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1.
PLoS One ; 19(1): e0291632, 2024.
Article in English | MEDLINE | ID: mdl-38295021

ABSTRACT

BACKGROUND: The binding of low-density lipoprotein (LDL) to proteoglycans (PGs) in the extracellular matrix (ECM) of the arterial intima is a key initial step in the development of atherosclerosis. Although many techniques have been developed to assess this binding, most of the methods are labor-intensive and technically challenging to standardize across research laboratories. Thus, sensitive, and reproducible assay to detect LDL binding to PGs is needed to screen clinical populations for atherosclerosis risk. OBJECTIVES: The aim of this study was to develop a quantitative, and reproducible assay to evaluate the affinity of LDL towards PGs and to replicate previously published results on LDL-PG binding. METHODS: Immunofluorescence microscopy was performed to visualize the binding of LDL to PGs using mouse vascular smooth muscle (MOVAS) cells. An in-cell ELISA (ICE) was also developed and optimized to quantitatively measure LDL-PG binding using fixed MOVAS cells cultured in a 96-well format. RESULTS: We used the ICE assay to show that, despite equal APOB concentrations, LDL isolated from adults with cardiovascular disease bound to PG to a greater extent than LDL isolated from adults without cardiovascular disease (p<0.05). CONCLUSION: We have developed an LDL-PG binding assay that is capable of detecting differences in PG binding affinities despite equal APOB concentrations. Future work will focus on candidate apolipoproteins that enhance or diminish this interaction.


Subject(s)
Atherosclerosis , Cardiovascular Diseases , Animals , Mice , Lipoproteins, LDL/metabolism , Proteoglycans/metabolism , Apolipoproteins B/metabolism , Protein Binding
2.
Nutrients ; 13(10)2021 Oct 17.
Article in English | MEDLINE | ID: mdl-34684636

ABSTRACT

Gestational high butterfat (HFB) and/or endocrine disruptor exposure was previously found to disrupt spermatogenesis in adulthood. This study addresses the data gap in our knowledge regarding transgenerational transmission of the disruptive interaction between a high-fat diet and endocrine disruptor bisphenol A (BPA). F0 generation Sprague-Dawley rats were fed diets containing butterfat (10 kcal%) and high in butterfat (39 kcal%, HFB) with or without BPA (25 µg/kg body weight/day) during mating and pregnancy. Gestationally exposed F1-generation offspring from different litters were mated to produce F2 offspring, and similarly, F2-generation animals produced F3-generation offspring. One group of F3 male offspring was administered either testosterone plus estradiol-17ß (T + E2) or sham via capsule implants from postnatal days 70 to 210. Another group was naturally aged to 18 months. Combination diets of HFB + BPA in F0 dams, but not single exposure to either, disrupted spermatogenesis in F3-generation adult males in both the T + E2-implanted group and the naturally aged group. CYP19A1 localization to the acrosome and estrogen receptor beta (ERbeta) localization to the nucleus were associated with impaired spermatogenesis. Finally, expression of methyl-CpG-binding domain-3 (MBD3) was consistently decreased in the HFB and HFB + BPA exposed F1 and F3 testes, suggesting an epigenetic component to this inheritance. However, the severe atrophy within testes present in F1 males was absent in F3 males. In conclusion, the HFB + BPA group demonstrated transgenerational inheritance of the impaired spermatogenesis phenotype, but severity was reduced in the F3 generation.


Subject(s)
Benzhydryl Compounds/toxicity , Butter , Dietary Fats/adverse effects , Infertility, Male/chemically induced , Maternal Exposure/adverse effects , Phenols/toxicity , Prenatal Exposure Delayed Effects/chemically induced , Spermatogenesis/drug effects , Animals , Diet, High-Fat/adverse effects , Disease Models, Animal , Endocrine Disruptors/toxicity , Epigenesis, Genetic , Estradiol , Female , Infertility, Male/genetics , Inheritance Patterns , Male , Maternal Nutritional Physiological Phenomena , Pregnancy , Prenatal Exposure Delayed Effects/genetics , Rats , Rats, Sprague-Dawley , Spermatogenesis/genetics , Testis/metabolism
3.
Article in English | MEDLINE | ID: mdl-33916482

