RESUMEN
Drug resistance constitutes a considerable problem in the therapy of acute myeloid leukemia (AML). In order to identify genes which might be related to drug resistance, we retrospectively studied gene expression patterns in blast populations of 14 patients with de novo AML, focusing on known or potential resistance mechanisms against cytosine arabinoside and anthracyclines. Following induction and postremission chemotherapy, 7 patients achieved a complete remission (CR) for more than 1 year, while 7 patients showed blast persistence (BP) after induction and salvage chemotherapy. Gene expression analysis was performed using RNA extracted from archived guanidine extracts and Affymetrix HGU133A gene chips. We utilized the Gene Ontology category Biological Process to select genes implicated in DNA metabolism, nucleoside and nucleotide metabolism and transport, reactive oxygen species metabolism, apoptosis and response to drugs and identified 32 differentially expressed genes. From this functional perspective, we found differences between the CR and BP groups with regard to nucleotide metabolism (PBEF1, G6PD; p = 0.048), apoptosis (TNFAIP3, TNFAIP8, MPO, BCL2A1, BAX, SON, BNIP3L; p = 0.039) and reactive oxygen species metabolism (SOD2, KIAA0179; p = 0.048). However, the attempt to construct a predictive model of chemoresistance failed. BP samples had a 2-fold higher expression of CD34 than CR samples. Thus, our findings are in line with reports describing differences in apoptosis resistance between CD34+ and CD34- blast populations. Taken together, our results suggest that drug resistance in AML is a heterogenous phenomenon that might be better defined by means of disturbed biological processes than by focusing on the alteration of the expression of distinct genes.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Leucemia Mieloide/genética , Proteínas de Neoplasias/biosíntesis , Células Madre Neoplásicas/metabolismo , Enfermedad Aguda , Adolescente , Adulto , Antígenos CD34/análisis , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/química , Nucleósidos/metabolismo , Nucleótidos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Inducción de Remisión , Terapia Recuperativa , Superóxidos/metabolismo , Resultado del TratamientoAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mieloma Múltiple/inmunología , Trasplante de Células Madre de Sangre Periférica , Terapia Combinada , Relación Dosis-Respuesta a Droga , Humanos , Inmunidad Celular/inmunología , Mieloma Múltiple/tratamiento farmacológico , Trasplante AutólogoRESUMEN
The present study aimed to evaluate the side-effects and efficacy of thalidomide in combination with an anthracycline-containing chemotherapy regimen in previously untreated myeloma patients. Thalidomide (400 mg/d) was combined with bolus injections of vincristine and epirubicin and oral dexamethasone (VED). Chemotherapy cycles were repeated every 3 wk until no further reduction in myeloma protein was observed, whereas the treatment with thalidomide was continued until disease progression. Thirty-one patients were enrolled, 12 patients were exclusively treated with thalidomide in combination with VED and 19 patients additionally received high-dose melphalan, for consolidation. Adverse events and response to therapy were assessed prior to treatment with high-dose chemotherapy. Response to thalidomide combined with VED was complete remission in six patients (19%), partial remission in 19 patients (61%), stable disease in five patients (16%), and progressive disease in one patient (3.2%). Grade 3 and 4 adverse events consisted of leukocytopenia in 10 patients (32%), and thrombocytopenia and anemia in one patient each (3.2%). Neutropenic infections grade 3 and 4 occurred in seven (23%) and three patients (9.7%), respectively, including two patients (6.5%) who died from septic shock. Deep vein thrombosis occurred in eight patients (26%), constipation in 20 patients (65%), and polyneuropathy in 20 patients (65%). The probability of event-free survival and overall survival in the whole group of patients at 36 months were 26 and 62%, respectively. In conclusion, the combination of thalidomide with VED appears to be highly effective in previously untreated patients with multiple myeloma, but it is associated with a high rate of thrombotic events, polyneuropathy, and neutropenic infections.