Inactivation of the Caenorhabditis elegans gene clk-1, which is required for ubiquinone biosynthesis, increases lifespan by an insulin signaling-independent mechanism. We find that homozygous inactivation of mclk1, the mouse ortholog of clk-1, yields ES cells that are protected from oxidative stress and damage to DNA. Moreover, in the livers of old mclk1 +/− mice, hepatocytes that have lost mclk1 expression by loss of heterozygosity undergo clonal expansion, suggesting that their resistance to stress allows them to outcompete cells that still express the gene. mclk1 +/− mice, whose growth and fertility are normal, also display a substantial increase in lifespan in each of three different genetic backgrounds. These observations indicate that the distinct mechanism by which clk-1/mclk1 affects lifespan is evolutionarily conserved from nematodes to mammals and is not tied to a particular anatomy or physiology.[Keywords: clk-1; mclk1; aging; loss of heterozygosity; reactive oxygen species; ubiquinone] Supplemental material is available at http://www.genesdev.org.

Maspin, a novel serine protease inhibitor (serpin), suppresses the growth and metastasis of breast tumor in vivo. However, the underlying molecular mechanism is unclear. In the current study, we report the ®rst evidence that endogenous maspin expression in mammary carcinoma cells MDA-MB-435 enhanced staurosporine (STS)-induced apoptosis as judged by the increased fragmentation of DNA, increased proteolytic inactivation of poly-[ADP-ribose]-polymerase (PARP), as well as the increased activation of caspase-8 and caspase-3. In parallel, recombinant maspin did not directly regulate the proteolytic activities of either caspase-3 or caspase-8 in vitro. Consistent with this result, maspin expressing normal mammary epithelial cells underwent more rapid STS-induced apoptosis as compared to breast carcinoma cells. Interestingly, maspin transfectant cells did not undergo spontaneous apoptosis in the absence of STS. Moreover, neither puri®ed maspin protein added from outside nor endogenous maspin secreted to the cell culture media sensitized cells to STS-induced apoptosis. To investigate the structural determinants of maspin in its apoptosis-sensitizing e ect, MDA-MB-435 cells were also transfected with maspin/PAI-1 and PAI-1/maspin chimeric constructs resulting from swapping the Nterminal and the C-terminal domains between maspin and PAI-1 (plasminogen activator inhibitor type 1). The resulting stable transfectant clones expressing maspin/ PAI-1 and PAI-1/maspin, respectively, did not undergo spontaneous apoptosis, and were similarly inhibited as maspin transfectant cells in motility assay. Interestingly, however, expression of both maspin/PAI-1 and PAI-1/ maspin in MDA-MB-435 cells failed to sensitize these cells to STS-induced apoptosis. Taken together, our evidence provides new insights into the complex molecular mechanisms of maspin that may suppress breast tumor progression not only at the step of invasion and motility, but also by regulating tumor cell apoptosis. The sensitizing e ect of maspin on apoptosis is to be contrasted by the pro-survival e ect of several other serpins.

