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PURPOSE: Conventional staging methods are inadequate to identify patients with stage II colon cancer (CC) who are at high risk of recurrence after surgery with curative intent. ColDx is a gene expression, microarray-based assay shown to be independently prognostic for recurrence-free interval (RFI) and overall survival in CC. The objective of this study was to further validate ColDx using formalin-fixed, paraffin-embedded specimens collected as part of the Alliance phase III trial, C9581. PATIENTS AND METHODS: C9581 evaluated edrecolomab versus observation in patients with stage II CC and reported no survival benefit. Under an initial case-cohort sampling design, a randomly selected subcohort (RS) comprised 514 patients from 901 eligible patients with available tissue. Forty-nine additional patients with recurrence events were included in the analysis. Final analysis comprised 393 patients: 360 RS (58 events) and 33 non-RS events. Risk status was determined for each patient by ColDx. The Self-Prentice method was used to test the association between the resulting ColDx risk score and RFI adjusting for standard prognostic variables. RESULTS: Fifty-five percent of patients (216 of 393) were classified as high risk. After adjustment for prognostic variables that included mismatch repair (MMR) deficiency, ColDx high-risk patients exhibited significantly worse RFI (multivariable hazard ratio, 2.13; 95% CI, 1.3 to 3.5; P < .01). Age and MMR status were marginally significant. RFI at 5 years for patients classified as high risk was 82% (95% CI, 79% to 85%), compared with 91% (95% CI, 89% to 93%) for patients classified as low risk. CONCLUSION: ColDx is associated with RFI in the C9581 subsample in the presence of other prognostic factors, including MMR deficiency. ColDx could be incorporated with the traditional clinical markers of risk to refine patient prognosis.
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Neoplasias del Colon/genética , Recurrencia Local de Neoplasia/genética , Anciano , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales de Origen Murino , Ensayos Clínicos Fase III como Asunto , Estudios de Cohortes , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Neoplasias del Colon/cirugía , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Estudios Multicéntricos como Asunto , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
A 15-gene prognostic signature for early-stage, completely resected, non-small-cell lung carcinoma, (which distinguishes between patients with good and poor prognoses) was clinically validated in prior studies. To achieve operational efficiencies, this study was designed to evaluate the assay's performance in RNA-stabilized tissue as an alternative to the fresh-frozen tissue format originally used to develop the assay. The percent concordance between matched tissue formats was 84% (95% Wilson CI, 70%-92%), a level of agreement comparable to the inherent reproducibility of the assay observed within biological replicates of fresh-frozen tissue. Furthermore, the analytical performance of the assay using the RNA-stabilized tissue format was evaluated. When compared to an accredited reference laboratory, the clinical laboratory achieved a concordance of 94% (95% Wilson CI, 81%-98%), and there was no evidence of bias between the laboratories. The lower limit of quantitation for the target RNA concentration was confirmed to be, at most, 12.5 ng/µL. The assay reportable range defined in terms of risk score units was determined to be -4.295 to 4.210. In a large-scale precision study, the assay showed high reproducibility and repeatability. When subjected to a maximal amount of genomic DNA, a potential contaminant, the assay still produced the expected results. The 15-gene signature was confirmed to produce reliable results and, thus, is suitable for its intended use.
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Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , ADN de Neoplasias/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Pulmonares/diagnóstico , ARN Neoplásico/química , Juego de Reactivos para Diagnóstico/normas , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/terapia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Adhesión en Parafina , Pronóstico , Sensibilidad y EspecificidadRESUMEN
A formalin-fixed paraffin-embedded tissue-based prognostic assay to assess the risk for recurrence in stage II colon cancer has recently been clinically validated. This study describes the analytical performance and quality control measures of the assay. The reportable range was determined to be [-1.129, 1.414] in risk score units. The accuracy was evaluated with a split sample comparison within the production lab and between the production lab and a reference lab. The concordance between the replicates within the production lab was 79% (95% confidence interval, 64%-91%). There was no evidence of bias, and the concordance was 78% (95% confidence interval, 61%-90%) between the labs. The lab-to-lab concordance was further evaluated by simulating risk scores from the full reportable range. The simulation suggested a higher concordance. The sensitivity study demonstrated that the percentage of tumor tissue did not impact the risk score and that RNA concentration of 9.5 ng/µL was a conservative determination of the analyte lower limit of quantification. From the precision study, the repeatability and reproducibility estimates were 0.1267 and 0.0548 in risk score units, respectively. Furthermore, multifaceted quality control measures were implemented, such as proper tissue processing steps, high-risk and low-risk controls, nontemplate control, and a gene expression-based classifier to evaluate the cDNA amplification kit, a key reagent in the assay. In conclusion, this study demonstrates the strong analytical performance of the assay and further supports its use as an objective standardized prognostic test for stage II colon cancer.
