Biomedical subjects

A Lafuente

Publications and source records attributed to A Lafuente.

At least 19 recordsLinked to original sources

PubMed2005

Toxic effects of cadmium on the regulatory mechanism of dopamine and serotonin on prolactin secretion in adult male rats.

This work was designed to analyze the effects of cadmium on the regulatory mechanism of prolactin in cadmium-exposed rats. Adult male rats were given cadmium at a dose of 25 ppm of cadmium chloride (CdCl2) in the drinking water for 1 month. At the end of the treatment, the rats were killed at six different time intervals throughout a 24 h cycle to measure circulating prolactin levels and dopamine and serotonin content in the median eminence and in both anterior and posterior pituitary. Control and cadmium-exposed animals exhibited significant time of day-dependent variations in plasma prolactin levels and in dopamine and serotonin concentration in all analyzed tissue. Cadmium exposure did not modify the mean values of the hormone around the clock but it modified the amplitude of the secretory peaks at 08:00 and 12:00 h. Cadmium decreased dopamine content in the median eminence, while increased its content in the posterior pituitary and induced a phase advanced peak at 20:00 h. On the other hand, serotonin content was not modified in the median eminence. Only differences in specific time points were observed, while serotonin concentration in anterior and posterior pituitary were increased in cadmium-treated rats. Differences in time peaks were also observed. The negative correlation between plasma prolactin and dopamine content in pituitary, and between plasma levels of the hormone and serotonin content in posterior pituitary, disappeared in cadmium-treated animals.

Animals↗

PubMed2005

Toxic effects of cadmium on GABA and taurine content in different brain areas of adult male rats.

This work assesses the possible changes in gamma amino butyric acid (GABA) and taurine content in the hypothalamus, the median eminence and striatum after the exposure to various doses of cadmium. Cadmium chloride (CdCl2) was administered in the drinking water at the doses of 5, 10, 25, 50 or 100 ppm to adult male rats for 1 month. In the anterior hypothalamus, taurine and GABA content decreased with the dose of 10 ppm of CdCl2 only. Cadmium exposure decreased both GABA and taurine content in mediobasal hypothalamus except for the 50 ppm dose. In posterior hypothalamus GABA and taurine content was not affected by cadmium treatment. As far as the median eminence, 5 or 10 ppm of CdCl2 increased taurine concentration, and at a dose of 5 ppm enhanced GABA content. A significant decrease of GABA and taurine concentration was seen in the striatum at any dose of cadmium used. The concentration of cadmium increased in the hypothalamus and in the striatum in animals receiving CdCl2 in the drinking water at doses of 25, 50 or 100 ppm. The results indicate that cadmium globally decreased GABA and taurine content in the brain areas studied through effects that were not dose dependent.

Animals↗

PubMed2004

Cadmium exposure differentially modifies the circadian patterns of norepinephrine at the median eminence and plasma LH, FSH and testosterone levels.

This work was designed to analyze the cadmium effects on time-of-day variations of norepinephrine (NE) content in median eminence and on plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone in adult male rats. Rats were given cadmium at a dose of 25 ppm of cadmium chloride (CdCl(2)) in the drinking water for 1 month. Significant 24-h changes of NE content in the median eminence, plasma LH and testosterone levels occurred in control animals. Cadmium exposure induced a phase advance of the nocturnal peak of NE content that was described in the control group, to 12h and increased its amplitude. However, the mean NE content was not changed by cadmium. Metal exposure abolished the daily pattern of plasma LH levels, although the mean levels of the hormone were not modified by cadmium. For testosterone, the metal increased the amplitude of its nocturnal peak and induced the appearance of another peak during the light phase at 12h, thus increasing the mean plasma levels of this hormone. An interaction between the metal and time for NE and plasma testosterone levels was observed. These data suggest that cadmium exerts differential effects at the median eminence, the pituitary and the testes, that may explain the changes in the 24-h pattern of plasma testosterone levels.

