DEVELOPMENTAL AND CELLULAR FUNCTIONS OF DELTA-CATENIN

Catenins have diverse and powerful roles in embryogenesis, homeostasis or disease progression, as best exemplified by the well-known beta-catenin. The less studied delta-catenin likewise contains a central Armadillo-domain. In common with other p120 sub-class members, it acts in a variety of intracellular compartments and modulates cadherin stability, small GTPase activities and gene transcription. In mammals, delta-catenin exhibits neural specific expression, with its knock-out in mice correspondingly producing cognitive defects and synaptic dysfunctions. My work instead employed the amphibian, Xenopus laevis, to explore delta-catenin’s physiological functions in a distinct vertebrate system. Initial isolation and characterization indicated delta-catenin’s expression in Xenopus. Unlike the pattern observed for mammals, delta-catenin was detected in most adult Xenopus tissues, although enriched in embryonic structures of neural fate as visualized using RNA in-situ hybridization. To determine delta-catenin’s requirement in amphibian development, I employed anti-sense morpholinos to knock-down gene products, finding that delta-catenin depletion results in developmental defects in gastrulation, neural crest migration and kidney tubulogenesis, phenotypes that were specific based upon rescue experiments. In biochemical and cellular assays, delta-catenin knock-down reduced cadherin levels and cell adhesion, and impaired activation of RhoA and Rac1, small GTPases that regulate actin dynamics and morphogenetic movements. Indeed, exogenous C-cadherin, or dominant-negative RhoA or dominant-active Rac1, significantly rescued delta-catenin depletion. Thus, my results indicate delta-catenin’s essential roles in Xenopus development, with contributing functional links to cadherins and Rho family small G proteins. In examining delta-catenin’s nuclear roles, I identified delta-catenin as an interacting partner and substrate of the caspase-3 protease, which plays critical roles in apoptotic as well as non-apoptotic processes. Delta-catenin’s interaction with and sensitivity to caspase-3 was confirmed using assays involving its cleavage in vitro, as well as within Xenopus apoptotic extracts or mammalian cell lines. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo-repeat 6 of delta-catenin, was identified through peptide sequencing. Cleavage thus generates an amino- (1-816) and carboxyl-terminal (817-1314) fragment each containing about half of the central Armadillo-domain. I found that cleavage of delta-catenin both abolishes its association with cadherins, and impairs its ability to modulate small GTPases. Interestingly, the carboxyl-terminal fragment (817-1314) possesses a conserved putative nuclear localization signal that I found is needed to facilitate delta-catenin’s nuclear targeting. To probe for novel nuclear roles of delta-catenin, I performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating its interaction with an uncharacterized KRAB family zinc finger protein I named ZIFCAT. My results indicate that ZIFCAT is nuclear, and suggest that it may associate with DNA as a transcriptional repressor. I further determined that other p120 sub-class catenins are similarly cleaved by caspase-3, and likewise bind ZIFCAT. These findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120 sub-family members, facilitating the coordinate modulation of cadherins, small GTPases and nuclear functions. Together, my work suggested delta-catenin’s essential roles in Xenopus development, and has revealed its novel contributions to cell junctions (via cadherins), cytoskeleton (via small G proteins), and nucleus (via ZIFCAT). Future questions include the larger role and gene targets of delta-catenin in nucleus, and identification of upstream signaling events controlling delta-catenin’s activities in development or disease progression.

Dasatinib inhibits the growth of molecularly heterogeneous myeloid leukemias.

PURPOSE: Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: We studied growth factor-dependent and growth factor-independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. RESULTS: Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at approximately 1 x 10(-9) mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at approximately 1 x 10(-6) mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI(50) of 5 x 10(-9) mol/L. Primary AML blast cells exhibited a growth inhibition of <1 x>10(-6) mol/L. Cell lines that showed growth inhibition at approximately 1 x 10(-6) mol/L showed a G(1) cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. CONCLUSIONS: Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects.

