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Biomedical subjects

I B Weinstein

Publications and source records attributed to I B Weinstein.

At least 91 records · Page 5Linked to original sources
PubMed1995

Deregulated expression of cyclin D1 and other cell cycle-related genes in carcinogen-induced rat mammary tumors.

Dysregulation of cyclin expression has been reported for several human malignancies, including breast cancer. To further investigate the role of cyclin genes in mammary tumorigenesis we analyzed the expression of cyclins D1, E and A and other cell cycle-related proteins in a series of nine N-methyl-N-nitrosourea-induced primary rat mammary tumors. Western blot analysis revealed a 10- to 15-fold increase in the level of cyclin D1 protein in most (7/9) of the tumors, when compared with normal rat mammary gland. The two tumors that did not show this increase also displayed negligible levels of the retinoblastoma protein. A moderate increase, 1.5- to 2-fold, in the level of cyclin E was observed in four tumors and three tumors displayed abnormal low molecular weight cyclin E-related proteins. None of the tumors showed amplification of the cyclin D1 or E genes when studied by Southern blot analysis. All nine tumors showed a 2- to 6-fold increase in the level of cyclin A protein. Most of the tumors also displayed a marked increase in levels of the CDK2 and CDK4 proteins. These changes did not appear to be simply a consequence of increased cell proliferation, as assessed by proliferating cell nuclear antigen analysis. Thus, aberrant expression of cyclins and other cyclin-related genes occurs frequently in mammary tumorigenesis in both rodents and humans.

Animals↗
PubMed1995

Activated ras genes occur in human actinic keratoses, premalignant precursors to squamous cell carcinomas.

UNLABELLED: BACKGROUND/DESIGN: The clonal theory of cancer predicts that transformed cells within a given tumor are derived from a single initiated precursor. Advancement of this precursor through various stages of tumor development occurs with the further accumulation of selective genetic and epigenetic lesions. Mammalian ras genes are important constituents of mitogenic signaling pathways, and when activated, they contribute to deregulated cellular growth. Activated ras genes play important roles in the development of certain skin tumors. Studies on a number of animal tumor model systems have shown that ras gene activation can be an early and perhaps initial event in the development of skin tumors. Activated ras genes are also found in a significant percentage of somatic human squamous cell carcinomas. To gain retrospective insight into the stages at which activated ras genes contribute to squamous cell carcinoma development, we investigated their incidence in actinic keratoses, premalignant precursors to squamous cell carcinomas. Using a nonradioactive polymerase chain reaction-based method developed in our laboratory, we examined a panel of 19 actinic keratoses and 33 squamous cell carcinomas for activated ras genes. RESULTS: DNA analysis revealed ras gene mutations in three (16%) of 19 actinic keratoses and in four (12%) of 33 squamous cell carcinomas. Activating mutations occurred at codon 12 of the K-ras gene, and codons 12, 13, and 61 of the H-ras gene. All positive actinic keratoses and squamous cell carcinomas occurred in sun-exposed regions. CONCLUSIONS: Activated ras genes can play important roles during early stages of squamous cell carcinoma development. Aberrant repair of UV-induced pyrimidine dimers is a likely cause of this activation.

Base Sequence↗
PubMed1994

Overexpression of human cyclin D1 reduces the transforming growth factor beta (TGF-beta) type II receptor and growth inhibition by TGF-beta 1 in an immortalized human esophageal epithelial cell line.

Cyclin D1 has been implicated in G1 cell cycle progression and is frequently amplified, overtranscribed, and oversynthesized in human tumors, including esophageal carcinomas. To further address the role of cyclin D1 in cell cycle control and tumorigenesis, we have stably transfected the human cyclin D1 in the nontumorigenic esophageal epithelial cell line HET-1A. These transfected cells, which express increased amounts of cyclin D1, have enhanced colony-forming efficiency and saturation density and are resistant to growth inhibition by TGF-beta 1 compared with the parental cell line or a control vector cell clone. The clones which express increased amounts of cyclin D1 exhibited a decrease in the amount of TGF-beta type II receptor, indicating a plausible mechanism for their diminished response to TGF-beta 1. Therefore, deregulated expression of the cyclin D1 gene can modulate the negative growth factor pathway of TGF-beta 1 and may disturb the control of epithelial cell proliferation in esophageal carcinogenesis.

Cell Cycle↗
PubMed1994

Overexpression of protein kinase C beta I in a murine keratinocyte cell line produces effects on cellular growth, morphology and differentiation.

