Organ defects of the Usp7K444R mutant mouse strain indicate the essential role of K63-polyubiquitinated Usp7 in organ formation.

BACKGROUND: K63-linked polyubiquitination of proteins have nonproteolytic functions and regulate the activity of many signal transduction pathways. USP7, a HIF1α deubiquitinase, undergoes K63-linked polyubiquitination under hypoxia. K63-polyubiquitinated USP7 serves as a scaffold to anchor HIF1α, CREBBP, the mediator complex, and the super elongation complex to enhance HIF1α-induced gene transcription. However, the physiological role of K63-polyubiquitinated USP7 remains unknown. METHODS: Using a Usp7K444R point mutation knock-in mouse strain, we performed immunohistochemistry and standard molecular biological methods to examine the organ defects of liver and kidney in this knock-in mouse strain. Mechanistic studies were performed by using deubiquitination, immunoprecipitation, and quantitative immunoprecipitations (qChIP) assays. RESULTS: We observed multiple organ defects, including decreased liver and muscle weight, decreased tibia/fibula length, liver glycogen storage defect, and polycystic kidneys. The underlying mechanisms include the regulation of protein stability and/or modulation of transcriptional activation of several key factors, leading to decreased protein levels of Prr5l, Hnf4α, Cebpα, and Hnf1ß. Repression of these crucial factors leads to the organ defects described above. CONCLUSIONS: K63-polyubiquitinated Usp7 plays an essential role in the development of multiple organs and illustrates the importance of the process of K63-linked polyubiquitination in regulating critical protein functions.

Overexpression of miR-199/214 is a distinctive feature of iron-induced and asbestos-induced sarcomatoid mesothelioma in rats.

Malignant mesothelioma (MM) is one of the most lethal tumors in humans. The onset of MM is linked to exposure to asbestos, which generates reactive oxygen species (ROS). ROS are believed to be derived from the frustrated phagocytosis and the iron in asbestos. To explore the pathogenesis of MM, peritoneal MM was induced in rats by the repeated intraperitoneal injection of iron saccharate and nitrilotriacetate. In the present study, we used microarray techniques to screen the microRNA (miR) expression profiles of these MM. We observed that the histological subtype impacted the hierarchical clustering of miR expression profiles and determined that miR-199/214 is a distinctive feature of iron saccharate-induced sarcomatoid mesothelioma (SM). Twist1, a transcriptional regulator of the epithelial-mesenchymal transition, has been shown to activate miR-199/214 transcription; thus, the expression level of Twist1 was examined in iron-induced and asbestos-induced mesotheliomas in rats. Twist1 was exclusively expressed in iron saccharate-induced SM but not in the epithelioid subtype. The Twist1-miR-199/214 axis is activated in iron saccharate-induced and asbestos-induced SM. The expression levels of miR-214 and Twist1 were correlated in an asbestos-induced MM cell line, suggesting that the Twist1-miR-199/214 axis is preserved. MeT5A, an immortalized human mesothelial cell line, was used for the functional analysis of miR. The overexpression of miR-199/214 promoted cellular proliferation, mobility and phosphorylation of Akt and ERK in MeT5A cells. These results indicate that miR-199/214 may affect the aggressive biological behavior of SM.

Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia.

