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Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer due to its highly metastatic nature. Melanomas harboring oncogenic BRAFV600E mutations combined with PTEN loss exhibit unrestrained PI3K/AKT signaling and increased invasiveness. However, the contribution of different AKT isoforms to melanoma initiation, progression, and metastasis has not been comprehensively explored, and questions remain about whether individual isoforms play distinct or redundant roles in each step. We investigate the contribution of individual AKT isoforms to melanoma initiation using a novel mouse model of AKT isoform-specific loss in a murine melanoma model, and we investigate tumor progression, maintenance, and metastasis among a panel of human metastatic melanoma cell lines using AKT isoform-specific knockdown studies. We elucidate that AKT2 is dispensable for primary tumor formation but promotes migration and invasion in vitro and metastatic seeding in vivo, whereas AKT1 is uniquely important for melanoma initiation and cell proliferation. We propose a mechanism whereby the inhibition of AKT2 impairs glycolysis and reduces an EMT-related gene expression signature in PTEN-null BRAF-mutant human melanoma cells to limit metastatic spread. Our data suggest that the elucidation of AKT2-specific functions in metastasis might inform therapeutic strategies to improve treatment options for melanoma patients.
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Canine hemangiosarcoma (HSA) is an aggressive cancer of endothelial cells with short survival times. Understanding the genomic landscape of HSA may aid in developing therapeutic strategies for dogs and may also inform therapies for the rare and aggressive human cancer angiosarcoma. The objectives of this study were to build a framework for leveraging real-world genomic and clinical data that could provide the foundation for precision medicine in veterinary oncology, and to determine the relationships between genomic and clinical features in canine splenic HSA. One hundred and nine dogs with primary splenic HSA treated by splenectomy that had tumour sequencing via the FidoCure® Precision Medicine Platform targeted sequencing panel were enrolled. Patient signalment, weight, metastasis at diagnosis and overall survival time were retrospectively evaluated. The incidence of genomic alterations in individual genes and their relationship to patient variables including outcome were assessed. Somatic mutations in TP53 (n = 44), NRAS (n = 20) and PIK3CA (n = 19) were most common. Survival was associated with presence of metastases at diagnosis and germline variants in SETD2 and NOTCH1. Age at diagnosis was associated with somatic NRAS mutations and breed. TP53 and PIK3CA somatic mutations were found in larger dogs, while germline SETD2 variants were found in smaller dogs. We identified both somatic mutations and germline variants associated with clinical variables including age, breed and overall survival. These genetic changes may be useful prognostic factors and provide insight into the genomic landscape of hemangiosarcoma.
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Enfermedades de los Perros , Hemangiosarcoma , Neoplasias del Bazo , Humanos , Perros , Animales , Hemangiosarcoma/genética , Hemangiosarcoma/veterinaria , Hemangiosarcoma/tratamiento farmacológico , Células Endoteliales , Estudios Retrospectivos , Enfermedades de los Perros/genética , Enfermedades de los Perros/tratamiento farmacológico , Neoplasias del Bazo/genética , Neoplasias del Bazo/veterinaria , Neoplasias del Bazo/tratamiento farmacológico , Genómica , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/uso terapéuticoRESUMEN
Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer, due to its highly metastatic nature. Melanomas harboring oncogenic BRAF V600E mutations combined with PTEN loss exhibit unrestrained PI3K/AKT signaling and increased invasiveness. However, the contribution of different AKT isoforms to melanoma initiation, progression, and metastasis has not been comprehensively explored, and questions remain whether individual isoforms play distinct or redundant roles in each step. We investigate the contribution of individual AKT isoforms to melanoma initiation using a novel mouse model of AKT isoform-specific loss in a murine melanoma model, and investigate tumor progression, maintenance, and metastasis among a panel of human metastatic melanoma cell lines using AKT-isoform specific knockdown studies. We elucidate that AKT2 is dispensable for primary tumor formation but promotes migration and invasion in vitro and metastatic seeding in vivo , while AKT1 is uniquely important for melanoma initiation and cell proliferation. We propose a mechanism whereby inhibition of AKT2 impairs glycolysis and reduces an EMT-related gene expression signature in PTEN-null BRAF-mutant human melanoma cells to limit metastatic spread. Our data suggest that elucidation of AKT2-specific functions in metastasis could inform therapeutic strategies to improve treatment options for melanoma patients.