The characterization of residential fungal spores and the relationship with housing characteristics in the Houston area

A number of indoor environmental factors, including bioaerosol or aeroallergen concentrations have been identified as exacerbators for asthma and allergenic conditions of the respiratory system. People generally spend 90% to 95% of their time indoors. Therefore, understanding the environmental factors that affect the presence of aeroallergens indoors as well as outdoors is important in determining their health impact, and in identifying potential intervention methods. This study aimed to assess the relationship between indoor airborne fungal spore concentrations and indoor surface mold levels, indoor versus outdoor airborne fungal spore concentrations and the effect of previous as well as current water intrusion. Also, the association between airborne concentration of indoor fungal spores and surface mold levels and the age of the housing structure were examined. Further, the correlation between indoor concentrations of certain species was determined as well. ^ Air and surface fungal measurements and related information were obtained from a Houston-area data set compiled from visits to homes filing insurance claims. During the sampling visit these complaint homes exhibited either visible mold or a combination of visible mold and water intrusion problems. These data were examined to assess the relationships between the independent and dependent variables using simple linear regression analysis, and independent t-tests. To examine the correlation between indoor concentrations of certain species, Spearman correlation coefficients were used. ^ There were 126 houses sampled, with spring, n=43 (34.1%), and winter, n=42 (33.3%), representing the seasons with the most samples. The summer sample illustrated the highest geometric mean concentration of fungal spores, GM=5,816.5 relative to winter, fall and spring (GM=1,743.4, GM=3,683.5 and GM=2,507.4, respectively). In all seasons, greater concentrations of fungal spores were observed during the cloudy weather conditions. ^ The results indicated no statistically significant association between outdoor total airborne fungal spore concentration and total living room airborne fungal spore concentration (β = 0.095, p = 0.491). Second, living room surface mold levels were not associated with living room airborne fungal spore concentration, (β= 0.011, p = 0.669). Third, houses with and without previous water intrusion did not differ significantly with respect to either living room (t(111) = 0.710, p = 0.528) or bedroom (t(111) =1.673, p = 0.162) airborne fungal spore concentrations. Likewise houses with and without current water intrusion did not differ significantly with respect to living room (t(109)=0.716, p = 0.476) or bedroom (t(109) = 1.035, p = 0.304) airborne fungal spore concentration. Fourth, houses with and without current water intrusion did not differ significantly with respect to living room (χ 2 (5) = 5.61, p = 0.346), or bedroom (χ 2 (5) = 1.80, p = 0.875) surface mold levels. Fifth, the age of the house structure did not predict living room (β = 0.023, p = 0.102) and bedroom (β = 0.023, p = 0.065) surface mold levels nor living room (β = 0.002, p = 0.131) and bedroom (β = 0.001, p = 0.650) fungal spore airborne concentration. Sixth, in houses with visually observed mold growth there was statistically significant differences between the mean living room concentrations and mean outdoor concentrations for Cladosporium (t (107) = 11.73, p < 0.0001), Stachybotrys (t (106)=2.288, p = 0.024, and Nigrosporia (t (102) = 2.267, p = 0.025). Finally, there was a significant correlation between several living room fungal species pairs, namely, Cladosporium and Stachybotrys (r = 0.373, p <0.01, n=65), Curvularia and Aspergillus/Penicillium (r = 0.205, p < 0.05, n= 111)), Curvularia and Stachybotrys (r = 0.205, p < 0.05, n=111), Nigrospora and Chaetomium (r = 0.254, p < 0.01, n=105) and Stachybotrys and Nigrospora (r = 0.269, p < 0.01, n=105). ^ This study has demonstrated several positive findings, i.e., significant pairwise correlations of concentrations of several fungal species in living room air, and significant differences between indoor and outdoor concentrations of three fungal species in homes with visible mold. No association was observed between indoor and outdoor fungal spore concentrations. Neither living room nor bedroom airborne spore concentrations and surface mold levels were related to the age of the house or to water intrusion, either previous or current. Therefore, these findings suggest the need for evaluating additional parameters, as well as combinations of factors such as humidity, temperature, age of structure, ventilation, and room size to better understand the determinants of airborne fungal spore concentrations and surface mold levels in homes. ^