Hypoxia and acidity provide microenvironment for selection under evolutionary pressure and proliferation in cancer cells. Carbonic anhydrases (CAs) are a superfamily of metalloenzymes present in all life kingdoms, equilibrating the reactions among CO2, bicarbonate and H+. CA9, a membrane-associated α-CA, has been a drug target for various cancers. Whereas iron is essential not only for cancer cells but also for all the lives on earth, little is known on the association among hypoxia, iron metabolism, extracellular acidity and redox regulation. Malignant mesothelioma (MM), an aggressive tumor with poor prognosis, is an intriguing model in that asbestos-associated pathogenesis includes excess iron environment during carcinogenesis. Re-analysis of rat asbestos-induced MM model revealed an inverse association between high CA9 expression and survival. Here we used human MMs to identify the molecular events surrounding CA9 from the viewpoint of iron metabolism. CA9 expression was significantly higher in MM cells than in MeT-5A mesothelial cells, which was further amplified under hypoxia (1%O2) with increased catalytic Fe(II). CA9 suppression by inhibitors (S4 and U104) decreased viability and migration of MM cells, accompanied by overexpression of TFRC, IREB1/2 and FPN1(SLC40A1) and by downregulation of FTH/FTL. This expressional pattern was similar to that of erastin-induced ferroptosis in the same cells. Furthermore, we observed mitochondrial fission and enhanced autophagy with increased catalytic Fe(II) in both mitochondria and lysosomes after CA9 inhibition, accompanied by increased peroxides, mitochondrial O2- and lipid peroxidation. The eventual cell death was significantly inhibited by deferoxamine, ferrostatin-1 and Z-VAD-FMK, suggesting a mixed cell death of ferroptosis and apoptosis. Therefore, CA9 plays a role in equilibrating among hypoxia, iron metabolism and redox regulation in MM cells.

Twist1 was detected in mesenchymal cells of mammary fibroadenoma and invasive components of breast carcinoma in rats.

Fibroadenoma (FA) is a common mammary fibroepithelial tumor. The tumor size of the FA is increased by estrogen, progesterone, prolactin, and pregnancy, whereas it decreases after menopause. These observations in humans indicate that FA is hormone dependent. In rats, the most common mammary neoplasm is also FA. Expression levels of Twist1, a transcriptional regulator of epithelial-mesenchymal transition, were examined in paraffin-embedded tissue sections of an experimental rat breast model to find physiological alternations coincident with reproductive hormonal changes. Twenty-three Fischer 344/Brown Norway F1 hybrid rats were used as 14- to 16-week-old adolescent rats (n=3), pregnant rats (n=4), and lactating rats (n=6) in addition to rats over 100-weeks-old that exhibited aging (n=3) and FA (n=7). Seventy-six cases of chemically induced breast carcinoma and two cases of FA in Sprague Dawley rats were also examined. Using tissue sections, we observed that Twist1-positive mesenchymal cells were predominantly located in the periductal region in adolescent and pregnant rats and in the terminal duct lobular unit in pregnant and elderly rats. Twist1 was also expressed diffusely in the mesenchymal cells of FA rats. Twist1-positive cancer-associated mesenchymal cells were found more frequently in the invasive components of breast carcinomas than in intraductal components. The expressions of Twist1 in mesenchymal cells were induced by physiological and pathological stimuli, suggesting the biological role of Twist1 in tissue structure. Further study may reveal the role of Twist1 in mesenchymal cells of mammary glands in rats.

Connective tissue growth factor-specific monoclonal antibody inhibits growth of malignant mesothelioma in an orthotopic mouse model.

Malignant mesothelioma is an aggressive neoplasm with no particularly effective treatments. We previously reported that overexpression of connective tissue growth factor (CTGF/CCN2) promotes mesothelioma growth, thus suggesting it as a novel molecular target. A human monoclonal antibody that antagonizes CTGF (FG-3019, pamrevlumab) attenuates malignant properties of different kinds of human cancers and is currently under clinical trial for the treatment of pancreatic cancer. This study reports the effects of FG-3019 on human mesothelioma in vitro and in vivo. We analyzed the effects of FG-3019 on the proliferation, apoptosis, migration/invasion, adhesion and anchorage-independent growth in three human mesothelioma cell lines, among which ACC-MESO-4 was most efficiently blocked with FG-3019 and was chosen for in vivo experiments. We also evaluated the coexistent effects of fibroblasts on mesothelioma in vitro, which are also known to produce CTGF in various pathologic situations. Coexistent fibroblasts in transwell systems remarkably promoted the proliferation and migration/invasion of mesothelioma cells. In orthotopic nude mice model, FG-3019 significantly inhibited mesothelioma growth. Histological analyses revealed that FG-3019 not only inhibited the proliferation but also induced apoptosis in both mesothelioma cells and fibroblasts. Our data suggest that FG-3019 antibody therapy could be a novel additional choice for the treatment of mesothelioma.

