Malignant mesotheliomas (MMs) are very aggressive tumors that respond poorly to standard chemotherapeutic approaches. The phosphatidylinositol 3-kinase (PI3K)/ AKT pathway has been implicated in tumor aggressiveness, in part by mediating cell survival and reducing sensitivity to chemotherapy. Using antibodies recognizing the phosphorylated/activated form of AKT kinases, we observed elevated phospho-AKT staining in 17 of 26 (65%) human MM specimens. In addition, AKT phosphorylation was consistently observed in MMs arising in asbestos-treated mice and in MM cell xenografts. Consistent with reports implicating hepatocyte growth factor (HGF)/Met receptor signaling in MM, all 14 human and murine MM cell lines had HGF-inducible AKT activity. One of nine human MM cell lines had elevated AKT activity under serum-starvation conditions, which was associated with a homozygous deletion of PTEN, the first reported in MM. Treatment of this cell line with the mTOR inhibitor rapamycin resulted in growth arrest in G1 phase. Treatment of MM cells with the PI3K inhibitor LY294002 in combination with cisplatin had greater efficacy in inhibiting cell proliferation and inducing apoptosis than either agent alone. Collectively, these data indicate that MMs frequently express elevated AKT activity, which may be targeted pharmacologically to enhance chemotherapeutic efficacy. These findings also suggest that mouse models of MM may be useful for future preclinical studies of pharmaceuticals targeting the PI3K/AKT pathway.

Lung cancers, malignant mesotheliomas (MM), and fibrosis are devastating diseases with limited treatment strategies, in part due to poorly-effective drug delivery to affected areas of lung. We hypothesized that acid-prepared mesoporous spheres (APMS) (1-2 microm diameter, 40 A pore size) might be effective vehicles for pulmonary chemotherapeutic drug delivery. To assess this, APMS, chemically modified with different surface molecules (lipid, a linker having a terminal amine group, a thiol group, or tetraethylene glycol [TEG]), were evaluated for uptake and possible cytotoxic effects after in vitro administration to murine alveolar epithelial Type II (C10) and human mesothelioma (MM) cells and after intrapleural or intranasal administration to C57Bl/6 mice. APMS coated with TEG (APMS-TEG) were most efficiently taken up by C10 and MM cells. The mechanism of cell uptake was rapid, actin-dependent, and did not involve clathrin- or caveolae-mediated mechanisms nor fusion of membrane-bound APMS with lysosomes. When injected intrapleurally in mice, APMS-TEG were taken up by both CD45-positive and -negative cells of the diaphragm, lung, and spleen, whereas APMS administered by the intranasal route were predominantly in lung epithelial cells and alveolar macrophages. After intrapleural or intranasal administration, APMS were nonimmunogenic and nontoxic as evaluated by differential cell counts and lactate dehydrogenase levels in bronchoalveolar and pleural lavage fluids. In the treatment of lung and pleural diseases, APMS-TEG may be useful tools to deliver chemotherapeutic drugs or molecular constructs.

Only a fraction of subjects exposed to asbestos develop malignant mesothelioma (MM), suggesting that additional factors may render some individuals more susceptible. We tested the hypothesis that asbestos and Simian virus (SV40) are cocarcinogens. Asbestos and SV40 in combination had a costimulatory effect in inducing ERK1͞2 phosphorylation and activator protein-1 (AP-1) activity in both primary Syrian hamster mesothelial cells (SHM) and primary human mesothelial cells (HM). Ap-1 activity caused the expression and activation of matrix metalloprotease (MMP)-1 and MMP-9, which in turn led to cell invasion. Experiments using siRNA and chemical inhibitors confirmed the specificity of these results. The same effects were observed in HM and SHM. Experiments in hamsters showed strong cocarcinogenesis between asbestos and SV40: SV40 did not cause MM, asbestos caused MM in 20% of hamsters, and asbestos and SV40 together caused MM in 90% of hamsters. Significantly lower amounts of asbestos were sufficient to cause MM in animals infected with SV40. Our results indicate that mineral fibers and viruses can be cocarcinogens and suggest that lower amounts of asbestos may be sufficient to cause MM in individuals infected with SV40.activator protein-1 ͉ ERK1͞2 ͉ matrix metalloprotease ͉ environmental carcinogenesis ͉ viral oncology M alignant mesothelioma (MM) is a malignancy of the mesothelial cells that form the serosal membranes that cover the chest and abdominal cavities. Median survival is Ϸ12 months, and MM causes 2,000-3,000 deaths per year in the USA and Ϸ1,000 deaths per year in the U.K (1). The continuing increase in the incidence of MM has been associated to the widespread use of asbestos in the past century. The mechanisms of asbestos carcinogenesis have been linked to the extracellular response regulated kinase ERK1͞2 and activator protein-1 (AP-1) pathways (2), to the activation of proinflammatory cytokines and NFB (3), and to the production of reactive mutagenic oxygen species by nearby lung macrophages exposed to asbestos (4).Only a fraction (Ϸ5%) of subjects exposed to high levels of asbestos develop MM. This finding suggests that additional factors, such as SV40 infection and genetic predisposition, may render some individuals more susceptible to asbestos carcinogenicity (1, 5). SV40 is a monkey DNA virus that induces MM in hamsters (6). Following this observation, numerous laboratories have detected SV40 in MM biopsies although the prevalence of SV40 varied in different studies from Ϸ6% to 60%, and some studies did not detect SV40 (7-11). SV40 contaminated human polio vaccines worldwide from 1955 until Ϸ1961. Epidemiological studies comparing cohorts born before or after 1961 detected an increased relative risk of 3 for MM in pre-1961 cohorts that included many individuals vaccinated with contaminated polio vaccines (12). However, the overall epidemiological evidence was considered inconclusive because of differences in ages among the cohorts studied and because it was uncertain that cohorts born after 1961 we...

To elucidate genes important in development or repair of asbestos-induced lung diseases, gene expression was examined in mice after inhalation of chrysotile asbestos for 3, 9, and 40 days. We identified changes in the expression of genes linked to proliferation (cyclin B2, CDC20, and CDC28 protein kinase regulatory subunit 2), inflammation (CCL9, CCL6, complement component 1, chitinase3-like 3, TNF superfamily member 10, and IL-1B), and matrix remodeling (MMP12, MMP3, integrin ␣X, and cathepsins K, Z, B, and S). The most highly induced gene at all time points was mclca3 (gob5), a putative calcium-activated chloride channel involved in the regulation of mucus production and/or secretion. Using histochemistry, we demonstrated accumulation of mucus and increased mClca3 protein in the bronchiolar epithelium of asbestos-exposed mice at all time points but peaking at 9 days. Cytokine levels (interleukin-1␤, interleukin-4, interleukin-6) in bronchoalveolar lavage fluid also increased at 9 days, suggesting Th2-mediated immunity may play a role in asbestosinduced mucus production. In contrast, levels of cathepsin K, a potent elastase, increased between 3 and 40 days at both the mRNA and protein levels, localizing primarily in CD45-positive leukocytes and interstitial cells. Identification of genes involved in lung injury and remodeling after asbestos exposure could aid in defining mechanisms of airborne particulate-

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