ABSTRACT

Poly- and perfluoroalkyl substances (PFAS) are manmade synthetic chemicals which have been in existence for over 70 years. Though they are currently being phased out, their persistence in the environment is widespread. There is increasing evidence linking PFAS exposure to health effects, an issue of concern since PFAS such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) bioaccumulate in humans, with a half-life of years. Many epidemiological studies suggest that, worldwide, semen quality has decreased over the past several decades. One of the most worrying effects of PFOS and PFOA is their associations with lower testosterone levels, similar to clinical observations in infertile men. This review thus focuses on PFOS/PFOA-associated effects on male reproductive health. The sources of PFAS in drinking water are listed. The current epidemiological studies linking increased exposure to PFAS with lowered testosterone and semen quality, and evidence from rodent studies supporting their function as endocrine disruptors on the reproductive system, exhibiting non-monotonic dose responses, are noted. Finally, their mechanisms of action and possible toxic effects on the Leydig, Sertoli, and germ cells are discussed. Future research efforts must consider utilizing better human model systems for exposure, using more accurate PFAS exposure susceptibility windows, and improvements in statistical modeling of data to account for the endocrine disruptor properties of PFAS.


Subject(s)
Alkanesulfonic Acids , Environmental Pollutants , Fluorocarbons , Infertility, Male , Alkanesulfonic Acids/toxicity , Caprylates/toxicity , Environmental Pollutants/toxicity , Fluorocarbons/toxicity , Humans , Infertility, Male/chemically induced , Infertility, Male/epidemiology , Male , Reproductive Health , Semen Analysis
4.
ACS Med Chem Lett ; 8(2): 163-167, 2017 Feb 09.
Article in English | MEDLINE | ID: mdl-28197305

ABSTRACT

New therapies are needed to eradicate androgen resistant, prostate cancer. Prostate cancer usually metastasizes to bone where the concentration of calcium is high, making Ca2+ a promising toxin. Ionophores can deliver metal cations into cells, but are currently too toxic for human use. We synthesized a new rotaxane (CEHR2) that contains a benzyl 15-crown-5 ether as a blocking group to efficiently bind Ca2+. CEHR2 transfers Ca2+ from an aqueous solution into CHCl3 to greater extent than alkali metal cations and Mg2+. It also transfers Ca2+ to a greater extent than CEHR1, which is a rotaxane with an 18-crown-6 ether as a blocking group. CEHR2 was more toxic against the prostate cancer cell lines PC-3, 22Rv1, and C4-2 than CEHR1. This project demonstrates that crown ether rotaxanes can be designed to bind a targeted metal cation, and this selective cation association can result in enhanced toxicity.

5.
Endocr Relat Cancer ; 24(2): 83-96, 2017 02.
Article in English | MEDLINE | ID: mdl-27998958

ABSTRACT

Humans are increasingly exposed to structural analogues of bisphenol A (BPA), as BPA is being replaced by these compounds in BPA-free consumer products. We have previously shown that chronic and developmental exposure to BPA is associated with increased prostate cancer (PCa) risk in human and animal models. Here, we examine whether exposure of PCa cells (LNCaP, C4-2) to low-dose BPA and its structural analogues (BPS, BPF, BPAF, TBBPA, DMBPA and TMBPA) affects centrosome amplification (CA), a hallmark of cancer initiation and progression. We found that exposure to BPA, BPS, DMBPA and TBBPA, in descending order, increased the number of cells with CA, in a non-monotonic dose-response manner. Furthermore, cells treated with BPA and their analogues initiated centrosome duplication at 8 h after release from serum starvation, significantly earlier in G-1 phase than control cells. This response was attended by earlier release of nucleophosmin from unduplicated centrosomes. BPA-exposed cells exhibited increased expression of cyclin-dependent kinase CDK6 and decreased expression of CDK inhibitors (p21Waf1/CIP1 and p27KIP1). Using specific antagonists for estrogen/androgen receptors, CA in the presence of BPA or its analogues was likely to be mediated via ESR1 signaling. Change in microtubule dynamics was observed on exposure to these analogues, which, for BPA, was accompanied by increased expression of centrosome-associated protein CEP350 Similar to BPA, chronic treatment of cells with DMBPA, but not other analogues, resulted in the enhancement of anchorage-independent growth. We thus conclude that selected BPA analogues, similar to BPA, disrupt centrosome function and microtubule organization, with DMBPA displaying the broadest spectrum of cancer-promoting effects.


