Viral T-antigen Research Articles

JC virus T-antigen expression induces chromosomal aberrations in human colorectal cells

JC virus T-antigen expression induces chromosomal aberrations in human colorectal cells

Reactivation of human neurotropic JC virus expressing oncogenic protein in a recurrent glioblastoma multiforme

Examination of the primary tumor of glioblastoma multiforme and its recurrence for their association with JC virus revealed that, while the viral genome is present in both initial and recurrent tumors, expression of the viral oncoprotein T-antigen occurs only in the recurrent tumor cells. Accordingly, the level of inducible cellular transcription factors, including the p65 subunit of NF-kappaB and YB-1, which have the ability to stimulate JCV gene expression, was found to be higher in the recurrent tumor cells. These observations suggest that induction of the regulatory factors after resection of the primary tumor may have reactivated JC virus gene expression and led to redevelopment of the tumor in brain.

Reactivation of human neurotropic JC virus expressing oncogenic protein in a recurrent glioblastoma multiforme

Examination of the primary tumor of glioblastoma multiforme and its recurrence for their association with JC virus revealed that, while the viral genome is present in both initial and recurrent tumors, expression of the viral oncoprotein T-antigen occurs only in the recurrent tumor cells. Accordingly, the level of inducible cellular transcription factors, including the p65 subunit of NF-κB and YB-1, which have the ability to stimulate JCV gene expression, was found to be higher in the recurrent tumor cells. These observations suggest that induction of the regulatory factors after resection of the primary tumor may have reactivated JC virus gene expression and led to redevelopment of the tumor in brain. Ann Neurol 2000;48:932–936

Dexamethasone-induced radioresistance occurring independent of human papilloma virus gene expression in cervical carcinoma cells.

Inactivation of p53 by binding to simian virus 40-T antigen (SV40-T) and human papilloma virus type 16 protein E6 (HPV 16 E6) in transfected human diploid fibroblasts causes enhanced radioresistance. The aim of this study was to investigate the role of HPV 18 E6 and E7 gene products with respect to radiosensitivity of two cervical carcinoma cell lines. The two cervical carcinoma lines C4-1 and SW 756 were used in which treatment with dexamethasone allows to modulate expression levels of HPV 18 E6 and E7 genes: upregulation in C4-1, downregulation in SW 756. Effects of treatment with dexamethasone on plating efficiency and radiosensitivity were assessed using a clonogenic assay. Treatment with dexamethasone increased plating efficiency of the C4-1 cells, but did not affect plating efficiency of SW 756 cells. Treatment with dexamethasone induced enhanced radioresistance in both cell lines. Thus in C4-1 cells the observed changes in radioresistance correlate to the enhancement in expression of HPV 18 genes E6/E7, whereas in SW 756, a reduced expression correlates negatively with the enhanced radioresistance. In C4-1 and SW 756 cells, treatment with dexamethasone induces radioresistance, and changes in expression levels of HPV 18 genes E6 and E7 do not correlate with the changes in radiosensitivity. Dexamethasone-induced radioresistance has previously been observed in HeLa cells, another human cervical carcinoma cell line. This leads us to speculate that dexamethasone-induced radioresistance may be important in certain clinical situations, and that therefore, the phenomenon deserves further study.

Loss of T-antigen sequences allows SV40-transformed human cells in crisis to acquire a senescent-like phenotype.

Normal human cells transfected with SV40 DNA exhibit an extended proliferative potential compared with controls, but they eventually enter a phase known as "crisis." During crisis, extensive cell death occurs and the cells exhibit some gene expression changes similar to senescent cells. This article presents results which indicate that crisis most likely depends on expression of the viral gene T-antigen. We have obtained a unique subpopulation of cells that have deleted the T-antigen gene and, rather than dying as cells do in crisis, remain viable and exhibit some senescent-like characteristics. We also found that the SV40 promoter is poorly expressed in senescent versus young cells. We hypothesize that decreased activity of the viral promoter may result in decreased expression of T-antigen, which is challenged by over-expression of the cell cycle inhibitors such as p21Sdi1. Conflicting signals to proceed/halt cells cycle progression result in the cell death associated with crisis.