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Cellular senescence is a carefully regulated process of proliferative arrest accompanied by functional and morphologic changes. Senescence allows damaged cells to avoid neoplastic proliferation; however, the induction of the senescence-associated secretory phenotype (SASP) can promote tumor growth. The complexity of senescence may limit the efficacy of anti-neoplastic agents, such as CDK4/6 inhibitors (Cdk4/6i), that induce a senescence-like state in tumor cells. The AKT kinase family, which contains three isoforms that play both unique and redundant roles in cancer progression, is commonly hyperactive in many cancers including melanoma and has been implicated in the regulation of senescence. To interrogate the role of AKT isoforms in Cdk4/6i-induced cellular senescence, we generated isoform-specific AKT knockout human melanoma cell lines. We found that the CDK4/6i Palbociclib induced a form of senescence in these cells that was dependent on AKT1. We then evaluated the activity of the cGAS-STING pathway, recently implicated in cellular senescence, finding that cGAS-STING function was dependent on AKT1, and pharmacologic inhibition of cGAS had little effect on senescence. However, we found SASP factors to require NF-κB function, in part dependent on a stimulatory phosphorylation of IKKα by AKT1. In summary, we provide the first evidence of a novel, isoform-specific role for AKT1 in therapy-induced senescence in human melanoma cells acting through NF-κB but independent of cGAS.
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PURPOSE: HER2 signaling functional activity may be important to measure in addition to HER2 protein quantification when identifying patients eligible for HER2 therapies. A HER2 Signaling Function (CELx HSF) Test for HER2-negative patients uses patient's live tumor cells on a biosensor to identify patients with abnormally high HER2-related signaling (HSFs+) likely to respond to anti-HER2 therapies. METHODS: The CELx HSF test was employed to: (1) characterize the sensitivity and specificity of the test to detect abnormal levels of HER2 signaling; (2) evaluate the inhibitory effectiveness of five different anti-HER2 therapies; (3) assess the correlation between CELx HSF test detection of abnormal HER2 signaling and response to HER2 therapy using xenograft models; and (4) confirm the prevalence of abnormal HER2 signaling amongst HER2-negative breast cancer patients (HER2-/HSFs+). RESULTS: HER2-/HSFs+ breast cancer patient samples were identified and showed sensitivity to five approved anti-HER2 therapies. Xenograft studies using both HER2+ and HER2- cell lines confirmed that CELx HER2 signaling status better predicts HER2 inhibitor efficacy than HER2 receptor status. In a study of 114 HER2-negative breast tumor patient samples, 27 (23.7%; 95% CI = 17-32%) had abnormal HER2 signaling (HSFs+). A ROC curve constructed with this dataset projects the CELx HSF Test would have greater than 90% sensitivity and specificity to detect the HER2-/HSFs+ patient population. CONCLUSIONS: The CELx HSF test is a well-characterized functional biomarker assay capable of identifying dynamic HER2-driven signaling dysfunction in tumor cells from HER2-negative breast cancer patients. This test has demonstrated efficacy of various HER2 targeted therapies in live tumor cells from the HSFs+ population and correlated the test result to HER2 drug response in mouse xenograft studies. The proportion of HER2-negative breast cancer patients found to have abnormal HER2 signaling in a 114 patient sample study, 20-25%, is significant. A clinical trial to evaluate the efficacy of anti-HER2 therapies in this patient population is warranted.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal/fisiología , Animales , Antineoplásicos/farmacología , Impedancia Eléctrica , Femenino , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) are well known for their capacity to drive necroptosis via mixed-lineage kinase-like domain (MLKL). Recently, RIPK1/3 kinase activity has been shown to drive inflammation via activation of MAPK signaling. However, the regulatory mechanisms underlying this kinase-dependent cytokine production remain poorly understood. In the present study, we establish that the kinase activity of RIPK1/3 regulates cytokine translation in mouse and human macrophages. Furthermore, we show that this inflammatory response is downregulated by type I interferon (IFN) signaling, independent of type I IFN-promoted cell death. Specifically, low-level constitutive IFN signaling attenuates RIPK-driven activation of cap-dependent translation initiation pathway components AKT, mTORC1, 4E-BP and eIF4E, while promoting RIPK-dependent cell death. Altogether, these data characterize constitutive IFN signaling as a regulator of RIPK-dependent inflammation and establish cap-dependent translation as a crucial checkpoint in the regulation of cytokine production.
