RESUMEN
Mutations in the p53 tumor suppressor gene are implicated in defective apoptotic response of tumors to genotoxic damage and, thus, are major determinants of resistance to a variety of anticancer agents. Because even melanomas harboring wild-type (wt) p53 show an abnormal response to radiation and p53 mutations occur late during melanoma progression, we investigated whether the effect of the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 is dependent on the p53 status in human C8161 melanoma cells. Upon treatment with oligonucleotide 4625, p53-mut C8161 cells showed earlier DNA damage, which occurred concomitantly with the reduction of bcl-2 and bcl-xL expression and the increase in the expression of proapoptotic bax. Loss of cell viability, bcl-2 down-regulation, and poly(ADP-ribose) polymerase cleavage, indicative of apoptosis, also occurred in wt p53 C8161 cells on treatment with oligonucleotide 4625. These effects, however, were mediated by strong induction of p53 without changes in p21 WAF1 expression in wt p53 cells, whereas a 70% decrease in p21 WAF1 expression was observed in mut p53 cells. In contrast to many other anticancer agents to which the apoptotic response is decreased because of p53 mutations, our data suggest that the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 effectively induces p53-independent apoptosis in human C8161 melanoma cells.
Asunto(s)
Apoptosis , Melanoma/patología , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/fisiología , División Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/efectos de los fármacos , Ciclinas/metabolismo , Fragmentación del ADN/efectos de los fármacos , Regulación hacia Abajo , Humanos , Mutación , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas , Proteínas/efectos de los fármacos , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína bcl-XRESUMEN
Metastatic tumors grow under conditions that restrict proliferation of non-metastatic, more differentiated cells. To investigate this prediction, we developed a simple adhesion-restrictive assay which allows proliferation of human metastatic C8161 melanoma, but prevents growth of neo 6.3/C8161 cells in which metastasis is suppressed by introduction of neo-tagged chromosome 6. We show that tyrosinase, a key enzyme in melanocytic cell differentiation, and expression of chromosome 6-encoded cell cycle modulators like p21WAF1 and cyclin D3 is selectively increased in C8161 tumors in which metastasisis is suppressed by chromosome 6. In the latter cells, growth arrest evidenced only under adhesion-restrictive conditions correlated with down-regulation of cyclin D3 and anti-apoptotic bcl-2. No comparable growth arrest or down-regulation was detected under comparable conditions in metastatic cells, which showed activation of invasion-associated MMP-9 92 kDa gelatinase B. Our data suggests that the metastasis-suppressing effects of chromosome 6 involving increased differentiation-associated tyrosinase and growth arrest on adhesion-restrictive substrates; are partly mediated by modulation of growth regulators, like p21WAF1 and cyclin D3.
Asunto(s)
Cromosomas Humanos Par 6 , Ciclinas/metabolismo , Regulación hacia Abajo , Melanoma/genética , Monofenol Monooxigenasa/metabolismo , Metástasis de la Neoplasia/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Supresión Genética , Regulación hacia Arriba , Animales , Apoptosis , Adhesión Celular , Diferenciación Celular , División Celular , Ciclina D3 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Gelatinasas/metabolismo , Humanos , Immunoblotting , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones SCID , Trasplante de Neoplasias , Células Tumorales CultivadasRESUMEN
The stability of p21(WAF1) and p53 is increased by UV radiation or proteasome inhibitors in normal and some tumor cells. However, p21(WAF1) can either stimulate in vitro assembly of active cyclin-kinase complexes at low concentrations or inhibit this activity at high concentrations. Also, ectopic p21(WAF1) over-expression has been reported to promote or suppress apoptosis, depending on the target cells. We have investigated changes in p21(WAF1) expression as a result of exposure to either 25 J/m(2) UV or 10 microM MG-115 proteasome inhibitor, both of which cause apoptosis in human C8161 melanoma cells. p21(WAF1) mRNA increased in response to UV irradiation but failed to accumulate at the protein level because of its early UV-activated degradation counteracted by proteasome inhibition. UV-mediated loss of p21(WAF1) protein preceding induction of p53 and cell death was greater in non-metastatic than in metastatic C8161 melanoma cells. No loss in p21(WAF1) occurred with apoptosis induced by 10 microM proteasome inhibitors MG-115 or lactacystin, mediated by over-expression of p21(WAF1). Our results suggest that conditions causing prolonged or permanent changes in basal levels of p21(WAF1) may impair its reversible cell-cycle checkpoint function, leading to irreversible growth arrest or cell death.
