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1.
We previously reported that increased DNA methylation was an important mechanism of silencing the p27 gene in some pituitary
tumor cell lines [1]. DNA methylation correlated inversely with p27 gene expression. The p27 and cyclin D2 genes are located
in the same region of mouse chromosome 6, rat chromosome 4, and human chromosome 12p13. Because both genes are located in
the same gene cluster, we investigated whether methylation was a principal mechanism regulating cyclin D2 as well as p27 expression
in rodent pituitary cell lines. Bisulfite genomic sequencing showed that the normally unmethylated cytosines of the p27 gene
in normal pituitary (NP) were extensively methylated in GH3 and GHRH-CL1 cells, but not in AtT 20, αT3-1 and LβT2 cells; but cyclin D2 was extensively inactivated in various pituitary tumor cell lines by increased DNA methylation. These abnormalities of methylation in p27 and cyclin
D2 genes occurred with different frequencies in five pituitary tumor cell lines with 100% (5/5) methylation of the cyclin
D2 gene and 40% (2/5) methylation of the p27 gene. Treatment with the methyl transferase inhibitor 5′-aza-2′-deoxycytidine
(AZAdC) increased expression of cyclin D2 and p27 in GH3 and GHRH-CL1 pituitary tumor cells. There was a correlation between
hypermethylation and gene expression. GH3 tumors implanted into Wistar-Furth rats in vivo did not change the methylation status of the p27 and cyclin D2 genes. These
data indicate a coordinately reduced expression of these two linked genes in most rodent pituitary tumor cell lines and suggest
that methylation of cyclin D2 and p27 might occur in a “hot spot” in this gene-rich cluster.
Supported in part by NIH CA 37231 and CA 42951 相似文献
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Pituitary hyperplasia and tumor growth are regulated by various hormones and growth factors. Estrogen (E2) stimulates pituitary cell proliferation and prolactin (PRL) production. Estrogen also regulates transforming growth factor-β
(TGF-β) effects in the pituitary. TGF-β in turn regulates various cell cycle proteins including p15 and p27Kip1 (p27). To better understand the regulatory role of growth factors and hormones in the cell cycle we analyzed cyclin D1, cyclin E, and p27 expression in normal and neoplastic rat pituitary cells. An in vitro analysis using cultured normal pituitary
cells and GH3 tumor cells and an in vivo analysis of estrogen-treated normal pituitary and implanted GH3 cells were performed. Semiquantitative RT-PCR was used to analyze mRNA expression for cyclin D1, cyclin E, and p27 in cultured pituitary cells and E2-treated pituitaries in vivo. Cyclin D1 and p27 were localized in the nuclei of normal pituitary cells by immunocytochemistry (ICC). Very weak or absent immunostaining
for cyclin D1 and p27 was present in GH3 cells. Both normal pituitary and GH3 cells had strong nuclear staining for cyclin E. Normal pituitary had a 20-fold greater amount of cyclin D1 mRNA and a 3-fold greater amount of p27 mRNA compared to GH3 cells, whereas GH3 cells had slightly (1.5-fold) more cyclin E than normal pituitary cells. E2 treatment in vivo stimulated cell proliferation and decreased cyclin D1 mRNA levels in normal pituitary. GH3 tumor cells, implanted subcutaneously in the same animal, showed increased proliferation after E2 treatment, but there was no change in cyclin D1 mRNA in GH3 cells. Cyclin E and p27 mRNA levels did not change significantly in normal pituitary or in GH3 cells after E2 treatment in vivo. Treatment of normal pituitary cells with 10−9
M TGF-β1 for 3 d in vitro led to significant decreases in cyclin D1 and p27 mRNAs (p < 0.05), whereas cyclin E levels were unchanged. These results indicate that cyclin D1 and p27 mRNAs are present at significantly higher levels in normal pituitary compared to GH3 cells, and that both E2 and TGF-β1 can downregulate cyclin D1 mRNA levels in normal pituitary cells, suggesting that these factors regulate G1 to S phase transition in pituitary cells.