Subject(s)
Benzhydryl Compounds/toxicity , Centrosome/drug effects , Endocrine Disruptors/toxicity , Microtubules/drug effects , Phenols/toxicity , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology
6.
Reprod Toxicol ; 68: 191-199, 2017 03.
Article in English | MEDLINE | ID: mdl-27658648

ABSTRACT

Exposure to xenoestrogens is a probable cause of male infertility in humans. Consumption of high-fat diets and exposure to bisphenol A (BPA) is pervasive in America. Here, we test the hypothesis that gestational exposure to high dietary fats and/or BPA disrupt spermatogenesis in adulthood. Sprague-Dawley rats were fed diets containing 10kcal% butter fat (AIN), 39kcal% butter fat (HFB), or 39kcal% olive oil (HFO), with or without BPA (25µg/kg body weight/day) during pregnancy. One group of male offspring received testosterone (T)- and estradiol-17ß (E2)-filled implants or sham-implants from postnatal day (PND)70-210. Another group was naturally aged to 18 months. We found that adult males with gestational exposure to BPA, HFB, or HFB+BPA, in both the aged group and the T+E2-implanted group, exhibited impairment of spermatogenesis. In contrast, gestational exposure to HFO or HFO+BPA did not affect spermatogenesis. Sham-implanted, gestational exposed groups also had normal spermatogenesis. Loss of ERα expression in round spermatids and premature expression of protamine-1 in diplotene spermatocytes were features associated with impaired spermatogenesis. Compared with the single-treatment groups, the HFB+BPA group experienced more severe effects, including atrophy.


Subject(s)
Benzhydryl Compounds/toxicity , Diet, High-Fat/adverse effects , Environmental Pollutants/toxicity , Maternal Exposure/adverse effects , Phenols/toxicity , Prenatal Exposure Delayed Effects/chemically induced , Spermatogenesis/drug effects , Animals , Butter , Dose-Response Relationship, Drug , Estradiol/administration & dosage , Estradiol/pharmacology , Female , Male , Olive Oil , Pregnancy , Prenatal Exposure Delayed Effects/physiopathology , Rats, Sprague-Dawley , Testosterone/administration & dosage , Testosterone/pharmacology
7.
Nanomedicine ; 13(2): 403-410, 2017 02.
Article in English | MEDLINE | ID: mdl-27520723

ABSTRACT

In this study, a development of a novel calcium phosphate-polymer hybrid nanoparticle system is reported.The nanoparticle system can co-encapsulate and co-deliver a combination of therapeutic agents with different physicochemical properties (i.e., inhibitors for microRNA-221 and microRNA-222 (miRi-221/222) and paclitaxel (pac)).miRi-221/222 are hydrophilic and were encapsulated with calcium phosphate by co-precipitation in a water-in-oil emulsion.The precipitates were then coated with an anionic lipid, dioleoylphosphatidic acid (DOPA), to co-encapsulate hydrophobic paclitaxel outside the hydrophilic precipitates and inside the same nanoparticle.The nanoparticles formed by following this approach had a size of about ≤100nm and contained both lipid-coated calcium phosphate/miRi and paclitaxel.This nanoparticle system was found to simultaneously deliver paclitaxel and miRi-221/222 to their intracellular targets, leading to inhibit proliferative mechanisms of miR-221/222 and thus significantly enhancing the therapeutic efficacy of paclitaxel.


Subject(s)
Antineoplastic Agents, Phytogenic/administration & dosage , Nanoparticles , Paclitaxel/administration & dosage , Triple Negative Breast Neoplasms/drug therapy , Calcium Phosphates , Cell Line, Tumor , Humans , MicroRNAs/antagonists & inhibitors , Polymers
8.
Data Brief ; 9: 812-817, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27872877

ABSTRACT

This data article contains supporting information regarding the research article entitled "High butter-fat diet and bisphenol A additively impair male rat spermatogenesis" (P. Tarapore, M. Hennessy, D. Song, J. Ying, B. Ouyang, V. Govindarajah, et al.,) [1]. Sprague-Dawley females were fed AIN, high fat butter, 17α-ethinyl estradiol, or high fat butter plus four bisphenol A doses (2500 µg/kg bw-d, 250 µg/kg bw-d, 25 µg/kg bw-d, and 2.5 µg/kg bw-d) before and during pregnancy. All diets were switched to AIN after the pups were born. Male offspring received testosterone (T)- and estradiol-17ß (E2)-filled implants from postnatal day 70-210 for 20 weeks (T+E2 rat model). The testes were weighed, and examined for impairments in spermatogenesis.