Partial purification and characterization of a protein kinase that is activated by nuclear localization signal peptides

A nuclear localization signal (NLS) is required for the transport of karyophilic proteins from the cytoplasm to the nucleus. In this study, NLS was examined in terms of its effect on diverse cellular functions such as protein phosphorylation reactions. When synthetic peptides containing the NLS of SV40 T-antigen were injected into the cytoplasm of Xenopus oocytes, and the oocytes incubated with [ 32P]phosphorus-containing medium, a 32 kDa protein was found to be preferentially phosphorylated in an NLS-dependent manner. The incubation of fractionated cytosolic extracts prepared from mouse Ehrlich ascites tumor cells with [ γ- 32P]ATP in the presence of the NLS peptides, results in the stimulation of the phosphorylation of several proteins. Similar in vitro stimulation was observed by other functional NLS peptides such as those of polyoma virus T-antigen and nucleoplasmin. Little or no stimulation, however, was detected for peptides of mutant type and reverse type NLS of SV40 T-antigen, and the C-terminal portion of lamin B. Using an in vitro assay, the phosphorylation activity was fractionated chromatographically and a fraction was obtained which contained a high level of activity. The fraction was found to contain three major proteins having molecular masses of 95, 70, and 43 kDa. The in vivo and in vitro results are consistent with the existence of a protein kinase, called NLS kinase, that is specifically activated by NLS peptides.

THE STATE OF IMMORTALIZED, TRANSFORMING AND SUPPRESSOR GENES IN RAT-CELLS, TRANSFECTED BY POLYOMAVIRUS LARGE-T AND HPV18 E6+E7 GENES

Several novel cell clones (A1-A6) derived from the rat embryo fibroblasts immortalized by the polyoma virus T-antigen (LT) gene and transformed by HPV18 E6 and E7 genes were explored. Using E6 or E7 peptide antisera we detected E6 and E7 proteins with approximate molecular masses of 16 kDa and 20 kDa, respectively. Monoclonal antibody to p53 PAb421 but not PAb240 precipitated different but appreciable amounts of p53 protein in all cell clones, indicating that wild-type p53 is expressed in these cells. So expression of HPV18 E6 protein in cells does not always lead to a complete reduction in p53 levels. The quantity of p53 protein in cell clones did not correlate with the level of polyoma virus large T-antigen expression. Variations in levels of p53 protein in clones did not influence on such cell biological properties as anchorage-independence and tumorigenicity for nude mice which were similar in all cell clones.

CHARACTERIZATION OF GLIOMA-CELLS DERIVED FROM HUMAN POLYOMAVIRUS-INDUCED BRAIN-TUMORS IN HAMSTERS

Intracerebral injection of human polyomavirus, JCV, into neonatal hamsters causes tumors of,glial origin. HJC is an established cell Line derived from a JCV-induced mixed hamster brain tumor with astrocytic and ependymal components. Flow cytometric and immunohistochemical analysis of HJC suggests that it is comprised of a mixed population of cells all of which contain the JCV early protein, T-antigen, in the nuclei. Five individual clonal lines, called HJC-15a to HJC-15e, were isolated by limiting dilution and were found to exhibit distinct morphological characteristics with 25-30% variation in their sizes. It was evident that each clone has unique growth rates, doubling times, and cell cycle parameters with different G(1), S, and G(2) phase times. All clonal cells showed the presence of the JCV early protein in the nucleus. Of interest was the observation from immunoprecipitation and Western analysis indicating qualitative and quantitative differences in the T-antigen isoforms produced in these cells. Similar to the parental clone, HJC-15b produced two distinct forms of JCV T-antigen isoforms, 88 kDa and 92 kDa proteins. In addition, HJC-15c was able to produce a 23-25 kDa protein which was recognized by anti-T-antigen antibody. The activity of cyclin-dependent kinases, in particular cdc2, was higher in HJC-15c than in the other cell lines. The data presented herein indicates that glioblastomas induced by viral T-antigen expression are composed of a multitude of distinct cells that possess a variety of different characteristics.

Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines.

