RESUMO
Activating mutations of RAS are thought to be early events in the evolution of thyroid follicular neoplasms. We used a doxycycline-inducible expression system to explore the acute effects of H-RAS12 on genomic stability in thyroid PCCL3 cells. At 2-3 days (first or second cell cycle) there was a significant increase in the frequency of micronucleation. Treatment of cells with YVAD-CHO inhibited RAS-induced apoptosis, but had no effect on micronucleation. The effects of H-RAS(V12) were mediated by activation of MAPK, as treatment with PD98059 at concentrations verified to selectively inhibit MEK1 reduced the frequency of prevalence of cells with micronuclei. In addition, doxycycline-inducible expression of a constitutively active MEK1, but not of a mutant RAC1, mimicked the effects of H-RAS(V12). The effects of H-RAS(V12) on genome destabilization were apparent even though the sequence of p53 in PCCL3 cells was confirmed to be wild-type. Acute activation of H-RAS(V12) evoked a proportional increase in both CREST negative and CREST positive micronuclei, indicating that both clastogenic and aneugenic effects were involved. H-RAS(V12) and activated MEK1 also induced centrosome amplification, and chromosome misalignment. Evidence that acute expression of constitutively activated RAS destabilizes the genome of PCCL3 cells is consistent with a mode of tumor initiation in which this oncogene promotes phenotypic progression by predisposing to large scale genomic abnormalities.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas de Saccharomyces cerevisiae , Neoplasias da Glândula Tireoide/genética , Proteínas ras/genética , Animais , Apoptose/efeitos da radiação , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Doxiciclina/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Raios gama , Regulação Neoplásica da Expressão Gênica , MAP Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mutação , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2 , Proteína Fosfatase 2C , Proteínas Serina-Treonina Quinases/genética , Ratos , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/metabolismo , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Mutations of ras are tumor-initiating events for many cell types, including thyrocytes. To explore early consequences after oncogenic Ras activation, we developed a doxycycline-inducible expression system in rat thyroid PCCL3 cells. Beginning 3-4 days after H-Ras(v12) expression, cells underwent apoptosis. The H-Ras(v12) effects on apoptosis were decreased by a mitogen-activated protein kinase kinase (MEK1) inhibitor and recapitulated by doxycycline-inducible expression of an activated MEK1 mutant (MEK1(S217E/S221E)). As reported elsewhere, acute expression of H-Ras(v12) also induces mitotic defects in PCCL3 cells through ERK (extracellular ligand-regulated kinase) activation, suggesting that apoptosis may be secondary to DNA damage. However, acute activation of SAPK/JNK (stress-activated protein kinase/Jun N-terminal kinase) through acute expression of Rac1(v12) also triggered apoptosis, without inducing large-scale genomic abnormalities. H-Ras(v12)-induced apoptosis was dependent on concomitant activation of cAMP by either TSH or forskolin, in a protein kinase A-independent manner. Thus, coactivation of cAMP-dependent pathways and ERK or JNK (either through H-Ras(v12), Rac1(v12), or MEK1(S217E/S221E)) is inconsistent with cell survival. The fate of thyrocytes within the first cell cycles after expression of oncogenic Ras is dependent on ambient TSH levels. If both cAMP and Ras signaling are simultaneously activated, most cells will die. Those that survive will eventually lose TSH responsiveness and/or inactivate the apoptotic cascade through secondary events, thus enabling clonal expansion.
Assuntos
Apoptose/genética , Genes ras , Transdução de Sinais , Glândula Tireoide/citologia , Glândula Tireoide/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , AMP Cíclico/metabolismo , Doxiciclina/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , MAP Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Ratos , Glândula Tireoide/efeitos dos fármacos , Tireotropina/metabolismo , Tireotropina/farmacologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
OBJECTIVES/HYPOTHESIS: To determine if metastatic squamous cell carcinoma with proliferative potential persists in N2 and N3 necks after conventional radiation. STUDY DESIGN: Retrospective case series. MATERIALS AND METHODS: We identified 17 patients from our head and neck tumor database who underwent organ-preserving radiotherapy for primary aerodigestive squamous cell cancer and N2-3 regional metastasis. Archival tissue from these 17 neck specimens was evaluated for routine histopathologic evidence of tumor, as well as immunohistochemically for cytokeratin and Ki-67 activity. An assay for apoptosis was also performed on 10 of the specimens. RESULTS: Routine H&E evaluation suggested metastatic cancer in 11 of 17 irradiated neck specimens. Cytokeratin immunostaining confirmed squamous cell carcinoma in these 11 necks as well as 1 additional specimen that had tested H&E negative. Ki-67 staining demonstrated proliferating tumor in 3 of 17 necks. The apoptosis assay confirmed regions of apoptosis in all of the specimens analyzed. CONCLUSIONS: The discovery of proliferating cancer cells in 3 of 17 irradiated specimens (18%) supports the practice of planned neck dissection after primary radiotherapy for patients with pretherapeutic N2+ metastatic disease.