9.
PLoS One ; 9(3): e90332, 2014.
Article in English | MEDLINE | ID: mdl-24594937

ABSTRACT

Human exposure to bisphenol A (BPA) is ubiquitous. Animal studies found that BPA contributes to development of prostate cancer, but human data are scarce. Our study examined the association between urinary BPA levels and Prostate cancer and assessed the effects of BPA on induction of centrosome abnormalities as an underlying mechanism promoting prostate carcinogenesis. The study, involving 60 urology patients, found higher levels of urinary BPA (creatinine-adjusted) in Prostate cancer patients (5.74 µg/g [95% CI; 2.63, 12.51]) than in non-Prostate cancer patients (1.43 µg/g [95% CI; 0.70, 2.88]) (p = 0.012). The difference was even more significant in patients <65 years old. A trend toward a negative association between urinary BPA and serum PSA was observed in Prostate cancer patients but not in non-Prostate cancer patients. In vitro studies examined centrosomal abnormalities, microtubule nucleation, and anchorage-independent growth in four Prostate cancer cell lines (LNCaP, C4-2, 22Rv1, PC-3) and two immortalized normal prostate epithelial cell lines (NPrEC and RWPE-1). Exposure to low doses (0.01-100 nM) of BPA increased the percentage of cells with centrosome amplification two- to eight-fold. Dose responses either peaked or reached the plateaus with 0.1 nM BPA exposure. This low dose also promoted microtubule nucleation and regrowth at centrosomes in RWPE-1 and enhanced anchorage-independent growth in C4-2. These findings suggest that urinary BPA level is an independent prognostic marker in Prostate cancer and that BPA exposure may lower serum PSA levels in Prostate cancer patients. Moreover, disruption of the centrosome duplication cycle by low-dose BPA may contribute to neoplastic transformation of the prostate.


Subject(s)
Benzhydryl Compounds/toxicity , Cell Division , Centrosome , Endocrine Disruptors/toxicity , Phenols/toxicity , Prostatic Neoplasms/chemically induced , Age of Onset , Benzhydryl Compounds/urine , Endocrine Disruptors/urine , Fluorescent Antibody Technique, Indirect , Humans , Male , Phenols/urine , Prostatic Neoplasms/pathology
10.
Neoplasia ; 15(11): 1262-71, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24339738

ABSTRACT

Alternative splicing of estrogen receptor ß (ERß) yields five isoforms, but their functions remain elusive. ERß isoform 5 (ERß5) has been positively correlated with better prognosis and longer survival of patients with breast cancer (BCa) in various clinical studies. In this study, we investigated the inhibitory role of ERß5 in BCa cells. Although ERß5 does not reduce proliferation of BCa cell lines MCF-7 and MDA-MB-231, its ectopic expression significantly decreases their survival by sensitizing them to doxorubicin- or cisplatin-induced apoptosis through the intrinsic apoptotic pathway. Moreover, we discovered Bcl2L12, which belongs to the Bcl-2 family regulating apoptosis, to be a specific interacting partner of ERß5, but not ERß1 or ERα, in an estradiol-independent manner. Knockdown of Bcl2L12 enhanced doxorubicin- or cisplatin-induced apoptosis, and this process was further promoted by ectopic expression of ERß5. Whereas Bcl2L12 was previously shown to inhibit apoptosis through binding to caspase 7, such interaction is reduced in the presence of ERß5, suggesting a mechanism by which ERß5 sensitizes cells to apoptosis. In conclusion, ERß5 interacts with Bcl2L12 and functions in a novel estrogen-independent molecular pathway that promotes chemotherapeutic Agent-Induced in vitro apoptosis of BCa cell lines.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Estrogen Receptor beta/metabolism , Muscle Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Apoptosis/genetics , Breast Neoplasms/genetics , Caspase 7/genetics , Caspase 7/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/pharmacology , Doxorubicin/pharmacology , Estrogen Receptor beta/genetics , Female , HEK293 Cells , Humans , MCF-7 Cells , Muscle Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/genetics
11.
J Biol Chem ; 288(35): 25038-25052, 2013 Aug 30.
Article in English | MEDLINE | ID: mdl-23857583