To establish in vivo gonadal tumor models and permanent lines of gonadal somatic cells we produced transgenic (TG) mice expressing the Simian virus (SV) 40 T-antigens (T-ag), driven by 6 or 2.1 kilobase fragments of the mouse inhibin alpha-subunit promoter. Hitherto, altogether 44 TG mice, one of which carried the shorter transgene, have produced gonadal tumors. Two founder females expressing the longer transgene, KK1 and KK3, and three established TG mouse lines were studied in detail. Penetrance of the phenotype in IT6-M and IT6-F mouse lines was 100% (tumors/TG: IT6-M 22/22, IT6-F 14/14). The T-ag mRNA was strongly expressed in the gonads, adrenal glands, pituitary, and brain. The KK-1 and KK-3 ovarian tumor cells immunostained with anti-SV40 large-T antibody. The KK-1 cells possessed high-affinity LH receptors [equilibrium association constant (Ka = 7.8 x 10(10) liters/mol] and responded to human CG by elevated cAMP and progesterone production. Also FSH slightly stimulated their cAMP and estradiol production (P < 0.01). These cells expressed cytochrome P450arom and inhibin alpha mRNA, but not cytochrome P450c17 alpha. In conclusion, the KK-1 cells are immortalized luteinizing granulosa cells expressing endogenous gonadotropin receptors, steroidogenic enzymes, and inhibin alpha. These cells will be useful in studies on the molecular aspects of granulosa cell function. The present study indicates that the 6-kilobase fragment of the inhibin alpha promoter described in this article contains the elements directing tissue-specific expression in vivo and is useful for targeted expression of other genes in the gonads.

Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin- responsive granulosa cell lines

Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin- responsive granulosa cell lines

Alterations in insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis in transformed osteoblastic cells.

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is secreted by a variety of osteoblastic cells and appears to be an integral component of bone cell physiology. We have previously reported that normal human osteoblast-like (hOB) cells secrete IGFBP-4 as well as a novel IGFBP-4 protease, which requires IGF for functional activity. In this study we assessed the IGFBP-4/IGFBP-4 protease system in transformed osteoblastic cells by Western ligand blotting and cell-free IGFBP-4 protease assays. Simian virus-40-immortalized hOB cells (HOBIT), human osteosarcoma cells (TE-85), and rat osteosarcoma cells (UMR 106-01, ROS 17/2.8) secrete IGFBP-4. In contrast to the rapid and dramatic proteolysis in hOB medium, medium conditioned by these cells had no apparent IGFBP-4 protease activity when assayed with exogenous IGF-II in culture or under cell-free conditions. Assayed in the presence of exogenous protease. HOBIT cells, but not the osteosarcoma cell lines, appeared to produce a cycloheximide-sensitive inhibitor of the IGFBP-4 proteolytic reaction. Transient cell transformation induced by incubating human osteoblasts transfected with a temperature-sensitive mutant of simian virus-40 T-antigen at the permissive temperature or by treating hOB cells with phorbol ester tumor promoters also resulted in inhibition of IGF-dependent IGFBP-4 proteolysis. Inhibition was observed if phorbol ester was added to the cultures at the time of medium change or after the protease had been expressed and secreted. Differences in IGFBP-4 proteolysis could not be accounted for by changes in IGFBP-4 messenger RNA expression or substrate levels. These data suggest that transformation is associated with alterations in the IGFBP-4/IGFBP-4 protease system in osteoblastic cells. Normal human osteoblasts secrete an IGF-dependent IGFBP-4 protease. The induction of an inhibitor of the IGF-dependent IGFBP-4 proteolytic reaction may be associated with early transformation processes. Fully tumorigenic bone cells expressed neither IGFBP-4 protease nor protease inhibitor activity.

Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons: a new tool for dissecting the molecular and cellular basis of LHRH physiology.