Animals↗

PubMed2004

Concentration of hydroxyproline in blood: a biological marker in occupational exposure to asbestos and its relationship with PiZ and PiS polymorphism in the alpha-1 antitrypsin gene.

BACKGROUND: Hydroxyproline (OHP) is one of the most abundant amino acids in collagen and, in general, it provides a good measure of overall collagen catabolism. METHODS: Asbestos workers suffering from asbestosis (cases n = 85); asbestos exposed workers without asbestosis (exposed controls, EC, n = 86), and non-exposed population (non-exposed controls, NEC, n = 122) were studied. The concentration of free OHP in whole blood was measured following the Pico-Tag procedure. RESULTS: Concentration of OHP in blood was significantly different in the three groups studied (P < 0.001), being higher in cases (19.8 +/- 14.7 micromol/L) than in EC (16 +/- 12.4) and NEC (13.5 +/- 6.7). When all individuals were grouped and stratified by the Pi*S and Pi*Z polymorphisms in the alpha-1-antitrypsin gene, the highest OHP levels were detected in the Pi*S homozygotes, one of the asbestosis-at risk-genotypes (Pi*S homozygotes, x = 24.5 +/- 11.7; Pi*S heterozygotes, x = 16.6 +/- 10.0; wild type, wt, x = 15.9 +/- 11.8). CONCLUSIONS: Blood OHP concentration could be used for monitoring human exposure to asbestos, either as a marker for occupational monitoring or as an additional clinical parameter in diagnostic exploration of asbestosis.

Alleles↗

PubMed2003

Effect of cadmium on 24-h variations in hypothalamic dopamine and serotonin metabolism in adult male rats.

This study was designed to analyze the possible cadmium effects on time-of-day variations of anterior, mediobasal, and posterior hypothalamic contents of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) content in adult male rats. Also DA and 5-HT metabolism, as expressed by the ratio 3,4-dihydroxyphenyl acetic acid (DOPAC) to DA and 5-hydroxyindoleacetic acid (5-HIAA) to 5-HT, respectively, were studied. Adult male rats were given cadmium at a dose of 25 ppm of cadmium chloride in drinking water for 1 month. Age-matched rats having access to cadmium-free water were used as controls. Weight gain for the whole period was not changed by cadmium exposure. The metal accumulated in the hypothalamus of rats. In the three hypothalamic regions, significant 24-h variations of NE and 5-HT concentration were found in controls, while DA content changed rhythmically in mediobasal hypothalamus only. Mean content of NE, 5-HT, and DA of anterior, mediobasal, and posterior hypothalamus decreased after cadmium exposure. After cadmium the 24-h pattern of NE changed only in mediobasal hypothalamus, whereas the metal changed significantly the pattern of 5-HT in all regions. DOPAC to DA and 5-HIAA to 5-HT ratios decreased and were differentially changed in all hypothalamic regions analyzed in cadmium-treated rats. There was a statistically significant relationship between time of administration of metal and time that the change took place in biogenic amines in the hypothalamus. These results indicate that cadmium may depress hypothalamic biogenic amine release.

Aging↗

PubMed2003

Effect of cadmium on lymphocyte subsets distribution in thymus and spleen.

This work was designed to analyze the possible dose dependent effects of cadmium on the distribution of lymphocyte subsets within the thymus and spleen. Cadmium accumulation was also evaluated in these tissues. For this purpose, adult male rats were exposed for one month to 0, 5, 10, 25, 50 or 100 ppm of cadmium chloride (CdCl2) in the drinking water. In both spleen and thymus, the B lymphocytes increased with the doses of 5 and 10 ppm of CdCl2, and decreased with the doses of 25-100 ppm. In spleen, the doses of 25 and 50 ppm decreased CD4+ cells and the doses of 5 and 10 ppm increased CD8+ cells, while the percentage of thymus T, CD4+, CD8+ and CD4(+)-CD8+ cells was not modified by cadmium treatment at any dose used in this study. After cadmium exposure, the metal was accumulated in the spleen only from the dose of 50 ppm on, and in the thymus, from the dose of 10 ppm on. In conclusion, although the accumulation of the metal is higher in thymus than in spleen, the metal affected CD4+ and CD8+ lymphocytes at the spleen but not at the thymus.

