SV40 Transformation Research Articles

Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes.

Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl-2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4', 6'-diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 +/- 3% (P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 +/- 2% (P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; approximately 11 +/- 1% of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 +/- 0.1% in untreated cholangiocytes (P < 0. 01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57% in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.

Sphingomyelin Synthase, a Potential Regulator of Intracellular Levels of Ceramide and Diacylglycerol during SV40 Transformation: DOES SPHINGOMYELIN SYNTHASE ACCOUNT FOR THE PUTATIVE PHOSPHATIDYLCHOLINE-SPECIFIC PHOSPHOLIPASE C?

Sphingomyelin synthase (SMS), an enzyme involved in sphingomyelin (SM) and ceramide metabolism, can potentially regulate, in opposite directions, the levels of ceramide and diacylglycerol. In this study SMS activity was investigated in normal and SV40-transformed human lung fibroblasts (WI38). The addition of [3H]C2-ceramide to cells resulted in a time-dependent formation of [3H]C2-SM. At 24 h after treatment, normal WI38 cells cleared 17% of [3H]C2-ceramide producing [3H]C2-SM, which accounted for 13% of total radioactivity. On the other hand, SV40-transformed cells cleared 45% of [3H]C2-ceramide and produced C2-SM, which accounted for 24% of total radioactivity. This enhanced production of C2-SM was also supported by an increase in the total SMS activity of cells (measured in vitro), such that SV40-transformed cells had SMS activity of 222 pmol/mg of protein/h, whereas wild type cells had 78 pmol/mg of protein/h of activity. Additional studies aimed at examining the SMS activity directed at ceramide produced in the plasma membrane. Treatment of cells with exogenous bacterial sphingomyelinase (SMase) for 25 min resulted in cleavage of 90-95% of total SM and the concomitant generation of ceramide. After bacterial SMase treatment, wild type WI38 cells cleared ceramide very slowly (19.2 pmol of ceramide/nmol of phosholipid Pi after 6 h of incubation) and hardly regenerated any SM. On the other hand, SV40-transformed cells cleared ceramide much faster (41.1 pmol/nmol of Pi after 6 h of incubation) and regenerated approximately 80% of the original SM. These results show that the enhanced SMS activity of transformed cells is particularly pronounced when ceramide is produced in the plasma membrane. Finally, several observations led us to consider the relationship of SMS to the "putative" phosphatidylcholine-specific phospholipase C (PC-PLC). We, therefore, tested the effects of D609, a purported PC-PLC-specific inhibitor on the activity of SMS. D609 inhibited SMS activity in vitro. In addition, cellular studies showed that SMS activity was dramatically inhibited by concentrations of D609 used previously to study PC-PLC (10-50 microg/ml). These results suggest SMS as an important biochemical target for D609, and they raise the distinct possibility that many of the roles of PC-PLC, especially in cell transformation, may be attributable to SMS.

Autonomy of the epithelial phenotype in human ovarian surface epithelium: Changes with neoplastic progression and with a family history of ovarian cancer

Epithelial ovarian carcinomas originate in the ovarian surface epithelium (OSE). In culture, OSE undergoes epithelio-mesenchymal conversion, an event mimicking a wound response, while ovarian carcinomas retain complex epithelial characteristics. To define the onset of this increased epithelial autonomy in ovarian neoplastic progression, we examined mesenchymal conversion in OSE from 25 women with no family histories (NFH-OSE) and 13 women with family histories (FH-OSE) of breast/ovarian cancer (including 8 with mutated BRCA1 or 17q linkage) and in 8 ovarian cancer lines. After 3-6 passages in monolayer culture, most NFH-OSE exhibited reduced keratin expression and high collagen type III expression. In contrast, keratin remained high but collagen expression was lower in p. 3-6 FH-OSE. This difference was lost in SV40-transformed lines, which all resembled FH-OSE. Most carcinoma lines remained epithelial and did not undergo mesenchymal conversion. In 3-dimensional (3-D) sponge culture, NFH-OSE cells dispersed and secreted abundant extracellular matrix (ECM). FH-OSE remained epithelial and did not secrete ECM. ECM production was also reduced in SV40-transformed lines. Carcinoma lines in 3-D formed epithelial cysts, aggregates and papillae and lacked ECM. Sponge contraction (a mesenchymal characteristic) was greater in NFH-OSE than in FH-OSE both before and after SV40 transformation and was absent in the cancer lines. Our results suggest that increased autonomy of epithelial characteristics is an early indicator of ovarian neoplastic progression and that phenotypic changes indicative of such autonomy are found already in overtly normal OSE from women with histories of familial breast/ovarian cancer.

