The role of functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) in normal human prostate epithelial cells and prostate cancer development

15-Lipoxygenase 2 (15-LOX2) is a recently cloned human lipoxygenase that shows tissue-restricted expression in prostate, lung, skin, and cornea. The protein level and enzymatic activity of 15-LOX2 have been shown to be down-regulated in prostate cancers compared with normal and benign prostate tissues. We report the cloning and functional characterization of 15-LOX2 and its three splice variants (termed 15-LOX2sv-a, 15-LOX2sv-b, and 15-LOX2sv-c) from primary prostate epithelial (NHP) cells. Western blotting with multiple NHP cell strains and prostate cancer (PCa) cell lines reveals that the expression of 15-LOX2 is lost in all PCa cell lines, accompanied by decreased enzymatic activity. 15-LOX2 is expressed at multiple subcellular locations, including cytoplasm, cytoskeleton, cell-cell border, and nucleus. Surprisingly, the three splice variants of 15-LOX2 are mostly excluded from the nucleus. To elucidate the relationship between nuclear localization, enzymatic activity, and tumor suppressive functions, we established PCa cell clones stably expressing 15-LOX2 or 15-LOX2sv-b. The 15-LOX2 clones express 15-LOX2 in the nuclei and possess robust enzymatic activity, whereas 15-LOX2sv-b clones show neither nuclear protein localization nor arachidonic acid-metabolizing activity. Interestingly, both 15-LOX2- and 15-LOX2sv-b-stable clones proliferate much slower in vitro when compared with control clones. When orthotopically implanted in nude mouse prostate, both 15-LOX2 and 15-LOX2sv-b suppress PC3 tumor growth in vivo. Finally, cultured NHP cells lose the expression of putative stem/progenitor cell markers, slow down in proliferation, and enter senescence. Several pieces of evidence implicate 15-LOX2 plays a role in replicative senescence of NHP cells: (1) promoter activity and the mRNA and protein levels of 15-LOX2 and its splice variants are upregulated in serially passaged NHP cells, which precede replicative senescence and occur in a cell-autonomous manner; (2) PCa cells stably expressing 15-LOX2 or 15-LOX2sv-b show a passage-related senescence-like phenotype; (3) enforced expression of 15-LOX2 or 15-LOX2sv-b in young NHP cells induce partial cell-cycle arrest and senescence-like phenotypes. Together, these results suggest that 15-LOX2 suppress prostate tumor development and do not necessarily depend on arachidonic acid-metabolizing activity and nuclear localization. Also, 15-LOX2 may serve as an endogenous prostate senescence gene and its tumor-suppressing functions might be associated with its ability to induce cell senescence. ^

Functional differences of beta-tubulin isotypes: A study of microtubule assembly and antimitotic drug resistance

Mammalian cells express 7 β-tubulin isotypes in a tissue specific manner. This has long fueled the speculation that different isotypes carry out different functions. To provide direct evidence for their functional significance, class III, IVa, and VI β-tubulin cDNAs were cloned into a tetracycline regulated expression vector and stably transfected Chinese hamster ovary cell lines expressing different levels of ectopic β-tubulin were compared for effects on microtubule organization, microtubule assembly and sensitivity to antimitotic drugs. It was found that all three isotypes coassembled with endogenous β-tubulin. βVI expression caused distinct microtubule rearrangements including microtubule dissociation from the centrosome and accumulation at the cell periphery; whereas expression of βIII and βVIa caused no observable changes in the interphase microtubule network. Overexpression of all 3 isotypes caused spindle malformation and mitotic defects. Both βIII and βIVa disrupted microtubule assembly in proportion to their abundance and thereby conferred supersensitivity to microtubule depolymerizing drugs. In contrast, βVI stabilized microtubules at low stoichiometry and thus conferred resistance to many microtubule destabilizing drugs but not vinblastine. The 3 isotypes caused differing responses to microtubule stabilizing drugs. Expression of βIII conferred paclitaxel resistance while βVI did not. Low expression of βIVa caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that βIVa may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of βVIa expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by increased paclitaxel binding, and drug supersensitivity is lost. From this study, I concluded that β-tubulin isotypes behave differently from each other in terms of microtubule organization, microtubule assembly and dynamics, and antimitotic drug sensitivity. The isotype composition of cell can impart subtle to dramatic effects on the properties of microtubules leading to potential functional consequences and opening the opportunity to exploit differences in microtubule isotype composition for therapeutic gain. ^

