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RNA interferences targeting the Fanconianemia/BRCA pathway upstream genes reversecisplatin resistance in drug-resistant lung cancer cellsChun-Hua Dai1, Jian Li2*, Ping Chen2, He-Guo Jiang2, Ming Wu3 and Yong-Chang Chen3

Abstract

Background: Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeuticefficacy of cisplatin is limited by the development of resistance.In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstreamgenes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells tocisplatin.

Result: Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. Theextent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cellswas significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL canreverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted inhigher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage.

Conclusion: Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin onsuppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.

Keywords: Chemoresistance, Cisplatin, Lung cancer, RNA interference, Fanconi anemia/BRCA pathway, Corecomplex

BackgroundCisplatin, a DNA cross-linking agent, is one of the mostcommonly used drugs in the treatment of lung cancer,including non-small cell lung cancer (NSCLC) and smallcell lung cancer (SCLC) [1, 2]. Although cisplatin-basedchemotherapy is used as the standard treatment for pa-tients with advanced NSCLC and SCLC, patients re-spond to the therapy had been more variable, andessentially all patients relapse due to acquired drug re-sistance [3, 4]. Thus, a great understanding of mecha-nisms of cisplatin resistance are essential to provide astrategy for sensitizing some drug-resistant tumor cellsand improve treatment of patients with lung cancer.

Cisplatin reacts with DNA to form intrastrand cross-links, which constitutes about 85–90 % of all lesions,and interstrand cross-links (ICLs) contributing about 1–3 % to the total lesion burden [5–7]. It is ordinarilythought that the intrastrand cross-links may be themajor type of DNA lesions responsible for the toxic ef-fects of cisplatin. However, due to its severe suppressingeffect on replication and transcription and the compli-cated character of its repair, the lower yield-formingICLs may greatly contribute to the toxicity of cisplatin[6, 7]. There is accumulating evidence that cellular re-sponse to cisplatin exposure include the activation ofmultiple DNA repair pathway and enhancement of DNAdamage repair process [7, 8]. It has been reported thatcisplatin resistance can arise from changes that increasea cell’s capacity to repair DNA damage [9, 10]. There-fore, DNA damage repair is one of main cisplatin resist-ant mechanisms. The DNA repair pathway that resolve

* Correspondence: [email protected] of Pulmonary Medicine, Affiliated Hospital of Jiangsu University,Zhengjiang 212001, ChinaFull list of author information is available at the end of the article

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Dai et al. Journal of Biomedical Science (2015) 22:77 DOI 10.1186/s12929-015-0185-4

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DNA ICLs, such as homologous recombination (HR)and translesion synthesis (TLS), are coordinated by aDNA damage response pathway termed the Fanconianemia (FA)/BRCA pathway [11].FA is a rare human genetic disease featured with se-

vere bone marrow failure, many congenital defects, andan extremely high cancer incidence [12]. Cells from pa-tients with FA display abnormally high sensitivity toDNA ICL-inducing agents, such as cisplatin and mito-mycin. Currently there are 16 verified FA genes thatmake up the FA/BRCA pathway [13–17]. The FA pro-teins encoded by these genes are thought to work co-operatively in a common signaling pathway to repairICLs [8, 11]. Upon DNA damage or upon entry into theS phase of the cell cycle, the core complex consists ofeight-protein (FANC-A, B, C, E, F, G, L, M) act as an E3ubiquitin ligase to monoubiquitinate FANCD2 and itsparalog FANCI [18]. Monoubiquitinated FANCD2/Icomplex is targeted to site of chromatin damage whereit recruits the downstream FA proteins (FANC-D1, N, J,O, P) and additional DNA repair proteins such asBRCA1. These downstream proteins participate in DNArepair by HR or TLC [8, 11, 17–20]. Monoubiquitinationand nuclear foci formation of FANCD2 are central stepsin the activation of FA/BRCA pathway [8, 11]. Cells de-fective in the upstream core complex are hypersensitiveto certain therapeutics, particularly ICL-inducing agents.The extent of hypersensitivity depends on the specificICL-inducing agent used [21, 22] and the defective FAprotein [23].It is realized that the integrity of the FA/BRCA path-

way is required for tumor resistance to cisplatin [8, 24].Consequently, the FA gene knockdown by RNA interfer-ence (RNAi) may sensitize tumor cells to cisplatin. Al-though inhibition of FANCF is known to result inFANCD2 ubiquitination inactivation and dysfunction ofthe FA/BRCA pathway, which enhance the sensitivity ofcancer cells to DNA cross-linking agents, such as cis-platin, mitomycin and melphalan in ovarian, breast can-cer, and myelomas [25–27], little is known about whichgene depletion in the upstream core complex is of astronger sensitizing effect to cisplatin in drug-resistancelung cancer cells. Therefore, we hypothesize that theknockdown of the different genes in the upstream corecomplex led to various degree sensitization to cisplatinin lung cancer cells. In this study, we test the impact ofFANCF, FANCL and/or FANCD2 knockdown by smallinterfering RNA (siRNA) on the FA/BRCA pathwayfunction and the sensitivity to cisplatin in human lungcancer cells A549 (an adenocarcinoma cell line), A549/DDP (a cisplatin-resistant A549 cell line), and SK-MES-1(a squamous cancer cell line). Our data show that thesensitization effect to cisplatin by depleting FANCF,FANCL and FANCD2 in A549/DDP cells was significantly

greater than in A549 cells. The knockdown of FANCL dis-play more significant sensitivity to cisplatin in A549/DDPcells, as opposed to the depletion of FANCF, comparableto that of the FANCD2, suggesting that the combinationof the gene therapy by depleting certain FA/BRCA path-way upstream gene and cisplatin has synergistic anti-tumor activity against cisplatin-resistant lung cancer cells.