The Fanconi Anemia Complementation Group C Gene (FAC) Suppresses Transformation of Mutant Fibroblasts by the SV40 Virus

Fanconi anemia (FA) is a heterogeneous genetic syndrome manifested by bone marrow failure and consisting of at least five complementation groups (A, B, C, D, E). Mutations in a gene termed FAC are responsible for the C complementation group, but the function of the FAC protein remains obscure. FA patients are also highly cancer-prone; the molecular basis for this susceptibility is unclear but has led to the hypothesis that the wild-type FA gene may act as a tumor suppressor. In vitro, mutant FA primary fibroblasts are 3- to 50-fold more sensitive than normal fibroblasts to transformation in culture by the SV40 virus. We confirmed this marked susceptibility to transformation of a FAC-mutant primary fibroblast cell line, GM449. We then introduced a copy of the wild-type FAC cDNA into GM449 cells using a recombinant adeno-associated virus (rAAV) vector. We found that GM449 cells transduced with a copy of the normal FAC cDNA by a FAC-rAAV vector were at least 10-fold less prone to form transformed foci. Diminished transformation potential of transduced cells was a specific effect of the FAC cDNA since GM449 cells transduced with a rAAV vector not containing FAC retained marked susceptibility to SV40 transformation.

Regulation of matrix metalloproteinases following cellular transformation

During progression towards malignancy, many tumor cells display changes in their repertoire of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). The recent finding that many members of the MMPs are regulated by protooncogenes may explain the frequent observation of changes in MMP gene expression during progression of many tumor types. While studies involving enzymatic assays of MMPs are usually confined to one or a few MMPs, reverse transcription-PCR (RT-PCR) permitted the analysis of seven members of the MMP family and two members of the TIMP family in several normal and transformed cell lines. RT-PCR permitted us to confirm the observation that MMP-9 is activated following transformation and also to observe the previously unreported activation of MMP-7 in SV40-transformed cells. It has previously been found that MMP-1, -2, -3, -8, and -9 are upregulated by phorbol esters; we have found that MMP-10 is also upregulated by phorbol esters. The phorbol ester upregulation of MMP-1, -3, and -10 was found to be abolished in cells transformed by SV40 virus. Several studies have shown that MMP-1 is upregulated by an integrin-mediated signal transduction pathway. This study demonstrates that MMP-3 and MMP-10 are also regulated by integrin-mediated signal transduction and that upregulation by this pathway is abolished following SV40 transformation. In summary, the more global view of MMP expression afforded by RT-PCR indicates that MMP-1, -3, and -10 are regulated by both integrin-mediated signal transduction and phorbol esters. While fibroblasts and transformed bone cells express several members of the MMP gene family, several other cell types do not express MMPs.

Identification of a renal cell line that constitutively expresses the kidney-specific high-affinity H+/peptide cotransporter.

In this study we describe for the first time the identification of a renal cell line that expresses the kidney-specific high-affinity H+/peptide cotransport system. The kidney cell line SKPT-0193 C1.2 was obtained by SV40 transformation of rat proximal tubular cells. The transport of the dipeptide glycylsarcosine (Gly-Sar) was studied in this cell line grown as a confluent monolayer on impermeable plastic supports. Uptake of the dipeptide was rapid and was stimulated sixfold by an inwardly directed H+ gradient, with optimal uptake occurring at an extracellular pH of 6.0. The uptake was markedly reduced by the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone whether measured at pH 7.5 or 6.0. Intracellular acidification of the cells by NH4Cl prepulse also reduced the uptake of glycylsarcosine. The dipeptide uptake was found to be mediated by a high-affinity transport system with a Michaelis-Menten constant (Kt) of 67 +/- 2 microM and a maximal transport velocity of 1.20 +/- 0.02 nmol.10 min-1.mg protein-1. Studied over a concentration range of 5 microM to 5 mM, there was no evidence for a second saturable transport component. Di- and tripeptides, but not glycine, were strong inhibitors of glycylsarcosine uptake, indicating that these peptides also interact with the transport system with high affinity. Northern blot analysis of poly(A)+RNA from these cells using cDNA probes specific for the human intestinal peptide transporter (PEPT 1) or the human kidney-specific peptide transporter (PEPT 2) revealed that the transport system expressed in these cells is PEPT 2. It is concluded that the SKPT-0193 C1.2 cell line constitutively expresses the kidney-specific high-affinity H+/peptide cotransporter described in the proximal tubular epithelial cells of the normal kidney.

