Altered gene expression profile in chemically induced rat mammary adenocarcinomas and its modulation by an aromatase inhibitor.

In the present study, competitive cDNA library screening (CCLS) and cDNA microarray analyses were employed to identify differentially expressed genes in methylnitrosourea-induced rat mammary adenocarcinomas. The preliminary screening of 100 000 plaques by CCLS identified 1217 clones with differential expression. Dot-blot analysis of the isolated clones verified differential expression in 471 distinct genes. Confirmation of these 471 genes was conducted by performing reverse transcription-polymerase chain reactions, and a total of 160 genes were confirmed after comparing six rat mammary adenocarcinomas and three normal rat mammary glands. Fifty-nine of these showed lower expression in the adenocarcinomas while the remaining 101 were overexpressed in the tumors. Employing a cDNA microarray containing 588 known genes revealed an additional 33 differentially expressed genes in these tumors. Importantly, most of the identified genes demonstrated relatively reproducible overexpression or underexpression in individual tumors. Many of the altered genes determined by cDNA microarray analysis were oncogenes, tumor suppressor genes, or genes involved in cell cycle control and apoptosis. CCLS identified many others not previously associated with mammary carcinogenesis, including a novel gene named RMT-7. Preliminary studies to determine the applicability of this gene expression approach for detecting potential biomarkers for cancer chemoprevention was evaluated in rat mammary tumors obtained from animals treated with vorozole, a potent aromatase inhibitor. When genes exhibiting differential expression as determined by CCLS or cDNA microarray analysis were examined in control and vorozole-treated tumors, expression of 19 genes was found to be modulated significantly in tumors treated with vorozole. Further investigations into these identified genes should contribute significantly to our understanding of the molecular mechanisms of rat mammary tumorigenesis. In addition, the identified genes may become useful targets for drug development and potential biomarkers for monitoring treatment and prevention of breast cancer in humans.

Expression of human endogenous retrovirus k envelope transcripts in human breast cancer.

PURPOSE: We investigated the expression of human endogenous retroviral (HERV) sequences in breast cancer. EXPERIMENTAL DESIGN: Reverse transcription-PCR (RT-PCR) was used to examine expression of the envelope (env) region of ERV3, HERV-E4-1, and HERV-K in breast cancer cell lines, human breast tumor samples, adjacent uninvolved breast tissues, nonmalignant breast tissues, and placenta. Expression of HERV transcripts was confirmed by Northern blot analysis and in situ hybridization (ISH). To evaluate coding potential, amplified HERV sequences were cloned into vectors for expression and sequence analysis. RESULTS: No expression of ERV3 or HERV-E4-1 RNA was detected in the analyzed breast samples. In contrast, HERV-K transcripts were detected in most breast cancer cell lines and many breast tumor tissues. Expression was detected in a small percentage of matched, uninvolved breast tissues and in placentas but not nonmalignant breast tissues. In HERV-K-positive breast cancer tissues, Northern blot analysis demonstrated full-length proviral and spliced env transcripts. ISH demonstrated expression of HERV-K transcripts in breast tumor cells but not in normal or uninvolved breast epithelial cells. Independently isolated clones of HERV-K env cDNA generated recombinant proteins of the expected size. Sequence analysis of env cDNA clones derived from four breast tumor samples revealed >97% identity with the type I HERV-K102, with no premature termination codons. Independent isolates from the same breast tumor sample showed nucleotide sequence differences, suggesting that multiple loci may be transcribed. CONCLUSIONS: These data indicate that HERV-K transcripts with coding potential for the envelope region are expressed frequently in human breast cancer.

Chronological loss of bone zinc during dietary zinc deprivation in neonatal pigs.