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Citocinas/metabolismo , Interferones/metabolismo , Biosíntesis de Proteínas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Citocinas/genética , Regulación hacia Abajo , Factor 4E Eucariótico de Iniciación/metabolismo , Femenino , Humanos , Inflamación/patología , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Cellular senescence has emerged as a potent tumor suppression mechanism that restrains proliferation of cells at risk for malignant transformation. Although senescent cells have permanently exited the cell cycle, their presence can have detrimental effects on the surrounding tissue, largely due to the development of the senescence-associated secretory phenotype (SASP). Here, we review the tumor-suppressive and tumor-promoting consequences of the senescence response, focusing on the SASP as a key mediator of this dichotomy. Accumulating evidence suggests that the persistence of senescent cells can exacerbate the development of a pro-inflammatory, immunosuppressive microenvironment that can favor tumorigenesis. Given that senescence of tumor and stromal cells is a frequent outcome of anti-cancer therapy, approaches that harness the growth inhibitory effects of senescence while limiting its detrimental effects are likely to have great clinical potential.
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Diferenciación Celular , Melanocitos/metabolismo , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Células de Schwann/metabolismo , Animales , Linaje de la Célula , Cruzamientos Genéticos , Humanos , Antígeno Ki-67/metabolismo , Melaninas/metabolismo , Melanocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Cresta Neural/metabolismo , Fenotipo , PigmentaciónRESUMEN
Akt1, a serine-threonine protein kinase member of the PKB/Akt gene family, plays critical roles in the regulation of multiple cellular processes, and has previously been implicated in lactation and breast cancer development. In this study, we utilized Akt1+/+ and Akt1-/- C57/Bl6 female mice to assess the role that Akt1 plays in normal mammary gland postnatal development and function. We examined postnatal morphology at multiple time points, and analyzed gene and protein expression changes that persist into adulthood. Akt1 deficiency resulted in several mammary gland developmental defects, including ductal outgrowth and defective terminal end bud formation. Adult Akt1-/- mammary gland composition remained altered, exhibiting fewer alveolar buds coupled with increased epithelial cell apoptosis. Microarray analysis revealed that Akt1 deficiency altered expression of genes involved in numerous biological processes in the mammary gland, including organismal development, cell death, and tissue morphology. Of particular importance, a significant decrease in expression of Btn1a1, a gene involved in milk lipid secretion, was observed in Akt1-/- mammary glands. Additionally, pseudopregnant Akt1-/- females failed to induce Btn1a1 expression in response to hormonal stimulation compared to their wild-type counterparts. Retroviral-mediated shRNA knockdown of Akt1 and Btn1a1 in MCF-7 human breast epithelial further illustrated the importance of Akt1 in mammary epithelial cell proliferation, as well as in the regulation of Btn1a1 and subsequent expression of ß-casein, a gene that encodes for milk protein. Overall these findings provide mechanistic insight into the role of Akt1 in mammary morphogenesis and function.
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Glándulas Mamarias Animales/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Western Blotting , Butirofilinas , Caseínas/genética , Caseínas/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Etiquetado Corte-Fin in Situ , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-akt/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Micron-sized particles of poorly soluble nickel compounds, but not metallic nickel, are established human and rodent carcinogens. In contrast, little is known about the toxic effects of a growing number of Ni-containing materials in the nano-sized range. Here, we performed physicochemical characterization of NiO and metallic Ni nanoparticles and examined their metal ion bioavailability and toxicological properties in human lung epithelial cells. Cellular uptake of metallic Ni and NiO nanoparticles, but not metallic Ni microparticles, was associated with the release of Ni(II) ions after 24-48 h as determined by Newport Green fluorescence. Similar to soluble NiCl2, NiO nanoparticles induced stabilization and nuclear translocation of hypoxia-inducible factor 1α (HIF-1α) transcription factor followed by upregulation of its target NRDG1 (Cap43). In contrast to no response to metallic Ni microparticles, nickel nanoparticles caused a rapid and prolonged activation of the HIF-1α pathway that was stronger than that induced by soluble Ni(II). Soluble NiCl2 and NiO nanoparticles were equally toxic to H460 human lung epithelial cells and primary human bronchial epithelial cells; metallic Ni nanoparticles showed lower toxicity and Ni microparticles were nontoxic. Cytotoxicity induced by all forms of Ni occurred concomitant with activation of an apoptotic response, as determined by dose- and time-dependent cleavage of caspases and poly (ADP-ribose) polymerase. Our results show that metallic Ni nanoparticles, in contrast to micron-sized Ni particles, activate a toxicity pathway characteristic of carcinogenic Ni compounds. Moderate cytotoxicity and sustained activation of the HIF-1α pathway by metallic Ni nanoparticles could promote cell transformation and tumor progression.