CAMP-Mediated Signals as Determinants for Apoptosis in Primary Granulosa Cells

Differentiation and luteinization of granulosa cells are induced by gonadotrophic hormones and other substances elevating intracellular levels of cyclic AMP (cAMP). We have investigated the correlation between the potency of these substances to enhance steroidogenesis and to induce apoptosis in primary granulosa cell cultures obtained from rat preovulatory follicles. The cAMP analog, 8-Br cAMP, induced apoptosis in more than 90% of the cell population within 15 h of incubation at 37°C in serum-free medium. The physiological stimulants of these cells, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which caused a moderate cAMP response in these cells, followed by a desensitization period, increased progesterone production by fourfold with no apparent effect on cell death. In contrast, forskolin, a potent activator of adenylate cyclase, stimulated both the cAMP and steroidogenic response by an order of magnitude greater than the gonadotropin stimulation, concomitantly with a pronounced increase in cell death (25%). Moreover, blocking of the cellular phosphodiesterase activity in forskolin-stimulated cells by isobutylmethylxanthine (IBMX), which maintains high levels of intracellular cAMP, led to further enhancement of cell death following 40 h of incubation (50%). Basic fibroblast growth factor (bFGF) and gonadotropin-releasing hormone (GnRH), which stimulated steroidogenesis in these cells in a cAMP-independent manner, did not promote cell death. Moreover, costimulation of the cells with forskolin and bFGF led to a substantial decrease in the incidence of apoptosis relative to forskolin alone. In order to examine whether the expression of tumor suppressor genes is involved in granulosa cell differentiation and apoptosis induced by cAMP, we examined the effect of cAMP in SV40 transformed granulosa cells, in which T-antigen expression is expected to block the activity of p53 as well as of the retinoblastoma gene product (pRB) and its related proteins. Cultures of three different cell lines established by SV40 transformation demonstrated resistance to 8-Br-cAMP- or forskolin plus IBMX-induced apoptosis, in contrast to the severe apoptotic response in primary cells. We suggest that stimulation of primary granulosa cells by high levels of cAMP catalyzes programmed cell death, while stimulation of the cells by gonadotropic hormones, which result in a moderate cAMP response, followed by desensitization to further stimulation, can prolong the lifespan of the luteinized granulosa cells. Moreover, one or more tumor suppressor proteins may mediate the cAMP generated signal leading to cell death.

Non-random jumping translocations as a result of SV40 large T-antigen expression in benign human tumor cells.

In an attempt to analyse the cytogenetic effects caused by SV40 large T-antigen expression in cells of human benign tumors we transfected cells of an uterine leiomyoma characterized by a primary reciprocal translocation t(12;14)(q15;q24) and a pleomorphic adenoma of the salivary gland with an inversion inv(12) (q15q24.1) using a construct coding for SV40 large and small T-antigen. The most interesting finding was not a generally destabilized karyotype, but the strictly non-random involvement of two chromosomal breakpoints, i.e. 5p13 and 10q11 in jumping translocations, never described before as a result of SV40 transformation. In addition we were able to show by non-radioactive in situ hybridization that there was no direct relationship between the integration site of the construct and the pre-disposition of 5p13 and 10q11 to somatic recombination. The jumping translocations with consistent breakpoints observed closely resemble the cytogenetic situation seen in a variety of human tumors with specific translocations. Based on the findings described here it is tempting to assume that the expression of SV40 large T-antigen can induce specific karyotypic alterations following an unknown trans-acting mechanism.

