Biomedical subjects
K Toyoshima
Publications and source records attributed to K Toyoshima.
Structure and function of the erbB gene family.
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Genetic alterations of the c-erbB-2 oncogene occur frequently in tubular adenocarcinoma of the stomach and are often accompanied by amplification of the v-erbA homologue.
We analyzed for alterations of the c-erbB-2 oncogene in 35 human stomach cancers and 8 cell lines derived from human stomach cancer. Amplification of c-erbB-2 was found in approximately 40% (5/13) of the tubular adenocarcinomas of the stomach examined, including 4 of 10 fresh tumors and one of 3 cell lines, but not in other histological types of stomach cancer examined (0/30), including 25 fresh tumors and 5 cell lines. This result strongly suggests that amplification of c-erbB-2 occurs frequently in tubular carcinomas in stomach cancer. Rearrangement of c-erbB-2 was also detected in one tubular adenocarcinoma. The rearranged fragment carried the 3' half, but not the 5' sequence, of the c-erbB-2 gene. Furthermore, one of the cellular homologues of v-erbA was amplified in 3 of 4 fresh tumors carrying the amplified c-erbB-2 gene. Both c-erbB-2 and the v-erbA homologue were expressed in all the stomach cancer cell lines tested.
[Recent progress in oncogene research].
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Sequence of membrane-associated thyroid hormone binding protein from bovine liver: its identity with protein disulphide isomerase.
The complementary DNAs of the bovine liver membrane-associated 3,5,3'-triiodo-L-thyronine binding protein with 55 k-dalton (T3BP) were cloned and the nucleotide sequences were determined. Monospecific antibodies against T3BP were used to screen a bovine liver cDNA library in lambda gtll. We analyzed the sequences of two cloned T3BP cDNAs. The clones encoded a polypeptide of 510 amino acid residues, including a signal peptide of 20 amino acid. Northern blot analysis of bovine and human RNA showed that the mRNAs encoding T3BP are 2.7 kilobase in length. Amino acid sequence of N-terminal and other three peptides isolated from purified T3BP were found in the predicted amino acid sequence from the cDNA sequence. The predicted amino acid sequence is closely homologous (93%) with that of rat protein disulphide isomerase (EC 5.3.4.1), which catalyzes the isomerization of the protein disulphide bonds and has been shown to play an important role in post-translational regulation. The results suggest that T3BP and protein disulphide isomerase should be the same protein.
c-yes and bcl-2 genes located on 18q21.3 in a follicular lymphoma cell line carrying a t(14;18) chromosomal translocation.
The c-yes-1 and bcl-2 genes have been independently mapped at the same position of the chromosome band, 18q21.3, which is a breakpoint in the t(14;18) chromosomal translocation. High-molecular-weight genomic DNAs and mRNA isolated from a follicular lymphoma cell line FL-18 and an Epstein-Barr virus harboring FL-18EB cell line, which carried the t(14;18), were analyzed. Although there was no detectable c-yes rearrangement, the chromosome 18-specific probe b (bcl-2) revealed one rearranged fragment after EcoRI, BamHI, PstI and SstI digestion. Therefore, breakage of chromosome 18 in the t(14;18) occurred at the SstI fragment containing the probe b sequence on the bcl-2 gene. The transcript of c-yes was undetectable, whereas active transcription of bcl-2 was observed in the FL-18 and FL-18EB cell lines at a higher level than in other types of lymphoma cell line. These data suggest that the c-yes-1 is not involved in the t(14;18), and the 2 genes located at 18q21.3 are independent sequences.
A Mr = 190,000 glycoprotein phosphorylated on tyrosine residues in epidermal growth factor stimulated KB cells is the product of the c-erbB-2 gene.
In human epidermoid carcinoma KB cells, a glycoprotein of Mr = 190,000 (gp190) has been shown to be phosphorylated on tyrosine residues upon EGF stimulation (Kadowaki et al., 1987, J. Biol. Chem. in press). Using a specific antibody to the c-terminal portion of the human c-erbB-2 gene product, we have found that gp190 is the human c-erbB-2 gene product which is structurally closely related to the epidermal growth factor (EGF) receptor. Since monoclonal antibody specific for the EGF receptor abolished both EGF binding to its receptor and tyrosine phosphorylation of the c-erbB-2 gene product, we have concluded that activation of EGF receptor tyrosine kinase activity upon EGF binding leads to the phosphorylation of the c-erbB-2 gene product on its tyrosine residues.