The present study demonstrates that the murine keratinocyte cell line 3PC expresses the Ca(2+)-insensitive isoforms of protein kinase C (PKC) delta, epsilon, zeta and (at both the mRNA and protein levels), but does not express the Ca(2+)-sensitive PKC isoforms alpha, beta or gamma. Recombinant retroviral gene transduction was used to develop derivatives of this cell line that stably express high levels of 1 PKC beta I-related transcripts and proteins, and have increased levels of Ca(2+)-stimulated PKC enzyme activity. Functional overexpression of the PKC beta I isoform in 3PC cells enhances both 12-O-tetradecanoyl phorbol-13-acetate-induced growth inhibition, and Ca(2+)-induced morphologic differentiation.

Animals↗
PubMed1994

Calcium reduces the increased fecal 1,2-sn-diacylglycerol content in intestinal bypass patients: a possible mechanism for altering colonic hyperproliferation.

Diacylglycerol (DAG) is a second messenger for protein kinase C, an enzyme with a key role in cellular signal transduction and growth control. In previous studies, it was demonstrated that DAG is produced by intestinal microflora. Bacterial DAG production is increased by bile acids and phospholipids, both of which may be precipitated by calcium. We have demonstrated that fecal total lipids, bile acids, and rectal epithelial proliferation are increased in intestinal bypass (IB) patients. Calcium was shown to alter fecal lipid composition and to reduce cell proliferation. In the present study, fecal DAG content and 14C-labeled DAG, 14C-phosphatidylcholine, and 14C-phosphatidylinositol metabolism were measured in 24-h stool collections in 15 stable IB patients before and after 3-month therapy with oral elemental calcium, 2.4 or 3.6 g/day. Fecal DAG concentration and output in IB patients were > 25- and > 200-fold greater than in normal controls. Oral calcium markedly reduced fecal DAG concentration and output and increased DAG, phosphatidylcholine, and phosphatidylinositol metabolism without enhancing DAG production. We conclude that fecal DAG content is markedly elevated post-IB and that calcium supplementation in these patients reduces fecal DAG and accelerates bacterial metabolism of DAG and its precursors. In separate studies, we have found that calcium supplementation also decreases rectal hyperproliferation in IB patients. Taken together, these findings suggest that a high luminal level of DAG enhances colonic cell proliferation and that calcium reduces cell proliferation in part by decreasing the level of DAG.

Calcium↗
PubMed1994

Growth inhibition of human melanoma-derived cells by 12-O-tetradecanoyl phorbol 13-acetate.

In vitro growth of 6 human melanoma-derived cell lines was inhibited markedly by the phorbol-ester tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of several isoforms of protein kinase C (PKC). Utilizing PKC isoform-specific antibodies in immunoblotting experiments, we found that the PKC alpha and PKC epsilon isoforms were expressed in all of the 6 melanoma cell lines tested, whereas the PKC beta isoform was expressed at detectable levels in only 2 of the 6 cell lines. The SK-Mel-173 melanoma cell line, which had relatively high levels of PKC beta mRNA and protein expression, and which was also the most sensitive to cell growth inhibition by TPA, was used to isolate clones whose growth was less inhibited by TPA. Immunoblotting experiments revealed that in parental SK-Mel 173 cells PKC beta was rapidly down-regulated to below detectable levels after treatment for 48 hr with TPA, but that in TPA-resistant variant clones there was negligible down-regulation of PKC beta by TPA. On the other hand, treatment of parental and TPA-resistant SK-Mel 173 cells with TPA led to partial down-regulation of PKC alpha in both cell lines. Total PKC enzyme activity was also greater in TPA-resistant cells than in parental SK-Mel 173 cells. Our results show that TPA might inhibit the growth of melanoma cells by causing down-regulation of specific isoforms of PKC that are required to maintain the growth of these cells.

Cell Division↗
PubMed1994

The effects of bile acids on phospholipase C activity in extracts of normal human colon mucosa and primary colon tumors.

