Angiotensin II type 1 receptor and ACTH receptor expression in human adrenocortical neoplasms

OBJECTIVE: Type 1 angiotensin II (Ang II) receptors transduce most of the known actions of Ang II, including steroidogenesis and trophic actions on the adrenal cortex. We investigated the type 1 Ang II receptor expression in adrenocortical tissues to define its regulation in adrenocortical neoplasms and to compare its expression with that of the ACTH receptor (ACTH-R). PATIENTS AND MEASUREMENTS: Poly A RNA was extracted from tumour tissue and electrophoresed through a 1.0% agarose gel, blotted and hybridized with alpha32P-CTP labelled PCR generated type 1 Ang II receptor cDNA probe. Receptor autoradiography was performed on slices from normal adrenals and tumour tissue by incubation with 125I-Sar1, Ile8-Ang II with and without pretreatment with cold Ang II or with the selective type 1 receptor antagonist losartan. RESULTS: Ang II type 1 receptor mRNA was high in cortisol producing (CPA; n = 5) and aldosterone producing (APA; n = 4) adenomas (normal adrenals 100 +/- 12% vs. 180 +/- 16% in CPA and 154 +/- 26% in APA, mean +/- SEM), but was low in nonfunctioning adenomas (NFA; n = 2; 2 +/- 1%). ACTH receptor mRNA followed a similar pattern (CPA 178 +/- 17, APA 196 +/- 30, NFA 0%, carcinomas 56 +/- 11%) with a good correlation between Ang II type 1 receptor and ACTH-R mRNA of r = 0.692, P = 0.0019. Receptor autoradiography in normal adrenals demonstrated Ang II type 1 receptors predominantly in the zona glomerulosa. In tumour tissue, mainly type 1 receptor expression was found confirming the Northern blot data. CONCLUSIONS: Angiotensin II type 1 receptor and ACTH receptor expression seems to be correlated with the functional status of adrenocortical tumours, suggesting regulation by similar factors. The predominant receptor expressed in adrenocortical tumours is the Angiotensin II type 1 receptor whereas type 2 receptor expression is minimal.     

Decreased expression of cyclic adenosine monophosphate-regulated aldose reductase (AKR1B1) is associated with malignancy in human sporadic adrenocortical tumors

The human aldose reductase, AKR1B1, participates in glucose metabolism and osmoregulation and is supposed to play a protective role against toxic aldehydes derived from lipid peroxidation and steroidogenesis that could affect cell growth/differentiation when accumulated. Adrenal gland is a major site of expression of AKR1B1, and we asked whether changes in its expression could be associated with adrenal disorders. Therefore, we examined AKR1B1 gene expression in human fetal adrenals, adrenocortical cell line, and tumors and compared the results with the expression of steroidogenic genes (StAR and CYP11A) and regulators of adrenal cortex development [steroidogenic factor-1 (SF-1) and dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1)]. Using specific antibodies, Northern blotting, and enzymatic assays, we present evidences that AKR1B1 detectable in 15-wk-old fetal glands is regulated by cAMP in NCI-H295 cells and thus that AKR1B1 is functionally related to the ACTH-responsive murine akr1b7/mvdp gene rather than to its direct ortholog, the mouse aldose reductase akr1b3 gene. Although low DAX1 expression in aldosterone-producing adenomas (n = 5) was confirmed (P < 0.05), no correlation was found between the expression of all other genes and the tumors endocrine activity. In contrast, relative abundance of AKR1B1 mRNA was decreased in adrenocortical carcinomas (n = 5; mean +/- sem, 0.95 +/- 0.2) when compared with adenomas (n = 12; 9.29 +/- 3.05; P < 0.001). Most (seven of eight) adrenocortical carcinomas (19.0 +/- 5.4) had very low relative AKR1B1 protein levels when compared with benign tumors (cortisol-producing adenomas, n = 5, 63.0 +/- 9.8; nonfunctional adenomas, n = 5, 58.0 +/- 10.4; aldosterone-producing adenomas, n = 4, 65.3 +/- 7.7; P < 0.001), Cushing's hyperplasia (n = 5, 54.6 +/- 5.3; P < 0.01), or normal adrenals (n = 4; 37.1 +/- 5.3; P < 0.001). These properties provide the first evidence that expression of cAMP-regulated AKR1B1 is decreased in adrenocortical cancer. This might take part in adrenal tumorigenesis and could be investigated as a marker of malignancy for the diagnosis of adrenal tumors.     

