Metabolic dysregulation and cancer risk program (MeDOC): a transdisciplinary approach to Obesity-Associated cancers.

With the escalating prevalence of obesity, the association between obesity and cancer is a growing public health concern. Obesity will soon surpass tobacco smoking as the most important preventable cause of cancer. Obesity-driven mechanisms can alter cell functions to induce metabolic changes, chronic inflammation, and insulin resistance that are believed to contribute to cancer risk and development; yet the specific underlying biological mechanisms of obesity-related cancer development are largely unknown. The Metabolic Dysregulation and Obesity Cancer Risk (MeDOC) Program is a trans-NCI research program supported by the Division of Cancer Control and Population Sciences, the Division of Cancer Biology, the Division of Cancer Prevention, and the Center to Reduce Cancer Health Disparities. The overall purpose of the MeDOC Program is to advance our understanding of the underlying mechanisms that connect obesity, metabolic dysregulation, and increased obesity cancer risk, as well as identify markers that will enhance cancer risk prediction, improve screening for high-risk individuals, and identify targets for preventive and therapeutic interventions for cancer interception or treatment. This report describes the funded research projects, the Coordinating Center, and the goals of the MeDOC Program.

Integrating sleep health into resilience research.

Sleep is a biological necessity that is a critical determinant of mental and physical well-being. Sleep may promote resilience by enhancing an individual's biological preparedness to resist, adapt and recover from a challenge or stressor. This report analyzes currently active National Institutes of Health (NIH) grants focussed on sleep and resilience, specifically examining the design of studies that explore sleep as a factor that promotes health maintenance, survivorship, or protective/preventive pathways. A search of NIH R01 and R21 research project grants that received funding in Fiscal Years (FY) 2016-2021 and focussed on sleep and resilience was conducted. A total of 16 active grants from six NIH institutes met the inclusion criteria. Most grants were funded in FY 2021 (68.8%), used the R01 mechanism (81.3%), were observational studies (75.0%), and measured resilience in the context of resisting a stressor/challenge (56.3%). Early adulthood and midlife were most commonly studied and over half of the grants focussed on underserved/underrepresented populations. NIH-funded studies focussed on sleep and resilience, or the ways in which sleep can influence an individual's ability to resist, adapt, or recover from a challenging event. This analysis highlights an important gap and the need to expand research focussed on sleep as a promotor of molecular, physiological, and psychological resilience.

Consensus Report of the 2015 Weinman International Conference on Mesothelioma.

On November 9 and 10, 2015, the International Conference on Mesothelioma in Populations Exposed to Naturally Occurring Asbestiform Fibers was held at the University of Hawaii Cancer Center in Honolulu, Hawaii. The meeting was cosponsored by the International Association for the Study of Lung Cancer, and the agenda was designed with significant input from staff at the U.S. National Cancer Institute and National Institute of Environmental Health Sciences. A multidisciplinary group of participants presented updates reflecting a range of disciplinary perspectives, including mineralogy, geology, epidemiology, toxicology, biochemistry, molecular biology, genetics, public health, and clinical oncology. The group identified knowledge gaps that are barriers to preventing and treating malignant mesothelioma (MM) and the required next steps to address barriers. This manuscript reports the group's efforts and focus on strategies to limit risk to the population and reduce the incidence of MM. Four main topics were explored: genetic risk, environmental exposure, biomarkers, and clinical interventions. Genetics plays a critical role in MM when the disease occurs in carriers of germline BRCA1 associated protein 1 mutations. Moreover, it appears likely that, in addition to BRCA1 associated protein 1, other yet unknown genetic variants may also influence the individual risk for development of MM, especially after exposure to asbestos and related mineral fibers. MM is an almost entirely preventable malignancy as it is most often caused by exposure to commercial asbestos or mineral fibers with asbestos-like health effects, such as erionite. In the past in North America and in Europe, the most prominent source of exposure was related to occupation. Present regulations have reduced occupational exposure in these countries; however, some people continue to be exposed to previously installed asbestos in older construction and other settings. Moreover, an increasing number of people are being exposed in rural areas that contain noncommercial asbestos, erionite, and other mineral fibers in soil or rock (termed naturally occurring asbestos [NOA]) and are being developed. Public health authorities, scientists, residents, and other affected groups must work together in the areas where exposure to asbestos, including NOA, has been documented in the environment to mitigate or reduce this exposure. Although a blood biomarker validated to be effective for use in screening and identifying MM at an early stage in asbestos/NOA-exposed populations is not currently available, novel biomarkers presented at the meeting, such as high mobility group box 1 and fibulin-3, are promising. There was general agreement that current treatment for MM, which is based on surgery and standard chemotherapy, has a modest effect on the overall survival (OS), which remains dismal. Additionally, although much needed novel therapeutic approaches for MM are being developed and explored in clinical trials, there is a critical need to invest in prevention research, in which there is a great opportunity to reduce the incidence and mortality from MM.

