IMPROVING QUANTITATIVE TREATMENT RESPONSE MONITORING WITH DEFORMABLE IMAGE REGISTRATION

Quantitative imaging with 18F-FDG PET/CT has the potential to provide an in vivo assessment of response to radiotherapy (RT). However, comparing tissue tracer uptake in longitudinal studies is often confounded by variations in patient setup and potential treatment induced gross anatomic changes. These variations make true response monitoring for the same anatomic volume a challenge, not only for tumors, but also for normal organs-at-risk (OAR). The central hypothesis of this study is that more accurate image registration will lead to improved quantitation of tissue response to RT with 18F-FDG PET/CT. Employing an in-house developed “demons” based deformable image registration algorithm, pre-RT tumor and parotid gland volumes can be more accurately mapped to serial functional images. To test the hypothesis, specific aim 1 was designed to analyze whether deformably mapping tumor volumes rather than aligning to bony structures leads to superior tumor response assessment. We found that deformable mapping of the most metabolically avid regions improved response prediction (P<0.05). The positive predictive power for residual disease was 63% compared to 50% for contrast enhanced post-RT CT. Specific aim 2 was designed to use parotid gland standardized uptake value (SUV) as an objective imaging biomarker for salivary toxicity. We found that relative change in parotid gland SUV correlated strongly with salivary toxicity as defined by the RTOG/EORTC late effects analytic scale (Spearman’s ρ = -0.96, P<0.01). Finally, the goal of specific aim 3 was to create a phenomenological dose-SUV response model for the human parotid glands. Utilizing only baseline metabolic function and the planned dose distribution, predicting parotid SUV change or salivary toxicity, based upon specific aim 2, became possible. We found that the predicted and observed parotid SUV relative changes were significantly correlated (Spearman’s ρ = 0.94, P<0.01). The application of deformable image registration to quantitative treatment response monitoring with 18F-FDG PET/CT could have a profound impact on patient management. Accurate and early identification of residual disease may allow for more timely intervention, while the ability to quantify and predict toxicity of normal OAR might permit individualized refinement of radiation treatment plan designs.

THE ASSOCIATION BETWEEN CANCER-RELATED-FATIGUE, RESPONSE TO FATIGUE TREATMENT, AND CYTOKINES AND THEIR RECEPTORS, IN PATIENTS WITH ADVANCED CANCER

Background: Inflammation is implicated in the development of cancer related fatigue (CRF). However there is limited literature on the mediators of inflammation (namely), cytokines and their receptors, associated with clinically significant fatigue and response to treatment. Methods: We reviewed 37 advanced cancer patients with fatigue (≥4/10), who participated in two Randomized Controlled Trials, of anti-inflammatory agents (Thalidomide and Dexamethasone) for CRF. Responders showed improvement in FACIT-F subscale at the end of study (Day 15). Baseline patient characteristics and symptoms were assessed by FACIT-F, ESAS; serum cytokines [IL-1β and receptor antagonist (IL-1RA), IL-6, IL-6R, TNF-α and sTNF-R1 and R2, IL-8, IL-10, IL-17] levels measured by Luminex. Data were analyzed using principal component analysis (PCA) [reporting cumulative variance (variance) for the first four components] to determine their association with fatigue and response to treatment. Results: Females were 54%. Mean (SD) was as follows for age, 61(14); baseline FACIT (F) scores, 21.4(8.6); ESAS Fatigue item, 6.5(1.9); and FACIT-F change, 6.4(9.7); ESAS (fatigue) change, -2 (2.41). Baseline median in pg/mL for IL-6, TNF-α, IL-1β were 31.9; 18.9; 0.55, respectively. Change in IL-6 negatively correlated with change in FACIT-F scores (p=0.02). Baseline CRF (FACIT-F score) was associated with IL-6, IL-6R and IL-17, Variance = 78% whereas IL-10, IL-1RA, TNF-α and IL-1β were associated with improvement of CRF, Variance=74%. Conversely, IL-6 and IL-8 were associated with no improvement or worsening of CRF, Variance= 93%. Conclusions: Change in IL-6 negatively correlated with change in FACIT-F scores. IL-6, IL-6R and IL-17 are associated with CRF while IL-6 and IL-8 were associated with no improvement of CRF. Further studies are warranted confirm our findings.

