Quantitative correlation between simian virus 40 T-antigen synthesis and late viral gene expression in permissive and nonpermissive cells.

The time-course of intranuclear Simian virus 40 (SV40) tumor (T) antigen synthesis and accumulation in permissive CV1 monkey cells and nonpermissive 3T3 mouse cells has been studied by immunofluorescence and cytofluorometry. CV1 cells accumulate T antigen continuously over a period of 48 h after infection, whereas in 3T3 cells the T-antigen content remains about constant and at a comparatively low level. Only those CV1 cells which have attained a threshold concentration of intranuclear T antigen synthesize viral capsid proteins (V antigen). In nonpermissive 3T3 cells, the T-antigen threshold value for the onset of V-antigen synthesis is higher than in CV1 cells and is never reached by infected cells. However, 3T3 cells microinjected with sufficient amounts of SV40 DNA easily surpass this value and behave permissively.

Chemotherapy resistance of mouse WAP-SVT/t breast cancer cells is mediated by osteopontin, inhibiting apoptosis downstream of caspase-3.

Impairment of the complex regulatory network of cell death and survival is frequently the reason for therapy resistance of breast cancer cells and a major cause of tumor progression. We established two independent cell lines from a fast growing mouse breast tumor (WAP-SVT/t transgenic animal). Cells from one line (ME-A cells) are sensitive to apoptotic stimuli such as growth factor depletion or treatment with antitumor agents (e.g. doxorubicin). Cells from the second line (ME-C cells), which carry a missense mutation at the p53 codon 242, are very insensitive to apoptotic stimuli. Co-cultivation experiments revealed that the ME-C cells mediate cell death resistance to the ME-A cells. Microarray and Western blot analysis showed that osteopontin (OPN) is selectively overexpressed by the ME-C cells. This glycoprotein is the most abundant protein secreted by the ME-C cells and we obtained strong indications that OPN is the main antiapoptotic factor. However, the OPN containing ME-C cell medium does not alter the expression level of pro- or antiapoptotic genes or known inhibitors of apoptosis (IAPs). Its signaling involves mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)1/2 as the kinase inhibitor PD98059 restores apoptosis but not the Akt inhibitor. In the ME-A cells, mitochondrial cytochrome c release occurs with and without external apoptotic stimuli. OPN containing ME-C cell medium does not prevent the mitochondrial cytochrome c release and caspase-9 processing. In serum starved ME-A cells, the OPN containing ME-C cell medium prevents caspase-3 activation. However, in doxorubicin-treated cells, although apoptosis is blocked, it does not inhibit caspase-3. This indicates that the ME-A cells distinguish between the initial apoptotic stimuli and that the cells possess a further uncharacterized control element acting downstream from caspase-3.

Methylation of single sites within the herpes simplex virus tk coding region and the simian virus 40 T-antigen intron causes gene inactivation.

In order to determine whether partial methylation of the herpes simplex virus (HSV) tk gene prevents tk gene expression, the HSV tk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesis, specific HSV tk gene segments were methylated, and the hemimethylated DNA molecules were microinjected into thymidine kinase-negative rat2 cells. Conversion of the hemimethylated DNA into symmetrical methylated DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expression was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the coding region, from +499 to +1309, were selectively methylated. This specific methylation pattern caused inactivation of the HSV tk gene, while methylation of the cytosine residues within the nucleotide sequence from +811 to +1309 had no effect on HSV tk gene activity. We also methylated single HpaII sites within the HSV tk gene using a specific methylated primer for in vitro DNA synthesis. We found that of the 16 HSV tk HpaII sites, methylation of 6 single sites caused HSV tk inactivation. All six of these "methylation-sensitive" sites are within the coding region, including the HpaII-6 site, which is 571 bp downstream from the transcription start site. The sites HpaII-7 to HpaII-16 were all methylation insensitive. We further inserted separately the methylation-sensitive HSV tk HpaII-6 site and the methylation-insensitive HpaII-13 site as DNA segments (32-mer) into the intron region of the simian virus 40 T antigen (TaqI site). Methylation of these HpaII sites caused inhibition of simian virus 40 T-antigen synthesis.

Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing.