Assuntos
Carcinoma de Células Escamosas/cirurgia , Neoplasias Hipofaríngeas/cirurgia , Excisão de Linfonodo , Neoplasias Orofaríngeas/cirurgia , Apoptose , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/secundário , Divisão Celular , Histocitoquímica , Humanos , Neoplasias Hipofaríngeas/patologia , Neoplasias Hipofaríngeas/radioterapia , Marcação In Situ das Extremidades Cortadas , Neoplasias Orofaríngeas/patologia , Neoplasias Orofaríngeas/radioterapia , Estudos RetrospectivosRESUMO
OBJECTIVE: To determine the safety and efficacy of an immunogenic gene therapy using a drug designed to produce expression of a foreign class I major histocompatibility complex protein in patients with head and neck cancer. DESIGN: Phase 1 prospective clinical trial. SETTING: Academic medical setting. PATIENTS: Nine patients with advanced head and neck squamous cell carcinoma who had failed conventional therapy and did not express HLA-B7, a class I major histocompatibility complex protein. INTERVENTION: Patients were treated with Allovectin-7 (Vical Inc, San Diego, Calif) by direct intratumoral injection. Allovectin-7 contains a plasmid complementary DNA complexed with a cationic lipid, which results in expression of HLA-B7. MAIN OUTCOME MEASURES: Patients were assessed for any toxic effects and for any change in tumor volume. Biopsy specimens obtained before and after therapy were evaluated by immunohistochemistry to detect HLA-B7 expression and with the terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay to detect any induction of apoptosis. RESULTS: There were no toxic effects of the gene therapy. In 4 of these 9 patients there was a partial response to treatment, evidenced by a gradual reduction in tumor size. One patient has remained alive for more than 17 months since commencing treatment, with no clinical evidence of disease but with persistent histological evidence of cancer. Analysis of the biopsy specimens from 2 of the patients who responded to therapy demonstrated HLA-B7 expression. The TUNEL assay demonstrated extensive apoptosis in both of these patients, suggesting that this may be the mechanism of tumor reduction. CONCLUSIONS: These data demonstrate the potential efficacy and lack of toxicity of this form of alloantigen gene therapy. A multi-institutional study has been initiated to expand on these findings.
Assuntos
Carcinoma de Células Escamosas/terapia , DNA , Terapia Genética/métodos , Antígeno HLA-B7/uso terapêutico , Neoplasias de Cabeça e Pescoço/terapia , Lipídeos/uso terapêutico , Plasmídeos/uso terapêutico , Adulto , Idoso , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/mortalidade , Terapia Combinada , DNA Recombinante , Feminino , Terapia Genética/efeitos adversos , Antígeno HLA-B7/efeitos adversos , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/mortalidade , Teste de Histocompatibilidade , Humanos , Imuno-Histoquímica , Lipídeos/efeitos adversos , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Plasmídeos/efeitos adversos , Estudos ProspectivosRESUMO
Centrosome amplification (CA) contributes to carcinogenesis by generating aneuploidy. Elevated frequencies of CA in most benign breast lesions and primary tumors suggest a causative role for CA in breast cancers. Clearly, identifying which and how altered signal transduction pathways contribute to CA is crucial to breast cancer control. Although a causative and cooperative role for c-Myc and Ras in mammary tumorigenesis is well documented, their ability to generate CA during mammary tumor initiation remains unexplored. To answer that question, K-Ras(G12D) and c-Myc were induced in mouse mammary glands. Although CA was observed in mammary tumors initiated by c-Myc or K-Ras(G12D), it was detected only in premalignant mammary lesions expressing K-Ras(G12D). CA, both in vivo and in vitro, was associated with increased expression of the centrosome-regulatory proteins, cyclin D1 and Nek2. Abolishing the expression of cyclin D1, Cdk4 or Nek2 in MCF10A human mammary epithelial cells expressing H-Ras(G12V) abrogated Ras-induced CA, whereas silencing cyclin E1 or B2 had no effect. Thus, we conclude that CA precedes mammary tumorigenesis, and interfering with centrosome-regulatory targets suppresses CA.