Methylation profile landscape in mesothelioma: possible implications in early detection, disease progression, and therapeutic options.

Malignant Pleural Mesothelioma (MPM) is an aggressive malignancy of the pleura associated with asbestos exposure. Incidence of MPM is expected to increase over the course of next decade in both Europe and the developing countries. Although significant progress has been made in terms of etiology and pathogenesis of this disease, currently available therapeutic options have not significantly improved the survival outcome of patients on standard chemotherapeutic regimens. Integrity of the cellular DNA is often altered in many cancers. Understanding of the molecular mechanisms that regulate cellular DNA alterations to facilitate cancer initiation and development has potential to allow better design of cancer cell inhibitory strategies. In this context, there is a need to explore the gamut of "omics" strategies to provide a comprehensive epigenetics profile for MPM. This chapter discusses the functional genomics and epigenetic patterns observed by various investigators studying MPM patient populations on global fronts, and attempts to present a holistic approach in combating this insidious disease. Here we provide investigators in this field with novel insights and methodologies used in other types of cancers that might have profound impact in the early detection, prognosis and potential therapeutic strategies for MPM.

Disulfiram suppresses growth of the malignant pleural mesothelioma cells in part by inducing apoptosis.

Dithiocarbamate compound Disulfiram (DSF) that binds with copper and functions as an inhibitor of aldehyde dehydrogenase is a Food and Drug Administration approved agent for treatment of alcoholism. Copper complexed DSF (DSF-Cu) also possesses anti-tumor and chemosensitizing properties; however, its molecular mechanisms of action remain unclear. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of DSF-Cu and the molecular mechanisms involved. DSF-Cu inhibited growth of the murine as well as human MPM cells in part by increasing levels of ubiquitinated proteins. DSF-Cu exposure stimulated apoptosis in MPM cells that involved activation of stress-activated protein kinases (SAPKs) p38 and JNK1/2, caspase-3, and cleavage of poly-(ADP-ribose)-polymerase, as well as increased expression of sulfatase 1 and apoptosis transducing CARP-1/CCAR1 protein. Gene-array based analyses revealed that DSF-Cu suppressed cell growth and metastasis-promoting genes including matrix metallopeptidase 3 and 10. DSF inhibited MPM cell growth and survival by upregulating cell cycle inhibitor p27Kip1, IGFBP7, and inhibitors of NF-κB such as ABIN 1 and 2 and Inhibitory κB (IκB)α and ß proteins. DSF-Cu promoted cleavage of vimentin, as well as serine-phosphorylation and lysine-63 linked ubiquitination of podoplanin. Administration of 50 mg/kg DSF-Cu by daily i.p injections inhibited growth of murine MPM cell-derived tumors in vivo. Although podoplanin expression often correlates with metastatic disease and poor prognosis, phosphorylation of serines in cytoplasmic domain of podoplanin has recently been shown to interfere with cellular motility and migration signaling. Post-translational modification of podoplanin and cleavage of vimentin by DSF-Cu underscore a metastasis inhibitory property of this agent and together with our in vivo studies underscore its potential as an anti-MPM agent.