$\beta\sb2$-adrenergic receptor desensitization, internalization and phosphorylation in response to full and partial agonists

The objective of this study is to test the hypothesis that partial agonists produce less desensitization because they generate less of the active conformation of the $\beta\sb2$-adrenergic receptor ($\beta$AR) (R*) and in turn cause less $\beta$AR phosphorylation by beta adrenergic receptor kinase ($\beta$ARK) and less $\beta$AR internalization. In the present work, rates of desensitization, internalization, and phosphorylation caused by a series of $\beta$AR agonists were correlated with a quantitative measure, defined as coupling efficiency, of agonist-dependent $\beta$AR activation of adenylyl cyclase. These studies were preformed in HEK-293 cells overexpressing the $\beta$AR with hemagglutinin (HA) and 6-histidine (6HIS) epitopes introduced into the N- and C-termini respectively. Agonists chosen provided a 95-fold range of coupling efficiencies, and, relative to epinephrine, the best agonist, (100%) were fenoterol (42%), albuterol (4.9%), dobutamine (2.5%) and ephedrine (1.1%). At concentrations of these agonists yielding $>$90% receptor occupancy, the rate and extent of the rapid phase (0-30 min) of agonist induced desensitization of adenylyl cyclase followed the same order as coupling efficiency, that is, epinephrine $\ge$ fitnoterol $>$ albuterol $>$ dobutamine $>$ ephedrine. The rate of internalization, measured by a loss of surface receptors during desensitization, with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like desensitization and internalization, $\beta$AR phosphorylation exhibited a dependency on agonist strength. The two strongest agonists epinephrine and fenoterol provoked 11 to 13 fold increases in the level of $\beta$AR phosphorylation after just 1 min, whereas the weakest agonists dobutamine and ephedrine caused only 3 to 4 fold increases in phosphorylation. With longer treatment times, the level of $\beta$AR phosphorylation declined with the strong agonists, but progressively increased with the weaker partial agonists. The major conclusion drawn from this study is that the occupancy-dependent rate of receptor phosphorylation increases with agonist coupling efficiencies and that this is sufficient to explain the desensitization, internalization, and phosphorylation data obtained.^ The mechanism of activation and desensitization by the partial $\beta$AR agonist salmeterol was also examined in this study. This drug is extremely hydrophobic and its study presents possibly unique problems. To determine whether salmeterol induces desensitization of the $\beta$AR its action has been studied using our system. Employing the use of reversible antagonists it was found that salmeterol, which has an estimated coupling efficiency near that of albuterol caused $\beta$AR desensitization. This desensitization was much reduced relative to epinephrine. Consistent with its coupling efficiency, it was found to be similar to albuterol in its ability to induce internalization and phosphorylation of the $\beta$AR. (Abstract shortened by UMI.) ^

Design and analysis for three level hierarchical structures with count response

In numerous intervention studies and education field trials, random assignment to treatment occurs in clusters rather than at the level of observation. This departure of random assignment of units may be due to logistics, political feasibility, or ecological validity. Data within the same cluster or grouping are often correlated. Application of traditional regression techniques, which assume independence between observations, to clustered data produce consistent parameter estimates. However such estimators are often inefficient as compared to methods which incorporate the clustered nature of the data into the estimation procedure (Neuhaus 1993).1 Multilevel models, also known as random effects or random components models, can be used to account for the clustering of data by estimating higher level, or group, as well as lower level, or individual variation. Designing a study, in which the unit of observation is nested within higher level groupings, requires the determination of sample sizes at each level. This study investigates the design and analysis of various sampling strategies for a 3-level repeated measures design on the parameter estimates when the outcome variable of interest follows a Poisson distribution. ^ Results study suggest that second order PQL estimation produces the least biased estimates in the 3-level multilevel Poisson model followed by first order PQL and then second and first order MQL. The MQL estimates of both fixed and random parameters are generally satisfactory when the level 2 and level 3 variation is less than 0.10. However, as the higher level error variance increases, the MQL estimates become increasingly biased. If convergence of the estimation algorithm is not obtained by PQL procedure and higher level error variance is large, the estimates may be significantly biased. In this case bias correction techniques such as bootstrapping should be considered as an alternative procedure. For larger sample sizes, those structures with 20 or more units sampled at levels with normally distributed random errors produced more stable estimates with less sampling variance than structures with an increased number of level 1 units. For small sample sizes, sampling fewer units at the level with Poisson variation produces less sampling variation, however this criterion is no longer important when sample sizes are large. ^ 1Neuhaus J (1993). “Estimation efficiency and Tests of Covariate Effects with Clustered Binary Data”. Biometrics , 49, 989–996^