After apoptosis or necrosis, macrophages clear dead cells by phagocytosis. Although this process is efficient, circulating nucleosomes can occur in certain diseases, presumably reflecting either increased production or impaired clearance. To investigate the generation of blood nucleosomes, graded numbers of apoptotic and necrotic cells were administered to healthy mice, and levels of blood nucleosomes and DNA were determined. Using Jurkat cells as a model, nucleosomes and DNA were detected in the blood after the administration of 10 8 apoptotic or necrotic cells per mouse by the intraperitoneal route. The kinetics of the response were similar for both types of cells. The role of macrophages was assessed by eliminating these cells with clodronate liposomes or silica. Although clodronate treatment alone produced a peak level of blood DNA, the subsequent administration of dead cells caused no change in DNA levels. In contrast, silica treatment alone did not elicit a blood DNA response, though this treatment limited the rise in DNA from administered cells. Molecular studies showed that the blood DNA following the administration of apoptotic or necrotic cells arose from the mouse and the Jurkat cells, and its size distribution was consistent with apoptosis. Together, these findings suggest that the generation of blood nucleosomes depends on macrophages, with apoptosis a concomitant of a high burden of dead and dying cells. IntroductionCell death is integral to the biology of the mammalian organism and varies in form and mechanism. Two types of cell death have been broadly defined. Apoptotic or programmed cell death is a regulated and orderly process mediated by a cascade of enzymes that rearrange and degrade cell constituents. Apoptosis occurs in physiologic and pathologic settings and can be triggered by a variety of stimuli that initiate a stereotyped death program. In contrast, necrotic or accidental cell death results from cell injury and is frequently a concomitant of inflammation, whether induced by physical or chemical means. [1][2][3][4] Death by necrosis does not involve the regulated action of enzymes and leads to cellular changes that differ in phenotype and structure from apoptotic death. Although this dichotomy may be an oversimplification, 5,6 it is nevertheless useful in assessing the impact of dead and dying cells on the organism.In addition to their structure and phenotype, apoptotic cells and necrotic cells may differ in their interaction with the immune system. As shown in in vitro studies, apoptotic cells are rapidly engulfed by mononuclear phagocytes that bind an array of cell surface molecules, including phosphatidylserine. Phosphatidylserine appears on the surface of the apoptotic cell with the loss of membrane symmetry. [7][8][9][10][11][12] In addition to signaling uptake by phagocytosis, phosphatidylserine binding may induce an antiinflammatory state because of the production of mediators such as transforming growth factor-␤ (TGF-␤). [13][14][15] Like apoptotic cells, necrotic cells are eng...

Several parameters may predict disease severity and overall survival in chronic lymphocytic leukemia (CLL). The purpose of our study of 190 CLL patients was to compare immunoglobulin heavy chain variable region (IgV H ) mutation status, cytogenetic abnormalities, and leukemia cell CD38 and Zap-70 to older, traditional parameters. We also wanted to construct a simple, inexpensive prognosis score that would significantly predict TTT and survival in patients at the time of diagnosis and help practicing clinicians. In univariate analyses, patients with higher clinical stage, higher leukocyte count at diagnosis, shorter leukocyte doubling time, elevated serum lactate dehydrogenase (LDH), unmutated immunoglobulin heavy chain variable region (IgV H ) genes, and higher CD38 had a shorter overall survival and time-to-treatment (TTT). CLL cell Zap-70 expression was higher in patients with unmutated IgV H , and those with higher Zap-70 tended to have shorter survival. IgV H 4-34 or IgV H 1-69 was the most common IgV H genes used (16 and 12%, respectively). Of those with IgV H 1-69, 86% had unmutated IgV H and had a significantly shorter TTT. A cytogenetic abnormality was noted in 71% of the patients tested. Patients with 11q22 del and 17p13 del or complex abnormalities were significantly more likely to have unmutated IgV H . We found that a prognostic score constructed using modified Rai stage, cellular CD38, and serum LDH (parameters easily obtained clinically) significantly predicted TTT and survival in patients at the time of diagnosis and performed as well or better than models using the newer markers. Am.

The EGFR monoclonal antibody cetuximab is the only approved targeted agent for treating head and neck squamous cell carcinoma (HNSCC). Yet resistance to cetuximab has hindered its activity in this disease. Intrinsic or compensatory HER3 signaling may contribute to cetuximab resistance. To investigate the therapeutic benefit of combining MM-121/SAR256212, an anti-HER3 monoclonal antibody, with cetuximab in HNSCC, we initially screened twelve HNSCC cell lines for total and phosphorylated levels of the four HER receptors. We also investigated the combination of MM-121 with cetuximab in preclinical models of HNSCC. Our results revealed that HER3 is widely expressed and activated in HNSCC cell lines. MM-121 strongly inhibited phosphorylation of HER3 and AKT. When combined with cetuximab, MM-121 exerted a more potent anti-tumor activity through simultaneously inhibiting the activation of HER3 and EGFR and consequently the downstream PI3K/AKT and ERK pathways in vitro. Both high and low doses of MM-121 in combination with cetuximab significantly suppressed tumor growth in xenograft models and inhibited activations of HER3, EGFR, AKT and ERK in vivo. Our current work is the first report on this new combination in HNSCC and supports the concept that HER3 inhibition may play an important role in future therapy of HNSCC. Our results open the door for further mechanistic studies to better understand the role of HER3 in resistance to EGFR inhibitors in HNSCC.

