Simian Virus 40 T-antigen Research Articles

TIGHTLY REGULATED HUMAN HEPATOCYTES AS A SOURCE OF A BIOARTIFICIAL LIVER

Purpose: One of the major limitations for establishing a novel bioartificial liver (BAL) is shortage of healthy human hepatocytes. In an effort to address this issue, we here describe a method to develop human hepatocyle cell lines with tight regulation. Methods: Human hepatocytes were transfected with a plasmid SV3neo containing the simian virus 40 T antigen (SV40T) gene followed by an introduction of the suicide herpes simplex virusthymidine kinase (TK) gene or transduced by a retroviral vector SSR#69 expressing the genes of TK and SV40T intervened by a pair of loxP recombination targets that were excisable by Cre/loxP-site specific recombination. Ganciclovir (GCV) sensitivity, the gene expression of differentiated liver functions, the efficacy of Cre/loxP recombination, and an in-vivo transplantation effect in animals were examined in SV40T-transdcued hepatocytes. Results: OUMS-29/TK, one of SV3neo-transformed hepatocytes, and NKNT-3, one of SSR#69-transduced hepatocytes, grew economically in culture with preserving adequate liver functions and were sensitive to 5 uM GCV. Intrasplenic transplantation of these cells prolonged the survival of 90% hepatectomized rats. A transient expression of Cre recombinase efficiently removed SV40T from NKNT-3 cells. Conclusion: We demonstrate here for the first time an attractive strategy for resolving the problem of donor liver shortage that currently limits the use of primary human hepatocytes for BAL therapy.

Species-specific elements in the large T-antigen J domain are required for cellular transformation and DNA replication by simian virus 40.

The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo.

Multipotency of a Bone Marrow Stromal Cell Line, TBR31-2, Established from ts-SV40 T Antigen Gene Transgenic Mice

Bone marrow is believed to contain multipotential stromal stem cells which can differentiate into osteoblasts, chondrocytes, adipocytes, and myoblasts (Prockop, D. J. Science 276, 71–74, 1997). Therefore, characterization and identification of the stem-like cell within the stromal cells are important to understand bone marrow function in relation to the hematopoietic microenvironment, and repair/regeneration of tissue defects. TBR31-2 cell, a bone marrow stromal cell line established from bone marrow of transgenic mice harboring temperature-sensitive (ts) simian virus (SV) 40T-antigen gene for immortality, is induced toward both adipocytic and osteogenic cells under conditions of the inactivation of T-antigen (Okuyama, R., Yanai, N., Obinata, M. Exp. Cell Res. 218, 424–429, 1995). In this work, using a semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis, mRNA expressions of tissue-specific differentiation markers for adipocyte (lipoprotein lipase), osteoblast (type I collagen and osteocalcin), chondrocyte (type II and X collagen), and muscle cell (desmin) were examined during a long-term culture of the cell. In addition, histochemical studies showed the appearance of adipocytic, osteoblastic, chondrocytic, and muscle cells during this long-term culture. Thus, TBR31-2, which has characteristics of an undifferentiated cell, has the potential to express the multipotential cell lineages. These results indicated that a multipotential progenitor cell including potential to differentiate into a muscle cell and which is situated in the mesenchymal cell lineage was first obtained.

SV40 virus transformation down-regulates endothelin receptor

Simian virus 40 (SV40) is an oncogenic DNA virus that induces malignant transformation. Endothelin (ET), a 21 amino acid peptide with mitogenic and anti-apoptotic effects, binds to G-protein coupled ET A and ET B receptors. This report examines the effect of SV40 transformation on the expression of ET receptors. Results from receptor binding and reverse transcription (RT)-polymerase chain reaction (PCR) studies show that human lung fibroblasts IMR90 and WI38 express both ET A and ET B receptors, and that the expression of both receptors is significantly down-regulated in IMR90-SV40 and WI38-SV40, cell lines derived from IMR90 and WI38 with SV40 virus transformation. Receptor binding and RT-PCR analysis of 3A(tPA-30-1), a cell line derived from human placenta that expresses a higher level of SV40 large T-antigen at the permissive temperature (33°C) than at the restrictive temperature (40°C), further demonstrates that there is an inverse correlation between the expression of SV40 T-antigen and the expression of ET receptor. ET-1 and fetal bovine serum stimulate DNA synthesis in non-transformed cells; however, proliferation of transformed cells is independent of either fetal bovine serum or ET-1. We conclude that SV40 transformation down-regulates the expression of ET receptors, and that expression of ET receptors is inversely correlated with expression of SV40 large T-antigen.