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Mieloma Múltiple/tratamiento farmacológico , Talidomida/administración & dosificación , Adulto , Anciano , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/efectos adversos , Dexametasona/administración & dosificación , Epirrubicina/administración & dosificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Inducción de Remisión , Talidomida/efectos adversos , Vincristina/administración & dosificaciónRESUMEN
We have used a new single-step real-time reverse transcription polymerase chain reaction (RT-PCR) method to quantify BCR-ABL transcripts, thereby estimating the relapse stage in chronic myeloid leukaemia patients after allogeneic transplants. In 402 samples from 172 patients, BCR-ABL expression was determined and normalized, using the GAPDH housekeeping gene product as an endogenous reference. In our real-time RT-PCR assay, serial dilutions of RNA of the K562 cell line remained positive down to 7.5 pg. The median normalized BCR-ABL amount differed significantly (P < 0.001) between the various disease stages and was 0.06% (range 0.001-1.55%), 3.2% (range 1.4-5.6%) and 21.5% (range 6.8 -827%) in 17 patients with a molecular relapse, in eight patients with a cytogenetic relapse and in 10 patients with a haematological relapse respectively.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Trasplante de Células Madre Hematopoyéticas , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/cirugía , Adolescente , Adulto , Análisis Citogenético , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , Recurrencia , Trasplante HomólogoRESUMEN
The detection of chimerism, residual molecular and cytogenetic disease following transplantation of peripheral blood stem cells (PBSCT) with a nonmyeloablative conditioning (n = 9) and the transplantation of highly purified CD34(+) stem cells (CD34(+) PBSCT) (n = 16) were compared to unmanipulated bone marrow transplantation (BMT) (n = 69) and unmanipulated PBSCT (n = 50) after myeloablative conditioning in patients with first chronic phase of chronic myelogenous leukemia (CML) (n = 137), second chronic phase of CML (n = 4), acute lymphoblastic leukemia (n = 2) and acute myeloid leukemia (n = 1). A molecular relapse (MR) as defined by two consecutive positive polymerase chain reaction assays for the detection of M-bcr-abl transcripts (n = 141) and cbfbeta-myh11 transcripts (n = 1) in a 4-week interval was found in 10 of 16 patients (63%) after CD34(+) PBSCT, and in 27 of 69 patients (39%) after BMT, whereas only three of 50 patients (6%) after PBSCT (P < 0.001) and one of eight patients (13%) after PBSCT with reduced conditioning suffered from a MR. A cytogenetic relapse occurred in five of 16 patients (31%) after CD34(+)PBSCT and 21 of 69 patients (30%) after BMT (NS) compared to two of 50 patients (4%) after PBSCT and none of the eight patients after PBSCT with reduced conditioning (P < 0.05). The lowest treatment-related mortality was seen in the 16 patients after CD34(+) PBSCT, who are all currently alive with a median follow-up of 15 months, whereas the survival rate for BMT, PBSCT and PBSCT with reduced conditioning were 65%, 63% and 58%, respectively. Multivariate analysis including all potential influential factors of post-transplant residual disease recurrence showed that patients after CD34(+) PBSCT had a significantly higher risk (two times) to develop a MR than patients after BMT (P < 0.03), whereas patients after unmanipulated PBSCT had a significant lower risk (eight times) for the occurrence of a MR post transplant (P < 0.001). Patients after BMT and CD34(+) PBSCT had the lowest rates of complete chimerism (CC) at 3 months after transplant. Only five of nine patients (55%) after CD34(+) PBSCT and 19 of 33 patients (58%) after BMT achieved CC compared to 19 of 22 (86%) patients after PBSCT and seven of eight (88%) patients after PBSCT with reduced conditioning (P < 0.05).