Proteomic identification and functional characterization of prohibitin 1 and 2 in T cell activation, expansion, survival and disease

Several immune pathologies are the result of aberrant regulation of T lymphocytes. Pronounced T cell proliferation can result in autoimmunity or hematologic malignancy, whereas loss of T cell activity can manifest as immunodeficiency. Thus, there is a critical need to characterize the signal transduction pathways that mediate T cell activation so that novel and rational strategies to detect and effectively control T cell mediated disease can be achieved. ^ The first objective of this dissertation was to identify and characterize novel T cell regulatory proteins that are differentially expressed upon antigen induced activation. Using a functional proteomics approach, two members of the prohibitin (Phb) family of proteins, Phb1 and Phb2, were determined to be upregulated upon activation of primary human T cells. Furthermore, their regulated expression was dependent upon CD3 and CD28 signaling pathways which synergistically increased their expression. In contrast to previous reports of Phb nuclear localization, both proteins were determined to localize to the mitochondrial inner membrane of human T cells. Additionally, novel Phb phosphorylation sites were identified and characterized using mass spectrometry, phosphospecific antibodies and site directed mutagenesis. ^ Prohibitins have been proposed to play important roles in cancer development however the mechanism of action has not been elucidated. The second objective of this dissertation was to define the functional role of Phbs in T cell activity, survival and disease. Compared to levels in normal human T cells, Phb expression was higher in the human tumor T cell line Kit225 and subcellularly localized to the mitochondrion. Ablation of Phb expression by siRNA treatment of Kit225 cells resulted in disruption of mitochondrial membrane potential and significantly enhanced their sensitivity to cell death, suggesting they serve a protective function in T cells. Furthermore, Q-RT-PCR analysis of human oncology cDNA expression libraries indicated the Phbs may represent hematological cancer biomarkers. Indeed, Phb1 and Phb2 protein levels were 6-10 fold higher in peripheral blood mononuclear cells isolated from malignant lymphoma and multiple myeloma patients compared to healthy individuals. ^ Taken together, Phb1 and Phb2 are novel phosphoproteins upregulated during T cell activation and transformation to function in the maintenance of mitochondrial integrity and perhaps energy metabolism, thus representing previously unrecognized intracellular biomarkers and therapeutic targets for regulating T cell activation and hematologic malignancies. ^

The impact of functional impairment on the survival of patients with Alzheimer's disease

Alzheimer's disease (AD), the most common form of dementia, is the fifth leading cause of death among U.S. adults aged 65 or older. Most AD patients have shorter life expectancy compared with older people without dementia. This disease has become an enormous challenge in the aging society and is also a global problem. Not only do families of patients with Alzheimer's disease need to pay attention to this problem, but also the healthcare system and society as a whole have to confront. In dementia, functional impairment is associated with basic activities of daily living (ADL) and instrumental activities of daily living (IADL). For patients with Alzheimer's disease, problems typically appear in performing IADL and progress to the inability of managing less complex ADL functions of personal care. Thus, assessment of ADLs can be used for early accurate diagnosis of Alzheimer's disease. It should be useful for patients, caregivers, clinicians, and policy planners to estimate the survival of patients with Alzheimer's disease. However, it is unclear that when making predictions of patient outcome according to their histories, time-dependent covariates will provide us with important information on how changes in a patient's status can effect the survival. In this study, we examined the effect of impaired basic ADL as measured by the Physical Self-Maintenance Scale (PSMS) and utilized a multistate survival analysis approach to estimate the probability of death in the first few years of initial visit for AD patients taking into consideration the possibility of impaired basic ADL. The dataset used in this study was obtained from the Baylor Alzheimer's Disease and Memory Disorders Center (ADMDC). No impaired basic ADL and older age at onset of impaired basic ADL were associated with longer survival. These findings suggest that the occurrence of impaired basic ADL and age at impaired basic ADL could be predictors of survival among patients with Alzheimer's disease. ^

Spectroscopic and functional investigations of the AMPA subtype of ionotropic glutamate receptors