1. Two LHRH neuronal cell lines were developed by targeted tumorigenesis of LHRH neurons in vivo. These cell lines (GN and GT-1 cells) represent a homogeneous population of neurons. GT-1 cells have been further subcloned to produce the GT1-1, GT1-3, and GT1-7 cell lines. While considerable information is accumulating about GT-1 cells, very little is currently known about the characteristics and responses of GN cells. 2. By both morphological and biochemical criteria, GT-1 cells are clearly neurons. All GT-1 cells immunostain for LHRH and the levels of prohormone, peptide intermediates, and LHRH in the cells and medium are relatively high. 3. GT-1 cells biosynthesize, process, and secrete LHRH. Processing of pro-LHRH appears to be very similar to that reported for LHRH neurons in vivo. At least four enzymes may be involved in processing the prohormone to LHRH. 4. LHRH neurons are unique among the neurons of the central nervous system because they arise from the olfactory placode and grow back into the preoptic-anterior hypothalamic region of the brain. Once these neurons reach this location, they send their axons to the median eminence. With respect to the immortalized neurons, GN cells were arrested during their transit to the brain. In contrast, GT-1 cells were able to migrate to the preoptic-anterior hypothalamic region but were unable correctly to target their axons to the median eminence. These problems in migration and targeting appear to be due to expression of the simian virus T-antigen. 5. While GT-1 cells are a homogeneous population of neurons, they are amenable to coculture with other types of cells. Coculture experiments currently under way should help not only to reveal some of the molecular and cellular cues that are important for neuronal migration and axonal targeting, but they should also highlight the nature of the cellular interactions which normally occur in situ. 6. GT-1 cells spontaneously secrete LHRH in a pulsatile manner. The interpulse interval for LHRH from these cells is almost identical to that reported for release of LH and LHRH in vivo. GT-1 cells are interconnected by both gap junctions and synapses. The coordination and synchronization of secretion from these cells could occur through these interconnections, by feedback from LHRH itself, and/or by several different compounds that are secreted by these cells. One such compound is nitric oxide. 7. GT-1 cells have Na+, K+, Ca2+, and Cl- channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of neuroepithelial tumors of the adrenal medulla in transgenic mice expressing a mouse hypothalamic growth hormone-releasing hormone promoter-simian virus-40 T-antigen fusion gene.

Expression of the mouse GH-releasing hormone (GRH) gene is restricted to neurons within the hypothalamus and to placenta. In an attempt to generate immortalized mouse hypothalamic neurons expressing GRH, the proximal 872-nucleotide segment of the 5'-flanking region of the hypothalamic mouse GRH gene was cloned by polymerase chain reaction and ligated to a 2.7-kilobase DNA sequence encoding the simian virus-40 (SV40) T-antigen, so that regulation of SV40 T-antigen expression was dependent on sequences within the mGRH 5'-flanking region. This region contains both TATA and CAAT boxes. The mouse GRH/SV40 T-antigen fusion gene was injected into 1-cell mouse embryos, and SV40 T-antigen incorporation in the mouse genome was found in 11 of 77 live births (3 males and 8 females). Although no evidence of hypothalamic tumors was found, all mice that expressed the transgene also developed tumors originating in the adrenal medulla. Gene copy number varied from 1-20 and was inversely proportional to survival, which ranged from 7-16 weeks. Corticosterone levels were normal. The male transgenic mice were fertile, and their progeny expressed the transgene and developed similar tumors. Microscopic examination of the tumors revealed a primitive neuroectodermal neoplasm that exhibited hematogenous and lymph node metastases and contained 100 ng norepinephrine, 2.85 ng epinephrine, and 1.1 ng dopamine/mg tumor tissue. Primary culture of dispersed tumor cells released norepinephrine into the medium (180 pg/ml.24 h). Cell lines from 2 tumors were established and exhibited characteristics similar to those of mixed neuroblastoma or primitive neuroectodermal tumors. In conclusion, the proximal 872 nucleotides of the hypothalamic mouse GRH promoter contain elements directing tissue-specific expression limited to early adrenal neuroectodermal cells. Other GRH DNA sequences appear to be required for restricted expression of mouse GRH within the hypothalamus.

Development of neuroepithelial tumors of the adrenal medulla in transgenic mice expressing a mouse hypothalamic growth hormone- releasing hormone promoter-simian virus-40 T-antigen fusion gene

Development of neuroepithelial tumors of the adrenal medulla in transgenic mice expressing a mouse hypothalamic growth hormone- releasing hormone promoter-simian virus-40 T-antigen fusion gene

Norepinephrine-dependent selection of brown adipocyte cell lines.