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Quimera por Trasplante/sangre , Análisis Actuarial , Adolescente , Adulto , Antígenos CD34 , Trasplante de Médula Ósea , Análisis Citogenético , Femenino , Proteínas de Fusión bcr-abl/genética , Trasplante de Células Madre Hematopoyéticas/normas , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Masculino , Persona de Mediana Edad , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , ARN Mensajero/análisis , Inducción de Remisión , Tasa de Supervivencia , Secuencias Repetidas en Tándem , Acondicionamiento Pretrasplante , Trasplante Homólogo/métodos , Trasplante Homólogo/normasRESUMEN
The feasibility of transplantation using highly purified G-CSF-mobilized peripheral blood CD34+ cells from HLA-identical sibling donors without prophylactic post-transplant immunosuppression was prospectively studied in 10 adult first chronic phase chronic myeloid leukemia (CML) patients with special reference to graft engineering performance and follow-up studies of minimal residual disease and immune reconstitution. CD34+ cells were enriched by clinical-scale magnetic-activated cell separation (MACS) using iron-dextran beads bound to monoclonal anti-CD34 antibody. Grafts contained a median of 9.7 (range 1.7-16.6) x 10(6) CD34+ cells per kilogram of recipient body weight with a purity between 94.5% and 98.3% (median 97.2%). The median number of transfused CD3+ T lymphocytes was 1.0 (range 0.5-8.5) x 10(4)/kg, corresponding to a log10 T lymphocyte depletion between 3.8 and 5.0 (median 4.6). All patients engrafted rapidly with a median duration to neutrophil counts >500/microl of 8 (range 8-19) days and to self-sustaining platelet counts >20,000/microl of 12 (range 9-25) days. Isolated skin acute graft-versus-host disease (GVHD) of stages I to II occurred in three patients. One patient developed secondary graft failure and was successfully salvaged by an unmanipulated blood stem cell graft from the same donor. All 10 patients are surviving in complete hematologic, cytogenetic and molecular remission (four patients after donor lymphocyte infusions) between 12 and 22 (median 16) months post transplant. In conclusion, transplantation of MACS-purified blood CD34+ cells from HLA-identical sibling donors in adult CML patients appears safe, effectively prevents acute GVHD without prophylactic post-transplant immunosuppression, and is capable of inducing complete cytogenetic and molecular remissions.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Terapia de Inmunosupresión , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Adolescente , Adulto , Femenino , Enfermedad Injerto contra Huésped/etiología , Prueba de Histocompatibilidad , Humanos , Separación Inmunomagnética , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Masculino , Persona de Mediana Edad , Neoplasia Residual , Estudios ProspectivosRESUMEN
The use of the real-time reverse-transcription polymerase-chain reaction (RT-PCR) method to quantify BCR-ABL transcripts before and after allogeneic transplant was prospectively studied in 65 patients with chronic myeloid leukemia (CML). The expression of the BCR-ABL transcript was determined and normalized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene product as an endogenous reference. In the single step real-time PCR assay, tenfold serial dilutions of cDNA of the K5652 cell line remained positive down to 100 pg cDNA only. However, molecular relapses of CML after transplant were only safely detectable when a nested real-time PCR assay was performed, which was able to detect 1-10 pg cDNA from a tenfold serial dilution. The median normalized BCR-ABL transcript level was measured as 0.004% in 17 patients with a molecular relapse, 0.4% in 7 patients with a cytogenetic relapse, 2.6% in 36 patients with a stable phase of CML, and 36% in 5 patients with a relapse in a blast crisis. The analyzed median normalized amount of BCR-ABL transcript differed significantly (P<0.001) between the various disease stages. In ten CML patients with relapse, the real-time PCR method was used to monitor the response of various immunotherapies as donor leukocyte infusions, withdrawal of immunosuppression, or interferon-alpha application. The results of the quantitative evaluation of BCR-ABL transcripts reflected very well the clinical effect of the different applied immunotherapies. The new real-time PCR method seems to be a suitable technique for the early detection of relapse after allogeneic transplant in patients with the BCR-ABL transcript. Its ability to distinguish between molecular and cytogenetic relapse (P<0.001) allows early therapeutic decisions.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mieloide de Fase Crónica/genética , Trasplante de Médula Ósea , Trasplante de Células Madre Hematopoyéticas , Humanos , Recurrencia Local de Neoplasia , ARN Mensajero/análisis , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y EspecificidadRESUMEN
