Race- and age-dependent alterations in global methylation of DNA in squamous cell carcinoma of the lung (United States).

OBJECTIVE: The current study investigated the race- and age-dependent alterations in global DNA methylation on the development and progression of squamous cell carcinomas (SCCs) of the lung. METHODS: Methylation status was evaluated in SCC and in the associated uninvolved bronchial mucosa (UBM) and epithelial hyperplasia (EH) of 53 Whites and 23 African Americans by using an antibody specific for 5-methylcytosine (5-mc). A low 5-mc score indicates global hypomethylation of DNA. RESULTS: 5-mc scores of SCC were significantly lower compared to 5-mc scores of UBM and EH in Whites (p < 0.05). In African Americans, 5-mc scores of SCCs were not significantly different from 5-mc scores of UBM and EH, suggesting an involvement of methylation in the development of SCCs in Whites, but not in African Americans. 5-mc scores were lower in younger subjects compared to older subjects in Whites. Since cancers in younger subjects tend to be more aggressive than cancers in older subjects, these observations may suggest that hypomethylation may have contributed to aggressiveness cancers of younger Whites. Hypomethylation of SCCs in White men was associated with shorter survival from the disease. CONCLUSIONS: These preliminary results suggest that the methylation status of DNA may affect the development, aggressiveness, and prognosis of SCCs in Whites.

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.

Omega-3 fatty acids decrease protein kinase expression in human breast cancer cells.

We report that 5-day exposure to physiological concentrations of eicosapentaenoic and docosahexaenoic acids resulted in a strong decrease in expression of the RIalpha regulatory subunit of protein kinase A and the PKC-alpha isozyme of protein kinase C in the human breast cancer cell line MDA-MB-231.

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.

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.

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.

Association of the C677T methylenetetrahydrofolate reductase mutation and elevated homocysteine levels with congenital cardiac malformations.

OBJECTIVE: The aim of this study was determine whether the cytosine-to-thymine mutation at base 677 of the gene for methylenetetrahydrofolate reductase (C677T MTHFR ), which has been associated with neural tube defects, is also associated with congenital cardiac malformations. STUDY DESIGN: Amniotic fluid homocysteine levels were measured and the presence or absence of the C677T MTHFR mutation in amniocytes was determined in stored amniotic fluid obtained from 26 pregnancies complicated by isolated (presumed multifactorial) fetal cardiac defects and from 116 normal pregnancies. RESULTS: The pregnancies affected by fetal cardiac defects had higher amniotic fluid homocysteine levels (1.7 +/- 1.7 vs 1.0 +/- 0.7 micromol/L; P =.07) and included more samples with homocysteine levels >90th percentile (27% vs 9%; P =.02) and more cases with the C677T MTHFR mutation (35% vs 13%; P =.01). Fifty percent of cases had either a high homocysteine level or the C677T MTHFR mutation (50% vs 20%; P =.003) and 12% had both (12% vs 0%; P =.0006). CONCLUSION: Fifty percent of these isolated congenital cardiac defects were associated with either the C677T MTHFR mutation or elevated amniotic fluid homocysteine levels, or both. This finding adds to what is already known about the multiple and complex biochemical and developmental functions of the homocysteine pathway.

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.

Localized folate and vitamin B-12 deficiency in squamous cell lung cancer is associated with global DNA hypomethylation.

We measured the concentrations of folate and vitamin B-12 in paired tissue samples of squamous cell cancer (SCC) and adjacent grossly normal-appearing uninvolved bronchial mucosa (from which SCC developed and also "at risk" of developing SCC) of the lung in 12 subjects to determine the involvement of these vitamins in 1) lung carcinogenesis and 2) global DNA methylation. The folate concentrations were significantly lower in SCCs than in uninvolved tissues (p = 0.03). The vitamin B-12 concentrations were also significantly lower in SCCs than in uninvolved tissues (p = 0.02). The radiolabeled methyl incorporation (inversely related to the degree of in vivo DNA methylation) was significantly higher in SCCs than in uninvolved tissues (p < 0.0001). The correlation between folate and radiolabeled methyl incorporation was inverse and statistically significant in SCCs (p = 0.03). The correlation between vitamin B-12 and radiolabeled methyl incorporation also was inverse and statistically significant in SCCs (p = 0.009). The relationship between tissue vitamin B-12 and DNA methylation was minimal in uninvolved tissues. The relationship between folate and DNA methylation, however, was inverse in uninvolved tissues. In the multiple regression models that included both vitamins, only folate was inversely associated with radiolabeled methyl incorporation in uninvolved and cancerous tissues. These results suggested that folate might be the limiting vitamin for proper DNA methylation in SCC as well as in tissues at risk of developing SCC. Several possible mechanisms of folate deficiency, including inactivation of the vitamin by exposure to carcinogens of cigarette smoke and underexpression or absence of folate receptor in SCCs and associated premalignant lesions, are discussed in light of these findings.

