Case-control study of candidate gene methylation and adenomatous polyp formation.

PURPOSE: Colorectal cancer (CRC) is one of the most common and preventable forms of cancer but remains the second leading cause of cancer-related death. Colorectal adenomas are precursor lesions that develop in 70-90 % of CRC cases. Identification of peripheral biomarkers for adenomas would help to enhance screening efforts. This exploratory study examined the methylation status of 20 candidate markers in peripheral blood leukocytes and their association with adenoma formation. METHODS: Patients recruited from a local endoscopy clinic provided informed consent and completed an interview to ascertain demographic, lifestyle, and adenoma risk factors. Cases were individuals with a histopathologically confirmed adenoma, and controls included patients with a normal colonoscopy or those with histopathological findings not requiring heightened surveillance (normal biopsy, hyperplastic polyp). Methylation-specific polymerase chain reaction was used to characterize candidate gene promoter methylation. Odds ratios (ORs) and 95 % confidence intervals (95% CIs) were calculated using unconditional multivariable logistic regression to test the hypothesis that candidate gene methylation differed between cases and controls, after adjustment for confounders. RESULTS: Complete data were available for 107 participants; 36 % had adenomas (men 40 %, women 31 %). Hypomethylation of the MINT1 locus (OR 5.3, 95% CI 1.0-28.2) and the PER1 (OR 2.9, 95% CI 1.1-7.7) and PER3 (OR 11.6, 95% CI 1.6-78.5) clock gene promoters was more common among adenoma cases. While specificity was moderate to high for the three markers (71-97 %), sensitivity was relatively low (18-45 %). CONCLUSION: Follow-up of these epigenetic markers is suggested to further evaluate their utility for adenoma screening or surveillance.

Chromatin structural transitions and the phenomenon of vitellogenin gene memory in chickens.

We have previously shown that the steroid hormone-mediated transcriptional activation of the chicken vitellogenin II gene (VTGII) in the liver is accompanied by a series of chromatin structural changes, including the formation of two sets of 5'-proximal nuclease-hypersensitive sites and the demethylation of a single 5'-flanking MspI site which lies within a region of DNA that recently has been shown by Jost and co-workers to specifically bind the estrogen receptor complex in vitro. To assay the stability and possible functional significance of these induced structural changes, we transiently activated the VTGII gene during embryonic development and then allowed the chickens to hatch and grow for various periods of time before analyzing their livers. By 7 weeks posthatching all of the induced 5'-flanking hypersensitive sites had decayed. Moreover, the loss of these sites occurred without consequence to the "memory effect," that is, these structural features did not need to be present in hormone withdrawn birds to allow this gene to be activated more rapidly in response to a secondary presentation of estradiol. Although the demethylation was more stable, it also appeared not to be the basis of the memory phenomenon. The birds that still exhibited memory after 25 weeks of hormone withdrawal were not more extensively demethylated within the receptor-binding site than were the birds which failed to show memory at this age. A similar uncoupling of these two parameters was also observed when embryos were first injected with submaximal doses of estradiol and then assayed 1 week after hatching; the chickens which acquired memory were not demethylated to any greater extent than those which did not acquire memory. Other parameters that may be relevant to memory are discussed.

Evidence that chicken CR1 elements represent a novel family of retroposons.

We report the first precise delineation of a chicken CR1 element and show that it is flanked by a 6-base-pair target site duplication that occurred when this repetitive element transposed. The 3' end of this CR1 element is defined by an 8-base-pair imperfect direct repeat, and we infer that this sequence represents the 3' end of all intact CR1 elements. In contrast, the 5' ends are not unique, and we argue that this variation existed at the time each element transposed. We also provide evidence that CR1 elements transposed into preferred target sites. CR1 elements therefore appear to represent a novel class of passive retroposons.

Mutational studies reveal a complex set of positive and negative control elements within the chicken vitellogenin II promoter.

