Bachelor of Science, University of Arizona (2004)
Doctor of Philosophy, University of Arizona (2010)
Exposure to organophosphate and carbamate pesticides can lead to neurotoxic effects through inhibition of cholinesterase enzymes. The paraoxonase (PON1) enzyme can detoxify oxon derivatives of some organophosphates. Lower PON1, acetylcholinesterase, and butyrylcholinesterase activities have been reported in newborns relative to adults, suggesting increased susceptibility to organophosphate exposure in young children. We determined PON1, acetylcholinesterase, and butyrylcholinesterase activities in Mexican-American mothers and their 9-year-old children (n=202 pairs) living in an agricultural community. We used Wilcoxon signed-rank tests to compare enzymatic activities among mothers and their children, and analysis of variance to identify factors associated with enzyme activities. Substrate-specific PON1 activities were slightly lower in children than their mothers; however, these differences were only statistically significant for the paraoxon substrate. We observed significantly lower acetylcholinesterase but higher butyrylcholinesterase levels in children compared with their mothers. Mean butyrylcholinesterase levels were strongly associated with child obesity status (body mass index Z scores >95%). We observed highly significant correlations among mother-child pairs for each of the enzymatic activities analyzed; however, PON1 activities did not correlate with acetylcholinesterase or butyrylcholinesterase activities. Our findings suggest that by age 9 years, PON1 activities approach adult levels, and host factors including sex and obesity may affect key enzymes involved in pesticide metabolism.
View details for DOI 10.1038/jes.2012.61
View details for Web of Science ID 000309989900013
View details for PubMedID 22760442
Nucleolin, the most abundant nucleolar phosphoprotein of eukaryotic cells, is known primarily for its role in ribosome biogenesis and cell proliferation. It is, however, a multifunctional protein that, depending on the cellular context, can drive either cell proliferation or apoptosis. Our laboratory recently demonstrated that nucleolin can function as a repressor of c-MYC transcription by binding to and stabilizing the formation of a G-quadruplex structure in a region of the c-MYC promoter responsible for controlling 85-90% of c-MYC's transcriptional activity. In this study, we investigate the structural elements of nucleolin that are required for c-MYC repression. The effect of nucleolin deletion mutants on the formation and stability of the c-MYC G-quadruplex, as well as c-MYC transcriptional activity, was assessed by circular dichroism spectropolarimetry, thermal stability, and in vitro transcription. Here we report that nucleolin's RNA binding domains 3 and 4, as well as the arginine-glycine-glycine (RGG) domain, are required to repress c-MYC transcription.
View details for DOI 10.1021/bi100509s
View details for Web of Science ID 000283833800006
View details for PubMedID 20932061
c-MYC is an important regulator of a wide array of cellular processes necessary for normal cell growth and differentiation, and its dysregulation is one of the hallmarks of many cancers. Consequently, understanding c-MYC transcriptional activation is critical for understanding developmental and cancer biology, as well as for the development of new anticancer drugs. The nuclease hypersensitive element (NHE) III(1) region of the c-MYC promoter has been shown to be particularly important in regulating c-MYC expression. Specifically, the formation of a G-quadruplex structure appears to promote repression of c-MYC transcription. This review focuses on what is known about the formation of a G-quadruplex in the NHE III(1) region of the c-MYC promoter, as well as on those factors that are known to modulate its formation. Last, we discuss the development of small molecules that stabilize or induce the formation of G-quadruplex structures and could potentially be used as anticancer agents.
View details for DOI 10.1146/annurev.pharmtox.48.113006.094649
View details for Web of Science ID 000274686300005
View details for PubMedID 19922264
myc is a proto-oncogene that plays an important role in the promotion of cellular growth and proliferation. Understanding the regulation of c-myc is important in cancer biology, as it is overexpressed in a wide variety of human cancers, including most gynecological, breast, and colon cancers. We previously demonstrated that a guanine-rich region upstream of the P1 promoter of c-myc that controls 85-90% of the transcriptional activation of this gene can form an intramolecular G-quadruplex (G4) that functions as a transcriptional repressor element. In this study, we used an affinity column to purify proteins that selectively bind to the human c-myc G-quadruplex. We found that nucleolin, a multifunctional phosphoprotein, binds in vitro to the c-myc G-quadruplex structure with high affinity and selectivity when compared with other known quadruplex structures. In addition, we demonstrate that upon binding, nucleolin facilitates the formation and increases the stability of the c-myc G-quadruplex structure. Furthermore, we provide evidence that nucleolin overexpression reduces the activity of a c-myc promoter in plasmid presumably by inducing and stabilizing the formation of the c-myc G-quadruplex. Finally, we show that nucleolin binds to the c-myc promoter in HeLa cells, which indicates that this interaction occurs in vivo. In summary, nucleolin may induce c-myc G4 formation in vivo.