A transgenic mouse carrying a simian virus-40 T-antigen gene under control of a mouse urinary protein promoter induced the formation of a brown fat tumor. In tissue culture, these tumor cells expressed the brown fat-specific mitochondrial uncoupling protein (Ucp) gene when stimulated by norepinephrine. To develop cell lines for the analysis of Ucp expression, we investigated growth conditions that would maintain expression. We found that the addition of 10(-7)-10(-6) M norepinephrine was critical for establishing cells with high Ucp expression, mitochondrial content, and adipogenesis. Norepinephrine exerts its effects by selectively stimulating the proliferation of brown fat cells. Results with receptor-specific agonists and antagonists indicate that norepinephrine interacts with the beta 1-adrenergic receptor to stimulate cell proliferation. The capacity of these brown fat cells to respond to norepinephrine by proliferating seems to be a manifestation of a normal physiological response of brown fat cells, because exposing an animal to the cold increases cell proliferation. Thus, this system indicates that two independent pathways for cell proliferation coexist in parallel, one based on transformation of the cell by the simian virus-40 T-antigen and the other on the normal response of the cell to stimulation by norepinephrine. Several clonal lines have been isolated that have similar levels of marker gene expression for adipogenesis and mitochondriogenesis, but differ in the level of Ucp expression. The results underscore the extreme sensitivity of mechanisms controlling Ucp expression and the fact that the expression of Ucp in brown fat is not coordinately linked to that of other nuclear genes which encode proteins associated with thermogenesis.

An immunohistochemical study of p53 and proliferating cell nuclear antigen expression in progressive multifocal leukoencephalopathy

Nuclear p53 immunoreactivity is demonstrated in infected oligodendroglia, as well as in a proportion of reactive and bizarre astrocytes, in seven progressive multifocal leukoencephalopathy (PML) biopsies. This likely represents binding to, and prolongation of the half-life of, wild-type p53 protein by JC virus T-antigen. Other possible mechanisms are considered. The same cells show proliferating cell nuclear antigen (PCNA) positivity, as do a proportion of morphologically normal oligodendroglia and astrocytes, reflecting proliferating populations of these glial sub-types. It is possible that functional inactivation of p53 in nonlytically infected astrocytes may allow neoplastic astrocyte clones to emerge. However, p53 and PCNA immunoreactivity per se cannot be regarded as indicative of neoplasia in PML, and caution must be exercised in the interpretation of such nuclear staining profiles.

Expression of viral T-antigen in pathological tissues from transgenic mice carrying JC-SV40 chimeric DNAs.

Immunostaining methods were used to detect viral T-antigen and the cellular protein p53 in pathological tissues obtained from transgenic mice carrying JC-SV40 hybrid viral DNAs. A transgenic mouse carrying the SV40 regulatory region and JC virus (JCV) T-antigen-coding sequences exhibited an SV40-characteristic choroid plexus papilloma that expressed JCV T-antigen and p53. JCV-associated pathology was observed in two other mice in which the JCV regulatory signals directed SV40 T-antigen-induced adrenal neuroblastomas and brain neoplastic cells. However, these mice also exhibited an SV40-characteristic osteosarcoma and abdominal lymphoma that contained SV40 T-antigen and p53-positive cells. Contrasting thymic pathology was observed in the two types of mice where the SV40 regulatory region directed a JCV T-antigen-induced thymoma in one mouse, and the JCV regulatory region directed SV40 T-antigen-induced thymic hypoplasia in two other mice.

Region- and age-specific differences in proglucagon gene expression in the central nervous system of wild-type and glucagon-simian virus-40 T- antigen transgenic mice

The gene encoding proglucagon is expressed in the A-cells of the endocrine pancreas and the L-cells of the gastrointestinal tract. Proglucagon-derived peptides have also been detected in different regions of the central nervous system; however, proglucagon mRNA transcripts have been localized predominantly to the brainstem and hypothalamus. We have studied proglucagon gene expression in the brain of mice using the reverse transcription-polymerase chain reaction technique. Proglucagon mRNA transcripts were detected in the cortex, cerebellum, and brainstem of embryonic day 19 mouse brain and in the cortex, cerebellum, brainstem, and hypothalamus of 2- to 12-week-old wild-type mice. Age-specific changes in proglucagon gene expression were observed, with a progressive decrease in the levels of proglucagon mRNA transcripts detected in the cortex of older mice. In contrast, a progressive increase in the levels of proglucagon mRNA transcripts was detected with increasing age in the brainstem. Proglucagon gene expression was also examined in the brain of glucagon-simian virus-40 (SV40) T-antigen transgenic mice. The levels of proglucagon gene expression in transgenic cerebellum and brainstem were relatively greater than those in wild-type mice on embryonic day 19, and increased levels of proglucagon mRNA transcripts were observed in transgenic cerebellum, brainstem, and hypothalamus at 2 weeks. In contrast, the levels of proglucagon mRNA transcripts were markedly reduced in the brain of 5-week-old transgenic mice. A reduction in the levels of SV40 T-antigen mRNA transcripts was also detected in the brain of transgenic mice at 5 weeks, but no suppression of the mRNA transcripts for the neuropeptide somatostatin was observed in 5-week-old transgenic brain. The results of these studies suggest that proglucagon mRNA transcripts are more widely distributed in the mouse central nervous system than previously demonstrated and that proglucagon gene expression in the brain appears to be regulated in a region- and age-specific manner. The coordinate region-specific expression and regulation of mRNA transcripts derived from the endogenous mouse proglucagon gene and a glucagon-SV40 T antigen transgene in the central nervous system of mice harboring a transgene containing 2.0 kilobases of rat proglucagon gene 5'-flanking sequences fused to the coding sequence of SV-4 large T-antigen suggest that neural-specific elements residing within the first 2.0 kilobases of glucagon gene 5'-flanking sequences are sufficient for the correct targeting and regulation of proglucagon gene expression in the brain.