Phlebotomy as a preventive measure for crocidolite-induced mesothelioma in male rats.

Malignant mesothelioma (MM) is a rare but socially important neoplasm due to its association with asbestos exposure. Malignant mesothelioma is difficult to diagnose at an early stage, yet there are no particularly effective treatments available at the advanced stage, thus necessitating efficient strategies to prevent MM in individuals already exposed to asbestos. We previously showed that persistent oxidative damage caused by foreign body reaction and affinity of asbestos both to hemoglobin and histones is one of the major pathogeneses. Accordingly, as an effective strategy to prevent asbestos-induced MM, we undertook the use of an iron chelator, deferasirox, which decreased the epithelial-mesenchymal transition in a crocidolite-induced rat MM model. However, this agent may show adverse effects. Here, we studied the effects of iron removal by phlebotomy as a realistic measure on the same rat model. We injected a total of 5 mg crocidolite i.p. to F1 hybrid rats between the Fischer-344 and Brown-Norway strains at the age of 6 weeks. We repeated weekly or biweekly phlebotomy of 6-8 mL/kg/time from 10 to 60 weeks of age. The animals were observed until 120 weeks. In male rats, phlebotomy significantly decreased the weight and nuclear grade of MM, and modestly reduced the associated ascites and the fraction of more malignant sarcomatoid subtype. Weekly phlebotomy prolonged long-term survival. Our results indicate that appropriate phlebotomy may be a practical preventive measure to attenuate the initiation and promotion capacity of asbestos towards MM by reducing iron in individuals exposed to asbestos.

Fenton reaction-induced renal carcinogenesis in Mutyh-deficient mice exhibits less chromosomal aberrations than the rat model.

Oxidative stress including iron excess has been associated with carcinogenesis. The level of 8-oxoguanine, a major oxidatively modified base in DNA, is maintained very low by three distinct enzymes, encoded by OGG1, MUTYH and MTH1. Germline biallelic inactivation of MUTYH represents a familial cancer syndrome called MUTYH-associated polyposis. Here, we used Mutyh-deficient mice to evaluate renal carcinogenesis induced by ferric nitrilotriacetate (Fe-NTA). Although the C57BL/6 background is cancer-resistant, a repeated intraperitoneal administration of Fe-NTA induced a high incidence of renal cell carcinoma (RCC; 26.7%) in Mutyh-deficient mice in comparison to wild-type mice (7.1%). Fe-NTA treatment also induced renal malignant lymphoma, which did not occur without the Fe-NTA treatment in both the genotypes. Renal tumor-free survival after Fe-NTA treatment was marginally different (P = 0.157) between the two genotypes. Array-based comparative genome hybridization analyses revealed, in RCC, the loss of heterozygosity in chromosomes 4 and 12 without p16INKA inactivation; these results were confirmed by a methylation analysis and showed no significant difference between the genotypes. Lymphomas showed a preference for genomic amplifications. Dlk1 inactivation by promoter methylation may be involved in carcinogenesis in both tumors. Fe-NTA-induced murine RCCs revealed significantly less genomic aberrations than those in rats, demonstrating a marked species difference.

Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma.

CD44 exists as a standard (CD44s) isoform and different variant isoforms (CD44v) due to alternative splicing. While the complex nature of these different isoforms has not been fully elucidated, CD44v expression has been shown to exert oncogenic effects by promoting tumor progression, metastasis and resistance of tumor cells to chemotherapy. One of the CD44v isoforms, CD44v8-10, was recently shown to protect cancer cells from oxidative stress by increasing the synthesis of glutathione (GSH). However, data regarding CD44 isoform expression in malignant mesothelioma (MM) are still lacking. Here, we show that most of the MM cell lines express both the CD44s and CD44v isoforms, in contrast to non-tumorigenic mesothelial cells, which express only CD44s. Moreover, we show here that these MM cell lines are positive for CD44 variable exon 9, with CD44v8-10 among the variant isoforms expressed. The expression of CD44 variable exon 9 was found to be statistically associated with NF2 inactivation, a common occurrence in MM. Knockdown of CD44 reduced the protein level of xCT, a cystine transporter, and increased oxidative stress. However, an increase in GSH was also observed and was associated with enhanced chemoresistance in CD44-knockdown cells. Increased GSH was mediated by the Nrf2/AP-1-induced upregulation of GCLC, a subunit of the enzyme catalyzing GSH synthesis. Our results thus suggest that the response to CD44 depletion is cell type-dependent and, in cases such as MM cells, compensatory pathway(s) might be activated rheostatically to account for the loss of CD44 and counteract enhanced oxidative stress.