Immunology and HIV expression in skin, in vitro, and in response to ultraviolet light

HIV can enter the body through Langerhans cells, dendritic cells, and macrophages in skin mucosa, and spreads by lysis or by syncytia. Since UVL induces of HIV-LTR in transgenic mice mid in cell lines in vitro, we hypothesized that UVB may affect HIV in people and may affect HIV in T cells in relation to dose, apoptosis, and cytokine expression. To determine whether HIV is induced by UVL in humans, a clinical study of HIV+ patients with psoriasis or pruritus was conducted during six weeks of UVB phototherapy, Controls were HIV-psoriasis patients receiving UVB and HIV+ KS subjects without UVB.Blood and skin biopsy specimens were collected at baseline, weeks 2 and 6, and 4 weeks after UVL. AIDS-related skin diseases showed unique cytokine profiles in skin and serum at baseline. In patients and controls on phototherapy, we observed the following: (1) CD4+ and CD8+ T cell numbers are not significantly altered during phototherapy, (2) p24 antigen levels, and also HIV plasma levels increase in patients not on antiviral therapy, (3) HIV-RNA levels in serum or plasma. (viral load) can either increase or decrease depending on the patient's initial viral load, presence of antivirals, and skin type, (4) HIV-RNA levels in the periphery are inversely correlated to serum IL-10 and (5) HIV+ cell in skin increase after UVL at 2 weeks by RT-PCR in situ hybridization mid we negatively correlated with peripheral load. To understand the mechanisms of UVB mediated HIV transcription, we treated Jurkat T cell lines stably transfected with an HIV-LTR-luciferase plasmid only or additionally with tat-SV-40 early promoter with UVB (2 J/m2 to 200 J/m2), 50 to 200 ng/ml rhIL-10, and 10 μg/ml PHA as control. HIV promoter activity was measured by luciferase normalized to protein. Time points up to 72 hours were analyzed for HIV-LTR activation. HIV-LTR activation had the following properties: (1) requires the presence of Tat, (2) occurs at 24 hours, and (3) is UVB dose dependent. Changes in viability by MTS (3-(4,5-dimethyhhiazol-2-y1)-5-(3-carboxymethoxyphonyl)-2-(4-sulfophenyl)-2H-tetrazolium) mixed with PMS (phenazine methosulfate) solution and apoptosis by propidium iodide and annexin V using flow cytometry (FC) were seen in irradiated Jurkat cells. We determined that (1) rhIL-10 moderately decreased HIV-LTR activation if given before radiation and greatly decreases it when given after UVB, (2) HIV-LTR activation was low at doses of greater than 70 J/m2, compared to activation at 50 J/m2. (Abstract shortened by UMI.)^

Comparison of compliance and immune response of drug users on the standard and accelerated hepatitis B vaccine schedules - Houston, Texas

