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Biomedical subjects

I B Weinstein

Publications and source records attributed to I B Weinstein.

At least 127 records · Page 7Linked to original sources
PubMed1991

A peptide from the GAP-binding domain of the ras-p21 protein as well as azatyrosine block ras-induced maturation of Xenopus oocytes.

The ras-oncogene-encoded p21 protein is known to produce malignant transformation of NIH 3T3 cells as well as maturation of Xenopus oocytes when microinjected into these cells. p21 protein is known to bind a GTPase activating protein (GAP) intracellularly; residues 32-45 have been implicated in interacting with GAP. We demonstrate here that a peptide corresponding to residues 35-47 of p21 as well as the antibiotic azatyrosine inhibit the ras-induced maturation of Xenopus oocytes in a dose-related manner upon microinjection. We have previously shown that this p21 peptide and azatyrosine could inhibit the effects of p21 protein on cell transformation and pinocytosis in NIH 3T3 cells. In the present study, in which we have extended these results to the oocyte system, we also demonstrate that both partially inhibit insulin-induced oocyte maturation, a process which is thought to involve activation of endogenous p21 protein; on the other hand, both agents fail to inhibit oocyte maturation induced by progesterone, which is known not to act through p21 protein activation. Control studies with other peptides and tyrosine analogues support the selective nature of these events. These results suggest that both the p21-related peptide and azatyrosine have potent anti-ras effects intracellularly.

Alanine↗
PubMed1991

The modulation of growth by HMBA in PKC overproducing HT29 colon cancer cells.

To examine whether protein kinase C (PKC) plays a role in mediating growth inhibitory effects of hexamethylene bisacetamide (HMBA) we compared a control H29 colon cancer cell line to a derivative, HT29-PKC7, that overexpresses high levels of PKC beta 1. We found that although HMBA markedly inhibited the growth of the control cells, no inhibition was seen with the HT29-PKC7 cells. On the other hand the tumor promoter 12-0-tetradecanoyl-phorbol-13 acetate inhibited the growth of HT29-PKC7 cells, but no inhibition was seen with the control cells. Maximum inhibition of the growth of both cell lines was obtained by combined treatment with HMBA and TPA. These results may be relevant to the use of HMBA in combination with other agents in the therapy of specific cancers.

Acetamides↗
PubMed1991

Immunological quantitation of levels of tissue inhibitor of metalloproteinase-1 in human colon cancer.

Treatment of rodent cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate activates protein kinase C, leading to increased expression of several genes, including a gene originally designated TPA-S1 or phorbin (M. D. Johnson, G. M. Housey, P. T. Kirschmeier, and I. B. Weinstein, Mol. Cell Biol., 7: 2821-2829, 1987). Sequence analysis of this cloned gene indicated homology with human erythroid-potentiating activity and tissue inhibitor of metalloproteinase (TIMP-1). Elevated levels of phorbin mRNA have been observed in human colon tumors (J. G. Guillem, M. F. Levey, L. L. Hsieh, M. D. Johnson, P. LoGerfo, K. A. Forde, and I. B. Weinstein, Mol. Carcinogen., 3: 68-74, 1990) and this increase correlated with the extent of invasion. To further investigate this phenomenon at the protein level, monoclonal antibodies were developed against the recombinant form of TIMP-1. A competitive enzyme-linked immunosorbent assay was developed for quantitation of the TIMP-1 protein in tissue extracts. Elevated levels of TIMP-1 protein were found in 31 human colon tumors, compared to paired samples of adjacent normal mucosa. In a subset of samples, previously analyzed for phorbin mRNA levels (n = 25), there was a good correlation between the abundance of TIMP-1 protein and phorbin mRNA. Immunoaffinity column purification of tumor extracts followed by Western blot analysis was used to confirm the enzyme-linked immunosorbent assay data. These results provide evidence that phorbin and TIMP-1 represent the same gene. In addition, the immunoassays we have developed may be useful in further studies on the role of TIMP-1 in human colon cancer.

Aged↗
PubMed1991

Cancer prevention: recent progress and future opportunities.

