Structure-function relation in retinoid regulated gene expression

Retinoids are known to inhibit proliferation of and induce terminal differentiation of many normal and transformed cells. It has been postulated that retinoids exert their effect by altering gene expression. HL-60 cells and macrophages both respond to retinoic acid action by the rapid induction of the enzyme tissue transglutaminase. The induction has been shown to be due to increased transcription of the transglutaminase gene. The first part of the dissertation studied the structure-function relationship of retinoid-regulated transglutaminase induction, differentiation and proliferation in HL-60 cells using retinoid analogs. The results indicated strict structural constraints and a strong structure-function correlation between transglutaminase induction and differentiation; those retinoids that induced transglutaminase also induced differentiation, those analogs that did not induce transglutaminase could not induce differentiation. The ability of the retinoids to induce transglutaminase in HL-60 cells was paralleled in macrophages. However, the antiproliferative effect of the retinoids displayed less stringent structural constraints than their differentiation- and transglutaminase-inducing properties. Specifically all the retinoids were able to inhibit proliferation to varying extents. It is concluded that the induction of transglutaminase and of differentiation by retinoids is mediated by receptors. While receptor mediation cannot be entirely ruled out, with the current data no definitive statement can be made about the antiproliferative activity of retinoids. Also, the concordance in the ability of the retinoids to induce transglutaminase and the ability to induce differentiation of HL-60 cells suggests that the former is an early response of the cells to retinoids and differentiation a later consequence on the same pathway. Using the induction of transglutaminase as an index of the direct, or primary, effect of retinoids on gene expression, the second part of the dissertation investigates, by 2D gel electrophoresis, the alteration in the rates of synthesis of other proteins in macrophages and HL-60 cells in response to short incubations with retinoic acid. Any changes in parallel with transglutaminase were taken to indicate proteins directly under the control of retinoic acid. It is concluded that retinoic acid regulates the expression of a circumscribed set of genes in a cell-specific manner. The results support the hypothesis that retinoids exert their multiple effects on myeloid cells, in part, by receptor-mediated alternations in gene expression. ^

Molecular basis of retinoid action: The role of retinoic acid receptors in retinoic acid-induced tissue transglutaminase expression

Retinoic acid has profound effects on the cellular growth and differentiation of a variety of cells. However, the molecular basis of retinoic acid action has, until recently, not been well understood. The identification of retinoic acid receptors which bear a high degree of homology to members of the steroid receptor super-family has dramatically altered our understanding of the biology of retinoids. The focus of this dissertation has been toward identification of retinoic acid binding proteins responsible for the effects of this molecule on gene expression.^ We have characterized in detail the retinoic acid-dependent induction of tissue transglutaminase gene expression in a myeloid cell line, human promyelocytic leukemia cells (HL-60 cells). Using cDNA probes specific for tissue transglutaminase, we have determined that the retinoic acid induced increase in enzyme level is due to an increase in the level of tissue transglutaminase mRNA. We have used this model as a probe to investigate the molecular basis of retinoid regulated gene expression.^ This thesis demonstrates that retinoic acid receptors are expressed in cells which induce tissue transglutaminase expression in response to retinoic acid. In Hl-60 cells retinoic acid-induced transglutaminase expression is associated with saturable nuclear retonic acid binding. Transcripts for both the alpha and beta forms of the retinoic acid receptors can be detected in these cells. Pretreatment of HL-60 cells with agents that potentiate retinoic acid-induced transglutaminase expression also modestly induced the alpha form of the retinoic acid receptor. Studies in macrophages and umbilical vein endothelial cells have also associated expression of the beta form of the retinoic acid with retinoic acid induced tissue transglutaminase expression.^ To investigate directly if retinoic acid receptors regulate retinoic acid-induced tissue transglutaminase expression we developed a series of stably transfected Balb-c 3T3 cells expressing different levels of the beta or gamma form of the retinoic acid receptor. These studies indicated that either the beta or gamma receptor can stimulate endogenous tissue transglutaminase expression in response to retinoic acid. These are among the first studies in the steroid field to describe regulation of an endogenous gene by a transfected receptor. ^

Studies of the expression of DNA repair related genes

This dissertation examines the biological functions and the regulation of expression of DNA ligase I by studying its expression under different conditions.^ The gene expression of DNA ligase I was induced two- to four-fold in S-phase lymphoblastoid cells but was decreased to 15% of control after administration of a DNA damaging agent, 4-nitroquinoline-1-oxide. When cells were induced into differentiation, the expression level of DNA ligase I was decreased to less than 15% of that of the control cells. When the gene of DNA ligase I was examined for tissue specific expression in adult rats, high levels of DNA ligase I mRNA were observed in testis (8-fold), intermediate levels in ovary and brain (4-fold), and low levels were found in intestine, spleen, and liver (1- to 2-fold).^ In confluent cells of normal skin fibroblasts, UV irradiation induced the gene expression of DNA ligase I at 24 and 48 h. The induction of DNA ligase I gene expression requires active p53 protein. Introducing a vector containing the wild type p53 protein in the cells caused an induction of the DNA ligase I protein 24 h after the treatment.^ Our results indicate that, in addition to the regulation by phosphorylation/dephosphorylation, cellular DNA ligase I activity can be regulated at the gene transcription level, and the p53 tumor suppresser is one of the transcription factors for the DNA ligase I gene. Also, our results suggest that DNA ligase I is involved in DNA repair as well as in DNA replication.^ Also, as an early attempt to clone the human homolog of the yeast CDC9 gene which has been shown to be involved in DNA replication, DNA repair, and DNA recombination, we have identified a human gene with mRNA of 1.7 kb. This dissertation studies the gene regulation and the possible biological functions of this new human gene by examining its expression at different stages of the cell cycle, during cell differentiation, and in cellular response to DNA damage.^ The new gene that we recently identified from human cells is highly expressed in brain and reproductive organs (BRE). This BRE gene encodes an mRNA of 1.7-1.9 kb, with an open reading frame of 1,149 bp, and gives rise to a deduced polypeptide of 383 amino acid residues. No extensive homology was found between BRE and sequences from the EMBL-Gene Banks. BRE showed tissue-specific expression in adult rats. The steady state mRNA levels were high in testis (5-6 fold), ovary and brain (3-4 fold) compared to the spleen level, but low in intestine and liver (1-2 fold). The expression of this gene is responsive to DNA damage and/or retinoic acid (RA) treatment. Treatment of fibroblast cells with UV irradiation and 4-nitroquinoline-1-oxide caused more than 90% and 50% decreases in BRE mRNA, respectively. Similar decreases in BRE expression were observed after treatment of the brain glioma cell line U-251 and the promyelocytic cell line HL-60 with retinoic acid. (Abstract shortened by UMI). ^

