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Biomedical subjects

I B Weinstein

Publications and source records attributed to I B Weinstein.

At least 145 records · Page 8Linked to original sources
PubMed1990

Identification of ultraviolet-inducible proteins that bind to a TGACAACA sequence in the polyoma virus regulatory region.

The exposure of mammalian cells to UV light or other DNA-damaging agents induces several responses which may provide cellular defense mechanisms and also play a role in carcinogenesis. Employing a 257-base pair DNA fragment from the polyoma virus that contains the origin of replication and regulatory region of this virus, we have identified a set of DNA-binding proteins that are induced in normal rat fibroblasts at 6-24 h after UV exposure. These proteins bind to a specific octamer sequence (TGACAACA) designated the "UV response element." Purification of these inducible proteins on a UV response element affinity column revealed a set of proteins, among which the major protein has a molecular weight of 40,000, which co-purify with c-fos but do not react with antibodies to c-jun-encoded proteins. These UV-induced proteins may, in concert with other cellular components, play a role in mediating specific cellular responses to DNA damage in mammalian cells.

Animals↗
PubMed1990

Production of diacylglycerol, an activator of protein kinase C, by human intestinal microflora.

Although dietary lipids have been implicated in colon cancer causation, the underlying mechanisms are not known. This paper indicates that when bacteria obtained from normal human feces are incubated with 14C-labeled phosphatidylcholine there is appreciable production of diacylglycerol (DAG), monoacylglycerol, and free fatty acid. Curiously, the production of DAG and monoacylglycerol, but not fatty acid, is strictly dependent on addition of certain bile acids to the incubation system. Among the bile acids tested deoxycholic acid is the most active. Assays of fecal specimens from 10 normal individuals demonstrate a 27-fold interindividual variation in the production of DAG in the in vitro assay system, and also in the absolute levels of DAG present in the same fecal samples. On the other hand, both parameters of DAG are quite constant in repeated fecal samples obtained from the same individual over a period of about 4 months. DAG is a normal physiological activator of protein kinase C, an enzyme that plays a key role in growth control and tumor promotion. We speculate, therefore, that DAG produced by the intestinal microflora might stimulate growth of colonic epithelial cells. Thus an interaction between dietary lipids, bile acids, and specific bacteria in the intestinal lumen could contribute to the risk of colon cancer development in humans.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

Restriction fragment length polymorphism of chromosome 3 (3p) and the epidermal growth factor receptor gene in human non-small cell lung carcinomas.

We have examined the possible loss of 3p alleles in lung tumor samples from 28 patients with non-small cell lung cancers (non-SCLC), using tumor adjacent lung tissue from the same patients as controls. Of the 14 patients with squamous cell carcinoma only 2 (14%) displayed constitutional heterozygosity at the 3p locus and the tumors of both of these cases did not show reduction to homozygosity. Of the 14 patients with adenocarcinomas, 50% had constitutional heterozygosity, and two of the tumors displayed a loss of heterozygosity. We have also examined restriction fragment length polymorphisms (RFLPs) of the epidermal growth factor (EGF) receptor gene in 29 non-SCLC tumor samples and in the tumor adjacent lung tissue samples obtained from the same cases. Digestion of the DNA samples with the BstEII enzyme and hybridization to a HER-A64-3 probe revealed four different types of polymorphic patterns. We did not, however, detect significant differences in the specific polymorphic bands between tumor and paired non-tumor lung tissues or between the different types of carcinomas.

Blotting, Southern↗
PubMed1990

Isolation of a complementary DNA encoding the catalytic subunit of protein kinase A and studies on the expression of this sequence in rat hepatomas and regenerating liver.

