Donald A. Baldwin

Differential Effects of Interleukin-2 and Interleukin-15 versus Interleukin-21 on CD4+ Cutaneous T-Cell Lymphoma Cells

In this study, we compared the effects of interleukin-2 (IL-2), IL-15, and IL-21 on gene expression, activation of cell signaling pathways, and functional properties of cells derived from CD4+ cutaneous T-cell lymphoma (CTCL). Whereas both IL-2 and IL-15 modulated, in a CTCL cell line, the expression of >1,000 gene transcripts by at least 2-fold, IL-21 up-regulated <40 genes. All three cytokines induced tyrosine phosphorylation of Jak1 and Jak3 in CTCL cell lines and native leukemic (Sezary) cells. However, only IL-2 and IL-15 strongly activated signal transducers and activators of transcription 5, phosphoinositide 3-kinase/Akt, and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase/ERK signaling pathways in the cell lines and mitogen-primed native cells. In contrast, IL-21 selectively activated signal transducers and activators of transcription 3. Whereas all three cytokines protected CTCL cells from apoptosis, only IL-2 and IL-15 promoted their proliferation. The effects of the cytokine stimulation were Jak3 kinase- and Jak1 kinase- dependent. These findings document the vastly different effect of IL-2 and IL-15 versus IL-21 on CTCL cells. They also suggest two novel therapeutic approaches to CTCL and, possibly, other CD4+ T-cell lymphomas: inhibition of the Jak1/Jak3 kinase complex and, given the known strong immunostimulatory properties of IL-21 on CD8+ T, natural killer, and B cells, application of this cytokine to boost an immune response against malignant CD4+ T cells.

Focus on RNA isolation: obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue

MicroRNAs (miRNAs) are present in all known plant and animal tissues and appear to be somewhat concentrated in the mammalian nervous system. Many different miRNA expression profiling platforms have been described. However, relatively little research has been published to establish the importance of upstream variables in RNA isolation for neural miRNA expression profiling. We tested whether apparent changes in miRNA expression profiles may be associated with tissue processing, RNA isolation techniques, or different cell types in the sample. RNA isolation was performed on a single brain sample using eight different RNA isolation methods, and results were correlated using a conventional miRNA microarray and then cross-referenced to Northern blots. Differing results were seen between samples obtained using different RNA isolation techniques and between microarray and Northern blot results. Another complication of miRNA microarrays is tissue-level heterogeneity of cellular composition. To investigate this phenomenon, miRNA expression profiles were determined and compared between highly-purified primary cerebral cortical cell preparations of rat primary E15-E18 neurons versus rat primary E15-E18 astrocytes. Finally, to assess the importance of dissecting human brain gray matter from subjacent white matter in cerebral cortical studies, miRNA expression profiles were compared between gray matter and immediately contiguous white matter. The results suggest that for microarray studies, cellular composition is important, and dissecting white matter from gray matter improves the specificity of the results. Based on these data, recommendations for miRNA expression profiling in neural tissues, and considerations worthy of further study, are discussed.

Diagnostic microRNAs in myelodysplastic syndrome

OBJECTIVEnThe myelodysplastic syndromes (MDS) are aging-associated disorders characterized by ineffective maturation of hematopoietic elements, which are often diagnostically challenging. This study identifies microRNAs (miRNA) and miRNA targets that might represent diagnostic markers for MDS.nnnMATERIALS AND METHODSnThis study utilized a total of 42 MDS samples and 45 controls. A discovery set of 20 frozen bone marrow mononuclear cell samples (10 MDS, 10 controls) was profiled on a custom Agilent miRNA microarray. Classifier miRNAs were validated in a separate set of 49 paraffin-embedded particle preparations by real-time polymerase chain reaction (24 MDS, 25 controls). Target prediction analysis was compared to a de novo transcriptional profile of MDS derived from the Microarray Innovations in Leukemia study. c-Myb and Sufu were further investigated by immunohistochemical stains on a set of 26xa0paraffin-embedded samples.nnnRESULTSnWe identified 13 miRNAs of interest from the discovery set, 8 of which proved statistically significant on real-time polymerase chain reaction verification. These eight miRNAs were then examined in an independent real-time polymerase chain reaction validation set. Notably, hsa-miR-378, hsa-miR-632, and hsa-miR-636 demonstrated particularly high discrimination between MDS and normal controls. Target prediction identified potential targets of miRNA regulation that correspond to many of the genes that characterize MDS. Immunohistochemical staining performed on a third validation set confirmed that c-Myb and Sufu are differentially expressed in MDS.nnnCONCLUSIONSnOur data utilize both discovery and validation sets and two complementary platforms to identify miRNAs associated with MDS. We have analyzed predicted targets and identified c-Myb and Sufu as potential diagnostic markers of MDS.

