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PURPOSE - Carbonyl reductases (CBRs) catalyze reduction of anthracyclines to cardiotoxic alcohol metabolites. Polymorphisms in CBR1 and CBR3 influence synthesis of these metabolites. We examined whether single nucleotide polymorphisms in CBR1 (CBR1 1096G>A) and/or CBR3 (CBR3 V244M) modified the dose-dependent risk of anthracycline-related cardiomyopathy in childhood cancer survivors.
PATIENTS AND METHODS - One hundred seventy survivors with cardiomyopathy (patient cases) were compared with 317 survivors with no cardiomyopathy (controls; matched on cancer diagnosis, year of diagnosis, length of follow-up, and race/ethnicity) using conditional logistic regression techniques.
RESULTS - A dose-dependent association was observed between cumulative anthracycline exposure and cardiomyopathy risk (0 mg/m(2): reference; 1 to 100 mg/m(2): odds ratio [OR], 1.65; 101 to 150 mg/m(2): OR, 3.85; 151 to 200 mg/m(2): OR, 3.69; 201 to 250 mg/m(2): OR, 7.23; 251 to 300 mg/m(2): OR, 23.47; > 300 mg/m(2): OR, 27.59; P(trend) < .001). Among individuals carrying the variant A allele (CBR1:GA/AA and/or CBR3:GA/AA), exposure to low- to moderate-dose anthracyclines (1 to 250 mg/m(2)) did not increase the risk of cardiomyopathy. Among individuals with CBR3 V244M homozygous G genotypes (CBR3:GG), exposure to low- to moderate-dose anthracyclines increased cardiomyopathy risk when compared with individuals with CBR3:GA/AA genotypes unexposed to anthracyclines (OR, 5.48; P = .003), as well as exposed to low- to moderate-dose anthracyclines (OR, 3.30; P = .006). High-dose anthracyclines (> 250 mg/m(2)) were associated with increased cardiomyopathy risk, irrespective of CBR genotype status.
CONCLUSION - This study demonstrates increased anthracycline-related cardiomyopathy risk at doses as low as 101 to 150 mg/m(2). Homozygosis for G allele in CBR3 contributes to increased cardiomyopathy risk associated with low- to moderate-dose anthracyclines, such that there seems to be no safe dose for patients homozygous for the CBR3 V244M G allele. These results suggest a need for targeted intervention for those at increased risk of cardiomyopathy.
The regulation of vitamin D receptor (VDR), a key mediator in the vitamin D pathway, in breast cancer etiology has long been of interest. We have shown here that the transcriptional repressor protein SLUG inhibits the expression of VDR in human breast cancer cells. To explore the possibility that SLUG regulates the VDR gene promoter, we cloned a 628bp fragment (-613 to +15) of the human VDR gene promoter. This region contains three E2-box sequences (CAGGTG/CACCTG), the classical binding site of SLUG. SLUG specifically inhibited VDR gene promoter activity. Chromatin-immunoprecipitation (ChIP) assays revealed that SLUG is recruited on the native VDR gene promoter along with the co-repressor protein CtBP1 and the effector protein HDAC1. These data suggests that SLUG binds to the E2-box sequences of the VDR gene promoter and recruits CtBP1 and HDAC1, which results in the inhibition of VDR gene expression by chromatin remodeling.
SLUG is a transcriptional repressor protein implicated to have major role in the oncogenesis and metastasis of human breast cells. We previously have shown by chromatin immunoprecipitation assay that human SLUG (hSLUG) is co-localized with the co-repressor protein CtBP1 as bound to the BRCA2 gene silencer [M.K. Tripathi, S. Misra, S.V. Khedkar, N. Hamilton, C. Irvin-Wilson,, C. Sharan, L. Sealy, G. Chaudhuri, J. Biol. Chem. 280 (2005) 17163-17171]. hSLUG was predicted to be binding directly to CtBP1 because of an apparent presence of CtBP1 binding site in its amino acid sequences. Here, we provide evidence through yeast two-hybrid and in vitro co-immunoprecipitation analyses that hSLUG does not directly interacts with hCtBP1. This observation will help in the study of the mode of action of hSLUG in human cells.
