Changes in miRNA Expression in a Model of Microcephaly

miRNAs function to regulate gene expression through post-transcriptional mechanisms to potentially regulate multiple aspects of physiology and development. Whole transcriptome analysis has been conducted on the citron kinase mutant rat, a mutant that shows decreases in brain growth and development. The resulting differences in RNA between mutant and wild-type controls can be used to identify genetic pathways that may be regulated differentially in normal compared to abnormal neurogenesis. The goal of this thesis was to verify, with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), changes in miRNA expression in Cit-k mutants and wild types. In addition to confirming miRNA expression changes, bio-informatics software TargetScan 5.1 was used to identify potential mRNA targets of the differentially expressed miRNAs. The miRNAs that were confirmed to change include: rno-miR-466c, mmu-miR-493, mmu-miR-297a, hsa-miR-765, and hsa-miR-1270. The TargetScan analysis revealed 347 potential targets which have known roles in development. A subset of these potential targets include genes involved in the Wnt signaling pathway which is known to be an important regulator of stem cell development.

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Characterization of E2F1's oncogenic, apoptotic and tumor-suppressive activities utilizing the K5 mouse model

Though E2F1 is deregulated in most human cancers by mutations of the p16-cyclin D-Rb pathway, it also exhibits tumor suppressive activity. A transgenic mouse model overexpressing E2F1 under the control of the bovine keratin 5 (K5) promoter exhibits epidermal hyperplasia and spontaneously develops tumors in the skin and other epithelial tissues after one year of age. In a p53-deficient background, aberrant apoptosis in K5 E2F1 transgenic epidermis is reduced and tumorigenesis is accelerated. In sharp contrast, K5 E2F1 transgenic mice are resistant to papilloma formation in the DMBA/TPA two-stage carcinogenesis protocol. K5 E2F4 and K5 DP1 transgenic mice were also characterized and both display epidermal hyperplasia but do not develop spontaneous tumors even in cooperation with p53 deficiency. These transgenic mice do not have increased levels of apoptosis in their skin and are more susceptible to papilloma formation in the two-stage carcinogenesis model. These studies show that deregulated proliferation does not necessarily lead to tumor formation and that the ability to suppress skin carcinogenesis is unique to E2F1. E2F1 can also suppress skin carcinogenesis when okadaic acid is used as the tumor promoter and when a pre-initiated mouse model is used, demonstrating that E2F1's tumor suppressive activity is not specific for TPA and occurs at the promotion stage. E2F1 was thought to induce p53-dependent apoptosis through upregulation of p19ARF tumor suppressor, which inhibits mdm2-mediated p53 degradation. Consistent with in vitro studies, the overexpression of E2F1 in mouse skin results in the transcriptional activation of the p19ARF and the accumulation of p53. Inactivation of either p19ARF or p53 restores the sensitivity of K5 E2F1 transgenic mice to DMBA/TPA carcinogenesis, demonstrating that an intact p19ARF-p53 pathway is necessary for E2F1 to suppress carcinogenesis. Surprisingly, while p53 is required for E2F1 to induce apoptosis in mouse skin, p19ARF is not, and inactivation of p19ARF actually enhances E2F1-induced apoptosis and proliferation in transgenic epidermis. This indicates that ARF is important for E2F1-induced tumor suppression but not apoptosis. Senescence is another potential mechanism of tumor suppression that involves p53 and p19ARF. K5 E2F1 transgenic mice initiated with DMBA and treated with TPA show an increased number of senescence cells in their epidermis. These experiments demonstrate that E2F1's unique tumor suppressive activity in two-stage skin carcinogenesis can be genetically separated from E2F1-induced apoptosis and suggest that senescence utilizing the p19ARF-p53 pathway plays a role in tumor suppression by E2F1. ^

Guinea Pigs: A Suitable Animal Model to Study Lipoprotein Metabolism, Atherosclerosis and Inflammation