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which may lead to the synthesis of two proteins. Generation of the three COT transcripts in rat does not depend on sex, development, fat feeding, the inclusion of the peroxisome proliferator diethylhexyl phthalate in the diet or hyperinsulinemia. In addition, trans-splicing was not detected in COT of other mammals, such as human, pig, cow and mouse, or in Cos7 cells from monkey. Rat COT exon 2 contains two purine-rich sequences. Mutation of the rat COT exon 2 upstream box does not affect the trans-splicing in vitro between two truncated constructs containing exon 2 and its adjacent intron boundaries. In contrast, mutation of the downstream box from the rat sequence (GAAGAAG) to a random sequence or the sequence observed in the other mammals (AAAAAAA) decreased trans-splicing in vitro. In contrast, mutation of the AAAAAAA box of human COT exon 2 to GAAGAAG increases trans-splicing. Heterologous reactions between COT exon 2 from rat and human do not produce trans-splicing. HeLa cells transfected with minigenes of rat COT sequences produced cis- and trans-spliced bands. Mutation of the GAAGAAG box to AAAAAAA abolished trans-splicing and decreased cis-splicing in vivo. We conclude that GAAGAAG is an exonic splicing enhancer that could induce natural trans-splicing in rat COT.

Hepatitis B virus X protein transcription activation domains are neither required nor sufficient for cell transformation.

The ability of the hepatitis B virus (HBV)-encoded X protein (HBx) to coactivate transcription of viral and cellular genes has been implicated in the development of HBV-related liver cancer. To dissect the transformation and the transcription activation properties of HBx, we generated REV2 cell lines expressing the wild-type and different truncated versions of the protein. Full-length HBx-expressing REV-2 cells display an altered morphology and form large colonies in soft agar. A similar transformation efficiency has been obtained with a truncated version of HBx, which contains only the first 50 NH2-terminal amino acids (HBx 1-50). In contrast, HBx mutants that lack the NH2-terminal segment but retain most of the transactivating function, as compared to the full length HBx, were unable to alter the growth characteristic of REV-2 cells. Furthermore, abrogation of full-length HBx transcriptional activation by the insertion of two amino acids (Arg-Pro) at position 68 did not affect REV-2 cells transformation. These results demonstrate that the transactivation activity of HBx is neither essential nor sufficient for tumor promotion.

Regulation of E2F4 mitogenic activity during terminal differentiation by its heterodimerization partners for nuclear translocation.

E2F/DP heterodimers play a pivotal role in the regulation of cell growth and differentiation. A decrease in E2F/DP activity occurs during cell cycle arrest and differentiation. However, very little is known about the specific role of the various E2F/DP members along the transition from proliferation to terminal differentiation. We have previously shown that E2F4 accounts for the vast majority of the endogenous E2F in differentiating muscle cells. Here, we show that E2F4, which lacks a nuclear localization signal (nls), is distributed in both the nucleus and the cytoplasm, in either asynchronously growing myoblasts or differentiated myotubes. E2F4 nuclear accumulation is induced by the binding in the cytoplasm with specific partners p107, pRb2/p130, and DP3delta, an nls-containing spliced form of DP3, which provide the nls. Although overexpression of E2F4/DP3delta reactivates the cell cycle in quiescent cells, the E2F4 nuclear accumulation induced by pRb2/p130 and p107 correlates with cell growth arrest Moreover, E2F4/DP3delta-induced cell cycle reactivation is efficiently counteracted by either p107 or pRb2/p130 overexpression. Reinduction in quiescent cells of DNA synthesis by E2F1/DP1 overexpression is abrogated by coexpression of pRb and is hampered by MyoD overexpression. Both pRb2/p130 and pRb, as well as MyoD, are up-regulated in myotubes. Accordingly, multinucleated myotubes, which are induced to reenter the S-phase by oncoviral proteins, are refractory to cell cycle reactivation by forced expression of E2F4/DP3delta or E2F1/DP1. Thus, E2F/DP repression represents only one of multiple redundant circuits that control the postmitotic state in terminally differentiated cells and that are targeted by adenovirus E1A and SV40 large T antigen.

Altered phosphorylation at specific sites confers a mutant phenotype to SV40 wild-type large T antigen in a flat revertant of SV40-transformed cells.

