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Biomedical subjects

C Borner

Publications and source records attributed to C Borner.

At least 37 records · Page 2Linked to original sources
PubMed1997

Overxpression of Bcl-2 inhibits UVA-mediated immediate apoptosiinrat 6 fibroblasts: evidence for the involvement of Bcl-2 as an antioxidant.

We examined the effect of broad spectrum UVA (320-380 nm) and UVB (290-320 nm) radiation on the induction of apoptosis in the rat 6 fibroblast cell line (R6). UVA, but not UVB, induces apoptosis in this cell line. The morphological changes and DNA ladders associated with apoptosis occurred within the first 4 h after UVA irradiation, a phenomenon referred to as "immediate" apoptosis. From previous studies, it is known that Bcl-2 inhibits most types of apoptotic cell death. Overexpression of mouse Bcl-2 in the R6 fibroblasts inhibited the UVA-induced immediate apoptosis. The induction of the heme oxygenase 1 (HO-1) gene by UVA is a general response to oxidative stress. As a marker of oxidative stress, we monitored the effect of Bcl-2 overexpression on the level of HO-1 mRNA accumulation after UVA irradiation. The results showed that the overexpression of Bcl-2 in the R6 fibroblasts strongly reduces the level of HO-1 induction from 12.5- to 4.9-fold. We propose that Bcl-2 expression inhibits UVA-induced immediate apoptosis via an antioxidant pathway, suppressing either the generation or effects of specific UVA-mediated reactive oxygen species.

Animals↗
PubMed1996

Diminished cell proliferation associated with the death-protective activity of Bcl-2.

The oncogene product Bcl-2 effectively spares cells from programmed cell death (apoptosis). The molecular mechanism underlying this death-protective activity has, however, remained enigmatic. Here we show that induction of Bcl-2 expression is consistently associated with a retardation of mammalian cell proliferation due to a prolongation of the G1 phase of the cell cycle. Whereas cells lacking Bcl-2 expression die from any point of the cell cycle in response to apoptotic agents, Bcl-2-overexpressing cells accumulate in the G0/G1 phase and are protected from cell death. Co-expression of Bax, a negative regulator of Bcl-2, reverts both the cell death protective and proliferation retarding activities of Bcl-2. Moreover, a Bcl-2 mutant defective in death protection does not affect cell division. These findings indicate that Bcl-2 contributes to cell survival by diminishing the rate of cell proliferation.

Animals↗
PubMed1996

Bcl-2 interrupts the ceramide-mediated pathway of cell death.

Ceramide, a product of sphingomyelin turn-over, has been proposed as a novel lipid second messenger with specific roles in mediating antiproliferative responses including apoptosis and cell cycle arrest. In this study, we examine the relationship between the ceramide-mediated pathway of growth suppression and the bcl-2 protooncogene. In ALL-697 leukemia cells, the addition of the chemotherapeutic agent vincristine resulted in a time-dependent growth suppression characterized by marked apoptosis. The effects of vincristine on cell death were preceded by a prolonged and sustained accumulation of endogenous ceramide levels reaching -10.4 pmol ceramide/nmol phospholipids at 12 hr following the addition of vincristine--an increase of 220% over vehicle-treated cells. Overexpression of bcl-2 resulted in near total protection of cell death in response to vincristine. However, the ceramide response to vincristine was not modulated by overexpression of bcl-2, indicating that bcl-2 does not interfere with ceramide formation. Overexpression of bcl-2 prevented apoptosis in response to ceramide, suggesting that bcl-2 acts at a point downstream of ceramide. On the other hand, bcl-2 did not interfere with the ability of ceramide to activate the retinoblastoma gene product or to induce cell cycle arrest, suggesting that the effects of ceramide on cell cycle arrest can be dissociated from the effects on apoptosis. These studies suggest that ceramide and bcl-2 partake in a common pathway of cell regulation. The results also cast ceramide as a gauge of cell injury rather than an "executor" of cell death with clearly dissociable biological outcomes of its action depending on downstream factors.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Bcl-2 overexpression blocks activation of the death protease CPP32/Yama/apopain.