Numerous animal models have been used to study diet effects on cholesterol and lipoprotein metabolism. However, most of those models differ from humans in the plasma distribution of cholesterol and in the processing of lipoproteins in the plasma compartment. Although transgenic or knock-out mice have been used to study a specific pathway involved in cholesterol metabolism, these data are of limited use because other metabolic pathways and responses to interventions may differ from the human condition.Carbohydrate restricted diets have been shown to reduce plasma triglycerides, increase HDL cholesterol and promote the formation of larger, less atherogenic LDL. However, the mechanisms behind these responses and the relation to atherosclerotic events in the aorta have not been explored in detail due to the lack of an appropriate animal model. Guinea pigs carry the majority of the cholesterol in LDL and possess cholesterol ester transfer protein and lipoprotein lipase activities, which results in reverse cholesterol transport and delipidation cascades equivalent to the human situation. Further, carbohydrate restriction has been shown to alter the distribution of LDL subfractions, to decrease cholesterol accumulation in aortas and to decrease aortic cytokine expression. It is the purpose of this review to discuss the use of guinea pigs as useful models to evaluate diet effects on lipoprotein metabolism, atherosclerosis and inflammation with an emphasis on carbohydrate restricted diets.

The INO80 chromatin remodeling complex is involved in the nucleotide excision repair pathway

The genomic DNA of eukaryotic cells is well organized into chromatin structures. However, these repressed structures present barriers that block the access of regulatory factors to the genome during various nuclear events. To overcome the obstacle, two major cellular processes, post-modification of histone tails and ATP-dependent chromatin remodeling, are involved in reconfiguring chromatin structure and creating accessible DNA. Despite the current research progress, much remains to be explored concerning the relationship between chromatin remodeling and DNA repair. Recently, one member of the ATP-dependent chromatin remodeling complexes, INO80, has been found to play a crucial role in DNA damage repair. However, the functions of this complex in higher eukaryotes have yet to be determined. The goal of my study is to generate a human somatic INO80 conditional knockout model and investigate the functions of Ino80 in damage repair.^ By homologous targeting of the INO80 locus in human HCT116 colon epithelial cells, I established a human somatic INO80 conditional knockout model. I have demonstrated that the conditional INO80 cells exhibited a sufficiently viable period when the INO80 protein is removed. Moreover, I found that loss of INO80 resulted in deficient UV lesion repair in response to UV while the protein levels of the NER factors such as XPC, XPA, XPD were not affected. And in vitro repair synthesis assay showed that the NER incision and repair synthesis activities were intact in the absence of INO80. Examination on the damage recognition factor XPC showed its recruitment to damage sites was impaired in the INO80 mutant cells. Loss of INO80 also led to reduced enrichment of XPA at the site of UV lesions. Despite the reduced recruitment of XPC and XPA observed in INO80 mutants, no direct interaction was detected. Meanwhile, direct interaction between INO80 and DDB1, the initial UV lesion detector, was detected by coimmunoprecipitation. UV-induced chromosome relaxation was reduced in cells devoid of INO80. These results demonstrate the INO80 complex may participates in the NER by interacting with DDB1 and having a critical role of in creating DNA accessibility for the nucleotide excision pathway. ^

A dual oxidase generates a protective oxidative burst during infection in C. elegans

Caenorhabditis elegans has recently been developed as a model system to study both pathogen virulence mechanisms and host defense responses. We have shown that C. elegans produces reactive oxygen species (ROS) in response to exposure to the important Gram-positive, noscomial pathogen, Enterococcus faecalis. We have also shown evidence of oxidative stress and upregulation of stress response after exposure to the pathogen. As in mammalian systems, this work shows that production of ROS for innate immune functions occurs via an NADPH oxidase. Specifically, reducing expression of a dual oxidase, Ce-duox1/BLI-3 causes a decrease in ROS production in response to E. faecalis. We also present evidence that reduction of expression of Ce-duox1/BLI-3 increases susceptibility to this pathogen, specifically when expression is reduced in the intestine and the hypodermis. This dual oxidase has previously been localized to the hypodermis, but we show that it is additionally localized to the intestine of C. elegans. To further demonstrate the protective effects of the pathogen-induced ROS production, we demonstrate that antioxidants that scavenge ROS, increase the sensitivity of the nematode to the infection, in stark contrast to their longevity-promoting effects under non-pathogenic conditions. In conclusion, we postulate that the generation of ROS by NADPH oxidases in the barrier epithelium is an ancient, highly conserved innate immune defense mechanism.^