Our objective is to identify molecular factors which contribute to the increased risk of smokers for oral squamous cell carcinoma (OSCC). In the present study, we investigated the effects of cigarette smoke condensate (CSC) on gene expression profiles in different human oral cell phenotypes: normal epidermal keratinocytes (NHEK), oral dysplasia cell lines (Leuk1 and Leuk2), and a primary oral carcinoma cell line (101A). We determined differential gene expression patterns in CSC-exposed versus non-exposed cells using high-density microarray RNA expression profiling and validation by quantitative real-time RT-PCR. A set of 35 genes was specifically up- or down-regulated following CSC treatment (25microg/ml for 24h) by at least 2-fold in any one cell type. Notably, five genes of the cytochrome P450 (CYP1A1, CYP1B1) and aldo-keto reductase (AKR1C1, AKR1C3, AKR1B10) families were highly increased in expression, some of them 15- to 30-fold. The timing and extent of induction for these genes differed among the four cell phenotypes. A potential biological interaction network for the CSC response in oral cells was derived from these data, proposing novel putative response pathways. These CSC-responsive genes presumably participate in the prevention or repair of carcinogen-induced DNA damage in tobacco-related oral carcinogenesis, and may potentially be exploited for determining the severity of exposure and for correcting mutagenic damage in exposed tissues of the oral cavity.
The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the non-dividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the -701 to -921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.
Human NAD(+)-dependent microsomal short-chain dehydrogenase/reductase RoDH-4 oxidizes all-trans-retinol, 13-cis-retinol and 3alpha-hydroxysteroids to corresponding retinaldehydes and 3-ketones. RoDH-4 behaves as an integral membrane protein, but its topology in the membrane is not known. Analysis of RoDH-4 polypeptide using algorithms for secondary structure predictions suggests that RoDH-4 contains four potential membrane-spanning domains: the N-terminal, the C-terminal, and the two central hydrophobic segments. To determine the role of each segment in association of RoDH-4 with the membrane, we prepared several expression constructs coding for truncated RoDH-4 polypeptides that lacked the putative membrane-spanning domains and expressed them in insect Sf9 cells using the Baculovirus system. Association of truncated RoDH-4 constructs with the microsomal membranes was analyzed by alkaline extraction and floatation in sucrose gradient. Catalytic activity of truncated RoDH-4 constructs was assayed using the 3alpha-hydroxysteroid androsterone as substrate. Truncated RoDH-4 that lacked the first thirteen amino acids of the N-terminal segment was partially active and exhibited the apparent K(m) value for androsterone similar to that of the wild-type enzyme. Removal of 23 N-terminal hydrophobic amino acids resulted in significant loss of activity and a 14-fold increase in the apparent K(m) value. Removal of the C-terminal 27 amino acid segment resulted in a approximately 600-fold increase in the apparent K(m) value. Each truncated mutant behaved as an integral membrane protein. Furthermore, protein that lacked all four hydrophobic segments remained associated with the membrane. Thus, the N-terminal and the C-terminal ends are both important for RoDH-4 activity and the removal of the putative transmembrane segments does not convert RoDH-4 into a soluble protein, suggesting additional sites of membrane interaction.
Retinoic acid is necessary for the maintenance of many lining epithelia of the body, such as the epithelium of the luminal surface of the uterus. Administration of estrogen to prepubertal rats induces in these epithelial cells the ability to synthesize retinoic acid from retinol, coincident with the appearance of cellular retinoic acid-binding protein, type two, which is normally present in these cells only at estrus in the mature, cycling animal. Here, we report the isolation, from a cDNA library prepared from uterine mRNA collected at the estrous stage and from a rat mammary adenocarcinoma cell line, of a cDNA that encodes a novel retinol dehydrogenase. A member of the short-chain alcohol dehydrogenase family, the encoded enzyme was capable of metabolizing retinol to retinal when expressed in cells after transfection of its cDNA. When cotransfected with the cDNA of human aldehyde 6, a known retinaldehyde dehydrogenase, the transfected cells synthesized retinoic acid from retinol. Immunohistochemical analysis revealed that the protein was present in the uterine lining epithelium of the mature animal only at estrus, coincident with the presence of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two. Consequently, this novel short-chain alcohol dehydrogenase is an excellent candidate for the retinol dehydrogenase that catalyzes the first step in retinoic acid biosynthesis that occurs in uterine epithelial cells.
All-trans-retinoic acid is a metabolite of vitamin A (all-trans-retinol) that functions as an activating ligand for a family of nuclear retinoic acid receptors. The intracellular levels of retinoic acid in tissues are tightly regulated, although the mechanisms underlying the control of retinoid metabolism at the level of specific enzymes are not completely understood. In this report we present the first characterization of the retinoid substrate specificity of a novel short-chain dehydrogenase/reductase (SDR) encoded by RalR1/PSDR1, a cDNA recently isolated from the human prostate (Lin, B., White, J. T., Ferguson, C., Wang, S., Vessella, R., Bumgarner, R., True, L. D., Hood, L., and Nelson, P. S. (2001) Cancer Res. 61, 1611-1618). We demonstrate that RalR1 exhibits an oxidoreductive catalytic activity toward retinoids, but not steroids, with at least an 800-fold lower apparent K(m) values for NADP+ and NADPH versus NAD+ and NADH as cofactors. The enzyme is approximately 50-fold more efficient for the reduction of all-trans-retinal than for the oxidation of all-trans-retinol. Importantly, RalR1 reduces all-trans-retinal in the presence of a 10-fold molar excess of cellular retinol-binding protein type I, which is believed to sequester all-trans-retinal from nonspecific enzymes. As shown by immunostaining of human prostate and LNCaP cells with monoclonal anti-RalR1 antibodies, the enzyme is highly expressed in the epithelial cell layer of human prostate and localizes to the endoplasmic reticulum. The enzymatic properties and expression pattern of RalR1 in prostate epithelium suggest that it might play a role in the regulation of retinoid homeostasis in human prostate.