ABSTRACT

Estrogen receptor (ER) ß1 and ERα have overlapping and distinct functions despite their common use of estradiol as the physiological ligand. These attributes are explained in part by their differential utilization of coregulators and ligands. Although Tip60 has been shown to interact with both receptors, its regulatory role in ERß1 transactivation has not been defined. In this study, we found that Tip60 enhances transactivation of ERß1 at the AP-1 site but suppresses its transcriptional activity at the estrogen-response element (ERE) site in an estradiol-independent manner. However, different estrogenic compounds can modify the Tip60 action. The corepressor activity of Tip60 at the ERE site is abolished by diarylpropionitrile, genistein, equol, and bisphenol A, whereas its coactivation at the AP-1 site is augmented by fulvestrant (ICI 182,780). GRIP1 is an important tethering mediator for ERs at the AP-1 site. We found that coexpression of GRIP1 synergizes the action of Tip60. Although Tip60 is a known acetyltransferase, it is unable to acetylate ERß1, and its coregulatory functions are independent of its acetylation activity. In addition, we showed the co-occupancy of ERß1 and Tip60 at ERE and AP-1 sites of ERß1 target genes. Tip60 differentially regulates the endogenous expression of the target genes by modulating the binding of ERß1 to the cis-regulatory regions. Thus, we have identified Tip60 as the first dual-function coregulator of ERß1.


Subject(s)
Estrogen Receptor beta/metabolism , Histone Acetyltransferases/metabolism , Response Elements/physiology , Transcription, Genetic/physiology , Transcriptional Activation/physiology , Acetylation , Carrier Proteins/genetics , Carrier Proteins/metabolism , Estrogen Receptor beta/genetics , HEK293 Cells , Histone Acetyltransferases/genetics , Humans , Lysine Acetyltransferase 5 , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism
12.
ACS Med Chem Lett ; 4(1): 27-31, 2013 Jan 10.
Article in English | MEDLINE | ID: mdl-23538490

ABSTRACT

Highly toxic bacterial ionophores are commonly used in veterinary medicine, but their therapeutic index is too narrow for human usage. With the goal of developing ionophores with a broader therapeutic index, we constructed highly derivatized synthetic ionophores. The toxicities of crown ether host-rotaxanes (CEHR's) against the SKOV-3 cell line were measured. The effect of Mg2+ or Ca2+ on toxicity was explored because changes in the intracellular concentration of these cations can cause cell death through apoptosis. We found Boc-CEHR is highly toxic and Arg-CEHR is slightly less toxic with IC50 values of 0.5 µM and 6 µM, respectively, in standard growth medium. Increasing the concentration of Ca2+ resulted in greater toxicity of the CEHRs, whereas increasing the concentration of Mg2+ was less effective on reducing IC50. Cell death occurs mainly through apoptosis. Although preliminary, these results suggest that the CEHRs deliver Ca2+ and perhaps Mg2+ into cells inducing apoptosis.

13.
Biochemistry ; 51(48): 9689-97, 2012 Dec 04.
Article in English | MEDLINE | ID: mdl-23106339

ABSTRACT

The increased mortality in prostate cancer is usually the result of metastatic progression of the disease from the organ-confined location. Among the major events in this progression cascade are enhanced cell migration and loss of adhesion. Moreover, elevated levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) found within the tumor microenvironment are hallmarks of progression of this cancer. To understand the role of nitrosative stress in prostate cancer progression, we investigated the effects of NO and iNOS on prostate cancer cell migration and adhesion. Our results indicate that ectopic expression of iNOS in prostate cancer cells increased the extent of cell migration, which could be blocked by selective ITGα6 blocking antibody or iNOS inhibitors. Furthermore, iNOS was found to cause S-nitrosylation of ITGα6 at Cys86 in prostate cancer cells. By comparing the activities of wild-type ITGα6 and a Cys86 mutant, we showed that treatment of prostate cancer cells with NO increased the level of ITGα6 heterodimerization with ITGß1 but not with ITGß4. Finally, S-nitrosylation of ITGα6 weakened its binding to laminin-ß1 and weakened the adhesion of prostate cancer cells to laminin-1. In conclusion, S-nitrosylation of ITGα6 increased the extent of prostate cancer cell migration, which could be a potential mechanism of NO- and iNOS-induced enhancement of prostate cancer metastasis.