CARP-1 functional mimetics are a novel class of small molecule inhibitors of malignant pleural mesothelioma cells.

Malignant pleural mesothelioma (MPM) is an asbestos-related thoracic malignancy that is characterized by late metastases, and resistance to therapeutic modalities. The toxic side-effects of MPM therapies often limit their clinical effectiveness, thus necessitating development of new agents to effectively treat and manage this disease in clinic. CARP-1 functional mimetics (CFMs) are a novel class of compounds that inhibit growth of diverse cancer cell types. Here we investigated MPM cell growth suppression by the CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited MPM cell growth, in vitro, in part by stimulating apoptosis. Apoptosis by CFM-4 involved activation of pro-apoptotic stress-activated protein kinases (SAPKs) p38 and JNK, elevated CARP-1 expression, cleavage of PARP1, and loss of the oncogene c-myc as well as mitotic cyclin B1. Treatments of MPM cells with CFM-4 resulted in depletion of NF-κB signaling inhibitor ABIN1 and Inhibitory κB (IκB)α and ß, while increasing expression of pro-apoptotic death receptor (DR) 4 protein. CFM-4 enhanced expression of serine-phosphorylated podoplanin and cleavage of vimetin. CFMs also attenuated biological properties of the MPM cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Both podoplanin and vimentin regulate processes of cell motility and invasion, and their expression often correlates with metastatic disease, and poor prognosis. The fact that phosphorylation of serines in the cytoplasmic domain of podoplanin interferes with processes of cellular motility, CFM-4-dependent elevated phosphorylated podoplanin and cleavage of vimentin underscore a metastasis inhibitory property of these compounds, and suggest that CFMs and/or their future analogs have potential as anti-MPM agents.

Withaferin A inhibits the proteasome activity in mesothelioma in vitro and in vivo.

The medicinal plant Withania somnifera has been used for over centuries in Indian Ayurvedic Medicine to treat a wide spectrum of disorders. Withaferin A (WA), a bioactive compound that is isolated from this plant, has anti-inflammatory, immuno-modulatory, anti-angiogenic, and anti-cancer properties. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of WA and the molecular mechanisms involved. WA inhibited growth of the murine as well as patient-derived MPM cells in part by decreasing the chymotryptic activity of the proteasome that resulted in increased levels of ubiquitinated proteins and pro-apoptotic proteasome target proteins (p21, Bax, IκBα). WA suppression of MPM growth also involved elevated apoptosis as evidenced by activation of pro-apoptotic p38 stress activated protein kinase (SAPK) and caspase-3, elevated levels of pro-apoptotic Bax protein and cleavage of poly-(ADP-ribose)-polymerase (PARP). Our studies including gene-array based analyses further revealed that WA suppressed a number of cell growth and metastasis-promoting genes including c-myc. WA treatments also stimulated expression of the cell cycle and apoptosis regulatory protein (CARP)-1/CCAR1, a novel transducer of cell growth signaling. Knock-down of CARP-1, on the other hand, interfered with MPM growth inhibitory effects of WA. Intra-peritoneal administration of 5 mg/kg WA daily inhibited growth of murine MPM cell-derived tumors in vivo in part by inhibiting proteasome activity and stimulating apoptosis. Together our in vitro and in vivo studies suggest that WA suppresses MPM growth by targeting multiple pathways that include blockage of proteasome activity and stimulation of apoptosis, and thus holds promise as an anti-MPM agent.

New insight into the molecular mechanisms of the biological effects of DNA minor groove binders.