MethodsCell lines and culture conditionsHuman lung caner cells A549 (an adenocarcinoma cellline), A549/DDP (a cisplatin-resistant A549 cell line),and SK-MES-1 (a squamous cancer cell line) were pur-chased from the Shanghai Institute for Biological Science(China). The cisplatin-resistant A549/DDP variant cellline was established by continuous exposure to increas-ing concentration of cisplatin for a 10 month period fol-lowing the methodology reported previously [28]. Thecharacteristics of A549/DDP cells have been describedpreviously [29]. The cells were grown in RPMI-1640complete medium supplemented with 10 % fetal calfserum (Gibco) in a humidified atmosphere containing5 % CO2 at 37 °C. A549/DDP cells medium additionallycontained 0.5 μg/ml cisplatin to maintain cells’ drug-resistant phenotype. Cells in the logarithmic phase ofgrowth were used for all experiments. A549/DDP cellswere cultured in complete media without cisplatin for5 days before performing experiments.

Small interfering RNA transfectionAll siRNA used in this study were purchased fromGuangzhou Riboio Co., Ltd (China). The siRNA se-quences used in this study are as follows: siRNAsagainst FANCF were siRNA-1 5′-GGUCAACGUUUGCACUAUG-3′, siRNA-2: 5′-GUACCUGGUCUUAGCAUCU-3′ and siRNA-3: 5′-CUUCGUAGUGGUGCAUUUA-3′; siRNAs against FANCL were siRNA-1 5′-GACAAGAGCTGTATGCTCT-3′, siRNA-2: 5′-CTTGCTGTGTGACTGTCAC-3′ and siRNA-3: 5′-CCAGGAAGCAACCACTTTC-3′; siRNAs against FANCD2were siRNA-1 5′-CCTCGTAGAAGCTTATCAA-3′,siRNA-2 5′-GGTTGGCTTAATAAATTCA-3′ and siRNA-3 5′-GCTCTTTCAACGTAAATTA-3′; and non-targeting control siRNA, 5′-UAGCGACUAAACACAUCAA-3′. The indicated siRNAs were synthesized byGuangzhou Riboio Co., Ltd (China). Cell transfectionwas performed using Lipofectamin 2000 according tothe manufacturer’s instructions. Cells were seeded infree-antibiotics media at a concentration of 1 × 105/well and cultured for 24 h before siRNA transfection.Black control, non-targeting control, siRNA-1, siRNA-2, and siRNA-3 groups were set up. Then cells weremixed with 5 μ/ Lipofectamin TM 2000 and 5 μ/siRNA, slightly waved and then incubated in 50 %

Dai et al. Journal of Biomedical Science (2015) 22:77 of 13

CO2 at 37 °C for 6 h, after which the medium waschanged to complete medium and incubation wascontinued for 48 h before harvesting cellular proteins.Efficacy of siRNA transfection was evaluated by west-ern blot and real-time quantitative RT–PCR. The tar-get siRNA was chosen for further investigation as ithad the highest intereference efficiency.All experiments in this study were performed in

triplicate.

Cell proliferation detectionThe cell Counting-kit (CCK-8) assays were performed tomeasure cell proliferation. A549 and A549/DDP cellswere seed at a density of 3 × 104 cells per well in 96-wellplates, and allowed to grow in the growth medium for24 h. Cells were then treated with 20 μg/ml cisplatin(He pharmaceutica Co., Ltd, China) with concentrationsordinally diluted by 50 %. There was a negative controlwith 0 μg/ml cisplatin. A blank group had no addedcells. The total medium volume of each well was 100 μl.Two day after drugs addition, 10 μl of cell proliferationreagent CCK-8 (DOJNDO, Japan) was added into mediain each well and the cell were incubated for 2 h at 37 °C.The absorbance of each well was determined with aspectrophotometer reading at a wavelength of 450 nm.Absorbance is assumed to be directly proportional tothe number of viable cells. The IC50 was calculated asthe cisplatin concentration that kills 50 % of cells of un-treated control.