PURPOSE: Although gene transfer with retroviral vectors has already been applied to patients as part of clinical protocols, low expression of transgenes in target cells still remains a problem. Therefore, we compared various retroviral vectors using different promoters and backbones for expression of the enhanced green fluorescent protein (EGFP) reporter gene in fibroblasts and CD34+ cells. METHODS: The N2A retroviral vector was used to test expression from the herpes simplex virus thymidine kinase promoter (vector N2A-TK-EGFP), a human phosphoglycerate kinase promoter (vector N2A-PGK-EGFP), and the SV40 promoter (vector N2A-SV-EGFP). Additional constructs used the spleen focus-forming virus (SFFV) long terminal repeat (LTR) as promoter and expressed EGFP alone (vector SFbeta1-EGFP) or EGFP and a downstream (vector SFbeta1-EGFP-IRES) or upstream (vector SFbeta1-IRES-EGFP) internal ribosomal entry site. RESULTS: For NIH 3T3 cells the fluorescence-activated cell sorting analysis revealed that the most active internal promoter was the SV40 promoter in the vector N2A-SV-EGFP (mean fluorescence intensity, MFI, 66.7 +/- 0.4), followed by N2A-PGK-EGFP (26.3 +/- 1.8 MFI), and N2A-TK-EGFP (4.8 +/- 0.1 MFI). Expression from the SFbeta1-EGFP vector (82.6 +/- 6.7 MFI) and the SFbeta1-EGFP-IRES vector (102.8 +/- 6.2 MFI) was higher than from SFbeta1-IRES-EGFP vector (15.5 +/- 1.8 MFI). In human CD34-positive cells, the EGFP expression from all vectors was considerably lower than in fibroblasts with the SFbeta1-EGFP vector still being four- to fivefold more active than the internal promoters tested. CONCLUSION: The SFFV LTR seems to allow a high expression of transgenes, as long as the transgene is not expressed downstream of an internal ribosomal entry site. Internal promoters may be useful for targeted gene expression in specific cell types, but the reduced level of expression from some internal promoters has to be taken into consideration.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/genética , Proteínas Luminiscentes/genética , Regiones Promotoras Genéticas , Retroviridae/genética , Células 3T3 , Animales , Antígenos CD34/metabolismo , Regulación hacia Abajo , Fibroblastos/inmunología , Fibroblastos/metabolismo , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas , Humanos , Indicadores y Reactivos , Ratones , Neoplasias/genética , Neoplasias/terapia , Fosfoglicerato Quinasa/genética , Virus 40 de los Simios/genética , Simplexvirus/enzimología , Timidina Quinasa/genética , Transducción GenéticaRESUMEN
A crucial function of the BCR-ABL chimeric gene in chronic myeloid leukemia is the prolongation of cell survival by inhibition of apoptosis. BCR-ABL expression confers cross-resistance to multiple genotoxic anticancer drugs by inhibition of the apoptotic response to DNA damage in association with cell cycle arrest at the G2-M restriction point. Previous reports indicated that BCR-ABL exerts its antiapoptotic effect against various apoptotic stimuli upstream to the cleavage and activity of caspase-3. Here we show that the adenovirus E1A protein induces substantial apoptosis in BCR-ABL expressing K562 and LAMA-84 leukemia cells. This apoptotic activity of E1A is accompanied by processing of caspase-3 and cleavage of poly(ADP-ribose) polymerase and can be significantly blocked by z-VAD-fmk Z-Val-Ala-Asp(OCH3)-CH2F and the caspase-3-specific inhibitor Z-DEVD-FMK Z-Asp(OCH3)-Glu-Val-Asp(OCH3)-CH2F. Moreover, E1A renders K562 cells, which are particularly resistant to cell death irrespective of the inducing agent, susceptible to induction of apoptosis by the chemotherapeutic agents etoposide and daunorubicin. Counteracting the DNA damage-induced inactivation of cdc2 kinase, E1A reverses the drug-induced G2-M arrest These results indicate that solitary delivery of E1A significantly antagonizes BCR-ABL-induced antiapoptotic functions and circumvents the inherent resistance to DNA damage-induced apoptosis, supporting the use of E1A in combination with chemotherapeutic agents as a promising therapeutic strategy for successful treatment of Philadelphia chromosome-positive leukemia in vivo.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Apoptosis/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Leucemia/metabolismo , Antibióticos Antineoplásicos/farmacología , Western Blotting , Proteína Quinasa CDC2/metabolismo , Caspasa 3 , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , División Celular , Daño del ADN/efectos de los fármacos , Daunorrubicina/farmacología , Relación Dosis-Respuesta a Droga , Etopósido/farmacología , Citometría de Flujo , Humanos , Células K562 , Leucemia/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Luciferasas/metabolismo , Microscopía de Contraste de Fase , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Péptidos/farmacología , Plásmidos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Adenoviral E1 A proteins exhibit a strong tumor-suppressive activity in human tumor cells. However, E1 A is capable of transforming rodent and human cells in cooperation with other oncoproteins, such as activated RAS. Thus, the therapeutic use of wild-type E1A harbors the principal risk of enhancing tumor malignancy. This prompted us to construct E1A 13S cDNA-derived mutants that were unable to transform baby mouse kidney cells in cooperation with E1B and to test their tumor-suppressive activity in BLM human melanoma cells. Anchorage-independent growth in soft agar was reduced for those cell lines expressing the E1AdelCR2 mutant, which lacks the entire conserved region 2 (CR2) sequences, or for cells expressing the E1AcR3Ex2 mutant, which contains CR3 plus exon 2 sequences. In contrast, cell lines expressing the entire E1A wild-type (E1AWT) or only the exon 2 sequences (E1AEx2) grew like the parental BLM cells. Moreover, inoculation of nude mice with BLM cells or cells expressing E1AEx2 revealed large tumors after 2 weeks. In contrast, tumors derived from E1AdelCR2- or E1ACR3Ex2-expressing cells exhibited a substantial delay in tumor growth accompanied by a loss of E1A expression in the outgrown tumors. Cell lines expressing E1AWT showed an intermediate phenotype. Thus, expression of CR3 plus exon 2 sequences is sufficient to enhance both the antioncogenic properties and the therapeutic safety of E1A in our system.