BACKGROUND: Bisbenzimides, or Hoechst 33258 (H258), and its derivative Hoechst 33342 (H342) are archetypal molecules for designing minor groove binders, and widely used as tools for staining DNA and analyzing side population cells. They are supravital DNA minor groove binders with AT selectivity. H342 and H258 share similar biological effects based on the similarity of their chemical structures, but also have their unique biological effects. For example, H342, but not H258, is a potent apoptotic inducer and both H342 and H258 can induce transgene overexpression in in vitro studies. However, the molecular mechanisms by which Hoechst dyes induce apoptosis and enhance transgene overexpression are unclear. METHODOLOGY/PRINCIPAL FINDINGS: To determine the molecular mechanisms underlying different biological effects between H342 and H258, microarray technique coupled with bioinformatics analyses and multiple other techniques has been utilized to detect differential global gene expression profiles, Hoechst dye-specific gene expression signatures, and changes in cell morphology and levels of apoptosis-associated proteins in malignant mesothelioma cells. H342-induced apoptosis occurs in a dose-dependent fashion and is associated with morphological changes, caspase-3 activation, cytochrome c mitochondrial translocation, and cleavage of apoptosis-associated proteins. The antagonistic effect of H258 on H342-induced apoptosis indicates a pharmacokinetic basis for the two dyes' different biological effects. Differential global gene expression profiles induced by H258 and H342 are accompanied by unique gene expression signatures determined by DNA microarray and bioinformatics software, indicating a genetic basis for their different biological effects. CONCLUSIONS/SIGNIFICANCE: A unique gene expression signature associated with H342-induced apoptosis provides a new avenue to predict and classify the therapeutic class of minor groove binders in the drug development process. Further analysis of H258-upregulated genes of transcription regulation may identify the genes that enhance transgene overexpression in gene therapy and promote recombinant protein products in biopharmaceutical companies. DATA DEPOSITION: The microarray data reported in this article have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no.GSE28616).

Antagonists of anaphase-promoting complex (APC)-2-cell cycle and apoptosis regulatory protein (CARP)-1 interaction are novel regulators of cell growth and apoptosis.

CARP-1/CCAR1, a perinuclear phosphoprotein, is a regulator of cell growth and apoptosis signaling. Although CARP-1 is a regulator of chemotherapy-dependent apoptosis, it is also a part of the NF-κB proteome and a co-activator of steroid/thyroid nuclear receptors as well as ß-catenin signaling. Our yeast two-hybrid screen revealed CARP-1 binding with the anaphase-promoting complex/cyclosome E3 ubiquitin ligase component APC-2 protein. CARP-1 also binds with anaphase-promoting complex/cyclosome co-activators Cdc20 and Cdh1. Following mapping of the minimal epitopes involved in CARP-1 binding with APC-2, a fluorescence polarization assay was established that indicated a dissociation constant (K(d)) of 480 nm for CARP-1/APC-2 binding. Fluorescence polarization assay-based high throughput screening of a chemical library yielded several small molecule antagonists of CARP-1/APC-2 binding, termed CARP-1 functional mimetics. CFM-4 (1(2-chlorobenzyl)-5'-phenyl-3'H-spiro[indoline-3,2'-[1,3,4]thiadiazol]-2-one), a lead compound, binds with and stimulates CARP-1 expression. CFM-4 prevents CARP-1 binding with APC-2, causes G(2)M cell cycle arrest, and induces apoptosis with an IC(50) range of 10-15 µm. Apoptosis signaling by CFM-4 involves activation of caspase-8 and -9 and caspase-mediated ubiquitin-proteasome pathway-independent loss of cyclin B1 and Cdc20 proteins. Depletion of CARP-1, however, interferes with CFM-4-dependent cell growth inhibition, activation of caspases, and apoptosis. Because CFM-4 also suppresses growth of drug-resistant human breast cancer cells without affecting the growth of human breast epithelial MCF-10A cells, elevating CARP-1 by CFM-4 and consequent apoptosis could in principle be exploited to further elucidate, and perhaps effectively target, often deregulated cell cycle pathways in pathological conditions, including cancer.

