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1.
Oncogene ; 26(20): 2840-50, 2007 May 03.
Article in English | MEDLINE | ID: mdl-17160024

ABSTRACT

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.


Subject(s)
Antigens, Polyomavirus Transforming/genetics , Apoptosis/genetics , Caspase 3/physiology , Drug Resistance, Neoplasm/genetics , Mammary Neoplasms, Animal/genetics , Milk Proteins/genetics , Osteopontin/physiology , Animals , Antibiotics, Antineoplastic/pharmacology , Culture Media, Serum-Free/pharmacology , Cytochromes c/metabolism , Doxorubicin/pharmacology , JNK Mitogen-Activated Protein Kinases/metabolism , Mammary Neoplasms, Animal/metabolism , Mammary Neoplasms, Animal/pathology , Mice , Mice, Transgenic , Mitochondria/metabolism , Models, Biological , Osteopontin/metabolism , Tumor Cells, Cultured
2.
Virology ; 305(1): 77-92, 2003 Jan 05.
Article in English | MEDLINE | ID: mdl-12504543

ABSTRACT

The HIV-1 Vpr protein harbors a nuclear localization signal in its N-terminal domain. A peptide bearing this domain and which is designated VprN has been used as a target to screen a phage display single chain Fv (scFv) library. Here we report the isolation of anti-VprN scFv fragments from this library. The purified scFv fragments were able to bind the VprN peptide in an ELISA-based system and to inhibit VprN-mediated nuclear import in permeabilized as well as in intact microinjected cells. Furthermore, the anti-VprN scFv fragments recognized the full-length recombinant Vpr protein and inhibited its nuclear import. The same scFv fragments did not inhibit nuclear import mediated by the nuclear localization signal of the SV40 large T-antigen demonstrating a specific effect. The use of the described inhibitory anti-VprN scFv fragments to study nuclear import of viral karyophilic proteins and their therapeutic potential is discussed.


Subject(s)
Active Transport, Cell Nucleus , Gene Products, vpr/immunology , HIV Antibodies/immunology , HIV-1/immunology , Nuclear Localization Signals/immunology , Animals , Complementarity Determining Regions , Gene Products, vpr/physiology , Humans , Immunoglobulin Fragments/immunology , Peptide Library , Tumor Cells, Cultured , vpr Gene Products, Human Immunodeficiency Virus
3.
FEBS Lett ; 507(3): 269-79, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11696354

ABSTRACT

Heterologous trans-splicing is a messenger RNA (mRNA) processing mechanism, that joins RNA segments from separate transcripts to generate functional mRNA molecules. We present here for the first time experimental evidence that the proximal segment of the HIV-nef RNA segment can be trans-spliced to both viral (e.g. SV40 T-antigen) and cellular transcripts. Following either microinjection of in vitro synthesized HIV-nef and SV40 T-antigen pre-mRNA or transfection of the HIV-nef DNA into T-antigen positive cells (CV1-B3; Cos7), it was found that recipient cells synthesized HIV-nef/T-antigen hybrid mRNA and protein molecules. To generate the hybrid mRNA, the cells utilized the 5' cryptic splice sites of the HIV-nef (5'cry 66 and 5'cry 74) and the SV40 T/t-antigen 3' splice site. To demonstrate that heterologous trans-splicing also occurs between the HIV-nef RNA and cellular transcripts, a cDNA library was established from HIV-nef positive CV1-B3 cells (CV1-B3/13 cells) and screened for hybrid mRNA molecules. Reverse transcription-PCR and Northern blot analysis revealed that a significant portion of the HIV-nef transcript is involved in heterologous trans-splicing. To date, eight independent HIV-nef/cellular hybrid mRNA molecules have been identified. Five of these isolates contain segments from known cellular genes (KIAA1454, PTPkappa, Alu and transposon gene families), while three hybrid segments contain sequences of not yet known cellular genes (genes 1-3).


