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Transgenic mice carrying the SV40 early region fused to the Drosophila hsp70 promoter developed smooth muscle and bone neoplasms. The smooth muscle tumors appeared in aged mice and were preferentially located on the muzzle or eyelids. Multiple neoplasms were often present and each appeared to be an independent proliferation. In contrast, the bone tumors typically developed in the petrous ridge and had all the features of osteogenic sarcomas, displaying distant metastasis and invasion of the brain. Cells in both types of tumors exhibited nuclear expression of SV40 T antigen. Mice homozygous for the transgene had a shorter latency for appearance of smooth muscle tumors and developed osteosarcomas more frequently than hemizygous mice. This model system implicates the cellular T antigen-binding proteins, such as Rb and p53, in the pathogenesis of bone and soft tissue neoplasms in mice.

We have cloned two isoforms of rat kidney prostaglandin E2 receptor EP3 subtype (rEP3A and rEP3B), which differ only in their cytosolic carboxyl-terminal tails (30 and 29 amino acids, respectively). The aim is to clarify the functional difference between two rEP3 receptor isoforms by examining formation of adenosine 3',5'-monophosphate (cAMP) and change in cytosolic free calcium ([Ca2+]i) in cultured cells transiently transfected with cloned rEP3A or rEP3B receptor cDNA. In immortalized renal distal tubule cells (TKC2), vasopressin (VP) stimulated cAMP formation, and the cAMP formation was significantly attenuated by a non-peptide VP receptor antagonist, OPC-31260. The VP-induced increase in cAMP formation was also attenuated by over-expression of rEP3A receptor but not that of rEP3B receptor. On the other hand, in COS-7 cells transfected with rEP3B receptor cDNA, PGE2 induced an increase in [Ca2+]i, but no increase in [Ca2+]i was observed in the cells transfected with rEP3A cDNA. In conclusion, rEP3A receptor is suggested to antagonize VP (V2) receptor by inhibiting cAMP formation, whereas rEP3B receptor is linked with Ca2+ messenger system.

To determine whether postmitotic neurons can be immortalized by oncogenic transduction, we used two approaches involving conditional expression of a temperature-sensitive SV40 large T antigen (Tts). Initially, Tts was introduced into E17 rat embryonal hippocampal cells that were then cultured at the non-permissive temperature to enrich for postmitotic pyramidal neurons, and subsequently cloned at the permissive temperature. One clonal line (HMR10-3) expressed neuron-specific proteins upon differentiation, was capable of generating action potentials, and formed synapses with primary rat neurons in co-culture. Replating of these postmitotic cells at the permissive temperature resulted in reversible loss of neurofilament expression. Conditionally immortalized cell lines were also generated from the brain of an adult mouse carrying an inducible Tts transgene. These lines proliferated in a T antigen-dependent manner and expressed neuron-specific proteins upon differentiation at the non-permissive temperature. These results suggest that postmitotic neurons can be induced to enter the cell cycle without losing their commitment to a neuronal lineage.

Previous studies have identified several host proteins (p53, p107, and p193), which form prominent complexes with SV40 T antigen in transformed cardiomyocytes. Expression of p53 and p107 was monitored during normal and pathologic growth in nontransformed murine myocardium. Both genes were expressed at relatively high levels in embryonic cardiomyocytes. Transcript levels decreased markedly during the process of cardiomyocyte terminal differentiation and were very low or undetectable in adult animals. In contrast, retinoblastoma transcripts were observed at low levels throughout myocardial development. None of the tumor suppressor genes examined were transcriptionally activated during acute myocardial overload or isoproterenol-induced myocardial hypertrophy. The potential role of tumor suppressor gene product expression in myocardial development and pathology is discussed.

In the murine IgH gene intronic enhancer (ENHiH), two major functional domains were reported. One is the E4/octomer region and another includes the microA and microB motifs. In the human ENHiH, it was reported that the HE2, which corresponds to the murine microB, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined the in vivo function of the E6, microA and HE2 motifs within the human ENHiH by using the transgenic mice technique. The microA and HE2 motifs together revealed almost the same enhancer function as the whole human ENHiH, but the E6 motif had lesser enhancer activity and tissue-specificity.

Two major roles have been defined for nitric oxide (NO): cell-cell communication mediated by the stimulation of cyclic guanosine 3',5'-monophosphate (cGMP) synthesis and cytotoxicity by direct or indirect interaction of the free radical NO with cellular targets. Thus, pathologic states might result from an alteration of NO pathways, e.g., by deregulated activity of NO synthase. To investigate this hypothesis, we introduced the murine-inducible NO synthase (iNOS) sequence into immortalized human bronchial epithelial cells (BEAS-2B). iNOS activity, measured by conversion of [14C]arginine to [14C]citrulline in the presence of 1 mM EGTA, was higher than 100 pmol/min/mg protein in early passages of iNOS-transfected cells but decreased with cell subculturing. No iNOS activity could be detected in control vector-transfected cells. NO stimulated cGMP production in iNOS-transfected cells, and this effect was inhibited by the iNOS inhibitor NG-monomethyl-L-arginine. In addition, NO production induced c-fos expression and did not interfere with clonal cell growth. These results suggest that BEAS-2B cells constitute a suitable model to study the consequences of iNOS activity on signal transduction pathways in bronchial epithelium.

Synergism between transcriptional activators is a powerful way of potentiating their function. Here we show that the glial POU domain protein Tst-1 (also known as Oct-6 and SCIP) and large tumor antigen (T antigen) synergistically increased transcription from both the early and the late promoters of papovavirus JC in glial cells. Synergism between both proteins did not require T-antigen-mediated DNA replication or direct binding of T antigen to the promoter. The ability of T antigen to functionally cooperate with Tst-1 was contained within its N-terminal region, shown by the fact that small tumor antigen (t antigen) could substitute for T antigen in transfection experiments. In addition to this functional synergism, a direct interaction between Tst-1 and T antigen was observed in vitro. Using deletion mutants of Tst-1 and T antigen, the POU domain of Tst-1 and the N-terminal region of T antigen were found to participate in this interaction. Because of the low levels of Tst-1 present in oligodendrocytes, synergism between Tst-1 and T antigen could be an important factor in establishing the lytic infection of oligodendrocytes by JC virus during the course of the fatal demyelinating disease progressive multifocal leukoencephalopathy.