The endogenous chicken vitellogenin II (VTGII) gene is transcribed exclusively in hepatocytes in response to estrogen. We previously identified two estrogen response elements (EREs) upstream of this gene. We now present an analysis of the VTGII promoter activated by these EREs in response to estrogen. Chimeric VTGII-CAT genes were cotransfected into LMH chicken hepatoma cells along with an estrogen receptor expression vector, and transient CAT expression was assayed after culturing the cells in the absence or presence of estrogen. An analysis of constructs bearing deletions downstream of the more proximal ERE indicated that promoter elements relevant to transcription in LMH cells extend to between -113 and -96. The relative importance of sequences within the VTGII promoter was examined by using 10 contiguous linker scanner mutations spanning the region from -117 to -24. Although most of these mutations compromised VTGII promoter function, one dramatically increased expression in LMH cells and also rendered the VTGII promoter capable of being activated by cis-linked EREs in fibroblasts cotransfected with an estrogen receptor expression vector. Gel retardation and DNase I footprinting assays revealed four factor-binding sites within this promoter. We demonstrate that three of these sites bind C/EBP, SP1, and USF (or related factors), respectively; the fourth site binds a factor that we denote TF-V beta. The biological relevance of these findings is suggested by the fact that three of these binding sites map to sites previously shown to be occupied in vivo in response to estrogen.

The major and minor chicken vitellogenin genes are each adjacent to partially deleted pseudogene copies of the other.

The major chicken vitellogenin gene (VTGII) has previously been cloned and sequenced. We now report the isolation of genomic clones that encompass a minor chicken vitellogenin gene (VTGIII) which is also expressed in the liver in response to estradiol. Our analysis reveals that a pseudogene for VTGII (psi VTGII) lies 1,426 base pairs upstream of this VTGIII gene. A reevaluation of published sequence data reveals that the converse is also true, namely, that a pseudogene for VTGIII (psi VTGIII) lies 1,345 base pairs downstream of the VTGII gene. Our results show that a 335-base-pair deletion has removed the psi VTGIII promoter and cap site but left residual estrogen response element in a region where nuclease-hypersensitive sites have been reported to be induced in response to estradiol.

Two functional estrogen response elements are located upstream of the major chicken vitellogenin gene.

We used a transient-expression assay to identify two estrogen response elements (EREs) associated with the major chicken vitellogenin gene (VTGII). Each element was characterized by its ability to confer estrogen responsiveness when cloned in either orientation next to a chimeric reporter gene consisting of the herpes simplex virus thymidine kinase promoter and the chloramphenicol acetyl transferase-coding region. Deletion analyses indicated that sequences necessary for the distal ERE resided within the region from -626 to -613 (nucleotide positions relative to the VTGII start site) whereas those necessary for the proximal ERE were within the region from -358 to -335. These distal and proximal elements contain, respectively, a perfect copy and an imperfect copy of the 13-base-pair sequence that is an essential feature of the EREs associated with two frog vitellogenin genes. These chicken VTGII EREs mapped near regions that were restructured at the chromatin level when the endogenous VTGII gene was expressed in the liver in response to estradiol. These data suggest a model for the tissue-specific expression of this estrogen-responsive gene.

Chicken vitellogenin gene-binding protein, a leucine zipper transcription factor that binds to an important control element in the chicken vitellogenin II promoter, is related to rat DBP.

We screened a chicken liver cDNA expression library with a probe spanning the distal region of the chicken vitellogenin II (VTGII) gene promoter and isolated clones for a transcription factor that we have named VBP (for vitellogenin gene-binding protein). VBP binds to one of the most important positive elements in the VTGII promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP was deduced from a nearly full length cDNA copy and was found to contain a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. Moreover, VBP is a stable dimer free in solution. A data base search revealed that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control.

DNA-binding specificity of the PAR basic leucine zipper protein VBP partially overlaps those of the C/EBP and CREB/ATF families and is influenced by domains that flank the core basic region.

