Mapping of polyomavirus middle T domain that is responsible for AP-1 activation.

Cell transformation by Polyomavirus middle T (MT) oncoprotein involves binding and activation of several cytoplasmic proteins that participate in growth factors-induced mitogenic signal transduction to the nucleus. We have previously reported that the AP-1 transcriptional complex is a target for MT during cell transformation. To analyse the interactions between MT and cellular proteins that are required for constitutive AP-1 activation, we compared wild type and transformation-defective MT mutant cell lines. High AP-1 activity, assessed by gel mobility shift assays, displayed by MT-overexpressing cells, is dependent on MT binding to phosphatidylinositol-3 kinase (P13K). Treatment with wortmannin (a specific P13K inhibitor) leads to decreased AP-1 activity. Supershift and Western blot analysis with specific antisera, indicate that JunB and cJun, but not cFos or FosB are present in the AP-1 complex. The results confirm the AP-1 complex as a downstream MT target and indicate that AP-1 activation may not be sufficient for cell transformation, since two transformation-defective MT mutants (250phe and MT322) display high AP-1 activity.

Contribution of hydrazines-derived alkyl radicals to cytotoxicity and transformation induced in normal c-myc-overexpressing mouse fibroblasts.

Several hydrazine derivatives (HD) tested so far have pharmacological activities, but many also have toxic side effects, including carcinogenesis. Their toxicity has been ascribed to carbocations (via formation of azoxy intermediates), alkyl radicals or reactive oxygen species. Cytotoxicity and transformation by carbocations is widely accepted, but the role of alkyl radicals is still questioned. We have investigated the cytotoxicity of HD to mouse fibroblasts in three activation systems in which enhanced alkyl radical formation is demonstrated by electron spin resonance/spin-trapping. Cytotoxicity was assayed by inhibition of [3H-methyl]thymidine uptake into DNA of Balb/c 3T3 and/or Myc 9E fibroblasts (normal Balb/c 3T3 cells over-expressing the c-myc proto-oncogene). Based on the results obtained in the cytotoxicity assays we also investigated the transforming potential of procarbazine (PCZ) and methylhydrazine (MeH) activated by horseradish peroxidase (HRP) using the Myc 9E cell line, which aims at the activation of a second cooperating oncogene. Our results show that: (i) cytotoxicity of HD to mouse fibroblasts is increased by HRP activation of MeH, phenelzine and PCZ, which displayed enhanced alkyl radical formation, but not of 1,2-dimethylhydrazine (DMH), which did not produce increased alkyl radical formation under these conditions; (ii) cytotoxicity of neutrophil-activated MeH (producing a 10-fold higher concentration of methyl radicals), is more pronounced than DMH; (iii) MeH and DMH activated by prolonged auto-oxidation in 24-h incubations have comparable cytotoxicity and alkyl radical formation; and (iv) PCZ and MeH activation by HRP to alkyl radicals increased the transformation induced in Myc 9E cells. Taken together, our results strongly support a role for hydrazine-derived alkyl radicals in HD-induced cytotoxicity and cell transformation.

Use of pEX and pGEX bacterial heterologous protein expression systems for recombinant oncoprotein production and antisera generation.

In order to study the role played by known and novel genes in growth control and neoplasia, we here compare the pEX and pGEX bacterial expression systems for recombinant oncoprotein production and for generation of specific antisera. The results of five pEX (MS2-c-Fos, MS2-Fra-1, MS2-JunD, bgal-c-Jun and bgal-JunB) and two pGEX [glutathione S-transferase (GSH)-JE/MCP-1 and GST-JunD] fusion-protein productions are presented. Higher (15-43-fold) yields are obtained with the pEX system, but only the pGEX system allows separation of the protein of interest from the fusion moiety by digestion with specific proteases. The degree of fusion-protein purification, as assessed by SDS/PAGE, is similar for both systems. Proteins produced by both systems were successfully used in the generation of specific antisera. The choice between the pEX and pGEX systems is dependent upon the specific recombinant protein produced.

Use of cDNA cloning to study the mechanism of action of glucocorticoid hormones at the molecular level.

We have previously described a dramatic phenomenon of phenotypic reversion (tumor to normal) caused by glucocorticoid hormones in C6/ST1 rat glioma cells, but not in their hormone-resistant C6/P7 counterpart. Blind cDNA cloning was adopted to search for glucocorticoid-regulated gene sequences responsible for this phenotype reversion. Differential hybridization and differential display of RNA were used in parallel to isolate a number of cDNA clones that were characterized by DNA sequencing and Northern blot analysis. This approach was coupled to the analysis of known growth control genes (oncogenes, tumor suppressor genes, cyclins, cyclin-dependent kinases, other kinases). Glucocorticoid target genes isolated from this cell system are likely to be good anti-tumor candidate molecules which can be used in tumor therapy and anti-tumor drug design.