A complementary DNA (cDNA) clone (B4) encoding the catalytic subunit of a cAMP-dependent protein kinase (PKAc) was isolated from a lambda gt10 rat brain cDNA library, using a synthetic oligonucleotide probe whose sequence was based on the known amino acid sequence of a bovine cardiac PKAc. Sequence analysis of this clone revealed a region of 1002 nucleotides which encodes a protein that is 92% homologous to amino acids 17-350 of the bovine cardiac PKAc protein. This clone lacks coding sequences for amino acids 1-16 of the latter protein. Nevertheless, it provided a useful probe to analyze expression of the related gene in a variety of systems. Northern blot analyses using a 32P-labeled probe prepared from a 0.6-kilobase PstI fragment of clone B4 revealed an abundant 4.6-kilobase band in rat brain RNA and lesser amounts of this 4.6-kilobase RNA in rat heart and liver. A 4.6-kilobase RNA was also detected in RNA samples obtained from mouse fibroblasts. This probe also detected homologous RNA in a variety of nonrodent species. In subsequent experiments, this cDNA was used as a probe to elucidate the role of PKAc in post-surgical hepatic regeneration and diethylnitrosamine-induced hepatomas in the rat. These experiments revealed that, following partial hepatectomy, PKAc mRNA is decreased 3-fold by 12 h, returning to normal by 72 h; hepatomas showed no consistent pattern of change in PKAc mRNA levels as compared to controls. Our results indicate that this cDNA encodes an isoform of PKAc which is distinct from PKAc-alpha isolated by Uhler et al. (Proc. Natl. Acad. Sci. USA, 83: 1300-1304, 1986) but highly homologous to PKAc-beta isolated by Showers and Maurer (J. Biol. Chem., 261: 16288-16291, 1986), that depression of cAMP-dependent protein phosphorylation may be an important mechanism in the regeneration of mature rat liver but is not a consistent alteration in chemically induced hepatoma, and that this cDNA is useful as a probe for the study of the role of PKAc gene expression in growth control, particularly in rodent species.

Amino Acid Sequence↗
PubMed1990

Characterization of a specific form of protein kinase C overproduced by a C3H 10T1/2 cell line.

Murine embryo fibroblasts (C3H 10T1/2) which were genetically engineered to overproduce the beta 1 isoform of protein kinase C (PKC-beta 1) were used to obtain homogeneous preparations of PKC-beta 1 for the purpose of characterizing the specific structural and functional properties of this isoform. Fractionation of PKC activity from these cells by hydroxyapatite chromatography produced one major peak, which represented 93% of the total cellular PKC activity and was not detected in control cells. This major peak of activity was shown by Western-blotting analysis with a beta 1-specific antiserum to be the overproduced beta 1-isoform, and exhibited a band at 77 kDa. The functional properties of the overproduced PKC-beta 1 were established with regard to phospholipid-dependence, Ca2(+)-dependence, responsiveness to a phorbol ester tumour promoter, activation by arachidonic acid (plus Ca2+), and inhibition by known PKC inhibitors. From these studies we conclude that PKC-beta 1 overproduced by C3H 10T1/2 cells exhibits the structural and functional properties previously ascribed to native PKC. Furthermore, these data provide the first definitive biochemical characteristics of this isoform of PKC.

Animals↗
PubMed1990

Inhibition of rodent protein kinase C by the anticarcinoma agent dequalinium.

Dequalinium has previously been shown to be an anticarcinoma agent (M. J. Weiss et al., Proc. Natl. Acad. Sci. USA, 84: 5444-5448, 1987). The present study demonstrates that it can inhibit protein kinase C-beta 1 isolated from an overproducing cell line with a 50% inhibitory concentration of 8-15 microM. Further examination of the inhibition by using structural analogues of dequalinium reveals that the length of the methylene bridge between the two quinaldinium moieties, the presence of the ring substituents, and the bipartite character of the compound each contributes to the inhibitory potency. Related studies show that the analogues display the same rank order of inhibitory potency when tested with the trypsin-generated catalytic fragment of the enzyme, indicating that dequalinium inhibits kinase activity through an interaction with the catalytic subunit. Further studies argue that the ability of a given analogue to inhibit phosphotransferase activity correlates with its ability to compete with [3H]phorbol-12,13-dibutyrate binding on the intact enzyme (50% inhibitory concentration of 2-5 microM). This suggests that the inhibitor is either binding directly to the regulatory subunit as well, or that due to its interaction with the catalytic subunit, dequalinium produces an indirect effect on sites defined by phorbol ester binding. Kinetic analysis revealed that inhibition is noncompetitive with respect to ATP or phosphatidylserine. Studies conducted with types I, II, and III rat brain isozymes, resolved by hydroxylapatite chromatography, demonstrate that dequalinium inhibits each of them with similar potency (50% inhibitory concentration of 11 microM) and imply that the site of contact on the enzyme is a highly conserved region. Morphology studies with dequalinium in intact cells demonstrate that the inhibitor can protect control cells against phorbol ester-induced morphology changes but cannot protect protein kinase C-overproducing cells, suggesting that an elevation in protein kinase C levels alone is sufficient to overturn the protection conferred by dequalinium. On the basis of these results, we propose that protein kinase C could be a critical in vivo target of dequalinium.

