RESUMO
A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.
Assuntos
Genes p53 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Timidilato Sintase/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Regulação da Expressão Gênica , Humanos , Substâncias Macromoleculares , Polirribossomos/química , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/química , Ratos , Ribonucleoproteínas/química , Timidilato Sintase/química , TransfecçãoRESUMO
Dephosphorylation of the RB protein has been reported to be associated with apoptosis. In contrast, we show that treatment of HL60 cells with etoposide or cytosine arabinoside or treatment of breast epithelial cells with alpha-FAS is associated with the cleavage of a 5 kDa fragment from the C-terminus of RB, resulting in a truncated product that we have designated as p100cl. This cleavage event coincides with the activation of cysteine proteases at the onset of apoptosis, is blocked by the addition of iodoacetamide to cells prior to the onset of apoptosis, and results in the expression of faster migrating protein species which can mimic dephosphorylated RB. The free 5 kDa fragment is detected only during apoptosis, predicts a cleavage site that we have mapped to a unique CPP32-like recognition sequence which is present at the C-terminus of all reported RB homologues, and results in a truncated RB protein with enhanced E2F binding affinity. While the causality for this cleavage event in the apoptotic process is still under investigation, our findings suggest distinct post-translational pathways for the RB product between cells examined during growth arrest (p105 hypophosphorylated RB) or apoptosis (p100cl).
Assuntos
Apoptose/fisiologia , Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Fragmentos de Peptídeos/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Cisteína Endopeptidases/metabolismo , Citarabina/farmacologia , Fatores de Transcrição E2F , Etoposídeo/farmacologia , Feminino , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Humanos , Fosforilação , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Receptor fas/fisiologiaRESUMO
Absent expression of the cyclin dependent kinase-inhibitor, p16INK4, is observed in a wide range of primary human cancers. Although homozygous deletions and point mutations have been reported in a subset of these tumors, the molecular basis for absent p16INK4 in other samples is unknown. We have examined 33 tumor cell lines and have shown that hypermethylation of a G:C-rich region within exon 1 of the CDKN2 gene was present in 100% of samples with wildtype RB expression and no detectable CDKN2 mutations. Treatment for at least 4 hours with the demethylating agent 5-aza 2'deoxycytidine, but not 5-azacytidine or 6-azacytidine, induces the prolonged expression of p16INK4 protein in each of these samples following a discrete 24-48 hour lag period. Consistent with the hypothesis that hypermethylation of the CDKN2 gene is a tumor-specific mechanism for gene inactivation, we observed hypomethylation at the exon 1 site exclusively in tumor lines that expressed p16INK4 or that had sustained inactivating point mutations within the CDKN2 open reading frame. These findings demonstrate a link between DNA methylation and the p16INK4:RB tumor suppressor pathway.
Assuntos
Azacitidina/análogos & derivados , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Metilases de Modificação do DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Sequência de Bases , Inibidor p16 de Quinase Dependente de Ciclina , DNA/metabolismo , Decitabina , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Metilação , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
The 357 amino acid open reading frame 1 (ORF-1), also designated DR7, within the SalI-L fragment of human herpesvirus 6 (HHV-6) exhibited transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter and increased HIV-1 replication (Kashanchi et al., Virology, 201, 95-106, 1994). In the current study, the SalI-L transforming region was localized to the SalI-L-SH subfragment. Several ORFs identified in SalI-L-SH by sequence analysis were cloned into a selectable mammalian expression vector, pBK-CMV. Only pBK/ORF1 transformed NIH3T3 cells. Furthermore, cells expressing ORF-1 protein produced fibrosarcomas when injected into nude mice, whereas control cells, expressing either no ORF-1 protein or C-terminal truncated (after residue 172) ORF-1 protein, were not tumorigenic. Western blot analysis of proteins extracted from the tumors revealed ORF-1 protein. Additional studies indicated that ORF-1 was expressed in HHV-6-infected human T-cells by 18 h. Co-immunoprecipitation experiments showed that ORF-1 protein bound to tumor suppressor protein p53, and the ORF-1 binding domain on p53 was located between residues 28 and 187 of p53, overlapping with the specific DNA binding domain. Functional studies showed that p53-activated transcription was inhibited in ORF-1, but not in truncated ORF-1, expressing cells. Importantly, the truncated ORF-1 mutant also failed to cause transformation. Analysis of several human tumors by PCR revealed ORF-1 DNA sequences in some angioimmunoblastic lymphadenopathies, Hodgkin's and non-Hodgkin's lymphomas and glioblastomas. The detection of ORF-1 sequences in human tumors, while not proof per se, is a prerequisite for establishing its role in tumor development. Taken together, the results demonstrate that ORF-1 is an HHV-6 oncogene that binds to and affects p53. The identification of both transforming and transactivating activities within ORF-1 is a characteristic of other viral oncogenes and is the first reported for HHV-6.