Asunto(s)
Apoptosis , Ciclinas/metabolismo , Cisteína Endopeptidasas/fisiología , Melanoma/patología , Complejos Multienzimáticos/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Humanos , Melanoma/metabolismo , Complejo de la Endopetidasa Proteasomal , ARN Mensajero/análisis , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Rayos UltravioletaRESUMEN
Terminal differentiation can result in either viable, non-proliferating or apoptotic cells. In B16 melanoma, millimolar L-tyrosine induces tyrosinase, a key enzyme for terminal pigmentation concurrent with either irreversible growth arrest at low cell density, or apoptosis at high cell density. Since the promoter for melanocyte-specific tyrosinase expression contains sites for the Sp1 transcription factor, we have investigated the relationship of Sp1-mediated GC-box DNA binding activity to growth control in undifferentiated and in terminally differentiated viable or apoptotic cells. Nuclear extracts from viable, differentiated cells showed increased retardation of GC box DNA sequence compared with that seen in proliferating cells or those reversibly arrested in early G(1) or late G(1) / S. In contrast, nuclear proteins from dying, differentiated cells showed loss of nuclear GC box DNA binding activity without decrease in binding to TTTGCGCG sequences recognized by the E2F transcription factor, which is known to interact with Sp1. However, cyto-plasmic fractions from apoptotic cells revealed phos-phatase-activated retardation of GC box DNA, which was not evident in similarly treated fractions from undifferentiated cells or sparse differentiated cells. Terminal differentiation also correlated with increase in a slow-migrating phosphorylated Sp1 isoform. Our data suggests that lack of nuclear Sp1/GC box DNA binding activity, may promote apoptosis by diminishing expression of survival-associated genes regulated by GC box DNA promoter sequences in dense terminally differentiated melanoma cells.
Asunto(s)
Apoptosis , ADN/metabolismo , Factor de Transcripción Sp1/metabolismo , Animales , Diferenciación Celular , División Celular , Fragmentación del ADN , Melanoma Experimental/patología , Ratones , Monofenol Monooxigenasa/biosíntesis , FosforilaciónRESUMEN
Since response to radiation and markers capable of distinguishing metastatic from non-metastatic cells are important, we now use high-stringency mRNA differential display with immune blotting and protein-activity assays, to identify genes induced after exposure to UV in human metastatic C8161 melanoma and its counterpart neo 6.3, in which metastatic ability is suppressed by introduction of neo-tagged chromosome-6 fragments. We cloned and sequenced a 600-bp cDNA 99% homologous to Cu/Zn superoxide dismutase, which was up-regulated after UV irradiation in both metastatic variants, and showed increased basal expression at the mRNA, protein and activity levels in non-metastatic cells. The latter cells also showed greater basal activity of chromosome-6-associated MnSOD, slower proliferation and greater UV-mediated inducibility of the p53 tumor-suppressor protein than did its metastatic counterpart. Our data suggest that suppression of metastatic ability by introduction of neo-tagged chromosome-6 fragments promotes basal expression of superoxide dismutases and increases inducibility of p53 in response to DNA damage.
