RESUMEN
The search for new prognostic indicators is especially important in the diagnosis and treatment of ovarian cancer because clinicopathologic criteria currently used to predict survival are largely inadequate. We examined 2 groups of patients with epithelial ovarian cancer, 1 group of long-term survivors (>5 years), and 1 group of short-term survivors (<2 years) for levels of expression of the cell cycle regulators p57(KIP2), cyclin D1, and cyclin E and their relationship with survival. Our findings show that p57(KIP2) is not associated with prognosis, in contrast to p27(KIP1) expression, which is previously shown to be positively associated with long-term survival in univariate analysis (P =.001). Cyclin E expression, in contrast to cyclin D1 expression, is marginally associated with short-term survival in univariate analysis for a group of 53 women. Among the short-term survivors, 15 (65%) of 23 were positive for cyclin E expression, compared with only 11 (37%) of 30 long-term survivors (P = 0.054). This association remained significant (P =.04) in a logistic regression analysis adjusted simultaneously for performance status and extent of residual disease, the 2 strongest predictors of survival in our study. We also found a significant difference in the frequency of the cyclin E staining pattern between nonserous and serous ovarian tumor subtypes (P =.0002). Immunostaining for levels of cyclin E and p27(KIP1) expression may have potential as prognostic markers in the management of ovarian cancer.
Asunto(s)
Adenocarcinoma/metabolismo , Ciclina D1/metabolismo , Ciclina E/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Ováricas/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Biomarcadores de Tumor/metabolismo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina , Supervivencia sin Enfermedad , Femenino , Humanos , Técnicas para Inmunoenzimas , Persona de Mediana Edad , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
Length alterations in microsatellite repeats, termed microsatellite instability (MSI), are found in 10-15% of sporadic colon, endometrial, and gastric cancers harboring defects in DNA mismatch repair (MMR) genes We used the microsatellite markers Big Adenine Tract (BAT) 26 and BAT-25 from the reference panel of five markers recommended by the National Cancer Institute to evaluate the incidence of MSI in 206 central nervous system tumors. We screened 102 pediatric and 104 adult cases representing 165 astrocytic and 41 nonastrocytic tumors. The overall incidence of MSI was 8% (16 of 206). All 16 tumors with MSI were found in pediatric rather than adult patients. MSI was associated with two distinct subtypes of pediatric tumors occurring in 27% (12 of 45) of WHO grade III and grade IV astrocytomas and 24% (4 of 17) of gangliogliomas We evaluated the difference in clinicopathological and genetic features among 45 high-grade pediatric astrocytomas by MSI status. The median survival for pediatric patients with MSI (n = 12) was 8 months compared with 15 months for those patients without MSI (n = 33; P = 0.18). The frequency of p53 gene mutations was 13% for pediatric patients with MSI (n = 8) compared with 47% for those patients without MSI (n = 19; P = 0.19). These results revealed a trend between MSI status and prog nosis and MSI status and frequency of p53 gene mutations. Our data suggest that pediatric high-grade astrocytomas can be attributed to two different genetic pathways: a MMR-deficient pathway and a MMR proficient pathway.