Use of cDNA cloning to study the mechanism of action of glucocorticoid hormones at the molecular level

We have previously described a dramatic phenomenon of phenotypic reversion (tumor to normal) caused by glucocorticoid hormones in C6/ST1 rat glioma cells, but not in their hormone-resistant C6/P7 counterpart. Blind cDNA cloning was adopted to search for glucocorticoid-regulated gene sequences responsible for this phenotype reversion. Differential hybridization and differential display of RNA were used in parallel to isolate a number of cDNA clones that were characterized by DNA sequencing and Northern blot analysis. This approach was coupled to the analysis of known growth control genes (oncogenes, tumor suppressor genes, cyclins, cyclin-dependent kinases other kinases). Glucocorticoid target genes isolated from this cell system are likely to be good anti-tumor candidate molecules which can be used in tumor therapy and anti-tumor drug design.

Polyomavirus-induced malignant transformation: comparative analysis of wild type and mutant middle T-overexpressing cell lines.

Polyomavirus, a DNA tumor virus, expresses three viral oncoproteins (large, middle and small T antigens), causes malignant transformation in cell culture and induces multiple tumors in vivo. The middle T (MT) antigen seems to play an essential role in transformation and tumorigenicity. The observation that MT-overexpressing cell lines are able to grow in the absence of PDGF (platelet-derived growth factor) led several laboratories to study the mechanism underlying MT-induced growth deregulation and the signal transduction pathway used by this viral oncoprotein. A number of cellular proteins were shown to be common to both the normal PDGF mitogenic pathway and the MT transforming pathway. The expression of some PDGF primary response genes (fos, jun, myc, JE, KC) was shown to be rendered constitutive by MT overexpression. Using MT mutants, important domains for binding and activation of cytoplasmic proteins were mapped. Wild type and mutant MT cell lines are used in our laboratory to analyze the expression and activity of the PDGF early response genes during cell transformation and correlate them with activation of specific cytoplasmic proteins. In addition to abrogating the PDGF requirement for growth, activation of cellular proteins caused by MT results in cell lines that have an altered morphology and are able to form colonies in agarose. These changes may be due to alterations in connexin 43 and other cell surface proteins.

Polyomavirus-induced malignant transformation: comparative analysis of wild type and mutant middles T-overexpressing cell lines

Polyomavirus, a DNA tumor virus, expresses three viral oncoproteins (large, middle and small T antigens), causes malignant transformation in cell culture and induces multiple tumors in vivo. The middle T (MT) antigen seems to play an essential role in transformation and tumori-genicity. The observation that MT-overexpressing cell lines are able to grow in the absence of PDGF (platelet-derived growth factor) led several laboratories to study the mechanism underlying MT-induced growth deregulation and the signal transduction pathway used by this viral oncoprotein. A number of cellular proteins were shown to be common to both the normal PDGF mitogenic pathway and the MT transforming pathway. The expression of some PDGF primary response genes (fos, jun, myc, JE, KC) was shown to be rendered constitutive by MT overexpression. Using MT mutants, important domains for binding and activation of cytoplasmic proteins were mapped. Wild type and mutant MT cell lines are used in our laboratory to analyze the expression and activity of the PDGF early response genes during cell transformation and correlate them with activation of specific cytoplasmic proteins. In addition to abrogating the PDGF requirement for growth, activation of cellular proteins caused by MT results in cell lines that have an altered morphology and are able to form colonies in agarose. These changes may be due to alterations in connexin 43 and other cell surface proteins.

Molecular genetic approach to cell proliferation control and neoplasia.

