Inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells

Our plain-English summary of Inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells, by P.A. Knobel, I.N. Kotov, E. Felley-Bosco, et al (Neoplasia, 2011).

The study

• Authors: P.A. Knobel, I.N. Kotov, E. Felley-Bosco, R.A. Stahel, T.M. Marti
• Journal: Neoplasia
• Published: 2011;13(10):961-70
• Cited by: 50 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

REV3 is the catalytic subunit of DNA translesion synthesis polymerase ζ. Inhibition of REV3 expression increases the sensitivity of human cells to a variety of DNA-damaging agents and reduces the formation of resistant cells. Surprisingly, we found that short hairpin RNA-mediated depletion of REV3 per se suppresses colony formation of lung (A549, Calu-3), breast (MCF-7, MDA-MB-231), mesothelioma (IL45 and ZL55), and colon (HCT116 +/-p53) tumor cell lines, whereas control cell lines (AD293, LP9-hTERT) and the normal mesothelial primary culture (SDM104) are less affected. Inhibition of REV3 expression in cancer cells leads to an accumulation of persistent DNA damage as indicated by an increase in phospho-ATM, 53BP1, and phospho-H2AX foci formation, subsequently leading to the activation of the ATM-dependent DNA damage response cascade. REV3 depletion in p53-proficient cancer cell lines results in a G(1) arrest and induction of senescence as indicated by the accumulation of p21 and an increase in senescence-associated β-galactosidase activity. In contrast, inhibition of REV3 expression in p53-deficient cells results in growth inhibition and a G(2)/M arrest. A small fraction of the p53-deficient cancer cells can overcome the G(2)/M arrest, which results in mitotic slippage and aneuploidy. Our findings reveal that REV3 depletion per se suppresses growth of cancer cell lines from different origin, whereas control cell lines and a mesothelial primary culture were less affected. Thus, our findings indicate that depletion of REV3 not only can amend cisplatin-based cancer therapy but also can be applied for susceptible cancers as a potential monotherapy.

In plain English

This was a laboratory study in cultured cells, not in people, and it did not study asbestos exposure. The authors used short hairpin RNA, delivered mainly by lentiviral particles, to lower levels of REV3, a protein that lets cells keep copying damaged DNA. They tested lung, breast, colon and mesothelioma cancer cell lines (the human ZL55 and rat IL45 mesothelioma lines), two control cell lines (AD293 and LP9-hTERT) and a normal mesothelial primary culture (SDM104). Lowering REV3 did not significantly reduce colony formation in AD293 (99% of mock-treated colonies), SDM104 (81%) or LP9-hTERT (98%). It did significantly reduce it in the cancer lines, for example to 27% in ZL55 and 4% in IL45. In HCT116 colon cancer cells this happened only at a higher virus dose. Cancer cells showed signs of persistent DNA damage and divided less. Cancer cells with working p53 showed signs of senescence, a lasting growth arrest. p53-deficient HCT116 cells instead stalled at the G2/M stage of cell division and more of them became aneuploid. The authors proposed that REV3 depletion could be applied as a potential monotherapy for susceptible cancers. They noted that the control lines might not fully represent normal cells.

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) cell-culture experiments: REV3 expression was reduced by shRNA and effects on colony formation, DNA damage markers, proliferation, cell cycle and senescence were measured
• Who was studied: Cell lines, not people: lung (A549, Calu-3), breast (MCF-7, MDA-MB-231), human mesothelioma (ZL55), rat mesothelioma (IL45) and colon (HCT116 with and without p53) cancer cell lines. Also the control cell lines AD293 and LP9-hTERT and the normal mesothelial primary culture SDM104. Work was done at University Hospital Zurich, Switzerland.
• Exposure: No asbestos or fiber exposure was studied. The experimental treatment was depletion of REV3 using short hairpin RNA (shREV3) delivered by lentiviral particles or plasmids, compared with mock treatment and a scrambled control (shSCR). Cisplatin (20 μM for 24 hours) was used in some experiments.
• Main results:
  • In the control cell line AD293, REV3 depletion did not significantly reduce colony formation (99% of mock-treated control colonies). The same was true for the primary mesothelial culture SDM104 (81%) and LP9-hTERT (98%).
  • REV3 depletion significantly suppressed colony formation in the cancer cell lines compared with mock-treated controls. Remaining colonies were A549 30%, Calu-3 57%, MDA-MB-231 47%, MCF-7 32%, human mesothelioma ZL55 27% and rat mesothelioma IL45 4%.
  • In HCT116 colon cancer cells, colony formation was significantly suppressed only with high-titer transduction (MOI 800). Remaining colonies were 49% in p53-proficient and 54% in p53-deficient HCT116.
  • In A549 cancer cells, REV3 depletion increased the average number of P-ATM foci per cell by a factor of 3.8 and γH2AX foci by a factor of 2.3 compared with mock control. Both are markers of DNA damage.
  • In the control cell line AD293, REV3 depletion did not significantly increase P-ATM, 53BP1 or γH2AX foci.
  • In A549 cells, micronuclei increased by a factor of 9 after REV3 depletion compared with mock control. Micronuclei are a sign of chromosomal instability.
  • Proliferation (BrdU incorporation) of A549 cells fell to 21% after REV3 depletion, compared with 37% in mock and 38% in scrambled controls. Replicating cells in AD293 and SDM104 were not diminished.
  • Senescence-associated β-galactosidase activity increased after REV3 depletion in p53-proficient IL45, A549 and HCT116 cells. In Table 1, senescent cells were 44.2% in shREV3 versus 2.8% in shSCR for IL45, and 16.1% versus 6.0% for A549. There was no increase in AD293, MDA-MB-231 or p53-deficient HCT116.
  • In p53-deficient HCT116 cells, REV3 depletion increased the fraction of cells in G2/M to 26%, compared with 17% in mock and 18% in scrambled controls. Aneuploid nondividing cells rose to 7%, compared with 3% and 4%.
  • No significant induction of apoptosis was observed in the control and cancer cell lines other than a slight increase in sub-G1 cells in p53-proficient A549 and HCT116.
• The authors' conclusion: The authors concluded that REV3 depletion suppressed growth of cancer cell lines while control cells were less affected. They proposed that REV3 depletion could amend cisplatin-based therapy and could be applied for susceptible cancers as a potential monotherapy. They also proposed that REV3 acts as a 'nononcogene addiction' gene in the cancer lines investigated.
• Limitations the authors state:
  • The control cell lines AD293 and LP9-hTERT are virally transformed or immortalized and might not fully represent normal cells.
  • Further studies are needed to clarify how normal cells tolerate the loss of REV3 function. Other groups found that REV3 knockout reduced growth of mouse embryonic fibroblasts.
  • The authors could not exclude that the alterations making cancer cells sensitive to REV3 depletion differ between the tested cancer cell lines.
  • Senescent cells that are not cleared by the immune system may remain dormant and represent a potential for tumor relapse.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Cisplatin.

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