Urokinase-type plasminogen activator receptor promotes proliferation and invasion with reduced cisplatin sensitivity in malignant mesothelioma.

Malignant mesothelioma (MM) is a rare neoplasm associated with asbestos exposure. The prognosis of MM is poor because it is aggressive and highly resistant to chemotherapy. Using a rat model of asbestos-induced MM, we found elevated urokinase-type plasminogen activator receptor (uPAR; Plaur) expression in rat tissues, which was associated with poor prognosis. The proliferation, migration and invasion of MM cells were suppressed by uPAR knockdown and increased by overexpression experiments, irrespective of urokinase-type plasminogen activator (uPA; Plau) levels. More importantly, we found that uPAR expression is associated with sensitivity to cisplatin in MM through the PI3K/AKT pathway, which was demonstrated with specific inhibitors, LY294002 and Akti-1/2. uPAR knockdown significantly increased sensitivity to cisplatin whereas its overexpression significantly decreased cisplatin sensitivity. Furthermore, sera and tissues from MM patients showed significantly high uPAR levels, which suggested the pathogenic role of uPAR in the tumor biology of human MM. In conclusion, our findings indicate that uPAR levels are associated with malignant characteristics and cisplatin sensitivity of MM. In addition to the potential use of uPAR as a prognostic marker, the combination of uPAR abrogation and cisplatin may reveal a promising therapeutic approach for MM.

Dual preventive benefits of iron elimination by desferal in asbestos-induced mesothelial carcinogenesis.

Asbestos-induced mesothelial carcinogenesis is currently a profound social issue due to its extremely long incubation period and high mortality rate. Therefore, procedures to prevent malignant mesothelioma in people already exposed to asbestos are important. In previous experiments, we established an asbestos-induced rat peritoneal mesothelioma model, which revealed that local iron overload is a major cause of pathogenesis and that the induced genetic alterations are similar to human counterparts. Furthermore, we showed that oral administration of deferasirox modified the histology from sarcomatoid to the more favorable epithelioid subtype. Here, we used i.p. administration of desferal to evaluate its effects on asbestos-induced peritoneal inflammation and iron deposition, as well as oxidative stress. Nitrilotriacetate was used to promote an iron-catalyzed Fenton reaction as a positive control. Desferal significantly decreased peritoneal fibrosis, iron deposition, and nuclear 8-hydroxy-2'-deoxyguanosine levels in mesothelial cells, whereas nitrilotriacetate significantly increased all of them. Desferal was more effective in rat peritoneal mesothelial cells to counteract asbestos-induced cytotoxicity than in murine macrophages (RAW264.7). Furthermore, rat sarcomatoid mesothelioma cells were more dependent on iron for proliferation than rat peritoneal mesothelial cells. Because inflammogenicity of a fiber is proportionally associated with subsequent mesothelial carcinogenesis, iron elimination from the mesothelial environment can confer dual merits for preventing asbestos-induced mesothelial carcinogenesis by suppressing inflammation and mesothelial proliferation simultaneously.

Malignant mesothelioma as an oxidative stress-induced cancer: An update.