Ion channels play a crucial role in the functioning of different systems of the body because of their ability to bridge the cell membrane and allow ions to pass in and out of the cell. Ionotropic glutamate receptors are one class of these important proteins and have been shown to be critical in propagating synaptic transmission in the central nervous system and in other diverse functions throughout the body. Because of their wide-ranging effects, this family of receptors is an important target for structure-function investigations to understand their mechanism of action. ^ α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one subtype of glutamate receptors and have been shown to be the primary receptors involved in rapid excitatory signaling in the central nervous system. Agonist binding to the extracellular ligand binding domain of these receptors causes various conformational changes that culminate in formation of the ion channel. Previous structural investigations have provided important information about their mechanism of action, including uncovering a relationship between the degree of cleft closure in the binding domain and activation of the receptor. However, what question remains unanswered is how specific interactions between the agonist and the protein interplay with cleft closure to mediate receptor activation. ^ To investigate this question, I applied a multiscale approach to investigate the effects of agonist binding on various levels. Vibrational spectroscopy was utilized to investigate molecular-level interactions in the binding pocket, and fluorescence resonance energy transfer (FRET) was employed to measure cleft closure in the isolated ligand binding domain. The results of these studies in the isolated binding domain were then correlated to activation of the full receptor. These investigations showed a relationship between the strength of the interaction at the α-amine group of the agonist and extent of receptor activation, where a stronger interaction correlated to a larger activation, which was upheld even when the extent of cleft closure did not correlate to activation. These results show that this interaction at the α-amine group is critical in mediating the allosteric mechanism of activation and provide a bit more insight into how agonist binding is coupled to channel gating in AMPA receptors. ^

Evaluation of the relationship between nutrition and functional independence measures among stroke patients undergoing inpatient rehabilitation

Cerebrovascular accidents (CVA) or strokes are now the third leading cause of death in the United States. Many who suffer strokes are admitted to rehabilitation centers in order to receive therapy to help rebuild and recovery function. Nutrition plays a significant role in rehabilitation patient outcomes, and is an essential part of comprehensive care. The purpose of this study is to determine if nutrition and diet consistency are directly and independently associated with changes in the Functional Independence Measure (FIM) scores in stroke patients in an acute rehabilitation unit. This study was a retrospective secondary analysis review of medical chart records, and included a total of 84 patients. Patients were divided into groups based on their admission diet: Regular, Dysphagia Advanced, Dysphagia Mechanically Altered, Dysphagia Pureed, and Nutrition Support. Measurements included admission and discharge Total, Motor, and Cognitive FIM scores; BMI, albumin and prealbumin; age, sex, and race. Patients did show a significant improvement in their FIM scores during their stay, with patients on Regular diets having the highest FIM scores. Patients who were more debilitated and had lower FIM scores were usually in one of the altered texture diet groups, or on nutrition support. Prealbumin and BMI were also the highest in patients who had high FIM scores. Patients who were admitted on an altered diet also tended to advance in their diets, which show improvement in overall function. It is crucial to continue to improve nutrition administration to this population to help prevent morbidity and mortality. Proper nutrition in the acute phase of stroke can lay the essential groundwork for recovery.^

Integrating diabetes self-management with the health goals of older adults: A qualitative exploration

Objective. This study investigates the life and health goals of older adults with diabetes, and explores the factors that influence their diabetes self-management. Methods: Qualitative in-depth interviews were conducted with 24 older adults with diabetes and other morbid conditions and/or their caregivers, when appropriate. ^ Results. Participants’ provided a consistent set of responses when describing life and health goals. Participants described goals for longevity, better physical functioning, spending time with family, or maintaining independence. Diabetes discordant conditions, but not diabetes, were seen as barriers to life goals for participants with functional impairments. Functionally independent participants described additional health goals that related to diabetes self-management as diabetes was seen often a barrier to life goals. Caregivers, co-morbid conditions, denial and retirement were among the factors that influenced initiation of diabetes self-management. ^ Conclusion. Participants endorsed health goals and diabetes self-management practices that they believed would help them accomplish their life goals. Functional capabilities and social support were key factors in the relationship between diabetes self-management and their broader goals. ^ Practice implications. When planning diabetes treatments, clinicians, patients and caregivers should discuss the relationship between diabetes self-management and health and life goals as well as the affects of functional limitations and caregiver support.^

Health information exchange: Using metrics to address quality of care and return on investment