A number of gene products involved in the control of cell proliferation fall into one of two classes: oncogenes and tumor suppressor genes. The same gene products have also been associated with malignant growth (tumors) caused by radiation, chemicals and tumor viruses. Here we describe our attempts to elucidate the molecular mechanisms underlying polyomavirus-induced cell transformation and the anti-tumor activity of glucocorticoid hormones. Wild type and mutant polyomavirus middle T (MT) overexpressing cell lines, generated with retroviral vector constructs, were used to investigate the role played by peptide growth factor primary response genes (fos, jun, myc, JE, KC) in viral transformation and to map the transduction pathway of the mitogenic signal of MT. Overexpression of MT leads to increased AP-1 (Fos/Jun) transcriptional complex activity. Transformation defective mutant analysis allowed the identification of sites in the MT molecule that are crucial for this activity. Two different approaches were used to investigate the molecular basis for glucocorticoids anti-tumor activity, namely: blind cloning of cDNAs and analysis of growth control genes in C6 glioma cell variants that are either hypersensitive (C6/ST1) or unresponsive to glucocorticoids (C6/P7). Four different glucocorticoid-regulated cDNA sequences were isolated using differential hybridization. A number of differentially expressed sequences were isolated from glucocorticoid-treated C6/ST1 cells by differential display (DDRT-PCR) and are currently being characterized. Expression of known growth control genes in C6/ST1 cells allowed the identification of important candidates for glucocorticoid hormone targets.

Molecular genetic approach to cell proliferation control and neoplasia

A number of gene products involved in the control of cell proliferation fall into one of two classes: oncogenes and tumor suppressor genes. The same gene products have also been associated with malignant growth (tumors) caused by radiation, chemicals and tumor viruses. Here we describe our attempts to elucidate the molecular mechanisms underlying polyomavirus-induced cell transformation and the anti-tumor activity of glucocorticoid hormones. Wild type and mutant polyomavirus middle T (MT) overexpressing cell lines, generated with retroviral vector constructs, were used to investigate the role played by peptide growth factor primary response genes (fos, jun, myc, JE, KC) in viral transformation and to map the transduction pathway of the mitogenic signal of MT. Overexpression of MT leads to increased AP-1 (Fos/Jun) transcriptional complex activity. Transformation defective mutant analysis allowed the identification of sites in the MT molecule that are crucial for this activity. Two different approaches were used to investigate the molecular basis for glucocorticoids anti-tumor activity, namely: blind cloning of cDNAs and analysis of growth control genes in C6 glioma cell variants that are either hypersensitive (C6/ST1) or unresponsive to glucocorticoids (C6/P7). Four different glucocorticoid-regulated cDNA sequences were isolated using differential hybridization. A number of differentially expressed sequences were isolated from glucocorticoid-treated C6/ST1 cells by differential display (DDRT-PCR) and are currently being characterized. Expression of known growth control genes in C6/ST1 cells allowed the identification of important candidates for glucocorticoid hormone targets.

Cloning of glucocorticoid-regulated genes in C6/ST1 rat glioma phenotypic reversion.

The C6 rat glioma cell line is response to glucocorticoid hormones. C6 variants that are hyper-responsive (ST1) and resistant (P7) to hormone treatment have been derived previously. Glucocorticoid treatment of ST1 cells leads to complete reversion of the transformed phenotype and loss of tumorigenic potential. Production of C type retrovirus particles is also induced by glucocorticoids in ST1 cells. Cloning of the genes regulated by glucocorticoids in this cell system was used here as a strategy to uncover the gene products involved in the transformed-to-normal phenotypic change. Construction of a cDNA library from glucocorticoid-treated ST1 cells and screening by differential hybridization resulted in the isolation of three cellular sequences that code for rat metallothioneins (C27 and C41) and alpha 1-acid glycoprotein (C36). Northern blot analysis revealed that expression of these genes was dramatically induced by hydrocortisone in ST1 but not in P7 cells. Viral genomic RNA was used to isolate and characterize retrovirus-related sequences that could also be responsible for the phenotypic reversion phenomenon.

Expression and characterization of mouse cFos protein using the baculovirus expression system: ability to form functional AP-1 complex with coexpressed cJun protein.

The products of the proto-oncogenes c-fos and c-jun play important roles in cell growth control, differentiation, and malignant transformation. Purified oncogenic proteins are essential tools in cell growth control studies. Here we describe the production of mouse cFos and cJun oncoproteins in Sf9 insect cells using the baculovirus expression system. The mouse c-fos cDNA was subcloned into two different baculovirus expression vectors, namely pVL1392 and pVLMH6, to generate, respectively, nonfusion and His-fusion cFos oncoproteins in insect cells. The products were characterized by immunofluorescence, immunoblotting, immunoprecipitation, and ability to bind to the in vitro translated JunB protein to form the AP-1 complex. A His-cJun fusion protein was also produced in insect cells using the pVLMH6 baculovirus vector. Coexpression of cFos and cJun in insect cells yielded functional AP-1 complexes as judged from gel retardation assays. The results point to the possibility of using insect cells for structural and functional studies of different Fos/Jun AP-1 complexes.

Isolation and characterization of c-fos recombinant baculovirus.