Malignant mesothelioma (MM) is a relatively rare cancer that occurs almost exclusively following respiratory exposure to asbestos in humans. Its pathogenesis is closely associated with iron overload and oxidative stress in mesothelial cells. On fiber exposure, mesothelial cells accumulate fibers simultaneously with iron, which either performs physical scissor function or catalyzes free radical generation, leading to oxidative DNA damage such as strand breaks and base modifications, followed by activation of intracellular signaling pathways. Chrysotile, per se without iron, causes massive hemolysis and further adsorbs hemoglobin. Exposure to indigestible foreign materials also induces chronic inflammation, involving consistent generation of free radicals and subsequent activation of NALP3 inflammasomes in macrophages. All of these contribute to mesothelial carcinogenesis. Genomic alterations most frequently involve homozygous deletion of INK4A/4B, and other pathways such as Hippo and TGF-ß pathways are also affected in MM. Recently, analyses of familial MM sorted out BAP1 as a novel responsible tumor suppressor gene, whose function is not fully elucidated. Five-year survival of mesothelioma is still ~8%, and this cancer is increasing worldwide. Connective tissue growth factor, a secretory protein creating a vicious cycle mediated by ß-catenin, has been recognized as a hopeful target for therapy, especially in sarcomatoid subtype. Recent research outcomes related to microRNAs and cancer stem cells also offer additional novel targets for the treatment of MM. Iron reduction as chemoprevention of mesothelioma is helpful at least in an animal preclinical study. Integrated approaches to fiber-induced oxidative stress would be necessary to overcome this currently fatal disease.

Rat model demonstrates a high risk of tremolite but a low risk of anthophyllite for mesothelial carcinogenesis.

Asbestos was abundantly used in industry during the last century. Currently, asbestos confers a heavy social burden due to an increasing number of patients with malignant mesothelioma (MM), which develops after a long incubation period. Many studies have been conducted on the effects of the asbestos types that were most commonly used for commercial applications. However, there are few studies describing the effects of the less common types, or minor asbestos. We performed a rat carcinogenesis study using Japanese tremolite and Afghan anthophyllite. Whereas more than 50% of tremolite fibers had a diameter of < 500 nm, only a small fraction of anthophyllite fibers had a diameter of < 500 nm. We intraperitoneally injected 1 or 10 mg of asbestos into F1 Fischer-344/Brown-Norway rats. In half of the animals, repeated intraperitoneal injections of nitrilotriacetate (NTA), an iron chelator to promote Fenton reaction, were performed to evaluate the potential involvement of iron overload. Tremolite induced MM with a high incidence (96% with 10 mg; 52% with 1 mg), and males were more susceptible than females. Histology was confirmed using immunohistochemistry, and most MMs were characterized as the sarcomatoid or biphasic subtype. Unexpectedly NTA showed an inhibitory effect in females. In contrast, anthophyllite induced no MM after an observation period of 550 days. The results suggest that the carcinogenicity of anthophyllite is weaker than formerly reported, whereas that of tremolite is as potent as major asbestos as compared with our previous data.

Connective tissue growth factor and ß-catenin constitute an autocrine loop for activation in rat sarcomatoid mesothelioma.

Due to the formerly widespread use of asbestos, malignant mesothelioma (MM) is increasingly frequent worldwide. MM is classified into epithelioid (EM), sarcomatoid (SM), and biphasic subtypes. SM is less common than EM but is recognized as the most aggressive type of MM, and these patients have a poor prognosis. To identify genes responsible for the aggressiveness of SM, we induced EM and SM in rats, using asbestos, and compared their transcriptomes. Based on the results, we focused on connective tissue growth factor (Ctgf), whose expression was significantly increased in SM compared with EM; EM itself exhibited an increased expression of Ctgf compared with normal mesothelium. Particularly in SM, Ctgf was a major regulator of MM proliferation and invasion through activation of the ß-catenin-TCF-LEF signalling pathway, which is autocrine and formed a positive feedback loop via LRP6 as a receptor for secreted Ctgf. High Ctgf expression also played a role in the epithelial-mesenchymal transition in MM. Furthermore, Ctgf is a novel serum biomarker for both early diagnosis and determining the MM prognosis in rats. These data link Ctgf to SM through the LRP6-GSK3ß-ß-catenin-TCF-Ctgf autocrine axis and suggest Ctgf as a therapeutic target.

Cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis through dysregulated adipocytokine production.