Alteration of oncogenic IGF-II gene methylation status associates with hepatocyte malignant transformation

BackgroundOncogenic insulin-like growth factor-II (IGF-II) is overexpressed in hepatocellular carcinoma (HCC). The present study aimed to analyze the dynamic alteration of IGF-II CpG site methylation status and its molecular mechanism in HCC progression.MethodsIGF-II alterations were observed in rat hepatocarcinogenesis models induced by 2-acetylaminofluorene. Liver IGF-II expression was compared by immunohistochemistry or tissue IGF-II specific concentration (nmol/mg protein). Status of human IGF-II promoter 3 (P3) or rat IGF-II P2 CpG site methylation was amplified by methylation-specific polymerase chain reaction (MSP). Serum IGF-II levels were quantitatively detected by an enzyme-linked immunosorbent assay.ResultsThe levels of hepatic IGF-II expression were significantly elevated in the HCC group (P < 0.001). The unmethylation rate of IGF-II P3 CpG sites was 100% in the HCC-, 52.5% in the paracancerous-, and none (0%) in the distal noncancerous-tissues. Abnormal IGF-II expression was related to differentiation degree, tumor invasion, and positive HBV-DNA (all P < 0.001), with a negative correlation between P3 methylation degree and IGF-II expression. There was a positive correlation between liver IGF-II specific concentration and circulating IGF-II level (r = 0.97, P < 0.001). Significantly negative correlation was found between IGF-II P2 CpG site methylation and circulating IGF-II (r_s = ?0.89, P < 0.001) or liver IGF-II level (r_s = ?0.84, P < 0.001).ConclusionsThe increase of serum IGF-II and the alteration of oncogenic gene IGF-II methylation may be biomarkers for HCC diagnosis and DNA methylation may be the therapeutic target of HCC.     

Inherited congenital adrenal hyperplasia in the rabbit: absent cholesterol side-chain cleavage cytochrome P450 gene expression.

We investigated adrenal steroidogenic enzymes, their activity and mRNA expression, and in vitro biosynthesis of an enzyme in rabbits with congenital adrenal hyperplasia (CAH; weight: CAH, 19 +/- 5 mg/adrenal; normal, 2.7 +/- 1.0 mg/adrenal). Serum pregnenolone (delta 5-P) levels in CAH newborn rabbits (12-36 h) were normal (mean/range, 438/51-2191 ng/dl), but corticosterone levels were low [0.05 +/- 0.05 microgram/dl; P less than 0.001 vs. normal (0.66 +/- 0.57)]. Serum Na+ levels in CAH newborn rabbits were in the normal range (143 +/- 30 meq/liter), but K+ levels were elevated [7 +/- 1.1 meq/liter; P less than 0.05 vs. normal (5.9 +/- 0.6 meq/liter)]. Minced normal adrenal tissue incubated with [3H] cholesterol (30-100 pmol/flask) and ACTH (100 mU/flask) produced [3H]delta 5-P (newborn, 21 and 45 fmol/100 mg; adult, 3 and 5 fmol/100 mg) and [3H]corticosterone (newborn, 23 fmol/100 mg; adult, 11.3 fmol/100 mg), but CAH adrenals produced no product (less than 1.3 fmol/100 mg). Adrenal mitochondria from normal newborn rabbits produced delta 5-P (4.4-7 nmol/mg protein), but CAH adrenals did not, while CAH adrenal mitochondria demonstrated over 4 times greater 11 beta-hydroxylase activity. A Western blot of adrenal homogenate from normal newborn rabbits revealed a cholesterol side-chain cleavage cytochrome P450 (P450scc)-immunoreactive species (mol wt, 53 x 10(3), but this species was absent in CAH adrenals; CAH adrenals had a normal adrenodoxin and intensified 17 alpha-hydroxylase cytochrome P450 (P450(17)alpha) band compared to normal adrenals. In vitro translation of RNA in a cell-free rabbit reticulocyte lysate system containing [35S] methionine yielded a precursor P450scc protein (mol wt, 58.5 x 10(3)) with normal adrenal RNA, but not with CAH adrenal RNA. P450scc mRNA was detected in all normal adrenals, but was not detected in all CAH adrenals. 21-Hydroxylase cytochrome P450 mRNA expression was detected at a similar level in both normal and CAH adrenals. We conclude that CAH in the rabbit is caused by inherited absent P450scc gene expression. The clinical, pathological, and biochemical manifestations of P450scc deficiency in the rabbit are nearly identical to the human disorder. Increased 11 beta-hydroxylase activity and increased P450(17)alpha on Western blot of CAH adrenals indicate altered gene expression of other steroidogenic enzymes due to CAH. Further molecular analysis of the P450scc gene in this animal CAH model will facilitate understanding of P450scc deficiency CAH.     