Human liver type pyruvate kinase: cDNA cloning and chromosomal assignment.
Pyruvate kinase (PK) has four isozymes (L,R,M1,M2) that are encoded mainly by two different genes. We isolated a cDNA clone from a Japanese adult liver lambda gt10 cDNA library by using a rat liver(L)-type PK cDNA probe. One positively hybridizing clone, hlPK-1, which contained a 1,049-base pair cDNA insert, was subjected to DNA sequence analysis. Comparisons of the sequence data with the rat PK cDNAs indicated that the cDNA encoded information for the carboxyl terminal 105 amino acids of a human L-type PK and a 3' untranslated region of 734 nucleotides. Furthermore, the karyotype analysis of several human-mouse hybrid cells and Southern blot analysis of DNAs of the hybrids with a hlPK-1 indicated that the human L-type PK gene is located on chromosome 1.
Oncogenes related to growth factor receptors.
Recent progress on analysis of functions of proto-oncogenes was discussed with the protein-tyrosine kinase group as an example. Many proto-oncogenes encode proteins related to regulation of cell growth and differentiation. Among those, the largest group is the protein kinase super family which includes growth factor receptors, non-receptor type protein-tyrosine kinases and serine-threonine kinases. They can be divided into subgroups according to their polypeptide and genomic structures. Members of each subgroup appeared to be originated from each single ancestral gene during evolutionary process even within the protein kinase super family.
Merkel-neurite complexes in the fungiform papillae of two species of monkeys.
Merkel-neurite complexes in tongues of Japanese and cynomolgus monkeys were examined by means of light and electron microscopy. Merkel-neurite complexes were found preferentially in the epithelium of fungiform papillae located at the tip of the tongue. It appears that the anterior fungiform papillae of the monkey are highly adapted for both taste and mechanical sensation.
Light and electron microscopical demonstration of c- erB-2 gene product-like immunoreactivity in human malignant tumors.
C-erbB-2 is a human protooncogene homologous with the well-known c-erbB. Genes and gene products of the EGF receptor and c-erbB are known to be closely related and to be closely homologous in their intracellular domain. Inspection of the deduced amino acid sequence suggested that the c-erbB-2 gene encodes a receptor for a yet unidentified growth factor. An immunohistological study was performed by introducing an antibody raised in the rabbit by immunization with a synthetic peptide corresponding to a part of the intracytoplasmic domain of predicted gene product. Specimens from 13 normal human organs, fresh frozen tissue from 41 surgically excised human malignant tumors and eight cell lines maintained in nude mice were studied. Positive staining was found in 4 of the 41 (9.8%) malignant tumors. All of the positive tumors were adenocarcinomas and two adenocarcinoma cell lines were also positive. Amongst the normal human tissues, epithelial cells in stomach, small and large intestine were faintly stained. When the positively stained cell lines were studied by immunoelectronmicroscopy, the reaction was most prominent in the membrane of microvilli, but part of the nuclear membrane, the endoplasmic reticulum and the outer cell membrane were also stained. DNA and mRNA blot assays, as well as our immunoprecipitation test, revealed that immunohistologically positive cell lines bore amplified c-erbB-2 DNA, c-erbB-2 mRNA and 185 kD protein which is supposed to be the gene product, while negative cell lines did not.
Monoamine-containing basal cells in the taste buds of the newt Triturus pyrrhogaster.
Monoamine-containing cells were examined by fluorescence histochemistry and electron microscopy. Two or three serotonin-like fluorescent cells were located just above the basal lamina and failed to reach the free surface of the taste bud. Ultrastructurally this cell type was characterized by the presence of dense-cored vesicles and finger-like cytoplasmic processes. Many characteristics of Merkel cells were present.
Viral oncogenes, v-yes and v-erbB, and their cellular counterparts.
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Characterization of the promoter region of the human c-erbB-2 protooncogene.