Knowledge gathered during the past few decades from the fields of cancer epidemiology, carcinogenesis, biochemistry, and molecular biology, much of which was communicated through the pages of the journal Cancer Research, provides powerful new strategies for cancer prevention. Indeed, I believe that it is possible to draft a blueprint for a comprehensive approach to cancer prevention, to be used as we approach the 21st century. This plan includes: (a) expanded epidemiological and laboratory studies to identify the specific causes of human cancers; (b) intervention studies, such as risk reduction by the cessation of cigarette smoking, dietary modifications, chemoprevention, and the development of vaccines for viral agents implicated in human cancer (i.e., hepatitis B, human papilloma virus, Epstein-Barr virus, and certain retroviruses), and (c) early detection and screening. Concepts and methods that have developed in the field of cellular and molecular biology will accelerate this progress. Indeed, we are currently in a very exciting phase of cancer research that brings together fields of cancer research which in the past were often disparate and disconnected, i.e., basic laboratory studies, epidemiology, prevention, and treatment. Thus, the astounding advances in our understanding of the molecular biology of cancer provide a unified concept of the nature of the cancer cell and also suggest new clinical approaches to cancer etiology, prevention, and treatment. Many of the biomarkers being developed for etiological studies, using the approach of molecular epidemiology, can also serve as biomarkers or intermediate end points to evaluate the efficacy of dietary intervention and chemoprevention studies. Some of these biomarkers will also be useful for evaluating the efficacy of cancer therapy. Furthermore, certain compounds being developed for cancer chemoprevention, e.g., the retinoids, may also be useful in the therapy of fully established neoplasms. It seems likely, therefore, that even before the year 2000 these powerful and unifying themes will accelerate advances in both cancer prevention and treatment, thus leading to major reductions in both the incidence and mortality of human cancer.

Humans↗
PubMed1991

Failure of wild-type or a mutant form of protein kinase C-alpha to transform fibroblasts.

A mutant form of the alpha-isoform of protein kinase C (PKC) was recently isolated from an ultraviolet radiation-induced murine fibrosarcoma cell line and reported to transform mouse BALB/c 3T3 fibroblasts on transfection. Four point mutations in the regulatory domain were assumed to be responsible for its oncogenicity and unusual preference for membrane localization. Here, we report that overexpression of the reported mutant PKC alpha complementary DNA in three fibroblast cell lines, including BALB/c 3T3, does not enable these cells to grow in soft agar or nude mice. In addition, this mutant PKC alpha form seems to be indistinguishable from the wild-type PKC alpha with respect to its dependence on cofactors, phorbol ester binding, subcellular distribution and its effects on growth and morphology. These results fail to confirm the previous study and indicate that overexpression of either the wild-type or the reported mutant form of PKC alpha does not transform rodent fibroblasts.

Animals↗
PubMed1991

Effects of a fecapentaene on protein kinase C.

Protein kinase C (PKC) is a Ca2(+)- and phospholipid-dependent serine and threonine protein kinase which binds and is activated by tumor promoters such as the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). PKC can be activated in vitro by phosphatidylserine (PS) plus Ca2+. We report here that the compound fecapentaene-12 can replace the requirement for PS in the activation of PKC by Ca2+. In addition, at low concentrations fecapentaene-12 can enhance the activation of PKC by Ca2+ and PS. It can also either enhance or inhibit activation of PKC by the tumor promoter teleocidin, depending on the assay conditions. These results are of interest since fecapentaene is known to be a potent mutagen that is produced by Bacteroides species present in the lumen of the human colon. The present studies raise the possibility that this compound might also play a role in colon cancer by altering the activity of PKC.

Animals↗
PubMed1991

A self-assembling protein kinase C inhibitor.

Previous studies have described a dicationic anticarcinoma agent that can chemically assemble in situ from monocationic phosphonium salts. The chemical combination of these monocationic precursors in the micromolar concentration range, occurring after their uptake by cells, was probably responsible for their synergistic inhibition of cell growth and for their selective cytotoxicity to Ehrlich ascites murine carcinoma cells relative to untransformed epithelial cells. Here, we report that the dicationic product that forms in this assembly reaction is an in vitro inhibitor of protein kinase C (PKC) alpha and beta 1 isoforms, exhibiting IC50 values of 20.4 microM and 35 microM, respectively. The monocationic precursors proved to be much weaker inhibitors of PKC (IC50 values greater than 200 microM). When PKC is exposed to combinations of the two precursors, the enzymatic activity decreases steadily as a function of time. Using dose-response data and HPLC kinetic studies, we show that when the two precursor compounds are added as a combination to PKC under these conditions, the rate of formation of the inhibitory product follows the observed time course of decline in PKC activity under identical conditions. We discuss the possibility that antiproliferative effects against carcinoma cells of the preformed dication and of the combined monocationic precursors involve inhibition of PKC.