Subject(s)
Gene Products, nef/genetics , Mammals/virology , RNA Splicing , Animals , Antigens, Polyomavirus Transforming/genetics , Cells, Cultured/virology , Chimera , HIV-1/genetics , Humans , Jurkat Cells , Microinjections , Molecular Sequence Data , Protein Biosynthesis , RNA, Messenger , nef Gene Products, Human Immunodeficiency Virus
4.
Oncogene ; 20(28): 3683-94, 2001 Jun 21.
Article in English | MEDLINE | ID: mdl-11439331

ABSTRACT

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.


Subject(s)
Gene Expression Regulation, Neoplastic , Genes, ras , Trans-Splicing , Animals , Enhancer Elements, Genetic , Exons , HeLa Cells , Humans , Introns , Rats
5.
Nucleic Acids Res ; 29(14): 3108-15, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11452036

ABSTRACT

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.


Subject(s)
Alternative Splicing , Carnitine Acyltransferases/genetics , Enhancer Elements, Genetic/genetics , Exons/genetics , Animals , Base Sequence , Blotting, Northern , COS Cells , Cattle , DNA, Complementary/chemistry , DNA, Complementary/genetics , Female , Gene Expression Regulation, Enzymologic , HeLa Cells , Humans , Intestines/enzymology , Liver/enzymology , Male , Mice , Molecular Sequence Data , Mutation , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Swine
6.
Oncogene ; 20(18): 2325-32, 2001 Apr 26.
Article in English | MEDLINE | ID: mdl-11402328

ABSTRACT

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.


Subject(s)
Antigens, Polyomavirus Transforming/physiology , Cell Transformation, Neoplastic , Mammary Glands, Animal/cytology , Mammary Neoplasms, Experimental/pathology , Retinoblastoma Protein/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Antigens, Polyomavirus Transforming/genetics , Binding Sites , Cell Differentiation/physiology , Cyclin D1/biosynthesis , Cyclin D1/physiology , Female , Gene Expression Regulation/physiology , Mammary Glands, Animal/immunology , Mammary Glands, Animal/physiology , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/immunology , Mice , Mice, Transgenic , Pregnancy , Retinoblastoma Protein/genetics , Tumor Suppressor Protein p53/genetics
7.
J Biol Chem ; 273(50): 33347-53, 1998 Dec 11.
Article in English | MEDLINE | ID: mdl-9837909

ABSTRACT

The hepatitis B virus-encoded HBx protein coactivates transcription of viral and cellular genes, and it is believed to play an important role in hepatitis B virus-related liver cancer. HBx has been shown to alter the coordinated balance between proliferation and programmed cell death, being able to either induce or block apoptosis. Here, we demonstrate for the first time that the HBx is a potent caspase 3 inhibitor. Rat fibroblasts (REV2) and hepatoma cells (Hep) synthesizing the HBx protein were resistant to various apoptotic stimuli such as growth factor depletion, tumor necrosis factor alpha, or anti-Fas antibodies administration. In these cells, HBx prevented DNA fragmentation and cell death in the absence of de novo protein synthesis, with a similar efficiency as the competitive caspase 3 substrates inhibitors VAD-FMK and DEVD-FMK. Protein extracts obtained from the HBx positive cells contained a very low caspase activity, and addition of anti-HBx antibody restored the endogenous caspase activity. To obtain a functional map of the anti-caspase activity of HBx, various cell lines were established that synthesized either N-terminally or C-terminally truncated HBx molecules. These gene dissection experiments revealed that the regions required for the anti-caspase activity overlap with the two known transactivation domains of HBx.


Subject(s)
Caspase Inhibitors , Trans-Activators/physiology , Amino Acid Sequence , Animals , Apoptosis , Base Sequence , Caspase 3 , Cell Line, Transformed , DNA Primers , Hydrolysis , Liver Neoplasms, Experimental/pathology , Poly(ADP-ribose) Polymerases/metabolism , Rats , Signal Transduction , Tumor Cells, Cultured , Viral Regulatory and Accessory Proteins
8.
Cancer Res ; 58(16): 3566-70, 1998 Aug 15.
Article in English | MEDLINE | ID: mdl-9721862

ABSTRACT

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.