The Rev2 cell line is a cellular revertant of the SV40 wild-type transformed rat cell line SV-52 [Bauer, M., Guhl, E., Graessmann, M. & Graessmann, A. (1987). J. Virol., 61, 1821-1827]. To characterize the level of cellular interference with the SV40 large T antigen (large T)-induced transformation pathway in Rev2 cells, we analysed the biological and biochemical properties of large T expressed in Rev2 cells. We found that Rev2 cells encoded an authentic wild-type large T, with regard to its sequence and its transforming functions. No differences were found in the metabolic stability of large T, or in complex formation with the cellular p53 protein, or in p53 metabolic stabilization. In contrast to SV-52 cells, Rev2 cells showed no association of large T with the chromatin fraction of isolated nuclei. This difference correlated with a reduced affinity of the Rev2 large T to SV40 DNA in vitro. The T proteins from both cell lines were phosphorylated at the same multiple sites. However, in Rev2 cells the phosphorylation of large T at specific serine -residues was significantly reduced. Thus the revertant phenotype of Rev2 cells may be due to an altered phosphorylation state of its large T protein, leading to altered nuclear localization and reduced transforming activity. The alterations of Rev2 large T properties and phosphorylation were very similar to the changes observed with mutant large T in FR(tsA58)A cells, an SV40 tsA58 N-type transformant, when the cells had reverted to the normal phenotype at the non-permissive growth temperature. Thus altered phosphorylation might provide a common structural basis for the biological inactivation of the large T proteins in these cells.

Breast cancer formation in transgenic animals induced by the whey acidic protein SV40 T antigen (WAP-SV-T) hybrid gene.

After injection of the whey acidic protein (WAP)-SV-T hybrid gene into fertilized mouse eggs, eight independent transgenic mouse lines were obtained. Females from three lines developed mammary carcinomas with high frequency, coinciding mostly with lactation. In contrast to the endogenous WAP gene, expression of the hybrid gene continued after lactation. The tumor cells had a very invasive growth characteristic. Tumor regression in vivo was not observed. However, after transfer into tissue culture 25% of the cells ceased to express the hybrid gene and acquired the growth characteristic of normal cells. It was possible to retransform these cells by injection of wild-type SV40 DNA, but not after transfer of the hybrid WAP-SV-T gene. Inactivation of the endogenous WAP and of the WAP-SV-T transgene did not correlate with DNA methylation.

SV40 T-antigen induces breast cancer formation with a high efficiency in lactating and virgin WAP-SV-T transgenic animals but with a low efficiency in ovariectomized animals.

The whey acid protein (WAP) is a major mouse milk protein and its gene expression is induced by various lactotrophic hormones (eg, estrogen, progesterone). Transgenic animals harboring the early SV40 coding region (T/t-antigen) under the transcriptional control of the WAP promoter develop breast cancer after the first lactation period. The tumor cells synthesize the SV40 T-antigen with a high efficiency indicating that WAP-SV-T expression escapes down-regulation after the lactation period. However about 5-10% of the tumors became T-antigen negative during tumor progression and WAP-SV-T expression was only demonstrable by PCR analysis. Both T-antigen positive and negative tumor cells expressed the estrogen and progesterone receptor at a comparable rate, indicating that hormone receptor levels do not determine expression of the WAP-SV-T transgene. Furthermore, WAP and WAP-SV-T gene expression are not restricted to the pregnancy-lactation period. Virgin animals also express both genes with a low efficiency and about 70% of these animals also developed T-antigen positive breast tumors. The tumor rate however was strongly reduced in ovariectomized animals, indicating that the ovary hormones play a critical role in breast cancer formation.

SV40 T/t-antigen induces premature mammary gland involution by apoptosis and selects for p53 missense mutation in mammary tumors.

We recently established transgenic animals (WAP-SV-T/t) carrying the early coding region of Simian Virus 40 (SV40) under the transcriptional control of the whey acidic milk protein promoter (WAP), which restricts the expression of the transgene to mammary gland epithelial cells (ME-cells). SV40 T/t-antigen synthesis causes premature mammary gland involution during late pregnancy by inducing apoptosis and leads to development of mammary tumors after the first lactation period in both p53 positive (WAP-SV-T/t) and p53 negative double transgenic animals (WAP-SV-T/t.p53-/-). The high apoptotic rate persists in all of the T/t-antigen positive breast tumor cells, as well as in established ME-tissue culture cell lines. ME-cells which spontaneously switch off the expression of the WAP-SV-T/t transgene do not undergo apoptosis. However, these cells again exhibit an extensive DNA fragmentation when SV40 T/t-antigen synthesis is reintroduced, which indicates that it is the expression of T/t antigen which is the critical factor for induction of apoptosis. In addition, we isolated several ME-cell lines from different breast tumors which have spontaneously lost the T/t-antigen yet remain maximally transformed. Strikingly, these cells contain a missense mutation of the p53 gene at codon 242 (p53(242)), which substitutes alanine for glycine. This mutation increases p53 stability and it reduces the transactivating function of p53, albeit without affecting the ability of the protein to interact with the DNA. This indicates that p53 missense mutations are selected for in breast tumors initially expressing T/t-antigen. Therefore, the p53(242) mutation is sufficient to maintain the transformed state after the ME-cells have switched off the WAP-SV-T/t transgene. Interestingly, the p53 minus state per se is not sufficient to induce ME-cell transformation since homozygous null mice for the p53 gene (p53-/-) fail to develop breast cancer.