The use of the baculovirus system to produce recombinant proteins is based on the high level of protein production and the possibility to obtain, in Spodoptera frugiperda insect cells, recombinant proteins with the post-translational modifications found in the native proteins. Here we describe the isolation and characterization of a recombinant baculovirus containing the mouse c-fos gene. The c-fos cDNA was subcloned into the pVL1392 baculovirus transfer vector. The recombinant plasmid (pVL1392.fos) was introduced into Sf9 insect cells by co-transfection with viral wild-type DNA. Upon selection and characterization of a viral recombinant clone, Sf9 cells were infected with this virus stock and the cFos protein expression was detected by immunological methods using an anti-cFos polyclonal antiserum.

Isolation and characterization of c-fos recombinant baculovirus

The use of the baculovirus system to produce recombinant proteins is based on the high level of protein production and the possibility to obtain, in Spodoptera frugiperda insect cells, recombinant proteins with the post-translational modifications found in the native proteins. Here we describe the isolation and characterization of a recombinant baculovirus containing the mouse c-fosgene. The c-fos cDNA was subcloned into the pVL1392 baculovirus transfer vector. The recombinant plasmid (pVL1392.fos) was introduced into Sf9 insect cells by co-transfection with viral wild-type DNA. Upon selection and characterization of a viral recombinant clone, SF9 cells were infected with this virus stock and the cFos protein expression was detected by immunological methods using an anti-cFos polyclonal antiserum

Glucocorticoid-regulated gene in transformed to normal phenotypic reversion.

Glucocorticoid hormones modulate the actions of peptide growth factors and constitute important therapeutic tools as anti-inflammatory and anti-tumor agents. The C6 rat glioma cell line responds to glucocorticoids with changes in morphology and growth block. The hyper-responsive ST1 cell variant displays a dramatic phenotypic reversion under the influence of these hormones. Thus, the transformed and tumorigenic cells reversibly change to a normal and non-tumorigenic phenotype. In addition, the cells also produce a C-type retrovirus. We used poly A+ mRNA from ST1 cells that had been treated with hydrocortisone to generate a cDNA library that was then screened, by differential hybridization, for glucocorticoid-responsive cellular sequences. The retroviral genomic RNA was used to generate a viral-specific probe. Cross hybridization led to the isolation of at least 4 cDNA clones of which 3 are cellular sequences and one corresponds to a retroviral gene. These clones were characterized by DNA sequencing and Northern blot hybridization analysis.

Induction of FOS and JUN proteins by adrenocorticotropin and phorbol ester but not by 3',5'-cyclic adenosine monophosphate derivatives.

We report the results of an extensive kinetic analysis of the effects of ACTH, cAMP derivatives (dibutyryl cAMP and 8-bromo-cAMP) and phorbol ester (phorbol-12-myristate-13-acetate) on the expression of fos and jun gene family members at the mRNA (Northern hybridization) and protein levels (immunoprecipitation and indirect immunofluorescence) in the mouse Y-1 adrenocortical cell line. FOS and JUN proteins are induced by ACTH independently of cell cycle stage. c-Fos, fos-B, fra-1, fra-2, c-jun, and jun-B genes are induced by ACTH, the kinetic profiles for mRNAs and respective protein products being similar, except for a 1-h protein delay. Jun-D mRNA is an exception, being constitutively expressed. However, JUN D protein is induced by ACTH. phorbol-12-myristate-13-acetate closely mimics these inductive effects of ACTH. On the other hand, cAMP derivatives are not effective in inducing the fos and jun genes, except for fra-2 mRNA, JUN D protein, and to some extent JUN B protein. Clearly, ACTH is endowed with the versatile capability of modulating fos and jun gene expression, suggesting that AP-1 transcription factors play a role in ACTH mechanisms of action. ACTH receptors are likely to activate signaling routes other than the classical cAMP/protein kinase A in order to induce FOS and JUN proteins.

PCR detection of Xbal polymorphism in the human Rb gene of retinoblastoma patients.