Health Information Exchange (HIE) will play a key part in our nation’s effort to improve healthcare. The evidence of HIEs transformational role in healthcare delivery systems is quite limited. The lack of such evidence led us to explore what exists in the healthcare industry that may provide evidence of effectiveness and efficiency of HIEs. The objective of the study was to find out how many fully functional HIEs are using any measurements or metrics to gauge impact of HIE on quality improvement (QI) and on return on investment (ROI).^ A web-based survey was used to determine the number of operational HIEs using metrics for QI and ROI. Our study highlights the fact that only 50 percent of the HIEs who responded use or plan to use metrics. However, 95 percent of the respondents believed HIEs improve quality of care while only 56 percent believed HIE showed positive ROI. Although operational HIEs present numerous opportunities to demonstrate the business model for improving health care quality, evidence to document the impact of HIEs is lacking. ^

Health status of Saudi women, the case of Yanbu al-Sinaiya

Detailed data on the health status of Saudi women are lacking. This cross sectional study attempts to provide a comprehensive description of the health status of Saudi females between the ages of 15-45 residing in Yanbu Al-Siniyah. The purpose is to assess women's needs for health services. The health status indicators are chronic tracer conditions, reported symptoms and multidimensional functioning levels. The generic functioning instrument of the Medical Outcome Study was used to estimate physical, social, and role functioning; degree of pain and health perceptions. The information was obtained by interviewing subjects and abstracting facts from their medical records. The results show functioning scores are in the "well health" range for physical, social, role and pain. Crowding and education have an equal or stronger effect of reducing functioning levels than the diagnosed tracer conditions. The highest prevalence conditions having a definite functional impact and diagnosed adequately in primary care are anemia, urinary tract infection, hemorrhoids, rheumatoid arthritis, caries and gingivitis. Reported symptoms strongly reducing function levels in this study are dyspnea, heart pain, incontinence, eye and skin problems, and joint ache. The impact of the reliability and validity of the measures used and other limitations of the results are discussed. Finally, some policy implications and suggestions for future study are presented. ^

Novel Use of Dual Anti-Inflammatory Therapy to Overcome Drug Resistance and Improve Functional Recovery Following Spinal Cord Injury

Over 1.2 million Americans are currently living with a traumatic spinal cord injury (SCI). Despite the need for effective therapies, there are currently no proven effective treatments that can improve recovery of function in SCI patients. Many therapeutic compounds have shown promise in preclinical models of SCI, but all of these have fallen short in clinical trials. P-glycoprotein (Pgp) is an active transporter expressed on capillary endothelial cell membranes at the blood-spinal cord barrier (BSCB). Pgp limits passive diffusion of blood-borne drugs into the CNS, by actively extruding drugs from the endothelial cell membrane. Pgp can become pathologically up-regulated, thus greatly impeding therapeutic drug delivery (‘multidrug resistance’). Importantly, many drugs that have been evaluated for the treatment of SCI are Pgp substrates. We hypothesized that Pgp-mediated drug resistance diminishes the delivery and efficacy of neuroprotective drugs following SCI. We observed a progressive, spatial spread of Pgp overexpression within the injured spinal cord. To assess Pgp function, we examined spinal cord uptake of systemically-delivered riluzole, a drug that is currently being evaluated in clinical trials as an SCI intervention. Blood-to-spinal cord riluzole penetration was reduced following SCI in wild-type but not Pgp-null rats, highlighting a critical role for Pgp in mediating spinal cord drug resistance after injury. Others have shown that pro-inflammatory signaling drives Pgp up-regulation in cancer and epilepsy. We have detected inflammation in both acutely- and chronically-injured spinal cord tissue. We therefore evaluated the ability of the dual COX-/5-LOX inhibitor licofelone to attenuate Pgp-mediated drug resistance following SCI. Licofelone treatment both reduced spinal cord Pgp levels and enhanced spinal cord riluzole bioavailability following SCI. Thus, we propose that licofelone may offer a new combinatorial treatment strategy to enhance spinal cord drug delivery following SCI. Additionally, we assessed the ability of licofelone, riluzole, or both to enhance recovery of locomotor function following SCI. We found that licofelone treatment conferred a significant improvement in hindlimb function that was sustained through the end of the study. In contrast, riluzole did not improve functional outcome. We therefore conclude that licofelone holds promise as a potential neuroprotective intervention for SCI.