Folate pathway polymorphisms and neuropsychological impairment after leukemia therapy

Neuropsychological impairment occurs in 20%-40% of childhood acute lymphoblastic leukemia (ALL) survivors, possibly mediated by folate depletion following methotrexate chemotherapy. We evaluated the relationship between two folate pathway polymorphisms and neuropsychological impairment after childhood ALL chemotherapy. Eighty-six childhood ALL survivors were recruited between 2004-2007 at Texas Children's Hospital after exclusion for central nervous system leukemia, cranial irradiation, and age<1 year at diagnosis. Neuropsychological evaluation at a median of 5.3 years off therapy included a parental questionnaire and the following child performance measures: Trail Making Tests A and B, Grooved Pegboard Test Dominant-Hand and Nondominant-Hand, and Digit Span subtest. We performed genotyping for polymorphisms in two folate pathway genes: reduced folate carrier (RFC1 80G>A, rs1051266) and dihydrofolate reductase (DHFR Intron-1 19bp deletion). Fisher exact test, logistic regression, Student's t-test, and ANOVA were used to compare neuropsychological test scores by genotype, using a dominant model to group genotypes. In univariate analysis, survivors with cumulative methotrexate exposure ≥9000 mg/m2 had an increased risk of attention disorder (OR=6.2, 95% CI 1.2 – 31.3), compared to survivors with methotrexate exposure <9000 mg/m2. On average, female survivors scored 8.5 points higher than males on the Digit Span subtest, a test of working memory (p=0.02). The RFC1 80G>A and DHFR Intron-1 deletion polymorphisms were not related to attention disorder or impairment on tests of attention, processing speed, fine motor speed, or memory. These data imply a strong relationship between methotrexate dose intensity and impairment in attention after childhood ALL therapy. We did not find an association between the RFC1 80G>A or DHFR Intron-1 deletion polymorphisms and long-term neuropsychological impairment in childhood ALL survivors.^

Expression study of the atherosclerosis mouse aorta reveals significant disturbance of calcium signaling pathway

Atherosclerosis is a complex disease resulting from interactions of genetic and environmental risk factors leading to heart failure and stroke. Using an atherosclerotic mouse model (ldlr-/-, apobec1-/- designated as LDb), we performed microarray analysis to identify candidate genes and pathways, which are most perturbed in changes in the following risk factors: genetics (control C57BL/6 vs. LDb mice), shearstress (lesion-prone vs. lesion-resistant regions in LDb mice), diet (chow vs. high fat fed LDb mice) and age (2-month-old vs. 8-month old LDb mice). ^ Atherosclerotic lesion quantification and lipid profile studies were performed to assess the disease phenotype. A microarray study was performed on lesion-prone and lesion-resistant regions of each aorta. Briefly, 32 male C57BL/6 and LDb mice (n =16/each) were fed on either chow or high fat diet, sacrificed at 2- and 8-months old, and RNA isolated from the aortic lesion-prone and aortic lesion-resistant segments. Using 64 Affymetrix Murine 430 2.0 chips, we profiled differentially expressed genes with the cut off value of FDR ≤ 0.15 for t-test, and q <0.0001 for the ANOVA. The data were normalized using two normalization methods---invariant probe sets (Loess) and Quantile normalization, the statistical analysis was performed using t-tests and ANOVA, and pathway characterization was done using Pathway Express (Wayne State). The result identified the calcium signaling pathway as the most significant overrepresented pathway, followed by focal adhesion. In the calcium signaling pathway, 56 genes were found to be significantly differentially expressed out of 180 genes listed in the KEGG calcium signaling pathway. Nineteen of these genes were consistently identified by both statistical tests, 11 of which were unique to the test, and 26 were unique to the ANOVA test, using the cutoffs noted above. ^ In conclusion, this finding suggested that hypercholesterolemia drives the disease progression by altering the expression of calcium channels and regulators which subsequently results in cell differentiation, growth, adhesion, cytoskeletal change and death. Clinically, this pathway may serve as an important target for future therapeutic intervention, and thus the calcium signaling pathway may serve as an important target for future diagnostic and therapeutic intervention. ^