The lower levels of specific cell cycle regulators in GH3 cells may explain the decreased regulatory control by E2 in GH3 tumor cells. 相似文献
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Epigenetic silencing through DNA and histone methylation of fibroblast growth factor receptor 2 in neoplastic pituitary cells
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Four members of the fibroblast growth factor receptor (FGFR) family of tyrosine kinases transduce signals of a diverse group of more than 23 fibroblast growth factor (FGF) ligands. Each prototypic receptor is composed of three immunoglobulin-like extracellular domains, two of which are involved in ligand binding. Alternative RNA splicing of one of two exons results in two different forms of the second half of the third immunoglobulin-like domain, the IIIb or IIIc isoforms. The contribution of each receptor and their isoforms in tumorigenesis remains unknown. In the pituitary, FGFR2 is expressed primarily as the IIIb isoform in normal adenohypophysial cells. In contrast, FGFR2 is significantly down-regulated in mouse corticotroph AtT20 tumor cells where the 5' promoter is methylated. Treatment of AtT20 cells with 5'-azacytidine resulted in FGFR2 re-expression, mainly as the FGFR2-IIIb isoform. Chromatin immunoprecipitation revealed evidence of histone methylation, but not of deacetylation, in the silencing of FGFR2 in AtT20 cells. Exposure of these cells to the cognate FGFR2-IIIb ligand FGF-7 resulted in diminished Rb phosphorylation and accumulation of p21 and p27, indicating diminished cell cycle progression. Examination of primary human pituitary adenomas revealed FGFR2 down-regulation in 52% (11 of 21) of samples and FGFR2 promoter DNA methylation in 45% (10 of 22) of samples. These data highlight the contribution from DNA and histone methylation as epigenetic mechanisms responsible for FGFR2 silencing in pituitary neoplasia. 相似文献
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《Genetics in medicine》2014,16(12):903-912
PurposeExpression of imprinted genes is regulated by DNA methylation of differentially methylated regions (DMRs). Beckwith–Wiedemann syndrome is an imprinting disorder caused by epimutations of DMRs at 11p15.5. To date, multiple methylation defects have been reported in Beckwith–Wiedemann syndrome patients with epimutations; however, limited numbers of DMRs have been analyzed. The susceptibility of DMRs to aberrant methylation, alteration of gene expression due to aberrant methylation, and causative factors for multiple methylation defects remain undetermined.MethodsComprehensive methylation analysis with two quantitative methods, matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing, was conducted across 29 DMRs in 54 Beckwith–Wiedemann syndrome patients with epimutations. Allelic expressions of three genes with aberrant methylation were analyzed. All DMRs with aberrant methylation were sequenced.ResultsThirty-four percent of KvDMR1–loss of methylation patients and 30% of H19DMR–gain of methylation patients showed multiple methylation defects. Maternally methylated DMRs were susceptible to aberrant hypomethylation in KvDMR1–loss of methylation patients. Biallelic expression of the genes was associated with aberrant methylation. Cis-acting pathological variations were not found in any aberrantly methylated DMR.ConclusionMaternally methylated DMRs may be vulnerable to DNA demethylation during the preimplantation stage, when hypomethylation of KvDMR1 occurs, and aberrant methylation of DMRs affects imprinted gene expression. Cis-acting variations of the DMRs are not involved in the multiple methylation defects.Genet Med16 12, 903–912. 相似文献
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Zbigniew Jab?onowski Edyta Reszka Jolanta Gromadzińska Wojciech W?sowicz Marek Sosnowski 《Archives of Medical Science》2011,7(3):512-516
Introduction
The aim of the study was to examine the frequency of methylation status in promoter regions of p16 and DAPK genes in patients with non-invasive bladder cancer.Material and methods
Forty-two patients (92.9% men, 73.8% smokers, 71.4% T1G1, 19.1% T1G2, 9.5% T1G3) and 36 healthy controls were studied. Isolation of genomic DNA from blood serum and methylation-specific PCR (MSP) were applied. Methylation status – methylated and unmethylated promoter regions of p16 and DAPK genes were analysed.Results
Seventeen out of 42 patients (40.5%) had the methylated p16 gene, while methylation of the DAPK gene was seen in 27 of 42 cases (64.3%). In 12 patients (28.6%) both analysed genes were methylated. A statistically significant (p = 0.046) higher frequency of DAPK gene methylation (71.4%) was observed in patients with lower grade (G1) bladder cancer.Conclusions
Detection of the aberrant hypermethylation of DAPK and p16 genes in blood DNA from non-invasive bladder cancer patients might offer an effective means for earlier auxiliary diagnosis of the malignancy. 相似文献7.