Interaction of the Single-stranded DNA-binding Protein Purα with the Human Polyomavirus JC Virus Early Protein T-antigen

Large T-antigen, the major regulatory protein encoded by polyomaviruses, including Simian Virus 40 (SV40) and JC virus (JCV), is a multifunctional phosphoprotein that is involved in many viral and cellular events. In addition to its integral role in viral replication and cellular transformation, T-antigen also regulates transcription of both viral and cellular genes. In particular, the viral late promoter has been used as a model for the analysis of T-antigen-mediated transcriptional activation. Earlier studies have demonstrated that the cellular protein Puralpha is able to attenuate the transcriptional activity of JCV T-antigen. We investigated the mechanism whereby Puralpha affects T-antigen function. Co-immunoprecipitation studies demonstrated that Puralpha and JCV T-antigen associate in vivo, and glutathione S-transferase affinity binding assays revealed that these two proteins interact in vitro. Moreover, we localized the sequences of Puralpha that are important for the interaction between Puralpha and JCV T-antigen. In addition, we demonstrated that Puralpha interacts with the SV40 T-antigen. Transient transfection studies demonstrated that Puralpha and JCV T-antigen interact functionally as well. More specifically, Puralpha and a deletion mutant that interacts with T-antigen attenuated T-antigen-mediated transcriptional activation. A Puralpha deletion mutant that is unable to interact with JCV T-antigen, however, was found to be incapable of abrogating JCV T-antigen transactivation. Taken together, these data demonstrate that Puralpha and T-antigen interact both physically and functionally and that this interaction modulates T-antigen-mediated transcriptional activation. The implication of these findings with respect to the cellular role of Puralpha is discussed.

Integration of SV40 in human osteosarcoma DNA.

Simian virus 40 (SV40) has been demonstrated in several types of tumors, including osteosarcoma, by polymerase chain reaction (PCR). We detected SV40 sequences by PCR, followed by hybridization, in nine of 35 osteosarcoma tumors and one of 11 osteosarcoma explants. PCR can detect fewer than one virus per cell but gives little detail of the gross structure and abundance of the virus. Analysis by Southern blotting of total DNA from ten osteosarcomas, positive for SV40 by PCR, found viral integration in half of these. Analysis showed integration of one to four copies per cell of rearranged SV40. No SV40 was detectable on blots of the remaining five SV40+ osteosarcomas, perhaps because of the lesser sensitivity of direct hybridization. Inactivation of the p53 and Rb tumor suppressors is a key activity of SV40 T-antigen. Unexpectedly, correlation of these findings with our prior studies indicated that five of ten osteosarcomas positive for SV40 DNA had mutations of p53, and two had deleted Rb. Apparently clonal integration with pre-existing alteration of a tumor suppressor gene, suggests that SV40 may play a role in the final conversion to malignant osteosarcoma.

Reinitiation of DNA synthesis and cell division in senescent human fibroblasts by microinjection of anti-p53 antibodies.

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the "young" morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.

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.