Animals↗

PubMed2002

PiS and PiZ alpha 1 antitrypsin polymorphism and the risk for asbestosis in occupational exposure to asbestos.

Alpha 1 antitrypsin is a highly polymorphic anti-elastase enzyme, especially active in the protection of alveoli and liver. Here we studied the distribution of two deficient alleles Pi*Z and Pi* S, in 194 asbestos workers, of whom 100 were asbestosis cases, and 94 were controls without disease (exposed controls, EC). A second group of controls without asbestos exposure (non-exposed controls, NEC; n=122) was also included. Multivariate analysis adjusted by age and smoking habit showed ninefold risk for asbestosis in Pi*Z heterozygous individuals and 5.9-fold risk for Pi*S homozygous although differences were only significant in the first case (cases vs. EC: OR 8.9; p=0.04). Considering both genotypes (Pi*Z heterozygous, Pi*S homozygous) we obtained an OR of 8 (p=0.01). Our results suggest that the alpha 1 antitrypsin polymorphisms, especially Pi*Z, could help to predict asbestosis risk and confirm the high prevalence of the Pi*S allele in Spain.

Adult↗

PubMed2002

Effects of oral cadmium exposure through puberty on plasma prolactin and gonadotropin levels and amino acid contents in various brain areas in pubertal male rats.

This work was undertaken to analyze if the effects of oral cadmium exposure through puberty, on plasma prolactin and gonadotropin levels are mediated by changes in amino acid contents in various brain areas in male rats. The contents of glutamate, glutamine, aspartate, GABA and taurine in the median eminence, anterior, mediobasal and posterior hypothalamus and prefrontal cortex in pubertal male rats exposed to 50 ppm of cadmium chloride (CdCl2) in the drinking waterfor 1 month (through puberty) were measured by high performance liquid chromatography (HPLC). Plasma prolactin, LH and FSH levels were measured by specific RIA methodology. Plasma prolactin levels decreased after cadmium exposure, whereas plasma levels of LH and FSH were not changed by the metal administration. After cadmium exposure, both glutamine and glutamate contents decreased in the median eminence and in anterior and posterior hypothalamus. Metal exposure also decreased aspartate content in anterior and posterior hypothalamus, but increased it in prefrontal cortex. GABA content decreased in any studied brain region after cadmium administration. Besides, the metal decreased taurine content in the median eminence, anterior and posterior hypothalamus and in prefrontal cortex. The results suggest that cadmium effects on plasma prolactin levels may be partially explained by the changes in aspartate, glutamate or taurine contents, but not by the decrease in GABA content in the brain regions studied.

Administration, Oral↗

PubMed2002

Possible role of glutamate, aspartate, glutamine, GABA or taurine on cadmium toxicity on the hypothalamic pituitary axis activity in adult male rats.

This work was designed to evaluate the possible changes in glutamate, aspartate, glutamine, GABA and taurine within various hypothalamic areas the striatum and prefrontal cortex after oral cadmium exposure in adult male rats, and if these changes are related to pituitary hormone secretion. The contents of glutamine, glutamate, aspartate, GABA and taurine in the median eminence, anterior, mediobasal and posterior hypothalamus, and in prefrontal cortex in adult male rats exposed to 272.7 micromol l(-1) of cadmium chloride (CdCl2) in the drinking water for one month. Cadmium diminished the content of glutamine, glutamate and aspartate in anterior hypothalamus as compared to the values found in the untreated group. Besides, there is a decrease in the content of glutamate, aspartate and taurine in the prefrontal cortex. The amino acids studied did not change in median eminence, mediobasal and posterior hypothalamus or the striatum by cadmium treatment. Plasma prolactin and LH levels decreased in rats exposed to the metal. These results suggest that (1) cadmium differentially affects amino acid content within the brain region studied and (2) the inhibitory effect of cadmium on prolactin and LH secretion may be partially explained by a decrease in the content of both glutamate and aspartate in anterior hypothalamus, but not through changes in GABA and taurine.