Reproducible alterations of DNA methylation at a specific population of CpG islands during blast formation of peripheral blood lymphocytes.

We investigated the changes in the methylation patterns of CpG islands associated with blast formation of human peripheral blood lymphocytes activated by anti-CD3 and interleukin-2 (IL-2), using restriction landmark genomic scanning with a methylation-sensitive restriction enzyme (RLGS-M) system. Of about 2,100 NotI spot/loci which were analyzed, only 10 showed changes, whereas drastic changes have been observed in cases of malignant and SV40 transformation. These changes were highly reproducible for samples from both the same and different individuals. Even the timing of the changes after cultivation was the same. Thus, we concluded that at least the genomic DNA methylation state in vivo was essentially retained in T blast cells activated in vitro by induction with IL-2 and anti-CD3, which are commonly used in biological experiments as well as clinical diagnosis and therapy.

Characterization of spontaneous and induced mutations in SV40-transformed normal and ataxia telangiectasia cell lines.

Spontaneous and induced mutations at the HPRT locus were analyzed in one normal (MRC5CV1) and one ataxia telangiectasia (AT5BIVA) SV40-transformed cell line derived from male donors. Multiplex PCR and Southern analyses revealed a high frequency of spontaneous deletion mutations that may be a consequence of the SV40 transformation. Four mutagens (ethyl methanesulfonate, bromodeoxyuridine, bleomycin, adriamycin), which differ in their types of primary DNA lesions, caused specific patterns of mutations. By using fluorescence in situ hybridization (FISH) techniques, we were able to show that more than 90% of the AT5BIVA cells contained two X chromosomes with HPRT alleles, while in more than 90% of the MRC5CV1 cells genomic hemizygosity for the HPRT gene was found. Taking into account these findings we found that the AT5BIVA cell line possesses spontaneous hypermutability as well as hypersensitivity and hypermutability to bleomycin (BLM) and adriamycin (AM). Both mutagens induced deletion mutations in both cell lines, but more complex mutations and larger deletions were found in AT5BIVA cells.

Alterations in oncogene expression and radiosensitivity in the most frequently used SV40-transformed human skin fibroblasts.

In comparison with primary cell cultures, SV40-transformed human skin fibroblasts, either from healthy donors or from patients suffering from ataxia-telangiectasia (AT) or xeroderma pigmentosum, are more resistant to the cytotoxic action of low LET 60cobalt gamma-rays as well as to high LET alpha-particles. Resistance factors calculated from D10's lie between 1.4 and 2.0. Northern blot analysis reveals spontaneous overexpression of the oncogenes c-myc, Ki-ras and c-raf and of the tumour suppressor gene p53 as a consequence of SV40 transformation. For c-myc, the increased expression is due to gene amplification and gene rearrangement. An even further increase in the expression of c-myc has been found for AT cells (AT5BI-VA) after moderate doses of 60cobalt gamma-irradiation. A possible correlation between SV40-induced changes in gene expression and cellular radioresistance is discussed.

Expression of a Candidate Ataxia-telangiectasia Group D Gene in Cultured Fibroblast Cell Lines and Human Tissues

A candidate gene for ataxia-telangiectasia group D (ATDC) has been cloned (Kapp et al. 1992), sequenced and found to be a member of a recently reported gene family (Leonhardt et al. 1994). Transcriptional behaviour of ATDC has been examined in a number of cell lines and human tissues using a 3.0 kb cloned cDNA as a probe. Three normal and two ataxia-telangiectasia (A-T) group D, non-transformed fibroblast cell lines produced a 2.4 kb mRNA transcript. The size of mRNA transcripts seen and the level of expression differ in different human tissues. Many tissues have multiple transcripts of ATDC and the most prominent transcripts observed were 3.0, 2.4 and 1.6 kb. Two out of three SV-40 transformed normal and one SV-40-transformed A-T group D fibroblast cell lines demonstrated no transcription of ATDC by RNA blotting analysis. These results suggest that SV-40 transformation may affect the expression of ATDC. Reverse transcription-polymerase chain reaction analysis showed that ATDC was expressed at low levels in all of these cells. Additional Northern blot analysis demonstrated that X-irradiation with 10 Gy had no effect on ATDC expression at 1, 4 and 24 h after irradiation in either SV-40-transformed normal or in SV-40-transformed A-T group D fibroblast cell lines. Further understanding of ATDC will require cloning of additional transcripts and studies of ATDC protein behaviour.