Effect of 5-aza-2'-deoxycytidine (Dacogen) on covalent histone modifications of chromatin associated with the epsilon-, gamma-, and beta-globin promoters in Papio anubis

OBJECTIVE: Treatment with the DNA demethylating drug 5-aza-2'-deoxycytidine (Dacogen; DAC) increased fetal hemoglobin and F cells to therapeutically significant levels in patients with sickle cell disease. To gain more insight into the mechanism of action of this drug and to increase our understanding of the relationship between DNA methylation and chromatin structure, we have determined the effect of DAC on covalent histone modifications of chromatin associated with the epsilon, gamma-, and beta-globin promoters in purified bone marrow erythroid cells of four baboons (P. anubis) pre- and posttreatment. RESULTS: Fetal hemoglobin increased from 6.45%+/-1.75% in pretreatment samples to 62.1%+/-7.94% following DAC. DNA methylation of three CpG sites within the epsilon-globin promoter and 5 CpG sites within the gamma-globin promoter decreased more than 50% following DAC treatment. Levels of RNA polymerase II, acetyl-histone H3, acetyl-histone H4, dimethyl-histone H3 (lys4), dimethyl-histone H3 (lys36), and dimethyl-histone H3 (lys79) associated with the epsilon-, gamma-, and beta-globin promoters were determined by chromatin immunoprecipitation of formaldehyde-fixed chromatin followed by real-time PCR. Dacogen treatment increased the association of RNA polymerase II, acetyl-histone H3, and acetyl-histone H4 with the gamma-globin promoter but did not significantly affect the association of dimethyl-histone H3 (lys4), dimethyl-histone H3 (lys36), and dimethyl-histone H3 (lys79) with the epsilon-, gamma-, and beta-globin gene promoters. CONCLUSION: These experiments illustrate the usefulness of the baboon model to investigate the mechanism of pharmacologic reactivation of fetal hemoglobin synthesis at the molecular level.     

Postnatal overexpression of insulin-like growth factor II in transgenic mice is associated with adrenocortical hyperplasia and enhanced steroidogenesis

