Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Oct 4;7(40):65157-65170.
doi: 10.18632/oncotarget.11214.

Co-inhibition of pol θ and HR genes efficiently synergize with cisplatin to suppress cisplatin-resistant lung cancer cells survival

Chun-Hua Dai  1 , Ping Chen  2 , Jian Li  2 , Tin Lan  3 , Yong-Chang Chen  3 , Hai Qian  3 , Kang Chen  2 , Mei-Yu Li  2
Affiliations

Co-inhibition of pol θ and HR genes efficiently synergize with cisplatin to suppress cisplatin-resistant lung cancer cells survival

Chun-Hua Dai et al. Oncotarget. .

Abstract

Cisplatin exert its anticancer effect by creating intrastrand and interstrand DNA cross-links which block DNA replication and is a major drug used to treat lung cancer. However, the main obstacle of the efficacy of treatment is drug resistance. Here, we show that expression of translesion synthesis (TLS) polymerase Q (POLQ) was significantly elevated by exposure of lung cancer cells A549/DR (a cisplatin-resistant A549 cell line) to cisplatin. POLQ expression correlated inversely with homologous recombination (HR) activity. Co-depletion of BRCA2 and POLQ by siRNA markedly increased sensitivity of A549/DR cells to cisplatin, which was accompanied with impairment of double strand breaks (DSBs) repair reflected by prominent cell cycle checkpoint response, increased chromosomal aberrations and persistent colocalization of p-ATM and 53BP1 foci induced by cisplatin. Thus, co-knockdown of POLQ and HR can efficiently synergize with cisplatin to inhibit A549/DR cell survival by inhibiting DNA DSBs repair. Similar results were observed in A549/DR cells co-depleted of BRCA2 and POLQ following BMN673 (a PARP inhibitor) treatment. Importantly, the sensitization effects to cisplatin and BMN673 in A549/DR cells by co-depleting BRCA2 and POLQ was stronger than those by co-depleting BRCA2 and other TLS factors including POLH, REV3, or REV1. Our results indicate that there is a synthetic lethal relationship between pol θ-mediated DNA repair and HR pathways. Pol θ may be considered as a novel target for lung cancer therapy.