Assuntos
Genes Reguladores/fisiologia , Oncogenes , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Células 3T3 , Animais , Fibrossarcoma/genética , Vetores Genéticos , Humanos , Camundongos , Camundongos Nus , TransfecçãoRESUMO
There are major differences between therapeutic tumor vaccines and chemotherapeutic agents that have important implications for the design of early clinical trials. Many vaccines are inherently safe and do not require phase I dose finding trials. Patients with advanced cancers and compromised immune systems are not good candidates for assessing either the toxicity or efficacy of therapeutic cancer vaccines. The rapid pace of development of new vaccine candidates and the variety of possible adjuvants and modifications in method of administration makes it important to use efficient designs for clinical screening and evaluation of vaccine regimens. We review the potential advantages of a wide range of clinical trial designs for the development of tumor vaccines. We address the role of immunological endpoints in early clinical trials of tumor vaccines, investigate the design implications of attempting to use disease stabilization as an end point and discuss the difficulties of reliably utilizing historical control data. Several conclusions for expediting the clinical development of effective cancer vaccines are proposed.
Assuntos
Antineoplásicos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Determinação de Ponto Final , Neoplasias/virologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Adjuvantes Imunológicos/uso terapêutico , Estudos de Coortes , Humanos , Hospedeiro Imunocomprometido , Neoplasias/tratamento farmacológico , Projetos de Pesquisa , Resultado do TratamentoAssuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/farmacologia , Quimioprevenção , Neoplasias/prevenção & controle , Infecções Bacterianas/complicações , Infecções Bacterianas/imunologia , Infecções Bacterianas/prevenção & controle , Comunicação Celular , Humanos , Neoplasias/imunologia , Linfócitos T/imunologia , Infecções Tumorais por Vírus/complicações , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/prevenção & controleRESUMO
The replication, or rep, gene of the human parvovirus, adeno-associated virus (AAV), is a pleiotropic effector of numerous viral functions. The rep gene trans-regulates viral DNA replication, mRNA transcription, and assembly of the infectious virion. In addition to its roles in the virus life cycle the rep gene also represses gene expression from viral or cellular transcription promoters in both transient and long-term assays. In this report we have investigated the ability of the rep gene to inhibit cellular transformation mediated by SV40 DNA or the adenovirus E1a and human ras oncogene pair. In DNA transfection assays, the complete AAV rep gene inhibited SV40 DNA and E1a/ras gene-mediated transformation of mouse fibroblasts. AAV DNA plasmids that expressed the Rep68/40 or Rep52/40 proteins alone did not suppress transformation. AAV DNA replication was not required for suppression. Due to the antiproliferative effect of the AAV rep gene, we propose that it acts a viral analogue of cellular anti-oncogenes and is a useful model system for studying the regulation of cellular proliferation.