Animals↗
PubMed1990

Growth factor-induced DNA synthesis in cells that overproduce protein kinase C.

In previous studies (Housey et al.: Cell 52:343-354, 1988), our laboratory demonstrated that a cell line R6-PKC3 that stably overproduces high levels of the beta 1 isoform of PKC displayed several abnormalities in growth control, and these phenotypic changes were also markedly enhanced when the cells were exposed to TPA. The present studies indicate that these cells also display marked changes in their response to certain growth factors. A striking finding was that several agents when tested alone in serum-free medium, including EGF, PDGF, TPA, teleocidin, and OAG, stimulated DNA synthesis in quiescent R6-PKC3 cells but had a negligible effect in quiescent R6-C1 cells, a vector control cell line with normal levels of PKC. R6-PKC3 cells also show an exaggerated response to very low concentrations of serum, when compared to R6-C1 control cells. These studies provide direct genetic evidence that alterations in cellular levels of PKC can markedly influence the responses of cells to specific growth factors.

Animals↗
PubMed1990

Increased levels of phorbin, c-myc, and ornithine decarboxylase RNAs in human colon cancer.

Our previous work on protein kinase C (PKC) and colon cancer has shown altered levels of PKC activity in human colon tumors, as well as activation of PKC by colon tumor promoters such as bile acids. To understand further the role of PKC in colon carcinogenesis, we analyzed the expression of phorbin, a gene induced by PKC activation, in a series of different stages of human colon tumors. As shown by northern blot analyses of poly (A)+ RNA, higher levels of phorbin RNA were seen in 26 colon tumor samples than in their adjacent normal colonic mucosa. There also appeared to be a correlation between the abundance of phorbin RNA in the tumors and the extent of invasion (tumor-to-normal tissue phorbin RNA ratio = 4.2, 8.0, and 11.9 for Dukes' A, B, and C, respectively). Phorbin RNA was also abundant in a human colon cancer line (HT29). We also examined the expression of other mitogen-responsive genes (c-myc, ODC, and beta-actin) in a set of 19 colon tumor samples. All tumors displayed significant (mean 3.8-fold) increases in the level of c-myc RNA compared with their adjacent normal colonic mucosa. About 47% and 16% of these tumor samples also showed increased levels of ODC (mean 3.1-fold) and beta-actin (mean 1.6-fold) RNA, respectively. The increased levels of c-myc, ODC, and beta-actin RNA did not correlate with the extent of tumor invasion. Taken together, these results demonstrate that human colon tumors usually display increased levels of both phorbin and c-myc RNAs. The marked increases in phorbin RNA suggest that this could serve as a useful biomarker in studies on human colon cancer.

Actins↗
PubMed1990

Factors influencing the expression of endogenous retrovirus-like sequences in Rat 6 cells.

Previous studies from this laboratory have demonstrated increased levels of expression of endogenous rat leukemia virus (RaLV) and 30S retrovirus-like sequences in liver and colon tumors induced by chemical carcinogens in rats. During the process of normal liver regeneration, the levels of RaLV RNA were dramatically increased, whereas the levels of 30S RNA did not change. The present study examined several factors that might influence the expression of these sequences in the Rat 6 embryo fibroblast cell line. Rat 6 cells in either log-phase or confluent cultures were treated with cycloheximide, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), or the activated carcinogen benzo[a]pyrene diol epoxide (BPDE) for various periods of time up to 48 h. Northern blot analyses of total RNA indicated that cells in untreated cultures in log phase had higher levels of RaLV and 30S RNAs than did confluent cells. Within 10 h cycloheximide (2 or 10 micrograms/mL) markedly increased the levels of RaLV and 30S RNAs in both log-phase and confluent cells. BPDE (100 ng/mL) induced a marked increase in the levels of RaLV RNA at 4 to 10 h, which returned to the basal level by 24 h in the log-phase cells; but no significant change was seen in the confluent cells. The level of 30S RNA also increased moderately in the BPDE-treated log-phase cells and was maximal at 24 h; but no change was seen in confluent cells. Treatment with TPA (100 ng/mL) induced no significant increase in either RaLV or 30S RNA levels in the log-phase or confluent cells. The exposure of Rat 6 cells to 5-azacytidine (3 microM for 24 h) led to a marked increase in the levels of both RaLV and 30S RNAs, which persisted during at least 15 subsequent passages in the absence of the drug. Thus, inhibition of protein synthesis, DNA damage, or hypomethylation can increase the expression of certain endogenous retrovirus-like sequences, but an activator of protein kinase C does not.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1990

Inducible cellular responses to ultraviolet light irradiation and other mediators of DNA damage in mammalian cells.