The C. elegans gene product ced-9 inhibits programmed cell death by negatively regulating the death-mediating protease ced-3. The mammalian homolog of ced-9 is the oncoprotein Bcl-2. Overexpression of Bcl-2 spares mammalian and nematodal cells from dying and prevents ectopic cell death in ced-9 loss-of-function mutants. Although Bcl-2 has been shown to act as an antioxidant under certain conditions, additional functions have emerged from studies under low oxygen pressure. Here we show that Bcl-2 overexpression impairs activation of the interleukin-1beta converting enzyme-related death protease CPP32/Yama/apopain, the mammalian homolog of ced-3. When U937 monocytes undergo programmed cell death in response to tumor necrosis factor alpha, the inactive CPP32 precursor is cleaved into its active forms. As a consequence poly(ADP ribose) polymerase, a major substrate of CPP32, is faithfully cleaved into a 85 kD fragment. Bcl-2 overexpressing cells are protected from tumor necrosis factor alpha-induced death and display neither CPP32 maturation nor PARP cleavage. The inhibitory effect of Bcl-2 on CPP32 activation is indirect since no physical interaction between the two proteins could be detected. These results indicate that Bcl-2 neutralizes an unknown cellular activator of CPP32 to save cells from programmed cell death.

Animals↗
PubMed1995

Two closely related isoforms of protein kinase C produce reciprocal effects on the growth of rat fibroblasts. Possible molecular mechanisms.

We have previously reported that two closely related protein kinase C (PKC) isoforms, PKC alpha and PKC beta I, had divergent effects on the growth and transformation of the same parental R6 rat embryo fibroblast cell line (Housey, G. M., Johnson, M. D., Hsiao, W.-L. W. O'Brian, C. A., Murphey, J. P., Kirschmeier, P., and Weinstein, I. B. (1988) Cell 52, 343-354; Borner, C., Filipuzzi, I., Weinstein, I. B., and Imber, R. (1991) Nature 353, 78-80). Whereas cells that overexpress PKC beta I lost anchorage dependence, grew to higher saturation densities, and generated small tumors when injected into nude mice, none of these properties were seen with cells that overexpress PKC alpha. In fact, the latter cells grew even slower and to lower saturation densities as compared to control cells. Here we investigate possible molecular mechanisms underlying the reciprocal effects of PKC alpha and PKC beta I. Overexpression of both isoforms enhanced 12-O-tetradecanoyl phorbol-13 acetate-induced expression of the growth regulatory genes c-jun, c-myc, and collagenase and enhanced feedback inhibition of epidermal growth factor receptor binding and cellular levels of diacylglycerol. However, the cells overexpressing PKC beta I differed from those overexpressing PKC alpha by displaying a decreased requirement for growth factors and by the production of a mitogenic factor. Thus, the basis for enhanced growth and transformation of cells overexpressing PKC beta I may be the establishment of an autocrine growth factor loop. These findings may be relevant to the roles of specific isoforms of PKC in carcinogenesis and tumor growth.

Animals↗
PubMed1995

Regulation of Fas(Apo-1/CD95)- and perforin-mediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl-2.

Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.

Animals↗
PubMed1994

The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.

Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.

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↗
PubMed1992

Expression of four protein kinase C isoforms in rat fibroblasts. Distinct subcellular distribution and regulation by calcium and phorbol esters.