The dermal papilla is a discrete group of cells at the base of the hair follicle and is implicated in controlling the hair growth cycle. Early passage dermal papilla cells can induce hair growth in vivo, but, upon further culturing, this property is lost. In order to study the events occurring in hair induction, a representative dermal papilla cell line was required. We have transfected passage 1 rat vibrissa dermal papilla cells with a polyomavirus large T gene encoding a temperature-sensitive T antigen, and generated permanent cell lines in which the immortalizing function can be switched off by temperature shift. The cells established without crisis, resembled cells in the starting population, and retained the aggregative properties of early passage dermal papilla cells. Growth studies were performed on the immortalized cell lines, which showed that transferring the cells to the restrictive temperature for the large T gene product resulted in cell senescence or quiescence, and changes in morphology. Implantation of cell pellets into the ears of immunologically compatible rats showed that the immortal cells retained hair-inductive ability. Cytokines are believed to have an important role in the control of hair growth. The pattern of cytokine gene expression in the immortal cell lines was compared with early passage dermal papilla cells and a non-hair-inducing dermal papilla cell line, using reverse transcriptase-polymerase chain reaction. Epidermal growth factor, tumour necrosis factor, and interleukin-1a were detected in the immortalized and non-hair-inducing dermal papilla cell lines, but were absent in passage 2 dermal papilla cells. All other cytokines examined were detected in all the cell types under study. These results demonstrate that the polyomavirus large Ttsa-immortalized dermal papilla cell lines are very similar to passage 2 dermal papilla cells and thus provide a good model for hair growth studies. Cytokine expression profiles indicate that the expression of several cytokines may be implicated in hair induction. Further studies are under way to investigate the relationship between cytokine expression and the hair growth cycle.

PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.

Males of the MT-PVLT-10 transgenic mouse line, which expresses the polyomavirus large T-antigen under the control of the metallothionein promoter, develop testicular adenomas and display seminal vesicle enlargement. Histological analysis of adenomatous testis indicates a predominance of Leydig cells, with few normal Sertoli cells or seminiferous tubules remaining. Primary cell cultures established from the testes of control nontransgenic animals (all ages) and young MT-PVLT-10 animals (before the appearance of any phenotype) quickly entered crisis and died. In contrast, permanent cell lines could be derived from pre- and postadenomatous testes from older MT-PVLT-10 mice. All primary cultures and cell lines expressed large T-antigen. A primary culture (D-37) derived from an MT-PVLT-10 male with normal testes but enlarged seminal vesicles has been maintained for over 2 years and experiments indicate that the D-37 culture is unable to form tumors in nude mice. In contrast, a primary culture (D-4) derived from adenomatous testes of an MT-PVLT-10 mouse is also immortal, but injection of this culture into nude mice consistently resulted in tumor formation. Cloning of the D-4 culture resulted in pure Sertoli or Leydig cell clones, neither of which could form tumor upon injection into nude mice. Injection of a mixture of both cell types did result in tumor formation, suggesting a dynamic interaction between these cell types in MT-PVLT-10-induced tumorigenesis.

We have expressed the simian virus 40 (SV40) large T antigen oncogene in erythroid tissues of mice to test its ability to immortilize erythroid cells. A transgene construct was built in which the SV40 large T antigen structural gene was linked to erythroid-specific enhancer and promoter sequences. The enhancer employed was the human beta-globin family microlocus control region, and the promoter sequences were derived from the human beta-globin promoter. Transgenic mice were generated and they expressed T antigen in the bone marrow and spleen cells. Yet, no hematopoietic neoplasia arose in these mice. Instead, after a lag period of 2-6 months, the mice developed soft tissue sarcomas and pancreatic islet-cell tumors that expressed high levels of T antigen.

The ability of simian virus 40 (SV40) large T antigen to catalyze the initiation of viral DNA replication is regulated by its phosphorylation state. Previous studies have identified the free catalytic subunit of protein phosphatase 2A (PP2Ac) as the cellular phosphatase which can remove inhibitory phosphoryl groups from serines 120 and 123. The catalytic C subunit exists in the cell complexed with a 65-kDa A subunit and one of several B subunits. To determine if any of the holoenzymes could activate T antigen, we tested the ability of the heterodimeric AC and two heterotrimeric ABC forms to stimulate T-antigen function in unwinding the origin of SV40 DNA replication. Only free catalytic subunit C and the heterotrimeric form with a 72-kDa B subunit (PP2A-T72) could stimulate T-antigen-dependent origin unwinding. Both the dimeric form (PP2A-D) and the heterotrimer with a 55-kDa B subunit (PP2A-T55) actively inhibited T-antigen function. We found that PP2A-T72 activated T antigen by dephosphorylating serines 120 and 123, while PP2A-D and PP2A-T55 inactivated T antigen by dephosphorylating the p34cdc2 target site, threonine 124. Thus, alterations in the subunit composition of PP2A holoenzymes have significant functional consequences for the initiation of in vitro SV40 DNA replication. The regulatory B subunits of PP2A may play a role in regulating SV40 DNA replication in infected cells as well.

A transgenic mouse line (EGF/Tag) has been established in which expression of SV40 T-antigen is directed by a 5.5 kb fragment of the 5'-flanking region of the mouse epidermal growth factor (EGF) gene. Of the two principal sites of EGF expression in mice, submaxillary gland and kidney, T-antigen mRNA and protein were detected in the former but not in the latter tissue of the EGF/Tag animals. T-antigen expression in the submaxillary gland was restricted to the EGF-producing cells of the granular convoluted tubules, and the oncoprotein induced hyperplasia of these cells. T-antigen levels were markedly higher in the submaxillary glands of male compared with female transgenic mice, suggesting that expression of the transgene was androgen-regulated, like the endogenous EGF gene. These results indicate that the 5.5 kb fragment upstream of the mouse EGF gene contains the DNA enhancer elements required for hormonally regulated expression in the submaxillary gland. Since the hyperplastic submaxillary glands of the EGF/Tag mice continue to synthesize EGF, these glands provide a tissue source from which it may prove possible to establish EGF-secreting cell lines for further in vitro studies of the mechanisms regulating expression of the EGF gene.