Nonresponsiveness of an SV40-transformed BALB/C-3T3 cell line to PDGF mitogenicity is correlated with an irreversible reduction in PDGF receptor number

The effect of SV40 transformation on the mitogenic responsiveness of Balb/c-3T3 cells to platelet-derived growth factor was examined. The SV40 transformed cell line (SV40-3T3) did not require exogenous PDGF for growth in a serum-free medium containing insulin, transferrin and high density lipoprotein as macromolecular supplements. The SV40-3T3 cells were found to have undetectable PDGF-stimulated PDGF receptor autophosphorylation and markedly reduced PDGF binding. PDGF-induced PDGF receptor autophosphorylation and PDGF binding of the normal 3T3 cells were not reduced by incubation with conditioned medium from SV40 transformed cells; nor could PDGF binding to SV40-3T3 be increased by an 8-hour incubation with 0.5 mM suramin, a compound known to prevent the binding of PDGF to its receptor. These data suggest that SV40-3T3 cells do not produce PDGF in quantities that are sufficient to down regulate their PDGF receptor and suggest that the reduced PDGF responsiveness and PDGF receptor number in SV40-3T3 cells may be a reflection of a more permanent, perhaps genetic, alteration.

Alterations of the glutathione cycle enzymes during and after SV40-transformation of human fibroblasts.

The activities of several enzymes involved in the antioxidant system of the cell were studied in parallel to cytogenetic alterations at various times after SV40 infection and transformation of human fibroblasts. At early passages after SV40 infection, glutathione reductase (GSR), glutathione peroxidase (GPX), glutathione transferase (GST) and glucose-6-phosphate dehydrogenase (G6PD) activities were decreased. This, associated with the low superoxide dismutase (SOD) and catalase activities previously noticed in these cells, suggested that they are in a highly pro-oxidant status. Although chromosomes carrying the genes encoding these enzymes are frequently underrepresented, there is no direct relationship between the number of chromosomes and enzyme activities. Except for GPX, all the activities tend to increase in established cell lines reaching levels comparable to those of non-transformed fibroblasts. The late increase of G6PD activity may correlate with the frequent duplication of the early replicating X. GSR seems to correlate with G6PD activity and GPX to SOD total activity. The most striking alterations affect mitochondrial and peroxisomal enzymes activities: SOD, GPX and catalase.

Decrease in catalase activity and loss of the 11p chromosome arm in the course of SV40 transformation of human fibroblasts.

The activity of catalase, a key enzyme in cell detoxication of oxygen derivatives, was studied in SV40 transformed human fibroblasts. A cytogenetic study was performed in parallel to establish a quantification of 11p arm on which the corresponding gene is mapped. mRNA amounts were determined by Northern blotting. At early passages, catalase activity strongly decreased whereas the corresponding mRNA was present. No deletions of 11p arms were detected. At later passages, catalase activity remained low. 11p arm deletions were frequent, and the amount of mRNA was decreased. In these late passages, the good correlation between the number of 11p arms and catalase activity suggested a gene dosage effect. It is assumed that the decrease of catalase activity provides a selective advantage for the transformed cells. This decrease is related to a post-transcriptional change of regulation at early passages and to the loss of the corresponding gene at later passages.

SV40 small t deletion mutants preferentially transform mononuclear phagocytes and B lymphocytes in vivo

Hamsters injected intracardiacally with wild-type SV40 developed diverse tumors, with no one type predominating. However, hamsters injected with small t deletion mutants of SV40 uniformly developed true histiocytic or B-cell lymphomas, both of which arise from rapidly dividing cell populations, with very few tumors of other types. Our results are consistent with in vitro studies which suggest a requirement for the small t function in SV40 transformation of nondividing cells.

