RESUMEN
We have previously reported that about 80% of acute myeloid leukaemia (AML) samples tested at diagnosis constitutively expressed cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). The present study compared CTLA-4 expression and function of leukaemic cells from AML patients at diagnosis with those from AML patients resistant to conventional chemotherapy. We also explored the possibility of targeting CTLA-4 for apoptosis induction in chemoresistant AML cells. AML cells either from untreated patients (n = 15) or in chemoresistant phase (n = 10) were analysed for CTLA-4 protein and transcript expression by flow cytometry and reverse transcription-polymerase chain reaction respectively. CTLA-4 expression was similar in untreated and in chemoresistant samples and was not associated with patients' clinical features. In chemoresistant AML cells, CTLA-4 transduced an apoptotic signal on engagement with its recombinant ligands r-CD80 and r-CD86, which induced an average of 71% and 62% apoptotic cells, respectively, at highest concentration. Apoptosis was equally induced in untreated leukaemic cells accompanied by cleavage of procaspase-8 and -3. Thus, this study provides the first evidence that killing of leukaemic cells from AML patients may be obtained by the engagement of CTLA-4 with its ligands, opening the way to a novel potential therapeutic approach based on triggering the CTLA-4 molecule to circumvent chemoresistance in AML.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Antígenos de Neoplasias/metabolismo , Apoptosis , Leucemia Mieloide/inmunología , Adulto , Anciano , Antígenos CD/genética , Antígenos de Diferenciación/genética , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígeno CTLA-4 , Caspasas/metabolismo , Resistencia a Antineoplásicos , Femenino , Humanos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/patología , Ligandos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transcripción Genética , Células Tumorales CultivadasRESUMEN
CTLA-4 (CD152) is a cell surface receptor that behaves as a negative regulator of the proliferation and the effector function of T cells. We have previously shown that CTLA-4 is also expressed on neoplastic lymphoid and myeloid cells, and it can be targeted to induce apoptosis. In our study, we have extended our analysis and have discovered that surface expression of CTLA-4 is detectable by flow cytometry on 30 of 34 (88%) cell lines derived from a variety of human malignant solid tumors including carcinoma, melanoma, neuroblastoma, rhabdomyosarcoma and osteosarcoma (but not in primary osteoblast-like cultures). However, by reverse transcriptase-PCR, CTLA-4 expression was detected in all cell lines. We have also found, by immunohistochemistry, cytoplasmic and surface expression of CTLA-4 in the tumor cells of all 6 osteosarcoma specimens examined and in the tumour cells of all 5 cases (but only weakly or no positivity at all in neighbouring nontumor cells) of ductal breast carcinomas. Treatment of cells from CTLA-4-expressing tumor lines with recombinant forms of the CTLA-4-ligands CD80 and CD86 induced apoptosis associated with sequential activation of caspase-8 and caspase-3. The level of apoptosis was reduced by soluble CTLA-4 and by anti-CTLA-4 scFvs antibodies. The novel finding that CTLA-4 molecule is expressed and functional on human tumor cells opens up the possibility of antitumor therapeutic intervention based on targeting this molecule.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Apoptosis/fisiología , Antígenos CD/metabolismo , Antígenos de Diferenciación/fisiología , Secuencia de Bases , Antígeno CTLA-4 , Línea Celular Tumoral , Cartilla de ADN , ADN Complementario , Citometría de Flujo , Humanos , Inmunohistoquímica , Ligandos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Dlx genes constitute a gene family thought to be essential in morphogenesis and development. We show here that in vertebrate cells, Dlx genes appear to be part of a regulatory cascade initiated by acute lymphoblastic leukemia (ALL)-1, a master regulator gene whose disruption is implicated in several human acute leukemias. The expression of Dlx2, Dlx3, Dlx5, Dlx6, and Dlx7 was absent in All-1 -/- mouse embryonic stem cells and reduced in All-1 +/- cells. In leukemic patients affected by the t(4;11)(q21;q23) chromosomal abnormality, the expression of DLX2, DLX3, and DLX4 was virtually abrogated. Our data indicate that Dlx genes are downstream targets of ALL-1 and could be considered as important tools for the study of the early leukemic cell phenotype.
Asunto(s)
Antígenos de Superficie/metabolismo , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 4/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Antígenos de Superficie/genética , Cartilla de ADN/química , Regulación hacia Abajo , Genes Homeobox , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre , Factores de Transcripción/genética , Translocación GenéticaRESUMEN
We investigated a possible relationship between apoptosis induction by fenretinide (4HPR) and the expression of a group of genes thought to be essential in morphogenesis and development, the DLX genes. We analyzed their expression under normal conditions or upon 4HPR stimulation in several tumor cell lines. We show that DLX2, DLX3 and DLX4 were expressed at higher levels in cell lines which where more sensitive to apoptotic induction, whereas DLX 5 and 6 appeared to segregate in a distinct functional compartment. Our data support the notion that DLX2, 3, 4 genes could participate in the control of 4HPR-mediated apoptosis, making them important molecules for the monitoring of therapy efficacy in cancer patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Unión al ADN/genética , Fenretinida/farmacología , Proteínas de Homeodominio/genética , Neoplasias/metabolismo , Factores de Transcripción/genética , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Neoplasias/genética , Neoplasias/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/análisis , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Proteínas de Unión al ARN , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Proteína bcl-XRESUMEN
Split hand/foot malformation type I (SHFM1) disease locus maps to chromosome 7q21.3-q22, a region that includes the distal-less-related (dll) genes DLX5 and DLX6. However, incomplete penetrance, variable expressivity, segregation distortion, and syndromic association with other anomalies have so far prevented the identification of the SHFM1 gene(s) in man. Here we show that the targeted double inactivation of Dlx5 and Dlx6 in the mouse causes in homozygous mutant animals bilateral ectrodactyly with a severe defect of the central ray of the hindlimbs, a malformation typical of SHFM1. This is the first evidence that the role of dll/Dlx genes in appendage development is conserved from insects to mammals and proves their involvement in SHFM1.