Administration, Oral↗

PubMed2001

Cadmium effects on dopamine turnover and plasma levels of prolactin, GH and ACTH.

This paper analyzes possible dopamine (DA) mediated cadmium effects on plasma levels of prolactin, growing hormone (GH) and adrenocorticotropic hormone (ACTH), and if these changes are related to metal accumulation. For that purpose, adult male rats were treated with 50 mg/L of CdCl2 in the drinking water for one month. Plasma levels of prolactin, ACTH and GH were measured by specific double antibody radioimmunoassays. DA was measured by high performance liquid chromatography using electrochemical detection. Cadmium content in the tissues was measured by atomic absorption spectometry with graphite furnace. Analysis was performed by using a T-Student test. Metal exposure increased DA content (34.79+/-3.06 vs. 18.2+/-2.88 pg/mg protein) and decreased its turnover (0.40+/-0.07 vs. 0.75+/-0.06) in posterior hypothalamus. Cadmium also decreased DA turnover in median eminence (0.48+/-0.15 vs. 1.50+/-0.63). Plasma levels of prolactin and GH decreased (2.4+/-0.11 vs. 3.1+/-0.15 ng/mL and 5.37+/-0.05 vs. 9.87+/-1.8 ng/mL respectively), while those of ACTH increased (2.73+/-0.14 vs. 1.7+/-0.16 ng/mL). Cadmium concentration increased in both hypothalamus (4.88+/-0.34 vs. 0.72+/-0.2 microg/g) and pituitary (22.82+/-4.57 vs. 5.02+/-1.25 microg/g) after the metal exposure. These results suggest that cadmium effects on the secretion of these hormones are not mediated by dopamine and might be correlated to the metal accumulation at pituitary level.

Adrenocorticotropic Hormone↗

PubMed2001

Alternate cadmium exposure differentially affects the content of gamma-aminobutyric acid (GABA) and taurine within the hypothalamus, median eminence, striatum and prefrontal cortex of male rats.

This work examines changes of gamma aminobutyric acid (GABA) and taurine contents in the hypothalamus, striatum and prefrontal cortex of the rat after an alternate schedule of cadmium administration. Age-associated changes were also evaluated, of those before puberty and after adult age. In control rats GABA content decreased with age in the median eminence and in anterior, mediobasal and posterior hypothalamus, prefrontal cortex and the striatum. Taurine content showed similar results with the exception of mediobasal hypothalamus and striatum, where no changes were detected. In pubertal rats treated with cadmium from 30 to 60 days of life, GABA content significantly decreased in all brain regions except in the striatum. When cadmium was administered from day 60 to 90 of life, GABA content was significantly changed in prefrontal cortex only compared with the age matched controls. Taurine content showed similar results in pubertal rats, with the exception of the median eminence and the mediobasal hypothalamus, neither of which showed a change. However, when cadmium was administered to rats from day 60 to 90 of life, taurine content only changed in prefrontal cortex compared with the age matched controls. These results suggest that cadmium differentially affects GABA and taurine contents within the hypothalamus, median eminence, striatum and prefrontal cortex as a function of age.

Animals↗

PubMed2001

Alternate cadmium exposure differentially affects amino acid metabolism within the hypothalamus, median eminence, striatum and prefrontal cortex of male rats.