Subject(s)
Cell Transformation, Viral , Hepatitis B virus , Trans-Activators/physiology , Transcriptional Activation/genetics , Viral Proteins/physiology , Antigens, Viral, Tumor/metabolism , Tumor Suppressor Protein p53/metabolism
9.
Oncogene ; 16(16): 2103-14, 1998 Apr 23.
Article in English | MEDLINE | ID: mdl-9572491

ABSTRACT

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.


Subject(s)
Antigens, Polyomavirus Transforming/metabolism , Apoptosis , Cell Transformation, Neoplastic , Mammary Neoplasms, Experimental/metabolism , Mutagenesis, Site-Directed , Tumor Suppressor Protein p53/genetics , Animals , Antigens, Polyomavirus Transforming/genetics , Cell Line , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Epithelial Cells/metabolism , Female , Mammary Glands, Animal , Mammary Neoplasms, Experimental/genetics , Mice , Mice, Transgenic , Pregnancy , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism
10.
Cancer Res ; 58(7): 1325-31, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9537223

ABSTRACT

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.


Subject(s)
DNA-Binding Proteins/physiology , Drosophila Proteins , Growth Substances/physiology , Muscles/cytology , Muscles/metabolism , Transcription Factors/physiology , Translocation, Genetic/physiology , Animals , Cell Cycle/physiology , Cell Nucleus/metabolism , Cells, Cultured , Cytoplasm/metabolism , DNA/biosynthesis , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Dimerization , E2F4 Transcription Factor , Genetic Vectors , Growth Substances/biosynthesis , Growth Substances/metabolism , Mice , Microinjections , Muscles/physiology , Subcellular Fractions/metabolism , Trans-Activators/biosynthesis , Trans-Activators/metabolism , Transcription Factors/biosynthesis , Transcription Factors/metabolism , Up-Regulation
11.
Gene ; 193(2): 163-72, 1997 Jul 09.
Article in English | MEDLINE | ID: mdl-9256073

ABSTRACT

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.


Subject(s)
Diptera/genetics , Genes, Insect , Insect Proteins , Salivary Proteins and Peptides/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary , Molecular Sequence Data , Peptide Fragments/genetics , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Transcription, Genetic
12.
Biol Chem ; 378(5): 393-405, 1997 May.
Article in English | MEDLINE | ID: mdl-9191026

ABSTRACT

The mammalian genome encodes a DNA cytosine-5-methyltransferase (MTase) of about 170 kDa that is apparently responsible for both de novo and maintenance methylation at CpG sites. Both methylation activities have to be regulated accurately to ensure correct developmental and cell type-specific gene activity. Distorted DNA methylation patterns have been associated with cell aging and diseases such as cancer and fragile X syndrome. Structural and functional in vitro studies of the mouse MTase have indicated that the enzyme has both a regulatory and a catalytic region located in the N-terminal and C-terminal parts of the protein, respectively. The regulatory region includes the nuclear localization signal (NLS), the sequence for DNA targeting and the Zn-binding domain. The catalytic domain carries the ten consensus sequence motifs specific for all known pro- and eukaryotic DNA cytosine-5-methyltransferases. In an attempt to separate regulatory and catalytic functions of the enzyme in vivo, we have tested various deletion mutations by means of transient and stable cell transfection experiments. Expression of the transgenes, all of which retained the C-terminal catalytic domain, was monitored by immunofluorescence staining, Northern blot analysis and SDS gel electrophoresis. Despite high levels of transgene expression, the truncated MTase molecules exhibited neither de novo nor maintenance methylation activity. These findings might indicate that in vivo, an efficient control mechanism prevents the ectopic activity of the DNA MTase that is structurally compromised in its N-terminal regulatory region.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , Gene Expression Regulation, Enzymologic/genetics , Mutation/genetics , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Blotting, Western , Catalysis , Cellular Senescence/genetics , DNA (Cytosine-5-)-Methyltransferases/chemistry , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Damage/genetics , Electrophoresis , Fluorescent Antibody Technique , Fragile X Syndrome/genetics , Mice , Molecular Sequence Data , Molecular Weight , Neoplasms/genetics , Templates, Genetic , Transfection , Zinc/metabolism
13.
Oncogene ; 14(10): 1171-84, 1997 Mar 13.
Article in English | MEDLINE | ID: mdl-9121766