We have recently shown that the nuclear mitotic apparatus protein (NuMA) is composed of at least three isoforms that differ mainly at the carboxy terminus, and are generated by alternative splicing of a common mRNA precursor from a single NuMA gene (J. Cell Sci. (1993) 104, 249-260). Transient expression of human NuMA-1 isoform (T33/p230) in Chinese hamster ovary polyoma (CHOP) cells showed that NuMA-1 was present in interphase nuclei and was concentrated at the polar regions of the spindle apparatus in mitotic cells. However, expression of two other isoforms (NuMA-m and -s) revealed a distinct subcellular localization. NuMA-m (U4/p195) and NuMA-s (U6/p194) were present in the interphase cytosol and appeared to be mainly located at the centrosomal region. When cells entered into mitosis, however, NuMA-m and -s moved to the mitotic spindle pole. Analysis of a series of linker scanning-mutants and NuMA/beta-galactosidase chimeric proteins showed that residues 1972-2007 of NuMA-1 constitute a novel nuclear localization signal (NLS) and residues 1538-2115 are necessary and sufficient for spindle association. Further analysis of the NLS by site-specific mutagenesis indicated that Lys1988 is essential for nuclear targeting, whereas Arg1984 is not. These results have allowed us tentatively to assign specific biological activities to distinct structural domains of the NuMA polypeptide.

Human endothelial cells were transfected with a plasmid containing the coding sequence of the large T protein of simian virus 40. Transfected cells were selected for their ability to grow in defined medium (DM). Several cell lines were derived and characterized in their response to endothelial cell growth supplement (ECGS), epidermal growth factor (EGF) and insulin (INS). In addition to cell lines that were dependent on these additives, others growing without any exogenous growth factor could be selected. No evidence of autocrine growth stimulation was found. For growth studies, a simple assay was used based on the acid phosphatase activity as a parameter for the cell number. Cell lines in defined medium showed less chromosome aberrations than those grown in serum-containing medium. Because of their long in vitro life span of about 100 generation doublings and defined medium requirements these cells represent valuable test material for all kinds of investigations on the vascular endothelium.

Physical and functional interactions of simian virus 40 (SV40) and polyomavirus large-T antigens with DNA polymerase alpha-primase were analyzed to elucidate the molecular basis for the species specificity of polymerase alpha-primase in viral DNA replication. SV40 T antigen associated more efficiently with polymerase alpha-primase in crude human extracts than in mouse extracts, while polyomavirus T antigen interacted preferentially with polymerase alpha-primase in mouse extracts. The apparent species specificity of complex formation was not observed when purified polymerase alpha-primases were substituted for the crude extracts. Several functional interactions between T antigen and purified polymerase alpha-primase, including stimulation of primer synthesis and primer elongation on M13 DNA in the presence or absence of the single-stranded DNA binding protein RP-A, also proved to be independent of the species from which polymerase alpha-primase had been purified. However, the human DNA polymerase alpha-primase was specifically required for primosome assembly and primer synthesis on SV40 origin DNA in the presence of T antigen and RP-A.

The oncogenic mouse polyoma virus encodes six proteins, two of which (the large and middle T antigens), when expressed together in primary rodent cells, generate an alteration of growth patterns commonly known as cellular transformation. The transformed cells grow with an unlimited life span and when introduced into immunocompromised mice rapidly form tumours. The large T gene product confers an indefinite growth potential on primary cells; however, the middle T gene product has been identified as bringing about the changes which ultimately allow the cell to form tumours. The 55 kDa middle T antigen has been shown to associate with a number of cellular enzymes involved in regulation of growth factor signalling pathways, all of which were identified as being components of the immunocomplexes that can be isolated from transformed cells using middle T antigen specific antibodies. Two-dimensional gels have assisted the search for less prominent species present in these complexes. These methods represents one approach to investigating associating proteins, and as such, select for those interactions that are stable under the conditions used. In order to explore the possibility that middle T antigen could form complexes with other cellular proteins given different conditions, recombinant middle T antigen was used in a series of "filter overlay" experiments.

Background: Expression of polyomavirus middle T antigen (PymT) rapidly induces endothelial tumors (hemangiomas) in mice, with an apparent single rate-limiting step. Because activation of Src-like kinases is thought to be an important component of PymT-induced transformation, we have analyzed the functional requirement for individual kinases in this process. This type of analysis has only recently become possible, with the generation of 'gene knock-out' mice lacking each of the kinase genes src, fyn and yes., Results: Hemangiomas develop efficiently in newborn mice lacking either src, fyn or yes after inoculation with a PymT-transducing retrovirus. In src- and fyn-deficient mice, the kinetics of induction and the histological properties of the tumors were indistinguishable from those in wild-type mice. In contrast, a reduced number of tumors arose in yes-deficient mice, with a significantly longer latency period. Transformed endothelial cell lines derived from the induced hemangiomas, however, did not differ in their morphological and tumorigenic properties from cell lines established previously from wild-type mice. Biochemical analysis of complexes between PymT and the Src-related kinases in these cell lines suggests that the Yes kinase is responsible for a significant amount of the PymT-associated kinase activity in transformed endothelial cells., Conclusion: We have demonstrated that inactivation of a single tyrosine kinase of the Src family in endothelial cells does not abrogate PymT-induced hemangioma formation. As the remaining kinases do not compensate for the absence of a family member by elevated kinase activity, the loss--which affects the transformation process to varying degrees--can be studied in this model system. Our studies suggest that the PymT-Yes kinase complex plays a major role in the tumor-initiating action of PymT.

An oncogene-carrying replication-defective retrovirus was used to establish immortalised lines of murine glial cells. Primary cultures of early postnatal cerebellar cells were infected with a retrovirus based on the Murine Moloney Leukemia Virus containing a temperature-sensitive mutant of the Simian Virus 40 large T antigen (SV40 T) oncogene and a gene coding for resistance to the antibiotic G418. Infected cells were selected in G418 and after several in vitro passages cells expressing the O4 antigen were established as a cell line. At a later time point O4-positive single-cell clones were established. Two different types of clones were obtained: 1) "plastic" clones consisting of cells which initially had a morphological and antigenic phenotype of young glial precursor cells but which gradually lost these features, and 2) "stable" cell clones including a clone with the immunological and electrophysiological characteristics of Schwann cells. Culture of the latter cells in the presence of 1 mM dibutyryl cyclic adenosine monophosphate for a period of at least 10 days induced a change in shape and a shift in antigen expression towards a more "differentiated" maturation stage. When the SV40 T O4-positive immortalised cell line isolated on the cell sorter was transplanted into demyelinated lesions in adult rats, cells were observed ensheathing axons and forming limited amounts of PNS-type myelin. Glial cells immortalised with a temperature-sensitive mutant of the SV40 T oncogene thus retain many physiological properties of their primary culture counterparts and can be induced to undergo limited differentiation in vitro and in vivo. These cell lines, which represent immature CNS glia or Schwann cells, are providing useful tools for investigating the role of cell surface antigens involved in neuron-glial interactions.