Three overlapping genomic clones that contain the 5'-terminal portion of the human c-erbB-2 gene (ERBB2) were isolated. The promoter region was identified by nuclease S1 mapping with c-erbB-2 mRNA. Seven transcriptional start sites were identified. DNA sequence analysis showed that the promoter region contains a "TATA box" and a "CAAT box" about 30 and 80 base pairs (bp), respectively, upstream of the most downstream RNA initiation site. Two putative binding sites for transcription factor Sp1 were identified about 50 and 110 bp upstream of the CAAT box, and six GGA repeats were found between the CAAT box and the TATA box. This region had strong promoter activity when placed upstream of the bacterial chloramphenicol acetyltransferase gene and transfected into monkey CV-1 cells. These data indicate that the promoter of the human c-erbB-2 protooncogene is different from that of the protooncogene c-erbB-1 (epidermal growth factor receptor gene), which does not contain either a TATA box or a CAAT box. Comparison of the promoter sequences and activities of the two protooncogenes should be helpful in analysis of the regulatory mechanism of expression of their gene products, which are growth-factor receptors.
Cell transformation by a virus containing a molecularly constructed gag-erbB fused gene.
A provirus DNA that contains a gag-erbB fused gene as the sole and transforming gene was molecularly constructed from plasmid pSRA2 containing the entire genome of Rous sarcoma virus and pAE7.7 containing the entire genome of an avian erythroblastosis virus (AEV), AEV-H. A virus containing the gag-erbB fused gene (GEV) was recovered from chicken embryo fibroblasts transfected with the proviral DNA and a helper virus DNA. GEV could transform chicken embryo fibroblasts as efficiently as could AEV-H. Anti-erbB and anti-gag sera immunoprecipitated a protein with a molecular weight of about 110,000 from GEV-transformed cells. The erbB and gag-erbB fused-gene products in AEV-H- and GEV-transformed cells were analyzed.
The yes-related cellular gene lyn encodes a possible tyrosine kinase similar to p56lck.
With v-yes DNA as the probe, a human cDNA library made from placental RNA was screened under relaxed conditions, and DNA clones derived from a novel genetic locus, termed lyn, were obtained. Nucleotide sequencing revealed that lyn could encode a novel tyrosine kinase that was very similar to mouse T-lymphocyte-specific tyrosine kinase p56lck and the v-yes protein as well as to the gene products of v-fgr and v-src. Northern hybridization analysis revealed that a 3.2-kilobase lyn mRNA was expressed in a variety of tissues of the human fetus. The pattern of lyn mRNA expression was different from those of related genes, such as yes and syn. Hybridization analysis of DNA from sorted chromosomes showed that the lyn gene is located on human chromosome 8 q13-qter.
Characterization of cDNA clones for the human c-yes gene.
Three c-yes cDNA clones were obtained from poly(A)+ RNA of human embryo fibroblasts. Sequence analysis of the clones showed that they contained inserts corresponding to nearly full-length human c-yes mRNA, which could encode a polypeptide of 543 amino acids with a relative molecular weight (Mr) of 60,801. The predicted amino acid sequence of the protein has no apparent membrane-spanning region or suspected ligand binding domain and closely resembles pp60c-src. Comparison of the sequences of c-yes and v-yes revealed that the v-yes gene contains most of the c-yes coding sequence except the region encoding its extreme carboxyl terminus. The region missing from the v-yes protein is the part that is highly conserved in cellular gene products of the protein-tyrosine kinase family.
Molecular cloning and characterization of an activated human c-raf-1 gene.
Results of previous studies have shown that a raf-related transforming DNA sequence is present in NIH 3T3 transformants that are derived from GL-5-JCK human glioblastoma DNA transfection. The transforming DNA was molecularly cloned by using cosmid vector pJB8 to determine its structure and origin. Analyses of selected clones revealed that the transforming DNA consisted of three portions of human DNA sequences, with the 3' half of the c-raf-1 gene as its middle portion. This raf region was about 20 kilobases long and contained exons 8 to 17 and the poly(A) addition site. RNA blot analysis showed that the raf-related transforming DNA was transcribed into 5.3-, 4.8-, and 2.5-kilobase mRNAs; the 2.5-kilobase transcript was thought to be the major transcript. Immunoprecipitation analyses revealed that a 44-kilodalton raf-related protein was specifically expressed in the NIH 3T3 transformants. The raf-related transforming DNA was considered to be activated when its amino-terminal sequence was truncated and the DNA was coupled with a foreign promoter sequence. On hybridization analysis of the original GL-5-JCK glioblastoma DNA, no rearrangement of c-raf-1 was detectable in the tumor DNA. The rearrangement of c-raf-1 may have occurred during transfection or may have been present in a small population of the original tumor cells as a result of tumor progression.