Animals↗
PubMed1991

Decreased levels of 1,2-sn-diacylglycerol in human colon tumors.

We have found that in 15 of 15 primary human colon tumors there was a significant decrease (by about 40%) in the levels of diacylglycerol when compared to paired adjacent normal mucosa samples. Assays on the same samples indicated that this decrease was seen both in tumors that did and did not display mutations in codon 12 of c-K-ras. These results, taken together with previous studies on protein kinase C, suggest that the protein kinase C signal transduction pathway is suppressed in human colon cancer.

Aged↗
PubMed1991

Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts.

We are using a Rat-6 fibroblast cell line that stably overexpresses the beta 1 isozyme of protein kinase C (PKC) to study regulation of phospholipid hydrolysis by PKC. Stimulation of control (R6-C1) or overexpressing (R6-PKC3) cells with phorbol ester results in an increase in diacylglycerol (DAG) mass with no increase in inositol phosphates, indicating that DAG is not formed by inositol phospholipid breakdown. A more dramatic DAG increase occurs in R6-PKC3 cells (4.0-fold over basal) compared to R6-C1 cells (1.5-fold over basal). To further define the source of DAG, phosphatidylcholine (PC) pools were labeled with [3H]myristic acid or with [3H]- or [32P]alkyllyso-PC and formation of labeled phosphatidylethanol, an unambiguous marker of phospholipase D activation, was monitored. Phorbol ester-stimulated phosphatidylethanol formation is 5-fold greater in the R6-PKC3 cell line. Formation of radiolabeled phosphatidic acid (PA) is also enhanced by PKC overexpression. In cells double-labeled with [3H]- and [32P]-alkyl-lysoPC, the 3H/32P ratio of PA and PC are identical 15 min after stimulation, suggesting that a phospholipase D mechanism predominates. In support of this, the PA phosphohydrolase inhibitor propranolol decreased phorbol 12-myristate 13-acetate-stimulated DAG formation by 72%. Increases in DAG and phosphatidylethanol were inhibited by the PKC inhibitors K252a and staurosporine. These results indicate that phospholipase D is regulated by the action of PKC. Enhanced phospholipase D activity may contribute to the growth abnormalities seen in PKC-overexpressing cells.

Alkaloids↗
PubMed1991

Two polychlorinated hydrocarbons cause phospholipid-dependent protein kinase C activation in vitro in the absence of calcium.

Polychlorinated hydrocarbons known to be nongenotoxic carcinogens were screened as activators of protein kinase C (PKC)-beta 1 either at high concentrations of Ca2+ or in the absence of Ca2+ (i.e., with 1 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N',-tetraacetic acid). Of those compounds tested, kepone and dicofol significantly stimulated PKC activity in the absence, but not the presence, of Ca2+. PKC activation was most pronounced in the presence of phosphatidylserine. Kepone and dicofol stimulated PKC activity 26% and 13%, respectively, as compared with the PKC activity (100%) stimulated by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Northern blot analysis of expression of TPA-inducible genes by kepone showed slight expression of phorbin and ornithine decarboxylase in murine embryo fibroblasts. Future studies are required to determine the relevance of PKC activation by kepone and dicofol to the known carcinogenicity of these compounds.

Animals↗
PubMed1991

Changes in expression of cellular oncogenes and endogenous retrovirus-like sequences during hepatocarcinogenesis induced by a peroxisome proliferator.

Previous studies have demonstrated that BR-931, a hepatic peroxisome proliferator, can induce liver tumours in mice and rats. Since alterations in gene expression may play a critical role in multistage hepatocarcinogenesis, the present studies examined the expression of the c-myc, c-H-ras, epidermal growth factor (EGF) receptor and ODC (ornithine decarboxylase) genes, as well as endogenous retrovirus-like sequences, in F344 rat liver during the first 8 weeks of feeding a 0.16% Br931 diet and in liver tumours induced by chronic feeding of this diet. Northern blot analysis of poly A + liver RNA samples showed an increase in the level of RNAs homologous to rat leukaemia virus (RaLV) but no significant change in the level of 30S-retrovirus related RNAs in the liver RNA samples obtained from rats during the first 8 weeks of feeding the diet containing BR931. An increase in the levels of c-myc, c-H-ras and ODC transcripts was also seen in the liver RNA samples from the treated rats. Of particular interest was a decrease in the abundance of EGF receptor transcripts in the liver RNA samples from rats fed the BR931 diet. Increased levels of RaLV, c-myc, and ODC RNAs were also seen in the tumours induced by BR931, but this was not the case for 30S and c-H-ras. The liver tumour samples also showed a decrease in EGF receptor RNA. These changes in cellular levels of specific RNAs resemble, in several respect, those we previously described in rodent liver during regeneration and tumour promotion, and also those seen in rodent hepatomas induced by other agents. Therefore, they may reflect a common profile of gene expression relevant to liver proliferation and carcinogenesis.