Percutaneous transluminal coronary angioplasty and coronary events in a biethnic Texas community, 1985--1990

The relationship between change in myocardial infarction (MI) mortality rate (ICD codes 410, 411) and change in use of percutaneous transluminal coronary angioplasty (PTCA), adjusted for change in hospitalization rates for MI, and for change in use of aortocoronary bypass surgery (ACBS) from 1985 through 1990 at private hospitals was examined in the biethnic community of Nueces County, Texas, site of the Corpus Christi Heart Project, a major coronary heart disease (CHD) surveillance program. Age-adjusted rates (per 100,000 persons) were calculated for each of these CHD events for the population aged 25 through 74 years and for each of the four major sex-ethnic groups: Mexican-American and Non-Hispanic White women and men. Over this six year period, there were 541 MI deaths, 2358 MI hospitalizations, 816 PTCA hospitalizations, and 920 ACBS hospitalizations among Mexican-American and Non-Hispanic White Nueces County residents. Acute MI mortality decreased from 24.7 in the first quarter of 1985 to 12.1 in the fourth quarter of 1990, a 51.2% decrease. All three hospitalization rates increased: The MI hospitalization rates increased from 44.1 to 61.3, a 38.9% increase, PTCA use increased from 7.1 to 23.2, a 228.0% increase, and ACBS use increased from 18.8 to 29.5, a 56.6% increase. In linear regression analyses, the change in MI mortality rate was negatively associated with the change in PTCA use (beta = $-$.266 $\pm$.103, p = 0.017) but was not associated with the changes in MI hospitalization rate and in ACBS use. The results of this ecologic research support the idea that the increasing use of PTCA, but not ACBS, has been associated with decreases in MI mortality. The contrast in associations between these two revascularization procedures and MI mortality highlights the need for research aimed at clarifying the proper roles of these procedures in the treatment of patients with CHD. The association between change in PTCA use and change in MI mortality supports the idea that some changes in medical treatment may be partially responsible for trends in CHD mortality. Differences in the use of therapies such as PTCA may be related to differences between geographical sites in CHD rates and trends. ^

Fasting blood glucose and 8-year mortality in the hypertension detection follow-up program population

This study analyzed the relationship between fasting blood glucose (FBG) and 8-year mortality in the Hypertension Detection Follow-up Program (HDFP) population. Fasting blood glucose (FBG) was examined both as a continuous variable and by specified FBG strata: Normal (FBG 60–100 mg/dL), Impaired (FBG ≥100 and ≤125 mg/dL), and Diabetic (FBG>125 mg/dL or pre-existing diabetes) subgroups. The relationship between type 2 diabetes was examined with all-cause mortality. This thesis described and compared the characteristics of fasting blood glucose strata by recognized glucose cut-points; described the mortality rates in the various fasting blood glucose strata using Kaplan-Meier mortality curves, and compared the mortality risk of various strata using Cox Regression analysis. Overall, mortality was significantly greater among Referred Care (RC) participants compared to Stepped Care (SC) {HR = 1.17; 95% CI (1.052,1.309); p-value = 0.004}, as reported by the HDFP investigators in 1979. Compared with SC participants, the RC mortality rate was significantly higher for the Normal FBG group {HR = 1.18; 95% CI (1.029,1.363); p-value = 0.019} and the Impaired FBG group, {HR = 1.34; 95% CI (1.036,1.734); p-value = 0.026,}. However, for the diabetic group, 8-year mortality did not differ significantly between the RC and SC groups after adjusting for race, gender, age, smoking status among Diabetic individuals {HR = 1.03; 95% CI (0.816,1.303); p-value = 0.798}. This latter finding is possibly due to a lack of a treatment difference of hypertension among Diabetic participants in both RC and SC groups. The largest difference in mortality between RC and SC was in the Impaired subgroup, suggesting that hypertensive patients with FBG between 100 and 125 mg/dL would benefit from aggressive antihypertensive therapy.^