Asunto(s)
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Transformación Celular Neoplásica/genética , Terapia Genética , Melanoma Experimental/terapia , Proteínas E1A de Adenovirus/metabolismo , Animales , Western Blotting , ADN de Neoplasias/análisis , Virus Defectuosos , Genes Supresores de Tumor , Humanos , Melanoma Experimental/metabolismo , Ratones , Ratones Desnudos , Reacción en Cadena de la Polimerasa , Transfección , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
Hemizygous deletions of the fragile histidine triad (FHIT) gene at human chromosome band 3p14.2 and down-regulation of its gene product are found in the majority of renal cell carcinomas (RCCs). Functional tumor suppressive activity of Fhit in renal cancer cells previously was observed in RCC cell line RC48, which lacks endogenous Fhit expression. To further investigate the potential role of FHIT as a tumor suppressor gene in RCC, we transfected FHIT cDNA expression constructs into RCC cell lines RCC-1 and SN12C, which show low-level expression of endogenous Fhit and reveal an intact von Hippel-Lindau (VHL) gene. Stable transfectants of both cell lines showed no alterations of cell morphology, proliferation kinetics, or cell cycle parameters in vitro. The FHIT gene transfer rate, however, was significantly lower in RCC-1 cells compared with SN12C cells, suggesting a selection against exogenous Fhit expression. In addition, in nude mouse assays, a significant delay of tumor formation was observed for FHIT-transfected RCC-1 cell lines, with outgrowing tumors demonstrating loss of Fhit expression in the majority of cells. In contrast, tumorigenicity of FHIT-transfected SN12C cell clones was not suppressed, despite stable transgene expression. In conclusion, our results demonstrate a selective tumor suppressive activity of Fhit in RCC cells in vivo and suggest that the susceptibility to suppression is not restricted to cancer cells with complete loss of Fhit expression.
Asunto(s)
Ácido Anhídrido Hidrolasas , Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Proteínas de Neoplasias , Proteínas/genética , Proteínas/metabolismo , Supresión Genética , Animales , Western Blotting , Ciclo Celular/genética , Cromosomas Humanos Par 3 , ADN Complementario/metabolismo , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Transfección , Células Tumorales CultivadasRESUMEN
Gene amplification is frequently present in human tumors, although specific target genes relevant to many amplified loci remain unidentified. An expression cloning assay enabled identification of a candidate oncogene derived from human chromosome 3p14.1. The cDNA retrieved from morphologically transformed cells contained the full-length protein coding region and detected an abundant transcript in the same cells. Sequence analysis revealed identity with the wild-type sequence of p44S10, a highly conserved subunit of the 26S proteasome that exhibits similarity to the Arabidopsis fus6/cop11 family of signaling molecules. p44S10 gene copy number and mRNA expression were increased in association with segmental 1.8 - 11-fold chromosomal gains in cutaneous malignant melanoma cell lines (5/13; 40%) and tumors (2/40; 5%), and in breast cancer MCF-7 cells. Likewise, malignant progression of human radial growth phase WM35 melanoma cells was associated with amplification and increased expression of endogenous p44S10, and increased expression of p44S10 was sufficient to induce proliferation of WM35 cells in vivo. The results demonstrate segmental copy number gains within chromosome 3p in cutaneous malignant melanoma and suggest that deregulation of a proteasome regulatory particle subunit may contribute to the malignant phenotype.