This research was conducted to measure the chronological changes in zinc concentrations of biopsied bone, hair, and plasma samples collected weekly during dietary zinc deprivation. Pigs 1-2 wk of age were fed a basal diet (< 1 microgram Zn/g) during a 1-wk depletion period and then assigned to one of three dietary regimens for 4 wk: a low-zinc diet (4 micrograms Zn/g) fed ad libitum, an adequate-zinc diet (100 micrograms/g) fed ad libitum, and an adequate-zinc diet restricted in intake to allow eight gain comparable with that of the low-zinc group. Bone zinc remained at approximately 120 micrograms/g dry wt for the control groups fed adequate zinc but steadily declined in pigs fed the low-zinc diet, leveling off at approximately 25% of the control values. Plasma and hair zinc concentrations also decreased but at a more rapid rate. Bone zinc is mobilizable in neonatal pigs, and biopsied bone zinc concentration is a reliable index of zinc status.

Eicosapentaenoic acid and epidermal growth factor modulation of human breast cancer cell adhesion.

Cancer cell adhesion to the subendothelial extracellular matrix (ECM) is an important step in metastasis formation. The effect of epidermal growth factor (EGF) and eicosapentaenoic acid (EPA) on adhesion of the highly metastatic MDA-MB-231 human breast cancer cell line to ECM components was examined in the present study. MDA-MB-231 cells exhibited a dose-dependent decrease in adhesion to Matrigel when treated with EGF. EGF and EPA, alone or in combination, decreased adhesion to Matrigel, fibronectin and type IV collagen. These results suggest that decreased adhesion to ECM substrates by combined EPA and EGF treatment may be the result of a common post-receptor signaling pathway.

Role of amniotic fluid homocysteine level and of fetal 5, 10-methylenetetrahydrafolate reductase genotype in the etiology of neural tube defects.

A mutation in the gene 5,10-methylenetetrahydrofolate reductase (MTHFR), leading to altered homocysteine metabolism, has been identified in parents and fetuses with fetal neural tube defects. We sought to determine which is of greater importance in fetal neural tube defect formation: the fetal MTHFR mutation or elevated amniotic fluid homocysteine level. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80), and from normal controls matched for race, month and year of amniocentesis, and maternal age. The presence or absence of the 677C-->T mutation of MTHFR was determined and homocysteine levels were measured; cases and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C-->T MTHFR mutation (44% vs 17%, P < or = 0.001). Cases were also significantly more likely than controls to have an amniotic fluid homocysteine level above the 90th centile (>1.85 micromol per liter); 27% vs 10%, P = 0.02. Thirty one cases and 12 controls had an abnormal genotype; however, amniotic fluid homocysteine levels were not significantly different between these two groups (6/31, or 19% of cases had an elevated homocysteine compared to 1/12, or 8% of controls; P = 0.65). In contrast, 40 cases and 60 controls had a normal genotype; the neural tube defect cases had significantly higher homocysteine levels (13/40, or 32% of cases had an elevated homocysteine level compared to only 6/60, or 10% of controls; P = 0.008). Although both abnormal fetal MTHFR genotype and abnormal amniotic fluid homocysteine concentration are significantly associated with neural tube defects, the association with amniotic fluid homocysteine concentration is significant regardless of the fetal MTHFR genotype. The relationship between maternal and fetal homocysteine metabolism is complex.

Amniotic fluid homocysteine levels, 5,10-methylenetetrahydrafolate reductase genotypes, and neural tube closure sites.

A specific gene mutation leading to altered homocysteine metabolism has been identified in parents and fetuses with neural tube defects (NTDs). In addition, current animal and human data indicate that spine closure occurs simultaneously in five separate sites that then fuse. We sought to determine whether either this mutation or abnormal amniotic fluid homocysteine levels are associated with all five neural tube closure sites. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80) and from normal controls matched for race, month and year of amniocentesis, and maternal age. Cases were categorized according to defect site by using all available medical records. The presence or absence of the 677C-->T mutation of 5, 10-methylenetetrahydrafolate reductase (MTHFR) gene was determined, and homocysteine levels were measured; case and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C-->T MTHFR mutation (44% vs. 17%, P < or = 0. 001). Likewise, cases were significantly more likely than controls to have amniotic fluid homocysteine levels >90th centile (>1.85 micromol/L), 27% vs. 10%, P = 0.02. Most (83%) of control cases had both normal MTHFR alleles and normal amniotic fluid homocysteine levels (normal/normal), whereas only 56% of NTD case were normal/normal (P = 0.001). When evaluated by defect site, only defects involving the cervical-lumbar spine, lumbosacral spine, and occipital encephalocele were significantly less likely to be normal/normal than controls (P = 0.007, 0.0003, and 0.007, respectively), suggesting a strong association with the 677C-->T allele. In contrast, anencephaly, exencephaly, and defects confined to the sacrum included many cases that had both normal MTHFR alleles and normal homocysteine and were not significantly different from controls. The 677C-->T MTHFR mutation and elevated homocysteine levels appear to be disproportionately associated with defects spanning the cervical-lumbar spine, lumbosacral spine, and occipital encephalocele. In contrast, anencephaly, exencephaly, and defects confined to the sacrum may not be related to altered homocysteine metabolism.