Asunto(s)
Neoplasias del Sistema Nervioso Central/genética , Repeticiones de Microsatélite/genética , Adolescente , Adulto , Factores de Edad , Astrocitoma/genética , Astrocitoma/mortalidad , Astrocitoma/patología , Neoplasias del Sistema Nervioso Central/mortalidad , Neoplasias del Sistema Nervioso Central/patología , Niño , Preescolar , Femenino , Humanos , Masculino , Pronóstico , Tasa de SupervivenciaRESUMEN
Members of the Bcl-2 family of proteins are key regulators of apoptosis. Some of these proteins undergo posttranslational modification, such as phosphorylation or proteolysis, that serves to alter their function. Caspases are known to cleave Bid, a proapoptotic family member, as well as Bcl-2 and Bcl-X(L), two prosurvival family members, which activate their cytotoxic activity resulting in the release of cytochrome c from mitochondria. Previously we showed that Bax was cleaved by calpain rather than by caspases from full-length 21 kDa to generate a cleavage fragment of 18 kDa. Since cleavage of Bid serves to activate its cytotoxic activity, we wanted to determine if the p18 form of Bax exhibited increased cytotoxicity compared to p21 Bax. Using a transient transfection system in human embryonic kidney 293T cells we show that the p18 form of Bax displays a more potent ability to induce cell death. The pancaspase inhibitor Z-VAD-fmk completely blocked apoptosis induced by p21 Bax but only partially inhibited apoptosis induced by p18 Bax. Cyclosporin A, an inhibitor of the mitochondrial permeability transition (PT) pore, had no effect on Bax-mediated apoptosis of 293T cells suggesting that apoptosis was independent of the PT. Thus cleavage of p21 Bax during apoptosis to the p18 form may serve to increase the intrinsic cytotoxic properties of this proapoptotic molecule and enhance its cell death function at the mitochondria.
Asunto(s)
Apoptosis , Canales Iónicos , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Línea Celular , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Modelos Biológicos , Peso Molecular , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/fisiología , Transfección , Proteína X Asociada a bcl-2RESUMEN
Classification of high grade astrocytomas of children into genetic subtypes similar to the adult remains to be defined. Here we report an extensive characterization of 29 high grade pediatric astrocytomas, 7 WHO grade III and 22 WHO grade IV, for genetic alterations frequently observed in high grade adult astrocytomas occurring in either the p53/MDM2/p14ARF or Rb/CDK4/p16INK4a tumor suppressor pathways. In addition, we have assessed the contribution of EGFR overexpression and amplification and LOH for chromosome 10, two genetic alterations commonly associated with the development of de novo adult glioblastoma for their roles in the development of de novo astrocytomas of childhood. Our results suggest two major differences in the genetic pathway(s) leading to the formation of de novo high grade astrocytomas in children compared with those of the adult. Our findings show preferential inactivation of the p53 tumor suppressor pathway in >95% of pediatric astrocytomas versus inactivation of the Rb tumor suppressor pathway in <25% of the same tumors. In addition, de novo high grade pediatric astrocytomas lack amplification of the EGFR gene compared with EGFR amplification in one-third of adult glioblastomas. Since drug treatments and gene therapy strategies exploit specific genetic alterations in tumor cells, our findings have important implications for the future development of treatments for high grade pediatric astrocytomas.
Asunto(s)
Neoplasias del Sistema Nervioso Central/genética , Receptores ErbB/genética , Amplificación de Genes , Silenciador del Gen , Genes Supresores de Tumor/genética , Glioblastoma/genética , Proteínas Nucleares , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor , Adolescente , Adulto , Proteínas Portadoras/genética , Neoplasias del Sistema Nervioso Central/metabolismo , Neoplasias del Sistema Nervioso Central/patología , Niño , Preescolar , Cromosomas Humanos Par 10/genética , Cromosomas Humanos Par 9/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Diagnóstico Diferencial , Receptores ErbB/metabolismo , Femenino , Eliminación de Gen , Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Masculino , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The INK4a-ARF locus encodes 2 separate proteins through differential splicing of alternative first exons to produce p16INK4a (exon 1alpha) and p14ARF (exon 1beta) products in human cells. The p16INK4a protein inhibits the cyclin D-dependent kinases (CDK) that control the phosphorylation of the Rb protein and cell proliferation. The p14ARF gene product can complex with and sequester the MDM2 protein within the nucleus, thus modulating the activity of the p53 protein. Loss of p16INK4a expression would disrupt the retinoblastoma (Rb)/p16INK4a/cyclin D-dependent kinase (CDK4) pathway, whereas loss of p14ARF expression would inactivate both the Rb and p53/ MDM2/p14ARF pathways through MDM2, which can complex with either Rb or p53. Loss of the p16INK4a gene on 9p21 has been documented in a wide range of human tumors, including one third of glioblastomas. However, in tumors showing homozygous loss of exon 2 of the p16INK4a gene, loss of exon 1beta of the p14ARF gene has not been established. In this study, we have assessed deletion of the p14ARF gene in 29 pediatric and 107 adult high-grade astrocytomas and 9 glioma cell lines, using multiplex PCR analysis for exon 1beta. We found homozygous deletions for exon 1alpha and exon 1beta in 3 of 29 (10%) of the pediatric cases (2 grade III, 1 grade IV), 25 of 107 (23%) of the adult cases (6 grade III and 19 grade IV), and 8 of 9 (89%) of the glioma cell lines. Therefore, loss of the INK4a-ARF locus in high-grade astrocytomas may contribute to the highly malignant behavior and treatment resistance of these tumors through elimination of multiple checkpoint cell cycle control proteins.