Asunto(s)
Cromosomas Humanos Par 6/fisiología , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Cutáneas/metabolismo , Superóxido Dismutasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Daño del ADN , ADN de Neoplasias/efectos de la radiación , Inducción Enzimática/efectos de la radiación , Humanos , Melanoma/genética , Melanoma/secundario , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Rayos UltravioletaRESUMEN
Because betulinic acid was recently described as a melanoma-specific inducer of apoptosis, we investigated whether this agent was comparably effective against metastatic tumors and those in which metastatic ability and 92-kD gelatinase activity had been decreased by introduction of a normal chromosome 6. Human metastatic C8161 melanoma cells showed greater DNA fragmentation and growth arrest and earlier loss of viability in response to betulinic acid than their non-metastatic C8161/neo 6.3 counterpart. These effects involved induction of p53 without activation of p21WAF1 and were synergized by bromodeoxyuridine in metastatic Mel Juso, with no comparable responses in non-metastatic Mel Juso/neo 6 cells. Our data suggest that betulinic acid exerts its inhibitory effect partly by increasing p53 without a comparable effect on p21WAF1.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Ciclinas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Melanoma/metabolismo , Triterpenos/farmacología , Proteína p53 Supresora de Tumor/biosíntesis , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Ciclina D1/metabolismo , Ciclina D3 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Daño del ADN , Resistencia a Antineoplásicos , Gelatinasas/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Melanoma/enzimología , Melanoma/genética , Metástasis de la Neoplasia , Triterpenos Pentacíclicos , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Ácido BetulínicoRESUMEN
Tumor-suppressor-gene products such as p53 and retinoblastoma (Rb) play an important role as negative regulators of cell-cycle progression, which is reciprocally favored by the availability of cyclin D1 and the E2F transcription factor. We now show that UV irradiation of B16 melanoma after prior exposure to the radiation sensitizer, bromodeoxyuridine (BUdR) leads to induction of p53 and DNA fragmentation, and concomitant decreases in Rb, E2F, cyclin D1, and cell viability, with no comparable effects on irradiated unsensitized cells. Our data suggest that over-expression of p53 correlates with down-regulation of E2F, cyclin D1 in inducing apoptosis.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Ciclina D1/biosíntesis , Proteínas de Unión al ADN , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Proteína de Retinoblastoma/biosíntesis , Factores de Transcripción/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Bromodesoxiuridina/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Muerte Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Daño del ADN , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/efectos de la radiación , Factores de Transcripción E2F , Regulación Neoplásica de la Expresión Génica , Melanoma Experimental/radioterapia , Ratones , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Células Tumorales Cultivadas/efectos de la radiación , Rayos UltravioletaRESUMEN
Characterization of genes expressed in normal cells and decreased in their malignant counterparts is important for detecting candidate tumor suppressor genes. We have now used comparative differential display of MRNAs from B16 melanoma and matched syngeneic normal melanocytes to detect a G0A gene expressed preferentially in resting G0 melanocytes compared to proliferating cells. Cloning and sequencing revealed no homology of G0A in the GenBank Database, suggesting that this is a new gene. Northern blot analysis with the cloned probe, confirmed about a five-fold higher expression in normal melanocytes compared to melanoma. Up-regulation of this gene was not detected by L-tyrosine induction of B16 melanoma terminal differentiation, but was seen in these cells, when exposed to the radiation sensitizer bromodeoxyuridine and subsequent UV radiation. Our differential expression data suggest that the G0A gene is important for melanocytic growth control and for the response of melanoma cells to radiation sensitizers.