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Células Epiteliales/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/efectos de los fármacos , Nanopartículas/administración & dosificación , Níquel/farmacocinética , Níquel/toxicidad , Apoptosis/efectos de los fármacos , Disponibilidad Biológica , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Humanos , Pulmón/citología , Pulmón/metabolismo , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Nanopartículas/química , Níquel/administración & dosificación , Níquel/química , Tamaño de la PartículaRESUMEN
BACKGROUND: Asbestos induces DNA and chromosomal damage, but the DNA repair pathways protecting human cells against its genotoxicity are largely unknown. Polymorphisms in XRCC1 have been associated with altered susceptibility to asbestos-related diseases. However, it is unclear whether oxidative DNA damage repaired by XRCC1 contributes to asbestos-induced chromosomal damage. OBJECTIVES: We sought to examine the importance of XRCC1 in protection against genotoxic effects of crocidolite and Libby amphibole asbestos. METHODS: We developed a genetic model of XRCC1 deficiency in human lung epithelial H460 cells and evaluated genotoxic responses to carcinogenic fibers (crocidolite asbestos, Libby amphibole) and nongenotoxic materials (wollastonite, titanium dioxide). RESULTS: XRCC1 knockdown sensitized cells to the clastogenic and cytotoxic effects of oxidants [hydrogen peroxide (H2O2), bleomycin] but not to the nonoxidant paclitaxel. XRCC1 knockdown strongly enhanced genotoxicity of amphibole fibers as evidenced by elevated formation of clastogenic micronuclei. Crocidolite induced primarily clastogenic micronuclei, whereas Libby amphibole induced both clastogenic and aneugenic micronuclei. Crocidolite and bleomycin were potent inducers of nuclear buds, which were enhanced by XRCC1 deficiency. Libby amphibole and H2O2 did not induce nuclear buds, irrespective of XRCC1 status. Crocidolite and Libby amphibole similarly activated the p53 pathway. CONCLUSIONS: Oxidative DNA damage repaired by XRCC1 (oxidized bases, single-strand breaks) is a major cause of chromosomal breaks induced by crocidolite and Libby amphibole. Nuclear buds are a novel biomarker of genetic damage induced by exposure to crocidolite asbestos, which we suggest are associated with clustered DNA damage. These results provide mechanistic evidence for the epidemiological association between XRCC1 polymorphisms and susceptibility to asbestos-related disease.
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Asbestos Anfíboles/toxicidad , Proteínas de Unión al ADN/deficiencia , Pulmón/efectos de los fármacos , Mutágenos/toxicidad , Mucosa Respiratoria/efectos de los fármacos , Asbesto Crocidolita/toxicidad , Carcinógenos/toxicidad , Línea Celular , Daño del ADN , Reparación del ADN , Humanos , Pulmón/metabolismo , Pruebas de Mutagenicidad , Mucosa Respiratoria/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
Mammalian females are endowed with a finite number of primordial follicles at birth. Immediately following formation of the primordial follicle pool, cohorts of follicles are either culled from the ovary or are recruited to grow until the primordial follicle population is depleted. The majority of ovarian follicles, including the oocytes, undergo atresia through apoptotic cell death. As PKB alpha/Akt1 is known to regulate apoptosis, we asked whether Akt1 functioned in the regulation of folliculogenesis in the ovary. Akt1(-/-) females display reduced fertility and abnormal estrous cyclicity. At Postnatal Day (PND) 25, Akt1(-/-) ovaries possessed a reduced number of growing antral follicles, significantly larger primary and secondary oocytes, and an increase in the number of degenerate oocytes. By PND90, there was a significant decrease in the number of primordial follicles in Akt1(-/-) ovaries relative to Akt1(+/+). In vivo granulosa cell proliferation was reduced, as were expression levels of Kitl and Bcl2l1, two factors associated with granulosa cell proliferation/survival. No compensation was observed by Akt2 or Akt3 at the mRNA/protein level. Significantly higher serum LH and trends for lower FSH and higher inhibin A and lower inhibin B relative to Akt1(+/+) females were observed in Akt1(-/-) females. Exposure to exogenous gonadotropins resulted in an increase in the number of secondary follicles in Akt1(-/-) ovaries, but few mature follicles. Collectively, our results suggest that PKB alpha/Akt1 plays an instrumental role in the regulation of the growth and maturation of the ovary, and that the loss of PKB alpha/Akt1 results in premature ovarian failure.