Expression of a fusion gene consisting of the mouse growth hormone-releasing hormone gene promoter linked to the SV40 T-antigen gene in transgenic mice

Limited information is available concerning the regulation of growth hormone-releasing hormone (GHRH) gene expression in the hypothalamus, largely because of the lack of a suitable cellular model. In an attempt to immortalize hypothalamic GHRH-producing neurons, we have generated a transgenic mouse model which expresses the simian virus 40 (SV40) T-antigen gene (Tag) under the control of the GHRH gene promoter. The transgene contains ≈5 kb of mouse GHRH gene sequences, including 3.5 kb of the 5′-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1, fused to the SV40 Tag gene. This construct was microinjected into fertilized oocytes. Fourteen of 96 mice born had integrated the transgene. These mice were fertile and showed no signs of central or peripheral tumors. The pattern of expression of the SV40 Tag gene was analyzed in four different transgenic lines by RT-PCR. The tissues tested include: hypothalamus, pituitary, cortex, cerebellum, spinal cord, adrenal, testis, spleen and lung. Transgene expression was consistently detected in the hypothalamus of all lines. In addition, SV40 Tag expression was also detected in the hypothalamus by Northern blot analysis in two of the transgenic lines. SV40 Tag expression was also detected in the testis of all transgenic lines by RT-PCR. This result was not expected since the GHRH gene sequences present in the transgene do not include the testis-specific transcription initiation site previously described. This suggests that GHRH gene expression in the mouse testis can be directed by regulatory sequences located downstream of the testis specific transcription start site. We conclude that the promoter region of the GHRH gene included in this construct contains the regulatory elements necessary to drive hypothalamic and testis expression in vivo . In addition, all mice from one of the transgenic lines developed cataracts in both eyes. SV40 Tag expression was detected not only in eyes with cataracts, but also, to a lesser extent, in eyes from other transgenic lines. Furthermore, the endogenous GHRH gene was found to be expressed in the eyes of normal mice.

Identification of a minimal promoter element of the mouse epidermal growth factor gene.

We have previously generated a transgenic mouse line (EGF/Tag) in which simian virus 40 (SV40) T-antigen expression is directed by the mouse epidermal growth factor (EGF) gene promoter. In these mice, cellular hyperproliferation is observed in the submaxillary gland associated with SV40 T-antigen expression. In addition, SV40 T-antigen-expressing tumours of prostatic origin are seen. We have now derived immortalized cell lines from these tissues and have used the cells to perform a functional analysis of the EGF gene promoter. Cells were transfected with EGF promoter/reporter constructs, and an element located between 51 and 35 bases upstream of the EGF mRNA start site required for basal activity of the promoter was identified. Electrophoretic mobility-shift analysis suggests that three proteins bind to this region, one of which is either Sp1 or a closely related protein.

Reexpression of the Type 1 Insulin-Like Growth Factor Receptor Inhibits the Malignant Phenotype of Simian Virus 40 T Antigen Immortalized Human Prostate Epithelial Cells1

Type 1 insulin-like growth factor receptor (IGF-1R) expression is decreased in prostate cancer compared to that in noncancerous prostate epithelium. We have demonstrated that as the simian virus 40 T antigen (SV40T) immortalized human prostate epithelial cell line, P69SV40T, undergoes transformation from a poorly tumorigenic to a malignant phenotype, the M12 subline, there is a significant decrease in IGF-1R expression. In the present study, we examine the effects of reexpression of the IGF-1R on the malignant phenotype of M12 cells. The IGF-1R was reexpressed in M12 cells using a retroviral vector containing a 7-kilobase coding sequence for the IGF-1R, LISN, to create several clones of the M12-LISN cell line. As a control, M12 cells were also infected with a retroviral vector (LNL6) without the 7-kilobase IGF-1R insert (M12-LNL6 clones). Functional assays were performed with two separate clones each of M12-LNL6 and M12-LISN cells. Each clone of M12-LISN cells regained the proliferative response to IGF that was lost in the transition from P69SV40T cells to M12 cells. In addition, M12-LISN clones had a significantly decreased growth rate compared to the M12-LNL6 cells when injected s.c. in athymic/nude mice (P < 0.001). Tumorigenicity, as assessed by anchorage-independent growth of colonies in soft agar, was also decreased by 75% in the M12-LISN clones compared to that in the M12-LNL6 control cells. These data demonstrate that reexpression of the IGF-1R in a malignant human prostate epithelial cell line results in decreased tumor growth and decreased anchorage-independent colony formation independent of an increased proliferative response to IGF. Reexpression of the IGF-1R may be associated with reacquisition of the regulation of cellular proliferative and differentiation functions mediated by the IGF-1R that are lost as prostate epithelial cells undergo conversion to a malignant phenotype.

