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S Gamou

Publications and source records attributed to S Gamou.

At least 37 records · Page 2Linked to original sources
PubMed1993

Further analyses of epitopes for human monoclonal anti-basement membrane zone antibodies produced by stable human hybridoma cell lines constructed with Epstein-Barr virus transformants.

We previously established Epstein-Barr virus (EBV)-transformed bullous pemphigoid (BP) patient lymphoblastoid cell lines, which produced human monoclonal anti-basement membrane zone antibodies. In the present study, we established two independent human-human hybridomas by fusion of these EBV transformants with a human B-cell line. These hybridomas, designated 5E-HY-4B and 10D-HY-8B, were very stable and showed a high yield of monoclonal antibody (MoAb) secretion. Each cell line was tetraploid and showed combined rearranged segments of immunoglobulin heavy-chain gene derived from both an EBV transformant and a parent cell. Immunoblot analysis showed that the 5E-HY-4B MoAb recognized the 230-kDa BP antigen but that the 10D-HY-8B MoAb did not show any reactivity. In contrast, both MoAbs precipitated the 230-kDa BP antigen with immunoprecipitation. These results indicate that the two MoAbs reacted with different epitopes on the 230-kDa BP antigen: a continuous epitope for the 5E-HY-4B MoAb and a conformation-dependent epitope for the 10D-HY-8B MoAb. This speculation was confirmed at the molecular level by the result that the fusion protein produced by a partial cDNA for the 230-kDa mouse BP antigen reacted with the 5E-HY-4B MoAb but not with the 10D-HY-8B MoAb. Furthermore, the study of the reactivity with fusion proteins of a series of deleted clones restricted the epitope for the 5E-HY-4B MoAb within the region with 114 amino acid residues in the C-terminal domain of the 230-kDa BP antigen.

Antibodies, Monoclonal↗
PubMed1992

[Epidermal growth factor and its receptor: the structure and function].

Epidermal growth factor (EGF) binds to the specific membrane receptor and subsequently activates the signal transduction pathway through intrinsic tyrosine kinase activity. To elucidate the mechanism in which structural alteration of the EGF may affect functional properties including its receptor binding ability, site-directed mutagenesis has been employed. The functional significance of structural characteristics of the EGF receptor has been studied also by mutant receptor constructs in the transfected cells. Recent progress on the studies of the EGF and EGF receptor interaction are reviewed.

Amino Acid Sequence↗
PubMed1991

Linkage analysis of affective disorder using DNA markers on chromosomes 11 and X.

We have investigated two pedigrees in an attempt to detect the putative linkages between affective disorder and c-Ha-ras-1 oncogene and the insulin gene on chromosome 11, or hypoxanthine phosphoribosyltransferase (HPRT) on X chromosome. The linkage between affective disorders and the markers on chromosomes 11 and X was ruled out with the assumption of no recombination.

Bipolar Disorder↗
PubMed1990

Regulation of the epidermal growth factor receptor gene expression in a morphological variant isolated from an epidermal growth factor receptor-deficient small cell lung carcinoma cell line.

Most cell lines derived from small cell lung carcinoma grow in an anchorage-independent manner; they neither possess epidermal growth factor binding activity nor express epidermal growth factor receptor (EGFR) mRNA. A variant AD320, which grew in an anchorage-dependent manner with altered morphology, was isolated from the small cell lung carcinoma cell line Lu134 by treatment with the demethylating agent 5-azacytidine. The analysis, using methylation-sensitive restriction enzymes, revealed that the methylation pattern was altered only in the structural region of the EGFR gene; EGFR mRNA and epidermal growth factor binding activity could be detected in the variant. In addition, drastic changes in gene expression including a decrease of creatine kinase B mRNA and an increase of c-myc mRNA were observed. The EGFR in the variant appeared to be an active part of the transmembrane signaling machinery since c-fos and c-jun mRNA accumulated after epidermal growth factor treatment, followed by EGFR and c-myc mRNA accumulation. A potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, also induced EGFR mRNA. Thus, the inducible regulatory mechanism for the EGFR gene was activated in the variant even though the EGFR gene was constitutively expressed.

Blotting, Northern↗
PubMed1989

Unique chromosomal location of amplified EGF receptor genes in EGF receptor-hyperproducing tumor cell line NA.

