Biomedical subjects

L Raptis

Publications and source records attributed to L Raptis.

At least 37 records · Page 2Linked to original sources

PubMed1997

Elimination of intercellular junctional communication requires lower Ras(leu61) levels than stimulation of anchorage-independent proliferation.

One of the effects of transformation by a variety of factors is a decrease in gap junctional, intercellular communication (GJIC). To investigate the role of the Ras oncogene product in gap junction closure, and to incorporate GJIC into the gamut of transformation-related properties which can be regulated by increasing levels of oncogene expression, a panel of murine C3H10T1/2 and 3T3L1 fibroblasts was constructed in which graded increments of Ras(leu61) could reliably be obtained. The inducibly activated Ras(leu61) protein substantially reduced or eliminated GJIC at levels much lower than those needed for neoplastic transformation, as indicated by acquisition of the ability to proliferate in the absence of anchorage. These results indicate that disruption of GJIC, although necessary, is not sufficient for neoplastic transformation in this system.

3T3 Cells↗

PubMed1997

Cellular ras gene activity is required for full neoplastic transformation by the large tumor antigen of SV40.

To investigate the role of the cellular ras gene product in neoplastic transformation by the SV40 large tumor antigen (SVLT), murine C3H10T1/2 cells were rendered deficient in Ras activity by transfection with inducible or constitutive antisense ras gene constructs or through the introduction of the dominant-negative mutant, ras(asn17). Consistent with previous results, SVLT-induced morphological transformation was unaffected by the down-regulation of c-ras gene product activity. On the other hand, colony formation in soft agar and tumorigenicity in nude mice were drastically reduced in c-Ras-deficient cells. In addition, SVLT expression in C3H10T1/2 cells led to increased c-Ras activity, as determined by an increase in the Ras-bound GTP/GTP + GDP ratio. These results suggest that c-Ras is required for full neoplastic transformation by SVLT.

Animals↗

PubMed1996

Cellular Ras partly mediates gap junction closure by the polyoma virus middle tumor antigen.

Endogenous, cellular Ras proteins (c-Ras) mediate the transforming action of the polyoma virus middle Tumor antigen (mT), which is accompanied by elimination of gap junctional, intercellular communication (GJIC). In this report we show that reducing the c-Ras content of murine C3H10T1/2 fibroblasts (10T1/2) through the expression of an anti-sense ras gene, increased GJIC by 60-80% mT totally eliminated GJIC in normal 10T1/2 cells but it reduced GJIC no more than 50% in the c-Ras deficient lines. These results indicate that endogenous c-Ras is at least partly responsible for the mT-induced gap junction closure.

Animals↗

PubMed1996

Ras is involved in gap junction closure in proliferating fibroblasts or preadipocytes but not in differentiated adipocytes.

A decrease in gap junctional, intercellular communication (GJIC) has been associated with cells neoplastically transformed by a variety of factors. To investigate the role of the Ras oncogene product in gap junction function, a panel of murine C3H10T1/2 (10T1/2) fibroblasts was constructed in which the levels of ras gene expression could be effectively up- or down-regulated. Intercellular communication was measured using a novel technique of in situ electroporation of adherent cells on a partly conductive slide. The introduction of increasing amounts of activated Ras(leu61) in mouse 10T1/2 fibroblasts proportionally reduced GJIC, while the downregulation of endogenous c-ras gene expression increased junctional permeability. These results indicate that Ras plays an important role in the junction closure pathway leading to the proliferation of normal cells. However, differentiation of c-Ras-deficient preadipocytes entirely abolished their initially extensive GJIC, indicating that junction closure in response to adipocytic differentiation is independent of Ras.

Adipocytes↗

PubMed1996

Down-regulation of cytokeratin 14 gene expression by the polyoma virus middle T antigen is dependent on c-Src association but independent of full transformation in rat liver nonparenchymal epithelial cells.