Subject(s)
Cell Movement , Integrin alpha6/metabolism , Integrin beta1/metabolism , Laminin/metabolism , Prostatic Neoplasms/pathology , Cell Adhesion , Cell Line, Tumor , Humans , Male , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolism , Prostatic Neoplasms/metabolism
14.
Cell Cycle ; 11(15): 2931-46, 2012 Aug 01.
Article in English | MEDLINE | ID: mdl-22833046

ABSTRACT

BRCA1, a product of a familial breast and ovarian cancer susceptibility gene, localizes to centrosomes and physically interacts with γ-tubulin, a key centrosomal protein for microtubule nucleation and anchoring at centrosomes. Here, we performed a rigorous analysis of centrosome localization of BRCA1, and found that BRCA1 is specifically associated with mother centrioles in unduplicated centrosomes, and daughter centrioles acquire BRCA1 prior to initiation of duplication, and thus duplicated centrosomes are both bound by BRCA1. We further found that BRCA1 suppresses centrosomal aster formation. In addition, we identified a new domain of BRCA1 critical for γ-tubulin binding, which confers not only its localization to centrosomes, but also its activity to suppress centrosomal aster formation.


Subject(s)
BRCA1 Protein/metabolism , Breast Neoplasms/metabolism , Centrioles/metabolism , Centrosome/metabolism , Tubulin/metabolism , BRCA1 Protein/genetics , Cell Line, Tumor , Female , Green Fluorescent Proteins , HeLa Cells , Humans , MCF-7 Cells , RNA Interference , RNA, Small Interfering , Spindle Apparatus/metabolism
15.
Steroids ; 77(7): 727-37, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22465878

ABSTRACT

Estrogen receptor (ER) ß was discovered over a decade ago. The design of most studies on this receptor was based on knowledge of its predecessor, ERα. Although breast cancer (BCa) has been a main focus of ERß research, its precise roles in breast carcinogenesis remain elusive. Data from in vitro models have not always matched those from observational or clinical studies. Several inherent factors may contribute to these discrepancies: (a) several ERß spliced variants are expressed at the protein level, and isoform-specific antibodies are unavailable for some variants; (b) post-translational modifications of the receptor regulate receptor functions; (c) the role of the receptor differs significantly depending on the type of ligands, cis-elements, and co-regulators that interact with the receptor; and (d) the diversity of distribution of the receptor among intracellular organelles of BCa cells. This review addresses the gaps in knowledge in ERß research as it pertains to BCa regarding the following questions: (1) is ERß a tumor suppressor in BCa?; (2) do ERß isoforms play differential roles in breast carcinogenesis?; (3) do nuclear signaling and extranuclear ERß signaling differ in BCa?; (4) what are the consequences of post-translational modifications of ERß in BCa?; (5) how do co-regulators and interacting proteins increase functional diversity of ERß?; and (6) how do the types of ligand and regulatory cis-elements affect the action of ERß in BCa?. Insights gained from these key questions in ERß research should help in prevention, diagnosis/prognosis, and treatment of BCa.


Subject(s)
Breast Neoplasms/physiopathology , Estrogen Receptor beta/physiology , Breast Neoplasms/pathology , Estrogen Receptor beta/genetics , Estrogen Receptor beta/metabolism , Female , Humans , Protein Processing, Post-Translational , RNA Splicing , Signal Transduction , Subcellular Fractions/metabolism
16.
ILAR J ; 53(3-4): 289-305, 2012.
Article in English | MEDLINE | ID: mdl-23744968