This work was designed to analyze the possible changes in glutamate, aspartate and glutamine content induced by cadmium exposure in the hypothalamus, striatum and prefrontal cortex of rats, using an alternate schedule of metal administration. Pubertal-adult differences were also evaluated. In adult control rats, glutamate and aspartate contents in the anterior hypothalamus decreased as compared to pubertal controls. After cadmium administration from day 30 to 60 of life, the content of anterior hypothalamic glutamate and aspartate diminished. In adult control animals, the glutamine content increased in mediobasal hypothalamus as compared to pubertal controls. After cadmium exposure from day 30 to 60 of life, the mediobasal glutamine content increased, and after cadmium treatment from day 60 to 90 of life, the mediobasal aspartate content decreased. In adult control rats the content of glutamine, glutamate and aspartate of the posterior hypothalamus decreased significantly. After cadmium administration in pubertal animals, posterior hypothalamic contents of glutamine, glutamate and aspartate diminished. Cadmium treatment of adult animals caused a decrease in glutamine content, as compared to controls. In adult control rats, only glutamate and aspartate content increased in the prefrontal cortex as compared to the values found in pubertal controls. When cadmium was administered to adult animals, only the aspartate content decreased. In the striatum, cadmium decreased the glutamine and aspartate contents when administered from day 60 to 90 of life. These data suggest that cadmium differentially affects amino acid metabolism in the hypothalamus, striatum and prefrontal cortex. Age-dependent effects of cadmium on these brain areas appeared to have occurred.

Aging↗

PubMed2001

Cadmium effects on hypothalamic-pituitary-testicular axis in male rats.

This study analyzes cadmium effects at the hypothalamic-pituitary-testicular axis. Male rats were given cadmium during puberty or adulthood. Cadmium exposure through puberty increased norepinephrine content in all hypothalamic areas studied, but not in the median eminence. Metal exposure increased serotonin turnover in median eminence and the anterior hypothalamus, while decreased it in mediobasal hypothalamus. Also, decreased plasma levels of testosterone were found. Cadmium exposure during adulthood increased norepinephrine content in posterior hypothalamus and decreased the neuro-transmitter content in anterior and mediobasal hypothalamus. Decreased circulating levels of luteinizing hormone (LH) and testosterone and increased plasma follicle stimulating hormone (FSH) levels were also observed. Cadmium accumulated in all analyzed tissues. Various parameters showed age-dependent changes. These data suggest that cadmium globally effects hypothalamic-pituitary-testicular axis function by acting at the three levels analyzed and that an interaction between cadmium exposure and age emerge.

Animals↗

PubMed2000

Possible estrogenic and/or antiandrogenic effects of methoxychlor on prolactin release in male rats.

Methoxychlor (MTX) is a pesticide currently used as a substitute for dichloro-diphenyl-trichloroethane (DDT). This organochloride insecticide has some estrogenic properties, and may modify the feedback mechanisms of steroids on the hypothalamus and pituitary. This work was undertaken to explore the possible effects of MTX on the episodic prolactin release and to analyze whether these effects are mediated by dopamine (DA), luteinizing hormone (LH), and/or testosterone. Adult male Sprague-Dawley rats were administered 25 mg/kg/day of MTX in sesame oil for 30 days. Control animals received vehicle only. The episodic prolactin release and plasma testosterone levels were measured as well as the dopamine (DA) content in the median eminence (ME) and in the anterior (AH), mediobasal (MBH), and posterior (PH) hypothalamus. The mean serum prolactin levels and absolute pulse amplitude of the hormone increased after the xenobiotic administration, whereas its relative pulse amplitude diminished. The frequency and duration of prolactin peaks and its half-life were not modified by the treatment with the pesticide. On the other hand, methoxychlor decreased the DA content in ME, increased it in AH, and did not change it in MBH or PH. MTX decreased plasma levels of LH and testosterone compared with controls. These data suggest estrogenic and antiandrogenic effects of MTX on the episodic prolactin secretion; the changes observed in prolactin release could be explained, at least in part, by some of the changes of DA at the hypothalamus and of LH at the pituitary, but not by changes of testosterone at the testicular level.