Assuntos
Centrossomo/metabolismo , Ciclina D1/fisiologia , Quinase 4 Dependente de Ciclina/fisiologia , Genes ras/fisiologia , Glândulas Mamárias Animais/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Apoptose/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Centrossomo/patologia , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Doença da Mama Fibrocística/genética , Doença da Mama Fibrocística/metabolismo , Genes ras/genética , Humanos , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Quinases Relacionadas a NIMA , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The generation of micronuclei is a reflection of DNA damage, defective mitosis, and loss of genetic material. The involvement of the MAPK pathway in mediating v-ras-induced micronuclei in NIH 3T3 cells was examined by inhibiting MAPK activation. Conversely, the MAPK pathway was constitutively activated by infecting cells with a v-mos retrovirus. Micronucleus formation was inhibited by the MAPK kinase inhibitors PD98059 and U0126, but not by wortmannin, an inhibitor of the Ras/phosphatidylinositol 3-kinase pathway. Transduction of cells with v-mos resulted in an increase in micronucleus formation, also consistent with the involvement of the MAPK pathway. Staining with the anti-centromeric CREST antibody revealed that instability induced by constitutive activation of MAPK is due predominantly to aberrant mitotic segregation, since most of the micronuclei were CREST-positive, reflective of lost chromosomes. A significant fraction of the micronuclei were CREST-negative, reflective of lost acentric chromosome fragments. Some of the instability observed was due to mitotic events, consistent with the increased formation of bi-nucleated cells, which result from perturbations of the mitotic spindle and failure to undergo cytokinesis. This chromosome instability, therefore, is a consequence of mitotic aberrations, mediated by the MAPK pathway, including centrosome amplification and formation of mitotic chromosome bridges.
Assuntos
Deleção Cromossômica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Oncogênica p21(ras)/fisiologia , Células 3T3 , Animais , Transformação Celular Viral , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Vírus da Leucemia Murina/fisiologia , Sistema de Sinalização das MAP Quinases , Camundongos , Micronúcleos com Defeito Cromossômico , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Mitose/genética , Proteínas Oncogênicas v-mos/genética , FosforilaçãoRESUMO
Interleukin-3 (Il-3) is a glycoprotein produced by a CD4+CD8- subpopulation of T-lymphocytes. Il-3 has been associated with the proliferation of bone marrow stem cells and their differentiation to granulocytes, macrophages, basophil/mast cells, megakaryocytes, erythroid cells, and neutrophils. The pBOR-Il-3 transgenic mice were developed by pronuclear microinjection to study how chemical insults modulate transcription of the Il-3 gene driven by a long-terminal repeat (LTR) of an endogenous retrovirus and to determine the biological consequences of interleukin-3 expression. We injected 5-azacytidine, a demethylating agent, to increase the LTR-driven expression of Il-3. Upon 5-azacytidine treatment, both the pBOR-Il-3 and the FVB/N nontransgenic controls developed thymic lymphomas. The pBOR-Il-3 mice developed thymic lymphomas at a higher frequency than the FVB/N mice. The thymic lymphoma cells were of a T-cell origin, as determined by T-cell receptor gene rearrangement analysis, and, in most cases, were of monoclonal origin. According to flow cytometric analysis of CD3, CD4, and CD8 cell surface markers, the thymic lymphoma cells did not lose their ability to differentiate, but the differentiation process was aberrant. Flow cytometric analyses also revealed that in pBOR-Il-3 mice the thymic lymphomas are mostly of a CD8+CD4- origin, whereas in the FVB/N group, the predominant type of thymic lymphoma is of a CD4+CD8- origin.
Assuntos
Interleucina-3/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Neoplasias do Timo/imunologia , Animais , Azacitidina/farmacologia , Complexo CD3/imunologia , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Carcinógenos/farmacologia , Modelos Animais de Doenças , Feminino , Rearranjo Gênico do Linfócito T , Incidência , Interleucina-3/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Neoplasias do Timo/genéticaRESUMO
Previous work has shown that the Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, we now show that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, we propose that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fase S/fisiologia , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F3 , Fator de Transcrição E2F4 , Fibroblastos/fisiologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities. E2F1, E2F2 and E2F3 belong to a subclass of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.