Hutchinson-Gilford progeria syndrome (HGPS) is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site and yields a truncated form of prelamin A called progerin. Small amounts of progerin are also produced during normal aging. Studies with mouse models of HGPS have allowed the recent development of the first therapeutic approaches for this disease. However, none of these earlier works have addressed the aberrant and pathogenic LMNA splicing observed in HGPS patients because of the lack of an appropriate mouse model. Here, we report a genetically modified mouse strain that carries the HGPS mutation. These mice accumulate progerin, present histological and transcriptional alterations characteristic of progeroid models, and phenocopy the main clinical manifestations of human HGPS, including shortened life span and bone and cardiovascular aberrations. Using this animal model, we have developed an antisense morpholino-based therapy that prevents the pathogenic Lmna splicing, markedly reducing the accumulation of progerin and its associated nuclear defects. Treatment of mutant mice with these morpholinos led to a marked amelioration of their progeroid phenotype and substantially extended their life span, supporting the effectiveness of antisense oligonucleotide-based therapies for treating human diseases of accelerated aging.

The identification of novel approaches to specifically target the DNA-damage checkpoint response in chemotherapy-resistant cancer stem cells (CSC) of solid tumors has recently attracted great interest. We show here in colon cancer cell lines and primary colon cancer cells that inhibition of checkpoint-modulating phosphoinositide 3-kinaserelated (PIK) kinases preferentially depletes the chemoresistant and exclusively tumorigenic CD133 1 cell fraction. We observed a time-and dose-dependent disproportionally pronounced loss of CD133 1 cells and the consecutive lack of in vitro and in vivo tumorigenicity of the remaining cells. Depletion of CD133 1 cells was initiated through apoptosis of cycling CD133 1 cells and further substantiated through subsequent recruitment of quiescent CD133 1 cells into the cell cycle followed by their elimination. Models using specific PIK kinase inhibitors, somatic cell gene targeting, and RNA interference demonstrated that the observed detrimental effects of caffeine on CSC were attributable specifically to the inhibition of the PIK kinase ataxia telangiectasia-and Rad3-related (ATR). Mechanistically, phosphorylation of CHK1 checkpoint homolog (S. pombe; CHK1) was significantly enhanced in CD133 1 as compared with CD133 2 cells on treatment with DNA interstrandcrosslinking (ICL) agents, indicating a preferential activation of the ATR/CHK1-dependent DNA-damage response in tumorigenic CD133 1 cells. Consistently, the chemoresistance of CD133 1 cells toward DNA ICL agents was overcome through inhibition of ATR/CHK1-signaling. In conclusion, our study illustrates a novel target to eliminate the tumorigenic CD133 1 cell population in colon cancer and provides another rationale for the development of specific ATR-inhibitors. STEM CELLS 2011;29:418-429 Disclosure of potential conflicts of interest is found at the end of this article.

Changes in gene expression are produced in cells as a consequence of virus infections. In the present work, we used proteomic technology to globally examine African swine fever virus (ASFV)-infected Vero cells searching for infection-associated proteins in order to determine target proteins for pathogenesis studies. We studied the alterations in cellular protein profile after ASFV infection by two-dimensional electrophoresis, identifying the modified cellular proteins by matrix-assisted laser desorption/ionization peptide mass fingerprinting. A total of twelve different over-expressed cellular proteins were unambiguously identified. The most significant changes were in redox-related proteins, nucleoside diphosphate kinases, heat shock proteins, members of the Ran-Gppnhp-Ranbd1 complex and apolipoproteins. These cellular protein modifications could represent distinct roles during infection related to apoptosis and transcriptional modulation mechanisms. The present study constitutes the first attempt to understand the dynamics of ASFV-host cell interactions by proteomics.

The breast cancer metastasis suppressor 1 (BRMS1) gene has been shown to suppress metastasis without affecting the growth of the primary tumor in mouse models. It has also been shown to suppress the metastasis of tumors derived from breast, melanoma, and, more recently, ovarian carcinoma (see ref 1). However, how BRMS1 exerts its metastasis suppressor function remains unknown. To shed light into its metastatic mechanism of action, the sensitive 2D-DIGE analysis coupled with MS has been used to identify proteins differentially expressed by either overexpressing (Mel-BRMS1) or silencing BRMS1 (sh635) in a melanoma cell line. After comparison of the protein profiles from WT, Mel-BRMS1, and sh635 cells, 79 spots were found to be differentially expressed. Mass spectrometry analysis allowed the unambiguous identification of 55 polypeptides, corresponding to 43 different proteins. Interestingly, more than 75% of the identified proteins were down-regulated in Mel-BRMS1 cells compared to WT. In contrast, all the identified proteins in sh635 cells extracts were up-regulated compared to WT. Most of the deregulated proteins are involved in cell growth/maintenance and signal transduction among other cell processes. Six differentially expressed proteins (Hsp27, Alpha1 protease inhibitor, Cofilin1, Cathepsin D, Bone morphogenetic protein receptor2, and Annexin2) were confirmed by immunoblot and functional assays. Excellent correlation was found between DIGE analysis and immunoblot results, indicating the reliability of the analysis. Available evidence on the reported functions of the identified proteins supports the emerging role of BRMS1 as negative regulator of the metastasis development. This work opens an avenue for the molecular mechanisms' characterization of metastasis suppressor genes with the aim to understand their roles.

A large body of evidence supports the hypothesis that proteasomal degradation of the growth suppressor p27(Kip1) (p27) facilitates mammalian cell cycle progression. However, very few studies have addressed the possibility of proteasome-independent mechanisms of p27 proteolysis. Here we provide evidence for a novel pathway of p27 degradation via the lysosome that is mediated by its interaction with the endosomal protein sorting nexin 6 (SNX6), a member of the sorting nexin family of vesicular trafficking regulators. p27 and SNX6 interact in vitro and in vivo in mammalian cells, partially colocalize in endosomes, and are present in purified endosomal fractions. Gain- and loss-of-function studies revealed that SNX6 induces endosomal accumulation of p27. Moreover, p27 is detected in lysosomes and inhibition of lysosome-dependent proteolysis impairs serum-mediated down-regulation of p27 in a SNX6-dependent manner. To validate the localization of p27 in these organelles, we analyzed several cell lines using two different anti-p27 antibodies, several organelle-specific markers [e.g., early endosome antigen 1, lysosomal-associated membrane protein (LAMP) 1, LAMP2, and LysoTracker], and overexpression of fluorescent p27 and SNX6. Remarkably, silencing of SNX6 attenuates p27 down-regulation in the G(1) phase of the mitotic cell cycle and delays cell cycle progression. We therefore conclude that, in addition to the proteasome-dependent pathway, SNX6-mediated endolysosomal degradation of p27 also contributes to cell cycle progression in mammalian cells.