The epidermal growth factor receptor (EGFR) gene was analyzed by in situ hybridization using a squamous cell carcinoma line NA, which has high numbers of EGF receptors and carries a 20-fold amplification of EGFR genes. NA cells are pseudotriploid (mode of chromosome number is 69) and have three copies of an apparently normal chromosome 7 together with several aberrant chromosomes. Strong hybridization signals were observed in the abnormal banding region of one of the aberrant chromosome, MH1, which has no structural homology to chromosome 7. This MH1 chromosome was lost in NA-derived variant lines that possess reduced numbers of EGF receptors. These results are in contrast to previous findings that EGFR gene amplification is associated with structural alterations of the short arm of chromosome 7 and provide new evidence in regard to the location of the amplified EGFR gene in tumor cells.

Cell Line↗
PubMed1989

Subcellular localization of the EGF receptor maturation process.

The glycosylation and the processing of the epidermal growth factor (EGF) receptor are suggested to play a crucial role(s) in the activation of ligand binding activity. To examine whether the receptor acquires EGF binding activity in the endoplasmic reticulum (ER) or in the Golgi complex, we carried out parallel kinetic analysis of the EGF binding activity and the intracellular transport of the newly synthesized receptor by immunoprecipitation with the anti-EGF receptor antibody B4G7 using the EGF receptor hyperproducing cell line NA. The kinetic analysis revealed that a receptor capable of binding EGF appeared after 30 to 60 min labeling with [35S]methionine. Pulse-chase experiments also indicated that the receptor capable of binding EGF appeared after a 30-min pulse with a 30-min chase. Subcellular fractionation analysis indicated that the newly synthesized receptor was present in the Golgi complex after labeling with [35S]methionine for 30 min. After a 30-min chase, the Mr 170K receptor appeared in the Golgi complex and plasma membrane. Thus, these results together indicated that after a 30-min pulse incubation a fraction of the EGF receptors have been transported from the ER to the Golgi complex; however, the receptor is unable to bind EGF. Although the EGF receptor appeared on the cell surface after a 30-min pulse with a 30-min chase, only half of the receptors are capable of binding EGF. Therefore, the EGF receptor acquires ligand binding activity at a late stage of the maturation process, most likely in the Golgi complex.

Animals↗
PubMed1989

Lung cancer cells often express high levels of protein kinase C activity.

We analyzed protein kinase C (PKC) activity in twenty-two tumor cell lines derived from lung, pancreas, stomach, tongue and vulva, and found that lung cancer cells often (9 out of 13) exhibit significantly higher PKC activity than other types of cancer cells. The PKC in these lung cancer cells was separated into one major and one minor peaks by a Mono Q column chromatography. The PKC in the major peak had an absolute requirement for Ca2+, phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate (TPA), as expected. However, the PKC in the minor peak did not require TPA for its activation. Hydroxyapatite column chromatography revealed that the PKC in the major peak is type III. These results indicate that in lung cancer cells type III PKC activity is often elevated compared to other types of cancer cells. The growth of many lung cancer cell lines was inhibited by TPA.

Animals↗
PubMed1988

Two independent mechanisms for escaping epidermal growth factor-mediated growth inhibition in epidermal growth factor receptor-hyperproducing human tumor cells.

Human squamous cell carcinoma cell lines often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGF. To investigate the mechanism of EGF-mediated inhibition of cell growth, variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGF receptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant isolated from Ca9-22 cells, CER-1, grew without being affected by EGF. CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicated an increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independent mechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.

Carcinoma, Squamous Cell↗
PubMed1988

Glycosylation of the epidermal growth factor receptor and its relationship to membrane transport and ligand binding.