THE FUNCTIONAL ORGANIZATION OF SYNAPTIC VESICLE POOLS IN A RETINAL BIPOLAR NEURON

Ribbon synapses are found in sensory systems and are characterized by ‘ribbon-like’ organelles that tether synaptic vesicles. The synaptic ribbons co-localize with sites of calcium entry and vesicle fusion, forming ribbon-style active zones. The ability of ribbon synapses to maintain rapid and sustained neurotransmission is critical for vision, hearing and balance. At retinal ribbon synapses, three vesicle pools have been proposed. A rapid pool of vesicles that are docked at the plasma membrane, and whose fusion is limited only by calcium entry, a releasable pool of ATP-primed vesicles whose size also correlates with the number of ribbon-tethered vesicles, and a reserve pool of non-ribbon-tethered cytoplasmic vesicles. However evidence of vesicle fusion at sites away from ribbon-style active zones questions this organization. Another fundamental question underlying the mechanism of vesicle fusion at these synapses is the role of SNARE (Soluble N-ethylmaleimide sensitive factor Attachment Protein Receptor) proteins. Vesicles at conventional neurons undergo SNARE complex-mediated fusion. However a recent study has suggested that ribbon synapses involved in hearing can operate independently of neuronal SNAREs. We used the well-characterized goldfish bipolar neuron to investigate the organization of vesicle pools and the role of SNARE proteins at a retinal ribbon synapse. We blocked functional refilling of the releasable pool and then stimulated bipolar terminals with brief depolarizations that triggered the fusion of the rapid pool of vesicles. We found that the rapid pool draws vesicles from the releasable pool and that both pools undergo release at ribbon-style active zones. To assess the functional role of SNARE proteins at retinal ribbon synapses, we used peptides derived from SNARE proteins that compete with endogenous proteins for SNARE complex formation. The SNARE peptides blocked fusion of reserve vesicles but not vesicles in the rapid and releasable pools, possibly because both rapid and releasable vesicles were associated with preformed SNARE complexes. However, an activity-dependent block in refilling of the releasable pool was seen, suggesting that new SNARE complexes must be formed before vesicles can join a fusion-competent pool. Taken together, our results suggest that SNARE complex-mediated exocytosis of serially-organized vesicle pools at ribbon-style active zones is important in the neurotransmission of vision.

Functional Analysis of Drosophila Integrator Complex in snRNA 3' End Processing

Uridine-rich small nuclear RNAs (U snRNAs) play essential roles in eukaryotic gene expression by facilitating the removal of introns from mRNA precursors and the processing of the replication-dependent histone pre-mRNAs. Formation of the 3’ end of these snRNAs is carried out by a poorly characterized, twelve-membered protein complex named Integrator Complex. In the effort to understand Integrator Complex function in the formation of the snRNA 3’ end, we performed a functional RNAi screen in Drosophila S2 cells to identify protein factors required for snRNA 3’ end formation. This screen was conducted by using a fluorescence-based reporter that elicits GFP expression in response to a deficiency in snRNA processing. Besides scoring the known Integrator subunits, we identified Asunder and CG4785 as additional core members of the Integrator Complex. Additionally, we also found a conserved requirement for Cyclin C and Cdk8 in both fly and human snRNA 3’ end processing. We have further demonstrated that the kinase activity of Cdk8 is critical for snRNA 3’ end processing and is likely to function independent of its well-documented function within the Mediator Cdk8 module. Taken together, this work functionally defines the Drosophila Integrator Complex and demonstrates a novel function for Cyclin C/Cdk8 in snRNA 3’ end formation. This thesis work has also characterized an important functional interaction mediated by a microdomain within Integrator subunit 12 (IntS12) and IntS1 that is required for the activity of the Integrator Complex in processing the snRNA 3’ end. Through the development of a reporter-based functional RNAi-rescue assay in Drosophila S2 cells, we analyzed domains within IntS12 required for snRNA 3’ end formation. This analysis unexpectedly revealed that an N-terminal 30 amino acid region and not the highly conserved central PHD finger domain, is required for snRNA 3’ end cleavage. The IntS12 microdomain (1-45) functions autonomously, and is sufficient to interact and stabilize the putative scaffold protein IntS1. Our findings provide more details of the Integrator Complex for understanding the molecular mechanism of snRNA 3’ end processing. Moreover, these results lay the foundation for future studies of the complex through the identification of a novel functional domain within one subunit and the identification of additional subunits.