Real-time quantitative-PCR analysisTotal RNA was extracted using a Trizol kit (Invitrogen,CA, USA), and the concentration and purity of the re-covered RNA were measured by UV-visible spectropho-tometer (Bio-Tek). Then cDNA was synthesized fromtotal RNA using MML-RT reverse transcriptase in ac-cordance with the manufacturr’s instructions.The real-time quantitative (RTQ)-PCR of FANCF,

FANCL, and FANCD2 mRNA and β-actin as internalcontrol were carried out on an ABI 7500 Thermal CyclerReal-time PCR system (Applied Biosystems, CA, USA),using the SYBR-Green I chemistry. Amplification primersof the three genes were synthesized by BioAsia Corpor-ation (Shanghai, China) as follows: primer sequences forFANCF were 5′-TGCTAACAGACTGGGGTCAAC-3′and 5′-TACAGGTCTCCAGGGCAGTTA-3′, and forFANCL, 5′-AAAGGCATTCTGGGATGTTTATG-3′ and5′-GAAAGAAGCACTCAGGAAGCAT-3′, and for FANCD2, 5′-CCUCGUAGAAGCUUAUCAADTDT-3′ and5′-DTDTGGAGCAUCUUCGAAUAGUU-3′ for β-actin,5′-TGACGTGGACATCCGCAAAG-3′ and 5′-CTGGAAGGTGGACAGCGAGG-3′. The cycling conditions havebeen described in detail in previous report [30]. Detectionof PCR products was accomplished by measuring the

emitting fluorescence (Rn) at the end of each reactionstep. Threshold cycle (Ct) corresponds with the cyclenumber required to detect a fluorescence signal above thebaseline. Relative quantification was calculated with the Ct(2—△△Ct) method [31].

Western blot assayWestern blot analyses were performed to detect FANCD2monoubiquitination and FANCF, FAMCL, FANCD2,cleaved-caspase-3, caspase-9, and poly ADP-ribose poly-merase (PARP) proteins. Total cell extracts were obtainedby direct lysis of cells in lysis buffer (50 mM Tris HCl,150 mM NaCl, 10 mM β-glycerophosphate, 1 % NP-40,1 μg/ml aprotinine, 1 μg/ml leupeptine, 1 μg/ml pepsta-tine, 0.5 mg/ml pefabloc, and 1 mM Na3VO4) boiled for5 min, and subjected to polyacrylamide SDS gel electro-phoresis (Sigma Co., USA). After electrophoresis, proteinswere transfer to Immobilonp-FL transfer membranes(Sigma Co., USA). The membranes were then blockedwith 5 % nonfat dried milk in PBS-T overnight at 4 °C andthen incubated at room temperature for 1 h or over-night at 4°Cin appropriate primary antibody diluted inPBS-T. After three times 5 min washes in PBS-T,membranes were then incubated for 1 h at roomtemperature in appropriate secondary antibody at adilution of 1:2000 in PBS-T + 0.5 % milk. Finally, blotswere washed five times for 5 min in PBS-T, and filmswere developed by using the ECL Western Blot Ana-lysis System (Beijing Sage Creation Technology Co.,Ltd, China). Immunoblots were probed with the fol-lowing primary antibodies: FANCF, FANCL, FANCD2(Santa Cruz and Rockland, USA), and cleaved-caspase-3, caspase-9, and PARP (Cell Signaling Tech-nology, USA).

Immunofluorescence analysisImmunofluorescence techniques were used to analyzeFANCD2 nuclear foci formation. Cells were seed onglass coverslips and incubated overnight before or aftertreatment with different concentration of cisplatin for24 h. Cells were then rinsed with PBS three times andfixed with 4 % paraformaldehyde in PBS for 15 min at4 °C. The fixed cells were permeabilized with 0.3 %Triton-x-100 (Sigma, USA) in PBS for 20 min on ice.After blocking in 30 % BAS for 1 h at room temperature,anti-FANCD2 antibodies (Santa Cruz, USA) were addedat dilution of 1:200. After overnight incubation at 4 °C,cells were washed three times with PBS and then incu-bated with fluorescent secondary antibodies (Sigma,USA) for 1 h at 37 °C. Then cells were rinsed with PBSthree times. Glass coverslips were mounted in Vecta-shied containing DAPI. Images were captured with aNikon fluorescence microscope and analyzed usingAdobe Photoshop software (Axanoae, USA).


Flow cytometry analysis of apoptosisThe percentage of apoptosis cells induced following cis-platin treatment was determined by flow cytometry withAnnexin V-FITC/PI staining. Exponential growing cellswere seeded in drug-free media at a concentration of1 × 105 cells/well overnight before experiment and thentreated with different concentration of cisplatin for 48 h.After treatment, the cells were harvested, washed twicewith PBS and resuspended in the provided binding buf-fer and subsequently staining with Annexin V-FITC (5-μg/ml) and incubated for 15 min in the dark at roomtemperature. Afterwards, the cells were analyzed by flowcytometry (BD Co., USA).

Statistical analysisStatistical analyses were performed using SPSS 16.0 ver-sion. All values were expressed as means ± standarderror of the mean (SEM). Comparisons between twogroups were conducted by unpaired t test. Group differ-ences resulting in P < 0.05 were considered to be statisti-cally significant.