Both naturally occurring and carcinogen-induced tumors display not only point mutations in cellular oncogenes but also more complex changes in cellular oncogenes and other cellular genes. For this and other reasons, it seems likely that DNA damage in mammalian cells can induce alterations in gene expression that may have both short and long term consequences in the target cell. The purpose of this review is to summarize current available information on inducible responses to UV-irradiation and other mediators of DNA damage in mammalian cells, and to provide some working hypotheses. We have divided these responses into three time frames, immediate (0-12 hours), early (12-48) and late (beyond 48 hours). Immediate responses include the action of DNA repair enzymes, some of which are induced as a consequence of DNA damage, and transient inhibition of DNA synthesis. Within the past few years considerable evidence has accumulated that during this immediate period there is increased expression of certain cellular oncogenes, proteases and proteins whose functions remain to be identified. It is of interest that the expression of some of these genes is also induced by certain growth factors, tumor promoters and heat shock. Alterations in gene expression during the subsequent "early" period (12-48 hrs.) have not been studied in detail, but it is during this period that one can detect increased replication of several types of viruses in cells that harbor these viruses. We have examined in detail the induction of asynchronous polyoma DNA replication (APR) in a rat fibroblast cell line carrying integrated copies of this DNA. We have obtained evidence that UV-irradiation of these cells leads to the synthesis of a 40 kd protein, within the first 1-24 hrs after irradiation, that binds to a specific sequence TGACAACA in the regulatory region of polyoma DNA. We suggest that this protein acts together with other proteins to induce APR and that this serves as a useful model for understanding the mechanisms responsible for amplification of cellular genes, a phenomenon often seen in malignant tumors. Finally, we discuss how the events occurring during the immediate and early periods following DNA damage might lead to late effects in the target cell that are stable and contribute to the genotype and phenotype of some of the progeny of these cells that are destined to become tumor cells.

Animals↗
PubMed1990

Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression.

By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the beta 1 isoform of rat protein kinase C (PKC). Two of these cell lines, PKC6 and PKC7, displayed an 11- to 15-fold increase in PKC activity when compared with the C1 control cell line that carries the vector lacking the PKC cDNA insert. Both of the overexpresser cell lines exhibited striking alterations in morphology when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Following exposure to TPA, PKC6 and PKC7 cells displayed increased doubling time, decreased saturation density, and loss of anchorage-independent growth in soft agar; but these effects were not seen with the C1 cells. Also, in contrast to the control cells, the PKC-overproducing cells failed to display evidence of differentiation, as measured by alkaline phosphatase activity, when exposed to sodium butyrate. In addition, the PKC-overexpresser cells displayed decreased tumorigenicity in nude mice, even in the absence of treatment with TPA. These results provide the first direct evidence that PKC can inhibit tumor cell growth. Thus, in some tumors, PKC might act as a growth-suppressor gene.

Animals↗
PubMed1990

Effects of specific fatty acids on cell transformation induced by an activated c-H-ras oncogene.

An increase in dietary lipid has been associated with an increase in the development of certain forms of cancer, notably breast and colon cancer, both in experimental animal studies and in human epidemiology studies. The underlying mechanisms are not, however, known with certainty. In the present studies we have examined whether certain specific fatty acids (FA) might act by enhancing the role of an activated oncogene in a model cell culture system. We found that when the rat fibroblast cell line Rat 6 was transfected with an activated human c-H-ras oncogene and the cells subsequently grown in medium supplemented with myristic acid, palmitic acid or stearic acid (20-80 microM) there was a marked enhancement of the number of transformed foci obtained. On the other hand arachidonic acid had a marked inhibitory effect in this transformation assay. However, this inhibitory effect can be partially reversed by indomethacin, an inhibitor of cyclo-oxygenase, at dose response manner. Control studies indicated that these results were not simply due to the effects of the FAs on growth of the Rat 6 cells or the process of transfection per se. Lipid analyses of cells grown in the presence of stearic acid indicated that the added FA was extensively incorporated into the major lipid classes of the cell and produced transient changes in lipid composition. This simple cell culture system may be useful for elucidating the mechanisms by which various dietary lipids and nutritional factors influence the carcinogenic process.