Single nucleotide polymorphisms (SNPs) associated with TGF-beta pathway and their significance in systemic sclerosis - A multilevel analysis

Systemic sclerosis (SSc) or Scleroderma is a complex disease and its etiopathogenesis remains unelucidated. Fibrosis in multiple organs is a key feature of SSc and studies have shown that transforming growth factor-β (TGF-β) pathway has a crucial role in fibrotic responses. For a complex disease such as SSc, expression quantitative trait loci (eQTL) analysis is a powerful tool for identifying genetic variations that affect expression of genes involved in this disease. In this study, a multilevel model is described to perform a multivariate eQTL for identifying genetic variation (SNPs) specifically associated with the expression of three members of TGF-β pathway, CTGF, SPARC and COL3A1. The uniqueness of this model is that all three genes were included in one model, rather than one gene being examined at a time. A protein might contribute to multiple pathways and this approach allows the identification of important genetic variations linked to multiple genes belonging to the same pathway. In this study, 29 SNPs were identified and 16 of them located in known genes. Exploring the roles of these genes in TGF-β regulation will help elucidate the etiology of SSc, which will in turn help to better manage this complex disease. ^

Elucidating the IGFBP2 signaling pathway in glioma development and progression

Diffuse gliomas are highly lethal central nervous system malignancies which, unfortunately, are the most common primary brain tumor and also the least responsive to the very few therapeutic modalities currently available to treat them. IGFBP2 is a newly recognized oncogene that is operative in multiple cancer types, including glioma, and shows promise for a targeted therapeutic approach. Elevated IGFBP2 expression is present in high-grade glioma and correlates with poor survival. We have previously demonstrated that IGFBP2 induces glioma development and progression in a spontaneous glioma mouse model, which highlighted its significance and potential for future therapy. However, we did not yet know the key physiological pathways associated with this newly characterized oncogene. We first evaluated human glioma genomics data harnessed from the publicly available Rembrandt source to identify major pathways associated with IGFBP2 expression. Integrin and ILK, among other cell migration and invasion-related pathways, were the most prominently associated. We confirmed that these pathways are regulated by IGFBP2 in glioma cells lines, and demonstrated that 1) IGFBP2 activates integrin α5β1, leading to the activation of key pathways important in glioma; 2) IGFBP2 mediates cell migration pathways through ILK; and 3) IGFBP2 activates NF-kB via an integrin α5 interaction. We then sought to determine whether this was a physiologically active signaling pathway in vivo by assessing its ability to induce glioma progression in the RCAS/tv-a spontaneous glioma mouse model. We found that ILK is a key downstream mediator of IGFBP2 that is required for the induction of glioma progression. Most significantly, a genetic therapeutic approach revealed that perturbation of any point in the pathway thwarted tumor progression, providing strong evidence that targeting the key players could potentially produce a significant benefit for human glioma patients. The elucidation of this signaling pathway is a critical step, since efforts to create a small molecule drug targeting IGFBP2 have so far not been successful, but a number of inhibitors of the other pathway constituents, including ILK, integrin and NF-kB, have been developed.

Genetic variants in the mammalian target of rapamycin (mTOR) signaling pathway as predictors of survival and clinical response in women with ovarian cancer