Polyoma virus middle T antigen (mT) transforms the T51B cell line and induces the loss of the cytokeratin 8 and 14 pair (CK8/CK14) present in these rat nonparenchymal liver epithelial cells (LECs), because of the selective down-regulation of CK14 gene expression. To identify the initial steps of the mT-induced signaling pathway(s) leading to this inhibition, T51B cells were transfected with vectors encoding the NG59, dl23, and 248M mT mutants, which are known to interact in a differential manner with c-Src, P13-kinase, and Shc. Immunofluorescence microscopy and Northern blot analysis showed a loss of cytokeratins in dl23 or 248M but not in NG59 mT mutant-containing cells. An in vitro kinase assay demonstrated that only the dl23 and 248M mT mutants could associate with c-Src. This c-Src-mediated action of mT on CK14 gene expression was further confirmed by adding the v-src gene product in T51B cells. The assessment of the transforming capacity of the mT mutants demonstrated that the NG59 and dl23 mT mutants were nontransformant, whereas the 248M mT mutant expressed an appreciable transforming activity. These results show that the down-regulation of CK14 gene expression by mT in the LEC line T51B is dependent on the association with the c-Src tyrosine kinase, but interestingly, this c-Src-mediated action of mT can occur in the absence of transformation. Furthermore, when coupled with recent data on the plasticity of LECs, the present findings provide the first essential element in our definition of the signaling pathway(s) that link growth/differentiation events with CK gene regulation in typical simple epithelial cells.

Animals↗

PubMed1993

High membrane-associated protein kinase C activity correlates to tumorigenicity but not anchorage-independence in a clone of mouse NIH 3T3 cells.

Anchorage-dependent, nontumorigenic rat F111 fibroblasts have a low level of membrane-associated protein kinase C (PKC) activity. After expression of the polyoma virus middle tumor antigen this activity increased, the cells grew in agar and formed tumors after injection into syngeneic rats or nude mice. Contrary to F111, a clone of mouse NIH 3T3 fibroblasts has a high membrane-associated PKC activity and is as tumorigenic as polyoma-transformed cells, although this clone is still anchorage-dependent. Therefore, membrane-associated PKC activation might be one of the signals leading to tumorigenicity but not necessarily anchorage-independence.

3T3 Cells↗

PubMed1993

Comparison of liposome fusion and electroporation for the intracellular delivery of nonpermeant molecules to adherent cultured cells.

Intracellular delivery of nonpermeant molecules to cultured cells in situ can be problematic. This work is a comparison between two methods of accomplishing this delivery; liposome-mediated delivery and electroporation. The final goal was to examine the effects of the glutathione S-transferase (GST) inhibitor Basilen Blue (BB) on glyceryl trinitrate biotransformation in porcine kidney epithelial (PK1) cells after intracellular delivery. Initial evaluation used the fluorescent markers carboxyfluorescein and lucifer yellow (LY). This was followed by biochemical analysis of glyceryl trinitrate biotransformation. Liposome-mediated delivery proved ineffective in spite of variations in the lipid composition of the liposomes and the use of an agglutinin and a fusogen. In contrast, electroporation was a very effective method for intracellular delivery of both lucifer yellow and basilen blue to the PK1 cells. The results show that in cells where Basilen Blue was introduced, there was a decrease in both the glyceryl trinitrate (GTN) biotransformation and ratio of glyceryl-1,2-dinitrate (1,2-GDN) to glyceryl-1,3-dinitrate (1,3-GDN) formed. The technique of in situ electroporation shows great promise for the assessment of a variety of nonpermeant molecules of pharmacological interest.

Animals↗

PubMed1991

Cellular ras gene activity is required for full neoplastic transformation by polyomavirus.

To investigate the role of ras gene activity in cellular transformation by polyomavirus, murine C3H10T1/2 cells were rendered ras deficient by transfection with an antisense ras gene construct. Ras deficiency resulted in a partial suppression of the polyomavirus-induced transformed phenotype. The production of viral middle T antigen and its association with pp60c-src, increased membrane-associated protein kinase C activity, and morphological transformation were unaffected by the downregulation of c-ras gene expression. On the other hand, stimulated proliferation, focus formation on confluent monolayers of normal cells, and colony formation in soft agar were all greatly reduced in cells containing reduced p21ras levels. It is concluded that ras gene activity is needed for full cell transformation by polyomavirus.

Animals↗

PubMed1991

Polyomavirus middle tumor antigen increases responsiveness to growth factors.