Get ur 60: A social marketing campaign to promote and facilitate physical activity among Central Texas middle school students

If allowed to continue unabated, the obesity epidemic may lead to the first decline in life expectancy in the developed world (Olshansky et al., 2005). Similar to the relationship between smoking habits in youth and adulthood, obesogenic dietary and physical activity habits in childhood may persist into adulthood (Kelder et al., 2002). Teaching children how to establish healthy eating habits and activity levels, as well as providing them the necessary resources to internalize and maintain these behaviors, may be the key to curbing this epidemic.^ A school-based obesity prevention approach is advantageous for many reasons including exposure to large captive audiences, reduced costs of sustainability and long-term maintenance, and generalizability of models and results across multiple populations. The effectiveness of school-based programs has been researched over the past 20 years, with promising results.^ Social marketing is a program-planning process that “facilitates the acceptance, rejection, modification, abandonment, or maintenance of particular behaviors” (Grier & Bryant, 2005). Social marketing has been shown to be effective in a variety of public health applications including improving diet, increasing physical activity, and preventing substance abuse. It is hypothesized that social marketing could further enhance the effectiveness of the Coordinated Approach To Child Health (CATCH) Central Texas Middle School Project, a school-based obesity prevention program.^ The development, implementation, and initial evaluation of the get ur 60 campaign, to promote the Center for Disease Control and Prevention (CDC) recommended sixty minutes of daily activity, is described in this paper. Various components of the get ur 60 campaign were assessed to evaluate the effectiveness of the campaign during the first semester of implementation. At the end of the spring semester focus groups were held to collect student reactions to the first semester of the get ur 60 campaign.^ The initial results from the first semester of get ur 60 have demonstrated that the campaign as designed was feasible to implement, accepted at all intervention schools, and resulted in a measure of success. ^

Systematic review of current trends of cardiovascular disease (CVD) and association between health insurance coverage and CVD mortality among women aged 60--65 in Texas in 2000--2011

Cardiovascular disease (CVD) is highly preventable, yet it is a leading cause of death among women in Texas. The primary goals of this research were to examine past and current trends of CVD, as well as identify whether there is an association between the insurance coverage and mortality from CVD among women aged 60–65 in Texas between 2000 and 2011. ^ The systematic review of the research is based on the guidelines and recommendations set by the Centre for Reviews and Dissemination for conducting reviews in health care. Over 47 citations of peer-reviewed articles from Ovid MEDLINE and PubMed databases and five websites were identified, of which 7 studies met inclusion criteria for the first systematic review to examine the trends of CVD in Texas. Ten citations of peer-reviewed articles from Ovid MEDLINE and PubMed databases and five web sites were reviewed for the second systematic review (to study the association between insurance coverage and cardiovascular health among Texas women 60–64 years of age), of which 3 studies met inclusion criteria and were included in the research. The results of the study highlighted key gaps in the existing literature and important areas for the further research, as well as determined directions for future public health CVD prevention programs in Texas. ^ Based on the conducted research, the major determinants of premature mortality among women attributed to cardiovascular disease are based on individual level characteristics, more specifically sex, age, race/ethnicity, and education. The results indicate that African American and non-Hispanic white women are more likely to have higher CVD mortality rates than Hispanic women due to higher prevalence of cardiac risk factors. The data also shows higher levels of mortality from CVD in the southeastern United States, with Texas ranking as the third state with the highest prevalence of CVD among women. According to the Texas Department of State Health Services, there are approximately 56,000 deaths caused by CVD annually in Texas, which represents about one death every ten minutes. Coronary artery disease and stroke were the causes of 31.2 percent of all female deaths in Texas in 2009, meaning that approximately 68 women die from any form of cardiac disease in Texas each day. ^ The data of the reviewed studies indicate that women' lack of health insurance was significantly associated with a higher prevalence of cardiovascular disease. The uninsured women were more likely to be unaware of their risk factors and more likely to have undiagnosed diabetes—a co-morbidity factor of CVD. One of the studies also reports strong correlation between state rates of uninsured and lower rates of preventive care. Given these strong correlations, those who were chronically uninsured were at a higher risk of mortality than the insured, due to prolonged periods of time without basic access to preventive and medical care. ^ Suggested recommendations to decrease CVD mortality rates in Texas are consistent with the existing literature and include state policy development that addresses elimination of health disparities, consideration of potential benefits of universal health coverage by the legislative policymakers, and maintenance of solid partnerships between public health agencies and hospitals to educate on, diagnose, and treat CVD among the female population in Texas. ^