The expression of polyomavirus large T antigen in stably transfected C2 myoblast cells inhibits terminal differentiation without inducing a transformed phenotype. In the present work, we report on the lifting of this inhibition by a mutation that prevents polyomavirus large T antigen from binding to the product of the retinoblastoma susceptibility gene (p105 RB). In contrast with cells containing wild-type large T, those with the Rb binding site mutant large T showed the same up-regulation of myosine heavy chain and myogenin mRNA expression as control cells. Furthermore, we correlate the cell cycle alteration induced by polyomavirus large T antigen expression with the inability of the cells to undergo terminal differentiation.

Revertant cell lines RD2005 and RXHO24 obtained from the reversion of the transformed cell lines TD2005 and TXHO24 produced Simian virus 40 large tumour antigen. Transformed cells resulted in the transfection of the FR3T3 cell line with the mutant plasmids of Simian virus (SV40) called D2005 and XHO24. The first plasmid D2005 encoded the large tumour (LT) antigen in its entire size. The second plasmid XHO24 encoded only a small portion: 19% of the LT-antigen. The analysis of the viral protein by immunoprecipitation showed the production of the LT protein in revertant cells. The LT oncogenes produced in revertant cells could not be expressed, because some cellular factors or some epigenetic and genetic events controlled their expression. The modulation of the expression of a viral oncogene could also be associated with alterations in the chromatin structures. This may explain the loss or the reacquisition of its activity, providing a useful dynamic heterogeneity model of tumoral progression in cancer cells.

The biosynthesis of myelin-associated glycolipids was studied in quiescent secondary cultures of Schwann cells and in rapidly proliferating population of transfected Schwann cells (TSC) by in vitro incorporation of [3H]galactose. The TSC demonstrated a marked increase (> 10-fold) in [3H]galactose incorporation when compared to quiescent Schwann cells. The level (or amount) of [3H]galactose incorporation into lipids is dependent upon the number of TSC in culture. The majority of 3H-labeled lipids were oligohexosylceramides (GL-2, GL-3, and GL-4). Substrates that inhibit TSC proliferation, collagen type I and Matrigel, and artificial basement membrane, decrease the [3H]galactose incorporation by 25% and 80%, respectively. Our results indicate that the synthesis of glucocerebroside and its homologs is associated with Schwann cell proliferation.

We transiently exposed SV 40 infected TC 7 cell cultures to a reduced O2 tension (4-8 h, about 200 ppm relative to 10(5) Pa total pressure). Under the hypoxic conditions, 'working' viral replication forks were greatly retarded or stopped, and initiation of daughter strand synthesis in further SV 40 DNA molecules was suppressed. Reoxygenation released an immediate burst of SV 40 replication which mainly consisted of a synchronous viral replication round. This synchronous in vivo replication began at the known origin of replication and proceeded at normal rates to the known termination region. Viral replicons seemed to accumulate under hypoxia in a state fully prepared to begin replication immediately after recovery of a normal pO2. The shut-down and sudden reactivation of DNA synthesis under hypoxia and reoxygenation, respectively, were not accompanied by changes of the phosphorylation state of large T antigen. The described synchronization procedure can be applied to optionally large SV 40 infected cell cultures.

We found that simian virus 40 (SV40), in addition to the SV40 early proteins large T antigen (large T) and small antigen (small t), codes for a third early protein with a molecular weight of 17 kDa. This protein (17kT) is expressed from an alternatively spliced third SV40 early mRNA, using a splice donor site at position 4425 and a splice acceptor site at position 3679 of the SV40 genome. The 17kT protein consists of 135 amino acids. Of these, 131 correspond to the amino-terminus of large T, while the four carboxy-terminal amino acids are unique and encoded by a different reading frame. 17kT mRNA, and the corresponding protein, were found in all SV40 transformed cells analyzed, as well as in SV40 infected cells. Transfection of a cDNA expression vector encoding the 17kT protein into rat F111 fibroblasts induced phenotypic transformation of these cells. The expression of the transforming amino-terminal domain of large T as an independent 17kT protein might provide a means for individually regulating the various functions associated with this domain.

Embryonic striatal cells were immortalized using the A58 temperature-sensitive allele of the SV40 large T antigen. Two cell lines, M213-2O and M26-1F, with gamma-aminobutyric acid (GABA)ergic properties were selected from 85 clones thus developed. M213-2O is a multipolar, polygonal cell line which expresses SV40 large T antigen and glutamate decarboxylase (GAD) at the permissive temperature (33 degrees C) and GAD and MAP-2 immunoreactivity at the nonpermissive temperature (39.3 degrees C). M26-1F has a fibroblast-like morphology and expresses SV40 large T antigen and GAD both at the permissive and nonpermissive temperatures and MAP-2 immunoreactivity at the nonpermissive temperature. Both lines contain GABA as measured by reversed-phase HPLC and M213-2O expresses nipecotic-sensitive [14C]GABA uptake.

Human fetal liver cells were cultured in serum free media enriched with hydrocortisone and infected with retrovirus containing SV40 large T gene. Replicating colonies with G-418 resistance developed in 50 days through 23 passages, while none grew in control dishes. After a crisis of 3 months duration, three colonies resumed active proliferation for over 15 months through 60 passages and became immortalized. These cells had epithelioid morphology, and were stained positively for CK-18. Southern blot and immunocytochemistry demonstrated the presence and expression of SV40 T-antigen in the immortalized cell lines. The cells expressed human albumin, especially in those while in cycle. Accumulation of p53 protein in the cell nuclei and strong expression of TGF-alpha as shown by immunocytochemistry explained at least partly the mechanism of unlimited growth of these immortalized human hepatocytes. These cells did not show anchorage-dependent growth in soft agar, nor did tumor form when inoculated into nude mice. These immortalized, differentiated, non-malignant human fetal hepatocyte lines may be useful for further studies.