Asunto(s)
Astrocitoma/genética , Astrocitoma/patología , Eliminación de Gen , Proteínas/genética , Adulto , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Preescolar , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Glioblastoma/genética , Glioma/genética , Homocigoto , Humanos , Inmunohistoquímica , Células Tumorales Cultivadas , Proteína p14ARF Supresora de TumorRESUMEN
We have previously demonstrated that calpain is responsible for the cleavage of Bax, a proapoptotic protein, during drug-induced apoptosis of HL-60 cells (Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078). Here we show the sequential activation of caspases and calpain during drug-induced apoptosis of HL-60 cells. Time course experiments using the topoisomerase I inhibitor 9-amino-20(S)-camptothecin revealed that cleavage of caspase-3 substrates poly(ADP-ribose) polymerase (PARP) and the retinoblastoma protein as well as DNA fragmentation occurred several hours before calpain activation and Bax cleavage. Pretreatment with the calpain inhibitor calpeptin blocked calpain activation and Bax cleavage but did not inhibit PARP cleavage, DNA fragmentation, or 9-amino-20(S)-camptothecin-induced morphological changes and cell death. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited PARP cleavage, DNA fragmentation, calpain activation, and Bax cleavage and increased cell survival by 40%. Interestingly, Z-VAD-fmk-treated cells died in a caspase- and calpain-independent manner that appeared morphologically distinct from apoptosis. Our results suggest that excessive or uncontrolled calpain activity may play a role downstream of and distinct from caspases in the degradation phase of apoptosis.
Asunto(s)
Apoptosis , Calpaína/metabolismo , Caspasas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3 , Cumarinas/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN , Dipéptidos/farmacología , Activación Enzimática , Células HL-60 , Humanos , Oligopéptidos/farmacologíaRESUMEN
This case-control study was designed to identify factors associated with long-term survival. We examined two groups of patients with epithelial ovarian cancer, one group of long-term survivors (> 5 years) and one group of short-term survivors (< 2 years), for levels of expression of p53 and p27KIP1 proteins (as both proteins have been shown to be independent prognostic markers in tumors other than ovary) and the relationship with patient survival. Our findings show that p27KIP1 expression, in contrast to p53 expression, is positively associated with long-term survival in univariate analysis (P = 0.001), in analyses stratified by residual disease (P = 0.02) or performance status (P = 0.02), the two strongest prognostic factors for ovarian cancer, as well as multivariate analysis (P = 0.002) adjusting simultaneously for age, tumor stage, residual disease, performance status, and grade of differentiation. Therefore, immunostaining for levels of p27KIP1 expression may have potential as a new prognostic factor in the management of ovarian cancer.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/fisiopatología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/fisiopatología , Proteínas Supresoras de Tumor , Adulto , Anciano , Biomarcadores de Tumor , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Pronóstico , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Deregulated expression of one or more growth control genes including p16, p53, EGF receptor (EGFR), MDM2 or Bcl-2 may contribute to the treatment resistance phenotype of GBM and generally poor patient survival. Clinically, GBM have been divided into two major groups defined by (1) histologic progression from a low grade tumor ("progressive" or "secondary" GBM) contrasted with (2) those which show initial clinical presentation without a prior history ("de novo" or "primary" GBM). Using molecular genetic analysis for p53 gene mutations together with immunophenotyping for overexpression of EGFR, up to four GBM variants can be distinguished, including the p53+/EGFR- progressive or the p53-/EGFR+ de novo variant. We examined the survival of 80 adult patients diagnosed