Internally repeated sequences in ras gene products.
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Animals↗
Biomedical subjects
T Hiwasa
Publications and source records attributed to T Hiwasa.
Isolation and characterization of a complementary DNA clone for a Mr 32,000 protein which is induced with tumor promoters in BALB/c 3T3 cells.
The synthesis of a protein of Mr 32,000 (p32) is enhanced by various tumor promoters, chemical carcinogens, metal salts, and heat shock in BALB/c 3T3 cells. We have isolated a complementary DNA (cDNA) clone for p32 from a lambda gt10 library of BALB/c 3T3 cells. The library was constructed from mRNA extracted from the cells treated with sodium arsenite, which stimulates the p32 expression most effectively among various agents so far tested. Having screened this library differentially with probes which represent induced and uninduced mRNA populations for p32, we first obtained a partial p32 cDNA clone and have subsequently succeeded in the isolation of a cDNA clone containing the entire coding sequence. RNA blot analysis has shown that p32 mRNA is induced as early as 0.5 h after the addition of 12-O-tetradecanoyl-phorbol-13-acetate or sodium arsenite. Computer-assisted comparison with GenBank data has revealed a striking similarity in the nucleotide sequences between cDNAs of p32 and rat heme oxygenase. These results strongly suggest that p32 is a mouse homolog of this enzyme.
Animals↗
Degradation of epidermal growth factor receptors by cathepsin L-like protease: inhibition of the degradation by c-Ha-ras gene products.
Extract of NIH3T3 mouse fibroblasts contains a protease which can cleave epidermal growth factor receptor (EGF receptor). This protease was tentatively named cathepsin X and purified to near homogeneity. The characteristics of cathepsin X were similar to those of cathepsin L and the proteolytic activity of cathepsin X was inhibited by c-Ha-ras gene products.
Animals↗
Inhibition of cathepsin L-induced degradation of epidermal growth factor receptors by c-Ha-ras gene products.
The inhibitory activities of c-Ha-ras gene products (p21s) toward several cysteine proteinases have been investigated. The activity of cathepsin L was inhibited by p21s most effectively while those of cathepsin B and papain were slightly inhibited by p21s. p21s did not show any inhibitory activity toward cathepsin H. In order to connect the protease-inhibitor activity of p21s with cell growth, the degradation of epidermal growth factor receptors (EGF-receptors) was investigated. EGF-receptors were preferentially cleaved by cathepsin L but not by cathepsin B or H. The cleavage of EGF-receptors by cathepsin L was inhibited by p21s dose-dependently. These results raise the possibility that p21s can suppress the degradation of growth-related proteins such as EGF-receptors and thereby affect cell growth.
Carcinoma, Squamous Cell↗
Possible involvement of protease inhibitor ras proteins in malignant transformation.
Based on our recent finding that human c-Ha-ras gene products (p21s) inhibit the activities of thiol proteinases, a possible new model for malignant transformation is proposed. The model involves a new concept that p21s can cause effects on the protein kinase activities of growth factor receptors by affecting down regulation of the receptors.
Cathepsin L↗
[Possible involvement of the protease-inhibitor activity of ras oncogene products in transformation].
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Amino Acid Sequence↗
Degradation of a cAMP-binding protein is inhibited by human c-Ha-ras gene products.
Incubation of the particulate fraction of cell extract prepared from NIH3T3 mouse fibroblasts resulted in preferential proteolytic degradation of a cAMP-binding protein. The proteolysis was inhibited by human c-Ha-ras gene products produced by Escherichia coli. The proteolysis was observed at pH 6 to 7, and inhibited by antipain and leupeptin. These results suggest that cAMP-binding proteins might be cleaved by thiol proteinases. In fact, c-Ha-ras gene products were proved to inhibit the cathepsin B-like activity present in the particulate fraction.
Animals↗
Defects of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in initiated clones derived from BALB/c 3T3 mouse fibroblasts.
Two-stage carcinogenesis is involved in the transformation of mouse fibroblasts BALB/c 3T3 cells. In order to investigate the role of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase at the stage of initiation, the following experiments were carried out: (a) two initiated clones (M14, M20) which exhibit 12-O-tetradecanoylphorbol-13-acetate-dependent growth in soft agar medium were isolated from cells treated with N-methyl-N'-nitro-N-nitrosoguanidine. The activity of cAMP-dependent protein kinase in M14 was reduced while that in M20 was similar to the level in parental cells. However, cAMP-binding activity to a regulatory subunit of cAMP-resistant clones were isolated from 4-nitroquinoline oxide- or ethyl methanesulfonate-treated cells. These clones have reduced activities in both cAMP-binding and cAMP-dependent protein kinase itself. Two of three cAMP-resistant clones were proved to be able to grow in soft agar medium only in the presence of 12-O-tetradecanoylphorbol-13-acetate.