Keywords: cisplatin-resistance; homologous recombination; lung caner cells; pol θ; translesion synthesis.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. A549/DR cells are resistant to cross-linking agents, and expression of FA, HR and TLS pathway factors are elevated compared with A549 and SK-MES-1 cells
A. A549, SK-MES-1, and A549/DR cells growing in 96-well plates were treated with cisplatin, carboplatin and BMN673 at the indicated dose. The CCK-8 assay was used to determine cell survival. B. and C. Total RNA was isolated from A549, SK-MES-1 and A5491DR cells. RNA was subjected to real time quantitative-PCR to determine the mRNA levels of the FA, HR and TLS pathway factors as the indicated. (★ compared with A549 and SK-MES-1 cells, P < 0.05; ◆ compared with A549 and SK-MES-1 cells, P < 0.01). D. and E. Whole cell lysate was prepared from the A549, SK-MES-1 and A 549/DR cells and subject to Western blot with specific antibodies as the indicated to determine the protein levels of various FA, HR and TLS pathway factors (◆ compared with A549 and SK-MES-1 cells, P < 0.05; ★ compared with A549 and SK-MES-1 cells, P < 0.01).
Figure 2
Figure 2. Expressions of POLQ were significantly increased in A549/DR cells compared with POLH, REV3, and REV1 by exposure of the cells to cisplatin
A. and C. Real-time quantitative-PCR was performed to determine mRNA expression of TLS pathway factors as indicated in A549/DR and A549 cells at different time points after cisplatin treatment. The expression of POLQ was normalized to GAPDH; the untreated control was set to one. (★ compared with POLH, REV3 and REV1, P < 0.05). B. and D. Protein expression of TLS pathway factors as the indicated was analyzed by Western blot using specific antibodies in whole cell lysate of A549/DR and A549 cells after cisplatin treatment. β-actin was used as loading control (◆ compared with Pol η, Pol ζ and REV1, P < 0.01).
Figure 3
Figure 3. The changes of sensitivity to cisplatin and BMN673 in A549/DR cells and A549 cells after transfections of siRNAs against to TLS pathway factors
A. Validation of siRNAs used in this study. Representative western blot showing POLQ, POLH, REV3 and REV1 expression in A549/DR and A549 cells. Cells were transfected with the indicated siRNAs for 48 hours. Whole cell lysates were prepared and subjected to Western blot for detecting the protein expressions of these factors. B. and D. A549/DR cell and C. and E. A549 cells growing in 96-well plates were transfected with various siRNA as indicated. Cell survival was determined by CCK-8 assay following cisplatin or BMN673 treatment. F. A549/DR cells depleted of POLQ, POLH, REV3 or REV1 exhibit a cisplatin-induced cell cycle arrest in S/G2 phases. The cells were exposure to 10 μm cisplatin and subject to cell cycle analysis 24h later by flow cytometry.
Figure 4
Figure 4. A549/DR cells depleted of POLQ, POLH, REV3 or REV1 display significant DNA damage response, and depletion of POLQ remarkably enhance RAD51 expression
A. and B. A549/DR and A549 cells were treated with indicated dose of cisplatin, and fixed and immunostained with γH2AX antibody. The percentage of cells with > 10 γH2AX foci was shown as the mean ± SEM from three independent experiments (★ compared with siREV3 and siREV1, P < 0.05). Additional representative images are shown in Supplementary Figure S2. C-F. siRNA transfected A549/DR and A549 cells were treated with cisplatin at indicated dose for 2 hours, cells were harvested and subject to Western blot with antibodies as indicated (◆ compared with siPOLH, siREV3 and siREV1, P < 0.005). G. siRNA transfected A549/DR cells were treated with indicated dose of cisplatin, and fixed and immunostained with RAD51 antibody. The percentage of cells with >10 RAD51 foci was quantified from Image Software (▲ compared with siPOLH, siREV3 and siREV1, P < 0.01).
Figure 5
Figure 5. Co-depletion of POLQ and FANCD2 or BRCA2 markedly increase sensitivity of A549/DR cells to cisplatin and BMN673 compared with double depletion of BRCA2 and POLH, or REV3, or REV1
A. Representative western blot showing BRCA2, RAD51, FAAP20 and FANCD2 expression in A549/DR cells after siRNA transfections. Expressions of Pol θ were markedly increased after transfection with siRNAs against FANCD2, FAAP20, BRCA2, and RAD51C. B. and C. Expressions of POLQ mRNA in A549/DR and A549 cells were significantly elevated after transfection with siRNAs against FANCD2, FAAP20, BRCA2, and RAD51C. Real-time quantitative-PCR was used to determine mRNA expressions. (★ compared with siControl, P < 0.001; ◆ compared with siControl, P < 0.01). D. and E. A549/DR cells were treated with cisplatin or BMN673 at the indicated dose following transfection with various siRNAs as indicated. Then cell survival was determined by the CCK-8 assay. F. and G. A549/DR cells were treated with cisplatin or BMN673 at the indicated dose following transfection with various siRNAs as indicated. The cells were then stained by crystal violet and total colonies were counted after two weeks. Colony numbers of control-treated cells were set as 100%. H. Co-depletion of BRCA2 and POLQ result in dramatically increased sub-G1 cells in response to cisplatin. A549/DR cells transfected with siRNAs as indicated were exposure to cisplatin, and subject to cell cycle analysis by flow cytometry.
Figure 6
Figure 6. Co-depletion of BRCA2 and POLQ in A549/DR cells caused strikingly cisplatin-induced cell cycle checkpoint response, and an inhibition of HR, and increased cisplatin-induced P-ATM and 53BP1-colocalized foci
A. A549/DR cells co-depleted of BRCA2 and POLQ display notably enhanced cisplatin-induced phosphorylation of H2AX, CHK1, CHK2 and KAP1 proteins, and B. show a significant decrease of percentage of GFP positive cells (★ compared with siBRCA2, siPOLQ, siBRCA2 +siPOLH, siBRCA2+siREV3 and siBRCA2+siREV1, P < 0.05), and C. a marked increase of cisplatin-induced, and D. BMN673-induced chromosomal aberrations (◆ compared with siBRCA2, siPOLQ and siBRCA2+siREV3, P < 0.05), and E. exhibit markedly increased percentage of cells with P-ATM and 53BP1-colocalized foci after cisplatin treatment (▲ compared with siBRCA2, siPOLQ, siBRCA2+siPOLH, siBRCA2+siREV3, and siBRCA2+siREV3, P < 0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fuertes MA, Castilla J, Alons C, Perez JM. Cisplatin biochemical mechanism of action: from cytotoxicity to induction of cell death through interconnections between apoptotic and necrotic pathways. Cur Med Chem. 2003;10:257–266. - PubMed
    1. Ahmad S. Platinum-DNA interactions and subsequent cellular processes controlling sensitivity to anticancer platinum complexes. Chem Biodivers. 2010;7:543–566. - PubMed
    1. Jung Y, Lippord SJ. Direct cellular response to platinum-induced DNA damage. Chem Rev. 2007;107:1387–1407. - PubMed
    1. Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat Rev Discov. 2005;4:307–320. - PubMed
    1. Stewart DJ. Mechanisms of resistance to cisplatin and carboplatin. Crit Rev Oncol Hematol. 2007;63:12–31. - PubMed

MeSH terms