with astrocytic GBM stratified by age category (>40, 41-60 or 61-80) to determine whether alterations in any one given growth control gene or whether different genetic variants of GBM (progressive versus de novo) were associated with different survival outcomes. Survival testing using Kaplan-Meier plots for GBM patients with or without altered expression of p16, p53, EGFR, MDM2 or Bcl-2 showed no significant differences by age group or by gene expression indicating a lack of prognostic value for GBM. Also the clinical outcome among patients with GBM showed no significant differences within each age category for any GBM variant including the progressive and de novo GBM variants indicating similar biologic behavior despite different genotypes. Using a pairwise comparison, one-third of the GBM with normal p16 expression showed accumulation of MDM2 protein and this association approached statistical significance (0.01 < P < 0.05) using the Bonferroni procedure. These GBM may represent a variant in which the p19ARF/MDM2/p53 pathway may be deregulated rather than the p16/cyclin D-CDK4/Rb pathway.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/genética , Glioblastoma/mortalidad , Proteínas Nucleares , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/fisiología , Receptores ErbB/genética , Femenino , Genes bcl-2 , Genes p16 , Genes p53 , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Polimorfismo Conformacional Retorcido-Simple , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Receptor ErbB-2/genética , SobrevidaRESUMEN
The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.
Asunto(s)
Apoptosis/efectos de los fármacos , Calpaína/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Ácido Aspártico/genética , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Calpaína/antagonistas & inhibidores , Calpaína/aislamiento & purificación , Camptotecina/análogos & derivados , Camptotecina/antagonistas & inhibidores , Camptotecina/farmacología , Muerte Celular/efectos de los fármacos , Extractos Celulares/química , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Dipéptidos/farmacología , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Células HL-60/ultraestructura , Humanos , Hidrólisis/efectos de los fármacos , Leucina/análogos & derivados , Leucina/farmacología , Datos de Secuencia Molecular , Mutación/genética , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Especificidad por Sustrato , Proteína X Asociada a bcl-2RESUMEN
bcl-2 protein expression was characterized in a series of 58 astrocytomas from 21 pediatric and 37 adult patients. As part of a continuing attempt to define relevant prognostic factors which may predict clinical outcome, we have determined the impact of bcl-2 accumulation in malignant astrocytes on the length of patient survival. Aberrant overexpression of bcl-2 protein in tumor cells was detected in 57% (12 of 21) of pediatric and 73% (27 of 37) of the adult cases. Among pediatric patients, the median survival in months showed no relationship with the incidence of bcl-2-positive tumors. Among the adult patients, a favorable prognostic indicator was low-tumor grade (P = 0.05). bcl-2-positive tumors occurred with similar frequencies in WHO grades III and IV of malignancy. When bcl-2 expression in tumor cells was tested as a variable to predict for patient survival, the 6 patients without bcl-2 expression among 23 adult patients with grade IV tumors had a shorter median survival. The same 58 tumors had been previously analyzed for alterations of p53:4 pediatric and 16 adult tumors had p53 gene mutations. There was no significant difference in median survival related to p53 gene status. There was no relationship between bcl-2 expression and p53 gene status: approximately equal numbers of tumors with either wild-type or mutant p53 were bcl-2 negative or bcl-2 positive. bcl-2 expression is high (40-100%) among other tumors of the central nervous system which also show low malignant potential. Up-regulation of bcl-2 in malignant astrocytes or constitutive expression in some tumor types may be a factor leading to a more favorable clinical outcome.