4-Nitroquinoline-1-oxide↗
c-Ha-ras gene products are potent inhibitors of cathepsins B and L.
c-Ha-ras proteins produced by Escherichia coli inhibited the activities of cathepsins B and L which had been partially purified from rat kidney. Furthermore, amino acid sequence homology between c-Ha-ras proteins and thiol proteinase inhibitors has been found.
Amino Acid Sequence↗
Altered properties of cAMP-dependent protein kinase in H-ras-transformed NIH3T3 cells.
cAMP-dependent protein kinase activity in the soluble fraction was decreased in both v-H-ras-transformed and activated-c-H-ras-transformed NIH3T3 cells as compared with that in NIH3T3 cells. Both of the elution profile of type II cAMP-dependent protein kinase from DEAE-cellulose and the electrophoretic behavior of its regulatory subunits in the particulate fraction of H-ras-transformed cells are different from those of control NIH3T3 cells. These results suggest that ras protein causes the alterations of some properties of cAMP-dependent protein kinases.
Animals↗
Increase in the synthesis of a Mr 32,000 protein in BALB/c 3T3 cells after treatment with tumor promoters, chemical carcinogens, metal salts, and heat shock.
The synthesis of a Mr 32,000 protein (p32) is enhanced as early as 2 h after the addition of tumor-promoting phorbol esters to BALB/c 3T3 cells. Among various compounds tested thus far, methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, L-ascorbic acid, sodium deoxycholate, p-tosyl-L-phenylalanine chloromethyl ketone, p-tosyl-L-lysine chloromethyl ketone, 3,3'-diaminobenzidine, and some of the metal salts stimulated the synthesis of p32 to varying extents. p32 might be one of the heat shock proteins because its synthesis was also stimulated by heat shock or sodium arsenite. The synergisms of the effects of different compounds on p32 synthesis suggest that the expression of a p32 gene is regulated through at least three pathways. Possible roles of protein kinases in p32 gene expression are discussed.
3,3'-Diaminobenzidine↗
Disorganization of microfilaments caused by tumor promoters in mouse fibroblasts.
The effect of tumor promoters on the organization of BALB/c 3T3 fibroblasts was examined by indirect immunofluorescence microscopy using anti-actin antibodies. Potent tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate, teleocidin, dihydroteleocidin B and debromoaplysiatoxin , in spite of their different chemical structures, all induced disruption of microfilaments. This change was still observed in the presence of actinomycin D.
Actins↗
Increase in the synthesis of a Mr 32,000 protein in BALB/c 3T3 cells treated with tumor-promoting indole alkaloids or polyacetates.
The synthesis of a unique protein with a molecular weight of 32,000 (p32) in BALB/c 3T3 cells has been shown previously to increase after treatment with potent tumor-promoting phorbol esters (Hiwasa et al., Proc. Natl. Acad. Sci. U. S. A., 79: 1800, 1982). In the present study, two new classes of tumor promoters which are structurally different from phorbol esters were investigated for their potencies to enhance p32 synthesis. Teleocidin, dihydroteleocidin B, and lyngbyatoxin A, which are indole alkaloid tumor promoters, enhanced p32 synthesis to the same extent that 12-O-tetradecanoylphorbol-13-acetate did. However, no increase was observed by treatment with the biologically inactive hydrolysate of teleocidin. Polyacetate tumor promoters such as aplysiatoxin and debromoaplysiatoxin also stimulated p32 synthesis, but their effective concentrations were higher than those of 12-O-tetradecanoylphorbol-13-acetate. When 3T3 cells were treated with a combination of two of the three tumor promoters, TPA, teleocidin, and aplysiatoxin, no synergistic effect of p32 synthesis was observed. This implies that these tumor promoters enhance the synthesis of p32 through the same mechanism.
Alkaloids↗
Tumor promoters increase the synthesis of a 32,000-dalton protein in BALB/c 3T3 cells.
The effect of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent tumor promoter, on the synthesis of proteins in BALB/c 3T3 mouse fibroblasts has been studied. When [35S]methionine-labeled proteins synthesized after the addition of TPA were analyzed by one- or two-dimensional polyacrylamide gel electrophoresis, the increased synthesis of a 32,000-dalton protein (designated p32) was noted as one of the earliest changes. The synthesis of p32 increased approximately 2-fold within 2 hr at a promoter concentration of 20 ng/ml. Phorbol 12,13-didecanoate, another potent tumor promoter, showed the same effect as TPA, whereas 4-O-methyl-TPA, which has little promoting activity, did not enhance the synthesis of p32 at the same concentration but did effect a slight increase at 1 microgram/ml. p32 differed immunologically from a major excreted protein discovered by Gottesman [Gottesman, M. M. (1978) Proc. Natl. Acad. Sci. USA 75, 2767-2771]. The increased rate of the synthesis of p32 was considered to be regulated at the transcriptional level, because the increase in synthesis of p32 was inhibited by actinomycin D, and TPA-treated cells were shown to contain a higher level of translatable mRNA coding for p32 than do control cells. A possible relationship between the increase in p32 synthesis and transformation is discussed.