Androgen Antagonists↗

PubMed2000

Pubertal and postpubertal cadmium exposure differentially affects the hypothalamic-pituitary-testicular axis function in the rat.

The effects of administration of cadmium on levels of hormones along the hypothalamic-pituitary-testicular axis were studied in rats. Male rats were treated subcutaneously from days 30 to 60 (pubertal rats) or from days 60 to 90 of life (postpubertal rats), with cadmium chloride (CdCl2) at a dose of 0.5 or 1 mg/kg, every 4 days in an alternate schedule, starting from the lower dose. Age-matched control rats received 0.3 m of saline subcutaneously every 4 days. The levels of norepinephrine (NE) increased on cadmium exposure in pubertal rats in all hypothalamic areas studied, but decreased in the median eminence. In contrast, in postpubertal rats the levels of NE only did not decrease in the posterior hypothalamus. Serotonin (5-HT) concentration in pubertal and postpubertal rats decreased in all hypothalamic regions, while serotonin turnover (measured by the ratio 5-hydroxyindolacetic acid/serotonin [5-HIAA/5-HT]) increased in the anterior hypothalamus. The serotonin metabolism was also increased in the median eminence in the pubertal and in the posterior hypothalamus in the postpubertal rats. Plasma levels of luteinizing hormone (LH) were not modified by cadmium in both age groups, but follicle stimulating hormone (FSH) levels decreased in postpubertal rats, but was not altered in pubertal rats. Plasma levels of testosterone increased in pubertal rats but decreased in postpubertal rats. Cadmium accumulation increased in the hypothalamus and testes in all the cadmium-treated animals, whereas in the pituitary accumulation of cadmium was found only in postpubertal rats. These data suggest that cadmium exerts age-dependent effects on the hypothalamic-pituitary-testicular axis function, and a disruption of the regulatory mechanisms of the hypothalamic-pituitary-gonadal axis emerges.

Animals↗

PubMed2000

Effects of subchronic alternating cadmium exposure on dopamine turnover and plasma levels of prolactin, GH and ACTH.

This study was undertaken to analyze if the effects of subchronic alternating cadmium exposure on pituitary hormone secretion are mediated by changes in dopamine turnover in an age dependent way or are directly correlated to cadmium accumulation at the hypothalamic-pituitary axis. Male rats were treated sc. from day 30 to 60 (prepubertal period) or from day 60 to 90 (adult age) of life, with cadmium chloride (CdCl2) at a dose of 0.5 and 1.0 mg kg(-1) bw, every 4th day in an alternate schedule, starting with the smaller dose. Dopamine (DA) turnover, expressed as the ratio of acid 3.3-dihidroxifenil acetic (DOPAC)/DA in various hypothalamic areas, the plasma levels of prolactin, growth hormone (GH) and adrenocorticotropic hormone (ACTH), and cadmium accumulation in the hypothalamus and pituitary were studied. Prepubertal cadmium exposure decreased DA content in all hypothalamic areas studied, although its turnover was not modified. A decrease in plasma ACTH levels with no changes in plasma prolactin and GH levels were found. Cadmium did not accumulate in pituitary while it increased in the hypothalamus. Metal exposure during adulthood decreased DA content in mediobasal and posterior hypothalamus, and its turnover in posterior hypothalamus and median eminence. It decreased plasma prolactin and ACTH levels but not those of GH. Cadmium concentration increased in both hypothalamus and pituitary. These results suggest that cadmium exposure produces age dependent changes on the secretory mechanisms of the pituitary hormones studied, related to the selective accumulation of the metal at both hypothalamic and hypophyseal level changes. However the effects of the metal are not mediated by dopamine.

Adrenocorticotropic Hormone↗

PubMed2000

NAD(P)H:quinone oxidoreductase-dependent risk for colorectal cancer and its association with the presence of K-ras mutations in tumors.