ResultsCisplatin resistant A549/DDP cells have enhancedfunction of FA/BRCA pathwayUsing CCK-8 assay, we compared cisplatin sensitivity inthe drug-sensitive and drug-resistant lung cancer cells.As expected, A549/DDP cells had increased prolifera-tion rates compared with their drug-sensitive parentalA549 cells 24 and 48 h after cisplatin treatment in aconcentration-dependent manner (Fig. 1a). The fullintegrity of the FA/BRCA pathway is needed for tumorcell resistance to ICL-inducing agents and activation

extent of FA/BRCA pathway depend on the level ofFANCD2 monoubiquitination [11]. To determine whetherthe activation degree of the FA/BRCA pathway is associ-ated with cisplatin resistance in A549/DDP cells, we testedFANCD2 monoubiquitination after treatment with variousconcentration of cisplatin in the two cells. The resultsshowed that the levels of FANCD2 monoubiquitina-tion induced by cisplatin in A549/DDP cells, as indi-cated by the ratio of FANCD2-L (monoubiquitinatedform of FANCD2) to FANCD2-S (unubiquitinated formof FANCD2), were much higher than those in A549 cells(Fig. 1b-d). Immunofluorescent techniques were used toanalyze FANCD2 nuclear foci formation, a hallmark ofFanconi anemia pathway activation. Cisplatin-inducedFANCD2 foci formation, as measured by percentage ofcells with greater than five foci, was found to be signifi-cantly increased in A549/DDP cells than in A549 cells(Fig. 2). These results indicate that A549/DDP cells havemore marked activation of FA/BRCA pathway and stron-ger capability of DNA damage repair as compared withA549 cells, and also imply that the FA upstream proteinparticipate in cisplatin resistance and upregulatory func-tion of the FA/BRCA pathway may contribute to en-hanced ICL repair capacity in A549/DDP cells.

Efficiency of FANCF, FANCL and FANCD2 geneknockdown by siRNAIn order to assess the role of FANCF, FANCL, andFANCD2 in the sensitivity of lung cancer cells to cis-platin, we designed three different siRNA sequences(siRNA-1, siRNA-2, siRNA-3) for each gene to knock-down the three genes respectively in A549 cells. The ex-pression levels of both protein and mRNA of them were

Fig. 1 The proliferation rates and FANCD2 monoubiquitination in A549 and A549/DDP cells treated with DDP. a The proliferation rates in A549/DDPcells 24 and 48 h after cisplatin treatment were significantly higher than those in A549 cells. b FANCD2 monubiquitination in A549 cells induced bycisplatin with different concentrations. c FANCD2 monubiquitination in A549/DDP cells induced by cisplatin with different concentrations. d The levelsof FANCD2 monoubiquitination in A549/DDP cells were markedly higher than those in A549 cells after cisplatin treatment


determined after the trasfection of different siRNAsfor 48 h. As shown in Fig. 3a, b, the FANCF-siRNA-3and FANCL-siRNA-3 significantly down-regurated bothmRNA and protein expression levels of FANCF andFANCL, compared with FANCF-siRNA-1 or -siRNA-2and FANCL-siRNA-1 or -siRNA-2. Therefore, FANCF-siRNA-3 and FANCL-siRNA-3 were selected for followedinvestigation. Meantime, western blot and QRT-PCR ana-lysis showed that the interference efficiency of FANCD2-siRNA-2 was the highest among three siRNAs againstFANCD2 (Fig. 3c), thus FANCD2-siRNA-2 was chosen asthe target siRNA for further experiments. In addition,there was no significant difference in expression levels ofthe three genes between the black control group and non-targeting control group.

Knockdown of FANCF, FANCL, or FANCD2 enhancesensitivity of A549 and SK-MES-1 cells to cisplatinUsing RNAi approach, we knocked down the expressionof FANCF, FANCL, and FANCD2 in A549 cells. Asshown in Fig. 4a, depletion of FANCF or FANCL indi-vidually sensitized A549 cells to cisplatin. Although cells

depleted of FANCL appear to be more sensitive to cis-platin than cells depleted of FANCF, difference in theIC50 between the two cells did not reach statistical sig-nificance (Table 1). Moreover, individual depletion ofFANCF or FANCL from these cells and the double de-pletion of both FANCF and FANCL resulted in compar-able hypersensitivity to cisplatin (Fig. 4a and Table 1).These data indicate that FANCF and FANCL function ina common pathway, or at least a partially overloppingpathway in the repair of DNA ICLs. The notion wasdemonstrated by the results of Western blot analysis,which shown that the level of FANCD2 monoubiquitina-tion induced by cisplatin was dramatically and similarlyreduced in A549 cells individually depleted of FANCFand FANCL (Fig. 4b, c). Furthermore, the double deple-tion of FANCF and FANCL did not further reduceFANCD2 monoubiquitination (Fig. 4b, c).Meanwhile, we assessed the impact of FANCD2 sta-

tus on the ability of DNA ICLs repair and the cellproliferation after treatment with cisplatin. As seen inFig. 4d, A549 cells lacking FANCD2 were remarkablysensitive to the antiproliferative effects of this ICLs