In vivo profiling of hypoxic gene expression in gliomas using the hypoxia marker EF5 and laser-capture microdissection

Hypoxia is a key determinant of tumor aggressiveness, yet little is known regarding hypoxic global gene regulation in vivo. We used the hypoxia marker EF5 coupled with laser-capture microdissection to isolate RNA from viable hypoxic and normoxic regions of 9L experimental gliomas. Through microarray analysis, we identified several mRNAs (including the HIF targets Vegf, Glut-1, and Hsp27) with increased levels under hypoxia compared with normoxia both in vitro and in vivo. However, we also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. Intriguingly, the mRNA levels of a substantial number of immunomodulatory and DNA repair proteins including CXCL9, CD3D, and RAD51 were found to be downregulated in hypoxic areas in vivo, consistent with a protumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic versus normoxic tumor regions. Moreover, CD8(+) T cells, which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo. This is the first study to analyze the influence of hypoxia on mRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors.

Gene expression profiling of porokeratosis demonstrates similarities with psoriasis

Background:u2002 Porokeratosis (PK) is a clinically heterogeneous entity associated with sharply demarcated, annular, or serpiginous lesions with a hyperkeratotic ridge. This disorder is associated with aberrant keratinocyte differentiation that histologically manifests as a stack of parakeratin termed the cornoid lamella; this structure represents the peripheral hyperkeratotic ridge of clinical lesions. Histologically, the keratinocytes forming the cornoid lamella demonstrate an altered differentiation program. However, the molecular basis of PK remains incompletely understood.

Image Defocus and Altered Retinal Gene Expression in Chick: Clues to the Pathogenesis of Ametropia

PURPOSEnBecause of the retinas role in refractive development, this study was conducted to analyze the retinal transcriptome in chicks wearing a spectacle lens, a well-established means of inducing refractive errors, to identify gene expression alterations and to develop novel mechanistic hypotheses about refractive development.nnnMETHODSnOne-week-old white Leghorn chicks wore a unilateral spectacle lens of +15 or -15 D for 6 hours or 3 days. With total RNA from the retina/(retinal pigment epithelium, RPE), chicken gene microarrays were used to compare gene expression levels between lens-wearing and contralateral control eyes (n = 6 chicks for each condition). Normalized microarray signal intensities were evaluated by analysis of variance, using a false discovery rate of <10% as the statistical criterion. Selected differentially expressed genes were validated by qPCR.nnnRESULTSnVery few retina/RPE transcripts were differentially expressed after plus lens wear. In contrast, approximately 1300 transcripts were differentially expressed under each of the minus lens conditions, with minimal overlap. For each condition, low fold-changes typified the altered transcriptome. Differentially regulated genes under the minus lens conditions included many potentially informative signaling molecules and genes whose protein products have roles in intrinsic retinal circadian rhythms.nnnCONCLUSIONSnPlus or minus lens wear induce markedly different, not opposite, alterations in retina/RPE gene expression. The initial retinal responses to defocus are quite different from those when the eye growth patterns are well established, suggesting that different mechanisms govern the initiation and persistence or progression of refractive errors. The gene lists identify promising signaling candidates and regulatory pathways for future study, including a potential role for circadian rhythms in refractive development.

Lack of TNFα expression protects anaplastic lymphoma kinase-positive T-cell lymphoma (ALK+ TCL) cells from apoptosis

Here we report that T-cell lymphomas characterized by the expression of anaplastic lymphoma kinase (ALK+ TCL) fail to express the TNFα and frequently display DNA methylation of the TNFα gene promoter. While only a subset of the ALK+ TCL-derived cell lines showed a high degree of the promoter methylation, all 6 showed low to nondetectable expression of the TNFα mRNA, and none expressed the TNFα protein. All 14 ALK+ TCL tissue samples examined displayed some degree of the TNFα promoter methylation, which was the most prominent in the distal portion of the the promoter. Treatment with a DNA methyltransferase inhibitor, 5′-aza-2′-deoxy-cytidine (5-ADC), reversed the promoter methylation and led to the expression of TNFα mRNA and protein. Furthermore, in vitro DNA methylation of the promoter impaired its transcriptional activity in the luciferase reporter assay. This impairment was seen even if only either distal or proximal portion were methylated, with methylation of the former exerting a more profound inhibitory effect. Notably, the ALK+ TCL cell lines uniformly expressed the type 1 TNFα receptor (TNF-R1) protein known to transduce the TNFα-induced pro-apoptotic signals. Moreover, exogeneous TNFα inhibited growth of the ALK+ TCL cell lines in a dose-dependent manner and induced activation of the members of the cell apoptotic pathway: Caspase 8 and caspase 3. These findings provide additional rationale for the therapeutic inhibition of DNA methyltransferases in ALK+ TCL. They also suggest that treatment with TNFα may be highly effective in this type of lymphoma.