Assuntos
Transformação Celular Viral , Vírus Defeituosos/genética , Genes ras , Proteínas Oncogênicas Virais/genética , Parvoviridae/genética , Proteínas Precoces de Adenovirus , Animais , Transformação Celular Viral/genética , Replicação do DNA , DNA Viral/fisiologia , Fibroblastos/microbiologia , Genes Virais , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/metabolismo , Vírus 40 dos Símios/genética , Transfecção , Células Tumorais CultivadasRESUMO
To study the molecular basis for the clinical phenotype of incomplete penetrance of familial retinoblastoma, we have examined the functional properties of three RB mutations identified in the germ line of five different families with low penetrance. RB mutants isolated from common adult cancers and from classic familial retinoblastoma (designated as classic RB mutations) are unstable and generally do not localize to the nucleus, do not undergo cyclin-dependent kinase (cdk)-mediated hyperphosphorylation, show absent protein "pocket" binding activity, and do not suppress colony growth of RB(-) cells. In contrast, two low-penetrant alleles (661W and "deletion of codon 480") retained the ability to localize to the nucleus, showed normal cdk-mediated hyperphosphorylation in vivo, exhibited a binding pattern to simian virus 40 large T antigen using a quantitative yeast two-hybrid assay that was intermediate between classic mutants (null) and wild-type RB, and had absent E2F1 binding in vitro. A third, low-penetrant allele, "deletion of RB exon 4," showed minimal hyperphosphorylation in vivo but demonstrated detectable E2F1 binding in vitro. In addition, each low-penetrant RB mutant retained the ability to suppress colony growth of RB(-) tumor cells. These findings suggest two categories of mutant, low-penetrant RB alleles. Class 1 alleles correspond to promoter mutations, which are believed to result in reduced or deregulated levels of wild-type RB protein, whereas class 2 alleles result in mutant proteins that retain partial activity. Characterization of the different subtypes of class 2 low-penetrant genes may help to define more precisely functional domains within the RB product required for tumor suppression.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Mutação , Penetrância , Proteína do Retinoblastoma/genética , Retinoblastoma/genética , Compartimento Celular , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Heterozigoto , Homozigoto , Humanos , Linhagem , Fosforilação , Ligação Proteica , Retinoblastoma/etiologia , Proteína do Retinoblastoma/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismoRESUMO
Previous studies have identified and characterized both murine in vivo and human in vitro T cell responses reflecting specific mutations in the ras proto-oncogenes at codon 12, 13, or 61. In an attempt to determine whether peptide epitopes reflecting point mutations in the ras oncogenes are immunogenic in humans for the production of CD4+ and/or CD8+ T cell responses, a phase I clinical trial was initiated in metastatic carcinoma patients whose primary tumors harbor mutations in the K-ras proto-oncogenes at codon 12. The peptides used here as immunogens, which were administered in Detox adjuvant, spanned the ras sequence 5-17 and reflected the amino acid substitution of glycine (Gly) at position 12 to aspartic acid (Asp), cysteine (Cys), or valine (Val). Three of eight evaluable patients have demonstrated peptide-specific cell-mediated immunity, as determined by the production of T cell lines resulting from the vaccination. First, an antigen (Ag)-specific, major histocompatibility complex (MHC) class II (DP)-restricted CD4+ T cell line was established in vitro from postvaccination lymphocytes of a non-small cell lung carcinoma patient whose primary tumor contained a Cys12 mutation when cultured on the immunizing peptide. Moreover, CD4+ proliferation was inducible against the corresponding mutant K-ras protein, suggesting productive T cell receptor recognition of exogenously processed Ag. Second, an Ag-specific, MHC class I (HLA-A2)-restricted CD8+ cytotoxic T lymphocyte (CTL) line was established in vitro from postvaccination lymphocytes of a colon carcinoma patient whose primary tumor contained an Asp12 mutation. To that end, a 10-mer peptide, nested within the 13-mer immunizing peptide, was identified [i.e., ras5-14(Asp12)], which was shown to bind to HLA-A2 and display specific functional capacity for expansion of the in vivo primed CD8+ CTL precursors. Third, both Ag-specific, MHC class II (DQ)-restricted CD4+ and MHC class I-restricted (HLA-A2) CD8+ T cell lines were generated from a single patient with duodenal carcinoma whose primary tumor contained a Val12 mutation when cultured on the immunizing 13-mer peptide or a nested 10-mer peptide [i.e., ras5-14(Val12)], respectively. Evidence is thus provided that vaccination with mutant ras oncogene peptides in adjuvant may induce specific anti-ras cellular immune responses, with no detectable cross-reactivity toward normal proto-ras sequences. Moreover, we have identified for the first time human HLA-A2-restricted, CD8+ CTL epitopes reflecting specific point mutations in the K-ras oncogenes at codon 12 which, in concert with the activation of the CD4+ T cell response, may have important implications for both active and passive immunotherapies in selected cancer patients.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Genes ras , Mutação Puntual , Adulto , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Códon/genética , Citotoxicidade Imunológica , Antígeno HLA-A2/metabolismo , Antígenos HLA-DQ/metabolismo , Humanos , Imunização , Técnicas In Vitro , Ativação Linfocitária , Masculino , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Proteínas ras/genética , Proteínas ras/imunologia , Proteínas ras/metabolismoRESUMO