Suraj Joshi Aman Kumar Ujaoney Payel Ghosh Deepti D. Deobagkar Bhakti Basu 《Research in microbiology》2021,172(1):103789
DNA methylation is ubiquitously found in all three domains of life. This epigenetic modification on adenine or cytosine residues serves to regulate gene expression or to defend against invading DNA in bacteria. Here, we report the significance of N6-methyladenine (6mA) to epigenetic immunity in Deinococcus radiodurans. Putative protein encoded by DR_2267 ORF (Dam2DR) contributed 35% of genomic 6mA in D. radiodurans but did not influence gene expression or radiation resistance. Dam2DR was characterized to be a functional S-adenosyl methionine (SAM)-dependent N6-adenine DNA methyltransferase (MTase) but with no endonuclease activity. Adenine methylation from Dam2DR or Dam1DR (N6-adenine MTase encoded by DR_0643) improved DNA uptake during natural transformation. To the contrary, methylation from Escherichia coli N6-adenine MTase (DamEC that methylates adenine in GATC sequence) on donor plasmid drastically reduced DNA uptake in D. radiodurans, even in presence of Dam2DR or Dam1DR methylated adenines. With these results, we conclude that self-type N6-adenine methylation on donor DNA had a protective effect in absence of additional foreign methylation, a separate methylation-dependent Restriction Modification (R-M) system effectively identifies and limits uptake of G6mATC sequence containing donor DNA. This is the first report demonstrating presence of epigenetic immunity in D. radiodurans. 相似文献
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Intracellular replicating DNA molecules (g9CsC1 = 1.725 g/cm3) of herpes simplex virus type 1 (HSV-1) were found to be transiently methylated. Primary rabbit kidney cells infected with HSV-1 (KOS strain) were labeled with l-[methyl-3H]methionine at different times during the virus growth cycle. Viral DNA was deproteinized, separated from cellular DNA by centrifugation in CsCl density gradients, and treated with NaOH to avoid any RNA contamination. Analysis of this intracellular viral DNA indicated that it was maximally methylated during 4–9 hr postinfection and the methylation of viral DNA started to decrease during later stages of infection. DNA from mature virions was not found to be methylated. The methylated base was identified as 5-methylcytosine. Nicotinamide, a potent acceptor of methyl groups, effectively inhibited the production of infectious virus particles at a concentration of 50 mM. These results suggest that methylation of HSV-1 DNA during active viral DNA synthesis is a prerequisite for infectious virus production. 相似文献
10.