The influence of postnatal insulin-like growth factor II (IGF-II) overexpression on adrenal growth and function was investigated in 3-month-old male phosphoenolpyruvate carboxykinase (PEPCK) promoter human IGF-II transgenic mice, which are characterized by 4-to 6-fold elevated postnatal IGF-II serum levels. Plasma corticosterone levels of PEPCK-IGF-II transgenic mice were 2-fold higher than in age- and sex-matched controls, both in the morning (7.4 +/- 1.5 vs. 17.8 +/- 3.9 ng/ml, P < 0.01) and in the evening (33.3 +/- 6.5 vs. 65.3 +/- 12 ng/ml, P < 0.01). When PEPCK-IGF-II transgenic mice were subjected to an ACTH challenge, corticosterone levels were stimulated 6-fold, to 396 +/- 17 ng/ml after 60 min, compared with 230 +/- 24 ng/ml in the control group. In contrast to corticosterone, plasma ACTH levels were similar in transgenic and control mice, excluding an indirect effect of IGF-II at the hypothalamic or pituitary level. In vitro, the basal and ACTH-induced corticosterone production of adrenal glands from transgenic mice was higher (2-fold and 1.8-fold, respectively) than that of control organs. However, when normalized for adrenal weight, the in vitro corticosterone secretion was similar in both groups. At autopsy, adrenal weights of transgenic mice were significantly greater than those of control adrenal glands (3.3 +/- 0.2 vs. 2.0 +/- 0.2 mg, P < 0.01, n = 10). Furthermore, a local expression of human IGF-II could be demonstrated in transgenic adrenal glands by RT-PCR, whereas in normal adult mice, no adrenal expression of IGF-II was detected. Stereological investigation of adrenal glands from another set of PEPCK-IGF-II transgenic mice and controls (6-month-old males) demonstrated that the increase in adrenal weight in transgenic mice is mainly caused by a 50% increase in the number of zona fasciculata cells, whereas cell volume and zonation of transgenic adrenal glands remained unchanged. In conclusion, our data indicate that postnatal overexpression of IGF-II induces an increased adrenal weight and elevated corticosterone serum levels, presumably by a direct mitogenic effect of IGF-II on adrenocortical fasciculata cells.     

Expression of inhibin alpha in adrenocortical tumours reflects the hormonal status of the neoplasm

Inhibins are gonadal glycoprotein hormones whose main endocrine function is to inhibit pituitary FSH secretion. In addition to testes and ovaries, other steroid-producing organs are sites of inhibin alpha subunit expression. To study the role of inhibins in human adrenal gland, we screened a panel of 150 adrenals (10 normal adrenals, 25 adrenocortical hyperplasias, 65 adrenocortical adenomas, 30 adrenocortical carcinomas and 20 phaeochromocytomas) for inhibin alpha expression. mRNA levels of inhibin alpha subunit were studied in 57 samples and all tissues were stained immunohistochemically with an inhibin alpha subunit-specific antibody. Inhibin alpha mRNA was detected in all adrenocortical tissues. Virilizing adenomas possessed a 10-fold higher median inhibin alpha mRNA expression than did normal adrenals. Bilaterally and nodularly hyperplastic adrenals and other than virilizing adrenocortical tumours had their median inhibin alpha mRNA levels close to those of normal adrenals. Immunohistochemically, inhibin alpha subunit was detectable in all normal and hyperplastic adrenals, as well as in 73% of the adrenocortical tumours. However, the percentage of inhibin alpha-positive cells varied greatly in different tumour types. The median percentage of positive cells was 10 in non-functional and Conn's adenomas, 30 in Cushing's adenomas and 75 in virilizing adenomas. In malignant adrenocortical tumours the median percentage of inhibin alpha-immunopositive cells was 20 in non-functional carcinomas, 30 in Conn's carcinomas, 65 in Cushing's carcinomas and 75 in virilizing carcinomas. All phaeochromocytomas were negative for inhibin alpha subunit both at the mRNA level and immunohistochemically. Our data show that inhibin alpha subunit is highly expressed in both normal and neoplastic androgen-producing adrenocortical cells, with less expression in cortisol-producing and hardly any in aldosterone-producing cells. This suggests a specific role for inhibins in the regulation of adrenal androgen production. We did not find any significant difference in inhibin alpha expression between benign and malignant adrenocortical tumours. Thus inhibin alpha gene does not seem to have a tumour suppressor role in human adrenal cortex.     

