Decitabine, differently from DNMT1 silencing, exerts its antiproliferative activity through p21 upregulation in malignant pleural mesothelioma (MPM) cells

Our plain-English summary of Decitabine, differently from DNMT1 silencing, exerts its antiproliferative activity through p21 upregulation in malignant pleural mesothelioma (MPM) cells, by S. Amatori, F. Papalini, R. Lazzarini, et al (Lung Cancer, 2009).

The study

• Authors: S. Amatori, F. Papalini, R. Lazzarini, B. Donati, I. Bagaloni, M.R. Rippo, A. Procopio, P.G. Pelicci, A. Catalano, M. Fanelli
• Journal: Lung Cancer
• Published: 2009;66(2):184-90
• Cited by: 30 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Malignant pleural mesothelioma (MPM) is a locally aggressive neoplasm, principally linked to asbestos fibres exposure. Strong evidences associate this pollutant with induction of DNA breaks, aberrant chromosomes segregation and important chromosomal rearrangements, considered crucial events in malignant transformation. A considerable contribution to cellular transformation in MPM is also given by the presence of high genomic instability, as well as by the increased DNA methylation, and consequent decreased expression, of tumor-suppressor genes. In this study we first demonstrated that MPM cells are characterized by a decreased methylation level of pericentromeric DNA sequences which can justify, at least in part, the genomic instability observed in this neoplasia. Concomitantly, we found a paradoxical increased expression of DNMT1, the most expressed DNA methyltransferases in MPM cells, DNMT3a and all five isoforms of DNMT3b. Thus, we compared two experimental strategies, DNMT1 silencing and usage of a demethylating agent (5-aza-2'-deoxycytidine or Decitabine), both theoretically able to revert the locally hypermethylated phenotype and considered potential future therapeutic approaches for MPM. Interestingly, both strategies substantially decrease cell survival of MPM cells but the antitumor activity of Decitabine, differently from DNMT1 silencing, is mediated, at least in part, by a p53-independent p21 upregulation, and is characterized by the arrest of MPM cells at the G2/M phase of the cell cycle. These results indicate that the two approaches act probably through different mechanisms and, thus, that DNMT1 silencing can be considered an effective alternative to Decitabine for cancer treatment.

In plain English

This laboratory study used cells grown in dishes, not people, and did not study asbestos exposure. The authors used a mesothelioma cell line (H28), two mesothelioma cell populations from patients, and normal mesothelial cells. Mesothelioma cells had less DNA methylation (a chemical tag) in a repeated DNA region, satellite 2, than normal cells. Patient-derived mesothelioma cells also had higher messenger RNA levels for the enzymes that add these tags. For one enzyme, DNMT1, levels were 4.4 to 7.2 fold higher, and it made up over 85–90% of the total. In H28 cells, stably reducing DNMT1 expression sharply cut living cells after 10 days; 8 days later none could be detected. The p53 and p21 proteins did not change. The drug Decitabine cut H28 survival below 50% of untreated cells after 6 days at three of five concentrations tested. It raised p21 but not p53, stalled cell division at the G2/M stage, and left cells unable to form colonies. Stably silencing p21 partly reduced the drug's effect on survival but not the stalling. The authors concluded the two approaches probably work differently.

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 (in vitro) study of cultured human mesothelioma and normal mesothelial cells, comparing DNMT1 gene silencing with Decitabine treatment
• Who was studied: The immortalized human malignant pleural mesothelioma cell line NCI-H28 (from the American Type Culture Collection), two primary malignant pleural mesothelioma cell populations (MES-SS 01, MES-PR 99) and primary normal mesothelial cells (HMC2), established from pleural effusions and tumor specimens. Most experiments used H28 cells.
• Exposure: No asbestos exposure was studied. Cells were either given the demethylating drug Decitabine (5-aza-2'-deoxycytidine) at a range of doses for up to 6 days, or had DNMT1 (and separately p21) stably silenced by lentivirus-based RNA interference using a short hairpin RNA.
• Main results:
  • Both primary mesothelioma cell populations and the H28 cell line showed decreased methylation of satellite 2 DNA compared with normal mesothelial cells.
  • DNMT expression was measured as mRNA levels by real-time quantitative RT-PCR. In primary mesothelioma cells compared with normal mesothelial cells, DNMT1 expression was increased 4.4 to 7.2 fold.
  • In primary mesothelioma cells compared with normal mesothelial cells, DNMT3a mRNA expression was increased 1.7 to 3.8 fold and DNMT3b 2 to 6.8 fold.
  • By RT-PCR relative quantification, DNMT1 was the most expressed DNA methyltransferase in mesothelioma cells, at more than 85–90% of the total.
  • In H28 cells with DNMT1 stably silenced, viable cells dropped sharply after 10 days, and after a further 8 days no viable cells could be monitored.
  • In DNMT1-silenced H28 cells, 49% of cells were hypodiploid after 16 days, with DNA fragmentation typical of programmed cell death.
  • In DNMT1-silenced H28 cells, p53 and p21 protein levels did not change, while p21 transcripts rose modestly (1.7 ± 0.19 fold compared with empty-vector cells).
  • After 6 days of Decitabine at the concentrations printed as 1, 5 and 10, H28 cell viability fell to less than 50% of untreated control cells.
  • In Decitabine-treated H28 cells, 10–15% of cells were hypodiploid after 6 days, with fewer cells in the G1 phase and more in the G2 phase.
  • H28 cells treated with Decitabine for 6 days could not form colonies when replated.
  • Decitabine increased p21 protein but not p53 protein in H28 cells. After 6 days, p21 transcripts were 12.34 ± 0.22 fold those of untreated H28 cells.
  • The authors reported comparable results in the two primary mesothelioma cell populations, but did not show the data.
  • Stably silencing p21 partially reduced Decitabine's effect on cell survival in H28 cells.
  • Silencing p21 did not prevent the Decitabine-induced G2/M arrest, which was even more pronounced in p21-silenced cells.
• The authors' conclusion: The authors concluded that DNMT1 silencing works against mesothelioma cells through a different mechanism from Decitabine. They suggested it could be an alternative or additional approach to cancer treatment, especially where demethylating agents cannot be used. They also concluded that p21 upregulation is not involved in the G2/M block caused by Decitabine.
• Limitations the authors state:
  • The authors stated that stable silencing cannot fully separate p21's role in the Decitabine response, because p21 is also needed for normal cell-cycle progression.
  • The authors noted that the lasting effect of Decitabine on the cells was shown only in the laboratory.

Topics

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Neoplasm, Malignant Transformation.

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