With the exception of ApoE4, genome-wide association studies have failed to identify strong genetic risk factors for late-onset Alzheimer's disease, despite strong evidence of heritability, suggesting that many low penetrance genes may be involved. Additionally, the nature of the identified genetic risk factors and their relation to disease pathology is also largely obscure. Previous studies have found that a cancer-associated variant of the cell cycle inhibitor gene p21cip1 is associated with increased risk of Alzheimer's disease. The aim of this study was to confirm this association and to elucidate the effects of the variant on protein function and Alzheimer-type pathology. We examined the association of the p21cip1 variant with Alzheimer's disease and Parkinson's disease with dementia. The genotyping studies were performed on 719 participants of the Oxford Project to Investigate Memory and Ageing, 225 participants of a Parkinson's disease DNA bank, and 477 participants of the Human Random Control collection available from the European Collection of Cell Cultures. The post mortem studies were carried out on 190 participants. In the in-vitro study, human embryonic kidney cells were transfected with either the common or rare p21cip1 variant; and cytometry was used to assess cell cycle kinetics, p21cip1 protein expression and sub-cellular localisation. The variant was associated with an increased risk of Alzheimer's disease, and Parkinson's disease with dementia, relative to age matched controls. Furthermore, the variant was associated with an earlier age of onset of Alzheimer's disease, and a more severe phenotype, with a primary influence on the accumulation of tangle pathology. In the in-vitro study, we found that the SNPs reduced the cell cycle inhibitory and anti-apoptotic activity of p21cip1. The results suggest that the cancer-associated variant of p21cip1 may contribute to the loss of cell cycle control in neurons that may lead to Alzheimer-type neurodegeneration.
Birt–Hogg–Dubé (BHD) syndrome, is a dominantly inherited familial cancer syndrome associated with susceptibility to renal cell carcinoma (RCC) caused by inactivating mutations in the folliculin (FLCN) gene. The precise functions of the FLCN gene product are still under investigation but RCC from BHD patients show loss of the wild-type allele consistent with a tumor suppressor gene function. In a search for potential synthetic-lethal targets for FLCN using a phosphatase siRNA library screening approach, we found that knockdown of SSH2 serine phosphatase (one of the three members of Slingshot family and previously implicated in actin reorganization) specifically induced Caspase3/7 activity in a dose-dependent manner (up to six-fold increase, 10 nM, 72 h) in two human FLCN-deficient cell lines (BHD-origin renal cell carcinoma UOK257 and thyroid carcinoma FTC133) but not in their folliculin expressing isogenic cell lines. SSH2 siRNA-induced knockdown was accompanied by increased expression of SSH1 and SSH3 (suggesting a compensatory regulatory mechanism among members of SSH family). FLCN-null cells exhibited evidence of dysregulated cofilin de/phosphorylation pathways. Knockdown of SSH2 in FLCN-null cells was associated with an alteration in cell cycle kinetics (20% increase in G1, 30% and 40% decrease in S and G2M, respectively). Combination treatment of multiple SSH family (SSH2 plus SSH1 and/or SSH3) siRNAs potentiated induction of Caspase3/7 activity and changes in the cell cycle kinetics. These data indicate that: (a) apoptotic cell death in FLCN-null cells can be triggered by SSH2 knockdown through cell cycle arrest; (b) SSH2 represents a potential therapeutic target for the development of agents for the treatment of BHD syndrome and, possibly, related tumors.
Considerable interest lies in the identification of novel anti-angiogenic compounds for cancer therapy. We have investigated whether dexrazoxane has anti-angiogenic properties and if so, the mechanism of the inhibition. The phenotypic effects of dexrazoxane on endothelial cell behaviour was investigated both in vitro using human umbilical vein endothelial cells (HUVECs) in cell proliferation, migration, cell cycle and aortic ring assays; and in vivo using the mouse angiogenesis subcutaneous sponge assay. Custom angiogenesis pathway microarrays were used to identify differentially expressed genes in endothelial cells after treatment with dexrazoxane vs a control. The differentially expressed genes were validated using real-time RT–PCR and western blotting; and the functional effect of one induced gene was confirmed using siRNA technology. Treatment of endothelial cells with dexrazoxane resulted in a dose–response inhibition of cell growth lasting for up to 5 days after a single dose of the drug. Dexrazoxane was inhibitory in the aortic ring tube forming assay and strongly anti-angiogenic in vivo in the rodent subcutaneous sponge model. The anti-angiogenic effect in the sponge was seen after systemic injection into the tail vein as well as after direct injection of dexrazoxane into the sponge. Treatment of microvascular endothelial cells in vitro with subtoxic doses of dexrazoxane stimulated thrombospondin-1 (THBS-1) secretion. Knockdown of THBS-1 with siRNA removed the angiogenesis inhibition effect of dexrazoxane, which is consistent with the anti-angiogenic and vascular normalising properties of the drug being principally mediated by THBS-1. We show that dexrazoxane administered in small repeated doses is strongly anti-angiogenic and that this activity is mediated by induction of the anti-angiogenic THBS-1 in endothelial cells.
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