The PAR subfamily of basic leucine zipper (bZIP) factors comprises three proteins (VBP/TEF, DBP, and HLF) that have conserved basic regions flanked by proline- and acidic-amino-acid-rich (PAR) domains and functionally compatible leucine zipper dimerization domains. We show that VBP preferentially binds to sequences that consist of abutted GTAAY half-sites (which we refer to as PAR sites) as well as to sequences that contain either a C/EBP half-site (GCAAT) or a CREB/ATF half-site (GTCAT) in place of one of the PAR half-sites. Since the sequences that we describe as PAR sites and PAR-CREB/ATF chimeric sites, respectively, were both previously described as high-affinity binding sites for the E4BP4 transcriptional repressor, we infer that these sequences may be targets for positive and negative regulation. Similarly, since the sequences that we describe as PAR-C/EBP and PAR-CREB/ATF chimeric sites are known to be high-affinity binding sites for C/EBP and CREB/ATF factors, respectively, we infer that these sites may each be targets for multiple subfamilies of bZIP factors. To gain insights regarding the molecular basis for the binding-site specificity of PAR factors, we also carried out an extensive mutational analysis of VBP. By substituting five amino acid residues that differ between the Drosophila giant bZIP factor and the vertebrate PAR bZIP factors, we show that the fork region, which bridges the basic and leucine zipper domains, contributes to half-site sequence specificity. In addition, we report that at least two domains amino terminal to the core basic region are required for VBP to bind to the full spectrum of PAR target sites. Thus, whereas direct base contacts may be restricted to basic-region residues (as indicated by GCN4-DNA crystal structures), several other domains also influence the DNA-binding specificity of PAR bZIP proteins.

A GATA-6 gene heart-region-specific enhancer provides a novel means to mark and probe a discrete component of the mouse cardiac conduction system.

The transcriptional programs that specify the distinct components of the cardiac conduction system are poorly understood, in part due to a paucity of definitive molecular markers. In the present study we show that a cGATA-6 gene enhancer can be used to selectively express transgenes in the atrioventricular (AV) conduction system as it becomes manifest in the developing multichambered mouse heart. Furthermore, our analysis of staged cGATA-6/lacZ embryos revealed that the activity of this heart-region-specific enhancer can be traced back essentially to the outset of the cardiogenic program. We provide evidence that this enhancer reads medial/lateral and anterior/posterior positional information before the heart tube forms and we show that the activity of this enhancer becomes restricted at the heart looping stage to AV myocardial cells that induce endocardial cushion formation. We infer that a deeply-rooted heart-region-specific transcriptional program serves to coordinate AV valve placement and AV conduction system formation. Lastly, we show that cGATA-6/Cre mice can be used to delete floxed genes in the respective subsets of specialized heart cells.

The chicken vitellogenin II gene is flanked by a GATA factor-dependent estrogen response unit.

The chicken vitellogenin II (VTGII) gene is flanked by an imperfect estrogen response element (ERE) at -350 and a perfect ERE at -620. In the present study we show that this imperfect ERE lies within an estrogen response unit (ERU) that requires a GATA factor and the estrogen receptor to function as an estrogen-dependent enhancer. We infer that GATA-6 contributes to the estrogen-dependent and liver-specific regulation of the endogenous VTGII gene since this is the predominant GATA factor expressed in adult liver. Our analysis of the VTGII ERU revealed four salient points. First, this ERU is comprised of an ERE and a bank of functionally redundant GATA-binding sites. Second, the GATA-6 transactivation domain is necessary (and sufficient, when tethered near the ERE) to render this ERU functional. Third, ERU enhancer activity is dependent on GATA 6, regardless of whether the resident ERE is imperfect or perfect. Fourth, in contrast to a report that the estrogen receptor antagonizes the activity of another GATA factor (GATA-1), we show that these two factors can function in a synergistic manner within the context of the VTGII ERU.

Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line.