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, consists of a family of at least eight distinct lipid-regulated enzymes. How the various PKC isozymes are regulated in vivo and how they couple to particular cellular responses is largely unknown. We have examined the expression and regulation of PKC isoforms in R6 rat embryo fibroblasts. Northern and Western blot analyses indicate that these cells express four PKC isoforms, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta; of which nPKC epsilon and nPKC delta are the most abundant. In agreement with the simultaneous presence of cPKC and nPKC isozymes, both Ca(2+)-dependent and -independent PKC activities were detected in extracts of these cells. cPKC alpha and nPKC zeta were predominantly localized in the cytosol when subcellular fractionation was carried out in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. When cell lysis was carried out in the presence of Ca2+, greater than 50% of cPKC alpha redistributed to the particulate fraction, whereas nPKC zeta remained in the cytosol. In contrast to cPKC alpha and nPKC zeta, 60-80% of nPKC epsilon and nPKC delta were located in a Ca(2+)-insensitive, membrane-bound form. Treatment of R6 cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA), resulted in the translocation of all four PKC isozymes to the membrane fraction, and the subsequent down-regulation of cPKC alpha, nPKC zeta, and nPKC delta, nPKC epsilon, however, was only partially down-regulated in response to long-term TPA exposure. Overproduction of exogenous cPKC beta I in R6 cells conferred partial resistance of nPKC delta to TPA-induced down-regulation and potentiated the resistance of nPKC epsilon to down-regulation. These results demonstrate that the multiple isoforms of PKC which coexist within a single cell type are differentially regulated by extra- and intracellular stimuli and may thereby influence growth control and transformation via distinct mechanisms.

Animals↗
PubMed1992

Expression of four protein kinase C isoforms in rat fibroblasts. Differential alterations in ras-, src-, and fos-transformed cells.

In the accompanying study (Borner, C.B., Guadagno, S. N., and Weinstein, I. B. (1992) J. Biol. Chem. 267, 12892-12899) we found that R6 embryo fibroblasts express four isoforms of PKC, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta whose subcellular distribution, activation, and down-regulation are differentially regulated. Furthermore, we demonstrated that overproduction of an exogenous cPKC beta I isoform in these cells (R6-PKC3) altered the TPA-induced down-regulation of nPKC delta and nPKC epsilon. In this paper we show that transformation of R6 or R6-PKC3 cells with a variety of different oncogenes results in differential alterations in expression of individual PKC isoforms. R6 or R6-PKC3 cells transformed by an activated c-H-ras oncogene displayed a marked increase in the expression of both cPKC alpha and nPKC delta, decreased expression of nPKC epsilon, and no change in the expression of nPKC zeta. These alterations occurred at both the mRNA and protein levels but did not significantly affect the subcellular distribution of any of the four isoforms. Studies using actinomycin D and nuclear run-off assays indicated that the increased expression of cPKC alpha in ras-transformed cells was due to increased de novo transcription rather than increased mRNA stability. Qualitatively similar, but less extensive changes in the expression of the four PKC isoforms were seen in v-fos-transformed R6-PKC3 cells. Decreased expression of nPKC epsilon was also seen in the v-src-transformed R6- and R6-PKC3 lines; however, the cellular level of cPKC beta I appeared to be a limiting factor in mediating the effects of v-src on the increased expression of cPKC alpha and nPKC delta. Interestingly, no major changes in the levels of expression of any of the four PKC isoforms were found when R6 cells were transformed by myc, neu/erb-B2, or mos oncogenes. These results demonstrate that transformation of R6 cells by the oncogenes ras, src, and fos differentially alter the expression of three isoforms of PKC in the same host cell, and they suggest that individual isoforms may play distinct roles in mediating cellular transformation by specific oncogenes.

Alkaloids↗
PubMed1992

Altered regulation of a major substrate of protein kinase C in rat 6 fibroblasts overproducing PKC beta I.