Altered global methylation of DNA: an epigenetic difference in susceptibility for lung cancer is associated with its progression.

Alterations in global DNA methylation have been observed in many cancers, but whether such alterations represent an epigenetic difference in susceptibility for the disease is unknown. The status of global DNA methylation also has not been reported in intact or specific types of cells involved in the carcinogenic process. To address these issues in lung carcinogenesis, we evaluated the status of global DNA methylation by using a monoclonal antibody specific for 5-methylcytosine (5-mc) in randomly selected lung specimens of 60 cigarette smokers who developed squamous cell carcinoma (SCC) and 30 cigarette smokers who did not. 5-mc immunostaining scores of DNA of SCC (0.61 +/- 0.42) and associated hyperplastic lesions (0.82 +/- 0.27) was significantly lower than those of DNA of histologically normal bronchial epithelial cells (0.99 +/- 0.52) and hyperplastic lesions (1.2 +/- 0.22) of noncancer specimens. The ratio of 5-mc scores between SCC and matched uninvolved bronchial epithelial cells was significantly associated with advanced stage and size of the tumor. The results suggest that alteration in global DNA methylation is an important epigenetic difference in susceptibility for the development of lung cancer. The reduced global DNA methylation in SCC compared with epithelial hyperplasia and its association with tumor size and disease stage is suggestive of its involvement in the progression of SCC. The results also indicate that normal methylation of DNA in epithelial hyperplastic lesions may prevent the transformation of these lesions to invasive cancer. If these results are confirmed, the status of DNA methylation in early lesions such as epithelial hyperplasia could be used to identify smokers who are at risk for the development of SCC.

Effect of angiotensin-converting enzyme gene polymorphism on pregnancy outcome, enzyme activity, and zinc concentration.

OBJECTIVE: To evaluate the effect of angiotensin-converting enzyme (ACE) genotypes on pregnancy outcome, the incidence of pregnancy-induced hypertension, and changes in blood pressure (BP) during pregnancy; and the relationship between plasma ACE activities and plasma and erythrocyte zinc concentrations in each genotype. METHODS: The subjects (n = 191) were selected from 580 indigent African-American pregnant women who enrolled toward the end of a trial to evaluate the effect of zinc supplementation on pregnancy outcome. This selection resulted in 93 subjects who received zinc and 98 who received placebo. Sample size was calculated with a 0.50 correlation coefficient between plasma ACE activities and zinc levels and a power of 80%. This calculation indicated that the sample size in each ACE genotype should be more than 28. Angiotensin-converting enzyme genotypes were identified using polymerase chain reaction. Blood pressure, plasma ACE activities, and plasma and erythrocyte zinc concentrations were measured at each prenatal clinical visit. RESULTS: Pregnancy outcome, the incidence of pregnancy-induced hypertension, and BP were not different among the three ACE genotypes. There was no significant correlation between plasma ACE activities and zinc concentrations. Zinc supplementation did not have a significant effect on either plasma ACE activities or zinc concentrations, probably because of the small sample size in our study. CONCLUSION: There was no effect of ACE gene polymorphism on pregnancy outcome, the incidence of pregnancy-induced hypertension, or changes in BP during pregnancy. Among each ACE genotype, plasma ACE activities did not correlate significantly with plasma zinc concentrations.