Phospholipase C (PLC) activity and its response to stimulation by bile acids was assayed in cellular extracts from 16 primary human colon tumors of various Duke's stages and paired adjacent normal mucosal samples. In the absence of bile acid, there was negligible degradation of phosphatidylinositol (PI) 1-stearoyl-2-[14C]-arachiodonoyl by tumor or normal tissue, but the addition of deoxycholic acid (DCA) or taurocholic acid (TCA) resulted in concentration-dependent and time-dependent stimulation of diacylglycerol (DAG) formation at optimal concentrations of 2 mM DCA and 4 mM TCA. Triton X-100 (0.125-1.0%) inhibited rather than enhanced the PI-degrading activity of these extracts, indicating that the stimulatory effects of DCA and TCA were not simply due to a detergent effect. Under the same assay conditions there was only a small amount of labeled monoacylglycerol or free arachidonic acid produced by extracts incubated in the absence or presence of DCA or TCA. No major differences in DAG production from PI were seen between paired samples of normal colon mucosa and primary colon tumors, in assays done in the presence of 2 mM TCA. Extracts from tumors in the distal part of the colon had higher activity than those from the proximal colon. This was also true for the extent of release of free arachidonic acid from labeled PI. Under the same conditions, labeled phosphatidylcholine or phosphatidylethanolamine did not serve as substrates for the colon mucosa or tumor extracts. Nor was there significant hydrolysis of the labeled DAG (1-stearoyl-2-14C-arachidonoylglycerol) by normal colon mucosa or tumor extracts, in the absence or presence of DCA or TCA. On the other hand, a low level of DAG lipase activity was detected in the presence of Triton X-100. These findings provide the first evidence that normal human colon mucosa and primary colon tumors contain a PI-specific PLC activity that is markedly stimulated by bile acids. Our results also suggest that bile acids may enhance colon carcinogenesis by acting on this enzyme system, thereby influencing signal transduction pathways in the target cells.

Aged↗
PubMed1994

Expression of multiple isoforms of protein kinase C in normal human colon mucosa and colon tumors and decreased levels of protein kinase C beta and eta mRNAs in the tumors.

Previous studies have suggested that protein kinase C (PKC) may play an important role in colon carcinogenesis and that human colon tumors have less total PKC enzyme activity than normal tissue. Because PKC is a multigene family that encodes for at least 11 distinct isoforms, in the study reported here we analyzed the expression of six of these isoforms at the mRNA level by northern blot hybridization in 22 pairs of primary colon tumors (of various stages), and adjacent normal mucosa samples. We found that the normal mucosa samples expressed the mRNAs of the following isoforms of PKC, in decreasing order of abundance: PKC delta > PKC eta > PKC alpha > PKC beta > PKC epsilon. There was no consistent difference in the levels of PKC alpha, PKC delta, and PKC epsilon mRNAs between the normal mucosa and the tumor samples. PKC gamma was expressed at a very low level in two of the colon tumors but could not be detected in the remaining tumors or any of the normal mucosa samples. The levels of both PKC beta and PKC eta mRNAs were significantly lower in the tumor samples than in the normal mucosa samples, and this was true of adenomas as well as Dukes' stage A, B, and C adenocarcinomas. Furthermore, the decrease in PKC eta mRNA appeared to be greater in the more poorly differentiated carcinomas. This finding is of interest because PKC eta is normally expressed in the more differentiated cells of epithelial tissues. The decreased levels of both PKC beta and PKC eta mRNAs occurred early in the multistage process of colon carcinogenesis, as it was also seen in adenomas. The functional significance of these changes remains to be determined.

Adenoma↗
PubMed1994

Molecular cloning of TPAR1, a gene whose expression is repressed by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA).

We previously isolated a partial cDNA sequence, termed TPAR1 (TPA repressed gene 1), from a cDNA library constructed from C3H10T1/2 mouse embryo fibroblasts treated with TPA, using a differential screening procedure. (M.D. Johnson et al. Mol. Cell. Biol. 7, 2821-2829, 1987). In the present study, we have cloned two corresponding full-length 1.9- and 3.4-kb cDNAs of TPAR1 from murine cDNA libraries. Sequence analysis of these TPAR1 cDNAs revealed that they encode 89 and 93 amino acid polypeptides, respectively, with a putative leader sequence and show significant homology with the human cytokine interleukin-8 (IL-8) and its superfamily. Genomic DNA isolation and structural characterization provide evidence that the TPAR1 mRNAs are transcribed from a single gene with alternative splicing. TPAR1 mRNAs are expressed ubiquitously among adult mouse tissues as three major transcripts, 1.9, 3.4, and 6.5 kb, whose expression depends on the tissue type. The levels of TPAR1 mRNAs were markedly decreased in fibroblasts following TPA treatment and also in serum-deprived quiescent fibroblasts stimulated by serum. The levels of TPAR1 mRNAs were dramatically down-regulated in regenerating rat liver when compared to normal adult liver. In addition, there was no detectable expression of TPAR1 in three rat hepatoma cell lines and several transformed fibroblast cell lines. Thus, the TPAR1 gene is a new member of the cytokine IL-8 superfamily, whose expression is down-regulated in rapidly dividing cells. Further studies are required to determine whether it plays a negative role in controlling cell proliferation and tumorigenesis.