Alterations of various components of the cell cycle regulatory machinery that controls the progression of cells from a quiescent to a growing state contribute to the development of many human cancers. Such alterations include the deregulated expression of G1 cyclins, the loss of function of activities such as those of protein p16INK4a that control G1 cyclin-dependent kinase activity, and the loss of function of the retinoblastoma protein (RB), which is normally regulated by the G1 cyclin-dependent kinases. Various studies have revealed an inverse relationship in the expression of p16INK4a protein and the presence of functional RB in many cell lines. In this study we show that p16INK4a is expressed in cervical cancer cell lines in which the RB gene, Rb, is not functional, either as a consequence of Rb mutation or expression of the human papillomavirus E7 protein. We also demonstrate that p16INK4a levels are increased in primary cells in which RB has been inactivated by DNA tumor virus proteins. Given the role of RB in controlling E2F transcription factor activity, we investigated the role of E2F in controlling p16INK4a expression. We found that E2F1 overexpression leads to an inhibition of cyclin D1-dependent kinase activity and induces the expression of a p16-related transcript. We conclude that the accumulation of G1 cyclin-dependent kinase activity during normal G1 progression leads to E2F accumulation through the inactivation of RB, and that this then leads to the induction of cyclin kinase inhibitor activity and a shutdown of G1 kinase activity.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Fatores de Transcrição/metabolismo , Animais , Proteínas de Transporte/metabolismo , Ciclo Celular , Linhagem Celular , Células Cultivadas , Ciclina D , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Inibidor p16 de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/antagonistas & inibidores , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Inibidores Enzimáticos , Feminino , Genes do Retinoblastoma , Células HeLa , Humanos , Queratinócitos , Masculino , Modelos Biológicos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Proteína 1 de Ligação ao Retinoblastoma , Pele/citologia , Fator de Transcrição DP1 , Neoplasias do Colo do ÚteroRESUMO
Mutations in the ras genes occur in 20% of all human cancers. These genes, in turn, produce mutated proteins that are unique to cancer cells, rendering them distinguishable from normal cells by the immune system. Thus, mutated Ras proteins may form potential targets for immune therapy. We conducted a phase I/pilot clinical trial in patients with advanced cancers to test the toxicity and the ability to induce an immune response by vaccination with 13-mer mutated Ras peptides reflecting codon 12 mutations. These peptides corresponded to each of the patient's own tumor Ras mutation. Patients were vaccinated monthly x3 subcutaneously with the specific Ras peptide along with Detox adjuvant (RiBi ImmunoChem Research, Inc., Hamilton, MT, U.S.A.) at one of five different peptide dose levels (100, 500, 1,000, 1,500, and 5,000 micrograms). Three out of 10 evaluable patients generated a mutant Ras specific CD4+ and/or CD8+ T-cell immune response. The CD8+ cytotoxic cells specific for Gly to Val mutation at codon 12 were capable of lysing an HLA-A2-matched tumor cell line carrying the corresponding mutant but not the wild-type ras gene. The treatment has been well tolerated with no evidence of serious acute or delayed systemic side effects on any of the five dose levels. We demonstrated that we can generate in cancer patients specific T-lymphocyte responses that detect single amino acid differences in Ras oncoproteins. Neither the immune responses nor the minor side effects seen were found to be dose dependent. This approach may provide a unique opportunity for generating a tumor-directed therapy. Also, in vitro stimulation of these cells with the corresponding peptide generated specific T-cell lines that could be used for adoptive immune therapy.
Assuntos
Vacinas Anticâncer/imunologia , Neoplasias/terapia , Proteínas ras/imunologia , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Ativação Linfocitária , Pessoa de Meia-Idade , Mutação , Neoplasias/imunologia , VacinaçãoRESUMO
We have previously reported the direct physical interaction between the human immunodeficiency virus (HIV) type I Tat protein and the basal transcription factor TBP/TFIID. Affinity chromatography demonstrated that wild-type Tat, but not a transactivation mutant of Tat, was capable of depleting TBP/TFIID from cell extracts. These experiments represented the first demonstration of a basal transcription factor that binds, in an activation-dependent manner, to Tat. We now report that the Tat-TBP interaction can be detected in HIV type 1-infected cells. The domain of TBP interacting with Tat has been mapped from amino acids 163 to 196 by using deletion and site-specific mutants of TBP. This domain of TBP, which includes the HI and S2 domains, is distinct from the H2 binding site for other activator proteins, such as E1A. The interaction of Tat with TFIID regulates the binding of accessory proteins to TFIID. Tat stabilizes the interaction of TFIID with TFIIA in a gel shift assay. In addition, Tat competes for Dr1 interaction with TBP. Our results suggest that the basal transcription factor TBP/TFIID represents an important regulatory molecule in HIV transcription.