Epidermal growth factor (EGF) receptor biosynthesis was examined in an oral squamous cell carcinoma line, NA, which overproduces the receptor to an even greater extent than the widely studied A431 cells. The EGF receptor of NA cells synthesized in the presence of tunicamycin had an apparent molecular weight of 130,000. The nascent protein in untreated cells was cotranslationally glycosylated to Mr 160,000 and further processed to Mr 170,000. The endo-beta-N-acetylglucosaminidase H (Endo H) digestion analysis revealed the presence of high mannose type oligosaccharide on the Mr 170,000 mature receptor. We extended the analysis by correlating the biosynthesis with the acquisition of binding activity. The unglycosylated Mr 130,000 receptor and the Mr 160,000 receptor seen after pulse-labeling had no EGF binding activity, whereas the Mr 160,000 receptor seen after chase-incubation and the Mr 170,000 receptor had binding activity. Thus, not only glycosylation but also some oligosaccharide processing is apparently necessary for the EGF binding. Treatment with processing inhibitors, such as monensin, swainsonine and 1-deoxynojirimycin, affected neither receptor transport to the plasma membrane nor binding activity. Inhibition by 1-deoxynojirimycin is thought to be incomplete since the surface receptor in treated cells had the same molecular weight as that in control cells. An Mr 160,000 receptor without binding activity accumulated in the intracellular fraction in the presence of brefeldin A, an inhibitor of intracellular transport. Thus, the EGF binding activity is thought to be acquired after the brefeldin A-sensitive process but prior to the swainsonine-sensitive mannose removal in NA cells.

Acetylglucosaminidase↗
PubMed1988

Methylation status of epidermal growth factor receptor gene in lung carcinoma cells.

The methylation status of the epidermal growth factor receptor (EGFR) gene was compared in cell lines from four major types of lung carcinoma, small cell lung carcinoma (SCLC), large cell lung carcinoma, squamous cell carcinoma and adenocarcinoma, in order to examine whether DNA methylation is responsible for the suppression of EGFR gene in SCLC cells. Southern blot analysis revealed that the structural region of the EGFR gene is methylated to various degrees regardless of the expression of EGF receptor on the surface. An 8-kilobase EcoRI fragment which contains the EGFR gene promoter region is readily digested with various methylation-sensitive restriction enzymes in all types of cells, indicating that the EGFR gene 5' region is barely methylated. Thus, a mechanism other than DNA methylation appears to control EGFR gene expression and the lack of EGFR gene expression in SCLC cells may be caused by a paucity of some transcription regulatory factor(s).

Adenocarcinoma↗
PubMed1988

Biosynthesis of the epidermal growth factor receptor in human squamous cell carcinoma lines: secretion of the truncated receptor is not common to epidermal growth factor receptor-hyperproducing cells.

The biosynthesis of the EGF receptor was examined in the epidermoid carcinoma cell line A431 and five novel cell lines from human squamous cell carcinomas possessing high numbers of EGF receptors. Newly synthesized EGF receptors were visualized by labeling with [35S]methionine and immunoprecipitation with a monoclonal anti-EGF receptor antibody. In addition, the processing of the EGF receptor and its intracellular transport was analyzed by distinguishing cell surface receptors from intracellular receptors and by treating cells with inhibitors such as tunicamycin, monensin and brefeldin A. These analyses revealed that in all the tumor cell lines the EGF receptor is synthesized as a glycosylated protein of Mr 160,000 which is converted to the receptor of Mr 170,000 through posttranslational glycosylation. The receptors of Mr 160,000 and 170,000 appeared to possess high mannose type oligosaccharide chains because endoglycosidase H treatment reduced their molecular weights by approximately 30,000. A431 was the only tumor cell line studied that secreted the truncated EGF receptor of Mr 110,000. In A431 cells, the truncated EGF receptor was generated from a protein of Mr 60,000 through tunicamycin- and monensin-sensitive glycosylation. A431 cells treated with monensin secreted the truncated receptor as a Mr 95,000 form.

Acetylglucosaminidase↗
PubMed1987

Molecular evidence for the lack of epidermal growth factor receptor gene expression in small cell lung carcinoma cells.

It has been shown that none of the small cell lung carcinoma (SCLC) cell lines possess epidermal growth factor (EGF) binding activity on their surface. We have examined several SCLC cell lines for the possibility that they may have EGF receptors but that the receptors are masked by an EGF-like protein factor(s), which may be produced by an autocrine mechanism. No evidence, however, was found for the production of such factors. We then used an EGF receptor complementary DNA to determine the state of the EGF receptor gene by Southern blot analysis. The receptor gene appears to be present in these cells in an intact, unrearranged form. These cells, however, were found to lack detectable levels of EGF receptor mRNA, suggesting a possible reason for the absence of EGF receptors on the cell surface. Furthermore, karyotype analysis revealed that SCLC cell lines Lu134 and H69 contained a morphologically normal chromosome 7, which carries the EGF receptor gene. Also, these SCLC cells contained the apparently normal chromosome 3 and exhibited the presence of c-raf-1 gene in an unrearranged form. Thus, the previously noted partial deletion of chromosome 3 is not necessarily common to the SCLC cells. Instead, the lack of EGF receptor is frequently found in SCLC cell lines and is distinct from the other types of lung cancer. We postulate that SCLC cells have some active regulatory mechanism which prevents the expression of EGF receptor gene.