Altered ganglioside composition in virally transformed rat embryo fibroblasts

The composition of gangliosides was examined in a normal rat embryo fibroblast cell line (REF52) and in two viral transformants: a polyoma transformant (REF52-PyMLV) and a simian viral 40 transformant (REF52-SV40). The distribution of gangliosides in the cell lines was determined using gas-liquid chromatography and high-performance thin-layer chromatography. N-acetylneuraminic acid was the predominant sialic acid species detected in the three cell lines. The total ganglioside concentration (μg/100 mg dry weight of cells) in the normal, PyMLV, and SV40 lines was 144.7 ± 10.4, 153.8 ± 9.2, and 86.1 ± 6.8, respectively. Gangliosides GM3, GM2, GM1, and GD1a were the major species in the normal and transformed lines. The distribution of these gangliosides, however, differed markedly between the normal and the transformed lines and also between the transformed lines themselves. The transformed cells also differed from the normal cells in growth rate, morphology, and social behavior. The cell line with highest GM3 content (PyMLV) formed islands, whereas the normal and SV40 cell lines, which had lower GM3 levels, grew as monolayers. The findings suggest that PyMLV and SV40 transformation can have multiple and different effects on cellular ganglioside distribution and growth behavior.

Pleomorphic adenoma cells vary in their susceptibility to SV40 transformation depending on the initial karyotype.

Chromosomal aberrations involving 8q12 or 12q13-15 characterize two cytogenetic subgroups of salivary gland pleomorphic adenomas. As the tumors of the two groups differ in their clinical and histologic characteristics, we decided to determine their susceptibility to SV40 transformation. We transfected cell cultures from 13 adenomas with aberrations involving 8q12 and from seven adenomas with involvement of 12q13-15 using an SV40 plasmid coding for the early region of the viral genome. Whereas all cultures with aberrations of 12q13-15 showed transformed foci, only 4 of the 13 cultures with 8q12 abnormalities showed foci of transformed cells. We also observed a much higher immortalization rate in the first group (3/7 vs. 1/13). All successfully transformed tumor cell cultures showed a relatively stable karyotype in the pre-crisis stage and a high mitotic index, were T-antigen positive, and had an extended life span in vitro.

Elevated cAMP is required for stimulation of eicosanoid synthesis by interleukin 1 and bradykinin in BALB/c 3T3 fibroblasts.

In Swiss 3T3 murine fibroblasts, interleukin 1 (IL-1) and bradykinin stimulate prostaglandin E2 (PGE2) synthesis. However, in the present study, we found that neither agonist stimulated PGE2 synthesis in BALB/c 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors for each agonist compared to Swiss 3T3 cells. When BALB/c 3T3 cells were preincubated with cAMP analogs, both IL-1 and bradykinin stimulated PGE2 synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL-1 and bradykinin stimulated PGE2 synthesis. Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, blocked the ability of cAMP or NECA to render cells responsive to IL-1 and bradykinin. In basal BALB/c 3T3 cells, bradykinin and IL-1 stimulated arachidonate release in the absence of cAMP, but little conversion of released arachidonate to PGE2 occurred. cAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV-T2 is a clonal line originating from BALB/c 3T3 transformed with SV-40. In these cells, IL-1 and bradykinin stimulated PGE2 synthesis despite basal intracellular cAMP concentrations similar to BALB/c, and cAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by cAMP in BALB/c 3T3 cells, and SV-40 transformation results in increased cyclooxygenase expression, apparently independent of cAMP.

An SV40 transformation revertant due to a host mutation: Isolation and complementation analysis

We have isolated an SV40 transformation revertant cell line, CLi L, by selection for normal cells whose growth is inhibited under low serum conditions. This line expresses a single, wild-type copy of large T antigen, yet is not transformed. It is not retransformable by transfection of SV40 DNA or infection with a recombinant retrovirus encoding large T antigen. Resistance to transformation therefore appears to be due to a cellular mutation. Fusion of CL1 L cells to normal 3T3 cells or SV40-transformed cells results in somatic cell hybrids that are transformed, indicating that resistance is recessive. In addition, fusion of CL1L cells to another SV40 transformation-resistant line, A27, results in transformed hybrids, indicating the existence of discrete complementation groups with respect to SV40 transformation.