A recently described chicken liver cell line, LMH, was characterized to evaluate responsiveness to estrogen. Expression of the endogenous apolipoprotein (apo) II gene was induced by 17 beta-estradiol when LMH cells were cultured with chicken serum. The response was low and yielded apoll mRNA at only 0.3% of the level seen in estrogenized rooster liver. Higher levels of apoll mRNA were achieved when LMH cells were transiently transfected with an expression plasmid for estrogen receptor. A transfected apoll gene was strongly expressed only when cotransfected with receptor. Expression of the endogenous vitellogenin (VTG) II gene was not detected. However, when cotransfected with a receptor expression plasmid, VTG II reporter plasmids were expressed in LMH cells in response to 17 beta-estradiol. These results suggest that estrogen responsiveness of LMH cells is limited by the availability of functional receptor. Low levels of estrogen receptor mRNA were detected in LMH cells, and receptor binding sites and mRNA were greatly increased following transient transfection with a receptor expression plasmid. Using this transient transfection protocol, several VTG II reporter plasmids were compared in LMH cells and chick embryo fibroblasts. A plasmid containing VTG II estrogen response elements linked to a heterologous promoter was regulated by estrogen in both cell types. In contrast, reporter plasmids containing the VTG II promoter were regulated by estrogen in LMH cells but were not expressed at all in chick embryo fibroblasts. These results suggest that regulation of the VTG II gene involves cell type-specific elements in addition to estrogen response elements.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of a chicken HMG2 cDNA clone and evidence for an HMG2-specific 3'-untranslated region.

HMG1 and HMG2 (high-mobility group proteins) are two of the most abundant nonhistone chromosomal proteins in higher eukaryotes. Mammalian HMG1 cDNA sequences have the unusual feature of being conserved not only over their coding regions, but also over large segments of their 3'-untranslated regions (3' UTRs) as well. In contrast, the only reported mammalian HMG2 cDNA clone has a distinct 3' UTR. We now report the isolation of a chicken HMG2 cDNA clone and show that it is markedly similar to the mammalian HMG2 cDNA clone over both its coding regions and 3' UTRs. We therefore infer that the 3' UTRs of the HMG1 and HMG2 genes are subject to distinct evolutionary pressures. Our data, along with published data, also serve to highlight 26 amino acid positions where HMG1 and HMG2 are distinctly conserved, and we note that trout HMG-T conforms to the HMG1 paradigm at most of these diagnostic positions.

Chicken repeat 1 (CR1) elements, which define an ancient family of vertebrate non-LTR retrotransposons, contain two closely spaced open reading frames.

Chicken repeat 1 (CR1) elements comprise a family of non-long terminal repeat (LTR) retrotransposons that have several noteworthy features. For example, whereas most other non-LTR elements have poly(A) tracts or other simple A-rich repeats at their 3' ends, the 3' ends of CR1 elements conform to the consensus [(CATTCTRT)(GATTCTRT)1-3]. CR1 elements also display an unusual bias for severe 5' truncations: only approx. 30 (out of a total of approx. 30 000) CR1 elements in the chicken genome include significant portions of the pol-like open reading frame (ORF) that we previously identified and partially sequenced [Burch et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8199-8203]. In the present study we derived a consensus sequence for this entire ORF (ORF2) as well as an upstream ORF (ORF1) and part of a 5' untranslated region (UTR). The conceptual translation product of ORF2 is predicted to contain an endonuclease domain in addition to a reverse transcriptase domain. These results suggest that CR1 elements retrotranspose using a "nick and prime" mechanism similar (but not identical) to other families of non-LTR elements.

Subfamilies of CR1 non-LTR retrotransposons have different 5'UTR sequences but are otherwise conserved.

CR1 elements and CR1-related (CR1-like) elements are a novel family of non-LTR retrotransposons that are found in all vertebrates (reptilia, amphibia, fish, and mammals), whereas more distantly related elements are found in several invertebrate species. CR1 elements have several features that distinguish them from other non-LTR retrotransposons. Most notably, their 3' termini lack a polyadenylic acid (poly A) tail and instead contain 2-4 copies of a unique 8 bp repeat. CR1 elements are present at approximately 100,000 copies in the chicken genome. The vast majority of these elements are severely 5' truncated and mutated; however, six subfamilies (CR1-A through CR1-F) are resolved by sequence comparisons. One of these subfamilies (i.e. CR1-B) previously was analyzed in detail. In the present study, we identified several full-length elements from the CR1-F subfamily. Although regions within the open reading frames and 3' untranslated regions of CR1-F and CR1-B elements are well conserved, their respective 5' untranslated regions are unrelated. Thus, our results suggest that new CR1 subfamilies form when elements with intact open reading frames acquire new 5' UTRs, which could, in principle, function as promoters.