Background. The mTOR pathway is commonly altered in human tumors and promotes cell survival and proliferation. Preliminary evidence suggests this pathway's involvement in chemoresistance to platinum and taxanes, first line therapy for epithelial ovarian cancer. A pathway-based approach was used to identify individual germline single nucleotide polymorphisms (SNPs) and cumulative effects of multiple genetic variants in mTOR pathway genes and their association with clinical outcome in women with ovarian cancer. ^ Methods. The case-series was restricted to 319 non-Hispanic white women with high grade ovarian cancer treated with surgery and platinum-based chemotherapy. 135 SNPs in 20 representative genes in the mTOR pathway were genotyped. Hazard ratios (HRs) for death and Odds ratios (ORs) for failure to respond to primary therapy were estimated for each SNP using the multivariate Cox proportional hazards model and multivariate logistic regression model, respectively, while adjusting for age, stage, histology and treatment sequence. A survival tree analysis of SNPs with a statistically significant association (p<0.05) was performed to identify higher order gene-gene interactions and their association with overall survival. ^ Results. There was no statistically significant difference in survival by tumor histology or treatment regimen. The median survival for the cohort was 48.3 months. Seven SNPs were significantly associated with decreased survival. Compared to those with no unfavorable genotypes, the HR for death increased significantly with the increasing number of unfavorable genotypes and women in the highest risk category had HR of 4.06 (95% CI 2.29–7.21). The survival tree analysis also identified patients with different survival patterns based on their genetic profiles. 13 SNPs on five different genes were found to be significantly associated with a treatment response, defined as no evidence of disease after completion of primary therapy. Rare homozygous genotype of SNP rs6973428 showed a 5.5-fold increased risk compared to the wild type carrying genotypes. In the cumulative effect analysis, the highest risk group (individuals with ≥8 unfavorable genotypes) was significantly less likely to respond to chemotherapy (OR=8.40, 95% CI 3.10–22.75) compared to the low risk group (≤4 unfavorable genotypes). ^ Conclusions. A pathway-based approach can demonstrate cumulative effects of multiple genetic variants on clinical response to chemotherapy and survival. Therapy targeting the mTOR pathway may modify outcome in select patients.^

Genetic variants within the Wnt/ B-catenin signaling pathway as indicators of bladder cancer clinical outcomes

The genetic factors that influence bladder cancer clinical outcomes are largely unknown. In this clinical outcomes study, I assessed genetic variations in the Wnt/β-catenin stem-cell pathway genes for association with recurrence and progression. A total of 230 SNPS in 40 genes from the Wnt/β-catenin pathway were genotyped in 419 histologically confirmed non-muscle invasive bladder cancer cases. Several significant associations were observed in the clinical outcomes analysis. Under the dominant model WNT8B: rs4919464 (HR: 1.55, 95% CI: 1.17-2.06, P=2.2x10-3) and WNT8B: rs3793771 (HR: 1.54, 95% CI: 1.09-1.62, P=4.6x10-3 ) were statistically significantly associated with an increase risk of recurrence while two other variants, APC2: rs11668593 (HR: 2.50, 95% CI: 1.43-4.35, P=1.2x10-3) and LRP5 : rs312778 (HR: 1.81, 95% CI: 1.23-2.65, P=2.7x10-3), were significantly associated with recurrence risk under the recessive model of inheritance. Four SNPs in the recessive model were associated with an increased risk of progression (AXIN2: rs1544427, LRP5: rs312778, AXIN1: rs370681, AXIN1: rs2301522). LRP5: rs312778 had the most significant increased risk of progression with a 2.68 (95% CI: 1.52-4.72, P=6.4x10-4)-fold increased risk. Stratification analysis based on treatment regimen (transurethral resection (TUR) and Bacillus Calmette-Guérin (BCG)) was also performed. Individuals with at least one variant in AXIN2: rs2007085 were found to have a 2.09 (95% CI: 1.24-3.52, P=5.4x10-3) -fold increased risk of recurrence in those that received TUR only, and no statistically significant effect was seen in those that received BCG. Individuals who received TUR with at least one variant in LEF1: rs10516550 were found to have a 2.26 (95% CI: 1.22-4.18, P=9.7x10-3)-fold increase risk of recurrence and no statistically significant effect was found in individuals who received BCG. Also, the recessive model of LRP6: rs2302684 in TUR only treatment was shown to have a 1.95 (95%CI: 1.18-3.21, P=8.8x10 -3)-fold increased risk of recurrence, and a suggested protective effect associated with a (HR: 0.83, 95% CI: 0.51-1.37, P=0.468) decreased risk of recurrence. Together, these findings implicate the Wnt/β-catenin stem-cell pathway as playing a role in bladder cancer clinical outcomes and have important implications for personalization of future treatment regimens. ^

ROLE OF MIR-19A RELEASED BY INFLAMMATORY BREAST CANCER CELLS IN THE REGULATION OF DENDRITIC CELL FUNCTIONS: IN VITRO MODEL OF CROSSTALK IN THE TUMOR MICROENVIRONMENT OF INFLAMMATORY BREAST CANCER