Malignant Transformation of CD4 + T Lymphocytes Mediated by Oncogenic Kinase NPM/ALK Recapitulates IL-2–Induced Cell Signaling and Gene Expression Reprogramming

Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4+ T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T cell lymphomas (TCLs), closely resembles cell activation induced by IL-2, the key cytokine supporting growth and survival of normal CD4+ T lymphocytes. Direct comparison of gene expression by ALK+ TCL cells treated with an ALK inhibitor and IL-2–dependent ALK− TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene-regulation pattern. Depending on the analysis method, up to 67% of the affected genes were modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT- and IL-2–signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes—CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4—was confirmed at the protein level. In both ALK+ TCL and IL-2–stimulated ALK− TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, whereas transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4+ T lymphocytes, at least in part, by using the pre-existing, IL-2–dependent signaling pathways.

MiRNA-9 and MiRNA-200a distinguish hemangioblastomas from metastatic clear cell renal cell carcinomas in the CNS.

Central nervous system (CNS) tumors in von Hippel–Lindau syndrome (VHL) include hemangioblastomas and metastatic clear cell renal cell carcinomas (Met CCRCC). While these tumors often show similar histologic features, differentiating them is of significant importance as Met CCRCC are higher‐grade tumors with worse prognosis. No single current immunohistochemical marker unequivocally differentiates between these two entities. MicroRNAs (miRNAs) are noncoding cellular small RNA molecules that play an important role in cancer. We hypothesized that hemangioblastomas and Met CCRCC display distinct miRNA signatures enabling their histologic differentiation. MiRNAs were profiled in 10 cases each of hemangioblastomas, Met CCRCC and primary CCRCC. Ten miRNAs had greater abundance (including miR‐9 (∼10‐fold) and miR‐135a (∼7‐fold)) and 39 miRNAs were lower [including miR‐200a (∼22‐fold) and miR‐200b (∼12‐fold)] in hemangioblastomas compared with Met CCRCC. Quantitative real‐time RT‐PCR in 20 hemangioblastomas and 13 Met CCRCC showed a 12‐fold increase in miR‐9 and a 15‐fold decrease of miR‐200a in hemangioblastomas compared with Met CCRCC. Finally, in situ hybridization for miR‐9 in 15 hemangioblastomas and 10 Met CCRCC confirmed these results. Our data suggest that miR‐9 and miR‐200a can distinguish between hemangioblastomas and Met CCRCC. Further, these results may also provide insight in understanding the biology of hemangioblastomas.

Abstract 5004: In vivo profiling of hypoxic mRNA and miRNA expression in gliomas and head and neck tumors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnHypoxia plays a key role in tumor aggressiveness and radiation resistance, yet little is known regarding hypoxic global gene regulation in vivo. Although the regulation of mRNA and miRNA expression by hypoxia has been investigated in isolation in vitro, it has not yet been analyzed directly in tumor samples.nnWe have used the hypoxia marker EF5 coupled with laser capture microdissection (LCM) to isolate RNA from viable hypoxic and normoxic regions of 9L experimental rat gliomas and from human head and neck (H&N) tumor samples. This was followed by microarray analysis of mRNA expression and comparison of this signature with that obtained from treating 9L cells with hypoxia in vitro. Through this, we have identified several mRNAs (including the HIF targets Vegf, Glut-1 and Hsp27) with increased levels under hypoxia compared to normoxia both in vitro and in vivo. We also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. An intriguing finding was the hypoxic-downregulation of a number of immunomodulatory and DNA repair proteins including CXCL9, CD3D and RAD51 in vivo, consistent with a pro-tumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic vs. normoxic tumor regions. Moreover, CD8+ T cells which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo.nnGlobal microRNA expression changes have been reported to occur in response to hypoxia in vitro. Hence, our second objective was to identify the cluster of miRNAs differentially expressed in hypoxic vs. normoxic regions of the human tumors using TaqMan® Array MicroRNA Cards. We employed the same technique used with the 9L tumors to analyze miRNA expression patterns in hypoxic vs. normoxic samples from H&N cancer patients who have been administered EF5 prior to surgical removal of the tumor. Consistent with published in vitro studies, miR-210 emerged as a major miRNA robustly induced in the hypoxic regions. We also confirmed miR-210 induction by an individual real time-PCR assay and found it to be upregulated > 2-fold in all hypoxic RNA samples. In addition, we have also found several miRNAs whose levels were upregulated and downregulated in hypoxic vs. normoxic areas. This is the first study to analyze the influence of hypoxia on mRNA and miRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5004. doi:10.1158/1538-7445.AM2011-5004