Phosphoinositide metabolism in a polyoma-BK-virus-transformed pancreatic islet cell line: evidence for constitutively activated phospholipase C.

We have characterized the phosphoinositide metabolism in a polyoma-BK-virus-transformed rat pancreatic islet cell line which has highly malignant characteristics, expresses viral T-antigen and has lost insulin-secreting capacity. After incorporation with [3H]inositol to isotopic equilibrium, all inositol metabolites were analyzed. When compared with normal pancreatic islets, increased levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), inositol 1,3,4-trisphosphates and inositol tetrakisphosphate (Ins-P4), and decreased levels of phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) were found. The Ins-1,4,5-P3/PIP2 ratio increased, whereas the PIP2/PIP ratio was not altered after the transformation. In the pancreatic islet cell line there was a stable accumulation of inositol phosphates at 3.3 mM glucose. Glucose, KCl, cholecystokinin (CCK) and carbachol with and without LiCl were all without effect on the accumulation of inositol phosphates. Somatostatin inhibited the accumulation of inositol phosphates but a Ca(2+)-free/EDTA solution did not. Preincubation with cholera toxin or pertussis toxin inhibited the accumulation of inositol phosphates at 3.3 mM glucose except for Ins-P4, whereas no effect was observed on the phosphoinositides. NaF stimulated the accumulation of inositol phosphates, with a concomitant decrease in the phosphoinositides, whereas neomycin was without effect on the inositol phosphates. In normal pancreatic islets, pertussis toxin inhibited the CCK-induced increase in Ins-1,4,5-P3, whereas no effect was seen at 3.3 mM glucose. Finally, pertussis toxin inhibited the CCK-induced increase in the Ins-1,4,5-P3/PIP2 ratio in normal pancreatic islets. The same inhibition was also found in the pancreatic islet cell line at 3.3 mM glucose. We conclude that in the transformed pancreatic islet cell line the phosphoinositide hydrolysis is constitutively activated at the level of phospholipase C, with a substantial loss of regulatory control.

Modulation of growth-related gene expression and growth inhibition by cyclic adenosine 3',5'-monophosphate-elevating agents in the insulin-producing cell line beta TC1.

We studied the involvement of the cAMP pathway in the regulation of beta TC1 cell growth with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the activator of adenylate cyclase forskolin. We examined the effect of the increase in cAMP content on the serum-induced resumption of the cell cycle of quiescent cells. IBMX and forskolin both inhibited the mitogenic effect of serum in a concentration-dependent manner. Intracellular cAMP levels were, respectively, enhanced 3.0- and 8.6-fold by IBMX (0.5 mM) and forskolin (20 microM) within 1 h. IBMX and forskolin were also inducers of insulin release, indicating that the growth-arrested beta TC1 cells have retained the essential characteristics of the normal differentiated beta-cells. The effects of IBMX and forskolin were correlated with a modulation of cell cycle-related gene expression. IBMX induced expression of the c-fos gene, which was further enhanced by the simultaneous addition of serum, whereas forskolin alone elicited maximal induction of this gene. Interestingly, c-jun expression was only enhanced with forskolin. We also studied the effects of IBMX and forskolin on the expression of the simian virus-40 T-antigen controlled by the rat insulin II promoter in beta TC1 cells. IBMX and forskolin inhibited the serum-induced accumulation of simian virus-40 T-antigen mRNA in quiescent as well as exponentially growing beta TC1 cells.