Inflammatory breast cancer (IBC) is a rare but very aggressive form of locally advanced breast cancer (1-6% of total breast cancer patients in United States), with a 5-year overall survival rate of only 40.5%, compared with 85% of the non-IBC patients. So far, a unique molecular signature for IBC able to explain the dramatic differences in the tumor biology between IBC and non-IBC has not been identified. As immune cells in the tumor microenvironment plays an important role in regulating tumor progression, we hypothesized that tumor-associated dendritic cells (TADC) may be responsible for regulating the development of the aggressive characteristics of IBC. MiRNAs can be released into the extracellular space and mediate the intercellular communication by regulating target gene expression beyond their cells of origin. We hypothesized that miRNAs released by IBC cells can induce an increased activation status, secretion of pro-inflammatory cytokines and migration ability of TADC. In an in vitro model of IBC tumor microenvironment, we found that the co-cultured of the IBC cell line SUM-149 with immature dendritic cells (iDCSUM-149) induced a higher degree of activation and maturation of iDCSUM-149 upon stimulation with lipopolysaccharide (LPS) compared with iDCs co-cultured with the non-IBC cell line SUM-159 (iDCSUM-159), resulting in: increased expression of the costimulatory and activation markers; higher production of pro-inflammatory cytokines (TNF-a, IL-6); and 3) higher migratory ability. These differences were due to the exosome-mediated transfer of miR-19a and miR-146a from SUM-149 and SUM-159, respectively, to iDCs, causing the downregulation of the miR-19a target genes PTEN, SOCS-1 and the miR-146a target genes IRAK1, TRAF6. PTEN, SOCS-1 and IRAK1, TRAF6 are important negative and positive regulator of cytokine- and TLR-mediated activation/maturation signaling pathway in DCs. Increased levels of IL-6 induced the upregulation of miR-19a synthesis in SUM-149 cells that was associated with the induction of CD44+CD24-ALDH1+ cancer stem cells (CSCs) with epithelial-to-mesenchymal transition (EMT) characteristics. In conclusion, in IBC tumor microenvironment IL-6/miR-19a axis can represent a self-sustaining loop able to maintain a pro-inflammatory status of DCs, leading to the development of tumor cells with high metastatic potential (EMT CSCs) responsible of the poor prognosis in IBC patients.

