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ABSTRACT: The aim of this study was to compare gene silencing in bovine zygotes when small interfering RNAs (siRNAs) were introduced into bovine zygotes by microinjection or lipid-based transfection. In Experiment 1, E-cadherin siRNA was injected at 100 or 375 µM and compared with PBS-injected and non-injected controls. Embryos were then cultured in vitro for 7 days and periodically assessed for development. For transfection, zona-free zygotes were incubated in transfection medium with siRNA for 1h at 39°C and then cultured to Day 7. Injection of PBS or 375 µM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5% and 45.5%) or the blastocyst stage (39.0 and 32.5%) compared with non-injected controls (62.9 and 45.0%, respectively; P<0.05). Messenger RNA abundance was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 and 375 µM, respectively, compared with controls (P<0.05). Transfection with 100 nM E-cadherin siRNA decreased development to the 8-cell stage (20.3 versus 53.0%) and blastocyst stage (7.2 versus 18.2%) compared with controls (P<0.05). Messenger RNA relative abundance was not different between controls (non-transfected or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100 and 200 nM E-cadherin siRNA led to a 72 and 38% reduction, respectively, in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P<0.05).
Reproduction Fertility and Development 05/2011; 23(4):534-43. · 2.11 Impact Factor
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Jenny A Watson,
Chris J Watson,
Ann-Maria McCrohan,
Kathryn Woodfine,
Miriam Tosetto,
Jennifer McDaid, Emma Gallagher,
David Betts,
John Baugh,
Jacintha O'Sullivan,
Adele Murrell,
R William G Watson,
Amanda McCann
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ABSTRACT: Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.
Human Molecular Genetics 08/2009; 18(19):3594-604. · 7.64 Impact Factor
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Orla Mc Cormack,
Wen Y Chung,
Patricia Fitzpatrick,
Fiachra Cooke,
Barbara Flynn,
Michele Harrison,
Edward Fox, Emma Gallagher,
Aloysius McGoldrick,
Peter A Dervan,
Amanda McCann,
Michael J Kerin
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ABSTRACT: Oestrogen receptor alpha (ER alpha) is traditionally measured on all breast tumour specimens to identify those patients more likely to respond to anti-oestrogens. Progesterone receptor (PR) status has contributed useful information in defining more responsive subgroups. PR negativity may be a marker for increased signalling through growth factor receptor tyrosine kinase pathways. Progesterone acts through two PRs, PRA and PRB. PRB, the functionally active PR, can be silenced by promoter hypermethylation.
Following DNA and RNA extraction from 94 breast carcinomas, the methylation status of the PRB promoter was assessed by sodium bisulphite modification and methylation sensitive PCR (MSP). A quantitative realtime PCR analysis (QRTPCR) was used to determine the levels of PRB mRNA expression. Protein expression was evaluated immunohistochemically with a commercially available PRB antibody.
76% of the primary breast carcinoma samples demonstrated a methylated band for PRB. PRB methylation significantly compromised total PR immunohistochemistry (IHC) expression (P = 0.03). PRB mRNA correlated positively with total PR IHC (r = 0.58, P = 0.04), ER alpha IHC (P = 0.02), and tumour grade (P = 0.01). PRB protein expression was significantly associated with a number of favourable prognostic variables including smaller (P = 0.004) lower grade (P = 0.007), ER alpha IHC positive tumours (P < 0.001), and tumours with a low Nottingham Prognostic Index (NPI) (P = 0.0008). PRB mRNA levels were significantly associated with better overall survival (P = 0.04) in a univariate analysis.
The majority of tumours were methylated for PRB. This did not directly compromise PRB expression suggesting that other factors may down regulate the PR gene. When PRB was expressed, it correlated with good prognostic markers and better overall survival.
Breast Cancer Research and Treatment 09/2008; 111(1):45-53. · 4.43 Impact Factor
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Maria Meehan,
Audrey Melvin, Emma Gallagher,
James Smith,
Alo McGoldrick,
Catherine Moss,
Steven Goossens,
Michèle Harrison,
Elaine Kay,
John Fitzpatrick,
Peter Dervan,
Amanda Mc Cann
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ABSTRACT: CTNNA3 (alpha-T-catenin) is imprinted with preferential monoallelic expression of the maternal allele in placental tissue. The allelic expression pattern of CTNNA3 in adult human cancer is unknown and warrants investigation as CTNNA3 stabilizes cellular adherence, a feature which if compromised could enable cells to acquire an increased capability to detach and invade. We document the frequency of monoallelic versus biallelic expression of CTNNA3 in urothelial carcinoma of the bladder (UCB) samples and compare the observed patterns with that found in the paired normal sample. DNA PCR reactions encompassing a transcribable SNP polymorphism within exon 12 of CTNNA3 were sequence analyzed to identify heterozygous cases. A total of 96 samples were analyzed and included 22 paired normal and tumor UCB cases, 38 formalin fixed paraffin embedded (FFPE) UCB samples consisting of 18 noninvasive pTa tumors and 20 lamina propria invasive pT1 tumors and 14 cell lines of various lineages. RT-PCR analysis of 35 heterozygous samples followed by sequence analysis allowed monoallelic versus biallelic patterns to be assigned. We have provided the first demonstration that CTNNA3 displays differing allelic expression patterns in UCB. Specifically, 35% (7/20) of informative UCB, showed monoallelic expression, a feature confined to the tumor, with normal urothelial samples displaying biallelic expression. Real time RT-PCR analyses, demonstrated a significantly lower (P = 0.00039) level of CTNNA3 in the tumor samples compared with the paired normals, all of which displayed biallelic expression. In conclusion, monoallelic and biallelic CTNNA3 expression patterns are demonstrable in tumor bladder tissue, whereas normal cases show only biallelic expression.
Genes Chromosomes and Cancer 07/2007; 46(6):587-93. · 3.31 Impact Factor