View details for DOI 10.1074/jbc.M109.018028
View details for Web of Science ID 000269180000048
View details for PubMedID 19581307
Sufficient levels of gene expression are required for effective gene therapy. One of the major obstacles in gene therapy is the low transgene expression obtained from currently available vector systems. To address this issue, we employed a transcriptional amplifier strategy in a single construct to enhance transgene expression. In the amplifier vectors (pHi-1 and pHi-2), the strong CMV promoter was used to drive a transcriptional factor, Tat, which could transactivate a second promoter (HIV1 LTR or HIV2 LTR) located in the same construct driving the gene of interest. Using the human interleukin-2 (IL-2) cytokine gene, our data showed that the pHi-1/2 amplifier vectors could produce significantly higher IL-2 levels in human lung cancer cells (A549) and breast cancer cells (MCF-7) than that obtained by directly using the CMV promoter alone. Injection of pHi-2-IL-2-modified Lewis Lung (LL/2) tumor clones led to significantly slower tumor growth and longer survival in mice compared to those injected with either CMV promoter driven IL-2 clones or the parental tumor cells. Our results demonstrated that the transcriptional amplifier-based expression cassettes could be very useful in applications where high levels of gene expression are difficult to achieve.
View details for Web of Science ID 000232026800007
View details for PubMedID 16153160
Umbilical cord blood (CB)-derived primitive hematopoietic stem progenitor cells (HSPC) are a promising source for stem cell-based gene therapy due to the reduced incidence and severity of graftversus- host disease (GVHD) after human leukocyte antigen (HLA)-disparate CB transplantation. Cell-surface markers such as CD34 and CD133 have been used in combination to enrich primitive HSPC for research and clinical applications. To understand the molecular characteristics of the CB HSPC, we compared the global gene expression of freshly isolated CB CD34+ CD133+ cells with their progenies using a cDNA microarray containing 22,000 human cDNA clones printed on a single chip. A total of 139 genes were differentially expressed between CB HSPC and their progenies. These transcripts included a number of known genes that might play roles in key functions of CB HSPC as well as many genes of unknown function. Among the genes showing the greatest differential expression levels in HSPC were: psoriasin 1, CRHBP, HDAC3, MLLT3, HBEX2, SPINK2, c-kit, H2BFQ, CD133, HHEX, TCF4, ALDH1A1, and FHL1. These data provide more information on the molecular phenotype of CB HSPC and may lead to the identification of new genes critical to stem cell function.
View details for Web of Science ID 000229217500010
View details for PubMedID 15910245
Many plant-derived isothiocyanates (ITCs), which occur in human diet, are potent cancer chemopreventive agents in animals. Among the anticarcinogenic mechanisms that have been revealed for ITCs is the inhibition of cell proliferation. We report that exposure of cancer cells to either allyl-ITC (AITC), benzyl-ITC (BITC), or phenethyl-ITC (PEITC) for only 3 h was long enough for the inhibition of cell growth, based on a comparison of IC50 values; regardless of the origin of cancer cells; and even in drug-resistant cells that overexpressed multidrug resistance associated protein-1 (MRP-1) or P-glycoprotein-1 (Pgp-1). In contrast, the inhibitory effect of another ITC, sulforaphane (SF), on these cells was highly time dependent. The finding that some ITCs could inhibit the proliferation of cancer cells in a largely time-independent manner is significant because ITCs that enter the human body are rapidly cleared through urinary excretion. Using human promyelocytic leukemia HL60/S as model cells, and focusing on AITC and BITC, we found that these ITCs modulated multiple cellular targets involved in proliferation, including the disruption of mitochondrial membrane potential, activation of multiple caspases, arrest of cell cycle progression, and induction of differentiation. Again, only a 3-h incubation of the cells with the ITCs was enough to exert their full effect on these targets. Taken together, our findings suggest that selected ITCs can rapidly initiate growth inhibition of cancer cells by simultaneously modulating multiple cellular targets, and their antiproliferative activity may be largely unaffected by their metabolism and disposition in vivo.
View details for Web of Science ID 000186054800012
View details for PubMedID 14578469
Six families of glutathione S-transferases (GSTs), which play an important role in cellular detoxification, are identified in human cells. We report that human keratinocytes and melanocytes express significant levels of GST activity, for which GSTP1-1 is mainly responsible. But, in contrast to previous reports that GSTP1-1 level increases in skin tumor tissues, GSTP1-1 expression does not increase in transformed keratinocytes and melanocytes in culture. Although the human GSTP1 gene carries in its 5'-flanking region multiple copies of the antioxidant response element (ARE), no increase in GSTP1-1 expression was observed after treatment of human keratinocytes (HaCaT) with ARE-mediated inducers. ARE is a cis-acting DNA element and stimulates the transcription of many genes. While studies suggest that an NF-kappaB binding site in the promoter region might suppress the ARE function, such a mechanism does not appear to exist in HaCaT cells. Moreover, although ras has been shown to stimulate the expression of human GSTP1-1, the effect of c-Ha-ras on GSTP1-1 expression in HaCaT cells appears limited.
View details for Web of Science ID 000180057900005
View details for PubMedID 12443843