Long intergenic non-coding RNA-p21 is associated with poor prognosis in chronic lymphocytic leukemia.

BACKGROUND: Long non-coding RNAs (LncRNAs) are RNA transcripts longer than 200 nucleotides. They are new players in transcriptional regulation and cancer research. LincRNA-p21 is a p53-regulated lncRNA involved in the p53 transcriptional network. It has an important role in regulating cellular proliferation and apoptosis. Chronic lymphocytic leukemia is derived by a typical defect in apoptosis and characterized by clonal proliferation and accumulation of mature B cells. The aim of the present study was to assess the expression pattern of the lincRNA-p21 and investigate its potential role as a new prognostic marker in CLL. METHODS: The study was conducted on 80 newly diagnosed CLL patients and 80 age- and sex-matched controls. The analysis of LincRNA-p21 and the p53 downstream proapoptotic target genes (MDM2, PUMA, BAX, and NOXA) was performed by real-time PCR. The cytogenetic abrasions and expression of ZAP70 and CD38 were detected by FISH and Flow cytometry, respectively. RESULTS: LincRNA-p21 was significantly downregulated in CLL patients compared to controls. The downstream proapoptotic targets were significantly downregulated in CLL patients and positively correlated with lincRNA-p21. Low expression of lincRNA-p21 was associated with poor prognostic markers (advanced stages of CLL, del 17p13, ZAP70, and CD38 expression), failure of complete remission, shorter progression free survival, and overall survival. Low lincRNA-p21 expression was independently prognostic for shorter time to treatment. CONCLUSION: Low expression of lincRNA-p21 demarcates a more aggressive form of CLL with poor prognosis. Therefore, it could be considered as a new prognostic marker to predict disease outcome in CLL.

Activation of spleen tyrosine kinase (Syk) at fertilization in Rhinella arenarum eggs.

Recently, we have provided evidence for the involvement of a cytosolic tyrosine-phosphorylatable 70 kDa oocyte protein in Rhinella arenarum (Anura: Bufonidae) fertilization. The aim of the present work was to characterize its phosphorylation, determine the identity of this protein and establish its biological role during the fertilization process. Tyrosine phosphorylation of the 70 kDa protein was not observed in eggs activated with the calcium ionophore A23187. Pretreatment of oocytes with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation of the 70 kDa protein. In order to identify this protein, we examined the presence in amphibian oocytes of non-receptor 70 kDa tyrosine kinase members of the Syk/Zap70 and Tec families by RT-PCR using degenerate primers. We found that R. arenarum oocytes contain the transcripts coding for Syk and Tec kinases. Western blot analysis confirmed the presence of Syk protein in unfertilized oocytes and eggs. Studies using phospho-Syk specific antibodies showed that fertilization rapidly (less than 10 minutes) induces phosphorylation on Syk tyrosine residues (352 and 525/526) that are necessary for the activation of the enzyme. Finally, specific inhibition of Syk with the R406 compound provoked a diminished fertilization score, thereby confirming a functional role of the Syk protein during R. arenarum fertilization. To our knowledge this is the first time that Syk is described as a player in the signaling cascade activated after fertilization.

Altered expression of degranulation-related genes in CD8+ T cells in human T lymphotropic virus type I infection.

Human T lymphotropic virus type I (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD8+ T cells may contribute to the protection or development of HAM/TSP. In this study we used SAGE methodology to screen for differentially expressed genes in CD8+ T cells isolated from HTLV-1 asymptomatic carriers (HAC) and from HAM/TSP patients to identify genes involved in HAM/TSP development. SAGE analysis was conducted by pooling samples according to clinical status. The comparison of gene expression profiles between HAC and HAM/TSP libraries identified 285 differentially expressed tags. We focus on cytotoxicity and cytokine-related genes due to their potential biological role in HTLV-1 infection. Our results showed that patients with HAM/TSP have high expression levels of degranulation-related genes, namely GZMH and PRF1, and of the cytoskeletal adaptor PXN. We found that GZMB and ZAP70 were overexpressed in HTLV-infected patients compared to the noninfected group. We also detected that CCL5 was higher in the HAM/TSP group compared to the HAC and CT groups. Our findings showed that CD8+ T cells of HAM/TSP patients have an inflammatory and active profile. PXN and ZAP70 overexpression in HTLV-1-infected patients was described for the first time here and reinforces this concept. However, although active and abundant, CD8+ T cells are not able to completely eliminate infected cells and prevent the development of HAM/TSP and, moreover, these cells might contribute to the pathogenesis of the disease by migrating to the central nervous system (CNS). These results should be further tested with biological functional assays to increase our understanding on the role of these molecules in the development of HTLV-1-related diseases.