Fig. 2 Immunofluorescence and quantification of cells exhibiting 5 or more FANCD2 foci in lung cancer cells. Cisplatin-induced FANCD2 fociformation in A549 (a) and A549/DDP cells (b) were inhibited markedly by knockdown of FANCF, FANCL, and FANCD2. c The graph below showquantified data of FANCD2 foci in the two cells. cisplatin-induced FANCD2 foci expressions were stronger in A549/DDP than in A549 cells


agent. Co-depletion of FANCD2 and FANCF, or andFANCL did not cause more profound sensitization tocisplatin (Fig. 4d, Table 1). Given that these geneknockdowns were highly effective, the results confirmthat FANCF and FANCL are epistatic of FANCD2and are involved in the same pathway to confer toler-ance of cisplatin-induced DNA damage. Consistently,co-depletion of both FANCD2 and FANCF, or FANCLproduced no additional decrease of FANCD2 monou-biquitination levels upon DNA damage induced bycisplatin, when compared with depletion of FANCD2alone (Fig. 4e, f ). Additionally, cisplatin-induced FANCD2foci formation was inhibited by depleting FANCF, FANCL,or FANCD2 in similar degree (Fig. 2a, c), which wereidentical with results of FANCD2 monoubiquitinationanalyses.SK-MES-1 cell is a lung squamous cancer cell which

display slight resistance to cisplatin (IC = 9.92 ± 0.35,Table 1) compared with A549 cell (IC = 4.97, Table 1).We examined the change of sensitivity in SK-MES-1cells by knocking down FANCF, FANCL, and FANCD2using siRNA transfection, and observed FANCD2 mono-ubiquitination levels. The results showed that individualand double knockdown of FANCF, or FANCL, orFANCD2 markedly enhanced the sensitivity of SK-MES-

1 cells to cisplatin, and reduced the levels of FANCD2monoubiquitination (Fig. 5a-f ), which are in line withthe findings in A549 cells.

Knockdown of FANCL results in higher cisplatin sensitivitycompared with knockdown of FANCF in A549/DDP cellsThe above-mentioned experimental results have shownthat FA/BRCA pathway involved the sensitivity to cis-platin in drug-resistant A549/DDP cells. We next ex-plored relative roles of FANCF and FANCL in thesensitivity to cisplatin by depleting FANCF only, FANCLonly, or both the two core complex components, andtesting cisplatin-induced cell proliferation in A549/DDPcells. As shown in Fig. 6a, siRNA mediated depletion ofFANCF or FANCL significantly sensitized A549/DDPcells to cisplatin. It is worth noting that cells depleted ofFANCL were more sensitive to cisplatin than those de-pleted of FANCF. As seen in Table 1, the IC50 value inFANCF depleted cells was significantly higher than thatin FANCL depleted cells (P < 0.001). But co-depletion ofFANCF and FANCL did not produce any additional sen-sitivity over that of FANCL. The differential sensitizationsuggests that FANCL has an epistatic yet more import-ant role than FANCF in cellular resistance to cisplatin.Moreover, western blot and immunofluorescence assay

Fig. 3 Expression of FANCF, FANCL and FANCD2 in A549 cells after siRNA transfection. The protein and mRNA relative expression levels of FANCF(a) and FANCL (b) were the lowest by using siRNA-3 transfection among three-different siRNA transfections. c The protein and mRNA expressioninhibitions of FANCD2 were the strongest by using siRNA-2 transfection among three-different siRNA transfections. BC: black control; NC:non-target control


revealed that individual or double depletion ofFANCF and FANCL resulted in significant decrease ofFANCD2 monoubiquitination levels (Fig. 6b, c) and in-hibition of FANCD2 foci formation (Fig. 2b, c).Additionally, we found that reduced extent of prolifer-

ation rate induced by cisplatin treatment in A549/DDPcells was significantly greater than in A549 cells afterknockdown of FANCF or FANCL (Fig. 4a and 6a,Table 1), suggesting that knockdown of FANCF orFANCL can reverse resistance of cisplatin-resistant lungcancer cells to cisplatin, whereas the knockdown of thetwo genes in cisplatin-sensitive lung cancer cells onlymoderately increase sensitivity to cisplatin.

Knockdown of FANCD2 lead to a similar cell sensitivity tothat of FANCL in A549/DDP cellsWe also evaluated the sensitizing effect of FACD2knockdown on cisplatin in A549/DDP cells. As shown inFig. 6d, depletion of FANCD2 remarkably reduced cellproliferation rate after cisplatin treatment. Nevertheless,depletion of FANCD2 alone, or co-depletion of FANCD2and FANCF, or and FANCL exhibited a similar reduc-tion in cell proliferation following cisplatin treatment.Consistent with the sensitization effects of these geneknockdowns on cisplatin, cells depleted of FANCD2alone, and cells co-depleted of both FANCD2 andFANCF, or FANCL, had nearly identical decrease of

Fig. 4 The effect of FA upstream gene knockdowns on proliferation and FANCD2 monoubiquitination in A549 cells. a Knockdowns of FANCF,FANCL, or both them inhibited the proliferation rates, and (b, c) reduced FANCD2 monoubiquitination levels after cisplatin treatment. dKnockdown of FANCD2, or double knockdown of FANCD2 and FANCF, or and FANCL suppressed the proliferation rate, and (e, f) droppedFANCD2 monoubiquitination levels after cisplatin treatment