Glucose-stimulated insulin secretion, glucose transport, glucose phosphorylation and glucose utilization have been characterized in the insulinoma cell line MIN6, which is derived from a transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta cells. Glucose-stimulated insulin secretion occurred progressively from 5 mmol/l glucose, reached the maximal level approximately seven-fold above the basal level at 25 mmol/l, and remained at this level up to 50 mmol/l. Glucose transport was very rapid with the half-maximal uptake of 3-O-methyl-D-glucose being reached within 15 s at 22 degrees C. Glucose phosphorylating activity in the cell homogenate was due mainly to glucokinase; the Vmax value of glucokinase activity was estimated to be 255 +/- 37 nmol.h-1.mg protein-1, constituting approximately 80% of total phosphorylating activity, whereas hexokinase activity constituted less than 20%. MIN6 cells exhibited mainly the high Km component of glucose utilization with a Vmax of 289 +/- 18 nmol.h-1.mg protein-1. Thus, glucose utilization quantitatively and qualitatively reflected glucose phosphorylation in MIN6 cells. In contrast, MIN7 cells, which exhibited only a small increase in insulin secretion in response to glucose, had 4.7-fold greater hexokinase activity than MIN6 cells with a comparable activity of glucokinase. These characteristics of MIN6 cells are very similar to those of isolated islets, indicating that this cell line is an appropriate model for studying the mechanism of glucose-stimulated insulin secretion in pancreatic beta cells.

The proliferation and differentiation programs of gut epithelial cells are expressed rapidly and perpetually along an anatomically well defined pathway. The mouse intestine thus provides an excellent in vivo model system to define the contributions of CCAAT enhancer binding protein alpha (C/EBP alpha) and related bZIP proteins to these processes. Immunocytochemical studies revealed that C/EBP alpha is produced in villus-associated enterocytes located in the duodenum and jejunum of adult mice. The protein is located in the cytoplasmic and nuclear compartments of these cells. C/EBP alpha is not detectable in proliferating and nonproliferating epithelial cells situated in small intestinal crypts nor is it evident in any gut epithelial cell lineage located in the ileum and colon. The related C/EBP beta and C/EBP delta proteins are not detectable by sensitive immunocytochemical methods in epithelial cells distributed along the duodenal-to-colonic axis. Developmental surveys indicate that C/EBP alpha is confined to postmitotic, villus-associated epithelial cells during conversion of the polyclonal intervillus epithelium to monoclonal crypts. Analyses of intestinal isografts reveal that these developmental stage-specific, lineage-specific, differentiation-dependent, and regional patterns of C/EBP alpha expression can be established and maintained in the absence of exposure to luminal contents. Transgenic mice containing nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene (I-FABP-1178 to +28) linked to the simian virus 40 large tumor antigen (T antigen) gene express T antigen in villus-associated enterocytes. This results in reentry of enterocytes into the cell cycle and a silencing of C/EBP alpha expression without an apparent effect on the accumulation of several markers of this lineage's terminal differentiation program or on gut morphogenesis. These findings indicate that there is a relationship between expression of C/EBP alpha in enterocytes and their exit from the cell cycle and suggest that I-FABP-1178 to +28/simian virus 40 T antigen transgenic mice could provide a screening assay for examining the role of C/EBP alpha in regulating the activity of genes known to be transcribed during differentiation of this gut epithelial cell lineage.

Primary cultures from neonatal optic nerve contain pluripotential O-2A progenitor cells that are capable of differentiating into oligodendrocytes, type-2 astrocytes or adult O-2A progenitors (O-2Aadult). Since primary optic nerve cultures contain a mixture of glial cell types of which only a small number are O-2A progenitors, experiments on cell lineage and differentiation carried out using these cultures are both intrinsically limited and difficult to interpret. Ideally, cells from a clonal cell population would provide the optimal starting material for biological studies. In this paper we describe the creation of an O-2A progenitor cell line using a retrovirus carrying a temperature-sensitive mutant SV40 large T antigen gene. This cell line has provided sufficient numbers of cells to allow analysis of their in vitro properties and their behaviour following transplantation into an in vivo environment. At the non-permissive temperature (39 degrees C), these cells differentiate into oligodendrocytes and type-2 astrocytes in a similar fashion to O-2A progenitor cells from primary cultures (O-2Aprim). When grown in media containing platelet-derived growth factor and basic fibroblast growth factor, the cell numbers can be expanded in culture without differentiating, consistent with the behaviour of O-2Aprim progenitor cells. By exploiting this property, it has been possible to culture large numbers of O-2A progenitors for in vivo analysis. In this study we have shown that transplantation of this O-2A cell line into glia-free areas in adult rat spinal cord results in differentiation of a proportion of cells into oligodendrocytes which are capable of myelinating axons. Furthermore, differentiation of O-2A cells into astrocytes was also observed, indicating that the bipotentiality of these cells in vitro can also be demonstrated in vivo.

The SV40 T-antigen-transfected human thyroid cell line SGHTL-34 was used to investigate the effect of thyrotropin (TSH), insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF) on c-fos and c-erbB/EGF receptor (EGF-R) mRNA expression and their role in human thyroid cell proliferation. EGF caused a transient 8- and 4-fold increase in c-fos mRNA level after 30 min in serum/hormone-deprived and in logarithmically growing cells, respectively. EGF was only mitogenic in the presence of serum, as measured by 3H-thymidine incorporation and cell counting. TSH had no detectable effect on c-fos mRNA expression and no mitogenic effect on the SGHTL-34 cells. IGF-1 showed no effect alone or in combination with EGF or TSH on either proliferation or c-fos mRNA expression. Our data suggest that increased c-fos mRNA levels are part of the mitogenic pathway, but are insufficient to engender a mitogenic response. SGHTL-34 cells produced high levels of transforming growth factor-alpha (TGF-alpha) and c-erbB/EGF-R mRNA, also seen in thyroid papillary carcinomas. The TGF-alpha protein was detected in conditioned medium from the SGHTL-34 cells, indicating that TGF-alpha may function as an autocrine growth factor. Our data show that the c-erbB/EGF-R mRNA level is regulated by growth factors and hormones in the SGHTL-34 cell line. The SGHTL-34 cells may therefore represent a useful model system for studying the role of TGF-alpha and EGF-R in thyroid carcinogenesis.