Animals↗
Phasing of nucleosomes in SV40 chromatin reconstituted in vitro.
Phasing of nucleosomes on SV40 DNA was studied by the reconstitution of chromatin from SV40 DNA form I and core histones. The reconstituted chromatin sedimented with increasing S values as the histone : DNA ratios increased, and the buoyant densities in CsCl decreased concomitantly. The average repeat lengths of nucleosomes in the chromatin reconstituted at ratios of 1.0 and 1.5 were estimated to be 168 and 143 base pairs, respectively, by electrophoretic analysis of DNA fragments generated by micrococcal nuclease digestion. The chromatin generated a series of DNA bands that differed in size by about 10 nucleotides upon DNase I digestion followed by heat-denaturation. Phasing of nucleosomes was probed by the use of single-site restriction endonucleases, EcoRI, BamH1, BglI, and HpaII: the latter two cleave DNA at and near the origin of DNA replication and transcription. The form I DNA in the chromatin reconstituted at ratios of 0.5, 1.0, and 1.5 was cleaved up to 60 to 80%, 20 to 60%, and 0 to 10%, respectively. Although the frequency of cleavage by these enzymes was not very different at the ratio 0.5, the BglI site became relatively more susceptible than the other sites at the ratio 1.0. At the ratio 1.5, the DNA was almost resistant to these enzymes, though a significant amount (10%) was cleaved by BglI. These results suggest that the origin is the site unfavorable for nucleosome phasing although the region can be almost completely covered with nucleosomes at higher histone : DNA ratios. The fraction of chromatin immunoprecipitated with anti-T serum after in vitro T antigen binding also decreased with increase in the histone : DNA ratios. The results suggest that T antigen binds preferentially to the internucleosomal region. T antigen preferentially bound to the chromatin reconstituted with the DNA fragment containing the origin. Inefficient phasing of nucleosomes at the origin of DNA replication may facilitate the binding of T antigen to the origin.
Antigens, Neoplasm↗
Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.
Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.
Animals↗
Differential sensitivity to mitomycin C between human RSa cell line and its derivative UVr-1.
To study cellular signaling factors responsible for the susceptibility of human cells to cell proliferation inhibition by anticancer drugs, human RSa cell line and its ultraviolet-resistant derivative UVr-1 were compared with respect to their sensitivity to the anti-proliferative effects of mitomycin C (MMC), 5-fluorouracil, nimustine (ACNU), cisplatin, pirarubicin (THP), bleomycin, methotrexate and ifosfamide. RSa cells were found to be highly sensitive to MMC by MTT assay compared to UVr-1 cells. The half maximum inhibition concentration of MMC against proliferation of RSa cells was approximately 100 ng/ml while that of UVr-1 cells was greater than 1 microgram/ml. There was no significant difference observed between RSa and UVr-1 cells in the sensitivity to other seven drugs examined. Analysis by flow cytometry revealed that the cell cycle of RSa was completely blocked at the G2/M phase 40 h after treatment with MMC at a concentration of 100 ng/ml whereas a substantial proportion of UVr-1 cells was not arrested at that phase even in the presence of MMC. Further immunoblot analysis on MMC-induced signal transduction showed that the amounts of phosphorylated ERK MAP kinases were increased in UVr-1 cells to a greater extent than those in RSa cells after treatment with MMC for longer than 2 h. However, the increase in p21Cip1 was observed in RSa cells 1 h after addition of MMC but was not observed in UVr-1 cells. These distinct signaling pathways might account for the differences in sensitivity to MMC between RSa and UVr-1 cells.
Antibiotics, Antineoplastic↗
Growth-suppressive activities of serine protease inhibitors, ONO-3403 and ONO-5046, toward normal and transformed murine fibroblasts.
ONO-3403 and ONO-5046 are potent synthetic protease inhibitors of trypsin and elastase, respectively. These compounds suppressed the proliferation of polyoma virus- and Kirsten sarcoma virus-transformed BALB/c 3T3 cells more effectively than their normal counterparts. SV40-transformed 3Y1 and v-Ha-ras-transformed NIH3T3 cells were also more sensitive to ONO-3403 and ONO-5046 than the parent normal cells. These results suggest that ONO-3403 and ONO-5046 are useful for selective suppression of the proliferation of rapidly growing transformed cells.
3T3 Cells↗