Silencing SMYD3 in hepatoma demethylates RIZI promoter induces apoptosis and inhibits cell proliferation and migration

AIM： To investigate the role of SMYD3 in hepatocellular carcinoma （HCC） development and progression and to verify whether its regulation activity was through RIZ1 inactivation. METHODS： Expression of SMYD3 in HCC cell lines and tissues were measured; silencing of SMYD3 by RNA interference （RNAi） was effectuated, hepatoma cell proliferation, migration and apoptosis were tested, with RIZl CpG promoter methylation, and corresponding mRNA expression were investigated. RESULTS： SMYD3 over-expression in HCC was associated with RIZl hypermethylation and mRNA down-expression. Suppression of SMYD3 expression de- methylated RIZl CpG promoter （P 〈 0.01） and increased RIZl mRNA expression （P 〈 0.01）. Consequently, SMYD3 down-expression with RIZl de-methylation strongly inhibited hepatoma cell growth （MTT inhibitory rates： Pgenesil-1-s1 60.95%± 7.97%, Pgenesil-1-s2 72.14% ± 9.68% vs Pgenesil-1-hk 6.89% ± 4.12%, P 〈 0.01） and migration （Pgenesil-1-s1 4.24% ± 1.58%, Pgenesil- 1-s1 4.87% ± 0.73% vs Pgenesil-1 19.03% ± 4.63%, Pgenesil-1-hk 19.95% ±5.21%, P 〈 0.01） and induced apoptosis （FCM subG1 phase Pgenesil-1-s1 19.07% ＋ 1.78%, Pgenesil-1-s2 17.68% ± 2.36% vs Pgenesil-1 0.47% ± 0.12%, Pgenesil-1-hk 1.46% ± 0.28%, P 〈 0.01. TUNEL-positive cells： Pgenesil-1-s1 40.24%± 5.18%, Pgenesil-1-s2 38.48% ± 4.65% vs Pgenesil-1 2.1B% - 1.34%, Pgenesil-1-hk 2.84%± 1.22%, P 〈 0.01） in HepG2 cells. CONCLUSION： These results demonstrate that SMYD3plays a critical role in the carcinogenesis and progression of HCC, The proliferation, migration induction and apoptosis inhibition activities of SMYD3 may be mediated through RIZl CpG promoter hypermethylation.     

Hypermethylation of CpG island in O6-methylguanine-DNA methyltransferase gene was associated with K-ras G to A mutation in colorectal tumor

AIM:To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression. METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas, 62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutant-allele-specific amplification was used to detect K-rasG to A point mutation in codon 12. RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P= 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation, respectively (P = 0.027, P<0.001). Thirteen of the 19 colorectal tumors with K-rasG to A point mutation in codon 12 had methylated MGMT(P= 0.011). The frequencies of K-rasG to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively. CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-rasG to A point mutation in colorectal tumor. The frequency of K-rasG to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.     

Epigenetic identification of ubiquitin carboxyl-terminal hydrolase L1 as a functional tumor suppressor and biomarker for hepatocellular carcinoma and other digestive tumors

The ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a carboxyl-terminal ubiquitin hydrolase regulating cellular ubiquitin levels, recently suggested as a tumor suppressor. However, the role of UCHL1 in hepatocellular carcinoma (HCC) is not clear. We investigated the expression and DNA methylation of the UCHL1 in primary HCC, liver metastases from digestive carcinomas, and primary digestive cancers. UCHL1 is expressed in all normal tissues and immortalized normal epithelial cell lines, but was low or silenced in 77% (10/13) of HCC cell lines, which is well correlated with its promoter methylation status. Methylation was further detected in 44% (12/27) of HCCs, but less in metastatic tumors generated from colorectal and stomach in the liver (19%, 3/16; P < 0.05). Methylation was also detected in primary digestive tumors, including 71% (22/31) of colon, 77% (53/69) of gastric, and 40% (18/45) of esophageal carcinomas, but none or occasionally in paired adjacent nontumor tissues. Detailed methylation analysis of 49 CpG sites at a 540-bp promoter region by bisulfite genomic sequencing confirmed the methylation. UCHL1 silencing could be reversed by chemical or genetic demethylation of the promoter, indicating direct epigenetic silencing. Restoring UCHL1 expression in silenced cell lines significantly inhibited their growth and colony formation ability by inhibiting cell proliferation, causing cell cycle arrest in G2/M phase and inducing apoptosis through the intrinsic caspase-dependent pathway. Moreover, UCHL1 directly interacts with p53 and stabilizes p53 through the ubiquitination pathway. CONCLUSION: Epigenetic inactivation of UCHL1 is common in primary HCCs and other digestive tumors. UCHL1 appears to be a functional tumor suppressor involved in the tumorigenesis of HCCs and other digestive cancers.     

