomas analyzed, relapse-free rates at 24 months were 55% in tumors with c-myc overexpression and 100% in case of tumors with no c-myc overexpression, respectively. The results suggest the possibility that activation of the c-myc oncogene is involved in tumor progression.

Suppression of anchorage-independent growth of human glioblastoma cell by major histocompatibility complex class I gene-transfection.

The host's immune system discriminates tumor cells from normal cells by recognizing the major histocompatibility complex (MHC) class I antigen expressed on the tumor cell membrane. However, the role of MHC class I antigen in tumor cells has not yet been clarified. In this study, the influence of MHC class I antigen expression on the tumorigenicity of a human glioblastoma cell line (KMG4) is examined. Barely detectable levels of MHC class I messenger ribonucleic acid were found to express in KMG4 cells by Northern blot analysis using mouse MHC class I (H-2Ld) and human leukocyte antigen (HLA)-B7 genes as probes. The H-2Ld gene connected at the downstream end of murine mammary tumor virus (MMTV)-promoter was cotransfected with the neomycine-resistant gene pSV2-neo into KMG4 cells, and the drug-resistant cells were selected. The KMG4 cells (KMG4-MMTV-Ld), which acquired the MHC class I gene were detected by Northern blot analysis with H-2Ld as the probe, and by immunohistochemistry using the H-2Ld-specific monoclonal antibody. Tumorigenicity, as determined by colony-forming ability in soft agar, was then compared between MHC class I-expressing KMG4-MMTV-Ld and nonexpressing control cells. The MHC class I-expressing cells were found to be deprived of colony-forming ability, indicating that MHC class I antigen could negatively influence the anchorage-independent cell growth of the human glioblastoma cell line KMG4.

Transformation-specific decrease of phosphorylation of 80K protein, a substrate of protein kinase C, in NIH3T3 cells.

Phosphorylation in normal and transformed NIH3T3 cells of the 80K protein, a specific substrate for protein kinase C, was compared by means of two-dimensional gel analysis. We obtained evidence that NIH3T3 cells transformed by the c-raf or H-ras oncogene maintained a decreased level of phosphorylation of the 80K protein, with or without phorbol ester (TPA)-stimulation, at all concentrations of serum tested while normal NIH3T3 cells maintained an elevated level of phosphorylation of the 80K protein. Furthermore, NIH3T3 cells transformed by N-ras, K-ras, src, mos or polyoma middle T antigen exhibited a decreased level of phosphorylation of the 80K protein. These events were confirmed by an analysis of a hormone-inducible H-ras transformant. Thus, phosphorylation of the 80K protein is inversely correlated with cellular transformation.

Changes in the patterns of RNA synthesis in early embryogenesis of Xenopus laevis.

We analyzed the accumulation of newly-synthesized heterogeneous mRNA-like RNA, 4 S RNA, 5 S RNA, snRNAs and rRNA before and after the midblastula transition (MBT) in Xenopus laevis embryogenesis. Based on the kinetics of the labeling, we concluded that the pattern of RNA synthesis in Xenopus embryogenesis changes following at least three characteristically different phases. The first phase is the pre-MBT stage, which is characterized by the synthesis of heterogeneous mRNA-like RNA, accompanied by the synthesis of small amounts of 4 S RNA, 5 S RNA and snRNAs. The second phase is the MBT stage which is characterized by a large activation (about 50-fold increase on a per cell basis) of 4 S RNA synthesis. The third phase is the post-MBT stage which is characterized by the commencement and increase in rRNA synthesis. We assume that RNA polymerases II, III and I are activated in this order in early Xenopus embryogenesis.

[Activated c-raf-1 gene from human stomach cancer].

We previously isolated a novel human transforming sequence from a primary stomach cancer and identified the gene as an activated version of the c-raf-1 gene which is the human homologue of v-raf, a viral oncogene encoding a serine/threonine-specific protein kinase. Of 57 kbp of the human sequence isolated, a region of 40 kbp was found to be the minimum functional unit for the transforming activity, because a cosmid clone harboring this region was capable of inducing foci upon transfection. The size of the transcript of the transforming c-raf-1 gene was estimated to be about 2.8kb. Analyses of cDNA clones of this gene revealed that the gene was generated by substitution of the 5'-sequence (exons 1-5) of the normal c-raf-1 gene with an unrelated human sequence. We identified a region in the genomic clone where the rearrangement had occurred. The rearranged EcoRI fragment was detected in all the primary transformants obtained from two independent transfections, suggesting that the recombination had occurred in the primary cancer. The substituted cDNA sequence is composed of an open reading frame, which joins to exon 6 of the c-raf-1 gene in an in-phase manner. The substituted open reading frame encodes an extremely hydrophobic polypeptide. Thus, the putative product of the transforming gene seems to have a hydrophobic stretch ahead of the ser/thr-protein kinase domain of the c-raf-1 gene product. These results suggest that the truncation or replacement of the amino-terminal domain of the c-raf-1 protein leads to constitutive activation of the protein kinase residing in the downstream domain.