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Biomarcadores de Tumor/genética , Neoplasias del Colon/diagnóstico , ADN de Neoplasias/análisis , Regulación Neoplásica de la Expresión Génica , Recurrencia Local de Neoplasia/diagnóstico , Juego de Reactivos para Diagnóstico/normas , Neoplasias del Colon/genética , Neoplasias del Colon/patología , ADN Complementario/análisis , ADN Complementario/genética , ADN de Neoplasias/genética , Formaldehído , Humanos , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Variaciones Dependientes del Observador , Adhesión en Parafina , Pronóstico , Control de Calidad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Fijación del TejidoRESUMEN
BACKGROUND: There is no method routinely used to predict response to anthracycline and cyclophosphamide-based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection. METHODS: DNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided. RESULTS: In a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval [Cl] =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population. CONCLUSIONS: A formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide-based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Daño del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Anemia de Fanconi/metabolismo , Adulto , Anciano , Antraciclinas/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/genética , Quimioterapia Adyuvante , Ciclofosfamida/administración & dosificación , Supervivencia sin Enfermedad , Epirrubicina/administración & dosificación , Anemia de Fanconi/genética , Femenino , Fluorouracilo/administración & dosificación , Humanos , Persona de Mediana Edad , Terapia Neoadyuvante/métodos , Oportunidad Relativa , Análisis de Secuencia por Matrices de Oligonucleótidos , Estudios ProspectivosRESUMEN
PURPOSE: Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples. PATIENTS AND METHODS: A gene signature was developed from a balanced set of 73 patients with recurrent disease (high risk) and 142 patients with no recurrence (low risk) within 5 years of surgery. RESULTS: The 634-probe set signature identified high-risk patients with a hazard ratio (HR) of 2.62 (P < .001) during cross validation of the training set. In an independent validation set of 144 samples, the signature identified high-risk patients with an HR of 2.53 (P < .001) for recurrence and an HR of 2.21 (P = .0084) for cancer-related death. Additionally, the signature was shown to perform independently from known prognostic factors (P < .001). CONCLUSION: This gene signature represents a novel prognostic biomarker for patients with stage II colon cancer that can be applied to FFPE tumor samples.
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Neoplasias del Colon/patología , Recurrencia Local de Neoplasia/patología , Adhesión en Parafina/métodos , Anciano , Neoplasias del Colon/genética , Femenino , Formaldehído , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Adhesión en Parafina/normas , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Fijación del TejidoRESUMEN
A role for BRCA1 in the direct and indirect regulation of transcription is well established. However, a comprehensive view of the degree to which BRCA1 impacts transcriptional regulation on a genome-wide level has not been defined. We performed genome-wide expression profiling and ChIP-chip analysis, comparison of which revealed that although BRCA1 depletion results in transcriptional changes in 1294 genes, only 44 of these are promoter bound by BRCA1. However, 27% of these transcripts were linked to transcriptional regulation possibly explaining the large number of indirect transcriptional changes observed by microarray analysis. We show that no specific consensus sequence exists for BRCA1 DNA binding but rather demonstrate the presence of a number of known and novel transcription factor (TF)- binding sites commonly found on BRCA1 bound promoters. Co-immunoprecipitations confirmed that BRCA1 interacts with a number of these TFs including AP2-α, PAX2 and ZF5. Finally, we show that BRCA1 is bound to a subset of promoters of genes that are not altered by BRCA1 loss, but are transcriptionally regulated in a BRCA1-dependent manner upon DNA damage. These data suggest a model, whereby BRCA1 is present on defined promoters as part of an inactive complex poised to respond to various genotoxic stimuli.
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Proteína BRCA1/fisiología , Transcriptoma , Proteína BRCA1/antagonistas & inhibidores , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Fisiológico/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. At present no reliable biomarkers are available to guide the management of this condition. Microarray technology may allow appropriate biomarkers to be identified but present platforms are lacking disease focus and are thus likely to miss potentially vital information contained in patient tissue samples. METHODS: A combination of large-scale in-house sequencing, gene expression profiling and public sequence and gene expression data mining were used to characterise the transcriptome of NSCLC and the data used to generate a disease-focused microarray - the Lung Cancer DSA research tool. RESULTS: Built on the Affymetrix GeneChip platform, the Lung Cancer DSA research tool allows for interrogation of ~60,000 transcripts relevant to Lung Cancer, tens of thousands of which are unavailable on leading commercial microarrays. CONCLUSION: We have developed the first high-density disease specific transcriptome microarray. We present the array design process and the results of experiments carried out to demonstrate the array's utility. This approach serves as a template for the development of other disease transcriptome microarrays, including non-neoplastic diseases.