ABSTRACT

This review focuses on how environmental factors through epigenetics modify disease risk and health outcomes. Major epigenetic events, such as histone modifications, DNA methylation, and microRNA expression, are described. The function of dose, duration, composition, and window of exposure in remodeling the individual's epigenetic terrain and disease susceptibility are addressed. The ideas of lifelong editing of early-life epigenetic memories, transgenerational effects through germline transmission, and the potential role of hydroxylmethylation of cytosine in developmental reprogramming are discussed. Finally, the epigenetic effects of several major classes of environmental factors are reviewed in the context of pathogenesis of disease. These include endocrine disruptors, tobacco smoke, polycyclic aromatic hydrocarbons, infectious pathogens, particulate matter, diesel exhaust particles, dust mites, fungi, heavy metals, and other indoor and outdoor pollutants. We conclude that the summation of epigenetic modifications induced by multiple environmental exposures, accumulated over time, represented as broad or narrow, acute or chronic, developmental or lifelong, may provide a more precise assessment of risk and consequences. Future investigations may focus on their use as readouts or biomarkers of the totality of past exposure for the prediction of future disease risk and the prescription of effective countermeasures.


Subject(s)
Epigenesis, Genetic/genetics , DNA Methylation/genetics , Disease Susceptibility/metabolism , Environmental Exposure/adverse effects , Epigenesis, Genetic/physiology , Histones/metabolism , Humans
17.
Mol Ther ; 19(2): 386-94, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21063391

ABSTRACT

Ovarian cancer is a highly metastatic and lethal disease, making it imperative to find treatments that target late-stage malignant tumors. The packaging RNA (pRNA) of bacteriophage phi29 DNA-packaging motor has been reported to function as a highly versatile vehicle to carry small interference RNA (siRNA) for silencing of survivin. In this article, we explore the potential of pRNA as a vehicle to carry siRNA specifically targeted to metallothionein-IIa (MT-IIA) messenger RNA (mRNA), and compare it to survivin targeting pRNA. These two anti-apoptotic cell survival factors promote tumor cell viability, and are overexpressed in recurrent tumors. We find that pRNA chimeras targeting MT-IIA are processed into double-stranded siRNA by dicer, are localized within the GW/P-bodies, and are more potent than siRNA alone in silencing MT-IIA expression. Moreover, knockdown of both survivin and MT-IIA expression simultaneously results in more potent effects on cell proliferation in the aggressive ovarian tumor cell lines than either alone, suggesting that therapeutic approaches that target multiple genes are essential for molecular therapy. The folate receptor-targeted delivery of siRNA by the folate-pRNA dimer emphasizes the cancer cell-specific aspect of this system. The pRNA system, which has the capability to assemble into multivalent nanoparticles, has immense promise as a highly potent therapeutic agent.


Subject(s)
Genetic Vectors/chemistry , Inhibitor of Apoptosis Proteins/genetics , Metallothionein/genetics , Ovarian Neoplasms/therapy , RNA/chemistry , Cell Line , Cell Line, Tumor , Cell Proliferation , Female , Flow Cytometry , Genetic Vectors/administration & dosage , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Ovarian Neoplasms/genetics , RNA/administration & dosage , RNA, Small Interfering/genetics , RNA, Small Interfering/physiology , Reverse Transcriptase Polymerase Chain Reaction , Survivin
18.
FEBS Lett ; 580(2): 399-409, 2006 Jan 23.
Article in English | MEDLINE | ID: mdl-16376875

ABSTRACT

Nucleophosmin (NPM) is a multifunctional phosphoprotein, being involved in ribosome assembly, pre-ribosomal RNA processing, DNA duplication, nucleocytoplasmic protein trafficking, and centrosome duplication. NPM is phosphorylated by several kinases, including nuclear kinase II, casein kinase 2, Polo-like kinase 1 and cyclin-dependent kinases (CDK1 and 2), and these phosphorylations modulate the activity and function of NPM. We have previously identified Thr(199) as the major phosphorylation site of NPM mediated by CDK2/cyclin E (and A), and this phosphorylation is involved in the regulation of centrosome duplication. In this study, we further examined the effect of CDK2-mediated phosphorylation of NPM by using the antibody that specifically recognizes NPM phosphorylated on Thr(199). We found that the phospho-Thr(199) NPM localized to dynamic sub-nuclear structures known as nuclear speckles, which are believed to be the sites of storage and/or assembly of pre-mRNA splicing factors. Phosphorylation on Thr(199) by CDK2/cyclin E (and A) targets NPM to nuclear speckles, and enhances the RNA-binding activity of NPM. Moreover, phospho-Thr(199) NPM, but not unphosphorylated NPM, effectively represses pre-mRNA splicing. These findings indicate the involvement of NPM in the regulation of pre-mRNA processing, and its activity is controlled by CDK2-mediated phosphorylation on Thr(199).