Solution structure of the origin DNA-binding domain of SV40 T-antigen.

The structure of the domain from simian virus 40 (SV40) large T-antigen that binds to the SV40 origin of DNA replication (T-ag-OBD131-260) has been determined by nuclear magnetic resonance spectroscopy. The overall fold, consisting of a central five-stranded antiparallel beta-sheet flanked by two alpha-helices on one side and one alpha-helix and one 3(10)-helix on the other, is a new one. Previous mutational analyses have identified two elements, termed A (approximately 152-155) and B2 (203-207), as essential for origin-specific recognition. These elements form two closely juxtaposed loops that define a continuous surface on the protein. The addition of a duplex oligonucleotide containing the origin recognition pentanucleotide GAGGC induces chemical shift changes and slows amide proton exchange in resonances from this region, indicating that this surface directly contacts the DNA.

Improved "activator trap" method for the isolation of transcriptional activation domains from random DNA fragments.

We have previously developed an "activator trap" method for selective isolation of activation domains from viral and cellular transcription factors. In this method, random sonicated DNA fragments were ligated next to the DNA binding domain (DBD) of the GAL4 factor in a plasmid that also contained a simian virus 40 (SV40) replication origin. A library of such random insert plasmids was transfected into a monkey cell line (CV-1-5GT), which had been stably transformed with a GAL4-inducible SV40 T-antigen gene. Chimeric GAL factors with a heterologous activation domain were harvested after selective replication in these CV-1-5GT cells. Here we report a simplification and generalization of the "activator trap" method. First, the time-consuming library construction step can be omitted by direct transfection of the sonicated DNA fragments and the linearized recipient plasmid vector into CV-1-5GT cells to obtain chimeric GAL-activation factors by in vivo ligations. Second, the dependence on CV-1-5GT cells can be bypassed by direct co-transfection of all components, including a plasmid carrying the T-antigen gene into cells other than CV-1-5GT. This latter step allows the application of the method to cultured human cells, as demonstrated with the human B-cell line BJA-B.

Simian virus 40 large T antigen alters the phosphorylation state of the RB-related proteins p130 and p107.

p130 and p107 are nuclear phosphoproteins related to the retinoblastoma gene product (pRb). pRb, p107, and p130 each undergo cell cycle-dependent phosphorylation, form complexes with the E2F family of transcription factors, and associate with oncoproteins of DNA tumor viruses, including simian virus 40 (SV40) large T antigen (TAg) and adenovirus ElA protein. The results of recent studies with mouse embryo fibroblasts (MEFs) lacking the retinoblastoma gene (Rb-1) have suggested that p130 and p107 may be important targets for SV40 large TAg-mediated transformation (J.B. Christensen and M.J. Imperiale, J. Virol. 65:3945-3948, 1995; J. Zalvide and J.A. DeCaprio, Mol. Cell. Biol. 15:5800-5810, 1995). In this report, we demonstrate that the expression of TAg affects the phosphorylation state of p130 and p107. In cells expressing wild-type TAg, only un(der)phosphorylated p130 and p107 were detected. To determine the domains within TAg that contribute to this effect on the phosphorylation of p130, we performed transient expression assays. While transiently expressed p130 was apparently phosphorylated normally, only un(der)phosphorylated p130 was detected when p130 was coexpressed with TAg. Using this assay, we found that the first 147 amino acids of TAg were sufficient to alter the phosphorylation state of p130. Within this region, the LXCXE domain of TAg, required for binding to the retinoblastoma family of proteins, was necessary but not sufficient to affect p130 phosphorylation. Residues within the first 82 amino acids of TAg were also required. TAg with mutations in the N terminus retained the ability to efficiently associate with p130 but did not affect its phosphorylation state. This demonstrates that the effect of SV40 TAg on p130 is not simply the result of binding and suggests that TAg has a novel effect on p130 and p107 that differs from its effect on pRb.

