SV40-induced expression of calretinin protects mesothelial cells from asbestos cytotoxicity and may be a key factor contributing to mesothelioma pathogenesis

Our plain-English summary of SV40-induced expression of calretinin protects mesothelial cells from asbestos cytotoxicity and may be a key factor contributing to mesothelioma pathogenesis, by T. Henzi, W.V. Blum, M. Pfefferli, et al (The American Journal of Pathology, 2009).

The study

• Authors: T. Henzi, W.V. Blum, M. Pfefferli, T.J. Kawecki, V. Salicio, B. Schwaller
• Journal: The American Journal of Pathology
• Published: 2009;174(6):2324-36
• Cited by: 28 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The calcium-binding protein calretinin has emerged as a useful marker for the identification of mesotheliomas of the epithelioid and mixed types, but its putative role in tumor development has not been addressed previously. Although exposure to asbestos fibers is considered the main cause of mesothelioma, undoubtedly, not all mesothelioma patients have a history of asbestos exposure. The question as to whether the SV40 virus is involved as a possible co-factor is still highly debated. Here we show that increased expression of SV40 early gene products in the mesothelial cell line MeT-5A induces the expression of calretinin and that elevated calretinin levels strongly correlate with increased resistance to asbestos cytotoxicity. Calretinin alone mediates a significant part of this protective effect because cells stably transfected with calretinin cDNA were clearly more resistant to the toxic effects of crocidolite than mock-transfected control cells. Down-regulation of calretinin by antisense methods restored the sensitivity to asbestos toxicity to a large degree. The protective effect observed in clones with higher calretinin expression levels could be eliminated by phosphatidylinositol 3-kinase (PI3K) inhibitors, implying an important role for the PI3K/AKT signaling (survival) pathway in mediating the protective effect. Up-regulation of calretinin, resulting from either asbestos exposure or SV40 oncoproteins, may be a common denominator that leads to increased resistance to asbestos cytotoxicity and thereby contributes to mesothelioma carcinogenesis.

In plain English

This was a laboratory cell-culture study, not a study of people. The authors worked with two clones of the human mesothelial cell line MeT-5A, which is immortalized with SV40 early region genes: an ATCC clone with strong large T antigen (Tag) and calretinin staining, and a Geneva (GE) clone with almost none. Stably transfecting GE cells with an SV40 early-gene plasmid raised both Tag and calretinin levels. Cells were exposed to NIEHS crocidolite asbestos, and viable-cell numbers were estimated by MTT assay, a measure reflecting both cell death and cell division. Across eight clones, resistance to crocidolite was positively correlated with calretinin expression (Pearson's r = 0.75, P = 0.032). Cells transfected with calretinin alone were, as a group, more resistant than the two mock-transfected clones, but not as resistant as the two most resistant SV40 clones. Lowering calretinin with antisense oligonucleotides or siRNA reduced the MTT signal of crocidolite-treated calretinin clones by 11 to 26 and 10 to 16 per cent respectively, compared with clones given a control oligonucleotide. PI3K inhibitors removed the protection only in high-calretinin clones.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: in vitro cell-culture (cell line transfection and cytotoxicity) experiments
• Who was studied: Human immortalized mesothelial MeT-5A cell clones (MeT-5A-ATCC and MeT-5A-GE), 18 SV40-transfected clones, 2 mock-transfected clones and 45 calretinin-transfected clones (10 analysed), studied in a laboratory in Fribourg, Switzerland
• Exposure: NIEHS crocidolite asbestos fibers added to cultures at 7.5, 10.0 or 12.5 micrograms per square centimetre (5 micrograms per square centimetre in inhibitor and AKT experiments, 2.5 in antisense experiments); plus transfection with SV40 early region genes or calretinin cDNA
• Main results:
  • Transfection of MeT-5A-GE cells with SV40 early genes raised both Tag and calretinin; clones with high Tag had considerably higher calretinin than untransfected GE cells, while mock clones M2 and M3 showed no increase
  • Across eight clones exposed to crocidolite at 10 micrograms per square centimetre for 5 days, resistance (a net measure of cell death and cell division) correlated positively with calretinin expression (Pearson's r = 0.75, P = 0.032)
  • The three SV40 clones with high calretinin (SV1, SV8, SV11) survived crocidolite better than the two SV40 clones with low calretinin (SV5, SV9), F1,3 = 108.0, P = 0.002
  • Calretinin-transfected clones as a group were more resistant to crocidolite than the two mock-transfected clones (F1,11 = 5.6, P = 0.038) but not as resistant as the two highly resistant SV40 clones SV1 and SV8 (F1,11 = 10.2, P = 0.009)
  • In calretinin clones exposed to crocidolite, antisense oligonucleotide treatment lowered the MTT signal by 11 to 26 per cent compared with cells treated with a control (nonsense) oligonucleotide (F1,3 = 28.0, P = 0.013), with no effect in control clones M2 and M3
  • Compared with control-oligonucleotide-treated cells, siRNA against calretinin reduced the MTT signal of the four calretinin clones by 10 to 16 per cent (F1,3 = 87.8, P = 0.003); absent in control clones M2 and M3
  • The PI3K inhibitor PI103 further increased crocidolite toxicity only in the high-calretinin SV40 clones (F1,3 = 35.7) and calretinin clones (F1,3 = 14.66); the ERK1/2 inhibitor PD98059 had no such effect, and the mTOR inhibitor rapamycin affected all clone groups equally
  • Short-term crocidolite exposure (5 micrograms per square centimetre for 1 hour) raised the pAKT/AKT ratio by a factor of 1.5, similarly in all three clone types
• The authors' conclusion: The authors concluded that SV40 early gene expression induces calretinin in mesothelial cells and that raised calretinin levels may be a common cause of increased resistance to asbestos cytotoxicity, acting through PI3K/AKT signalling and possibly contributing to mesothelioma development
• Limitations the authors state:
  • The authors stated that cell culture models can identify proteins involved in transformation but that a protein's role in tumorigenesis in vivo needs verification, for example in animal models
  • The authors stated that calretinin up-regulation alone cannot fully account for the protection seen with SV40 early gene expression
  • The authors stated that the mechanism by which calretinin might contribute to mesothelioma tumorigenesis remains unknown
  • The authors noted the reversal by antisense was incomplete, attributing this to the maximum achievable knock-down

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite, Mesothelioma.

Medical terms in this study, explained in our Lexicon: SV40, Calretinin, Mesothelial Cells, Mesothelioma, Epithelioid, Asbestos Exposure, Crocidolite, Carcinogenesis.

Related studies

• Calretinin is essential for mesothelioma cell growth/survival in vitro: a potential new target for malignant mesothelioma therapy? — W. Blum, B. Schwaller (2013)
• Effect of enhanced expression of connexin 43 on sunitinib-induced cytotoxicity in mesothelioma cells — M. Uzu, H. Sato, et al. (2015)
• Low calretinin expression and high neutrophil-to-lymphocyte ratio are poor prognostic factors in patients with malignant mesothelioma undergoing extrapleural pneumonectomy — S.C. Kao, S. Klebe, et al. (2011)
• Overexpression or absence of calretinin in mouse primary mesothelial cells inversely affects proliferation and cell migration — W. Blum, L. Pecze, et al. (2015)
• Long-term exposure of mesothelial cells to SV40 and asbestos leads to malignant transformation and chemotherapy resistance — A.L. Cleaver, K. Bhamidipaty, et al. (2014)
• Calretinin. A selective marker of normal and neoplastic mesothelial cells in serous effusions — M.C. Barberis, M. Faleri, et al. (1997)

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