Amino Acid Sequence↗
PubMed1994

Suppression of mitogenic activity by stable expression of the regulatory domain of PKC beta.

The amino-terminal regulatory domain portion of each protein kinase C (PKC) family member (which in the case of PKC beta 1 includes the pseudosubstrate, C1, V1 and C2 domains) plays an important role in regulating the kinase activity of the carboxyl-terminal catalytic domain. To examine the possibility that this regulatory domain region (designated 'PAT') might have biological functions independent of the catalytic domain, we have developed derivatives of R6 cells which stably express a truncated PKC beta 1 cDNA that encodes the amino-terminal 317 amino acids, including the entire regulatory domain. These R6-plPAT cells express abundant amounts of a 38 kDa protein which binds a labeled phorbol ester, but lacks protein kinase activity. In contrast to the 79 kDa PKC beta 1 holoenzyme which, when overexpressed in R6 cells, is found mostly in the cytosol, the 38 kDa PAT protein is predominantly associated with the particulate subcellular fraction. Furthermore, the PAT protein fails to show down-regulation following treatment of R6-plPAT cells with 12-O-tetradecanoylphorbol-13-acetate (TPA). Evidence is also presented that TPA-stimulated growth is suppressed in R6-plPAT cells. These findings suggest that the PKC beta 1 regulatory domain could be involved in the suppression of mitogenic signaling.

Animals↗
PubMed1994

Effects of increased expression of protein kinase C on radiation-induced cell transformation.

The in vitro oncogenic transformation of C3H 10T1/2 cells by ionizing radiation is known to be enhanced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). It is also known that the activation of protein kinase C (PKC) by TPA is an important step in its tumor-promoting effect. In the present study, we examined the effects of overexpression of a specific isoform of PKC, PKC beta 1 on gamma-ray-induced transformation of 10T1/2 cells. In addition, the effects of overexpression of PKC beta 1 on the malignant phenotype of a previously transformed 10T1/2 cell line were also evaluated. Derivatives of 10T1/2 cells that stably overexpress PKC beta 1 were obtained by transduction with the retroviral expression vector pMV7 carrying the rat PKC beta 1 cDNA sequence. We found that the parental 10T1/2 cells and a control cell line 10T1/2 MV7, which carried only the pMV7 vector without the cDNA insert, expressed dose-dependent transformation frequencies when exposed to gamma-rays. On the other hand, concurrently treated PKC-overexpressing cells that had an 11-fold increase in enzyme activity (PKC-4 cells) failed to yield any morphologically identifiable foci. Cell lines that expressed lower levels of PKC beta 1 were partially resistant to transformation by gamma-rays. Clonogenic survival data indicated that this observation was not due to radioresistance per se. Thus, overexpression of PKC beta 1 did not appear to function as an endogenous substitute for TPA in promoting radiation-induced transformation. Furthermore, overexpression of PKC did not reverse the transformation phenotypes in tumorigenic 10T1/2 cells once it was established. These findings are discussed with respect to the specific roles of individual isoforms of PKC in growth control.

Animals↗
PubMed1993

Calcium-dependent activation of protein kinase C. The role of the C2 domain in divalent cation selectivity.

Activation of certain isoforms of protein kinase C (cPKCs) requires Ca2+ and is associated with a conserved C2 domain that is not present in Ca(2+)-independent isoforms (nPKCs). The site(s) of Ca2+ binding and the role of the C2 domain have not been previously identified. We have analyzed phosphatidylserine-dependent Ca2+ binding to fusion proteins expressed in Escherichia coli that carry various modifications in the regulatory region of cPKC beta 1 or nPKC epsilon. Ca2+ is bound mainly to the C1 domain of PKC beta 1, but the C2 domain confers specificity for Ca2+ binding when compared with Mg2+ and Mn2+. We propose that in cPKCs there is selective binding of Ca2+ to a pocket formed by the C1 and C2 domains. This induces a change in conformation that activates the enzyme. In nPKCs, the cation binding pocket is less specific for Ca2+ because it lacks the C2 domain. Therefore, divalent cations like Mg2+ can bind to it, thereby abrogating the requirement of Ca2+ for enzyme activation.

Animals↗
PubMed1993

Amplification and overexpression of cyclin D1 in human hepatocellular carcinoma.