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.

The accumulation of ascorbic acid by squamous cell carcinomas of the lung and larynx is associated with global methylation of DNA.

BACKGROUND: Cigarette smokers are known to have lower concentrations of circulating ascorbic acid than nonsmokers. In contrast, there is evidence that the extracellular fluid lining of the alveolus, which comes in close contact with cigarette smoke, and the alveolar macrophages of smokers are enriched with ascorbic acid. The clinical significance of these observations is unknown. METHODS: The authors measured the ascorbic acid concentrations and radiolabeled methyl incorporation (which is inversely related to the degree of DNA methylation in vivo) of paired samples of squamous cell carcinoma (SCC) and adjacent uninvolved mucosa of the lung and larynx (n = 22). RESULTS: Cancerous tissues had significantly higher ascorbic acid concentrations (mean +/- standard deviation [SD, 485 +/- 77; median, 483 ng/mg protein) compared with their matched uninvolved tissues (mean +/- SD, 151 +/- 52; median, 72 ng/mg protein; P < 0.0001). The radiolabeled methyl incorporation was significantly higher in cancerous tissues (mean +/- SD, 31,419 +/- 2629; median, 31,416 counts per minute [CPM]/microg DNA) compared with their matched uninvolved tissues (mean +/- SD, 11,883 +/- 1567; median, 11,444 CPM/microg DNA; P < 0.0001). The Spearman correlation between ascorbic acid concentrations and radiolabeled methyl incorporation by DNA in SCCs was inverse and statistically significant (r = -0.58, P = 0.008), indicating a beneficial effect of accumulated ascorbic acid in global methylation of DNA. In the uninvolved tissues, this correlation was inverse but statistically not significant (r = -0.20, P =0.35). CONCLUSIONS: Cancerous tissues of the lung and larynx demonstrated their ability to accumulate ascorbic acid. The accumulation of ascorbic acid by these tissues seemed to facilitate global methylation of DNA.

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.

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.

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.

59Fe is retained from an elemental 59Fe powder supplement without effects on 65Zinc, 47Calcium and 67Copper in young pigs.

In vivo counting with the use of a germanium detector evaluated the retention of an elemental 59Fe powder supplement while measuring potential interactions with zinc, calcium and copper. Effects of dietary iron and zinc on in vivo retentions of 59Fe, 65Zn, 67Cu and 47Ca were studied in young pigs. In Experiment 1, 4-d-old piglets fed a cereal-based diet were randomly assigned to one of four treatment groups (2 x 2 factorial arrangement, n = 5 per group). Variables were dietary iron source (either elemental iron or FeSO4, each at 100 mg iron/kg diet) and the dosage form of radioactive iron (either elemental 59Fe powder or 59FeSO4). Experiment 2 (2 x 3 factorial arrangement) was performed using two levels of iron (100 and 200 mg/kg, as elemental iron) and three levels of zinc (25, 50 and 100 mg/kg). Piglets were also dosed with 47Ca, 65Zn and 67Cu; all radioisotopes were measured for 8 d. Apparent absorption of elemental 59Fe powder was 13 +/- 1%, whereas 59Fe sulfate was significantly (P < 0.05) higher at 26 +/- 1%. The FeSO4 diet decreased 65Zn retention in Experiment 1, in contrast to the elemental iron diet, which did not have this effect in either experiment. Apparent 65Zn absorption averaged 44 +/- 2, 35 +/- 1 and 27 +/- 2% for the three levels of zinc (25, 50 and 100 mg/kg), respectively. Retention of 47Ca was not affected by dietary iron or zinc; retention of 67Cu was not affected by dietary iron. The data demonstrate good bioavailability of elemental iron without effects on zinc, copper and calcium.

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.

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.

Characterization of the mouse dihydrolipoamide dehydrogenase (Dld) gene: genomic structure, promoter sequence, and chromosomal localization.

The mouse dihydrolipoamide dehydrogenase (Dld) gene has been cloned, characterized, and mapped. This nuclear gene encodes a mitochondrial protein that is shared among several alpha-keto acid dehydrogenase complexes and the glycine cleavage system. The Dld gene is contained within an approximately 21-kb region and consists of 14 exons ranging in size from 69 to 521 nucleotides. The open reading frame codes for a preprotein of 509 amino acids with a predicted mature protein of 474 amino acids that is highly conserved among mammalian species (> 90% identical). Primer extension analyses have shown the gene to have transcription initiation sites with tissue-specific differences in relative utilization. The 5' flanking region is G-C rich and lacks a TATA box, but does contain initiator element and multiple transcription factor-binding consensus sequences. Northern blot analysis shows that the Dld mRNA in various tissues is approximately 2.4 kb in size. The Dld gene has been localized to the proximal region of chromosome 12, approximately 21 cM from the centromere.