CHOP2 cells, a subline of the Chinese hamster ovary (CHO) cell glycosylation mutant Lec2 which expresses polyoma virus large T antigen, was used as the host cell line to select cDNAs conferring resistance to the toxic effects of ricin. Glycoconjugates in CHOP2 cells are deficient in sialic acid and therefore the cells are hypersensitive to ricin, a galactose-binding lectin. CHOP2 cells acquiring cDNA that either corrected the Lec2 mutation or created a Lec2/Lec1 phenotype were expected to show a selective growth advantage in ricin-containing medium. After a single cycle of transfection with a lymphoid cDNA library in pCDM8 followed by ricin selection, the the predominant cDNA recovered from the cells was antisense N-acetyl-glucosaminyltransferase I (GlcNAc-TI) which conferred a Lec2/Lec1 phenotype. cDNA encoding GlcNAc-TI in a sense orientation was also enriched by transfecting a cDNA library into CHOP-1 cells, a mutant deficient in this enzyme, and selecting in medium containing ConA lectin. The results show that cell selection with toxic agents can be used to expression-clone both antisense and sense cDNA sequences from bi-directional cDNA libraries in the pCDM8.

Expression of simian virus 40 (SV40) large T antigen efficiently immortalizes and transforms primary cells. We previously reported that a hybrid polyomavirus-SV40 large T antigen, PyT1-521-SVT336-708, binds to both p53 and pRb but does not transform an established rat cell line (J. J. Manfredi and C. Prives, J. Virol. 64:5250-5259, 1990). Here we show that this hybrid large T antigen is capable of immortalizing primary rat cells. Plasmids that express resistance to G418 sulfate and either SV40 large T antigen or PyT1-521-SVT336-708 were transfected into primary rat embryo fibroblasts, and cell lines were established. The cell lines that expressed PyT1-521-SVT336-708 were not fully transformed but did exhibit altered growth properties. Although these PyT1-521-SVT336-708-expressing lines did not form foci, they did grow in low serum and grew to a high saturation density; these cell lines also formed colonies in soft agar, but their colonies were much smaller than those seen with an SV40 large-T-antigen-expressing line. PyT1-521-SVT336-708 also demonstrated the ability to cooperate with activated Ha-ras to form foci on primary rat embryo fibroblasts. Surprisingly, two types of morphologies in such lines were observed: refractile and spindle shaped. Although there was no correlation between T-antigen level and morphology, all lines that displayed refractile morphology expressed high levels of p21ras. Since the p53 binding activity of PyT1-521-SVT336-708 appears to be intact, these results suggest that there are functions residing in the amino end of SV40 large T antigen which are necessary for full transformation that are missing from the amino end of polyomavirus large T antigen. Conversely, conferring the ability to bind to p53 on an amino-terminal fragment of polyomavirus large T antigen, although not enough to allow full transformation function, does increase its oncogenic activity in saturation density and soft agar growth assays.

The large T antigen of polyomavirus (PyLT) efficiently immortalizes rodent fibroblasts, but, unlike SV40 T antigen, it is not sufficient to achieve complete oncogenic transformation. We analysed a series of transgenic mouse families that express the PyLT protein under control of the viral enhancer-promoter region. In all of them, the transgene was expressed in the seminiferous epithelium of the testis (Sertoli and germ cells), with no pathological consequences during most of the animals' lives. However, every old male developed large bilateral tumours of the testes, generated by the proliferation of Sertoli cell derivatives. Cell lines could be readily established both from the tumours and from the still apparently normal testis before the onset of tumoral growth. They retained in vitro morphological and ultrastructural features characteristic of Sertoli cells. But, in addition to this major Sertoli component, the maintenance of a cellular contingent of germinal origin was suggested by the expression of genes that are normally transcribed during the premeiotic and early meiotic stages of spermatogenesis (LDH-X, Hox1.4 and c-kit). The two cell types remained tightly associated, even at late passages in culture, and could not be separated by conventional cloning procedures. This association in culture of the two cell types whose interaction is critical for spermatogenesis may provide a useful tool for its molecular analysis.

Among their biological properties, several oxysterols display a stronger toxicity towards tumor cells than towards normal cells. Water-soluble phosphodiesters of 7 beta-hydroxycholesterol (JB69 and XA29), that were proved to retain a specific antitumoral activity in vitro, have been recently developed, allowing in vivo studies. They have been assayed on transgenic mice expressing the Large T antigen of SV40 in their liver and developing systematically hepatocarcinoma within 8 months. We show that JB69 and XA29 administered intraperitoneally into transgenic mice, before the onset of adenoma, may prevent or delay the tumor development. Consequently, oxysterol derivatives might constitute new efficient prodrugs against naturally occurring tumors.

Regulatory elements of the mouse gamma F-crystallin gene were used to derive transgenic mice expressing SV40 large T antigen in terminally differentiating fiber cells of the ocular lens. The resulting gamma F-crystallin-T antigen mice developed either malignant or regressive lens tumors in a strain-dependent fashion. Developmental and RNA analyses revealed that in both 'tumor-progressing' and 'tumor-regressing' mouse strains expression of the transgene blocked morphological differentiation of lens fibers without appreciably affecting gamma-crystallin gene expression, a marker of terminal lens fiber cell differentiation. Strain-dependent differences in tumorigenic outcome could be correlated with both subtle differences in transgene expression and the ability of tumor cells to escape from the normal confines of the lens. The results implicate the importance of cellular environment to malignant tumor development and provide insight into those features of normal lens ontogeny that may render the lens refractory to the development of spontaneous tumors.

The phenylethanolamine N-methyltransferase promoter directs the expression of the SV40 T antigen to subsets of amacrine and horizontal neurons of the retina in a line of transgenic mice. T antigen expression begins in these cells during the first postnatal week. The horizontal cells appear to develop normally for another week but then begin to die. Subsequently, most of the horizontal cells disappear from the central and mid retina, resulting in loss of the outer plexiform layer and absence of ribbon synapses between the photoreceptors and bipolar cells. Neuronal transformation occurs only in the peripheral retina. These experiments indicate that horizontal neurons are heterogeneous with respect to susceptibility to transformation and that T antigen expression in a subset of horizontal neurons can be a direct cause of neuronal cell death. Furthermore, critical interdependencies exist between horizontal neurons after retinal neurogenesis is complete.

