Ravi Kothapalli

Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression

Large granular lymphocyte (LGL) leukemia is characterized by the expansion of antigen-activated cytotoxic T lymphocytes. These leukemic cells are resistant to Fas-mediated apoptosis despite expressing high levels of Fas. We found that leukemic LGL from 19 patients displayed high levels of activated STAT3. Treatment of leukemic LGL with the JAK-selective tyrosine kinase inhibitor AG-490 induced apoptosis with a corresponding decrease in STAT-DNA binding activity. Moreover, using an antisense oligonucleotide approach to diminish STAT3 expression, we found that Fas sensitivity was restored in leukemic LGL. AG-490-induced apoptosis in leukemic LGL was independent of Bcl-xL or Bcl-2 expression. However, we found that the Bcl-2-family protein Mcl-1 was significantly reduced by AG-490 treatment. Activated STAT3 was shown to bind an SIE-related element in the murine mcl-1 promoter. Using a luciferase reporter assay, we demonstrated that v-src overexpression in NIH3T3 induced STAT3-dependent transcriptional activity from the mcl-1 promoter and increased endogenous Mcl-1 protein levels. We conclude that STAT3 activation contributed to accumulation of the leukemic LGL clones. These findings suggest that investigation should focus on novel strategies targeting STAT3 in the treatment of LGL leukemia.

Microarray results: how accurate are they?

BackgroundDNA microarray technology is a powerful technique that was recently developed in order to analyze thousands of genes in a short time. Presently, microarrays, or chips, of the cDNA type and oligonucleotide type are available from several sources. The number of publications in this area is increasing exponentially.ResultsIn this study, microarray data obtained from two different commercially available systems were critically evaluated. Our analysis revealed several inconsistencies in the data obtained from the two different microarrays. Problems encountered included inconsistent sequence fidelity of the spotted microarrays, variability of differential expression, low specificity of cDNA microarray probes, discrepancy in fold-change calculation and lack of probe specificity for different isoforms of a gene.ConclusionsIn view of these pitfalls, data from microarray analysis need to be interpreted cautiously.

Characterization of a human sphingosine-1-phosphate receptor gene (S1P5) and its differential expression in LGL leukemia.

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder often associated with autoimmune disease. A central feature of this disease is dysregulation of apoptosis. In order to identify differentially expressed genes in LGL leukemia, microarray analysis was performed. We found many differentially expressed genes including several expression sequence tags (ESTs). As a systematic study, we selected one up-regulated EST (GenBank Accession number N47089) and further investigated. An LGL leukemia library was screened using this EST as a probe and a full-length sequence for a novel gene was identified. The deduced amino acid sequence revealed that the novel gene encodes a G-protein-coupled receptor gene that exhibits 86% identity with rat sphingosine-1-phosphate receptor (edg-8/nrg-1). This gene is present in brain, spleen, and peripheral blood mononuclear cells (PBMC) and is overexpressed in leukemic LGL.

Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template.

In order to identify the reverse transcriptase activity in sera and conditioned media from peripheral blood mononuclear cells (PBMCs) of large granular lymphocyte leukemia patients product enhanced reverse transcriptase activity (PERT) assays were performed using bacteriophage MS2 RNA as a template. All samples obtained from conditioned media of virus-infected cell lines as well as PBMCs of lymphocytic leukemia patients and normal healthy individuals tested positive with this assay. Therefore the validity of the assay was questioned. Careful evaluation of the assay revealed that some of the essential reagents used, such as Taq DNA polymerase and RNase inhibitor contained indigenous amplifiable DNA. DNase I treatment of Taq DNA polymerase before PCR reduced the product significantly. Moreover, no false positive results were observed when encephalomyocarditis virus RNA was used instead of MS2 RNA as the template. These results suggest a need for caution when using bacteriophage MS2 RNA as the template in PERT assays to confirm the presence of retroviral infection or for identification of novel retroviruses.

Induction of apoptosis in large granular lymphocyte (Lgl) Leukemia by jak tyrosine kinase inhibitor AG-490: Role of STAT3-regulated MCL-1

Abstract Large granular lymphocyte (LGL) leukemia is characterized by the expansion of antigen-activated cytotoxic T lymphocytes. These leukemic cells are resistant to Fas-mediated apoptosis despite expressing high levels of Fas. We found that leukemic LGL from 19 patients displayed constitutively activated STAT3. Treatment of leukemic LGL from 11 patients with the JAK-selective tyrosine kinase inhibitor, AG-490, induced apoptosis with a corresponding decrease in STAT-DNA binding activity. AG-490-induced apoptosis in leukemic LGL was independent of Bcl-X L or BCl-2 expression. However, we found that the Bcl-2-family protein, Mcl-1, was significantly reduced by AG-490 treatment. Activated STAT3 was shown to bind an SIE-related element in the murine mcl-1 promoter. Moreover, using a luciferase reporter assay, we demonstrated that v-src expression in NIH3T3 induced STAT3-dependent transcriptional activity from the mcl-1 promoter. We conclude that one mechanism involved in AG-490-induced apoptosis in leukemic LGL may be mediated by inhibition of STAT3-regulated Mcl-1 expression. These findings suggest that AG-490 warrants further study as a possible therapeutic modality for the treatment of LGL leukemia.