Assuntos
Proteínas de Bactérias , Cromossomos Humanos Par 17 , Genes p53/genética , Íntrons/genética , Polimorfismo de Fragmento de Restrição , Sequência de Bases , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Reação em Cadeia da Polimerase , População Branca/genéticaRESUMO
With the perspective of bone marrow purging in autologous transplantation, we investigated the cytotoxicity of the anti-T cell immunotoxin (IT) WT1-ricin A (anti-CD7) to malignant T cells obtained from patients with T cell acute lymphoblastic leukaemia or lymphoma. The cytotoxic efficacy of IT was based on the extent of protein synthesis inhibition. Cytotoxicity of IT to malignant T cells showed a dependency on antigen density comparable to the T cell lines GH1, CEM, Jurkat, HSB-2 and HPB-ALL and was enhanced considerably in the presence of 6 mM ammonium chloride. The ultimate proof of cell kill can only be obtained from clonogenic assays; however, culturing of malignant T cells was not feasible. Therefore these assays were performed with the cell line CEM that expresses comparable amounts of CD7 antigen as malignant T cells of most patients. More than 6-logs of CEM appeared to be eliminated after incubation with 10(-8) M WT1-ricin A. Immunotoxins are only effective after entering the target cell. The pattern of internalization of the IT was determined by means of 125I-WT1. After internalization the CD7 antigen was re-expressed on the cell membrane. This enables a long incubation period resulting in an increased elimination of malignant T cells. Even after 16 h the IT was still accumulated intracellularly. This pattern of continuous uptake of IT was reflected in a gradually increasing cytotoxicity with incubation time. Effective bone marrow purging can be carried out without adverse effects on progenitor cells with 10(-8) M WT1-ricin A. At that concentration the antibody binding capacity was saturated. We showed that the protein synthesis inhibition in malignant T cells by WT1-ricin A is comparable to the inhibition in T cell lines and that high amounts of CEM cells can be killed. These data suggest that cell lines can be used to test the efficacy of IT to malignant T cells. WT1-ricin A appears to be very potent for the purging of autologous bone marrow from patients with T cell malignancies.