ABSTRACT

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.


Subject(s)
CDC2-CDC28 Kinases , Carrier Proteins , Cell Cycle Proteins , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , DNA-Binding Proteins , Muscle, Skeletal/metabolism , MyoD Protein/physiology , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/metabolism , Animals , Cell Differentiation/physiology , Cell Line , Cyclin-Dependent Kinase 2 , DNA/metabolism , E2F Transcription Factors , E2F4 Transcription Factor , G1 Phase/physiology , Mice , Mice, Inbred C3H , Muscle, Skeletal/cytology , MyoD Protein/genetics , Resting Phase, Cell Cycle/physiology , Retinoblastoma-Binding Protein 1 , Transcription Factor DP1 , Up-Regulation
14.
EMBO J ; 16(2): 369-83, 1997 Jan 15.
Article in English | MEDLINE | ID: mdl-9029156

ABSTRACT

The nuclear phosphoprotein p300 is a new member of a family of 'co-activators' (which also includes the CREB binding protein CBP), that directly modulate transcription by interacting with components of the basal transcriptional machinery. Both p300 and CBP are targeted by the adenovirus E1A protein, and binding to p300 is required for E1A to inhibit terminal differentiation in both keratinocytes and myoblasts. Here we demonstrate that, in differentiating skeletal muscle cells, p300 physically interacts with the myogenic basic helix-loop-helix (bHLH) regulatory protein MyoD at its DNA binding sites. During muscle differentiation, MyoD plays a dual role: besides activating muscle-specific transcription, it induces permanent cell cycle arrest by up-regulating the cyclin-dependent kinase inhibitor p21. We show that p300 is involved in both these activities. Indeed, E1A mutants lacking the ability to bind p300 are greatly impaired in the repression of E-box-driven transcription, and p300 overexpression rescues the wild-type E1A-mediated repression. Moreover, p300 potentiates MyoD- and myogenin-dependent activation of transcription from E-box-containing reporter genes. We also provide evidence, obtained by microinjection of anti-p300 antibodies, that p300 is required for MyoD-dependent cell cycle arrest in either myogenic cells induced to differentiate or in MyoD-converted C3H10T1/2 fibroblasts, but is dispensable for maintenance of the postmitotic state of myotubes.


Subject(s)
Cell Cycle , Gene Expression Regulation, Developmental , Muscle Development , Muscle, Skeletal/growth & development , MyoD Protein/physiology , Nuclear Proteins/physiology , Trans-Activators , Transcription Factors/physiology , Animals , Blotting, Western , Cell Differentiation , DNA/metabolism , E1A-Associated p300 Protein , Helix-Loop-Helix Motifs , Mice , Microinjections , Myogenin/metabolism
15.
Mol Cell ; 1(1): 35-45, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9659901

ABSTRACT

PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD-DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program.