Asunto(s)
Astrocitoma/genética , Neoplasias Encefálicas/genética , Expresión Génica , Genes p53 , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Adolescente , Adulto , Astrocitoma/metabolismo , Astrocitoma/mortalidad , Astrocitoma/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Niño , Preescolar , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Tasa de SupervivenciaAsunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 10/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores , Animales , Southern Blotting , Humanos , Células Híbridas/metabolismo , Hibridación Fluorescente in Situ , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Ratones , Proto-Oncogenes MasRESUMEN
Loss of p16 expression can occur via homozygous deletion, point mutation, or hypermethylation of exon 1. Astrocytomas representing all World Health Organization (WHO) grades of malignancy were analyzed in a correlative study using multiplex polymerase chain reaction (PCR) analysis to detect deletions of the p16 gene together with immunohistochemistry to detect loss of the protein in archival specimens of the same tumors. Homozygous deletions of p16 were detected in 29% (15 of 52) of WHO grade 3 and 4 tumors. Immunostaining for p16 protein was present in 26 tumors retaining the p16 gene and absent in 11 tumors with deletions of the p16 gene. A close correlation was found between the two detection methods, with all tumors lacking immunostaining showing homozygous loss of the p16 gene. Astrocytomas exhibiting inactivation of the p16 gene most often contained p53 gene mutations or amplified epidermal growth factor receptor genes, genetic characteristics associated with both the progressive and de novo tumor variants. Immunohistochemical evaluation may be a useful, rapid method to screen astrocytomas for loss of p16 gene expression, regardless of the underlying mechanism leading to p16 gene inactivation.
Asunto(s)
Astrocitoma/genética , Neoplasias Encefálicas/genética , Proteínas Portadoras/genética , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Inmunohistoquímica/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Astrocitoma/patología , Neoplasias Encefálicas/patología , Proteínas Portadoras/química , Niño , Preescolar , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Análisis Mutacional de ADN , Femenino , Eliminación de Gen , Humanos , Masculino , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
C57BL/6J mice were treated with N-methylnitrosourea (NMU) and the evolution of leukemic T-cells clones into frank thymic lymphomas was followed in 42 animals using serology of T-cell markers, rearrangements of the T-cell receptor genes gamma, beta1 and beta2 and detection of carcinogen-induced Ki-ras mutations and trisomy of chromosome 15. During the latent period, multiple populations of T-cell clones were present in the thymus, many contained trisomy 15, but few had detectable Ki-ras mutations. Since most frank lymphomas consisted of a single T-cell clone with both a mutation of Ki-ras and trisomy 15, the results imply that these two events are critical for the evolution of T-cell clones from the preleukemic phase to a more malignant disease stage. Progression to frank lymphomas is coincident with changes in the expression pattern of the T-cell growth factor interleukin-2 receptor, which may play a role in the selection, expansion and thymus-independent growth of a T-cell clone.
Asunto(s)
Linfoma/patología , Mutación , Neoplasias del Timo/patología , Animales , Carcinógenos , Células Clonales , Femenino , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T , Linfoma/inducido químicamente , Linfoma/genética , Metilnitrosourea , Ratones , Ratones Endogámicos C57BL , Neoplasias del Timo/inducido químicamente , Neoplasias del Timo/genética , TrisomíaRESUMEN
A novel B-cell derived (Bcd) oncogene has been isolated from the peripheral blood lymphocytes of one B-cell chronic lymphocytic leukemia (B-CLL) patient using DNA transfer and a mouse tumorigenicity assay. The Bcd proto-oncogene was activated by a truncation in the 5' UTR. It predicts for two open reading frames (ORFs). ORF1 consists of 240 bp that would encode 80 amino acids, while the major ORF2 consists of 648 bp capable of coding for 216 amino acids. Predicted peptide sequence of ORF2 contained a zinc finger domain which showed significant homology to GC box binding proteins BTEB2 and SP1. Transfection of an expression vector containing ORF2 but not full length cDNA was able to transform NIH3T3 cells and induce tumors in nude mice. Bcd mRNA transcripts of < or = 2.6 kb were selectively expressed in PBL and testis of healthy individuals. Within the PBL, Bcd gene expression was restricted to CD19+ B-cells and absent from CD14+ monocytes and T-cells. Bcd transcripts were detected in all normal PBL samples tested but not in several malignant human B-cell lines and not in 50% of B-cells from B-CLL patients. However, stimulation of B-cells from B-CLL patients under conditions which induced differentiation into plasma cells was associated with induction of Bcd gene expression. The Bcd gene may therefore play an important role in B-cell growth and development.