Animals↗
PubMed1990

Transformation by a ras oncogene causes increased expression of protein kinase C-alpha and decreased expression of protein kinase C-epsilon.

Rat embryo fibroblasts and liver epithelial cell lines normally express two isoforms of protein kinase C (PKC), PKC alpha and PKC epsilon. Derivatives of these cells transformed by an activated human c-H-ras oncogene display a several-fold increase in expression of PKC alpha and a concomitant decrease in PKC epsilon, at both the protein and mRNA levels. Similar changes are seen when the transformed phenotype is induced by Zn2+ in cells carrying the activated ras oncogene under the control of a metallothionein promoter. Studies using cell lines that express very high levels of PKC beta 1, studies using a specific inhibitor of PKC (CGP 41251), and studies in which PKC activity is down-regulated by treatment with a phorbol ester tumor promoter provide evidence that the effects of the ras oncogene on the expression of PKC alpha and PKC epsilon are mediated mainly through a PKC-independent pathway. The present results provide the first evidence that transformation of cells by an oncogene can alter the relative expression of specific isoforms of PKC. It is possible that these changes contribute to the malignant phenotype of these cells.

Animals↗
PubMed1989

Altered expression of retrovirus-like sequences and cellular oncogenes in mice fed methyl-deficient diets.

Methyl-deficient (lipotrope-deficient) diets enhance liver carcinogenesis in rodents. Although the mechanisms responsible for the cancer-promoting activity of such diets have not been identified, they have been observed to cause impaired immune response, alterations in methylation of liver RNA and DNA, and enhanced susceptibility to oxidative damage. Since alterations in gene expression may also play a critical role, the present studies examined the expression of the c-myc, c-H-ras, epidermal growth factor receptor, and ornithine decarboxylase genes, as well as endogenous retrovirus-like sequences, in C57BL/6J x C3H/HeJ F1 mouse liver during the first 2 weeks of feeding of a methyl-deficient diet. The kinetics of liver cell proliferation was investigated in parallel. Increased [3H]thymidine incorporation into liver DNA was found at day 4 and reached a maximum at days 7-11 after commencement of the methyl-deficient diet, when compared to age-matched mice fed a complete diet. Northern blot analysis of polyadenylated liver RNA samples indicated an increase in the levels of RNA homologous to Moloney murine leukemia virus and intracisternal A particle sequences but no significant change in the level of VL30 retrovirus-related RNA in the samples from mice fed methyl-deficient diets. A marked increase in the levels of c-myc and a slight increase in the levels of ornithine decarboxylase and c-H-ras transcripts were seen in the liver RNA samples from the treated mice. Of particular interest was a decrease in the abundance of epidermal growth factor receptor transcripts in the liver RNA samples from the treated mice. These changes in cellular levels of specific RNA resemble, in several respects, those we have previously described in rodent liver during regeneration and tumor promotion and also those seen in rodent hepatomas. They may reflect, therefore, a common profile of gene expression relevant to cell proliferation.

Animals↗
PubMed1989

Effects of triiodothyronine and tamoxifen on cell transformation induced by an activated c-Ha-ras oncogene.

We have found that the thyroid hormone 3,5',3'-triiodo-L-thyronine stimulates the transformation of Rat 6 fibroblasts when these cells are transfected with an activated human c-Ha-ras oncogene (T24). 3,5',3'-Triiodo-L-thyronine did not further augment the stimulation of oncogene-induced transformation obtained with a phorbol ester tumor promoter (12-O-tetradecanoylphorbol-13-acetate) or a factor from fetal calf serum. On the other hand, tamoxifen, an antiestrogen that also inhibits protein kinase C, markedly inhibited Ha-ras-induced cell transformation in the presence of 12-O-tetradecanoylphorbol-13-acetate or fetal calf serum. Time course studies and Southern blot analyses of DNAs isolated from transformed foci provided evidence that 3,5',3'-triiodo-L-thyronine and tamoxifen do not exert their effects simply by enhancing or inhibiting integration of the transfected oncogene into cellular DNA. These findings indicate that hormonal factors can modulate the ability of an activated Ha-ras oncogene to transform cells. They may be relevant to the process of multistage carcinogenesis in vivo.

Animals↗