Analysis of CD38 and ZAP70 mRNA expression among cytogenetic subgroups of Iranian chronic-lymphocytic-leukemia patients.

Chromosomal abnormalities and ZAP70 expression profile are two major independent prognostic markers in B-cell chronic lymphocytic leukemia. We investigated a possible correlation between these two markers. ZAP70 expression using real-time RT-PCR was examined in 20 B-cell chronic lymphocytic leukemia patients with del13q14, 13 patients with del11q22, 15 patients with trisomy 12, and 16 patients with no detected chromosomal abnormalities. Molecular analysis revealed that ZAP70 expression in the del13q subgroup was the same as in the control group, while it increased 2.78-fold in the del11q subgroup and 2.95-fold in the trisomy 12 subgroup, compared to the 15 cases in the control group. Comparison of the mean and standard deviation of the ZAP70 expression profile within the subgroups showed it to be highly variable among the individuals of the del11q and trisomy 12 subgroups, versus tight clustering for the del13q subgroup. Therefore, there is a correlation between del13q aberration, which has good prognosis with normal levels of ZAP70 expression. Due to a high degree of variation, no conformity is seen for del11q and trisomy 12 subgroups, making this grouping poor for prognostic discrimination. As a result, neither of these markers can serve as sole discriminators to determine the course of the disease; the use of both markers improves prognostic assessment.

MFI ratio estimation of ZAP-70 in B-CLL by flow cytometry can be improved by considering the isotype-matched antibody signal.

INTRODUCTION: The IgV(H) mutational status of B-cell chronic lymphocytic leukemia (B-CLL) is of prognostic value. Expression of ZAP-70 in B-CLL is a surrogate marker for IgV(H) unmutated (UM). As determination of IgV(H) mutational status involves a methodology currently unavailable for most clinical laboratories, it is important to have available a reliable technique for ZAP-70 estimation in B-CLL. Flow cytometry (FC) is a convenient technique for this purpose. However, there is still no adequate way for data analysis, which would prevent the assignment of false positive or negative expression. METHODS: We have modified the currently most accepted technique, which uses the ratio of the mean fluorescent index (MFI) of B-CLL to T cells. The MFI for parallel antibody isotype staining is subtracted from the ZAP-70 MFI of both B-CLL and T cells. We validated this technique comparing the results obtained for ZAP-70 expression by FC with those obtained with quantitative PCR for the same patients. RESULTS: We applied the technique in a series of 53 patients. With this modification, a better correlation between ZAP-70 expression and IgV(H) UM was obtained. CONCLUSIONS: Thus, the MFI ratio B-CLL/T cell corrected by isotype is a reliable analysis technique to estimate ZAP-70 expression in B-CLL.

Bortezomib induces different apoptotic rates in B-CLL cells according to IgVH and BCL-6 mutations.

BACKGROUND: B-cell chronic lymphocytic leukemia (B-CLL) is a remarkably heterogeneous disorder. Some patients have an indolent disease whereas others undergo a more agressive presentation needing treatment. New therapeutics approaches are necessary for the treatment of B-CLL. Bortezomib (Btz), is a proteasome inhibitor, currently undergoing clinical trials whose function, at least in part, by stabilizing the IkappaBalpha protein and inhibiting NFkappaB activation. OBJECTIVE: The objective of this work was to study the effects of Btz on isolated human B-CLL cells, in vitro, and to correlate the differential rates of apoptosis induction with biological variables. MATERIAL AND METHODS: 31 B-CLL samples, from patients in stage A of Binet were used for this study, and the apoptotic effect of Btz on these cells was measured. RESULTS: Our data show that Btz treatment of B-CLL cells induces apoptosis in a time and dose-dependent manner. The apoptosis induction is mediated in part by inhibition of NFkappaB and is dependent on caspases activation. Interesting, in IgVH mutated cells, Btz have statistically significant differences in their in vitro activity on B-CLL cells according to their BCL-6 mutational status. CONCLUSIONS: Btz is a promising pharmacologic agent for the treatment of B-CLL, but its efficacy seems to be related to IgVH and BCL-6 mutational status, therefore, it could be interesting to further investigate the mechanisms involved in the different behavior of the cells in response to apoptosis induction by this drug.