RESUMEN
PURPOSE: We investigated whether BRCA1 mRNA expression levels may represent a biomarker of survival in sporadic epithelial ovarian cancer following chemotherapy treatment. EXPERIMENTAL DESIGN: The effect of loss of BRCA1 expression on chemotherapy response in ovarian cancer was measured in vitro using dose inhibition assays and Annexin V flow cytometry. Univariate and multivariate analyses were done to evaluate the relationship between BRCA1 mRNA expression levels and survival after chemotherapy treatment in 70 fresh frozen ovarian tumors. RESULTS: We show that inhibition of endogenous BRCA1 expression in ovarian cancer cell lines results in increased sensitivity to platinum therapy and decreased sensitivity to antimicrotubule agents. In addition, we show that patients with low/intermediate levels of BRCA1 mRNA have a significantly improved overall survival following treatment with platinum-based chemotherapy in comparison with patients with high levels of BRCA1 mRNA (57.2 versus 18.2 months; P = 0.0017; hazard ratio, 2.9). Furthermore, overall median survival for higher-BRCA1-expressing patients was found to increase following taxane-containing chemotherapy (23.0 versus 18.2 months; P = 0.12; hazard ratio, 0.53). CONCLUSIONS: We provide evidence to support a role for BRCA1 mRNA expression as a predictive marker of survival in sporadic epithelial ovarian cancer.
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Proteína BRCA1/biosíntesis , Resistencia a Antineoplásicos/genética , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , ARN Mensajero/análisis , Antineoplásicos/uso terapéutico , Apoptosis/genética , Proteína BRCA1/genética , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Western Blotting , Femenino , Citometría de Flujo , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/mortalidad , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/mortalidad , Reacción en Cadena de la Polimerasa , Análisis de SupervivenciaRESUMEN
BACKGROUND: BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors. METHODS: We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) (the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA (siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided. RESULTS: Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors (95% confidence interval [CI] = 2.6-fold to 40.1-fold, P = .0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector (T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: >10(-5) versus 8.0 x 10(-9) M [95% CI = 3.1 x 10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha: >10(-5) versus 4.9 x 10(-8) M [95% CI = 2.0 x 10(-9) to 3.9 x 10(-6) M]). CONCLUSIONS: BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.
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Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/química , Neoplasias de la Mama/genética , Estradiol/análogos & derivados , Moduladores de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/deficiencia , Silenciador del Gen , Genes BRCA1 , Mutación , Northern Blotting , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Femenino , Fulvestrant , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Inmunoprecipitación , ARN Mensajero/análisis , ARN Interferente Pequeño , Proyectos de Investigación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
BRCA1 encodes a tumor suppressor gene that is mutated in the germ line of women with a genetic predisposition to breast and ovarian cancer. BRCA1 has been implicated in a number of important cellular functions including DNA damage repair, transcriptional regulation, cell cycle control, and ubiquitination. Using an Affymetrix U95A microarray, IRF-7 was identified as a BRCA1 transcriptional target and was also shown to be synergistically up-regulated by BRCA1 specifically in the presence of IFN-gamma, coincident with the synergistic induction of apoptosis. We show that BRCA1, signal transducer and activator of transcription (STAT)-1, and STAT2 are all required for the induction of IRF-7 following stimulation with IFN-gamma. We also show that the induction of IRF-7 by BRCA1 and IFN-gamma is dependent on the type I IFNs, IFN-alpha and IFN-beta. We show that BRCA1 is required for the up-regulation of STAT1, STAT2, and the type I IFNs in response to IFN-gamma. We show that BRCA1 is localized at the promoters of the molecules involved in type I IFN signaling leading to their up-regulation. Blocking this intermediary type I IFN step using specific antisera shows the requirement for IFN-alpha and IFN-beta in the induction of IRF-7 and apoptosis. Finally, we outline a mechanism for the BRCA1/IFN-gamma regulation of target genes involved in the innate immune response, which is dependent on type I IFN signaling.