Effect of high retinoid doses on RAR-beta mRNA expression in female B6D2F1 mice.

The expression of retinoid receptors is altered during the development of several types of cancer. In the present study, we determined the influence of high dietary concentrations of 4-hydroxyphenylretinamide (4-HPR) and 13-cis-retinoic acid (13-cis-RA) on RAR-beta mRNA expression in female mice. Expression of liver and lung RAR-beta RNA increased with increasing levels of dietary retinoid (both 4-HPR and 13-cis RA). Bladder RAR-beta mRNA levels, however, were significantly decreased in mice fed 13-cis RA or 4-HPR. These results suggest that feeding high levels of retinoids to mice results in tissue-specific elfects on expression of RAR-beta mRNA.

Methylenetetrahydrofolate reductase (MTHFR) polymorphism increases the risk of cervical intraepithelial neoplasia.

Although cervical cancer is a common female cancer, little attention has been given to genetic susceptibility factors. The present case-control study was undertaken to examine MTHFR polymorphism as a potential molecular marker of cervical intraepithelial neoplasia (CIN) susceptibility and to relate the findings to smoking, HPV infection, ethnicity, parity and oral contraceptive use, which are known risk factors for cervical cancer. A base change from C to T at the nucleotide position 677 of the MTHFR gene results in substitution of valine (GTC) for alanine (GCC). The homozygous normal (Ala/Ala), homozygous mutant (Val/Val), and heterozygous mutant (Ala/Val) genotypes for the MTHFR gene were determined in cervical tissues of 64 cases of CIN lesions and 31 controls. The genotype frequencies of both Val/Val (17%) and Ala/Val (56%) were significantly higher in subjects with CIN lesions compared to controls with Val/Val (10%) and Ala/Val (39%), (trend p = 0.03). The results suggested a significantly increased CIN risk with an alanine to valine substitution at amino acid 223 of MTHFR with an odds ratio of 2.9 (95% confidence interval: 1.2-7.9, p = 0.02). Age, ethnicity, smoking and oral contraceptive use were weakly and nonsignificantly associated with CIN risk. HPV infection was associated with a statistically nonsignificant threefold increase in CIN risk. Parity and MTHFR genotype displayed a strong interaction. Neither nulliparous women with MTHFR polymorphism nor parous women without the polymorphism were at higher risk than women who did not have children and were MTHFR homozygous normal (the reference category). Women with mutant MTHFR genotype who had children, however, showed a significantly higher risk of CIN, with an odds ratio of 23 (95% confidence interval: 2.3-225) as compared to the reference category. No other factors displayed such a strong pattern of interaction. Since MTHFR polymorphism and pregnancy increases folate requirements and can impair folate status, this association could reflect an inadequate response of mutant MTHFR genotype carriers to the increased demand for folate imposed by pregnancy. Tissue folate deficiency, in turn, could increase the risk of CIN in the affected women.

Iron compounds catalyze the oxidation of 10-formyl-5,6,7,8 tetrahydrofolic acid to 10-formyl-7,8 dihydrofolic acid.

We have previously demonstrated that 10-formyl-7,8-dihydrofolic acid (10-HCO-H2folate) is a better substrate for mammalian aminoimidazolecarboxamide ribotide transformylase (EC 2.1.2.3) than is 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate) (J.E. Baggott, G.L. Johanning, K.E. Branham, C.W. Prince, S.L. Morgan, I. Eto, W.H. Vaughn, Biochem. J. 308, 1995, 1031-1036). Therefore, the possible metabolism of 10-HCO-H4folate to 10-HCO-H2folate was investigated. A spectrophotometric assay for the oxidation of 10-HCO-H4folate to 10-HCO-H2folate which measures the disappearance of reactant (decrease in absorbance at 356 nm after acidification of aliquots of the reaction solution), is used to demonstrate that iron compounds catalyze the oxidation of 10-HCO-H4folate to 10-HCO-H2folate in the presence and absence of ascorbate. Chromatographic separation of the 10-HCO-H2folate product from the reaction mixture, its UV spectra, a microbiological assay and an enzymatic assay established that the iron-catalyzed oxidation product of 10-HCO-H4folate was 10-HCO-H2folate; without substantial side reactions. The inhibition of this iron-catalyzed oxidation by deferoxamine, apotransferrin and mannitol and the stimulation by citrate and EDTA indicated of a mechanism involving a reaction of 10-HCO-H4folate with hydroxyl radicals (*OH) generated by Fenton chemistry. The presence of "free iron" (e.g., Fe3+ citrate) in bile, cerebrospinal fluid and intracellularly suggest that this oxidation could occur in vivo and that 10-HCO-H4folate may be a *OH scavenger.