The study of immortalization and other alterations associated with neoplastic transformation of endometrial stromal cells is important to understanding the development of uterine sarcomas and mixed tumors. Because stromal cells are important regulators of associated epithelial cells, alterations in the regulation of stromal cell proliferation that influence epithelial cells may also contribute to the development of endometrial carcinomas. To study immortalization and associated phenotypic and genetic alterations of human endometrial stromal cells, cultures were transfected with a plasmid containing an ori-, temperature-sensitive mutant SV40, A209 (tsSV40). Morphologically transformed colonies were selected and propagated at the permissive temperature until they entered 'crisis'. In contrast to human fibroblasts, every clone tested was immortalization competent. The frequency of immortalization was approximately 1 x 10(-6). One uncloned and six cloned cell lines escaped from crisis and appear to be immortal. Two clones, M4 and B10T1, were selected for further study. Immortalization is conditional; proliferative arrest occurs at the restrictive temperature for large T antigen function. Furthermore, withdrawal of the large T antigen results in expression of the senescent phenotype of enlarged, flattened cells. Colony-forming efficiency at the restrictive temperature was undetectable. Immortalization is also associated with several genetic alterations. The DNA content of tsSV40 transfected cells was either diploid or tetraploid in the precrisis stage of proliferation, but became aneuploid upon immortalization. Several structural rearrangements of chromosomes were detected in the immortalized stromal cells which differ from those found in SV40 immortalized fibroblasts. Although their capacity for anchorage-independent proliferation (AIP) is variable, tsSV40-immortalized endometrial stromal cells have a higher capacity for AIP than their tsSV40-transfected progenitor cells in the period of proliferation prior to 'crisis'.

We established a human osteoblastic cell line immortalized by simian virus 40 (SV40) in vitro, and designated it SV-HFO. Immunocytochemically, the cells were positive for SV40 large T-antigen, vimentin and osteocalcin, but negative for keratin and epithelial membrane antigen. The cells had characteristic morphologic and ultrastructural features of osteoblasts, produced alkaline phosphatase, and synthesized osteocalcin, the levels of which were elevated by treatment of the cells with 1a,25-dihydroxyvitamin D3. The cells proliferated and showed such osteoblastic properties even under serum-free conditions. The cells grew in soft agar, but did not form tumors when transplanted into athymic nude mice. Karyotypic analysis by the Q-banding technique showed that these cells were of human origin. The SV-HFO cell line is expected to serve as a suitable model for studying metabolism and carcinogenesis in human bone.

To study the effect of SV40 large T-antigen expression in myelin-forming cells of both the central and peripheral nervous system, a series of transgenic mice were generated expressing the SV40 large T-antigen under control of the myelin basic protein (MBP) promoter. Two neurologic phenotypes, designated A and B, appeared among individual transgenic founders and their progeny. The A mice developed a severe action tremor at about 10 days of age that progressed into periods of convulsions and early death by three to four weeks of age. In contrast, the B mice exhibited a progressive hindlimb ataxia and had a more normal lifespan. The A mice displayed hypomyelinating lesions in the central nervous system (CNS), whereas the B mice had lesions in either the peripheral nervous system (PNS) alone or in both the PNS and CNS. Immunohistochemical staining of spinal cord sections of a type A mouse showed a substantial depletion in MBP. Moreover, T-antigen-positive cells appeared predominantly in white matter tracts as randomly distributed single cells. Double labeling immunocytochemistry demonstrated that some of these T-antigen-positive cells were positive for oligodendrocyte differentiation markers MBP and O4. Thus, T-antigen expression appeared to coincide with a terminal stage of oligodendrocyte differentiation.

These studies examined cell cycle progression and quantitative changes in T-antigen following infection by SV40. Single cells were assayed by multiparameter flow cytometric analysis (FCM) for DNA content and T-antigen expression. Conditions were used which permitted permissive, semi-permissive, and non-permissive cells to be monitored through two rounds of DNA synthesis induced by SV40. The permissive cells included the monkey kidney cell lines; CV-1, Vero and BSC-1 and the COS-1 and COS-7 which are CV-1 cells transformed with an origin defective SV40. The non-permissive cell strains included mouse embryo fibroblasts, Chinese hamster fibroblasts, and IMR-90, a human diploid fibroblast. Cell types differed in the maximal amount of T-antigen expressed per cell. Additionally, all cell types expressed a limited quantity of T-antigen for each cell cycle phase and the quantity increased in each successive phase. The level in each phase was increased only two-fold when 100 times more virus was used. Thus, for an infected population the quantity of T-antigen was dependent on cell cycle distribution. High levels of T-antigen were not required for permissive infection; however, permissive cells were distinguished from non-permissive cells by the G2 levels. Permissive G2 cells had more than double the T-antigen content expressed in G1, while nonpermissive G2 cells had less than a two-fold increase over G1 levels. The appearance of cells with tetraploid DNA content and the failure to undergo mitosis correlated to the higher T-antigen levels in the G2 of the permissive cells. Two other strains of SV40, 776, and VA45 exhibit similar values for T-antigen expression and movement into tetraploid DNA content. This study establishes the levels of T-antigen correlated to the cell cycle and cell type.

Transgenic mice that express a glucagon gene-simian virus-40 large T-antigen (GLUTag) fusion gene develop neuroendocrine carcinoma of the large bowel. This glucagon-producing tumor was implanted sc and reproducibly formed tumors in nude mice. The transplanted GLUTag tumor expressed large amounts of proglucagon mRNA transcripts, and the levels of proglucagon mRNA transcripts remained constant during 2-8 weeks of tumor growth. The posttranslational processing of proglucagon in the transplantable tumor resembled that detected in the original transgenic tumor, with the liberation of glicentin, oxyntomodulin, glucagon, glucagon-like peptide (1-37) [GLP-1-(1-37)] and GLP-1-(7-37). Tumor-bearing mice demonstrated progressive elevations in the plasma levels of proglucagon-derived peptides. Elevated plasma levels of glucagon-like immunoreactive peptides and immunoreactive glucagon were associated with a marked reduction in the levels of pancreatic glucagon mRNA transcripts by 4 weeks, and after 8 weeks of tumor growth, the levels of glucagon mRNA transcripts in the pancreas were not detectable by Northern blot analysis. Synthesis of the proglucagon-derived peptides was also significantly suppressed at 4-8 weeks in the pancreas of tumor-bearing animals. Histological examination of the endocrine pancreas in mice carrying the GLUTag tumor for 6-8 weeks demonstrated a marked reduction in the number and size of the islets of Langerhans and a disproportionately greater decrease in the number of cells exhibiting glucagon immunoreactivity. By electron microscopy, the residual A-cells were small, compressed at the periphery of the islets, and had poorly developed cytoplasmic organelles. In contrast, no changes in mouse glucagon gene expression or islet morphology were detected in control animals without tumors or mice carrying a sc v-jun-induced fibrosarcoma. The suppression of pancreatic A-cell function and islet size in mice with elevated plasma levels of the proglucagon-derived peptides raises the possibility that a proglucagon-derived peptide may participate in a negative feedback loop, inhibiting expression of the glucagon gene in the A-cells of the endocrine pancreas.