Bcr: A conditional inhibitor of Bcr -Abl oncoprotein

Chronic myelogenous leukemia (CML) is characterized cytogenetically by the presence of the Philadelphia chromosome and clinically by the clonal expansion of the hematopoietic stem cells and the accumulation of large numbers of myeloid cells. Philadelphia chromosome results from the reciprocal translocation between chromosomes 9 and 22 [t(9;22)(324;q11)], which fuses parts of the ABL proto-oncogene to 5′ portions of the BCR gene. The product of the fused gene is Bcr-Abl oncoprotein. Bcr-Abl oncoprotein has elevated protein tyrosine kinase activity, and is the cause of Philadelphia chromosome associated leukemias. The Bcr sequence in the fusion protein is crucial for the activation of Abl kinase activity and transforming phenotype of Bcr-Abl oncoprotein. Although the Bcr-Abl oncoprotein has been studied extensively, its normal counterpart, the Bcr protein, has been less studied and its function is not well understood. At this point, Bcr is known to encode a novel serine/threonine protein kinase. In Bcr-Abl positive leukemia cells, we found that the serine kinase activity of Bcr is impaired by tyrosine phosphorylation. Both the Bcr protein sequences within Bcr-Abl and the normal cellular Bcr protein lack serine/threonine kinase activity when they become phosphorylated on tyrosine residues by Bcr-Abl. Therefore, the goal of this study was to investigate the role of Bcr in Bcr-Abl positive leukemia cells. We found that overexpression of Bcr can inhibit Bcr-Abl tyrosine kinase activity, and the inhibition is dependent on its intact serine/threonine kinase function. Using the tet repressible promoter system, we demonstrated that Bcr when induced in Bcr-Abl positive leukemia cells inhibited the Bcr-Abl oncoprotein tyrosine kinase. Furthermore, induction of Bcr also increased the number of cells undergoing apoptosis and inhibited the transforming ability of Bcr-Abl. In contrast to the wild-type Bcr, the kinase-inactive mutant of Bcr (Y328F/Y360F) had no effects on Bcr-Abl tyrosine kinase in cells. Results from other experiments indicated that phosphoserine-containing Bcr sequences within the first exon, which are known to bind to the Abl SH2 domain, are responsible for observed inhibition of the Bcr-Abl tyrosine kinase. Several lines of evidence suggest that the phosphoserine form of Bcr, which binds to the Abl SH2 domain, strongly inhibits the Abl tyrosine kinase domain of Bcr-Abl Previously published findings from our laboratory have also shown that Bcr is phosphorylated on tyrosine residue 177 in Bcr-Abl positive cells and that this form of Bcr recruits the Grb2 adaptor protein, which is known to activate the Ras pathway. These findings implicate Bcr as an effector of Bcr-Abl's oncogenic activity. Therefore based on the findings presented above, we propose a model for dual Function of Bcr in Bcr-Abl positive leukemia cells. Bcr, when active as a serine/threonine kinase and thus autophosphorylating its own serine residues, inhibits Bcr-Abl's oncogenic functions. However, when Ber is tyrosine phosphorylated, its Bcr-Abl inhibitory function is neutralized thus allowing Bcr-Abl to exert its full oncogenic potential. Moreover, tyrosine phosphorylated Bcr would compliment Bcr-Abl's neoplastic effects by the activation of the Ras signaling pathway. ^

Role of janus tyrosine kinase -3 (JAK3) and signal transducer and activator of transcription (STAT5) pathway in T lymphocyte activation

T cell activation and expansion is essential for immune response against foreign antigens. However, uncontrolled T cell activity can be manifested as a number of lymphoid derived diseases such as autoimmunity, graft versus host disease, and lymphoma. The purpose of this research was to test the central hypothesis that the Jak3/Stat5 pathway is critical for T cell function. To accomplish this objective, two novel Jak3 inhibitors, AG490 and PNU156804, were identified and their effects characterized on Jak3/Stat5 activation and T cell growth. Inhibition of Jak3 selectively disrupted primary human T lymphocyte growth in response to Interleukin-2 (IL-2), as well as other γ c cytokine family members including IL-4, IL-7, IL-9, and IL-15. Inhibition of Jak3 ablated IL-2 induced Stat5 but not TNF-α mediated NF-κβ DNA binding. Loss of Jak3 activity did not affect T cell receptor mediated signals including activation of p56Lck and Zap70, or IL-2 receptor a chain expression. To examine the effects of Jak3/Stat5 inhibition within a mature immune system, we employed a rat heart allograft model of Lewis (RT1 1) to ACI (RT1a). Heart allograft survival was significantly prolonged following Jak3/Stat5 inhibition when rats were treated with AG490 (20mg/kg) or PNU156804 (80mg/kg) compared to non-treated control animals. This effect was synergistically potentiated when Jak3 inhibitors were used in combination with a signal 1/2 disrupter, cyclosporine, but only additively potentiated with another signal 3 inhibitor, rapamycin. This suggested that sequential inhibition of T cell function is more effective. To specifically address the role of Stat5 in maintaining T cell activity, novel Stat5 antisense oligonucleotides were synthesized and characterized in vitro. Primary human T cells and T-cell tumor lines treated with Stat5 antisense oligonucleotide (7.5 μM) rapidly underwent apoptosis, while no changes in cell cycle were observed as measured by FACS analysis utilizing Annexin-V-Fluorescein and Propidium iodide staining. Evidence is provided to suggest that caspase 8 and 9 pathways mediate this event. Thus, Stat5 may act rather as a negative regulator of apoptotic signals and not as a positive regulator of cell cycle as previously proposed. We conclude that the Jak3/Stat5 pathway is critical for γc cytokine mediated gene expression necessary for T cell expansion and normal immune function and represents an therapeutically relevant effector pathway to combat T cell derived disease. ^