Table 1 The 50 % inhibitory concentration (IC50) of cisplatin in A549 and A549/DDP cells 48 h after drug treatment

Cell Control siFANCF siFANCL siFANCF+ siFANCD2 siFANCD2+ siFANCD2+

siFANCL siFANCF siFANCL

A549 4.97 ± 0.32 3.05 ± 0.24a 2.46 ± 0.21b 2.44 ± 0.27 b 2.48 ± 0.31 b 2.45 ± 0.18 b 2.40 ± 0.23 b

A549/DDP 34.15 ± 0.42 4.43 ± 0.28c 2.51 ± 0.33c, d 2.48 ± 0.25 c, d 2.54 ± 0.17 c, d 2.52 ± 0.22 c, d 2.47 ± 0.19 c, d

SK-MES-1 9.92 ± 0.35 3.47 ± 0.25c 2.68 ± 0.29c 2.45 ± 0.32c 2.58 ± 0.23c 2.50 ± 0.33c 2.46 ± 0.35c

acompared with control, p = 0.009; bcompared with control, p = 0.001; ccompared with control, p < 0.001; dcompared with siFANCF, p < 0.001


FANCD2 monoubiquitination (Fig. 6e, f ). Moreover, theIC50 value of FANCD2 knockdown cells was similar tothat of FANCL depleted cells, but significantly lowerthan that of FANCF depleted cells (P < 0.001) (Table 1).Taken together, these results further support the ideathat FANCF and FANCL are epistatic to FANCD2, andFANCF and FANCL function in a common pathway, butFANCL may play a more important role in facilitatingFANCD2 monoubiquitination.

Knockdown of FA upstream proteins promotes apoptosisof A549/DDP cellsTo explore whether effect of proliferative inhibition incisplatin-resistant lung cancer cells by depleting FANCFor FANCL was associated with apoptosis, we detectedthe apoptosis rate of the A549/DDP cells induced by cis-platin following depletion of the two genes. As shown inFig. 7, compared with the control, depletion of FANCF

or FANCL in A549/DDP cells significantly increased thepercentage of both early and late apoptosis cells aftercisplatin treatment in a concentration-dependent man-ner (Fig. 7a-d, P = 0.001 and P < 0.001). Furthermore, thepercentages of apoptosis cells in FANCL depleted cellswere markedly higher than those in FANCF depletedcells, when treatment with 10 μg/ml and 20 μg/ml cis-platin (Fig. 7d, P = 0.001 and P < 0.001), indicating thatknockdown of FANCF or FANCL by siRNA could syner-gize the effect of cisplatin on inducing apoptosis ofcisplatin-resistant lung cancer cells through suppressingFA/BRCA pathway. To investigate if the apoptosiscaused by depleting FANCF or FANCL is caspase-dependent, we analyzed caspase-dependent apoptpsis-associated proteins by western blot. The results showedthat depletion of FANCF and FANCL clearly increasedthe levels of cleaved caspase-3, caspase-9, and PARPproteins in A549/DDP cells (Fig. 8a, b), indicating that

Fig. 5 The effect of FA upstream gene knockdowns on proliferation and FANCD2 monoubiquitination in SK-MES-1 cells. a Knockdowns of FANCF,FANCL, or both them inhibited the proliferation rates, and (b, c) reduced FANCD2 monoubiquitination levels after cisplatin treatment. (d) Knockdownof FANCD2, or double knockdown of FANCD2 and FANCF, or and FANCL suppressed the proliferation rate, and (e, f) dropped FANCD2monoubiquitination levels after cisplatin treatment


knockdown of FANCF and FANCL activate caspase-dependent apoptosis.

DiscussionIncreasing evidences indicate that cancer cell resistanceto cross-links agents, such as cisplatin, is linked to thefunctional status of the FA/BRCA pathway [8, 11, 25–27], albeit the exact mechanism of this resistance to thechemical crosslinker remains to be fully elucidated. Inthis study, we confirm that upregulation of FANCD2monoubiquitination and activation of FA/BACA path-way contributes to acquired resistance to cisplatin forlung cancer A549/DDP cells. Moreover, we show thatknockdown of FANCF and/or FANCL by siRNA potenti-ates sensitivity to cisplatin in cisplatin-sensitive and -re-sistant lung cancer cells.Within the FA/BRCA pathway, eight upstream FA

protein and some FA-associated proteins form aligase core complex that monoubiquitinates both com-ponents of the ID complex, FANCD2 and FANCI, inresponse to DNA damage and replication stress duringS phase. The FA core complex mainly comprises three