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Proteína BRCA1/metabolismo , Inmunidad Innata/genética , Factor 7 Regulador del Interferón/genética , Interferón Tipo I/metabolismo , Interferón gamma/farmacología , Apoptosis , Proteína BRCA1/análisis , Línea Celular Tumoral , Proliferación Celular , Humanos , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/farmacología , Regiones Promotoras Genéticas , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Regulación hacia ArribaRESUMEN
We have demonstrated previously that certain members of a series of novel pyrrolo-1,5-benzoxazepine (PBOX) compounds potently induce apoptosis in a variety of human chemotherapy-resistant cancer cell lines and in primary ex vivo material derived from cancer patients. A better understanding of the molecular mechanisms underlying the apoptotic effects of these PBOX compounds is essential to their development as antineoplastic therapeutic agents. This study sought to test the hypothesis that proapoptotic PBOX compounds target the microtubules. We show that a representative proapoptotic PBOX compound, PBOX-6, induces apoptosis in both the MCF-7 and K562 cell lines. An accumulation of cells in G2/M precedes apoptosis in response to PBOX-6. PBOX-6 induces prometaphase arrest and causes an accumulation of cyclin B1 levels and activation of cyclin B1/CDK1 kinase in a manner similar to that of two representative antimicrotubule agents, nocodazole and paclitaxel. Indirect immunofluorescence demonstrates that both PBOX-6 and another pro-apoptotic PBOX compound, PBOX-15, cause microtubule depolymerization in MCF-7 cells. They also inhibit the assembly of purified tubulin in vitro, whereas a nonapoptotic PBOX compound (PBOX-21) has no effect on either the cellular microtubule network or on the assembly of purified tubulin. This suggests that the molecular target of the pro-apoptotic PBOX compounds is tubulin. PBOX-6 does not bind to either the vinblastine or the colchicine binding site on tubulin, suggesting that it binds to an as-yet-uncharacterised novel site on tubulin. The ability of PBOX-6 to bind tubulin and cause microtubule depolymerization confirms it as a novel candidate for antineoplastic therapy.
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Apoptosis/efectos de los fármacos , Oxazepinas/farmacología , Pirroles/farmacología , Tubulina (Proteína)/metabolismo , Antineoplásicos Fitogénicos/metabolismo , Antineoplásicos Fitogénicos/farmacología , Sitios de Unión , Unión Competitiva , Western Blotting , Proteína Quinasa CDC2/metabolismo , Carbamatos/farmacología , División Celular , Línea Celular Tumoral , Colchicina/metabolismo , Colchicina/farmacología , Ciclina B/metabolismo , Ciclina B1 , Relación Dosis-Respuesta a Droga , Fase G2 , Humanos , Células K562 , Metafase/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Oxazepinas/metabolismo , Pirroles/metabolismo , Factores de Tiempo , Vinblastina/metabolismo , Vinblastina/farmacologíaRESUMEN
Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.
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Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Oxazepinas/farmacología , Pirroles/farmacología , Receptores de Estrógenos/análisis , Animales , Ciclo Celular/efectos de los fármacos , Supervivencia Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/fisiología , Receptores de Estrógenos/fisiología , Células Tumorales CultivadasRESUMEN
The present study examines the molecular mechanisms by which a member of a novel series of pyrrolo-1,5-benzoxazepines, PBOX-21, induces G1 arrest in 1321N1 cells. PBOX-21-induced G1 arrest is preceded by both a decrease in CDK2 kinase activity, which is critical for the G1/S transition, and a downregulation in cyclin D(3) protein expression levels, suggesting that these two events may be crucially involved in the mediation of the cell cycle arrest. The decrease in CDK2 activity may be due to an observed decrease in CDK2 protein levels following PBOX-21 treatment. Coinciding with the arrest is a reduction in the activity of CDK4, due to either the observed PBOX-21 induced downregulation in CDK4 expression, or a reduction in complex formation between cyclin D(3)-CDK4 leading to a decrease in the levels of active cyclin D(3)-CDK4 complexes with kinase activity. The level of CDK6 activity was also seen to be reduced following PBOX-21 treatment, also possibly due to a reduction in complex formation with cyclin D(3). However, this reduction in CDK6 kinase activity was not seen until after PBOX-21-induced G1 arrest has reached its maximum, and therefore may be viewed as a consequence of, and a method of maintaining the PBOX-21-induced arrest, rather than a cause. Also in parallel with the G1 arrest elicited by PBOX-21 is an upregulation in the universal CDK inhibitor, p21. Furthermore, the retinoblastoma protein (Rb), a substrate of CDK2 and CDK6, whose phosphorylation is necessary for cell cycle progression, becomes hypophosphorylated. These results indicate that PBOX-21 exerts its growth inhibitory effects through the modulation of the expression and activity of several key G1 regulatory proteins.