Amplification of the chromosome 11q13 region occurs in several types of human cancer including esophageal, breast, lung, bladder and hepatocellular carcinoma (HCC). The gene cyclin D1 maps to this region in close proximity to two proto-oncogenes hst-1 and int-2. We previously demonstrated that cyclin D1 was not only amplified but also overexpressed in about 30% of human esophageal cancers. To investigate the role of cyclin D1 in human hepatocellular carcinoma (HCC), DNA from 30 HCC and 5 control liver tissues from Taiwan and also the HCC cells lines HepG2 and Hep3B, were examined for amplification of the cyclin D1 gene. A 3 to 20-fold amplification was found in 4 of the 30 (13%) HCC samples but not in any of the 5 control tissues or the 2 cell lines. Immunohistochemical analysis of cyclin D1 indicated overexpression of this protein in tumors that displayed gene amplification. Weak or negative staining was observed in the other HCC samples as well as in the control tissues and cell lines. These data suggest that increased expression of cyclin D1 may play an important role in the development of a subset of human HCC, perhaps by perturbing normal control of the cell cycle.

Blotting, Northern↗
PubMed1993

Altered expression of the cyclin D1 and retinoblastoma genes in human esophageal cancer.

We have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin D1 gene. We also examined 36 of these 50 carcinomas for expression of the cyclin D1 and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3- to 10-fold amplification of the cyclin D1 gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin D1 protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin D1 gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show amplification of the cyclin D1 gene and expressed only low levels of the cyclin D1 protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin D1 gene.

Blotting, Southern↗
PubMed1993

The regulatory domain of protein kinase C beta 1 contains phosphatidylserine- and phorbol ester-dependent calcium binding activity.

Certain isoforms of protein kinase C (PKC) require both Ca2+ and phospholipid for optimum activity. However, little is known about the nature of the interaction between PKC and Ca2+. The present study demonstrates that the isolated regulatory domain of PKC beta 1, when synthesized as a fusion protein in Escherichia coli, binds 45Ca2+ with high affinity, but only in the presence of phosphatidylserine or 12-O-tetradecanoyl-phorbol-13-acetate. This binding is highly selective for Ca2+ since it is preferentially inhibited by excess non-radioactive Ca2+ when compared with the cations Mg2+, Mn2+, Na+, or K+. It appears, therefore, that the binding of Ca2+ to PKC requires a complex tertiary structure in the regulatory domain.

Base Sequence↗
PubMed1993

A comparison of techniques for introducing macromolecules into living cells.

Flow cytometry has been used to evaluate several techniques for introducing macromolecules into large numbers of living cells. One technique is cell fusion with red blood cell ghosts loaded with a fluorescent reporter molecule (RBCF). The second technique, termed osmotic lysis of pinosomes (OLP), involves a brief exposure of cells to a hypertonic solution containing the reporter molecule; subsequently, a hypotonic media is added which lyses the pinosomes formed during the hypertonic treatment. A third technique, scrape loading (SL), involves the creation of transient holes in the cell membrane through the application of mechanical forces, which allows for the passage of reporter molecules into cells. A comparison of these techniques is presented here. OLP appears to offer several advantages: It is a simple procedure, virtually all cells are fluorescently labelled, and it is capable of loading larger amounts of material more uniformly into cells while maintaining excellent viability.

Cell Fusion↗
PubMed1993

Correlation of p53 mutations with epidermal growth factor receptor overexpression and absence of mdm2 amplification in human esophageal carcinomas.

Esophageal carcinomas from 24 patients, most of whom were smokers and consumed alcoholic beverages daily, were analyzed for mutations in exons 5-8 of the p53 tumor suppressor gene. Mutations were identified by polymerase chain reaction amplification and direct sequencing in 12 of 24 (50%) of the samples; almost half of the mutations were at A:T base pairs. Nuclear accumulation of p53 protein, determined by immunohistochemistry with the CM-1 polyclonal antibody, was observed in all cases in which a missense mutation in the p53 gene was detected. None of the 24 carcinomas had amplification of the mdm2 gene, an alternate pathway to p53 loss of function. Alterations involving three other cancer-related genes associated with human esophageal carcinogenesis, c-erbB-1/epidermal growth factor receptor (EGFR), c-myc, and retinoblastoma (Rb), were examined by Southern blot or immunohistochemical analysis in the same sample set to explore the possibility of a link between oncogene activation and loss of tumor suppressor function. While no associations were observed between amplification of the c-myc or EGFR genes and p53 abnormalities, a significant correlation (P < 0.01) was seen between the presence of p53 mutation and EGFR overexpression. Absence of Rb protein, measured immunohistochemically, was observed in four tumors, none of which had aberrations of the p53 gene.

Carcinoma, Squamous Cell↗