Curcumin suppresses growth of mesothelioma cells in vitro and in vivo, in part, by stimulating apoptosis.

Malignant pleural mesothelioma (MPM) is an aggressive, asbestos-related malignancy of the thoracic pleura. Although, platinum-based agents are the first line of therapy, there is an urgent need for second-line therapies to treat the drug-resistant MPM. Cell cycle as well as apoptosis pathways are frequently altered in MPM and thus remain attractive targets for intervention strategies. Curcumin, the major component in the spice turmeric, alone or in combination with other chemotherapeutics has been under investigation for a number of cancers. In this study, we investigated the biological and molecular responses of MPM cells to curcumin treatments and the mechanisms involved. Flow-cytometric analyses coupled with western immunoblotting and gene-array analyses were conducted to determine mechanisms of curcumin-dependent growth suppression of human (H2373, H2452, H2461, and H226) and murine (AB12) MPM cells. Curcumin inhibited MPM cell growth in a dose- and time-dependent manner while pretreatment of MPM cells with curcumin enhanced cisplatin efficacy. Curcumin activated the stress-activated p38 kinase, caspases 9 and 3, caused elevated levels of proapoptotic proteins Bax, stimulated PARP cleavage, and apoptosis. In addition, curcumin treatments stimulated expression of novel transducers of cell growth suppression such as CARP-1, XAF1, and SULF1 proteins. Oral administration of curcumin inhibited growth of murine MPM cell-derived tumors in vivo in part by stimulating apoptosis. Thus, curcumin targets cell cycle and promotes apoptosis to suppress MPM growth in vitro and in vivo. Our studies provide a proof-of-principle rationale for further in-depth analysis of MPM growth suppression mechanisms and their future exploitation in effective management of resistant MPM.

Novel cis-trans interactions are involved in post-transcriptional regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 mRNA.

BACKGROUND: A variety of pathways target CDKI p21WAF1/CIP1 expression at transcriptional, post-transcriptional as well as translational levels. We previously found that cell growth suppressing retinoid CD437 enhanced expression of p21WAF1/CIP1 and DNA damage inducible GADD45 proteins in part by elevating their mRNA stability. RESULTS: Here, we investigated molecular mechanisms of CD437-dependent post-transcriptional regulation of p21WAF1/CIP1 expression. By utilizing MDA-MB-468 HBC cells expressing chimeric rabbit beta-globin-p21WAF1/CIP1 transcripts we mapped multiple CD437-responsive sequences located within positions 1195 to 1795 of the 3'-untranslated region of p21WAF1/CIP1 mRNA. Several cytoplasmic proteins present in MDA-MB-468, MCF-7 HBC as well as HL-60R leukemia cells bound specifically, in vitro, with these CD437-responsive sequences. CD437 treatment of cells resulted in elevated binding of ~85 kD and ~55 kD cytoplasmic proteins with putative CD437-responsive sequences. A 12 nt RNA sequence (5'-UGUGGUGGCACA-3') present within CD437-responsive region of p21WAF1/CIP1 mRNA displayed specific and elevated binding with the above noted proteins. Treatment of cells with ActD or CHX prior to CD437 exposure did not abrogate RNA-protein interactions. However, treatment of cytoplasmic protein extracts with proteinase K or alkaline phosphatase resulted in loss of RNA-protein interactions. CONCLUSIONS: CD437 regulates cell growth in part by regulating stability of p21WAF1/CIP1 mRNA that involves specific RNA-protein interactions that are phosphorylation-dependent, while not requiring nascent transcription or protein synthesis.

A novel mechanism of cell growth regulation by Cell Cycle and Apoptosis Regulatory Protein (CARP)-1.