Modulation of breast cancer cell adhesion by unsaturated fatty acids.

Triglycerides, which are major constituents of dietary fat, contain a mixture of saturated and unsaturated fatty acids. One newly recognized function of unsaturated fatty acids is modulation of cell adhesion to components of the extracellular matrix. Alterations in cell adhesiveness or cell adhesion molecule expression accompany the onset of a number of diseases including arthritis, atherosclerosis, and cancer. Cell adhesion is necessary for the metastatic spread of cancer cells to new organs. Circulating cancer cells adhere to endothelial cells and the underlying subendothelial basement membrane as an initial step in the process of invading target organs during metastasis. Several recent studies have provided convincing evidence that unsaturated fatty acids and their metabolites influence adhesion of cultured human cancer cells to individual components of the basement membrane. These unsaturated fatty acid effects appear to be dependent in some instances on the expression of specific cell surface adhesion molecules. Unsaturated fatty acids influence the development of metastases in animal tumor models by largely unexplored mechanisms; the possibility that cell adhesion is involved in this process has not been thoroughly investigated. Future studies of unsaturated fatty acid effects on cell adhesion molecule expression in breast cancer patients should reveal the clinical relevance of the studies reviewed here.

Immunohistochemical evaluation of global DNA methylation: comparison with in vitro radiolabeled methyl incorporation assay.

The in vitro radiolabeled methyl incorporation assay, a commonly used technique to evaluate global methylation of DNA, has some disadvantages and limitations. The purpose of the present study was to compare the results of global DNA methylation evaluated by radiolabeled methyl incorporation (CPM/microg of DNA) with immunohistochemical staining of the same tissue sections with a monoclonal antibody developed against 5-methylcytosine used (5-mc). We archival specimens of squamous cell cancer (SCC) of the human lung with a matched uninvolved specimen (n = 18 pairs) and 18 lung specimens from subjects without lung cancer (noncancer specimens) to make this comparison. The immunostaining for 5-mc was reported as a percentage of cells positive for staining as well as a weighted average of the intensity score. The results suggested that both radiolabeled methyl incorporation assay and immunostaining for 5-mc can be used to demonstrate hypomethylation of DNA in SCC tissues compared to matched uninvolved tissues. An advantage of immunostaining, however, is its ability to demonstrate hypomethylation of SCC compared to adjacent bronchial mucosa on the same archival specimen, obviating the need to use sections from both SCC and matched uninvolved tissues. Only by using the immunostaining technique were we able to document a statistically significant difference in DNA methylation between SCC and noncancer tissues. We conclude that the immunostaining technique has advantages over the radiolabeled methyl incorporation assay and may be best suited for evaluation of global DNA methylation when the methylation status of cancer cannot be normalized by methyl incorporation of normal tissues or when the number of samples available for evaluation is small.

Unsaturated fatty acid effects on human breast cancer cell adhesion.