Asunto(s)
Adenosina Trifosfatasas/genética , Cisteína Endopeptidasas/genética , Amplificación de Genes , Melanoma/enzimología , Melanoma/genética , Complejos Multienzimáticos/genética , Proteínas Oncogénicas/genética , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Animales , Línea Celular Transformada , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 20 , Cromosomas Humanos Par 3 , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Progresión de la Enfermedad , Activación Enzimática/genética , Humanos , Melanoma/patología , Melanoma Experimental/enzimología , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Proteínas Oncogénicas/aislamiento & purificación , Proteínas Oncogénicas/metabolismo , Péptido Hidrolasas/genética , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Ratas , Análisis de Secuencia de ADN , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
The purpose of this study was to evaluate the long-term outcome of interferon (IFN) alfa treatment in patients with Philadelphia chromosome-positive chronic myeloid leukemia (CML). Between 1984 and 1990, a total of 71 patients with newly diagnosed CML had been enrolled into two consecutive IFN trials at our institution. Follow-up extended to December 1998, resulting in a median observation period for surviving patients of 11.4 years. The median survival time from diagnosis was 5.9 years. A plateau in the actuarial survival curve was found from 8.2 to 12.3 years following diagnosis with a projected 10-year survival rate of 32%. 'Landmark' studies showed a significant survival advantage for patients with karyotype responses. Of 68 patients accessible to calculation of the Hasford score, three were in the high risk group, 24 belonged to the medium risk group, and 41 had low risk features. The majority of cytogenetic responders including all eight assessable patients in complete cytogenetic remission were in the low risk group. Achieving a cytogenetic remission was found to provide a survival advantage also for patients with low risk disease. Of the seven patients surviving more than 11 years, six were in continuous complete cytogenetic remission. Their favorable outcome appears to translate into an out-flattening of the survival curve for the 71 single center patients presented. It will be of interest to see whether prolonged follow-ups of the large multicentric randomized trials will similarly show a subset of long-term surviving patients with ongoing IFN-induced remission.
Asunto(s)
Antineoplásicos/uso terapéutico , Factores Inmunológicos/uso terapéutico , Interferón-alfa/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Adulto , Antineoplásicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Crisis Blástica/epidemiología , Femenino , Estudios de Seguimiento , Humanos , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/efectos adversos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/efectos adversos , Interferón gamma/administración & dosificación , Interferón gamma/uso terapéutico , Cariotipificación , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Tablas de Vida , Masculino , Cromosoma Filadelfia , Pronóstico , Proteínas Recombinantes , Inducción de Remisión , Riesgo , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Consistent deletion of DNA sequences in chromosomal band 3p21 observed in a variety of human tumors suggests the presence of one or more tumor suppressor genes within this region. Previously, we reported on the construction of two distinct cosmid contigs and our identification of several new genes within 3p21.1. In our search for tumor suppressor genes from this region, we have cloned a gene that we have called DRR 1 (downregulated in renal cell carcinoma). The gene was first mapped to 3p21.1 by fluorescence in situ hybridization analysis. Further analysis of yeast artificial chromosome clones in 3p14.2-p21.1 refined its localization. DRR 1 spans about 10 Kb of genomic DNA with a 3.5-Kb mature transcript. The putative protein encoded by this gene is 144 amino acids and includes a nuclear localization signal and a coiled domain. The gene showed loss of expression in eight of eight renal cell carcinoma cell lines, one of seven ovarian cancer cell lines, one of one cervical cancer cell line, one of one gastric cancer cell line, and one of one non-small-cell lung cancer cell line. Southern blot analysis did not show any altered bands, indicating that gross structural changes or deletions did not cause the loss of expression. This gene was also found to have reduced expression in 23 of 34 paired primary renal cell carcinomas. Mutational analysis detected three polymorphic sites within the gene, but no point mutations were identified in the 34 primary tumors. However, we did detect base substitutions in 4 of 12 cell lines that had undetectable expression of the gene. We also transfected the gene into DRR 1-negative cell lines and observed clear growth retardation. Our results suggest that loss of expression of the DRR 1 gene may play an important role in the development of renal cell carcinoma and possibly other tumors. Genes Chromosomes Cancer 27:1-10, 2000.