BACKGROUND: CARP-1/CCAR1, a perinuclear phospho-protein, regulates signaling by adriamycin, steroids, or growth factors. However, intracellular events that regulate CARP-1-dependent cell growth are not fully understood. RESULTS: Here we investigated whether CARP-1 is involved in signaling induced by the protein kinase A inhibitor H89. Treatments of human breast cancer cells with H89 resulted in apoptosis that involved enhanced CARP-1 threonine phosphorylation and expression. Depletion of CARP-1, on the other hand, abrogates apoptosis induced by H89. CARP-1 binds with signal transducer TAZ and over-expression of TAZ inhibits apoptosis by CARP-1. CARP-1 (651-759) interacts with a novel, N-terminal epitope of TAZ. H89 treatment stimulates threonine phosphorylation of CARP-1 (651-759), while substitution of threonine667 to alanine interferes with its binding with TAZ and apoptosis by H89. In addition, expression of wild type or CARP-1 (651-759) causes loss of c-myc expression due, in part, to suppression of c-myc transcription. CONCLUSIONS: CARP-1 threonine667 regulates H89-dependent signaling by a novel pathway that involves modulation of CARP-1 interaction with TAZ and transcriptional down-regulation of c-myc.

Targeted proteasome inhibition by Velcade induces apoptosis in human mesothelioma and breast cancer cell lines.

INTRODUCTION: Thoracic malignancies and human breast cancer (HBC) continue to be aggressive solid tumors that are poor responders to the existing conventional standard chemotherapeutic approaches. Malignant pleural mesothelioma (MPM) is an asbestos-related tumor of the thoracic pleura that lacks effective treatment options. Altered ubiquitin proteasome pathway is frequently encountered in many malignancies including HBC and MPM and thus serves as an important target for therapeutic intervention strategies. Although proteasome inhibitor Velcade (Bortezomib) has been under clinical investigation for a number of cancers, limited preclinical studies with this agent have thus far been conducted in HBC and MPM malignancies. PURPOSE: To study the biological and molecular responses of MPM and HBC cells to Velcade treatments, and to identify mechanisms involved in transducing growth inhibitory effects of this agent. METHODS: Flow-cytometric analyses coupled with western immunoblotting and gene-array methodologies were utilized to determine mechanisms of Velcade-dependent growth suppression of five MPM (H2595, H2373, H2452, H2461, and H2714) and two breast cancer (MDA MB-468, SKBR-3) cell lines. RESULTS: Our data revealed significant reduction in cell growth properties that were dose and time dependent. Velcade treatment resulted in G2M phase arrest, increased expression of cyclin-dependent kinase inhibitor p21 and pro-apoptotic protein Bax. Pretreatment of mesothelioma cells with Velcade showed synergistic effect with cisplatin combination regimens. High-throughput gene expression profiling among Velcade treated and untreated mesothelioma cell lines resulted in identification of novel transducers of apoptosis such as CARP-1, XAF1, and Troy proteins. CONCLUSIONS: Velcade targets cell cycle and apoptosis signaling to suppress MPM and HBC growth in part by activating novel transducers of apoptosis. This pilot study has paved way for further in-depth analysis of the downstream target molecules associated with presensitization of mesothelioma cells in finding effective therapeutic treatment options for both mesothelioma and recalcitrant breast cancers.

Protumorigenic role of HAPLN1 and its IgV domain in malignant pleural mesothelioma.