We have shown previously that the stable overproduction of protein kinase C beta I (cPKC beta I) in rat 6 (R6) embryo fibroblasts results in multiple cellular growth abnormalities. To characterize the pathways through which cPKC beta I acts to exert its effects, we have undertaken a biochemical analysis of the cell line R6-PKC3. The subcellular distribution of cPKC beta I in unstimulated R6-PKC3 cells was approximately 80% cytosolic and approximately 20% membrane bound, and treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in translocation and down-regulation of an appreciable fraction of the cPKC beta I enzyme. However, long term TPA treatment was not sufficient to down-regulate all of the overproduced enzyme from both the cytosolic and membrane fractions. Two-dimensional gel analysis of 32P-labeled cellular phosphoproteins from either untreated or TPA-treated cultures revealed only minor qualitative differences between R6-PKC3 cells and a vector control cell line, R6-C1. On the other hand, several quantitative differences in the level of phosphorylation of discrete protein spots were seen. The most prominent phosphoprotein was a previously described 80/87-kDa protein designated MARCKS (myristoylated alanine-rich C kinase substrate). Compared with R6-C1 cells, R6-PKC3 cells exhibited a 2-3-fold increase in the basal level of phosphorylation of MARCKS and after treatment with TPA, displayed a dramatic prolongation in phosphorylation of this protein. Additionally, treatment of R6-PKC3 cells with TPA led to a prolonged increase in both the cytosolic and total cellular level of the MARCKS protein and a pronounced decrease in the level of MARCKS mRNA. Taken together, these results indicate that overproduction of cPKC beta I markedly alters several parameters of the MARCKS protein which may be responsible, at least in part, for the altered phenotype of these cells.

Animals↗
PubMed1992

c-myc protein distribution. Neoplastic tissues of the human colon.

There is an extensive literature documenting the increased or deregulated expression of the c-myc oncogene in human malignancies. The authors have recently devised a sensitive immunocytochemical method for studying the tissue localization of c-myc protein in tissue sections of human colon. We have compared nuclear c-myc staining using a polyclonal rabbit anti-c-myc antibody and a mouse monoclonal myc antibody NCM II 274. Microscopic observation of the tissue specific pattern of c-myc protein distribution shows that nuclear staining intensity varies in normal and neoplastic crypt cell nuclei in parallel with morphologic criteria of neoplasia. These studies yield further information on the usefulness of c-myc protein as a prognostic indicator.

Cell Nucleus↗
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↗
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

Biosynthesis and posttranslational modifications of protein kinase C in human breast cancer cells.

Several forms of protein kinase C with molecular masses of 74-, 77-, and 80-kDa were detected in subcellular fractions of human breast cancer MDA-MB-231 cells which express the alpha-type protein kinase C. Several lines of evidence indicated that the 74-kDa is the precursor of the 77- and 80-kDa protein kinase C forms. (i) Pulse-labeling experiments revealed that protein kinase C is synthesized on membranes as a 74-kDa protein that can be chased into the 77- and the 80-kDa protein kinase C forms. (ii) The primary translation product of protein kinase C displayed an apparent molecular size of 74-kDa as determined by in vitro translation of poly(A)+ RNA from MDA-MB-231 cells. (iii) Incubation with serine/threonine-specific protein phosphatases (potato acid phosphatase and phosphatase 1 or 2A) resulted in the complete dephosphorylation of the 77-kDa to the 74-kDa protein kinase C form. Protein kinase C appears to be synthesized in membranes as an unphosphorylated and presumably inactive 74-kDa form that is converted into the active 77- and 80-kDa protein kinase C by post-translational modification involving at least two phosphorylation steps. The first phosphorylation is probably achieved by a specific, yet unidentified, "protein kinase C kinase" since the 74-kDa protein kinase C species did not undergo autophosphorylation and was neither a substrate for the purified protein kinase C, S6 kinase, phosphorylase kinase, casein kinase II, nor for the catalytic subunit of cAMP-dependent protein kinase. Except for phosphorylase kinase and the catalytic subunit of the cAMP-dependent protein kinase, phosphorylation of the 77-kDa protein kinase C form with purified protein kinase C (autophosphorylation), S6 kinase or casein kinase II shifted the molecular mass of the 77-kDa protein kinase C to 80-kDa. Prolonged exposure of MDA-MB-231 cells to phorbol 12-myristate 13-acetate not only leads to a complete down-regulation of protein kinase C activity but also to an accumulation of 74-kDa protein kinase C due to a retarded conversion of the 74-kDa into the 77- and 80-kDa protein kinase C forms in these cells. Our data indicate that tumor promoters additionally interfere with the posttranslational processing that converts the 74-kDa protein kinase C precursor into the 77- and 80-kDa forms of the enzyme.

Breast Neoplasms↗