Like many other human cancers, the development of malignant mesothelioma is closely associated with a chronic inflammatory condition. Both macrophages and mesothelial cells play crucial roles in the inflammatory response caused by asbestos exposure. Here, we show that adipocytes can also contribute to asbestos-induced inflammation through dysregulated adipocytokine production. 3T3-L1 preadipocytes were differentiated into mature adipocytes prior to use. These cells took up asbestos fibers (chrysotile, crocidolite and amosite) but were more resistant to asbestos-induced injury than macrophages and mesothelial cells. Expression microarray analysis followed by reverse transcription-PCR revealed that adipocytes respond directly to asbestos exposure with an increased production of proinflammatory adipocytokines [e.g. monocyte chemoattractant protein-1 (MCP-1)], whereas the production of anti-inflammatory adipocytokines (e.g. adiponectin) is suppressed. This was confirmed in epididymal fat pad of mice after intraperitoneal injection of asbestos fibers. Such dysregulated adipocytokine production favors the establishment of a proinflammatory environment. Furthermore, MCP-1 marginally promoted the growth of MeT-5A mesothelial cells and significantly enhanced the wound healing of Y-MESO-8A and Y-MESO-8D human mesothelioma cells. Our results suggest that increased levels of adipocytokines, such as MCP-1, can potentially contribute to the promotion of mesothelial carcinogenesis through the enhanced recruitment of inflammatory cells as well as a direct growth and migration stimulatory effect on mesothelial and mesothelioma cells. Taken together, our findings support a potential cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis.

Intraperitoneal administration of tangled multiwalled carbon nanotubes of 15 nm in diameter does not induce mesothelial carcinogenesis in rats.

Multiwalled carbon nanotubes (MWCNTs) have attracted public attention not only for their potential applications in engineering and materials science but also for possible environmental risks. MWCNTs share similar properties with asbestos, a definite human carcinogen causing malignant mesothelioma (MM), in that they are both biopersistent thin fibers with a high aspect ratio. Certain types of MWCNTs do induce MM in animal experiments. Though there are many different types of MWCNTs awaiting use in industry, there is little evidence about what types of MWCNTs present a high risk for MM in vivo. We have previously shown that the diameter of MWCNTs is one of the critical factors for mesothelial injury, which eventually leads to MM. Because of the extensive commercial use of MWCNTs, the properties of MWCNTs that determine carcinogenic activity should be clarified. Here we report that a high dose (10 mg) of a tangled form of pristine MWCNT (with a diameter of 15 nm) did not induce MM after intraperitoneal administration in rats, which were followed for up to 3 years after injection. This observation strengthens our previous finding that the rigidity, diameter, length and surface properties of MWCNTs are important factors in MM induction in vivo.

Deferasirox induces mesenchymal-epithelial transition in crocidolite-induced mesothelial carcinogenesis in rats.

Asbestos was used worldwide in huge quantities in the past century. However, because of the unexpected carcinogenicity to mesothelial cells with an extremely long incubation period, many countries face this long-lasting social problem. Mesothelioma is often diagnosed in an advanced stage, for which no effective therapeutic protocols are yet established. We previously reported on the basis of animal experiments that the major pathology in asbestos-induced mesothelial carcinogenesis is local iron overload. Here, we undertook to find an effective strategy to prevent, delay, or lower the malignant potential of mesothelioma during asbestos-induced carcinogenesis. We used intraperitoneal injections of crocidolite to rats. We carried out a 16-week study to seek the maximal-tolerated intervention for iron reduction via oral deferasirox administration or intensive phlebotomy. Splenic iron deposition was significantly decreased with either method, and we found that Perls' iron staining in spleen is a good indicator for iron reduction. We injected a total of 10 mg crocidolite at the age of six weeks, and the preventive measures were via repeated oral administration of 25 to 50 mg/kg/d deferasirox or weekly to bimonthly phlebotomy of 4 to 10 mL/kg/d. The animals were observed until 110 weeks. Deferasirox administration significantly increased the fraction of less malignant epithelioid subtype. Although we found a slightly prolonged survival in deferasirox-treated female rats, larger sample size and refinement of the current protocol are necessary to deduce the cancer-preventive effects of deferasirox. Still, our results suggest deferasirox serves as a potential preventive strategy in people already exposed to asbestos via iron reduction.