Werner syndrome (WS) mouse embryonic fibroblasts (MEFs) can spontaneously escape from senescence and become immortalized, either tumorigenic or non-tumorigenic. Our data revealed a single p53N236S point mutation in the tumorigenic cell lines, which was correlated with the down-regulation of p21Waf1/Cip1. p16Ink4a expression was significantly decreased in all immortalized cell lines.Bisulfate sequencing indicated that the p16Ink4a gene was methylated in the tumorigenic cells. Exogenous overexpression of p21Waf1/Cip1 demethylated p16Ink4a and restored its expression, which induced cell growth arrest and senescence. While in non-tumorigenic immortalized cells, the Ink4a loci and adjacent genomic DNA were found to be deleted.These data suggest that the loss of p16Ink4a function by either genomic DNA deletion or methylation have been adopted by senescent WS MEFs escaping from senescence, with distinct tumorigenic consequences. The fact that cells that had escaped senescence via the spontaneous biallelic deletion of the Ink4a loci could not form tumors suggests that the functional loss of p16Ink4aper se might not be sufficient for tumorigenesis; most likely, it is a byproduct and passenger mutation. The mutations in factors regulating p16Ink4a methylation might be the driver mutation. These findings shed light on the strategy of anti-aging by regulating p16Ink4a expression. 相似文献
11.
DNA methylation, which most commonly occurs at the C5 position of cytosines within CpG dinucleotides, is one of several epigenetic mechanisms that cells use to control gene expression. The importance of DNA methylation in a variety of biological processes (i.e., embryonic development, cellular proliferation and differentiation, chromosome stability) has led to a demand for a precise and efficient method to determine the exact DNA methylation status. Bisulfite genomic sequencing is regarded as a gold-standard technology for detection of DNA methylation as it provides a qualitative, quantitative and efficient approach to identify 5-methylcytosine at single base-pair resolution. To optimize the final results of the bisulfite genomic sequencing protocol, numerous modifications have been explored and have significantly improved the sensitivity and accuracy of this procedure. The aim of this methodological report is to give an overview of the bisulfite genomic sequencing protocol, discussing the critical methodological aspects. Since we are interested in studying the methylation status of specific genes involved in T cell development, we applied the bisulfite genomic sequencing to the study of the CD8A T cell co-receptor gene to determine whether the CGIs of this gene were subjected to methylation in different types of tissues. The results show that CD8A gene is differentially methylated depending on the tissue. In conclusion, we described a bisulfite genomic sequencing protocol that can be successfully used for the quantitative analysis of CpG island methylation of specific genes. 相似文献
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Sneha Arya M.A. Majaid S.D. Shwetha K. Sravani A. Arivazhagan S. Sampath V. Santosh 《Pathology, research and practice》2014
Background
Very little literature exists on frequency of MGMT methylation status in pituitary adenoma. We assessed the frequency of MGMT methylation and protein expression in pituitary adenoma and correlated with patients’ age group and prognosis.Methods
Tumor tissues from 30 patients with pituitary adenoma were evaluated for MGMT promoter methylation status by methylation specific PCR method. All tumors were also immunostained with MIB-1, anti-p53 and anti-MGMT antibodies.Results
MGMT methylation status was noted in 40% of cases (7/20 non-functioning adenomas and 5/10 functioning adenomas). MGMT protein expression on IHC was noted in 72.2% of unmethylated tumors, while only 41.6% of methylated tumors demonstrated protein expression. The mean labeling index of MGMT protein was higher in unmethylated tumors as compared to the methylated group, though the difference was not statistically significant (18.6% vs 8.8%; p = 0.131). Tumor regrowth occurred in four unmethylated tumors as compared to none in methylated group. Even though there was no correlation between patient age and MGMT methylation status (p = 0.823), we noted that the frequency of methylation in middle age patients (between 30 and 59 yrs) was 64.7%, which was higher compared to other age groups.Conclusion
This is the first study from India showing MGMT promoter methylation status with protein expression in pituitary adenoma. We noted that tumor regrowth was higher in unmethylated tumors. 相似文献14.