Chronic ethanol or nicotine treatment results in partial cross-tolerance between these agents.

Female DBA/2Ibg mice were treated chronically (21 days) with ethanol- or dextrin-containing liquid diets or infused chronically with nicotine (8 mg/kg/h) or saline for 10 days. The responses of these animals to challenge doses of ethanol (2.5 g/kg) or nicotine (1 or 2 mg/kg) were measured using a test battery consisting of respiration rate, acoustic startle response, Y-maze crosses and rears, heart rate and body temperature. Chronic ethanol-treated animals were tolerant to the effects elicited by a challenge dose of ethanol on four of the six measures and were cross-tolerant to nicotine's effects on the acoustic startle test. Chronic nicotine-treated animals were tolerant to nicotine's effects on five of the six measures and cross-tolerant to ethanol's effects on heart rate and body temperature. Thus, partial cross-tolerance between ethanol and nicotine exists. Chronic nicotine treatment resulted in significant increases in L-[3H]-nicotine binding in six of seven brain regions and in alpha-[125I]-bungarotoxin binding in three of seven brain regions. Chronic ethanol treatment failed to alter the binding of either ligand. Therefore, the cross-tolerance that develops between ethanol and nicotine is not totally dependent on alterations in the number of brain nicotinic receptors.

Geomagnetic disturbances are associated with reduced nocturnal excretion of a melatonin metabolite in humans.

The effects of geomagnetic disturbances on urinary excretion of the melatonin metabolite, 6-hydroxymelatonin sulfate (6-OHMS), were studied in conjunction with 60 Hz magnetic field (MF) and ambient light exposure in 132 electric utility workers. Geomagnetic activity was assessed using a local (equivalent amplitude or A(K), Boulder, CO) and global (average antipodal or aa) index. Personal exposures to 60 Hz MFs and light were obtained using data-logging meters. The relationship between geomagnetic activity and 6-OHMS was assessed with adjustment for age, light exposure, and month of participation. Mean overnight 6-OHMS excretion was lower on days when the 36-h A(K) or aa values exceeded 30 nT. A greater reduction in 6-OHMS excretion was observed when increased geomagnetic activity was combined with elevated 60 Hz MF or reduced ambient light exposures.

Melatonin metabolite excretion among cellular telephone users.

PURPOSE: The relationship between cellular telephone use and excretion of the melatonin metabolite 6-hydroxymelatonin sulfate (6-OHMS) was evaluated in two populations of male electric utility workers (Study 1, n=149; Study 2, n=77). MATERIALS AND METHODS: Participants collected urine samples and recorded cellular telephone use over 3 consecutive workdays. Personal 60-Hz magnetic field (MF) and ambient light exposures were characterized on the same days using EMDEX II meters. A repeated measures analysis was used to assess the effects of cellular telephone use, alone and combined with MF exposures, after adjustment for age, participation month and light exposure. RESULTS: No change in 6-OHMS excretion was observed among those with daily cellular telephone use >25 min in Study 1 (5 worker-days). Study 2 workers with >25 min cellular telephone use per day (13 worker-days) had lower creatinine-adjusted mean nocturnal 6-OHMS concentrations (p=0.05) and overnight 6-OHMS excretion (p=0.03) compared with those without cellular telephone use. There was also a linear trend of decreasing mean nocturnal 6-OHMS/creatinine concentrations (p=0.02) and overnight 6-OHMS excretion (p=0.08) across categories of increasing cellular telephone use. A combined effect of cellular telephone use and occupational 60-Hz MF exposure in reducing 6-OHMS excretion was also observed in Study 2. CONCLUSIONS: Exposure-related reductions in 6-OHMS excretion were observed in Study 2, where daily cellular telephone use of >25 min was more prevalent. Prolonged use of cellular telephones may lead to reduced melatonin production, and elevated 60-Hz MF exposures may potentiate the effect.