Metamorphosis of mesothelial cells with active horizontal motility in tissue culture.

Mesothelial cells, which have diverse roles in physiology and pathology, constitute the mesothelium along with connective tissue and the basement membrane; the mesothelium serves to shield the somatic cavities. After mesothelial injury, mesothelial cells undergo tissue recovery. However, the mechanism of mesothelial regeneration remains poorly understood. In this study, we used confocal time-lapse microscopy to demonstrate that transformed mesothelial cells (MeT5A) and mouse peritoneal mesothelial cells can randomly migrate between cells in cell culture and in ex vivo tissue culture, respectively. Moreover, peritoneal mesothelial cells changed their morphology from a flattened shape to a cuboidal one prior to the migration. Conversely, MDCKII epithelial cells forming tight cell-cell contacts with one another do not alter the arrangement of adjacent cells during movement. Our evidence complements the current hypotheses of mesothelial regeneration and suggests that certain types of differentiated mesothelial cells undergo morphological changes before initiating migration to repair injured sites.

Iron overload signature in chrysotile-induced malignant mesothelioma.

Exposure to asbestos is a risk for malignant mesothelioma (MM) in humans. Among the commercially used types of asbestos (chrysotile, crocidolite, and amosite), the carcinogenicity of chrysotile is not fully appreciated. Here, we show that all three asbestos types similarly induced MM in the rat peritoneal cavity and that chrysotile caused the earliest mesothelioma development with a high fraction of sarcomatoid histology. The pathogenesis of chrysotile-induced mesothelial carcinogenesis was closely associated with iron overload: repeated administration of an iron chelator, nitrilotriacetic acid, which promotes the Fenton reaction, significantly reduced the period required for carcinogenesis; massive iron deposition was found in the peritoneal organs with high serum ferritin; and homozygous deletion of the CDKN2A/2B/ARF tumour suppressor genes, the most frequent genomic alteration in human MM and in iron-induced rodent carcinogenesis, was observed in 92.6% of the cases studied with array-based comparative genomic hybridization. The induced rat MM cells revealed high expression of mesoderm-specific transcription factors, Dlx5 and Hand1, and showed an iron regulatory profile of active iron uptake and utilization. These data indicate that chrysotile is a strong carcinogen when exposed to mesothelia, acting through the induction of local iron overload. Therefore, an intervention to remove local excess iron might be a strategy to prevent MM after asbestos exposure.

Diameter and rigidity of multiwalled carbon nanotubes are critical factors in mesothelial injury and carcinogenesis.

Multiwalled carbon nanotubes (MWCNTs) have the potential for widespread applications in engineering and materials science. However, because of their needle-like shape and high durability, concerns have been raised that MWCNTs may induce asbestos-like pathogenicity. Although recent studies have demonstrated that MWCNTs induce various types of reactivities, the physicochemical features of MWCNTs that determine their cytotoxicity and carcinogenicity in mesothelial cells remain unclear. Here, we showed that the deleterious effects of nonfunctionalized MWCNTs on human mesothelial cells were associated with their diameter-dependent piercing of the cell membrane. Thin MWCNTs (diameter ∼ 50 nm) with high crystallinity showed mesothelial cell membrane piercing and cytotoxicity in vitro and subsequent inflammogenicity and mesotheliomagenicity in vivo. In contrast, thick (diameter ∼ 150 nm) or tangled (diameter ∼ 2-20 nm) MWCNTs were less toxic, inflammogenic, and carcinogenic. Thin and thick MWCNTs similarly affected macrophages. Mesotheliomas induced by MWCNTs shared homozygous deletion of Cdkn2a/2b tumor suppressor genes, similar to mesotheliomas induced by asbestos. Thus, we propose that different degrees of direct mesothelial injury by thin and thick MWCNTs are responsible for the extent of inflammogenicity and carcinogenicity. This work suggests that control of the diameter of MWCNTs could reduce the potential hazard to human health.