Differential regulation by SV40 T-antigen binding at site I defines two distinct classes of nucleosome-free promoter.

T-antigen binding site I has been shown previously to play a role in regulating the proportion of Simian Virus 40 (SV40) chromosomes containing a nuclease hypersensitive promoter region. In order to determine whether these changes in nuclease hypersensitivity were a result of changes in the proportion of SV40 chromosomes, which contained a nucleosome-free promoter region, SV40 chromosomes were visualized by electron microscopy. SV40 chromosomes were prepared from cells infected with either wild-type or mutant virus lacking T-antigen binding site I, and the chromosomes were analyzed by electron microscopy for the presence of nucleosome-free regions. Both the wild-type and mutant chromatin were found to contain heterogeneous nucleosome-free promoter regions consisting of small (3-4 times the normal internucleosomal distance) and large (greater than four times the normal internucleosomal distance) regions. Quantification of the proportion of chromosomes containing each type of region indicated that deletion of site I resulted in a 25% increase in the proportion of chromosomes containing a large nucleosome-free region but had no effect on the proportion of chromosomes containing a small nucleosome-free region. The results indicate that there are two distinct classes of nucleosome-free promoter region in SV40 chromatin and that T-antigen binding to site I is specifically involved in the regulation of only the class that contains a large nucleosome-free region.

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.

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.

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.

Simian virus 40 T antigen as a carrier for the expression of cytotoxic T-lymphocyte recognition epitopes

Simian virus 40 (SV40) large T antigen can immortalize a wide variety of mammalian cells in culture. We have taken advantage of this property of T antigen to use it as a carrier for the expression of cytotoxic T-lymphocyte (CTL) recognition epitopes. DNA sequences corresponding to an H-2Db-restricted SV40 T-antigen site I (amino acids 205 to 215) were translocated into SV40 T-antigen DNA at codon positions 350 and 650 containing EcoRI linkers. An H-2Kb-restricted herpes simplex virus glycoprotein B epitope (amino acids 498 to 505) was also expressed in SV40 T antigen at positions 350 and 650. Primary C57BL/6 mouse kidney cells were immortalized by transfection with the recombinant and wild-type T-antigen DNA. Clonal isolates of cells expressing chimeric T antigens were shown to be specifically susceptible to lysis by CTL clones directed to SV40 T-antigen site I and herpes simplex virus glycoprotein B epitopes, indicating that CTL epitopes restricted by two different elements can be processed, presented, and recognized by the epitope-specific CTL clones. Our results suggest that SV40 T antigen can be used as a carrier protein to express a wide variety of CTL epitopes.

Conditionally immortalized intestinal epithelial cells: novel approach for study of differentiated enterocytes

Clonal cell lines have been established from primary fetal rat intestinal epithelial cells by stable transfection with plasmids containing either the simian virus 40 (SV40) large T-antigen gene under the control of the heavy metal inducible metallothionein promoter (pMTWt) or the thermolabile SV40 T-antigen gene under the control of the SV40 early promoter (pZipSVtsa58). pMTWt-transfected cells produced sufficient T-antigen to allow them to proliferate both when the metallothionein promoter was induced and uninduced. No differences were observed in the pattern of intestinal epithelial markers expressed when the cells were cultured in the presence or absence of inducing agent (zinc). In contrast, fetal rat intestinal epithelial cells transfected with pZipSVtsa58 were immortalized conditionally; cells proliferated at 32 degrees C but ceased to proliferate between 48 and 72 h of culture at 39 degrees C. Four of these cell lines were characterized in detail; they showed microvilli and tight junctions as well as dome formation and expressed functional and biochemical markers of intestinal epithelial cells, including keratins 8, 19, and 21, aminopeptidase N, and dipeptidyl peptidase IV. One cell line, 2/4/A1, expressed in addition a low level of lactase and sucrase-isomaltase. The amount and/or activity of some of these markers changed during the switch from the proliferative to the nonproliferative state (switch from culture at 32 to 39 degrees C), resulting in a more differentiated phenotype and mimicking similar changes taking place during intestinal epithelial cell differentiation in vivo.