The middle tumor antigen (mT) of polyomavirus is unable to transform a clone of NIH 3T3 cells to anchorage independence (L. Raptis and J.B. Bolen, J. Virol. 63:753-758, 1989). However, this oncogene increased the responsiveness of these cells to the growth factors (alpha-like and beta-type transforming growth factors) produced by cells possessing the whole transforming region of polyomavirus. This resulted in the growth of NIH 3T3 cells, expressing mT under control of the dexamethasone-regulatable mouse mammary tumor virus promoter, in agar medium supplemented with these growth factors upon addition of the inducer. Therefore, mT, a transforming oncogene, is able to enhance the responsiveness of established cells to growth factors, a property previously attributed primarily to myc and other establishment type oncogenes.

Animals↗

PubMed1991

Polyoma virus middle tumor antigen stimulates membrane-associated protein kinase C at lower levels than required for phosphatidylinositol kinase activation and neoplastic transformation.

Twelve independent rat F111 cell lines expressing the polyoma virus middle tumor antigen (mT) under control of the dexamethasone-regulatable MMTV-LTR promoter were assayed for levels of membrane-associated protein kinase C (PKC) activity. Low background levels of mT antigen expression (approximately 2%), although insufficient for transformation, triggered a dramatic increase in PKC activity. Under the same conditions, levels of the mT-associated phosphatidylinositol kinase activity were low, indicating that this kinase might be a factor limiting transformation in this cell system.

1-Phosphatidylinositol 4-Kinase↗

PubMed1990

Transforming signals generated by the polyoma virus tumor antigens.

The secretion of a threshold amount of SAGF can elicit one of the major features of the transformed phenotype, anchorage-independent growth. It appears that, like other cells, NIH 3T3 cells make and secrete SAGF, but not enough to enable them to proliferate in soft agar. The stream of signals from polyoma virus MTAg:pp60c-src:PI-kinase complexes attached to the inner surface of the cell membrane is not enough to raise SAGF secretion to the threshold level for agar growth, but these signals can powerfully enhance the responsiveness to added SAGF. Only when NIH 3T3 cells express all three polyoma T antigens do they secrete enough SAGF and become responsive enough to the factor to form colonies in soft agar. This is in contrast to F111 cells which produce adequate amounts of, and respond to, SAGF after MTAg expression alone. The reasons for this difference remain to be investigated. Obviously, factors expressed during the course of the spontaneous establishment are different for these two lines and they affect the cells' response to MTAg. Clearly, knowing how polyoma virus transforms cells will require a deeper understanding of the deregulation of membrane signaling enzymes by the viral MTAg and the identification and characterization of the autocrine SAGF's that mediate at least part of the neoplastic response to the viral signals.

Animals↗

PubMed1990

Electroporation of adherent cells in situ.

A simple, rapid, and reproducible procedure for the introduction of macromolecules into adherent mammalian cells by electroporation is described. Cells were growing on a glass surface coated with electrically conductive, optically transparent indium-tin oxide at the time of pulse delivery. Several factors affected the optimal voltage for permeation of a given line including the metabolic state of the cells and their degree of spreading onto the conductive growth surface. Careful control of the electric field strength resulted in almost 100% of the cells containing introduced antibodies without any detectable change in the length of their division cycle. Higher voltages were required for the stable expression of DNA than for the introduction of antibodies, resulting in a significant rate of cell death.

Animals↗

PubMed1989

Polyomavirus transforms rat F111 and mouse NIH 3T3 cells by different mechanisms.

Polyomavirus middle tumor antigen (mT) was expressed in a line of mouse NIH 3T3 cells under control of the dexamethasone-regulatable mouse mammary tumor virus promotor. Contrary to rat F111 cells which were rendered anchorage independent by mT expression alone (L. Raptis, H. Lamfrom, and T.L. Benjamin, Mol. Cell. Biol. 5:2476-2487, 1985), mT-producing NIH 3T3 cells were unable to grow in agar even after full mT induction. The mT:pp60c-src-associated phosphatidylinositol kinase was activated in these cells to a degree similar to that in fully transformed cells expressing the small and large T antigens, in addition to mT. We therefore propose that the stimulation of this phosphatidylinositol kinase, although apparently necessary, is not sufficient for transformation of NIH 3T3 cells by polyomavirus.

1-Phosphatidylinositol 4-Kinase↗

PubMed1988

Protein kinase C increases the activity of the polyoma virus middle T antigen-associated phosphatidylinositol kinase.