Hedgehog通路中Ptchl基因在人胃癌中高甲基化状态分析

目的 研究Hedgehog通路中Ptchl基因表达及其甲基化状态在胃癌发生中的变化.方法 分别抽提10例胃癌组织及其癌旁＞3 cm组织和胃癌细胞株AGS的总RNA和基因组DNA.实时定量逆转录多聚酶链反应(QRT-PCR)检测Ptch基因的mRNA表达.应用软件分析Ptchl基因5'调控序列的CpG岛状态,亚硫酸氢盐测序PCR(BSP)分析甲基化水平.结果 对Ptchl mRNAla转录体转录起始位点(计为0点)上游-3950 bp和下游+2050 bp进行CpG岛分析,发现存在两个CpG岛,第1个为-1139 bp～+860 bp,第2个为+875 bp～+1692 bp.以第1个CpG岛的-870 bp～+229 bp区段内的19个CpG位点的BSP测序结果显示,胃癌细胞株AGS全部发生甲基化,胃癌组织中甲基化程度为16%～100%,平均64%±32%,癌旁组织甲基化程度为0%～42%,平均13%±14%,两组问差异有统计学意义(P＜0.05).Spearman秩相关分析发现,Ptchl基因甲基化同其表达呈负相关(r=-0.558,P=0.011).结论 Ptchl基因高甲基化参与胃癌的发生,可能为胃癌新的肿瘤标志物.     

Metallothionein protein and minichromosome maintenance protein-2 expression in adrenocortical tumors

AimSome resected adrenal-confined adrenocortical carcinomas metastasize and others not. The present study was designed to evaluate the expression of metallothionein protein (MT) and minichromosome maintenance protein-2 (MCM2) in adrenocortical carcinomas and adrenocortical adenomas, and to test the correlation between this and adrenocortical carcinoma aggressiveness.Materials and methodsThe study comprised 14 patients operated on for adrenocortical carcinoma, 15 operated on for adrenocortical adenoma and 2 with normal adrenals. Hematoxylin-eosin staining was used for histological evaluation under light microscopy, and sequential sections were used for MCM2 and MT staining.ResultsIn normal adrenals, positive staining was weak for MT and zero for MCM2. Rates of positive staining for MT and MCM2 were significantly higher in adrenocortical carcinomas than in adrenocortical adenomas (P = 0.008 and P < 0.001, respectively). In adrenocortical carcinomas, a significant positive correlation was found between MCM2 staining and Weiss revisited score (P = 0.022) but not for Weiss score, and a significant positive correlation was found between MCM2 and mitotic rate on histology (P = 0.033). MCM2 but not MT staining was also shown to correlate significantly with stage IV carcinoma (P = 0.008 and P = 0.165, respectively).ConclusionMCM2 and MT are overexpressed in adrenocortical carcinoma, and MCM2 expression correlates significantly with metastatic disease.     

High expression of cyclin E and G1 CDK and loss of function of p57KIP2 are involved in proliferation of malignant sporadic adrenocortical tumors