Polyunsaturated fatty acids influence several steps involved in metastasis formation in animal tumor models. During the process of metastasis from the primary site, tumor cells adhere to the endothelium and underlying basement membrane before extravasation and secondary growth. The purpose of this study was to determine the effect of unsaturated fatty acids on adhesion of human breast cancer cell lines to components of the basement membrane. Cells were cultured in low-serum medium for five days with or without added unsaturated fatty acids. Adhesion assays were conducted by incubating cells with basement membrane substrates coated on 96-well plates, washing to remove nonadherent cells, and staining adherent cells with crystal violet. Linoleic acid (LA) and eicosapentaenoic acid increased adhesion of the metastatic cell line MDA-MB-231 to Matrigel and type IV collagen, while eicosapentaenoic acid decreased adhesion of the less metastatic cell line SK-BR-3 to these two basement membrane substrates. Oleic acid increased adhesion of MDA-MB-231 cells to Matrigel and fibronectin. Nordihydroguaiaretic acid and high concentrations of indomethacin, each of which inhibits the lipoxygenase pathway of arachidonate metabolism, were effective in reversing the stimulatory effect of LA on MDA-MB-231 cell adhesion. A protein kinase C inhibitor likewise suppressed the increase in adhesion observed when MDA-MB-231 cells were incubated in media with added LA. Unsaturated fatty acids modified the adhesive properties of human breast cancer cell lines in vitro, and LA appeared to increase human breast cancer cell adhesion to extracellular matrix components by activating lipoxygenase and/or protein kinase C pathways.

10-Formyl-dihydrofolic acid is bioactive in human leukemia cells.

The bioactivity of 10-formyl-7,8-dihydrofolic acid and 10-formyl-folic acid was determined in human leukemia (CCRF-CEM) cells grown in a folate-depleted medium containing methotrexate. Excess 10-formyl-7,8-dihydrofolic acid, (but not 10-formyl folic acid) supported the growth of these cells, but it was less potent than5-formyl-5,6,7,8-tetrahydrofolic acid (a control). 10-formyl-7, 8-dihydrofolic acid (not 10-formyl folic acid) was active as substrate for aminoimidazole carboxamide ribotide transformylase and dihydrofolate reductase. This is the first experimental evidence that 10-formyl-7,8-dihydrofolic acid is a bioactive folate in mammalian cells. These experiments and several other lines of evidence in the literature suggest that 10-formyl-folic acid must be metabolized to bioactive folate by enteric bacteria before it can be utilized by the vertebrate host.

Effect of zinc deficiency and food restriction in rats on erythrocyte membrane zinc, phospholipid and protein content.

Zinc deficiency in rats increases the osmotic fragility of erythrocytes, suggesting a structural defect in the plasma membrane. The purpose of this study was to determine the effect of zinc deficiency on membrane components that might be responsible for the increased fragility. Immature male rats were fed for 3 wk a zinc-deficient (0.5 mg Zn/kg) or a control (100 mg Zn/kg) diet either ad libitum or pair-fed. Red blood cell membranes were analyzed for zinc after wet ashing in nitric-perchloric acid. The fatty acid composition of membrane phosphatidylcholine and phosphatidylethanolamine was determined by gas-liquid chromatography, and the protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane zinc concentration was decreased by zinc deficiency, but returned to control levels upon repletion with a zinc-adequate diet for 3 d. The increased osmotic fragility was also restored to the control level during the same repletion period, suggesting that zinc is an important determinant of membrane stability. The phospholipid fatty acid composition was altered by zinc deficiency, but these changes were not rapidly reversed by repletion. The membrane cholesterol:phospholipid ratio was also increased by zinc deficiency. Zinc status had no influence on the membrane protein profiles of the components visualized by staining. Only the concentration of membrane zinc correlated with osmotic fragility during zinc depletion and repletion.

Overview of pup in a cup model: hepatic lipogenesis in rats artificially reared on a high-carbohydrate formula.

The artificial rearing technique allows nutritional investigations to be conducted in rat pups during a critical period that previously has been inaccessible to researchers. The technique will be useful for identifying dietary components contributing to metabolic adaptations in the preweaning period as well as "metabolic imprinting" or permanent metabolic effects in adult rats resulting from early dietary intervention. Artificially reared rat pups fed a formula high in carbohydrate-derived energy in the preweaning period have the following characteristics compared with pups fed a high-fat formula or reared naturally: (i) a higher level of plasma insulin, (ii) an increased hepatic lipogenic capacity and (iii) precocious induction of hepatic malic enzyme. The results also show that early exposure to a high-carbohydrate diet in the preweaning period predisposes the rat to an increased lipogenic capacity in liver and adipose tissues and to the development of obesity later in adult life.