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Carcinoma de Células Renales/genética , Cromosomas Humanos Par 3/genética , Genes Supresores de Tumor , Neoplasias Renales/genética , Proteínas Nucleares , Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Carcinoma de Células Renales/metabolismo , Mapeo Cromosómico , Clonación Molecular , Análisis Mutacional de ADN , ADN Complementario , Regulación hacia Abajo/genética , Femenino , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Neoplasias Renales/metabolismo , Datos de Secuencia Molecular , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Biosíntesis de Proteínas , Proteínas/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
It has been shown repeatedly that exposure of G(1) cells unifilarily labelled with 5-bromodeoxyuridine (BrdU) to X-rays leads to sister chromatid exchanges (SCE) when the cells are allowed to grow for one further cycle in the absence of BrdU. It has been suggested that damage induced by ionizing radiation does not lead to 'true' SCE and that the observed SCE are 'false', resulting from structural chromosomal aberrations, especially interstitial inversions. We used a painting probe for the p14 region of human chromosome 3 and anti-BrdU antibodies to analyse the frequency of radiation-induced SCE in that chromosome. This method allowed us to discriminate between para- and pericentric 'true' and 'false' SCE. Our results indicate that most radiation-induced SCE do not result from inversions.
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Inversión Cromosómica , Cromosomas Humanos Par 3 , Linfocitos/efectos de la radiación , Intercambio de Cromátides Hermanas , Adolescente , Bromodesoxiuridina/análisis , Células Cultivadas , Cromosomas Humanos Par 3/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Fase G1 , Humanos , Linfocitos/metabolismo , MasculinoRESUMEN
BACKGROUND: Alterations of chromosome region 3p14 are observed in numerous human malignancies. Because the pattern of allelic losses suggests the existence of at least one tumor suppressor gene within this region, we established a library of yeast artificial chromosomes (YACs) containing contiguous human 3p14 sequences to permit a search for tumor suppressor loci within the 3p14 region by use of functional complementation. METHODS: YACs specific for human chromosome region 3p14 were transduced by spheroplast fusion into cells of the human nonpapillary renal carcinoma cell line RCC-1, which shows a cytogenetically detectable 3p deletion and is tumorigenic in nude mice. RESULTS: We identified a 3p14.2-specific YAC clone, located in the vicinity of the fragile histidine triad (FHIT) gene (but toward the telomere), that is capable of inducing sustained suppression of tumorigenicity in nude mice and of activating cellular senescence in vitro. Among 23 mice given injections of RCC-1 cells containing this YAC, 16 (70%) remained tumor free for at least 6 months, whereas tumor formation occurred after a median of 6 weeks in control mice given injections of either RCC-1 parental cells or a revertant cell line (in which the YAC had lost all human sequences) or RCC-1 parental cells containing other, unrelated YACs. Similar results were obtained following microcell-mediated transfer of the entire human chromosome 3. CONCLUSION: These data provide strong evidence for the existence of a novel tumor suppressor locus adjacent to the previously identified candidate tumor suppressor gene, FHIT, in 3p14.2. Positional cloning of the novel suppressor element within the 3p14.2-specific YAC and the sequence's molecular and functional characterization should add to the understanding of the pathogenesis of renal cell carcinoma and other human tumors that exhibit 3p14 aberrations.