Despite the availability of hepatitis B vaccine for over two decades, drug users and other high-risk adult populations have experienced low vaccine coverage. Poor compliance has limited efforts to reduce transmission of hepatitis B infection in this population. Evidence suggests that immunological response in drug users is impaired compared to the general population, both in terms of lower seroprotection rates and antibodies levels.^ The current study investigated the effectiveness of the multi-dose hepatitis B vaccine and compared the effect of the standard and accelerated vaccine schedules in a not-in-treatment, drug-using adult population in the city of Houston, USA.^ A population of drug-users from two communities in Houston, susceptible to hepatitis B, was sampled by outreach workers and referral methodology. Subjects were randomized either to the standard hepatitis vaccine schedule (0, 1-, 6-month) or to an accelerated schedule (0, 1-, 2-month). Antibody levels were detected through laboratory analyses at various time-points. The participants were followed for two years and seroconversion rates were calculated to determine immune response.^ A four percent difference in the overall compliance rate was observed between the standard (73%) and accelerated schedules (77%). Logistic regression analyses showed that drug users living on the streets were twice as likely to not complete all three vaccine doses (p=0.028), and current speedball use was also associated with non-completion (p=0.002). Completion of all three vaccinations in the multivariate analysis was also correlated with older age. Drug users on the accelerated schedule were 26% more likely to achieve completion, although this factor was marginally significant (p=0.085).^ Cumulative adequate protective response was gained by 65% of the HBV susceptible subgroup by 12-months and was identical for both the standard and accelerated schedules. Excess protective response (>=100 mIU/mL) occurred with greater frequency at the later period for the standard schedule (36% at 12-months compared to 14% at six months), while the greater proportion of excess protective response for the accelerated schedule occurred earlier (34% at 6 months compared to 18% at 12-months). Seroconversion at the adequate protective response level of 10 mIU/mL was reached by the accelerated schedule group at a quicker rate (62% vs. 49%), and with a higher mean titer (104.8 vs. 64.3 mIU/mL), when measured at six months. Multivariate analyses indicated a 63% increased risk of non-response for older age and confirmed the existence of an accelerating decline in immune response to vaccination manifesting after 40 years (p=0.001). Injecting more than daily was also highly associated with the risk of non-response (p=0.016).^ The substantial increase in the seroprotection rate at six months may be worth the trade-off against the faster antibody titer decrease and is recommended for enhancing compliance and seroconversion. Utilization of the accelerated schedule with the primary objective of increasing compliance and seroconversion rates during the six months after the first dose may confer early protective immunity and reduce the HBV vulnerability of drug users who continue, or have recently initiated, increased high risk drug use and sexual behaviors.^

Antibody response to Panton-Valentine Leukocidin, a Staphylococcus aureus virulence factor, in pediatric patients from Texas Children's Hospital in Houston, Texas

Staphylococcus aureus is an important human pathogen of global health significance, whose frequency is increasing and whose persistence and versatility allow it to remain established in communities worldwide. An observed significant increase in infections, particularly in children with no predisposing risk factors or medical conditions, led to an investigation into pediatric humoral immune response to Panton-Valentine Leukocidin (PVL) and to other antigens expressed by S. aureus that represent the important classes of virulence activities. Patients who were diagnosed with staphylococcal infections were enrolled (n=60), and serum samples collected at the time of admission were analyzed using ELISA and Western blot to screen for immune response to the panel of recombinant proteins. The dominant circulating immunoglobulin titers in this pediatric population were primarily IgG, were specific, and were directed against LukF and LukS, while suppression of other important virulence factors in the presence of PVL was suggested. Patients with invasive infections (osteomyelitis, pneumonia or myositis) had higher titers against LukF and LukS compared to patients with non-invasive infections (abscesses, cellulitis or lymphadenitis). In patients with osteomyelitis, antibody responses to LukF and LukS were higher than antibody responses to any other virulence factor examined. This description of immune response to selected virulence factors of S. aureus caused by isolates of the USA300 lineage in children is novel. Antibody titers also correlated with markers of inflammation. The significance of these correlations remains to be understood.^

Characterization of cytochrome P450 4F subfamily: Response in traumatic brain injury and gene regulation

CYP4F enzymes metabolize endogenous molecules including arachidonic acid, leukotrienes and prostaglandins. The involvement of these eisosanoids in inflammation has led to the hypothesis that CYP4Fs may modulate inflammatory conditions after traumatic brain injury (TBI). In rat, TBI elicited changes in mRNA expression of CYP4Fs as a function of time in the cerebrum region. These changes in CYP4F mRNA levels inversely correlated with the cerebral leukotriene B4 (LTB4) level following injury at the same time points. TBI also resulted in changes in CYP4F protein expression and localization around the injury site, where CYP4F1 and CYP4F6 immunoreactivity increased in surrounding astrocytes and CYP4F4 immunoreactivity shifted from endothelia of cerebral vessels to astrocytes. The study with rat primary astrocytes indicated that pro-inflammatory cytokines TNFα and IL-1β could affect the transcription of CYP4Fs to a certain degree, whereas the changing pattern in the primary astrocytes appeared to be different from that in the in vivo TBI model.^ In addition, the regulation of CYP4F genes has been an unsolved issue although factors including cytokines and fatty acids appear to affect CYP4Fs expression in multiple models. In this project, HaCaT cells were used as an in vitro cellular model to define signaling pathways involved in the regulation of human CYP4F genes. Retinoic acids inhibited CYP4F11 expression, whereas cytokines TNFα and IL-1β induced transcription of CYP4F11 in HaCaT cells. The induction of CYP4F11 by both cytokines could be blocked by a JNK specific inhibitor, indicating the involvement of the JNK pathway in the up-regulation of CYP4F11. Retinoic acids are known to function in gene regulation through nuclear receptors RARs and RXRs. The RXR agonist LG268 greatly induced transcription of CYP4F11, whereas RAR agonist TTNPB obviously inhibited CYP4F11 transcription, indicating that the down-regulation of CYP4F11 by retinoic acid was mediated by RARs, and that inhibition of CYP4F11 by retinoic acid may also be related to the competition for RXR receptors. Thus, the CYP4F11 gene is regulated by signaling pathways including the RXR pathway and the JNK pathway. In contrast, the regulation mechanism of other CYP4Fs by retinoic acids appears to be different from that of CYP4F11.^