Subject(s)
Acetyl-CoA C-Acyltransferase/metabolism , Acetyltransferases/metabolism , Cell Cycle Proteins/metabolism , Muscle Fibers, Skeletal/cytology , Muscle, Skeletal/cytology , Saccharomyces cerevisiae Proteins , Trans-Activators , Animals , Antigens, Viral, Tumor/pharmacology , CREB-Binding Protein , Cell Differentiation/physiology , Cells, Cultured , E1A-Associated p300 Protein , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/physiology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Histone Acetyltransferases , Mice , Multienzyme Complexes/metabolism , Muscle Fibers, Skeletal/chemistry , Muscle Fibers, Skeletal/enzymology , Muscle, Skeletal/chemistry , Muscle, Skeletal/enzymology , MyoD Protein/metabolism , Nuclear Proteins/metabolism , Nuclear Proteins/pharmacology , RNA Polymerase II/metabolism , Transcription Factors/metabolism , Transcription Factors/pharmacology , Transcriptional Activation/physiology , p300-CBP Transcription Factors
16.
FEBS Lett ; 394(2): 227-32, 1996 Sep 30.
Article in English | MEDLINE | ID: mdl-8843169

ABSTRACT

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.


Subject(s)
RNA Splicing , RNA, Complementary/metabolism , RNA, Viral/metabolism , Simian virus 40/genetics , Animals , Antigens, Polyomavirus Transforming/genetics , Blotting, Northern , Cell Line , Cell Nucleus/metabolism , DNA Primers , Electrophoresis, Agar Gel , Exons/genetics , Fluorescent Antibody Technique , Genes, Viral , Microinjections , Polymerase Chain Reaction , Protein Biosynthesis , RNA, Complementary/chemical synthesis , Rats
17.
FEBS Lett ; 394(2): 233-6, 1996 Sep 30.
Article in English | MEDLINE | ID: mdl-8843170

ABSTRACT

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.


Subject(s)
Antigens, Polyomavirus Transforming/genetics , RNA Caps , RNA Precursors/metabolism , RNA Splicing , RNA, Viral/metabolism , Simian virus 40/genetics , Animals , Antigens, Polyomavirus Transforming/analysis , Blotting, Northern , Cell Nucleus/metabolism , Cells, Cultured , DNA Primers , Electrophoresis, Agar Gel , Fluorescent Antibody Technique , Microinjections , Microscopy, Fluorescence , Polymerase Chain Reaction , Protein Biosynthesis , RNA Precursors/chemical synthesis , RNA Precursors/genetics , RNA, Complementary/genetics , RNA, Complementary/metabolism , RNA, Viral/genetics , Rats
18.
Nucleic Acids Res ; 24(9): 1653-61, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8649982

ABSTRACT

The early SV40 BstXI-BamHI (Bst/Bam) DNA fragment encodes exclusively for the second exon of the large T-antigen and contains the intact small t-antigen intron. Rat cells transformed by the p14T, a construct that carries the Bst/Bam DNA fragment as a tail-to-head tandem duplication, synthesize a truncated T-antigen (T1-antigen) without having a direct equivalent at the DNA level. Formation of the T1-mRNA occurs by means of two distinct mechanisms: alternative-tandem-cis-splicing and trans-splicing. To generate the T1-mRNA the cells utilize a cryptic 5' splice site, located within the second exon of the large T-antigen and the regular small t-antigen 3' splice site. Since these splice sites are in an inverted order two Bst/Bam transcripts are required to generate one T1-mRNA molecule. For alternative-tandem-cis-splicing the cells utilize a 4.4 kb pre-mRNA that contains the sequence of the entire Bst/Bam tandem repeat. The proximal Bst/Bam segment provides the 5' donor splice site and the distal segment the 3' acceptor site. This requires that the pre-mRNA not be cleaved after the RNA polymerase II has passed the polyadenylation signal of the proximal Bst/Bam DNA segment. Synthesis of the 4.4 kb pre-mRNA was demonstrable by RT-PCR but not by Northern blot analysis. For trans-splicing, the cells utilize two separate pre-mRNA molecules. One transcript provides the cryptic 5' splice donor site and the other the 3' splice acceptor site. To demonstrate this a three base pair deletion was introduced into the proximal Bst/Bam segment of the p14T DNA (p14Tdelta-3) as a marker, destroying the recognition site for Pf/MI restriction enzyme. This deletion allowed the differentiation between the proximal and distal Bst/Bam segment. RT-PCR analysis and DNA sequencing confirmed that the p14Tdelta-3 transformed cells generate the T1-mRNA by intra- and inter-molecular RNA splicing.