K. Koike Z. X. Zhang Y. Sakamoto A. Miyake M. Inoue 《American journal of reproductive immunology (New York, N.Y. : 1989)》1998,39(3):217-222
PROBLEM: Cytokine-induced neutrophil chemoattractant (CINC) was reported to influence anterior pituitary hormone release. We recently found that Unkei-to, one of the Japanese Kampo medicines, stimulated CINC secretion by rat anterior pituitary cells and the pituitary folliculo-stellate (FS)-like cell line (TtT/GF). Therefore, the effect of Unkei-to on growth hormone (GH) secretion by rat anterior pituitary cells was investigated. METHOD OF STUDY: Dispersed normal anterior pituitary cells, the folliculo-stellate-like cell line TtT/GF, and the GH3 cell were used to test the effect of Unkei-to on GH secretion. In reconstitutive coculture experiments, TtT/GF cells were mixed with GH3 cells at a ratio (TtT/GF cells: GH3 cells) of 1:99. From this mixture, cells were seeded onto plates at a density of 104 cells/well and were cultured for 5 days. The cells were then used in the experiments. RESULTS: Unkei-to at 20 μg/ml significantly inhibited GH secretion by normal anterior ptuitary cells within 12 hr of incubation. In contrast Unkei-to stimulated GH secretion by GH3 cells in a time- and dose-dependent manner, suggesting that an accessory cell type was involved. To assess the contribution of CINC as a paracrine factor, an experiment using a reconstitutive coculture system was performed, and Unkei-to was found to inhibit GH secretion when GH3 cells were cocultured with TtT/GF cells. The addition of anti-CINC antibody to the reconstitutive coculture system antagonized Unkei-to-inhibited GH secretion. CONCLUSION: CINC, which was secreted from FS cells by Unkei-to, may be responsible for mediating the inhibitory effect of Unkei-to on GH secretion by rat anterior pituitary cells. 相似文献
15.
Bradford Coffee Kasinathan Muralidharan William E Highsmith Pablo Lapunzina Stephen T Warren 《Genetics in medicine》2006,8(10):628-634
PURPOSE: Beckwith-Wiedemann Syndrome is caused by defects in imprinted gene expression at 11p15. Currently, quantitative Southern analysis using DNA methylation-sensitive restriction enzymes is used in molecular diagnosis of this syndrome. METHODS: We describe a rapid and highly quantitative test for assessing DNA methylation at 11p15 using sodium bisulfite treatment of genomic DNA coupled with quantitative TaqMan methylation-sensitive polymerase chain reaction. RESULTS: TaqMan MSP can assess DNA methylation at both differentially methylated region (DMR)1 and DMR2 at 11p15. In addition, by using TaqMan MSP we were able to determine the parent of origin of a duplication of 11p15 by quantification of both DMR1 and DMR2 DNA methylation. CONCLUSION: TaqMan MSP method is a robust and rapid method for detecting changes in DNA methylation that compares favorably to the current standard of Southern blot for DNA methylation analysis. Assessment of DMR1 and DMR2 provides the most comprehensive assay for methylation defects in Beckwith Wiedemann Syndrome, accounting for more than 70% of the cases. The advantages of TaqMan MSP are that it requires less DNA and that it is rapid, less labor-intensive, and amenable to high-throughput analysis. Moreover, this approach can be modified to assess DNA methylation changes anywhere in the genome. 相似文献
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Rauch T Pfeifer GP 《Laboratory investigation; a journal of technical methods and pathology》2005,85(9):1172-1180
Hypermethylation of CpG islands is a phenomenon commonly observed during the development and progression of human tumors. Detection of methylated-CpG islands in easily accessible biological materials such as serum has the potential to be useful for the early diagnosis of cancer. Most currently used methods for detecting methylated-CpG islands are based on sodium bisulfite conversion of genomic DNA, followed by PCR reactions. Here we describe a method, methylated-CpG island recovery assay (MIRA) that does not depend on the use of sodium bisulfite but has similar sensitivity and specificity as bisulfite-based approaches. Methyl-CpG-binding domain proteins, such as methyl-CpG-binding domain protein-2 (MBD2), have the capacity to bind specifically to methylated DNA sequences. In the MIRA procedure, sonicated genomic DNA isolated from cells or tissue is incubated with a matrix containing glutathione-S-transferase-MBD2b in the presence of methyl-CpG-binding domain protein 3-like-1, a binding partner of MBD2 that increases the affinity of MBD2 for methylated DNA. Specifically bound DNA is eluted from the matrix and gene-specific PCR reactions are performed to detect CpG island methylation. Methylation can be detected using 1 ng of DNA or 3000 cells. MIRA is a specific and sensitive, but not laborious, technique that can be clinically useful in the detection and diagnosis of any DNA methylation-associated disease, including cancer. 相似文献
19.