The SV40 small t-antigen prevents mammary gland differentiation and induces breast cancer formation in transgenic mice; truncated large T-antigen molecules harboring the intact p53 and pRb binding region do not have this effect.

We report here for the first time, that the SV40 small t-antigen inhibits mammary gland differentiation during mid-pregnancy and that about 10% of multiparous WAP-SVt transgenic animals develop breast tumors with latencies ranging from 10-17 months. Cyclin D1 is deregulated and over expressed in the small t-antigen positive mammary gland epithelial cells (ME-cells) and in the breast tumor cells. SV40 small t-antigen immortalized ME-cells (t-ME-cells) exhibit a strong intranuclear cyclin D1 staining, also in the absence of external growth factors and the cells continue to divide for several days without serum. In addition, the expression rate of cyclin E and p21(Waf1) but not of p53 is increased. Coimmunoprecipitation experiments revealed that p21(Waf1) is mainly associated with the cyclin D/CDK4 but not with the cyclin E/CDK2 complex. WAP-SVT transgenic animals exhibit an almost regular mammary gland development until late pregnancy but the majority of the ME-cells are eliminated by apoptosis during the early lactation period. Tumor formation is delayed and less efficient than in T/t-antigen positive animals. Sequestration of p53 and pRb by the N-terminal truncated T-antigen molecules (T1-antigen and T2-antigen) does not affect mammary gland differentiation and the transgenic animals (WAP-SVBst-Bam) do not develop breast tumors.

Modulation in vitro of H-ras oncogene expression by trans-splicing.

In man, activated N-, K- and H-ras oncogenes have been found in around 30% of the solid tumours tested. An exon known as IDX, which has been described previously and is located between exon 3 and exon 4A of the c-H-ras pre-mRNA, allows an alternative splicing process that results in the synthesis of the mRNA of a putative protein named p19. It has been suggested that this alternative pathway is less tumorigenic than that which results in the activation of p21. We have used the mammalian trans-splicing mechanism as a tool with which to modulate this particular pre-mRNA processing to produce mRNA similar to that of mature p19 RNA. The E4A exon of the activated H-ras gene was found to be a good target for external trans-splicing. We reprogrammed the rat carnitine octanoyltransferase exon 2 to specifically invade the terminal region of H-ras. Assays performed with this reprogrammed trans-exon showed that the trans-splicing product was obtained in competition with cis-splicing of the D intron of the H-ras gene, and was associated with concomitant down-modulation of D intron cis-splicing. We also found that the exon 4A of the human c-H-ras gene underwent successive trans-splicing rounds with an external exon.

MyoD prevents cyclinA/cdk2 containing E2F complexes formation in terminally differentiated myocytes.

Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream effectors of 'pocket' proteins are the components of the E2F family of transcription factors, which regulate the G1/S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undifferentiated and differentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G0/G1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G1/S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1/S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes. In differentiating C2C12 cells, E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream effector p21 are induced. Thus, during myogenic differentiation, formation of E2F4 and pRb2/p130 containing complexes is an early event, but not enough on its own to prevent the reactivation of DNA synthesis. Using a subclone of C3H10T1/2 mouse fibroblasts stably expressing Estrogen Receptor-MyoD (ER-MyoD) chimerae, we found that estrogen directed MyoD activation prevents the reassociation of cyclinA/cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle. Our data indicate that, in differentiating myocytes, one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation.

The second large simian virus 40 T-antigen exon contains the information for maximal cell transformation.

Microinjection of early simian virus 40 (SV40) DNA fragments has shown that maximal transformation of rat cells (Ref 52) is a property of the second SV40 T-antigen exon. Expression of this particular T-antigen region was obtained by coinjection of the Taq/Bam DNA fragment with the early promoter/enhancer HpaII/BglI fragment. Microinjection of the DNA fragment mixture induced two categories of transformants; namely, maximally and minimally transformed cells. The maximally transformed cells synthesize two Taq/Bam-specific polypeptides, and the minimally transformed cells only the lower molecular weight form. Both types of transformants contain the cellular p52 protein at high concentrations. Furthermore, maximal transformation of Ref 52 cells requires the carboxy terminus of the T-antigen. Cells transformed by microinjection of the SV40 Pst A-fragment display different parameters of maximally transformed cells but not anchorage-independent growth.

Molecular characterization of the DNA puff C-8 gene of Rhynchosciara americana.