NAD(P)H:quinone oxidoreductase (NQO1) is a polymorphic enzyme involved in the detoxification of potentially mutagenic and carcinogenic quinones. The homozygous C609T NQO1 genotype resulting in loss of reductase activity is found in 2-20% of individuals. In the present study, the NQO1-dependent risk for sporadic colorectal cancer (CRC) was studied in 247 incident CRC cases and 296 hospital-based controls recruited during 1996-1997. Four subgroups of cases were studied: (i) all CRCs; (ii) a molecular CRC subgroup (n = 117, cases with molecular tumor analyses); (iii) within the molecular subgroup those tumors with K-ras mutations in codon 12 (CRC K12); (iv) within the molecular subgroup those tumors with K-ras mutations in codon 13 (CRC K13). The C609T NQO1 genotype was found to be twice as prevalent in all CRC patients (6.8%) compared with controls (3%) and six times more common in the subset CRC K12 (20%). Multivariant analyses in the overall population of 247 cases and 296 controls showed a significant age and gender adjusted risk for CRC associated with the C609T NQO1 genotype (OR 2.9, 95% CI 1.19-6.97; P = 0.01) or with any variant genotype (the low activity allele frequency, i.e. heterozygotes plus homozygotes) (OR 1.41, 95% CI 1.02-1.92; P = 0.03). Within cases of the molecular subgroup (n = 117) the C609T NQO1 genotype was associated with the presence of K-ras codon 12 mutation (OR 6.5 95%, CI 1.39-34.9; P = 0.003). Logistic regression showed an age and gender adjusted risk for K-ras codon 12 mutant CRC associated with the C609T NQO1 genotype (OR 10.5, 95% CI 2.99-36.7; P: = 0.0002) or with any variant NQO1 genotype (OR 2.23, 95% CI 1.23-4.00; P = 0.007) compared with the control group. Genetically determined variations in NQO1 may modify the risk for CRC and these risks may be greatest for tumors containing K-ras codon 12 mutations. CRC with K-ras codon 12 mutations may represent a distinct and etiologically more homogeneous subtype of the disease, which may be associated with toxicants that are metabolized via a NQO1-dependent pathway.

Age Factors↗

PubMed2000

Correlations of partial and extensive methylation at the p14(ARF) locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors.

p14(ARF) is a putative tumor suppressor gene thought to modify the levels of p53. CpG sites within the 5'-flanking region and exon 1beta of p14(ARF) are targets of aberrant methylation and transcriptional silencing in human colorectal cancer (CRC). Here we have developed methylation-specific polymerase chain reaction (MSPCR) methods to detect methylation of CpG sites in p14(ARF) in CRC cell lines and primary CRC tumors, and correlated p14(ARF) mRNA expression with methylation in CRC cell lines using competitive quantitative reverse transcription-polymerase chain reaction methods. Ten CRC cell lines were studied; three (DLD-1, HCT15 and SW48) showed extensive methylation and six (Colo320, SW480, HT29, Caco2, SW837 and WiDr) were unmethylated; the other cell line, LoVo, showed partial methylation that affected exon 1beta but not the immediate upstream CpG sites. p14(ARF) mRNA was expressed at extremely low levels in fully methylated cell lines and at 10(4)- to 10(5)-fold higher levels in unmethylated cell lines. p14(ARF) expression in the partially methylated LoVo cell line was intermediate. Treatment of LoVo cells with 2 microM 5-aza-2'-deoxycytidine for 72 h was associated with marked (100-fold) induction of mRNA levels. Of 119 primary CRCs, 18% contained p14(ARF) methylation, although partial methylation was the most common pattern observed (in 67% of methylated tumors). Methylation of p14(ARF) was often accompanied by p16(INK4a) methylation; however, 50% of p14(ARF) methylated tumors contained unmethylated p16(INK4a). Methylation at p14(ARF) was associated with female gender, greater age, proximal anatomic location and poor differentiation, but not stage at diagnosis. A two-step MSPCR method for assaying p14(ARF) methylation in human tumors is described.

Adult↗