Carcinoma, Small Cell↗
PubMed1987

Change in metabolic turnover is an alternate mechanism increasing cell surface epidermal growth factor receptor levels in tumor cells.

The biosynthesis and metabolic turnover of the epidermal growth factor (EGF) receptor was examined in a human pancreatic carcinoma cell line, UCVA-1. This cell line has been shown to possess a much higher level of EGF receptors than is expected solely from receptor gene/mRNA dosage. Analysis of the biosynthesis using metabolic labeling, immunological quantitation, and inhibitor treatment revealed that the naked EGF receptor in UCVA-1 cells is a protein of Mr 130,000 that is matured consecutively as a Mr 160,000 and 170,000 glycoprotein through post-translational glycosylation. Analysis of the metabolic turnover using pulse-chase labeling and inhibitor treatment revealed that the rate of EGF receptor synthesis in UCVA-1 cells was similar to that in two squamous cell carcinoma cell lines, NA and Ca9-22, which also have high numbers of EGF receptors, but because of gene amplification. In contrast, the rate of receptor degradation in UCVA-1 cells was significantly slower than in the other two cell lines. These results suggest that the retarded metabolic turnover may constitute a unique mechanism for elevating cell surface EGF receptor levels in some tumor cells independent of gene amplification.

Carcinoma, Squamous Cell↗
PubMed1986

Adipocyte differentiation of 3T3-L1 cells in serum-free hormone-supplemented media: effects of insulin and dihydroteleocidin B.

We previously established a serum-free hormone-supplemented medium for the induction of adipocyte differentiation of 3T3-L1 cells (Gamou, S. and N. Shimizu. in "Growth and Differentiation of Cells in Defined Environment", H. Murakami et al., ed., Kodansha/Springer-Verlag, pp. 173-178, 1985). Under those conditions the stage of the cell's commitment to adipocyte differentiation was separated from the stage of expression of the adipocyte phenotype. In the current study, the relationship between cell division of the growth-arrested 3T3-L1 cells and their entry into the differentiation program was examined by autoradiography at the individual cell level. It was found that cells treated with the inducers dexamethasone and 1-methyl-3-isobutylxanthine went through DNA synthesis (S phase) prior to lipid accumulation and that insulin enhanced this differentiation process. Under these serum-free hormone-supplemented conditions, the tumor promoter dihydroteleocidin B was found to be a strong inhibitor of adipocyte differentiation.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Molecular mechanisms involved in increasing epidermal growth factor receptor levels on the cell surface.

Various tissues and cell lines were analyzed to determine factors which regulate the epidermal growth factor (EGF) receptor level on the cell surface. At least 3 mechanisms are postulated to potentially control the EGF receptor level: gene amplification, receptor mRNA levels, and receptor half-life. The abnormal activation of any or all of these mechanisms may contribute to the high level of EGF receptors observed in squamous cell carcinomas.

Carcinoma, Squamous Cell↗
PubMed1985

Relationship between production of epidermal growth factor receptors, gene amplification, and chromosome 7 translocation in variant A431 cells.

Synthesis of the epidermal growth factor (EGF) receptor has been analyzed in a series of variant A431 human epidermoid carcinoma cell clones reported to contain different amounts of EGF binding sites. The amount of EGF receptor protein, quantitated by immunoaffinity chromatography, and EGF receptor mRNA, quantitated by cDNA hybridization, were closely correlated to the extent of EGF receptor gene amplification. This correlation existed in variants selected for reduced EGF receptors and in revertants from those variants with increased EGF receptors. There was also a correlation between the frequency of translocation of chromosome 7, containing the EGF receptor gene, and EGF receptor protein. These results support gene amplification as the mechanism enhancing A431 cell EGF receptor protein and determining growth responses.

Carcinoma, Squamous Cell↗