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Carcinoma de Células Renales/genética , Cromosomas Humanos Par 3 , Genes Supresores de Tumor , Neoplasias Renales/genética , Animales , Senescencia Celular , Cromosomas Artificiales de Levadura , Técnicas de Transferencia de Gen , Prueba de Complementación Genética , Histidina/genética , Humanos , Pérdida de Heterocigocidad , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Tumorales CultivadasAsunto(s)
Cromosomas Humanos Par 3/genética , Proteínas Contráctiles/genética , Proteínas de Microfilamentos/genética , Mapeo Cromosómico , Cromosomas Artificiales de Levadura/genética , Cartilla de ADN/genética , Filaminas , Humanos , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa , Transcripción GenéticaRESUMEN
The detection of residual molecular and cytogenetic disease was prospectively compared in patients with Philadelphia-chromosome (Ph1) positive first chronic phase chronic myelogenous leukemia (CML) who underwent allogeneic transplantation of unmanipulated peripheral blood stem cells (PBSCT) (n = 29) or bone marrow (BM) (n = 62) using genotypically HLA-identical sibling donors or partially HLA-matched extended family donors. A molecular relapse (MR), as defined by two consecutive positive polymerase chain reaction (PCR) assays for the detection of M-bcr-abl transcripts in a 4-week interval, was found in two of 29 (7%) patients after PBSCT compared with 20 of 62 (32%) patients after bone marrow transplantation (BMT). This corresponds to a 4-year molecular relapse estimate (+/- standard error) of 7% +/- 5% after PBSCT and of 44% +/- 8% after BMT (P <.009). With identical follow-up periods of survivors in both patient subsets between 6 and 55 months (median, 28 months), 14 of the 20 patients with MR after BMT progressed to an isolated cytogenetic (n = 10) or a hematologic (n = 4) disease recurrence, resulting in a 4-year cytogenetic relapse estimate of 47% +/- 11%, while none of the patients after PBSCT has so far relapsed (P <.006). Multivariate analysis including all potential influencial factors of posttransplant disease recurrence identified the source of stem cells (P <.02) as the only independent predictor of molecular relapse. In conclusion, this prospective comparison of molecular and cytogenetic residual disease demonstrates that peripheral blood stem cell transplants have a more pronounced activity against residual CML cells than bone marrow transplants. Prospective randomized trials comparing PBSCT and BMT in patients with first chronic phase Ph1-positive CML are strictly required to further substantiate differences in the antileukemic activity of the two stem cell sources.
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Trasplante de Médula Ósea , Trasplante de Células Madre Hematopoyéticas , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Leucemia Mieloide de Fase Crónica/terapia , Trasplante Homólogo , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/sangre , Niño , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Proteínas de Fusión bcr-abl/sangre , Alemania/epidemiología , Humanos , Incidencia , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mieloide de Fase Crónica/sangre , Leucemia Mieloide de Fase Crónica/patología , Masculino , Persona de Mediana Edad , Neoplasia Residual , Células Madre Neoplásicas/química , Células Madre Neoplásicas/ultraestructura , Estudios Prospectivos , Recurrencia , Riesgo , Acondicionamiento Pretrasplante , Resultado del TratamientoRESUMEN
The demonstration of the Philadelphia (Ph) chromosome in B lymphocytes from patients with chronic myelogenous leukemia (CML) has provided evidence that the disorder originates in a pluripotent progenitor cell. Divergent results, however, exist as to the degree of contribution of clonally derived cells to the B-cell compartment. To address this issue, B lymphocytes were selected from the blood of seven patients in the chronic phase of Ph-positive CML and were examined with dual-color fluoresence in situ hybridization for the presence of the Ph translocation. The purity of the B-cell preparations ranged from 88% to 97% (mean 93%). The Ph translocation was detected in 22-34% (mean, 27%) of the sorted B cells. There was no evidence that the duration of the disease affects the ratio of Ph-positive and -negative B cells. In summary, clonally derived circulating B lymphocytes were present in all patients studied but made only minor contribution to this compartment.
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Linfocitos B/patología , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Adulto , Antígenos CD19/biosíntesis , Linfocitos B/química , Células Clonales/patología , Femenino , Proteínas de Fusión bcr-abl/genética , Humanos , Hibridación Fluorescente in Situ/métodos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Antígenos Comunes de Leucocito/biosíntesis , MasculinoRESUMEN
Immortalization and tumorigenic transformation of many human cell types, including human uroepithelial cells (HUCs), are frequently associated with loss of genetic material from the short arm of chromosome 3 (3p). In addition, losses of 3p have been observed in many human cancers including renal cell carcinoma, lung cancer, breast cancer, and bladder cancer. Genetic studies suggest that there are at least two regions on 3p in which tumor suppressor genes might be located, but the precise location of these genes is not known. We studied chromosome 3 losses that were specifically associated with immortalization of five independent human papilloma virus 16 (HPV16) E6- or E7-transformed HUCs. Cytogenetic analysis showed that the smallest common region of deletion was 3p14.1-->14.2. Fluorescence in situ hybridization using a 3p13-->14-specific yeast artificial chromosome (YAC) contig showed the precise localization of the breakpoints to be in 3p13 and 3p14.2, thus defining the smallest common overlap of 3p deletions in HPV16 E6- or E7-immortalized HUCs. These results suggest the presence in this region of genes involved in the control of senescence in vitro and possibly tumorigenesis in vivo.