Asunto(s)
ADN Complementario/genética , ADN de Neoplasias/genética , Leucemia Linfocítica Crónica de Células B/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/genética , Transactivadores , Células 3T3 , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Secuencia de Bases , Clonación Molecular , Reordenamiento Génico , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
We investigated the relationship among chemosensitivity to drug-induced apoptosis in vitro, the presence of p53 gene mutations, and the expression of bcl-2 and bax proteins in B-cells from B-cell chronic lymphocytic leukemia (B-CLL) patients. Apoptosis was induced with a camptothecin analogue, 9-amino-20(s)-camptothecin, or a purine analogue, fludarabine. Cell death was monitored by propidium iodide staining and FACS analysis. Drug-induced apoptosis in B-CLL cells was p53-independent. Immunoblot analysis of bcl-2 and bax expression revealed a correlation between drug-induced apoptosis and the ratio of endogenous levels of bcl-2 to bax proteins. B-CLL cells with none to low bcl-2/bax ratios were drug-sensitive as compared to cells with intermediate to high ratios that were drug-resistant (P = 0.015). Prior to drug treatment, bax protein migrated as a single species of 21 kDa. Following drug-induced apoptosis, anti-bax specific protein complexes of 36-42 kDa were up-regulated. Using two-dimensional gel electrophoresis, bax complexes were disrupted under reducing conditions to reveal homo- and heterodimers of 18 and 21 kDa suggesting that disulfide interactions were required for complex formation. The de novo appearance of the 18 kDa anti-bax specific protein together with its increased expression in drug-sensitive B-CLL B-cells undergoing cell death suggests a role for this protein in the regulation of apoptosis.
Asunto(s)
Apoptosis/fisiología , Genes p53/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Mutación , Proteínas Proto-Oncogénicas/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Resistencia a Antineoplásicos/fisiología , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Mercaptoetanol/farmacología , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2 , Regulación hacia Arriba , Proteína X Asociada a bcl-2RESUMEN
The type I interferons induce an anti-viral state and suppress cell growth. The p135tyk2 non-receptor tyrosine kinase appears to initiate, at least in part, the type I interferon signal transduction pathway, and thereby activates type I interferon-dependent gene expression. To determine if p135tyk2 can suppress growth and/or tumorigenesis, derivatives of the tyk2 gene were introduced into the tumorigenic cell line Daudi. Transfectants expressing a tyk2 construct missing the carboxy-terminal 22 amino acids cloned with a greatly reduced efficiency in soft agar and displayed a partial decrease in the ability to form tumors in athymic mice. In addition, transfectants producing a kinase deficient version of tyk2 show an increase in both growth rate and agar cloning efficiency, suggesting that the inactive kinase can act in a dominant-negative manner. Surprisingly, the carboxyl-terminal deleted protein lacks both auto-kinase activity, and activity towards a putative substrate, even though it induces a phenotype which is precisely the opposite of that produced by another kinase-deficient tyk2 mutant containing an altered ATP binding site. Thus, while these results add tyk2 to a growing list of interferon-alpha regulated proteins that might be able to suppress tumor formation, the biochemical basis of this activity remains unknown.