A novel method for evaluating an interaction effect of correlated continuous covariates in a linear model

Interaction effect is an important scientific interest for many areas of research. Common approach for investigating the interaction effect of two continuous covariates on a response variable is through a cross-product term in multiple linear regression. In epidemiological studies, the two-way analysis of variance (ANOVA) type of method has also been utilized to examine the interaction effect by replacing the continuous covariates with their discretized levels. However, the implications of model assumptions of either approach have not been examined and the statistical validation has only focused on the general method, not specifically for the interaction effect.^ In this dissertation, we investigated the validity of both approaches based on the mathematical assumptions for non-skewed data. We showed that linear regression may not be an appropriate model when the interaction effect exists because it implies a highly skewed distribution for the response variable. We also showed that the normality and constant variance assumptions required by ANOVA are not satisfied in the model where the continuous covariates are replaced with their discretized levels. Therefore, naïve application of ANOVA method may lead to an incorrect conclusion. ^ Given the problems identified above, we proposed a novel method modifying from the traditional ANOVA approach to rigorously evaluate the interaction effect. The analytical expression of the interaction effect was derived based on the conditional distribution of the response variable given the discretized continuous covariates. A testing procedure that combines the p-values from each level of the discretized covariates was developed to test the overall significance of the interaction effect. According to the simulation study, the proposed method is more powerful then the least squares regression and the ANOVA method in detecting the interaction effect when data comes from a trivariate normal distribution. The proposed method was applied to a dataset from the National Institute of Neurological Disorders and Stroke (NINDS) tissue plasminogen activator (t-PA) stroke trial, and baseline age-by-weight interaction effect was found significant in predicting the change from baseline in NIHSS at Month-3 among patients received t-PA therapy.^

Multivariate methods for correlated effects sizes

Current statistical methods for estimation of parametric effect sizes from a series of experiments are generally restricted to univariate comparisons of standardized mean differences between two treatments. Multivariate methods are presented for the case in which effect size is a vector of standardized multivariate mean differences and the number of treatment groups is two or more. The proposed methods employ a vector of independent sample means for each response variable that leads to a covariance structure which depends only on correlations among the $p$ responses on each subject. Using weighted least squares theory and the assumption that the observations are from normally distributed populations, multivariate hypotheses analogous to common hypotheses used for testing effect sizes were formulated and tested for treatment effects which are correlated through a common control group, through multiple response variables observed on each subject, or both conditions.^ The asymptotic multivariate distribution for correlated effect sizes is obtained by extending univariate methods for estimating effect sizes which are correlated through common control groups. The joint distribution of vectors of effect sizes (from $p$ responses on each subject) from one treatment and one control group and from several treatment groups sharing a common control group are derived. Methods are given for estimation of linear combinations of effect sizes when certain homogeneity conditions are met, and for estimation of vectors of effect sizes and confidence intervals from $p$ responses on each subject. Computational illustrations are provided using data from studies of effects of electric field exposure on small laboratory animals. ^

Validity of using item response theory to analyze data from the Work Limitations Questionnaire