HER3 (ErbB3) is a unique member of the human epidermal growth factor receptor (EGFR) family (ErbB family). It functions only through dimerization with other members of the ErbB family and modulates activity and sensitivity to targeted cancer therapies. This paper briefly describes the mechanism of HER3 in signal transduction and its potential role in acquired resistance to EGFR- and HER2-targeted therapies. We also consider recent developments in HER3-targeting therapeutics and their combination with inhibitors of other ErbB members in clinical applications.

This studay is to construct a lentiviral vector harbouring an RNA interference (RNAi) sequence that targets the gene encoding the human high-mobility group nucleosomal binding protein 1 (NSBP1); to study its role in inducing G2/M phase arrest and apoptosis in prostate cancer (PCa) DU145 cells; and to assess the effect of its knockdown on cell proliferation in vitro and in vivo. RNAi was applied to knock down NSBP1 expression in the PCa cell line DU145 by lentiviral plasmids producing an NSBP1 small hairpin RNA. After NSBP1 knockdown in DU145 cells, the growth rate of cells was analyzed by MTT, and G2/M cell cycle arrest and apoptosis were assessed using a FACScalibur flow cytometer. Tumour growth was assessed in nude mice. The mRNA and protein expression levels of NSBP1, cyclin B1 and Bcl-2 were analysed in vitro and in vivo by reverse-transcriptase polymerase chain reaction and Western blotting. Knockdown of NSBP1 resulted in a 22.6% decrease in the growth rate of cells compared with the PscNC lentivirus control cells at 96 h, decreased tumour growth in nude mice, and the induction of G2/M cell cycle arrest (8.78%) and apoptosis (2.19-fold). Consistent with the cell cycle arrest and apoptosis, the mRNA and protein expression levels of cyclin B1 and Bcl-2 were decreased. In conclusion, knockdown of NSBP1 causes a statistically significant inhibition of the in vitro and in vivo growth of the PCa cell line DU145. Growth suppression is at least partially due to NSBP1 knockdown-induced G2/M cell cycle arrest and apoptosis. The present data provide the evidence that the NSBP1 knockdown-induced G2/M phase arrest and apoptosis may result from negative regulation of cyclin B1 and Bcl-2 by NSBP1, with the resulting reduced expression of these proteins.

BACKGROUND:To understand the mechanism of frequent and early lymph node metastasis in high-risk human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC), this study investigated whether b-catenin is regulated by the HPV oncoprotein and contributes to OPSCC metastasis. METHODS: Expression levels of p16, b-catenin, and epidermal growth factor receptor (EGFR) were examined in OPSCC samples (n 5 208) by immunohistochemistry. The expression and subcellular localization of b-catenin and EGFR activation were also studied in HPV-positive and HPV-negative head and neck squamous cell carcinoma cell lines with western blot analysis. HPV16 E6 small interfering RNA was used to elucidate the effect of the HPV oncoprotein on bcatenin translocation. The involvement of EGFR in b-catenin translocation was confirmed by treatment with erlotinib. Moreover, the invasive capacity was evaluated after HPV16 E6/E7 repression. RESULTS: The results showed that the membrane weighted index of b-catenin was inversely correlated with p16 positivity (P <.001) and lymph node metastasis (P 5.026), whereas nuclear staining of bcatenin was associated with p16-positive OPSCC (P <.001). A low level of membrane b-catenin expression was significantly associated with disease-free and overall survival (P <.0001 in both cases). Furthermore, the membrane weighted index of EGFR was inversely correlated with p16 positivity (P <.001) and positively correlated with membrane b-catenin (P <.001). The in vitro study showed that HPV16 E6 repression led to reductions of phospho-EGFR and nuclear b-catenin, which were also observed after erlotinib treatment, and inhibition of invasion. CONCLUSIONS: The findings suggest that HPV16 E6 mediates the translocation of b-catenin to the nucleus, which may be regulated by activated EGFR. Cancer 2015;121:214-25.