Study on the effect of metformin in patients of metastatic renal cell cancer with diabetes receiving tyrosine kinase inhibitor therapy

Objective: The primary objective of our study was to study the effect of metformin in patients of metastatic renal cell cancer (mRCC) and diabetes who are on treatment with frontline therapy of tyrosine kinase inhibitors. The effect of therapy was described in terms of overall survival and progression free survival. Comparisons were made between group of patients receiving metformin versus group of patients receiving insulin in diabetic patients of metastatic renal cancer on frontline therapy. Exploratory analyses were also done comparing non-diabetic patients of metastatic renal cell cancer receiving frontline therapy compared to diabetic patients of metastatic renal cell cancer receiving metformin therapy. ^ Methods: The study design is a retrospective case series to elaborate the response rate of frontline therapy in combination with metformin for mRCC patients with type 2 diabetes mellitus. The cohort was selected from a database, which was generated for assessing the effect of tyrosine kinase inhibitor therapy associated hypertension in metastatic renal cell cancer at MD Anderson Cancer Center. Patients who had been started on frontline therapy for metastatic renal cell carcinoma from all ethnic and racial backgrounds were selected for the study. The exclusion criteria would be of patients who took frontline therapy for less than 3 months or were lost to follow-up. Our exposure variable was treatment with metformin, which comprised of patients who took metformin for the treatment of type 2 diabetes at any time of diagnosis of metastatic renal cell carcinoma. The outcomes assessed were last available follow-up or date of death for the overall survival and date of progression of disease from their radiological reports for time to progression. The response rates were compared by covariates that are known to be strongly associated with renal cell cancer. ^ Results: For our primary analyses between the insulin and metformin group, there were 82 patients, out of which 50 took insulin therapy and 32 took metformin therapy for type 2 diabetes. For our exploratory analysis, we compared 32 diabetic patients on metformin to 146 non-diabetic patients, not on metformin. Baseline characteristics were compared among the population. The time from the start of treatment until the date of progression of renal cell cancer and date of death or last follow-up were estimated for survival analysis. ^ In our primary analyses, there was a significant difference in the time to progression of patients receiving metformin therapy vs insulin therapy, which was also seen in our exploratory analyses. The median time to progression in primary analyses was 1259 days (95% CI: 659-1832 days) in patients on metformin therapy compared to 540 days (95% CI: 350-894) in patients who were receiving insulin therapy (p=0.024). The median time to progression in exploratory analyses was 1259 days (95% CI: 659-1832 days) in patients on metformin therapy compared to 279 days (95% CI: 202-372 days) in non-diabetic group (p-value <0.0001). ^ The median overall survival was 1004 days in metformin group (95% CI: 761-1212 days) compared to 816 days (95%CI: 558-1405 days) in insulin group (p-value<0.91). For the exploratory analyses, the median overall survival was 1004 days in metformin group (95% CI: 761-1212 days) compared to 766 days (95%CI: 649-965 days) in the non-diabetic group (p-value<0.78). Metformin was observed to increase the progression free survival in both the primary and exploratory analyses (HR=0.52 in metformin Vs insulin group and HR=0.36 in metformin Vs non-diabetic group, respectively). ^ Conclusion: In laboratory studies and a few clinical studies metformin has been proven to have dual benefits in patients suffering from cancer and type 2-diabetes via its action on the mammalian target of Rapamycin pathway and effect in decreasing blood sugar by increasing the sensitivity of the insulin receptors to insulin. Several studies in breast cancer patients have documented a beneficial effect (quantified by pathological remission of cancer) of metformin use in patients taking treatment for breast cancer therapy. Combination of metformin therapy in patients taking frontline therapy for renal cell cancer may provide a significant benefit in prolonging the overall survival in patients with metastatic renal cell cancer and diabetes. ^