Animals↗
PubMed1991

A novel, plasmid-based system for studying gene rearrangements in mammalian cells.

We have developed a plasmid-based system for isolating gene rearrangements in mammalian cells by selection for reversion of a promoterless drug resistance gene. pNH4 contains the selectable marker gene neo under the control of the herpes simplex virus, thymidine kinase (tk) promoter and, upstream and in the opposite orientation, a dormant promoterless hygromycin B resistance gene (hph) that can be expressed following rearrangement events. An NIH 3T3 cell line stably transfected with pNH4 that has a spontaneous frequency of generation of Hphr colonies of approximately 10(-8) was isolated. Treatment of this line with ethyl methanesulfonate raised the frequency of Hphr colony formation approximately 100-fold. Approximately 60% (21 of 35) of ethyl methanesulfonate-induced Hphr clones showed rearrangements detectable by Southern blot analysis within a 40-kb region surrounding the integrated construct, including a nonhomologous recombination event and, possibly, a large insertion. Additionally, three Hphr clones showed evidence of gene amplification. Northern (RNA) blot analysis of hph mRNA suggests that the rearrangements may provide a function that allows the tk promoter to initiate transcription off the opposite strand, thus yielding hph transcripts. Cell lines harboring pNH4, or modifications of it, may be valuable for studying recombination mechanisms responsible for the various types of genetic rearrangements found in cancer cells.

Animals↗
PubMed1991

Nonmutagenic mechanisms in carcinogenesis: role of protein kinase C in signal transduction and growth control.

There is accumulating evidence that the multistage carcinogenic process is associated with the progressive acquisition of mutations in cellular proto-oncogenes and in growth-suppressor genes. At the same time, several types of evidence indicate that nongenotoxic agents and epigenetic events also play an important role in the evolution of tumors. One of the most intensively studied nongenotoxic agents is the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and related compounds. Since TPA appears to exert its biologic effects through protein kinase C (PKC), a key enzyme in signal transduction, we have studied this enzyme in considerable detail. Our strategy has been to perturb signal transduction by developing cell lines that overexpress the beta 1 isoform of PKC. Such derivatives of rat fibroblasts display alterations in morphology and growth factors, altered expression of c-myc, ornithine decarboxylase, and phorbin, and increased susceptibility to transformation by certain oncogenes, H-ras, myc, and fos. These findings provide direct genetic evidence that PKC plays a critical role in growth control and the action of certain growth factors, tumor promoters, and oncogenes. In related studies, we have characterized the beta 1 isoform that is overproduced in the above cell systems in terms of its biochemical, kinetic, and immunologic properties. The enzyme has several properties characteristic of native PKCs. A surprising finding is that c-H-ras-transformed derivatives of the cells that overexpress PKC beta 1 display a several-fold increase in the expression of the endogenous alpha 1 isoform of PKC and a decrease in the expression of the endogenous epsilon isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Rapid and sensitive nonradioactive detection of mutant K-ras genes via 'enriched' PCR amplification.

We have developed a rapid and highly sensitive nonradioactive method for the detection of a mutant codon 12 human c-K-ras allele in the presence of as many as 10(4) copies of the wild type codon 12 allele. This sensitivity is achieved by selective polymerase chain reaction (PCR) amplification of mutant K-ras gene sequences employing a two stage procedure. The first stage entails the amplification of both K-ras mutant and wild type codon 12 sequences, followed by a selective restriction enzyme digestion of only wild type sequences. The second stage involves a subsequent amplification of undigested amplified fragments, enriched in mutant codon 12 sequences. These products are subject to restriction length polymorphism analysis for the detection of point mutations at codon 12. This technique is rapid, nonradioactive, and eliminates the need for either oligonucleotide hybridization or DNA sequencing. Variations of this selective amplification procedure may prove promising for the detection of specific point mutations in heterogenous cell populations.