We have mapped the only transcription unit known to be present in the C-8 DNA puff of Rhynchosciara americana and describe the isolation and sequence of a cDNA clone, pRa C-8-22, which contains a nearly complete copy of the mRNA transcribed from this DNA puff and part of the sequence of genomic clone BSC8-0.9, which contains the promotor region and the remainder of the transcription unit. The characteristics of the protein predicted from the ORF present in the cDNA indicate that it is unique and secreted.

Molecular characterization of the C-3 DNA puff gene of Rhynchosciara americana.

We have mapped a region of about 33 kb which includes the transcription unit of the C-3 DNA puff gene of Rhynchosciara americana. The C-3 TU and a region extending approximately 800 bp upstream of the C-3 promoter were characterized. The TU is composed of three exons and produces a 1.1-kb mRNA whose level in salivary glands increases with the expansion of the C-3 puff. The C-3 messenger appears to undergo rapid deadenylation resulting in an RNA of about 0.95 kb which can still be observed in gland cells 15 h after the puff has regressed. The 1.1-kb mRNA codes for a 32.4-kDa, predominantly alpha-helical polypeptide with three conserved parallel coiled-coil stretches. The aa composition and structure of this polypeptide suggests that it is secreted and contributes to the formation of the cocoon in which the larvae pupate. The region upstream of the promoter contains several A-rich sequences with similarity to the ACS of yeast which might have a role in the initiation of replication/amplification.

In vitro synthesized SV40 cRNA is trans-spliced after microinjection into the nuclei of mammalian cells.

We present for the first time experimental evidence that in vitro synthesized RNA (cRNA) is trans-spliced after microinjection into the nuclei of mammalian tissue culture cells. The template used for cRNA synthesis was the early SV40 BstXl/BamHI DNA fragment. This DNA fragment encodes exclusively for the second T-antigen exon and contains the intact small t-antigen intron. To generate the corresponding mRNA (T1-mRNA) by trans-splicing, the cells utilize a 5' cryptic splice site located within the second T-antigen exon of one cRNA molecule which is spliced to the small t-antigen 3' splice site of another cRNA molecule. Formation of the T1-mRNA by trans-splicing was confirmed by RT-PCR analysis and DNA sequencing. Efficient trans-splicing required that competitive small t-antigen cis-splicing be inhibited by deletion of the small t-antigen 5' splice site. The T1-mRNA was not generated when the cryptic 5' splice site was mutated.

Methylation of the SV40 HpaII site does not affect late viral gene expression in microinjected tissue culture cells.

Intranuclear coinjection of the late SV40 KpnI/BclI DNA fragment and the early promotor/enhancer HpaII/BglI DNA segment into permissive monkey and non-permissive mouse cells allows late SV40 gene expression without T-antigen synthesis and DNA replication. These conditions were chosen to analyse the effect of DNA methylation on V-antigen synthesis detached from the process of DNA replication. We found that in vitro methylation of a single cytosine nucleotide proximal to the major late mRNA cap site by the HpaII methylase does not block capsid protein synthesis. This result is in contrast to reported data obtained in Xenopus laevis oocyte injection experiments [(1982) Proc. Natl. Acad. Sci. USA 79, 5142-5146].

After microinjection hemimethylated DNA is converted into symmetrically methylated DNA before DNA replication.

In this investigation we analysed the maintenance methylation activity of the mammalian cell DNA methyltransferase by microinjection of hemimethylated HSV-tk DNA into thymidine kinase-negative rat 2 cells. We found that the hemimethylated DNA was efficiently converted into symmetrical methylated molecules before DNA replication. Furthermore, integration of the trans-DNA into the host genome is an early event after gene transfer.

SV40 T1-mRNA trans-splicing and translation requires that the in vitro synthesized cRNA is capped before microinjection.

The purpose of this investigation was to study the effect on cap structure for trans-splicing in mammalian cells. The early SV40 Bst/Bam pre-mRNA (cRNA) was synthesized in vitro in both capped (cap-Bst/Bam-cRNA) and non-capped (Bst/Bam-cRNA) versions and microinjected into the nuclei of TC7 cells. Trans-splicing was monitored by immunofluorescence staining (T1-antigen) and by RT-PCR analysis. Cap-Bst/Bam-cRNA was trans-spliced with high efficiency, but not the Bst/Bam-cRNA molecules. Northern blot analysis revealed that both the capped and uncapped cRNA molecules had similar stability in the microinjected cells. The coinjected m7G(5')ppp(5')G cap analog did not inhibit the trans-splicing reaction in vivo and did not prevent nuclear export of the mRNA.