Asunto(s)
Daño del ADN , Regulación Neoplásica de la Expresión Génica , Melanocitos , Melanoma/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Bromodesoxiuridina/farmacología , Diferenciación Celular/efectos de los fármacos , Clonación Molecular , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Genes Supresores de Tumor/genética , Melanocitos/citología , Melanocitos/efectos de la radiación , Datos de Secuencia Molecular , ARN Mensajero/análisis , ARN Neoplásico/análisis , Fármacos Sensibilizantes a Radiaciones/farmacología , Fase de Descanso del Ciclo Celular , Análisis de Secuencia de ADN , Células Tumorales Cultivadas , Tirosina/farmacología , Rayos UltravioletaRESUMEN
Differentially regulated expression of activators and inhibitors of cyclin-dependent kinases (cdks) modulate cell cycle progression. In normal fibroblasts, these complexes consist of the cdk inhibitor p21WAF1/PCNA/G1 cyclin/cdk. We now show that bromodeoxyuridine (BrdUrd), a thymidine analogue and radiation sensitizer, inhibits growth and activity of cyclin A-cdk2 kinase in metastatic C8161 and nonmetastatic neo 6.3/C8161 human melanoma cells. Inhibition is not due to altered levels of cyclin D or catalytic cdk2 but involves a decrease in cyclin A and proliferating cell nuclear antigen, paralleled by higher levels of p21WAF1 without increases in p53. In contrast to serum starvation, which prevents accumulation of cyclins A and D in normal fibroblasts, such treatment did not down-regulate either cyclin in these melanoma cells, implying an aberrant control for G1 cyclins in these tumor cells. However, cyclin A was decreased by BrdUrd, suggesting that this pyrimidine analogue arrests melanoma cells at a G1 transition point, unlike that of serum starvation. This is the first report indicating that the antitumor therapeutic action of BrdUrd may be mediated by a p53-independent reciprocal effect on activators and inhibitors of cdk kinases.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclinas/metabolismo , Inhibidores Enzimáticos/metabolismo , Melanoma/patología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Proteínas Sanguíneas/farmacología , Western Blotting , Bromodesoxiuridina/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/agonistas , Ciclinas/efectos de los fármacos , Humanos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/fisiología , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
To identify cyclins specifically associated with control of melanoma cell proliferation, we now compared expression of cyclin A, reported to be a marker for hematological malignancies, with that of cyclin D and its cdk4 kinase partner. All these proteins were expressed in proliferating B16 melanoma. However, L-tyrosine which induces melanoma terminal differentiation, selectively decreased cyclin D with no comparable effect on cdk4 or cyclin A. A 2-hour exposure of the cells to the tyrosine phosphatase inhibitor, sodium vanadate, further decreased cyclin D from differentiated cells, suggesting that tyrosine phosphorylation regulates cyclin D turnover. Addition of serum to starved cells also revealed that tyrosine did not block the early cyclin D increase associated with serum stimulation, but accelerated its subsequent loss. Our data suggest that cyclin D decrease with melanoma terminal differentiation could be an alternative mode of growth arrest even in cells harbouring a mutant or transcriptionally silent cdk4 inhibitor tumor suppressor p16ink4 gene. These results also imply that cyclin D may be useful as a target and as a prognostic marker in melanoma therapy.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/biosíntesis , Ciclinas/biosíntesis , Melanocitos/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Proteínas Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas , Tirosina/farmacología , Animales , Biomarcadores de Tumor/análisis , Diferenciación Celular/efectos de los fármacos , División Celular , Ciclina D1 , Quinasa 4 Dependiente de la Ciclina , Fase G1 , Melanocitos/citología , Ratones , Ratones Endogámicos C57BL , Fase S , Vanadatos/farmacologíaRESUMEN
Malignant transformation is frequently accompanied by changes in the cytoarchitecture of adherent cells, which may be influenced by fluctuations in actin gene expression. We now show that normal melanocytes express a 5 fold higher level of actin mRNA than their melanoma counterparts. Induction of terminal melanogenesis did not increase actin in melanoma cells. However, culture with the thymidine analog, Bromodeoxyuridine, increased actin expression in undifferentiated but not in differentiating melanoma. Cell detachment assays and cell shape comparisons revealed a direct correlation of actin mRNA with increased melanoma cell adhesion rather than with differentiation-mediated suppression of tumor growth.