subunits, including FANCB-FANCL-FAAP100 (B-L-100),FANCC-FANCE-FANCF (C-E-F) and FANCA-FANCG-FAAP20 (A-G-20) [32–37]. It is generally thought that asingle mutation in any of the FA upstream genes thatmake up the core complex prevents the FANCD2 monou-biquitination event from occurring. FANCF had been im-plicated in the physical stability of the majority of othercore complex proteins, FANCC, FANCE, FANCG andFANCA by several groups and functions as a adaptor thatis essential for the integrity of the core complex and themonoubiquitination of FANCD2 [34, 38]. FANCL is con-sidered a key component of the FA core complex and rep-resent the E3 ligase necessary for ubiquitin conjugation tolysine 561 of FANCD2 and lysine 523 of FANCI [39, 40].The depletion of FANCF gene results in the inhibition ofFANCD2 monoubiquitination and the enhancement ofsensitivity to DNA cross-linking agents (melphalan, cis-platin and mitomycin) in myeloma, ovarian and breastcancer cells [25–27]. In line with these reports, we resultsshow that knockdown of FANCF sensitized drug-sensitiveand -resistant lung cancer cells to cisplatin and diminishedthe levels of FANCD2 monoubiquitination. Similarly,

Fig. 6 The effect of FA upstream gene knockdowns on proliferation and FANCD2 monoubiquitination in A549/DDP cells. a Knockdown ofFANCF, FANCL, or both them markedly inhibited the proliferation rates, and (b, c) reduced FANCD2 monoubiquitination levels after treatmentwith cisplatin. (d) Knockdown of FANCD2, or double knockdown of FANCD2 and FANCF, or and FANCL significantly suppressed the proliferationrates, and (e, f) reduced FANCD2 monoubiquitination levels after treatment with cisplatin


Fig. 7 (See legend on next page.)


knockdown of FANCL increase sensitivity to cisplatin inthe lung cancer cells. However, the sensitization effect tocisplatin by depleting FANCF or FANCL in A549/DDPcells was more significant as compared with A549 andSK-MES-1 cells. For instance, the IC50 values of A549/DDP cells transfected with FANCL-siRNA were reducedfrom 34.15 μg/ml pre-transfection to 2.51 μg/ml post-transfection, whereas the IC50 values of A549 cells pre-and post-transfection were 4.97 μg/ml and 2.46 μg/ml, re-spectively (Table 1). Also, the dropped extent of FANCD2monoubiquitination levels in A549/DDP cells was greaterthan in A549 cells following depletion of FANCF and/orFANCL. The results suggest that enhanced E3 ligase activ-ity of the core complex in A549/DDP cells might be re-sponsible for cisplatin resistance, and the suppression ofthe core complex ligase activity and downregulation ofFANCD2 monoubiquitnation by siRNA targeting FANCFor FANCL can reverse resistance of cisplatin-resistantlung cancer cells to cisplatin.In this study, another interesting finding is that knock-

down of FANCL led to more dramatic sensitization

effect on cisplatin compared with knockdown of FANCFin A549/DDP cells. The IC50 value of depleted FANCFA549/DDP cells was markedly higher than that ofdepleted-FANCL cells 48 h after cisplatin treatment.Moreover, apoptosis rates of A549/DDP cells depleted ofFANCL were significantly elevated following cisplatintreatment, not only compared with those of control, butalso compared with those depleted of FANCF. But co-depletion of FANCF and FANCL did not have a signifi-cantly additive impact on sensitivity to cisplatin. Thisphenomenon was different from A549 cells, in which in-dividual knockdown of FANCF and FANCL, or in com-bination, resulted in comparable cisplatin sensitivity andlevels of FANCD2 monoubiquitination. These findingsuggest that FANCL may play a notably important rolefor acquired resistance to cisplatin in A549/DDP cells,although FANCF and FANCL were epistatic and act inthe common FA/BRCA pathway.A recent study shown that not all the components of

the core complex contribute equally to cellular resist-ance against DNA damage [23], even though the study

(See figure on previous page.)Fig. 7 Knockdown of FANCF and FANCL promote the apoptosis induced by cisplatin in A549/DDP cells. a The apoptosis rates of control cells, and (b)cells transfected with FANCF-siRNA, and (c) cells transfected with FANCL-siRNA after cisplatin treatment. Early apoptotic cells are in the lower rightquadrant; late apoptotic cells are in the upper right quadrant. d The apoptosis rates in cells transfected with FANCF-siRNA or FANCL-siRNA weresignificantly higher than in control cells in a concentration-dependent manner. In addition, the apoptosis rates of cells transfected with FANCL-siRNAhave dramatic increase as compared with cells transfected with FANCF-siRNA following treatment of 10 μg/ml and 20 μg/ml cisplatin