Measuring value in cancer-related health care: A case study in head and neck cancer

In the current climate of escalating health care costs, defining value and accurately measuring it are two critical issues affecting not only the future of cancer care in particular but also the future of health care in general. Specifically, measuring and improving value in cancer-related health care are critical for continued advancements in research, management, and overall delivery of care. However, in oncology, most of this research has focused on value as it relates to insurance industry and payment reform, with little attention paid to value as the output of clinical interventions that encompass integrated clinical teams focusing on the entire cycle of care and measuring objective outcomes that are most relevant to patients. ^ In this study, patient-centered value was defined as health outcomes achieved per dollar spent, and calculated using objective functional outcomes and total care costs. The analytic sample comprised patients diagnosed with three common head and neck cancers—cancer of the larynx, oral cavity, and oropharynx—who were treated in an integrated tertiary care center over an approximately 10-year period. The results of this study provide initial empirical data that can be used to assess and ultimately to help improve the quality and value of head and neck cancer care, and more importantly they can be used by patients and clinicians to make better-informed decisions about care, particularly what therapeutic services and outcomes matter the most to patients.^

CONFORMATIONAL DYNAMICS OF K-RAS AND H-RAS PROTEINS: IS THERE FUNCTIONAL SPECIFICITY AT THE CATALYTIC DOMAIN?

Ras proteins serve as crucial signaling modulators in cell proliferation through their ability to hydrolyze GTP and exist in a GTP “on” state and GTP “off” state. There are three different human Ras isoforms: H-ras, N-ras and K-ras (4A and 4B). Although their sequence identity is very high at the catalytic domain, these isoforms differ in their ability to activate different effectors and hence different signaling pathways. Much of the previous work on this topic has attributed this difference to the hyper variable region of Ras proteins, which contains most of the sequence variance among the isoforms and encodes specificity for differential distribution in the membrane. However, we hypothesize that sequence variation on lobe II of Ras catalytic domain alters dynamics and leads to differential preference for different effectors or modulators. In this work, we used all atom molecular dynamics to analyze the dynamics in the catalytic domain of H-ras and K-ras. We have also analyzed the dynamics of a transforming mutant of H-ras and K-ras and further studied the dynamics of an effectorselective mutant of H-ras. Collectively we have determined that wild type K-ras is more dynamic than H-ras and that the structure of the effector binding loop more closely resembles that of the T35S Raf-selective mutant, possibly giving us a new view and insight into the v mode of effector specificity. Furthermore we have determined that specific mutations at the same location perturb the conformational equilibrium differently in H-ras and K-ras and that an enhanced oncogenic potential may arise from different structural perturbations for each point mutation of a specific isoform.

Identification and characterization of cellular genes differentially expressed in a subset of tumors with non -functional WT1

Wilms tumor (WT) or nephroblastoma is a genetically heterogeneous pediatric renal tumor that accounts for 6–7% of all childhood cancers in the U.S. WT1, located at 11p13, is the sole WT gene cloned to date. Additional genomic regions containing genes that play a role in the development of Wilms tumor include 11p15, 7p, 16q, 1p, 17q and 19q. This heterogeneity has made it extremely difficult to develop an understanding of the pathways involved in the development of WT, even in the 5–20% of tumors that show mutations at the WT1 locus. My research addresses this gap in our current comprehension of the development of WT. ^ I have used two complementary approaches to extend the current understanding of molecular changes involved in the development of WT. In order to minimize complexities due to genetic heterogeneity, I confined my analysis to the WT1 pathway by assessing those genetically defined tumors that carry WT1 mutations. WT1 encodes a zinc finger transcription factor, and in vitro studies have identified many genes that are potentially regulated in vivo by WT1. However, there is very little in vivo data that suggests that they are transcriptionally regulated endogenously by WT1. In one approach I assessed the role of WT1 in the in vivo regulation of PDGFA and IGF2, two genes that are strong contenders for endogenous regulation by WT1. Using primary tissue samples, I found no correlation between the level of RNA expression of WT1 with either PDGFA or IGF2, suggesting that WT1 does not play a critical role in their expression in either normal kidney or WT. ^ In a parallel strategy, using differential display analysis I compared global gene expression in a subset of tumors with known homozygous inactivating WT1 mutations (WT1-tumors) to the gene expression in a panel of appropriate control tissues (fetal kidney, normal kidney, rhabdoid tumor and pediatric renal cell carcinoma). Transcripts that are aberrantly expressed in this subset of Wilms tumors are candidates for endogenous transcriptional regulation by WT1 as well as for potentially functioning in the development of WT. By this approach I identified several differentially expressed transcripts. I further characterized two of these transcripts, identifying a candidate WT gene in the process. I then performed a detailed analysis of this WT candidate gene, which maps to 7p. Future studies will shed more light on the role of these differentially expressed genes in WT. ^