Circadian and infradian rhythms in mood.

The aim of this study was to assess any variation in positive, negative and total affect recorded longitudinally; to compare the results with those from prior transverse or hybrid population studies, based on the same or a different method of mood rating; and to test for any association of mood with cardiovascular, hormonal and geophysical variables monitored concomitantly. The study approach was as follows. A clinically healthy 34-year-old man filled out the positive and negative affective scale (PANAS) questionnaire five times a day for 86 days. Systolic (S) and diastolic (D) blood pressure (BP) and heart rate (HR) were also measured automatically at 30-minute intervals with an ambulatory monitor from May 19 to June 29, 2000, while different endpoints of heart rate variability (HRV) were also determined at 5-minute intervals from beat-to-beat electrocardiogram (ECG) monitoring for 42 days between May 3 and June 14, 2000, with only short interruptions while the subject took a shower and changed ECG tapes. Saliva samples were collected at the times of mood ratings for one month for later determination of melatonin and cortisol concentrations. Intervals of 24 hours of the record of each variable displaced in increments of 24 hours were analyzed by chronobiologic serial section at a trial period of 24 hours to assess the circadian characteristics as they changed from one day to another. Estimates of the midline-estimating statistic of rhythm (MESOR) and circadian amplitude and acrophase obtained on consecutive days were correlated among variables to assess any associations. The findings were as follows. Overall, a circadian rhythm was demonstrated for all variables. A positive association was noteworthy between the circadian amplitude of negative affect and the MESOR of both SBP (r= 0.363; P= 0.029) and DBP (r= 0.389; P= 0.019), suggesting that BP is raised in the presence of large swings in negative affect. Needing further validation was a weak association between the MESOR of negative affect and the circadian amplitude of SBP (r= - 0.272; P = 0.108), suggesting a lowering of the circadian SBP amplitude in the presence of a strong negative affect. Of further interest was the lack of a statistically significant relation between positive and negative affect, not only in terms of the MESOR but also in terms of the circadian amplitude.

Tolerance to and cross tolerance between ethanol and nicotine.

Female DBA mice were subjected to one of four treatments: ethanol-containing or control diets, nicotine (0.2, 1.0, 5.0 mg/kg/hr) infusion or saline infusion. After removal from the liquid diets or cessation of infusion, the animals were challenged with an acute dose of ethanol or nicotine. Chronic ethanol-fed mice were tolerant to the effects of ethanol on body temperature and open field activity and were cross tolerant to the effects of nicotine on body temperature and heart rate. Nicotine infused animals were tolerant to the effects of nicotine on body temperature and rotarod performance and were cross tolerant to the effects of ethanol on body temperature. Ethanol-induced sleep time was decreased in chronic ethanol- but not chronic nicotine-treated mice. Chronic drug treatment did not alter the elimination rate of either drug. Chronic ethanol treatment did not alter the number or affinity of brain nicotinic receptors whereas chronic nicotine treatment elicited an increase in the number of [3H]-nicotine binding sites. Tolerance and cross tolerance between ethanol and nicotine is discussed in terms of potential effects on desensitization of brain nicotinic receptors.

A diallel analysis of nicotine-induced hypothermia.

The hypothermic responses of mice that occur after acute injection of nicotine show genetic influences. The body temperatures of mice of all five strains tested decreased after injection of either 0.75 or 1.5 mg/kg nicotine, but mice of the C3H strain were less affected than were those of the DBA, BALB, or C57BL strains. Mice of the A strain were the most sensitive to nicotine's effects. Genetic effects on nicotine-induced hypothermia were further examined using a five-by-five diallel cross. Additive genetic variance occurred at both nicotine doses. Substantial dominance variance, including directional dominance toward a large hypothermic response induced by injection of a low dose of nicotine (0.75 mg/kg), suggested that an intense response to a low drug dose is adaptive. The directional dominance was absent after treatment with a high dose (1.5 mg/kg) of the drug. Epistatic interactions occurring in crosses involving C57BL mice were pronounced.