Subject(s)
Cell Nucleus/metabolism , Nuclear Proteins/metabolism , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Threonine/metabolism , Animals , Antibodies, Phospho-Specific/metabolism , Cells, Cultured , Cyclin A/metabolism , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Nucleophosmin , Phosphorylation , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
19.
FEBS Lett ; 579(29): 6621-34, 2005 Dec 05.
Article in English | MEDLINE | ID: mdl-16297385

ABSTRACT

Nucleophosmin (NPM)/B23 is a multifunctional protein, involving in a wide variety of basic cellular processes, including ribosome assembly, DNA duplication, nucleocytoplasmic trafficking, and centrosome duplication. It has previously been shown that NPM/B23 localizes to centrosomes, and dissociate from centrosomes upon phosphorylation by Cdk2/cyclin E. However, detail characterization of centrosomal association of NPM/B23 has been hampered by the lack of appropriate antibodies that efficiently detects centrosomally localized NPM/B23, as well as by apparent loss of natural behavior of NPM/B23 when tagged with fluorescent proteins. Here, by the use of newly generated anti-NPM/B23 antibody, we conducted a careful analysis of centrosomal localization of NPM/B23. We found that NPM/B23 localizes between the paired centrioles of unduplicated centrosomes, suggesting the role of NPM/B23 in the centriole pairing. Upon initiation of centrosome duplication, some NPM/B23 proteins remain at mother centrioles of the parental centriole pairs. We further found that inhibition of Crm1 nuclear export receptor results in both accumulation of cyclin E at centrosomes and efficient dissociation of NPM/B23 from centrosomes.


Subject(s)
Karyopherins/metabolism , Nuclear Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Antibodies, Monoclonal , Cell Cycle , Centrioles/metabolism , Centrosome , Cyclin E/metabolism , Epitope Mapping , Immunohistochemistry , Karyopherins/antagonists & inhibitors , Karyopherins/physiology , Mice , Nuclear Proteins/immunology , Nuclear Proteins/physiology , Nucleophosmin , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/physiology , Exportin 1 Protein
20.
Cancer Res ; 65(11): 4568-77, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15930274

ABSTRACT

The liver exhibits an exquisitely controlled cell cycle, wherein hepatocytes are maintained in quiescence until stimulated to proliferate. The retinoblastoma tumor suppressor, pRB, plays a central role in proliferative control by inhibiting inappropriate cell cycle entry. In many cases, liver cancer arises due to aberrant cycles of proliferation, and correspondingly, pRB is functionally inactivated in the majority of hepatocellular carcinomas. Therefore, to determine how pRB loss may provide conditions permissive for deregulated hepatocyte proliferation, we investigated the consequence of somatic pRB inactivation in murine liver. We show that liver-specific pRB loss results in E2F target gene deregulation and elevated cell cycle progression during post-natal growth. However, in adult livers, E2F targets are repressed and hepatocytes become quiescent independent of pRB, suggesting that other factors may compensate for pRB loss. Therefore, to probe the consequences of acute pRB inactivation in livers of adult mice, we gave adenoviral-Cre by i.v. injection. We show that acute pRB loss is sufficient to elicit E2F target gene expression and cell cycle entry in adult liver, demonstrating a critical role for pRB in maintaining hepatocyte quiescence. Finally, we show that liver-specific pRB loss results in the development of nuclear pleomorphism associated with elevated ploidy that is evident in adult mice harboring both acute and chronic pRB loss. Together, these results show the crucial role played by pRB in maintaining hepatocyte quiescence and ploidy in adult liver in vivo and underscore the critical importance of delineating the consequences of acute pRB loss in adult animals.


Subject(s)
Liver/physiology , Ploidies , Retinoblastoma Protein/deficiency , Animals , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Cell Growth Processes/genetics , DNA-Binding Proteins/genetics , E2F Transcription Factors , Gene Silencing , Genes, Retinoblastoma/genetics , Hepatocytes/cytology , Hepatocytes/physiology , Liver/cytology , Liver/metabolism , Mice , Mice, Knockout , Retinoblastoma Protein/genetics , Retinoblastoma Protein/physiology , Transcription Factors/genetics
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