The solution structure of the 1,N(2)-propanodeoxyguanosine (PdG) adduct was determined in a 3-base hairpin loop formed by d(CGCGGTXTCCGCG) (X = PdG). This sequence is contained within the Salmonella typhimurium hisD3052 gene, a hotspot for frameshift mutagenesis. PdG provides a structural model for the primary adduct induced in DNA by malondialdehyde, the 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one (M(1)G) lesion. The solution structure of the PdG-containing hairpin was refined by molecular dynamics calculations restrained by a combination of NMR-derived distances and dihedral angles, using a simulated annealing protocol. The structure of the PdG-modified hairpin consisted of a five-base-pair stem and a three-base loop consisting of T(6), X(7), and T(8). T(6) projected into the minor groove of the stem adjacent to G(4). The modified base X(7) stacked on top of the duplex stem and wedged between bases T(8) and C(9). The PdG moiety was oriented such that the imidazole proton was facing the minor groove of the stem and the exocyclic protons projected into the major groove. The structure of the adducted hairpin was compared with the structure of the corresponding unmodified oligodeoxynucleotide, and was found to be similar. There was a minor difference in the backbone angles of the G and PdG Hairpins at the phosphate linkage between G(5) and T(6) involving the G(5) epsilon angle and T(6) alpha and beta angles. The PdG-modified hairpin exhibited an increase in T(m) of approximately 2 degrees C compared to the unmodified hairpin. The structural and thermodynamic similarities suggested that PdG does not stabilize this hairpin and thus may not promote its extrusion in duplex DNA. The structural results are correlated with the results of site-specific mutagenesis experiments in the same sequence, which do not show evidence of frameshift mutations associated with hairpin loop formation. The geometry of this three-base loop is similar to that of other DNA hairpins containing three-base loops, and suggests a common motif for the folding of these loops.
The structure of the 1,N(2)-Propanodeoxyguanosine (PdG) adduct was determined at pH 5.2 in the oligodeoxynucleotide duplex 5'-d(CGCGGTXTCCGCG)3'.5'-d(CGCGGACACCGCG)-3' (X = PdG). This sequence, referred to as the -TXT- sequence, is contained within the Salmonella typhimurium hisD3052 gene and contains a palindrome, representing a potential hotspot for frameshift mutagenesis. PdG provides a model for the primary adduct induced in DNA by malondialdehyde, the 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one (M(1)G) lesion. The solution structure was refined by molecular dynamics calculations restrained by a combination of NMR-derived distances and dihedral angles, using a simulated annealing protocol. PdG introduced a localized perturbation into the sequence at base pair X(7).C(20), which was pH-dependent. At neutral pH, conformational exchange resulted in spectral line broadening, and it was not possible to determine the structure. A stable structure was observed at pH 5.2 in which PdG rotated about the glycosyl bond into the syn conformation. This placed the exocyclic moiety into the major groove of the duplex. PdG formed a protonated Hoogsteen pair with nucleotide C(20) in the complementary strand. The pseudorotation of the deoxyribose at C(20) was altered to an approximately equal blend of C2'-endo and C3'-endo structures. However, these made little difference in the overall structure of the modified oligodeoxynucleotide. The structure was compared to that of PdG in the 5'-d(CGCXCGGCATG)-3'.5'-(CATGCCGCGCG)-3' sequence (the -CXC- sequence) at pH 5.8 [Singh, U. S., Moe, J. G., Reddy, G. R., Weisenseel, J. P., Marnett, L. J., and Stone, M. P. (1993) Chem. Res. Toxicol. 6, 825-836]. A sequence effect was observed. When PdG was placed into the -TXT- sequence at low pH, the structural perturbation was limited to the X(7).C(20) base pair. In contrast, when PdG was placed into the -CXC- sequence at low pH, both the modified base pair and its 3'-neighbor base pair were disrupted. The results are discussed in the context of differential outcomes for site-specific mutagenesis and replication bypass experiments when PdG was placed in the -TXT- and -CXC- sequences, respectively.