Subject(s)
Alternative Splicing/genetics , Antigens, Polyomavirus Transforming/genetics , RNA Splicing/genetics , Simian virus 40/immunology , Animals , Antigens, Polyomavirus Transforming/biosynthesis , Base Sequence , Cell Line, Transformed , Exons/genetics , Molecular Sequence Data , RNA Precursors/biosynthesis , RNA Precursors/genetics , RNA, Messenger/biosynthesis , Rats , Repetitive Sequences, Nucleic Acid/genetics , Sequence Deletion/genetics
19.
EMBO J ; 15(9): 2236-48, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8641289

ABSTRACT

p300 is a nuclear phosphoprotein likely to be involved in the control of cell growth. Here we show that SV40 large T antigen (Tag) forms a specific complex with p300. In various Tag-expressing cell lines, the affinity of Tag for p300 was restricted to a newly identified unphosphorylated but ubiquitinated form of the protein. Further, Tag did not associate with p300 in an SV40 Tag-producing cell line (REV2) in which the original transformed phenotype (SV52) is reverted. Biochemical studies demonstrate that both the phosphorylation and the ubiquitination profile of p300 are altered in REV2 with respect to the wild-type fully transformed SV52 parental cells, wherein Tag-p300 complexes are readily detected. In contrast to Tag, the adenovirus early expression product E1a interacts with both phosphorylated and unphosphorylated forms of p300. In addition, when REV2 cells were infected with adenovirus, E1a-p300 complexes were detected, suggesting that the p300 expressed in REV2 has lost the affinity for Tag, but not for E1a. We then compared the ability of Tag and E1a to affect the transcription levels of the cAMP-responsive promoter (CRE), which is modulated in vivo by p300, in REV2 cells. We found that Tag repressed the CRE promoter in all of the cell lines in which Tag-p300 complexes were detected, but not in REV2 cells. In contrast, E1a efficiently inhibited CRE-directed transcription in this cell line. The data thus indicate that the different specificities exhibited by Tag and E1a towards the various forms of p300 are reflected in vivo as a difference in the ability of these viral oncoproteins to modulate the expression of CRE-containing genes.


Subject(s)
Adenovirus E1A Proteins/metabolism , Antigens, Polyomavirus Transforming/metabolism , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Cell Line , Chlorocebus aethiops , HeLa Cells , Humans , Phosphorylation , Protein Processing, Post-Translational , Ubiquitins/metabolism
20.
FEBS Lett ; 380(3): 215-8, 1996 Feb 19.
Article in English | MEDLINE | ID: mdl-8601427

ABSTRACT

Transgenic animals of the line 8 contain the WAP-SV-T transgene. Females of this line synthesise the SV40 T-antigen in mammary gland epithelial cells during pregnancy and the lactation period. All females are 'milk-less' and the offspring have to be nursed by foster mothers. The reason for this phenomenon is a premature apoptosis during late pregnancy. Nontheless a significant number of mammary epithelial cells escape apoptosis and all transgenic females devlop breast cancer after the first lactation period.


Subject(s)
Antigens, Polyomavirus Transforming/physiology , Apoptosis , Mammary Glands, Animal/cytology , Pregnancy, Animal , Animals , Antigens, Polyomavirus Transforming/genetics , Epithelial Cells , Female , Lactation , Mammary Glands, Animal/physiology , Mammary Neoplasms, Experimental/etiology , Mice , Mice, Transgenic , Milk Proteins/genetics , Pregnancy , Promoter Regions, Genetic
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