Maternal loss of heterozygosity (LOH) of the 11p15 region and overexpression of the insulin-like growth factor (IGF)-II gene are associated with the malignant phenotype in sporadic adrenocortical tumors. In the imprinted 11p15 region, the p57KIP2 gene is maternally expressed and encodes a cyclin-dependent kinase (CDK) inhibitor involved in G1/S phase of the cell cycle. We hypothesized that maternal LOH in malignant adrenocortical tumors could be responsible for loss of p57KIP2 gene expression and, thus, could favor progression through the cell cycle. We investigated 3 normal adrenals, 31 adrenocortical tumors [11 tumors with normal expression of the IGF-II gene (mainly benign) and 20 with IGF-II gene overexpression (mainly malignant)], and the human adrenocortical tumor cell line NCI H295R for expression of the p57KIP2 gene, G1 cyclins (cyclin D2 and E) and G1 CDK (CDK2, CDK3 and CDK4) protein contents and for kinase activity of G1 cyclin-CDK complexes. The expression of p57KIP2, G1 cyclins, and G1 CDKs in benign tumors was similar to that in normal adrenal tissues, as were kinase activities of G1 cyclin-CDK complexes. By contrast, abrogation of the p57KIP2 gene expression and increased expression of G1 cyclins (cyclin E) and G1 CDKs (CDK2 and CDK4) were associated with high activity of G1 cyclin-CDK complexes in malignant tumors and in the H295R cell line. These data suggest that the p57KIP2 gene might act as a tumor suppressor gene in adrenocortical tumors. Maternal LOH with duplication of the paternal allele or pathological functional imprinting of the 11p15 region are responsible for loss of expression of the p57KIP2 gene and increased expression of the IGF-II gene. Consequently, both events favor cell proliferation in malignant adrenocortical tumors.     

胃癌组织RASSF1A甲基化对其mRNA及蛋白表达的影响

目的研究RASSF1A基因启动子区甲基化对胃癌组织中RASSF1AmRNA及蛋白表达的影响。方法RT-PCR方法检测54例胃癌及癌旁正常组织RASSF1AmRNA表达,甲基化特异性PCR方法检测RASSF1A启动子区CpG岛甲基化状态;Westernblot方法检测20例胃癌及癌旁正常组织中RASSF1A蛋白表达。结果(1)RASSF1AmRNA和蛋白表达水平在胃癌组织中明显低于癌旁正常组织(A值分别为0·2589±0·2407比0·5448±0·2971,P<0·0001;0·1874±0·0737比0·6654±0·2201,P<0·0001);(2)RASSF1A在胃癌组织和正常组织中的甲基化频率分别为66·7%和14·8%,差异有统计学意义(P<0·0001);(3)在胃癌组织中,甲基化组RASSF1AmRNA的表达明显低于非甲基化组(0·1384±0·1142比0·5018±0·2463,P<0·0001)。结论胃癌组织RASSF1AmRNA和蛋白表达缺失或低下,与其启动子甲基化程度增高显著相关。     

Increased and persistent circulating insulin-like growth factor II in neonatal transgenic mice suppresses developmental apoptosis in the pancreatic islets

In rats, a proportion of pancreatic beta-cells are deleted by apoptosis in the second week of postnatal life and replaced by endocrine cell neogenesis from pancreatic ductal epithelium. This coincides with a reduction in pancreatic insulin-like growth factor II (IGF-II) expression, and IGF-II has been shown to act as a beta-cell survival factor in vitro. To examine whether IGF-II regulates beta-cell apoptosis in vivo, an IGF-II transgenic mouse model was used in which mouse IGF-II is overexpressed in skin, gut, and uterus driven by a keratin promoter, so that circulating IGF-II is retained postnatally. Mice were killed between postnatal days 7 and 26, and the pancreas was examined histologically. Apoptotic cells were visualized by the terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling method, and proliferating cells were examined by immunohistochemistry for proliferating cell nuclear antigen. In nontransgenic mice, serum IGF-II was absent by 26 days, but mean (+/-SEM) values were 45+/-9 ng/ml (n = 5) in transgenic animals. A 2- to 3-fold rise in islet cell apoptosis was seen in normal animals between days 11 and 16, but this was substantially decreased in IGF-II transgenic mice (day 11; control, 12+/-1%; transgenic, 6+/-1%; P < 0.01; n = 5). Consequently, islets from IGF-II transgenic mice had a significantly greater mean area from days 11-16, but the proportions of beta- and alpha-cells and circulating insulin levels were not changed. Islet cell DNA synthesis was increased in transgenic mice on days 13 and 16. The total islet number per section did not alter. The results show that a persistent presence of circulating IGF-II postnatally alters endocrine pancreatic ontogeny in the mouse and largely prevents the wave of developmental apoptosis that precipitates beta-cell turnover in neonatal life.