Bcr-Abl-mediated resistance to apoptosis is independent of constant tyrosine-kinase activity.

Bcr-Abl is one of the most potent antiapoptotic molecules and is the tyrosine-kinase implicated in Philadelphia (Ph) chromosome-positive leukemia. It is still obscure how Bcr-Abl provides the leukemic cell a strong resistance to chemotherapeutic drugs. A rational drug development produced a specific inhibitor of the catalytic activity of Bcr-Abl called STI571. This drug was shown to eliminate Bcr-Abl-positive cells both in vitro and in vivo, although resistant cells may appear in culture and relapse occurs in some patients. In the study described here, Bcr-Abl-positive cells treated with tyrosine-kinase inhibitors such as herbimycin A, genistein or STI571 lost their phosphotyrosine-containing proteins, but were still extremely resistant to apoptosis. Therefore, in the absence of tyrosine-kinase activity, Bcr-Abl-positive cells continue to signal biochemically to prevent apoptosis induced by chemotherapeutic drugs. We propose that secondary antiapoptotic signals are entirely responsible for the resistance of Bcr-Abl-positive cells. Precise determination of such signals and rational drug development against them should improve the means to combat Ph chromosome-positive leukemia.

HIV-1 long terminal repeat modulation by glucocorticoids in monocytic and lymphocytic cell lines.

Glucocorticoid gene regulation can be carried out through direct binding of glucocorticoid receptor to glucocorticoid responsive elements (GRE), regulating directly gene transcription and modulating some signaling pathways. The human immunodeficiency virus type 1 (HIV-1) expression can be activated by different immunomodulators through binding of particular nuclear factors to its long terminal repeat (LTR). In order to investigate the effect of glucocorticoids in pathways that activate HIV-1 expression, we transfected promonocyte (U937) and T lymphocyte (CEM-T4) cell lineages with a plasmid containing the chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 LTR. In U937 cells, dexamethasone (DEX) downregulates CAT expression induced by either phorbol myristate acetate (PMA), tumor necrosis factor alpha (TNFalpha) or granulocyte/macrophage-colony stimulating factor (GM-CSF). In CEM-T4 cells the CAT activity was slightly upregulated by DEX following the induction by either PMA or TNFalpha. Interestingly, in both cell lines transactivation of this reporter gene by transactivator protein (TAT) was downregulated by DEX. When the CAT gene was under control of HIV-1 enhancer isolated from its LTR background, the CAT activity induced by PMA was not affected by the presence of glucocorticoids. In all experiments, comparable data were obtained when DEX was replaced by hydrocortisone (HC). Our results show that, depending on the cell line, glucocorticoids can differently affect HIV-1 expression, probably by interfering in cellular pathways involved in virus expression. Moreover, the target of this regulation in LTR is probably not the enhancer region itself.

Identification of the site in the Syk protein tyrosine kinase that binds the SH2 domain of Lck.

The Syk protein tyrosine kinase (PTK) is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. Upon cross-linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues and enzymatically activated. We and others have found that the Lck kinase, a member of the Src family of PTKs, binds through its Src homology (SH) 2 domain to tyrosine phosphorylated Syk and to the related Zap kinase. Here we report that this interaction is direct and identify the two tandem tyrosines at the autophosphorylation site of Syk, Tyr518, and Tyr519, as the binding site for the SH2 domain of Lck. Mutation of either or both tyrosines to phenylalanines abrogated binding, while mutation of a second repetition of the motif at Tyr539 and Tyr540, or of the three tyrosines in the C terminus of Syk, did not. The SH2 domain of Lck bound the autophosphorylation site only when both Tyr518 and Tyr519 were phosphorylated. In intact cells the binding of the SH2 domain of Lck correlated with the ability of Syk to induce tyrosine phosphorylation of cellular proteins.

Normal expression of the serologically defined H-Y antigen in Leydig cell hypoplasia.

H-Y antigen, the proposed inducer of testicular organogenesis, was determined serologically in 3 patients with male pseudohermaphroditism due to Leydig cell hypoplasia, a pathological model with lack of Leydig cell differentiation but normal seminiferous tubule embryogenesis. One patient was the offspring of consanguineous parents and 2 siblings presented as women with a lack of breast development and primary amenorrhea. Gonads were palpable in the inguinal canal, except for the right intra-abdominal testis in 1 patient. Two patients had female external genitalia and 1 had partial labial fusion. Karyotypes were 46XY. Gonadotropin levels were elevated, and testosterone was low and failed to increase after stimulation with human chorionic gonadotropin. Testosterone precursors were not elevated. Testicular histology showed absence of mature Leydig cells but relatively preserved seminiferous tubules. Family history was consistent for autosomal recessive inheritance. H-Y antigen expression measured by an enzyme-linked immunosorbent assay was normal, indicating that lack of other inductive factors for Leydig cell differentiation are responsible for Leydig cell hypoplasia.