PURPOSE: Tumor extracellular matrix (ECM) plays a crucial role in cancer progression mediating and transforming host-tumor interactions. Targeting the ECM is becoming an increasingly promising therapeutic approach in cancer treatment. We find that one of the ECM proteins, HAPLN1, is overexpressed in the majority of mesotheliomas. This study was designed to characterize the protumorigenic role of HAPLN1 in mesothelioma. EXPERIMENTAL DESIGN: Overexpression of HAPLN1 was assessed and validated on a large set of normal/mesothelioma specimens on the RNA and protein levels. We also analyzed DNA copy number alterations in the HAPLN1 genomic locus using the array-based comparative genomic hybridization representational oligonucleotide microarray analysis tool. Tumorigenic activities of the HAPLN1 domains were evaluated in vitro on mesothelioma cells transfected with HAPLN1-expressing constructs. RESULTS: We found that HAPLN1 is 23-fold overexpressed in stage I mesothelioma and confirmed it for 76% samples (n = 53) on RNA and 97% (n = 40) on protein levels. The majority of lung cancers showed no differential expression of HAPLN1. Analysis of DNA copy number alterations identified recurrent gain in the 5q14.3 HAPLN1 locus in approximately 27% of tumors. Noteworthy, high expression of HAPLN1 negatively correlated with time to progression (P = 0.05, log-rank test) and overall survival (P = 0.006). Proliferation, motility, invasion, and soft-agar colony formation assays on mesothelioma cells overexpressing full-length HAPLN1 or its functional domains strongly supported the protumorigenic role of HAPLN1 and its SP-IgV domain. CONCLUSION: Overexpression of HAPLN1 and its SP-IgV domain increases tumorigenic properties of mesothelioma. Thus, targeting the SP-IgV domain may be one of the therapeutic approaches in cancer treatment.

Schlafen 3, a novel gene, regulates colonic mucosal growth during aging.

Although aging is associated with increased proliferation and decreased apoptosis in the colonic mucosa of Fischer 344 rats, the regulatory mechanisms are poorly understood. Gene expression profiling (Illumina platform) was carried out in freshly isolated colonic mucosal cells from young (4-6 mo old) and aged (22-24 mo old) Fischer 344 rats. Sixty-six genes were differentially expressed in the colonic mucosa between young and old animals (P<0.05). In particular, the expression of schlafen 3, a negative regulator of proliferation, was decreased by 8- to 10-fold in the colonic mucosa of aged rats. Administration of wortmannin, which inhibited colonic mucosal proliferation in the colonic mucosa of aged rats, stimulated the expression of schlafen 3, indicating a growth regulatory role of this gene. To further determine the growth regulatory properties of schlafen 3 gene, schlafen 3 cDNA was transfected in colon cancer HCT-116 cells. This resulted in a 30-40% inhibition of cellular growth, accompanied by decreased expression of PCNA and cyclin D1 and reduced phosphorylation of retinoblastoma protein. In conclusion, our present study demonstrates that several genes involved in proliferation and apoptosis are differentially expressed in the colonic mucosa of young and aged rats. Schlafen 3, a novel negative regulator of growth, which is markedly downregulated in the colonic mucosa of the aged, may play a role in regulating colonic mucosal growth during aging.

Soluble mesothelin-related peptide level elevation in mesothelioma serum and pleural effusions.

BACKGROUND: Soluble mesothelin-related peptide (SMRP) is a potential marker for malignant pleural mesothelioma (MPM), which may be useful for screening high-risk asbestos-exposed individuals. METHODS: We evaluated SMRP in serum from MPM patients (n = 90), lung cancer patients (n = 170), age and tobacco-matched asbestos-exposed individuals (n = 66), and in MPM pleural effusions (n = 45), benign effusions (n = 30), and non-MPM effusions (n = 20) using the MesoMark enzyme-linked immunosorbent assay kit (Fujirebio Diagnostics, Malvern, PA). Receiver operating characteristic (ROC) curves were used to define true and false positive rates at various cutoffs. RESULTS: Mean serum SMRP levels were higher in MPM compared with lung cancer (5.67 +/- 0.82 nM [mean +/- standard error of the mean vs 1.99 +/- 0.43 nM, p < 0.001), and stage I MPM SMRP levels (n = 12; 2.09 +/- 0.41 nM) were significantly higher than those in asbestos-exposed individuals (0.99 +/- 0.09 nM, p = 0.02, respectively). Stage 2 to 4 SMRP serum levels were significantly higher than those for stage 1 MPM. The area under the ROC curve for serum SMRP was 0.81 for differentiating MPM and asbestos-exposed individuals; cutoff = 1.9 nM (sensitivity = 60%, specificity = 89%). The MPM pleural effusion SMRP was significantly higher than benign or other non-MPM pleural effusions (65.57 +/- 11.33 nM vs 27.46 +/- 11.25 nM [p = 0.003] and 18.99 +/- 7.48 nM [p = 0.044], respectively). CONCLUSIONS: These data support SMRP as a promising marker for MPM in both serum and pleural effusion fluid, and justify prospective screening studies of SMRP in combination with other markers for screening of asbestos-exposed cohorts.