A serum-dependent and two serum-independent variants of the Bowes melanoma cell line, RPMI7272, were transfected with plasmids containing a geneticin-resistance (neo) gene transcribed by the HSV thymidine kinase promoter and an SV40 T antigen gene under control of the mouse metallothionein I promoter. T-antigen increased the cloning efficiency of the serum-dependent cell line in soft-agar more than 50-fold, but cloning efficiency of serum-independent lines was not increased. Trypsinization of serum-independent lines required 100 times lower concentrations of trypsin than serum-dependent cells. Human metal-inducible T-antigen-producing (HMT) melanoma cells supported replication of transfected plasmids containing an SV40 origin of replication. Transient expression of interferon or plasminogen activator from such plasmids was 40-fold higher than in untransformed melanoma cells and could be enhanced 30-fold more by stimulation of transcription of the T antigen gene with cadmium chloride. HMT cells can be grown in suspension and thus may represent an attractive alternative to monkey kidney COS cells.

The proglucagon gene is expressed in A cells of the pancreas and L cells of the large and small intestine. Transgenic mice expressing SV40 large T antigen under the control of proglucagon regulatory sequences develop neuroendocrine carcinoma of the large intestine. To determine the consequences of coexpression of SV40 large T antigen and proglucagon in different cell types, the levels of proglucagon mRNA transcripts and proglucagon-derived peptides were determined in tumor-bearing transgenic mice and in age-matched paired controls. Plasma levels of proglucagon-derived peptides (glicentin, oxyntomodulin, and glucagon, as determined by high pressure liquid chromatography and radioimmunoassay) were markedly elevated in association with tumor growth (p < 0.001). Northern blot analysis demonstrated that the increased concentration of proglucagon-derived peptides was associated with significant inhibition of the endogenous proglucagon gene in pancreas, and to a lesser extent, small intestine. Concomitantly, the concentrations of proglucagon-derived peptides fell to 1-10% of control values in pancreas (p < 0.001) and to 62% of control values in small intestine (p < 0.001). Analysis of proglucagon-derived peptides in mice of different ages demonstrated that tumor growth was associated with a switch in the post-translational processing of proglucagon. Compared with normal mouse intestine, tumors contained increased proportions of glucagon and glucagon-like peptide-1(7-37) relative to glicentin, oxyntomodulin, and glucagon-like peptide-1(1-37). The results of these studies provide evidence for humorally-mediated tissue-specific inhibition of proglucagon gene expression.

Mullerian inhibiting substance (MIS) is a glycoprotein hormone expressed by Sertoli cells that induces the regression of Mullerian ducts during development of the male reproductive tract. Transgenic mice carrying a fusion gene composed of human MIS transcriptional regulatory sequences linked to the SV40 T-antigen gene specifically develop testicular tumors composed of a cell type histologically resembling the Sertoli cell. The lack of pathology at other sites suggests tissue-restricted expression of the transgene. A cell line derived from one of the testicular tumors has been established that continues to express markers associated with Sertoli cells, such as transferrin, sulfated glycoprotein-2, and inhibin-beta B. The cell line does not express detectable levels of inhibin-alpha, MIS, or FSH receptor. However, the cells have retained forskolin responsiveness. As adult Sertoli cells cannot be propagated in vitro, the availability of an immortal cell line displaying features characteristic of normal Sertoli cells should aid in subsequent analyses of the biology of this cell type.

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.

To study the mechanism by which SV40 large T antigen transforms cells under physiological conditions, we analysed several mutant forms of T antigen for their ability to induce cell proliferation and tumorigenesis in transgenic mice. These mutant proteins, which differ in their ability to form complexes with the tumor suppressors pRB and p53, were analysed under conditions in which wild-type T antigen induces choroid plexus papillomas as a result of uniform proliferation of the entire choroid plexus epithelium. The results presented here show that binding of T antigen to p53 is not required for induction of choroid plexus tumors. However, tumorigenesis does appear to require the binding of T antigen to pRB/p107. An additional activity, resident in the amino-terminal one-fifth of the protein, may also play a role. These experiments indicate the importance of whole-animal assays in determining the molecular basis of transformation, since each of these mutants possessed similar transformation phenotypes in culture but showed distinct phenotypes in the choroid plexus of the animal.

The effects of sodium butyrate on simian virus 40 early gene expression were determined in SV40-transformed human embryonic lung fibroblasts (SVWI-38). Northern blot analysis and nuclear run-off transcription studies revealed that treatment of cells with millimolar concentrations of sodium butyrate (2.5 to 10 mM) resulted in increased levels of SV40 early gene transcripts, with a concomitant increase in their corresponding proteins (large-T and small-t antigens). Although sodium butyrate treatment enhanced the expression of the early genes, it was associated with a reduction in cell growth and total protein synthesis, as measured by cell number and incorporation of 3H-leucine into macromolecules, respectively. Immunoprecipitation of 35S-labelled cellular proteins with anti-p53 and anti-T antibodies revealed that the level of the cellular protein, p53, declined markedly in the presence of sodium butyrate. Furthermore, in control cells only 30% of the p53 was complexed with large-T antigen, whereas in butyrate-treated cells all the p53 was complexed with large-T antigen. The increased early gene expression was not due to altered methylation patterns, gene amplification, or rearrangement of the integrated SV40 genome. Sodium butyrate treatment did, however, result in the appearance of a new nuclear protein which bound specifically to a SV40 promoter fragment containing large-T antigen binding sites I and II.

C57BL/6 (B6) mice (H-2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag-specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2-10 micrograms purified, soluble T Ag and boosted with an injection of 2 micrograms T Ag 2-4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6-derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag-encoding expression vector. Effector cells of this response were H-2 class I-restricted CD3+ CD4-CD8+ CTL. The magnitude of the anti-T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti-T Ag CTL response of SV40-infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.