Luis?Carretero Francisco?Barros Pablo?Miranda Jorge?Fernández-Trillo Angeles?Machín Pilar?de la?Pe?a Pedro?Domínguez
While the thyrotropin-releasing hormone (TRH) effect of raising intracellular Ca2+ levels has been shown to rely on Gq/11 and PLC activation, the molecular mechanisms involved in the regulation of ERG K+ channels by TRH are still partially unknown. We have analysed the effects of βγ scavengers, Akt/PKB inactivation, and TRH
receptor (TRH-R) overexpression on such regulation in native and heterologous expression cell systems. In native rat pituitary
GH3 cells β-ARK/CT, Gαt, and phosducin significantly reduced TRH inhibition of rERG currents, whereas in HEK-H36/T1 cells permanently expressing
TRH-R and hERG, neither of the βγ scavengers affected the TRH-induced shift in V
1/2. Use of specific siRNAs to knock Akt/PKB expression down abolished the TRH effect on HEK-H36/T1 cell hERG, but not on rERG
from GH3 cells. Indeed, wortmannin or long insulin pretreatment also blocked TRH regulation of ERG currents in HEK-H36/T1 but not
in GH3 cells. To determine whether these differences could be related to the amount of TRH-Rs in the cell, we studied the TRH concentration
dependence of the Ca2+ and ERG responses in GH3 cells overexpressing the receptors. The data indicated that independent of the receptor number additional cellular factor(s)
contribute differently to couple the TRH-R to hERG channel modulation in HEK-H36/T1 cells. We conclude that regulation of
ERG currents by TRH and its receptor is transduced in GH3 and HEK-H36/T1 cell systems through common and different elements, and hence that the cell type influences the signalling
pathways involved in the TRH-evoked responses. 相似文献
20.
Disruption of the gene for the cyclin dependent kinase inhibitor (CDKI) p27/kip1 results in pituitary corticotroph hyperplasia while diminished expression of this protein has been described in aggressive human pituitary tumors. We have previously shown that 1,25-vitamin D3 (VD) hypophosphorylates p27 and interferes with the degradation of this CDKI in thyroid carcinoma cells. In this study we investigated whether VD/EB1089 can induce p27 accumulation and cause growth arrest of pituitary corticotroph cells. VD and EB1089 exhibited a significant reduction in AtT20 corticotroph but not PRL235 lactotroph cell growth. These changes were accompanied by selective accumulation of p27 in AtT20 but not in PRL235 cells. As p27 levels are highly dependent on protein degradation, we examined the effect of VD/EB1089 on p27 association with factors that target this CDKI to the proteasome. VD/EB1089 significantly restricted the association of p27 with Skp2 as well as with cyclin dependent kinase 2 (CDK2). As the tumor suppressor and phosphatase PTEN has been implicated in p27 regulation, we tested whether the effects of VD/EB1089 on p27 accumulation in corticotrophs could be mediated through this pathway. VD/EB1089 did not appreciably alter PTEN expression. Moreover, transfection of PTEN did not influence the effect of VD on p27 accumulation in corticotrophs. We conclude that VD/EB1089 can selectively arrest pituitary corticotroph growth and induce p27 accumulation.This effect is mediated at least partially through diminished p27 association with Skp2 and with CDK2. In contrast to other cell systems, PTEN does not participate in the regulation of corticotroph p27 and is not involved in mediating the effect of VD on p27 in these cells. Our findings highlight p27 and VD analogs as targets for manipulation and drug development respectively in the treatment of inoperable corticotroph adenomas. 相似文献