The Work Limitations Questionnaire (WLQ) is used to determine the amount of work loss and productivity which stem from certain health conditions, including rheumatoid arthritis and cancer. The questionnaire is currently scored using methodology from Classical Test Theory. Item Response Theory, on the other hand, is a theory based on analyzing item responses. This study wanted to determine the validity of using Item Response Theory (IRT), to analyze data from the WLQ. Item responses from 572 employed adults with dysthymia, major depressive disorder (MDD), double depressive disorder (both dysthymia and MDD), rheumatoid arthritis and healthy individuals were used to determine the validity of IRT (Adler et al., 2006).^ PARSCALE, which is IRT software from Scientific Software International, Inc., was used to calculate estimates of the work limitations based on item responses from the WLQ. These estimates, also known as ability estimates, were then correlated with the raw score estimates calculated from the sum of all the items responses. Concurrent validity, which claims a measurement is valid if the correlation between the new measurement and the valid measurement is greater or equal to .90, was used to determine the validity of IRT methodology for the WLQ. Ability estimates from IRT were found to be somewhat highly correlated with the raw scores from the WLQ (above .80). However, the only subscale which had a high enough correlation for IRT to be considered valid was the time management subscale (r = .90). All other subscales, mental/interpersonal, physical, and output, did not produce valid IRT ability estimates.^ An explanation for these lower than expected correlations can be explained by the outliers found in the sample. Also, acquiescent responding (AR) bias, which is caused by the tendency for people to respond the same way to every question on a questionnaire, and the multidimensionality of the questionnaire (the WLQ is composed of four dimensions and thus four different latent variables) probably had a major impact on the IRT estimates. Furthermore, it is possible that the mental/interpersonal dimension violated the monotonocity assumption of IRT causing PARSCALE to fail to run for these estimates. The monotonicity assumption needs to be checked for the mental/interpersonal dimension. Furthermore, the use of multidimensional IRT methods would most likely remove the AR bias and increase the validity of using IRT to analyze data from the WLQ.^

Role of TRP Channels in Mediating the Calcium Signaling Response of Brain Endothelial Cells to Mechanical Stretch

Traumatic brain injury (TBI) often results in disruption of the blood brain barrier (BBB), which is an integral component to maintaining the central nervous system homeostasis. Recently cytosolic calcium levels ([Ca2+]i), observed to elevate following TBI, have been shown to influence endothelial barrier integrity. However, the mechanism by which TBI-induced calcium signaling alters the endothelial barrier remains unknown. In the present study, an in vitro BBB model was utilized to address this issue. Exposure of cells to biaxial mechanical stretch, in the range expected for TBI, resulted in a rapid cytosolic calcium increase. Modulation of intracellular and extracellular Ca2+ reservoirs indicated that Ca2+ influx is the major contributor for the [Ca2+]i elevation. Application of pharmacological inhibitors was used to identify the calcium-permeable channels involved in the stretch-induced Ca2+ influx. Antagonist of transient receptor potential (TRP) channel subfamilies, TRPC and TRPP, demonstrated a reduction of the stretch-induced Ca2+ influx. RNA silencing directed at individual TRP channel subtypes revealed that TRPC1 and TRPP2 largely mediate the stretch-induced Ca2+ response. In addition, we found that nitric oxide (NO) levels increased as a result of mechanical stretch, and that inhibition of TRPC1 and TRPP2 abolished the elevated NO synthesis. Further, as myosin light chain (MLC) phosphorylation and actin cytoskeleton rearrangement are correlated with endothelial barrier disruption, we investigated the effect mechanical stretch had on the myosin-actin cytoskeleton. We found that phosphorylated MLC was increased significantly by 10 minutes post-stretch, and that inhibition of TRP channel activity or NO synthesis both abolished this effect. In addition, actin stress fibers formation significantly increased 2 minutes post-stretch, and was abolished by treatment with TRP channel inhibitors. These results suggest that, in brain endothelial cells, TRPC1 and TRPP2 are activated by TBI-mechanical stress and initiate actin-myosin contraction, which may lead to disruption of the BBB.

Regulation of Bcl-2 expression by the product of the prostate apoptosis response gene -4, Par-4

Elevated expression levels of the bcl-2 proto-oncogene have been correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t (14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer, there being no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2 expressing, androgen-independent prostate cancer cell lines, by all trans-retinoic acid treatment but this did not occur in the androgen-dependent cell lines expressing low levels of Bcl-2. Changes in localization of Par-4, and an induction in the expression of WT1 protein accompanied the decrease in the Bcl-2 protein and transcript following all trans-retinoic acid treatment, in the androgen-independent prostate cancer cell line. In stable clones expressing ectopic Par-4 we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1 binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1 binding site on the bcl-2 promoter. ^