Progressive aberrant methylation of the RASSF1A gene in simian virus 40 infected human mesothelial cells

Our plain-English summary of Progressive aberrant methylation of the RASSF1A gene in simian virus 40 infected human mesothelial cells, by S. Toyooka, M. Carbone, K.O. Toyooka, et al (Oncogene, 2002).

The study

• Authors: S. Toyooka, M. Carbone, K.O. Toyooka, M. Bocchetta, N. Shivapurkar, J.D. Minna, A.F. Gazdar
• Journal: Oncogene
• Published: 2002;21(27):4340-4
• Cited by: 73 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Mesotheliomas are tumors arising from mesothelial cells and are associated with asbestos exposure and approximately 50% contain simian virus 40 (SV40) DNA sequences. SV40 infection of human mesothelial cells (HM) causes early cellular immortalization and late transformation. Aberrant methylation is a major mech-anism for loss of function of tumor suppressor genes (TSGs). We recently reported that of seven genes frequently methylated in epithelial tumors, only RASSF1A gene was frequently methylated in mesotheliomas, and its methylation was correlated with loss of RASSF1A expression and the presence of SV40. We studied whether SV40 infection of normal HM induces aberrant methylation of the genes previously studied in mesotheliomas. Of six infected foci examined at early passages (passages 8-30) there was no methylation of the seven genes examined. Of two foci examined at late passages (passages 51-86) after the appearance of morphological changes suggestive of transformation, methylation and loss of expression of RASSF1A was detected. Sequencing of the CpG dense region around the transcription start site and semi-quantitative real-time methylation specific PCR (MSP) assay for RASSF1A methylation demonstrated progressive methylation during late passages. Exposure to the demethylating agent 5-aza-2'-deoxycytidine restored RASSF1A expression, while exposure to the histone deacetylation inhibitor trichostatin A had no effect. These data, together with our previous findings, support a causal relationship between SV40 infection, progressive RASSF1A methylation and its silencing, and the pathogenesis of mesothelioma.

In plain English

This laboratory study used cultured human mesothelial cells (which line the chest and abdomen) infected with simian virus 40 (SV40), a monkey virus. The cells were not exposed to asbestos. The authors followed six groups of infected cells (foci) as they were repeatedly re-grown (passaged). They tested seven genes for methylation, a chemical change that can switch genes off. In early passages (8 to 30), a test called methylation-specific PCR found none of the seven genes methylated. Two foci were tested at later passages (51 to 86), when they looked like transformed cells. In both, a gene called RASSF1A had become methylated and was no longer active. The authors sequenced 58 sites near the start of the RASSF1A gene and averaged the results over 10 copies per sample. In one focus, the share of these sites that were methylated rose from 0.3% at passage 12 to 86% at passage 86. A drug that removes methylation (5-aza-2'-deoxycytidine) switched the gene back on; trichostatin A did not. The authors suggested that methylation is the main way RASSF1A is silenced in these cells. They also concluded that their findings support a role for SV40 in mesothelioma.

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: Laboratory (cell culture) study of SV40-infected human mesothelial cells followed over serial passages
• Who was studied: Six foci of SV40-infected human mesothelial cells were tested at early passages. Two of these foci (F1 and F4) were also tested at later passages 51 to 86. Uninfected primary mesothelial cell cultures were used for comparison.
• Exposure: Infection with simian virus 40 (SV40). No asbestos exposure was part of the experiment.
• Main results:
  • By methylation-specific PCR, RASSF1A methylation was absent in early passages of all six foci. The other six genes tested were not methylated at any passage.
  • In both foci tested at later passages (F1 and F4, passages 51 to 86), the RASSF1A gene was methylated.
  • Sequencing covered 58 CpG sites in the RASSF1A 5' flanking region, with 10 clones per passage. It showed minimal methylation early and rising methylation later. In focus 1, the mean share of methylated sites was 0.3% at passage 12, 58% at passage 51, 76% at passage 70 and 86% at passage 86. In focus 4, it was 0.3% at passage 11 and 75% at passage 60.
  • In late passages, the RASSF1A and RASSF1F transcripts were not expressed. The RASSF1C transcript was expressed in all passages tested of foci F1, F3 and F4.
  • The demethylating drug 5-aza-2'-deoxycytidine restored RASSF1A expression in late passages of foci 1 and 4. The histone deacetylase inhibitor trichostatin A did not.
• The authors' conclusion: The authors suggested that methylation, rather than histone deacetylation, is the major mechanism silencing RASSF1A in SV40 transformation of these cells. They concluded that their findings support the role of SV40 in the pathogenesis of malignant mesothelioma. They also suggested that RASSF1A methylation and silencing may be a useful approach for early detection.

Topics

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

Medical terms in this study, explained in our Lexicon: Mesothelial Cells, Mesothelioma, Asbestos Exposure, SV40, Tumor Suppressor Gene.

Related studies

• Aberrant methylation profile of human malignant mesotheliomas and its relationship to SV40 infection — M. Suzuki, S. Toyooka, et al. (2005)
• Gemcitabine reactivates epigenetically silenced genes and functions as a DNA methyltransferase inhibitor — S.G. Gray, A.M. Baird, et al. (2012)
• CpG island methylation and expression of tumour-associated genes in lung carcinoma — R. Dammann, M. Strunnikova, et al. (2005)
• Simian virus 40 transformation, malignant mesothelioma and brain tumors — F. Qi, M. Carbone, et al. (2011)
• Human mesothelial cells are unusually susceptible to simian virus 40-mediated transformation and asbestos cocarcinogenicity — M. Bocchetta, I. Di Resta, et al. (2000)
• Aberrant methylation of the CDKN2a/p16INK4a gene promoter region in preinvasive bronchial lesions: a prospective study in high-risk patients without invasive cancer — A. Lamy, R. Sesboüé, et al. (2002)

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