Asunto(s)
Linfoma de Burkitt/enzimología , Interferón-alfa/farmacología , Mutación , Proteínas Tirosina Quinasas , Proteínas/genética , Animales , Northern Blotting , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , División Celular , Inducción Enzimática , Genes Supresores de Tumor , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fenotipo , Biosíntesis de Proteínas , Transducción de Señal , TYK2 Quinasa , Transfección , Células Tumorales Cultivadas/enzimología , Ensayo de Tumor de Célula MadreRESUMEN
The spectrum and pattern of p53 mutations detected in 42 cases of B-cell chronic lymphocytic leukemia (B-CLL) were analyzed, and several interesting features were noted. Codon 209 in the p53 gene may be a new hot-spot for p53 mutation in B-CLL disease. Four of the 42 (10%) reported B-CLL p53 mutations occurred at codon 209 versus none in 214 cases of other lymphoid malignancies screened for p53 mutations (P = 0.0006). Transversion mutations predominated at codon 273 rather than the transition mutations that are known to occur at this CpG site. Four of six (67%) B-CLL cases had transversions at codon 273 compared with two of 17 (12%) of all other lymphoid tumors examined (P = 0.02). In addition, over 65% of the p53 mutations detected in B-CLL showed a strand bias for p53 mutations on the untranscribed DNA strand. This feature of DNA strand bias is notable in cancers of the lung, esophagus, and head and neck, which may result from high exposure to carcinogens. This spectrum of p53 mutations in B-CLL together with the high frequency of transversion mutations and DNA strand bias may implicate environmental carcinogens associated with p53 gene damage in some B-CLL patients.
Asunto(s)
Genes p53/genética , Leucemia Linfocítica Crónica de Células B/genética , Codón , Análisis Mutacional de ADN , Humanos , Leucemia/genética , Linfoma/genética , MutaciónRESUMEN
B-cell chronic lymphocytic leukemia (B-CLL) samples were screened for alterations in multiple tumor suppressor genes (p53 (17p13), p16INK4 (9p21), and disrupted in B-cell malignancy (DBM) (13q14) by using polymerase chain reaction-based assays. Eleven percent (11 of 96) of the B-CLL cases analyzed in this study and a previous study had mutations in the p53 gene. In contrast, analysis of the p16 gene showed none of 80 B-CLL cases had mutations and five cases (6%) had homozygous deletions. Deletions of 13q14 (DBM) occurred in 18% (17 of 96) of patients surveyed. Thus, 28 of 96 cases showed an alteration in one or more of the three tumor suppressor loci examined. However, cases with p53 mutations rarely showed simultaneous loss of DBM. Our results suggest that inactivation of the tumor suppressor genes p53 and DBM may be mutually exclusive, thus providing alternate pathways for tumor development in B-CLL patients.
Asunto(s)
Eliminación de Gen , Genes Supresores de Tumor , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Secuencia de Bases , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Exones , Homocigoto , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaAsunto(s)
Anticuerpos Antineoplásicos/genética , Antígenos de Neoplasias/genética , Subgrupos de Linfocitos B/metabolismo , Antígenos CD5/genética , Células Clonales/metabolismo , Regulación Leucémica de la Expresión Génica , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina M/genética , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/patología , Anciano , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos , Subgrupos de Linfocitos B/patología , Células Clonales/patología , Reordenamiento Génico de Cadena Pesada de Linfocito B , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Persona de Mediana Edad , Mutación , Reacción en Cadena de la PolimerasaRESUMEN
N-Methylnitrosourea (NMU)-induced codon 12 Ki-ras mutations were analyzed in premalignant thymic lymphomas from C57BL/6J mice by using a selective polymerase chain reaction amplification strategy. The frequency of codon 12 Ki-ras mutations was 67% (16 of 24) in NMU-treated animals with premalignant stage I disease. Previously, animals with different stages of disease had been analyzed for cytogenetic changes and for mutations in the p53 tumor suppressor gene. The genetic changes observed were early-activating codon 12 G35-->A transition mutations of the Ki-ras gene, followed closely by trisomy 15 and infrequent mutation of the p53 gene late in tumor development. The consistent and early detection of Ki-ras mutations in NMU-treated animals but not in untreated controls suggests that the mutations result from direct carcinogen exposure. Alternate pathways of NMU-induced thymic lymphomagenesis were implicated. One pathway involved putative NMU-induced mutations in other, non-ras oncogenes that cooperate with trisomy 15 to produce similar T-cell tumors. The frequency of p53 gene mutations in human and murine T-cell tumors is similar but low.