Exposure of polyoma virus-transformed fibroblasts to the protein kinase C-stimulating phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) is known to increase the transforming potential of the virus's middle T antigen. Here it is shown that this TPA treatment also stimulates an 85 kDa phosphatidylinositol kinase associated with the middle T antigen. Since activation of this kinase is known to be necessary, although not by itself sufficient for the transformation of cells by polyoma virus, bursts of protein kinase C activity, triggered by TPA or various cellular receptors, might enhance the oncogenicity of polyoma virus by stimulating this middle T antigen-associated phosphatidylinositol kinase.

Animals↗

PubMed1987

Calcium, cyclic AMP and protein kinase C--partners in mitogenesis.

Evidence is steadily mounting that the proto-oncogenes, whose products organize and start the programs that drive normal eukaryotic cells through their chromosome replication/mitosis cycles, are transiently stimulated by sequential signals from a multi-purpose, receptor-operated mechanism (consisting of internal surges of Ca2+ and bursts of protein kinase C activity resulting from phosphatidylinositol 4,5-bisphosphate breakdown and the opening of membrane Ca2+ channels induced by receptor-associated tyrosine-protein kinase activity) and bursts of cyclic AMP-dependent kinase activity. The bypassing or subversion of the receptor-operated Ca2+/phospholipid breakdown/protein kinase C signalling mechanism is probably the basis of the freeing of cell proliferation from external controls that characterizes all neoplastic transformations.

Animals↗

PubMed1986

Protein kinase C stimulation increases the transforming ability of the polyoma virus middle T antigen.

Exposure of dexamethasone-treated cells of the mT-1 line of F111 rat cells bearing a dexamethasone-inducible polyoma virus middle T (mT) antigen gene to very low concentrations of the protein kinase C-stimulating phorbol ester TPA increased the association of mT antigen with the cellular pp60c-src tyrosine protein kinase, as indicated by an increased phosphorylation of tyrosine residues of mT in mT:pp60c-src complexes precipitated from extracts of the TPA-treated cells by anti-mT antibodies. This TPA (hence probably protein kinase C)-enhanced association of mT with pp60c-src was accompanied by a large increase in the transforming ability of mT as indicated by a much enhanced ability of TPA-treated mT-1 cells producing submaximal levels of mT to proliferate while suspended in semi-solid medium and to form foci on confluent monolayers of normal F111 cells. NRCC NO: 26558.

Antigens, Polyomavirus Transforming↗

PubMed1986

Protein kinase C promotes the phosphorylation of immunoprecipitated middle T antigen from polyoma-transformed cells.

Stimulation of protein kinase C in polyoma virus-transformed cells increased the phosphorylation of tyrosine residues of the viral middle T (mT) antigen in mT:pp60c-src complexes precipitated by anti-mT antibodies. This increase might have been due to a stimulation of the complex's pp60c-src tyrosine kinase activity or to an increased ability of the mT protein to be phosphorylated by pp60c-src. These observations suggest that cellular protein kinase C might control the ability of polyoma virus to transform its host cell.

Animals↗

PubMed1986

Effects of naloxone and pimozide on initiation and maintenance measures of free feeding.

Latency to initiate and duration of eating of 18 daily 5-pellet meal segments was measured in 22-h food-deprived rats under conditions of pimozide (1 mg/kg, i.p.) and naloxone (2.5, 5.0 and 10.0 mg/kg, i.p.) pretreatment. Neither drug increased latency on the first or second day of testing; pimozide increased mean latency slightly on the third test. Both drugs slowed duration scores dramatically; the slowing was progressive both within and across test sessions in the case of pimozide; it was dose-dependent and progressive within (but not across) sessions in the case of naloxone. In each case, shifts in mean duration scores. In each case, shifts in mean duration score reflected an increase in score variance caused by an increase in the number and extremity of long duration scores. In each case best scores under the drug condition were equal to best scores under control conditions; indeed, very fast latency scores were slightly more frequent under each drug on the first day of testing. Thus each drug decreased the probability of moderate scores but neither drug caused a simple change in the ability to make occasional fast responses. While the effects of the two drugs were similar in some ways, naloxone, unlike pimozide, did not completely suppress feeding.

Animals↗