Fig. 8 Caspase-3, caspase-9, and PARP expressions were detected by Western blot in A549/DDP cells. a The expressions of cleaved caspase-3,caspase-9, and PARP induced by cisplatin were elevated after siFANCF transfection. b The expressions of cleaved caspase-3, caspase-9, and PARPinduced by cisplatin were increased after siFANCL transfection


results deviates from a general idea that losing any oneof the core comlex components results in completeelimination of FANCD2 activation and disintegration ofthe core complex. The study reported that the subunitsin core complex are of different function and the overallintegrity of the core complex is sustained when certainFA proteins are removed. Among these subunits, FANCB,L and FAAP100 constitute a catalytic subunit that is abso-lutely required for the E3 ligase activity of the core com-plex, whereas the A-G-20 and C-E-F subunits providenon-redundant and ancillary functions that help the cata-lytic subunit bind chromatin or site of DNA damage [23].Also, other recent studies suggest a key role of FANCL asthe E3 ligase in the monoubiquitination of FANCD2 andactivation of FA/BRCA pathway. For instance, DT40 cells(a chicken somatic cell) lacking FANCL, B, or FAAP100completely lost the ability to monoubiquitinate FANCD2,implying that subunit is indispensable for FANCD2 mon-ubiquitination. However, FANCD2 monoubiquitinationactivity in cells depleted of FANCC and FANCF was com-parable to the wild–type cells [37]. Furthermore, the studyfound that in comparison to wild-type cells, residualchromatin-associated FANCD2 monoubiquitination wasstill present in the cells lacking FANCA, FANCG, FANCF,or FANCC [37]. Therefore, loss of different FA core com-ponents produces variable impact on the activation of theFA/BRCA pathway and correspondingly variable cellularsensitivity to cross-linking agents. Perhaps these findingsmay explain our results that depletion of FANCL had amore potent sensitization on A549/DDP cells than knock-down of FANCF. However, it is unclear why there was nosignificant difference for the sensitivity of A549 cells tocisplatin between knockdown of FANCF and FANCL. Butit is known that cisplatin-induced DNA cross-links mightbe repaired by FANCF and FANCL which are componentsof FA core complex. FANCF might play a minor role inthe repair pathway for cisplatin-induced damage, whereasFANCL play a crucial role as mentioned above. The differ-ence for repair capacity of DNA damage between FANCFand FANCL might be more significant in cisplatin-resistant lung cancer cells. Further study is required to as-certain the precise mechanism that DNA repair activitiesof FA/BRCA pathway in cisplatin-resistant cells differfrom cisplatin-sensitive cells.It is known that monoubiquitinated FANCD2-I (ID)

complex is at the heart of the FA/BRCA pathway. Themonoubiquitination of FANCD2 at lysine 561 is essentialfor the downstream function of the pathway in DNAdamage repair [41]. Cells expressing k561R FANCD2,which is a mutant form of FANCD2 and cannot bemonoubiquitinated, have increased sensitivity to cross-linking agent compared with wild-type FANCD2 [41].Consistent with previous reports using other cells [42,43], siRNA-mediated depletion of FANCD2 potently

sensitized A549, A549/DDP and SK-MES-1 cells to cis-platin, which is similar to the sensitization of the threecells to cisplatin by depleting FANCL. As expected, wedid not observe further sensitization of the three cellsto cisplatin upon treatment with double depletion ofFANCD2 and FANCF, or FANCL, further supportingthe notion that FANCD2, FANCF and FANCL act ina common DNA repair pathway.Cisplatin-based combination chemotherapy is the first-

line therapy for SCLC and advanced NSCLC. Despiteconsiderable success in treatment of lung cancer, the ef-fectiveness of cisplatin in clinics has been limited by theresistance that cancer cells developed during the courseof chemotherapy. Thus, overcoming tumor cells resist-ance to cisplatin using RNAi targeting the FA upstreamgenes in combination with cisplatin is a promising strat-egy for treating lung cancer. With recent advance ingene therapy techniques, improvements of in vivo RNAidelivery methodologies indicate that adjuvant siRNAtherapy may be achievable in accessible tumor sites [44].Further investigations are warranted to determine whetherthe gene therapy by targeting the FA/BRCA pathway com-bined with chemotherapy can used as an effective noveltherapeutic regimen for patients with lung cancer,particular those with tumor relapse or progressionafter chemotherapy.

ConclusionIn summary, we herein report that depletion of FANCF,FANCL, or FANCD2 by using RNAi could reverse cis-platin resistance in cisplatin-resistant lung cancer cellsthrough inhibition of FA/BRCA pathway. Moreovre,knockdown of FANCL resulted in higher cisplatin sensi-tivity and dramatically elevated apoptosis rates comparedwith knockdown of FANCF, indicating that FANCL playan important role in the repair of cisplatin-induced DNAdamage. Targeting of the FA/BRCA pathway may consti-tute a potential treatment modality for lung cancer.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsC-HD and JL contributed to the conception of design of the study, analysis ofdata, and wrote the manuscript. PC, H-GJ, and MW performed the experimentsand the statistical analysis. Y-CC participated in the design of the study, analysisof data, and revised the manuscript. All authors read and approved the finalmanuscript.

AcknowledgementsThis research was supported by Center of Experimental Medicine, AffiliatedHospital of Jiangsu University.

Author details1Department of Radiation Oncology, Affiliated Hospital of Jiangsu University,Zhengjiang 212001, China. 2Department of Pulmonary Medicine, AffiliatedHospital of Jiangsu University, Zhengjiang 212001, China. 3Institute ofMedical Science, Jiangsu University, Zhengjiang 212013, China.


Received: 4 June 2015 Accepted: 10 September 2015

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