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Infertilidad Femenina/etiología , Oocitos/crecimiento & desarrollo , Folículo Ovárico/crecimiento & desarrollo , Proteínas Proto-Oncogénicas c-akt/deficiencia , Animales , Peso Corporal , Cruzamiento , Ciclina D/análisis , Ciclina D/genética , Estradiol/sangre , Ciclo Estral , Femenino , Masculino , Ratones , Ratones Noqueados , Oocitos/citología , Tamaño de los Órganos , Folículo Ovárico/química , Ovario/química , Ovario/metabolismo , Ovario/patología , Progesterona/sangre , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/fisiología , ARN Mensajero/análisis , Maduración Sexual/fisiología , Esteroides/biosíntesisRESUMEN
Human diseases associated with exposure to asbestos fibers include pleural fibrosis and plaques, pulmonary fibrosis (asbestosis), lung cancer, and diffuse malignant mesothelioma. The critical determinants of fiber bioactivity and toxicity include not only fiber dimensions, but also shape, surface reactivity, crystallinity, chemical composition, and presence of transition metals. Depending on their size and dimensions, inhaled fibers can penetrate the respiratory tract to the distal airways and into the alveolar spaces. Fibers can be cleared by several mechanisms, including the mucociliary escalator, engulfment, and removal by macrophages, or through splitting and chemical modification. Biopersistence of long asbestos fibers can lead to inflammation, granuloma formation, fibrosis, and cancer. Exposure to synthetic carbon nanomaterials, including carbon nanofibers and carbon nanotubes (CNTs), is considered a potential health hazard because of their physical similarities with asbestos fibers. Respiratory exposure to CNTs can produce an inflammatory response, diffuse interstitial fibrosis, and formation of fibrotic granulomas similar to that observed in asbestos-exposed animals and humans. Given the known cytotoxic and carcinogenic properties of asbestos fibers, toxicity of fibrous nanomaterials is a topic of intense study. The mechanisms of nanomaterial toxicity remain to be fully elucidated, but recent evidence suggests points of similarity with asbestos fibers, including a role for generation of reactive oxygen species, oxidative stress, and genotoxicity. Considering the rapid increase in production and use of fibrous nanomaterials, it is imperative to gain a thorough understanding of their biologic activity to avoid the human health catastrophe that has resulted from widespread use of asbestos fibers.
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Nanoestructuras/envenenamiento , Animales , Amianto/farmacocinética , Amianto/envenenamiento , Amianto/toxicidad , Humanos , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/metabolismo , Ratones , Nanoestructuras/toxicidad , Nanoestructuras/ultraestructuraRESUMEN
SV40 virus has emerged as a potential cofactor with asbestos in the development of diffuse malignant mesothelioma, but its precise role in the pathogenesis of this tumor is unclear. SV40 large T antigen is known to inactivate cellular proteins involved in DNA damage and senescence, including p53 and pRb. We hypothesize that SV40 oncoproteins will sensitize mesothelial cells to DNA damage induced by asbestos or chemotherapeutic agents. SV40 oncoprotein expression in murine mesothelial cell lines enhanced spontaneous and asbestos-induced double-strand breaks, indicated by gamma-H2AX foci, and potentiated micronucleus formation. Mesothelial cells exposed to asbestos or bleomycin for 96 h acquired senescent-like morphology and displayed elevated senescence-associated beta-galactosidase activity, reduced bromodeoxyuridine (BrdUrd) incorporation, and reduced colony formation. SV40 oncoprotein expression abrogated the senescent phenotype, and transfected cell lines showed an increase in both BrdUrd incorporation and colony formation after prolonged DNA damage. Murine mesothelial cell lines lacking wild-type p53 due to a point mutation or gene rearrangement also failed to senesce in response to asbestos or chemotherapeutic agents. In addition, stress-induced senescence in human mesothelial cell lines was impaired by SV40 oncoprotein expression (MeT-5A), p53 small interfering RNA, or spontaneous p53 mutation (REN). These studies suggest that exposure to DNA-damaging agents can induce senescence in both murine and human mesothelioma cell lines and suggest a major, although not exclusive, role for p53 in this response. SV40 virus may contribute to mesothelioma progression by impairing stress-induced senescence, in part through p53 inactivation, thereby favoring survival and proliferation of mesothelial cells that have sustained DNA damage.