Cell growth inhibition by okadaic acid involves gut-enriched Kruppel-like factor mediated enhanced expression of c-Myc.

Human breast cancer (HBC) cell growth suppression by okadaic acid (OA) was previously found to involve elevated expression of oncogenes c-myc and c-fos and apoptosis. Since, c-Myc influences diverse pathways of cell growth, we hypothesized that elevated levels of c-Myc are involved in HBC growth suppression. Here, we investigated whether induction of c-Myc by OA or protein synthesis inhibitor cycloheximide contributed to HBC growth inhibition and the mechanisms involved. OA, cycloheximide, or the chemotherapeutic drug Taxol suppressed HBC cell growth. However, OA or cycloheximide treatments over 6 or 10 h, respectively, induced c-Myc expression. Depletion of c-Myc, on the other hand, resulted in enhanced HBC cell viabilities when exposed to OA or cycloheximide, but not by Taxol. OA induced c-myc transcription by targeting an 80-bp region from positions -11 to +70, relative to the P1 transcription start of mouse c-myc promoter. Gel mobility shift assays revealed binding of HBC cell nuclear proteins to the OA-responsive c-myc promoter fragment, whereas binding of one complex was elevated in the case of the OA-treated or cycloheximide-treated HBC cell nuclear extracts. Database search revealed presence of a consensus sequence for zinc finger protein gut-enriched Kruppel-like factor (GKLF) in OA-responsive region of the c-myc promoter. Mutation of GKLF consensus sequences abrogated OA responsiveness of the c-myc promoter, and OA treatments caused enhanced expression of GKLF in HBC cells. Thus, OA-dependent attenuation of HBC growth is accomplished, in part, by zinc finger transcription factor GKLF-mediated enhanced transcription of c-myc.

DNA methylation profile of 28 potential marker loci in malignant mesothelioma.

Patients with malignant mesothelioma (MM), an aggressive cancer associated with asbestos exposure, usually present clinically with advanced disease and this greatly reduces the likelihood of curative treatment. MM is difficult to diagnose without invasive techniques; the development of non-invasively detectable molecular markers would therefore be highly beneficial. DNA methylation changes in cancer cells provide powerful markers that are potentially detectable non-invasively in DNA shed into bodily fluids. Here we examined the methylation status of 28 loci in 52 MM tumors to investigate their potential as molecular markers for MM. To exclude candidate MM markers that might be positive in biopsies/pleural fluid due to contaminating surrounding non-tumor lung tissue/DNA, we also examined the methylation of these markers in lung samples (age- or environmentally induced hypermethylation is frequently observed in non-cancerous lung). Statistically significantly increased methylation in MM versus non-tumor lung samples was found for estrogen receptor 1 (ESR1; p = 0.0002), solute carrier family 6 member 20 (SLC6A20; p = 0.0022) and spleen tyrosine kinase (SYK; p=0.0003). Examination of associations between methylation levels of the 28 loci and clinical parameters suggest associations of the methylation status of metallothionein genes with gender, histology, asbestos exposure, and lymph node involvement, and the methylation status of leucine zipper tumor suppressor 1 (LZTS1) and SLC6A20 with survival.