Adenocarcinoma↗
PubMed1991

Effects of overexpression of ornithine decarboxylase (ODC) on growth control and oncogene-induced cell transformation.

The enzyme ornithine decarboxylase (ODC) has been implicated in the control of cell growth, differentiation and tumor promotion. To further elucidate its precise role a murine ODC cDNA was inserted into the retrovirus-derived vector pMV7 and retrovirus-like particles were used to infect NIH3T3 and rat 6(R6) fibroblasts. Derivatives were obtained that stably express a 5-40 fold increase in ODC enzyme activity. Despite these high levels of enzyme activity the cells retained a normal morphology and displayed no major changes in growth properties in monolayer culture or in agar suspension. On the other hand, R6 cells that expressed high levels of ODC displayed a marked increase in susceptibility to morphologic transformation by an activated c-H-ras oncogene. These results provide the first evidence that ODC can cooperate with an activated oncogene in the process of cell transformation. Although the mechanism is not known, these findings may be relevant to the multistage carcinogenic process.

Animals↗
PubMed1991

The roles of specific isoforms of protein kinase C in growth control and human colon cancer.

Protein kinase C (PKC) consists of a family of lipid-regulated enzymes which play a pivotal role in signal transduction. Studies with the cell line R6-PKC3, a derivative of R6 rat fibroblasts that overexpresses PKC beta 1, provide direct evidence that this isoform of PKC influences cell morphology, growth control, the production of an autocrine growth factor, and the action of an activated H-ras oncogene. Analysis of 32P-labelled phosphoproteins indicates that R6-PKC cells display increased phosphorylation of a 80/87 kDa protein (designated MARCKS), and after treatment with TPA they display a dramatic prolongation in the phosphorylation and in the cytosolic accumulation of this protein. These alterations in MARCKS may be responsible, at least in part, for the altered growth properties of R6-PKC3 cells. We have also examined the expression of endogenous isoforms of PKC in R6 cells and oncogene-transformed derivatives. Normal R6 cells express four isoforms of PKC, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta; nPKC delta and nPKC epsilon are the most abundant. nPKC epsilon and nPKC delta have an unusual distribution since 60-80% is membrane-bound. In response to TPA, the cytosolic levels of all four PKC isozymes were recruited to the membrane fraction. Prolonged treatment of R6 cells with TPA caused total loss of cPKC alpha, nPKC delta and nPKC zeta but only a 60% reduction in nPKC epsilon.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line, Transformed↗
PubMed1991

1 alpha,25-dihydroxyvitamin D3-induced regulation of protein kinase C gene expression during HL-60 cell differentiation.

The human promyelocytic leukemia cell line HL-60 differentiates in vitro when treated with various inducers. It has previously been shown that protein kinase C (PKC) isozymes are modulated during granulocytic differentiation of HL-60 cells induced by dimethyl sulfoxide or retinoic acid (M. Makowske, R. Ballester, Y. Cayre, and O.M. Rosen, J. Biol. Chem., 263: 3402-3410, 1988; K. Hashimoto, A. Kishimoto, H. Aihara, I. Yasuda, K. Mikawa, and Y. Nishizuka, FEBS Left., 263: 31-34, 1990). HL-60 responds to 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) or to 12-O-tetradecanoylphorbol-13-acetate by giving rise to monocytic cells. In the present study, we demonstrate that treatment of HL-60 cells with 1,25-(OH)2D3 causes dramatic increases in PKC-alpha and PKC-beta protein levels detected by immunoblotting with PKC isoform-specific antibodies and in Ca(2+)- and phospholipid-dependent protein kinase activity. We also observed a transient increase in the steady-state levels of PKC-alpha and PKC-beta mRNA species in Northern blotting experiments, with maximal induction occurring 48 h after addition of 1,25-(OH)2D3. Analyses of 1,25-(OH)2D3-induced PKC mRNA expression by nuclear run-on transcription experiments suggest that the observed increases in PKC mRNA levels may occur by a posttranscriptional mechanism(s). In contrast to the transient increases in PKC mRNA levels, the increases in PKC Mr 80,000 protein species and in PKC enzyme activity were progressive in HL-60 cells treated with 1,25-(OH)2D3 between 1 and 5 days, thus implying the existence of a further up-regulation of PKC proteins occurring at the translational and/or posttranslational levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitriol↗