Inactivation of the Rb (retinoblastoma) tumor suppressor gene is associated with hereditary and sporadic cases of retinoblastoma and other Rb-related tumors. Early diagnosis and genetic counseling heavily depend on practical methods for the detection of Rb deletions and mutations in high-risk families. Here we report on the use of a pair of primers in polymerase chain reaction (PCR) to amplify a 945-bp fragment from intron 17 of the Rb gene (T.L. McGee, G.S. Cowley, D.W. Yandell and T.P. Dryja, 1990, Nucleic Acid Research, 18: 207). Xbal digestion of the PCR product reveals 2 allelic versions: a single 945-bp fragment (allele 1) or 2 fragments of 315 and 630 bp (allele 2). We used total genomic DNA (blood and tumors) to investigate the power of this PCR-Rb-Xbal-RFLP in the identification of both segregation and loss of heterozygosity of the Rb gene. In one family studied (family 1A) in which 2 generations were affected, it was possible to localize the mutated Rb gene to Xbal-Rb allele 2. The assay of loss of heterozygosity of the Rb gene is available for all Xbal-Rb allele 1-2 individuals, so that analyses may be applied in large scale investigation of the participation of Rb gene in tumor development. We conclude that PCR-Rb-Xbal-RFLP is a practical and powerful tool for oncology research and genetic counseling.

PCR detection of Xbal polymorphism in the human Rb gene of retinoblastoma patients

Inactivation of the Rb (retinoblastoma) tumor suppressor gene is associated with hereditary and sporadic cases of retinoblastoma and other Rb-related tumors. Early diagnosis and genetic counseling heavily depend on practical methods for the detection of Rb deletions and mutations in high-risk families. Here we report on the use of a pair of primers in polymerase chain reaction (PCR) to amplify a 945-bp fragment from intron 17 of the Rb gene (T.L. McGee, G.S. Cowley, D.W. Yandell and T.P. Dryja, 1990, Nucleic Acid Research, 18: 207). Xbal digestion of the PCR product reveals 2 allelic versions: a single 945-bp fragment (allele 1) or 2 fragments of 315 and 630 bp (allele 2). We used total genomic DNA (blood and tumors) to investigate the power of this PCR-Rb-Xbal-RFLP in the identification of both segregation and loss of heterozygosity of the Rb gene. In one family studied (family 1A) in which 2 generations were affected, it was possible to localize the mutated Rb gene to Xbal-Rb allele 2. The assay of loss of heterozygosity of the Rb gene is available for all Xbal-Rb allele 1-2 individuals, so that analyses may be applied in large scale investigation of the participation of Rb gene in tumor development. We conclude that PCR-Rb-Xbal-RFLP is a practical and powerful tool for oncology research and genetic counseling

Secretion of a neuropeptide-metabolizing enzyme similar to endopeptidase 22.19 by glioma C6 cells.

An endopeptidase capable of metabolizing a number of neuropeptides and generating [Met5] and [Leu5] enkephalin from enkephalin-containing peptides is secreted by glioma C6 cells. This neutral endopeptidase that is likely to be a thiol protease, has a Mr of 71KDa and is effective only towards oligopeptides. Its specificity towards neuropeptides is identical to that of soluble endopeptidase 22.19. Moreover, when a partially purified preparation of enkephalin-generating enzyme secreted by glioma C6 cells was submitted to immunoblotting, an antiserum against purified brain endopeptidase 22.19 recognized a single band at Mr of 71 KDa. These data suggest that the soluble endopeptidase 22.19 may be secreted by glioma C6 cells thus allowing its participation in the biotransformation of opioid peptides in the CNS.

Downregulation of JE and KC genes by glucocorticoids does not prevent the G0----G1 transition in BALB/3T3 cells.

The effects of glucocorticoid hormones on the expression of the growth factor-inducible genes JE, KC, and c-myc were analyzed in parental BALB/3T3 and polyomavirus middle-T antigen-transfected cell lines. Northern (RNA) blot hybridization and run-on transcription analysis showed that (i) glucocorticoid hormones selectively inhibit JE and KC expression at the transcriptional level and (ii) the downregulatory effect of glucocorticoids on JE and KC expression is partial for serum-stimulated and middle T antigen-transformed cells and total for quiescent and exponentially growing cells. Gel mobility assays using AP-1 oligonucleotides showed a positive correlation between glucocorticoid downregulating effect and presence of the AP-1 complex. JE and KC downregulation by means of the AP-1 complex may play a role in the actions of glucocorticoids as anti-inflammatory and antitumor agents. The ability of glucocorticoids to downregulate JE and KC was used to investigate the relevance of these genes to the mitogenic response to serum growth factors. Hydrocortisone did not alter the basal DNA synthesis level displayed by quiescent 3T3 cells, but it potentiated both the mitogenic effect of platelet-derived growth factor and c-myc induction by serum growth factors. Upon serum restimulation, untreated and dexamethasone-treated quiescent 3T3 cultures entered the S phase after an identical time lag (G1). These results suggest that (i) JE and KC are not necessary for the G0----G1----S transition and (ii) c-myc overexpression is likely to be the basis for the potentiating effect of glucocorticoids on serum growth factors.