Aberrant signaling in T-cell acute lymphoblastic leukemia: biological and therapeutic implications

T-cell acute lymphoblastic leukemia (T-ALL) is a biologically heterogeneous disease with respect to phenotype, gene expression profile and activation of particular intracellular signaling pathways. Despite very significant improvements, current therapeutic regimens still fail to cure a portion of the patients and frequently implicate the use of aggressive protocols with long-term side effects. In this review, we focused on how deregulation of critical signaling pathways, in particular Notch, PI3K/Akt, MAPK, Jak/STAT and TGF-beta, may contribute to T-ALL. Identifying the alterations that affect intracellular pathways that regulate cell cycle and apoptosis is essential to understanding the biology of this malignancy, to define more effective markers for the correct stratification of patients into appropriate therapeutic regimens and to identify novel targets for the development of specific, less detrimental therapies for T-ALL

Regulation of RKIP Function by Helicobacter pylori in Gastric Cancer

Helicobacter pylori (H. pylori) is a gram-negative, spiral-shaped bacterium that infects more than half of the world’s population and is a major cause of gastric adenocarcinoma. The mechanisms that link H. pylori infection to gastric carcinogenesis are not well understood. In the present study, we report that the Raf-kinase inhibitor protein (RKIP) has a role in the induction of apoptosis by H. pylori in gastric epithelial cells. Western blot and luciferase transcription reporter assays demonstrate that the pathogenicity island of H. pylori rapidly phosphorylates RKIP, which then localizes to the nucleus where it activates its own transcription and induces apoptosis. Forced overexpression of RKIP enhances apoptosis in H. pylori-infected cells, whereas RKIP RNA inhibition suppresses the induction of apoptosis by H. pylori infection. While inducing the phosphorylation of RKIP, H. pylori simultaneously targets non-phosphorylated RKIP for proteasome-mediated degradation. The increase in RKIP transcription and phosphorylation is abrogated by mutating RKIP serine 153 to valine, demonstrating that regulation of RKIP activity by H. pylori is dependent upon RKIP’s S153 residue. In addition, H. pylori infection increases the expression of Snail, a transcriptional repressor of RKIP. Our results suggest that H. pylori utilizes a tumor suppressor protein, RKIP, to promote apoptosis in gastric cancer cells

A quantitative assay to study the lipid selectivity of membrane-associated systems using solution NMR

The activity of membrane proteins and compounds that interact with the membrane is modulated by the surrounding lipid composition. However, there are no simple methods that determine the composition of these annular phospholipids in eukaryotic systems. Herein, we describe a simple methodology that enables the identification and quantification of the lipid composition around membrane-associated compounds using SMA-nanodiscs and routine $^1H-^{31}P NMR$

Heat shock protein-90-alpha, a prolactin-STAT5 target gene identified in breast cancer cells, is involved in apoptosis regulation

Introduction The prolactin-Janus-kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) pathway is essential for the development and functional differentiation of the mammary gland. The pathway also has important roles in mammary tumourigenesis. Prolactin regulated target genes are not yet well defined in tumour cells, and we undertook, to the best of our knowledge, the first large genetic screen of breast cancer cells treated with or without exogenous prolactin. We hypothesise that the identification of these genes should yield insights into the mechanisms by which prolactin participates in cancer formation or progression, and possibly how it regulates normal mammary gland development. Methods We used subtractive hybridisation to identify a number of prolactin-regulated genes in the human mammary carcinoma cell line SKBR3. Northern blotting analysis and luciferase assays identified the gene encoding heat shock protein 90-alpha (HSP90A) as a prolactin-JAK2-STAT5 target gene, whose function was characterised using apoptosis assays. Results We identified a number of new prolactin-regulated genes in breast cancer cells. Focusing on HSP90A, we determined that prolactin increased HSP90A mRNA in cancerous human breast SKBR3 cells and that STAT5B preferentially activated the HSP90A promoter in reporter gene assays. Both prolactin and its downstream protein effector, HSP90α, promote survival, as shown by apoptosis assays and by the addition of the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in both untransformed HC11 mammary epithelial cells and SKBR3 breast cancer cells. The constitutive expression of HSP90A, however, sensitised differentiated HC11 cells to starvation-induced wild-type p53-independent apoptosis. Interestingly, in SKBR3 breast cancer cells, HSP90α promoted survival in the presence of serum but appeared to have little effect during starvation. Conclusions In addition to identifying new prolactin-regulated genes in breast cancer cells, we found that prolactin-JAK2-STAT5 induces expression of the HSP90A gene, which encodes the master chaperone of cancer. This identifies one mechanism by which prolactin contributes to breast cancer. Increased expression of HSP90A in breast cancer is correlated with increased cell survival and poor prognosis and HSP90α inhibitors are being tested in clinical trials as a breast cancer treatment. Our results also indicate that HSP90α promotes survival depending on the cellular conditions and state of cellular transformation

Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype.

The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; Stat5a delta 750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a delta 750 mutant. The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that Stat5a delta 750 and Stat5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters

IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line

International audienceActivation of the Jak/STAT pathway by cytokines has been shown to regulate differentiation, proliferation or apoptosis in hematopoeitic cells. Among the Stat proteins, STAT5 is activated by a broad range of cytokines. In order to study the role of STAT5 in hematopoietic cells, we stably expressed a dominant negative form of STAT5 (STAT5A delta749) in the IL-3 dependent bone marrow derived Ba/F3 cell line. Ba/F3 cells expressing STAT5A delta749 were found to be more sensitive to apoptosis than parental or control Ba/F3 cells after IL-3 withdrawal. Analysis of the expression of the cell death regulators, Bcl-2 and Bcl-x, revealed that the level of Bcl-x was lower in Ba/F3 cells expressing STAT5A delta749 than in control cells. IL-3 regulation of Bcl-x expression at protein and mRNA levels was impaired in these cells while that of Bcl-2 expression was unaffected. We further demonstrated that the Bcl-x gene promoter contained a proximal STAT consensus sequence that bound STAT5. Transactivation of a Bcl-x gene promoter reporter construct by STAT5 was observed in Ba/F3 cells. Introduction of a mutation in the STAT binding site abolished this transactivation. These data indicate that Bcl-x is probably a STAT5 target gene. They also support the involvement of STAT5 in hematopoietic cell survival