Cancer Investigation, 29:347–352, 2011ISSN: 0735-7907 print / 1532-4192 onlineCopyright C© 2011 Informa Healthcare USA, Inc.DOI: 10.3109/07357907.2011.568566

ORIGINAL ARTICLE: CELLULAR AND MOLECULAR BIOLOGY

Interferon-α Induces G1 Cell-Cycle Arrest in Renal Cell Carcinoma CellsVia Activation of Jak-Stat Signaling

Donghao Shang,1∗ Peiqian Yang,1∗ Yuting Liu,2 Jian Song,1 Fengbo Zhang,1 and Ye Tian1

Department of Urology, Friendship Hospital, Capital Medical University, Beijing, China,1 Department of Pathology, CapitalMedical University, Beijing, China2

The purpose of this study was to clarify the mechanism of IFN-αresistance in RCC. The effects of IFN-α on induction ofapoptosis and cell-cycle arrest were analyzed by flowcytometric analysis. Jak-Stat pathway components induced byIFN-α was evaluated using Western blotting. The resultssuggested that IFN-α caused growth inhibition of RCC cell linesvia arrest in the G1 phase without inducing apoptosis. Theresistance of RCC to IFN-α was associated with the lowexpression of Stat1. This study indicated that the Jak-Statpathway should be considered a primary target for improvingthe response of RCC to IFN-α treatment.

Keywords: IFN-α, Cell cycle arrest, Jaks-Stat, Renal cellcarcinoma

INTRODUCTION

Renal cell carcinoma (RCC) is the most common cancer ofthe adult kidney, and survival is poor once metastatic dis-ease develops, with a 5-year survival rate of approximately20% (1). RCC is also resistant to conventional chemother-apy (2). At present, interferon-alpha (IFN-α) is the first-lineagent for treating some cancers, and treatment regimens us-ing IFN-α have been applied to overcome RCC. This ap-proach has demonstrated therapeutic response rates around4–33% (3). Recent studies reveal that IFN-α may mediate an-titumor effects either indirectly by modulating immunomod-ulatory and anti-angiogenic responses or directly via antipro-liferation effects, which are associated with the cellular differ-entiation of tumor cells (4).

IFN-α exerts its effects by binding to cell surface receptorsand thus activating members of the Janus kinase (Jak) family.Activated Jak1 or tyrosine kinase 2 (Tyk2) can phosphorylatesignal transducers and activators of transcription (Stats), andactivated Stats can then translocate to the nucleus and inter-act with specific regulatory elements to induce transcriptionof specific target genes (5). Despite the relative beneficial ef-fects of IFN-α for treatment of RCC, the mechanism formingthe basis of the observed growth suppression and sensitivity

∗These authors contributed equally to the work.Correspondence to: Ye Tian, Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China. email:tianye6251@163.com

of RCC to IFN-α is still not completely clear. Thus, a betterunderstanding of the mechanism that is responsible for thecellular response to IFN-α would undoubtedly lead to the im-proved use of IFN-α for treatment of RCC.

In the present study, we investigated the antiproliferativeeffect of IFN-α in vitro, and low expression of Stat1 was con-firmed to be associated with the resistance of RCC to IFN-αtreatment. These results demonstrated that therapy restoringStat1 expression could be useful for improving the responserate of RCC to IFN-α therapy.

MATERIALS AND METHODS

Cell lines and reagentsFive RCC cell lines, ACHN, Caki-1, A498, NC65, andKY/RC-17, were cultured in complete medium consist-ing of RPMI-1640 (Gibco Biocult, Glasgow, Scotland, UK)supplemented with 25 mM HEPES, 2 mM L-glutamine,1% nonessential amino acids, 100 units/mL penicillin,100 µg/mL streptomycin, and 10% heat-inactivated fetalbovine serum. Cell lines were maintained as monolayers on10-cm plastic dishes and incubated in a humidified atmo-sphere containing 5% CO2 at 37◦C. IFN-α (Intron A R©, re-combinant IFN-α2b; Schering-Plough Co., Kenilworth, NJ,USA) was used.

WST-1 assayThe effects of IFN-α on RCC cells were determined usingthe WST-1 assay. Exponentially growing cells were harvestedand seeded at 2,000 cells/well in a 96-well microtiter plate.After 4 hr of incubation, IFN-α or penicillin/streptomycin-free medium (untreated control) were added, followed bycontinuous incubation for 48 hr. Ten microliters of WST-1(Roche, Penzberg, Germany) was added to each well, and theincubation was continued for another 2 hr. The absorbancewas measured with a microculture plate reader (Immunore-ader, Japan Intermed Co., Tokyo, Japan) at 450 nm. Thepercentage cytotoxicity was calculated using the followingformula:% cytotoxicity = [1 − (absorbance of experimental

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Table 1. Primer Sequences Used in the Present Study

Gene Forward Primer (5′→3′) Reverie Primer (5′→3′) Length of PCR Product (bp)

IFN-AR1 GCAGCACTACTTACGTCATG TACGCGGAGAAGGTAAATTC 312IFN-AR2 AACGTTGTTCAGTTGCTCAC ACTGCTTGCTCATCACTGTG 309IFN-AR2 isoform 1 TTTTGATAGCATTGGTCTTG AGTTTTGGAGTCATCTCATTAT 807IFN-AR2 isoform 2 CACCAGAGTTTGAGATTGT GCTGTACTGTTTGCTTTATTT 628GAPDH GAAGGTGAAGGTCGGAGTC GAAGATGGTGATGGGATTTC 226

1. Total RNA was isolated using an RNeasy mini kit (Qiagen, Germany), and a first-strand cDNA synthesis kit (Amersham Biosciences, UK) was used for reverse transcription.Polymerase chain reaction (PCR) conditions were set according to the manufacturer’s instructions, and the expected size of the PCR products was confirmed by agarose gelelectrophoresis. All primer sets used in the present study are shown in Table 1.2. mRNA expression of the IFN-α receptor in renal cell carcinoma cell lines. IFN-AR1, AR2, isoform 1, and isoform 2 of IFN-AR2 expression were examined by reversetranscription (RT)–polymerase chain reaction (PCR). Despite the different susceptibilities of the RCC cells to IFN-α, the expression of IFN-α receptor 1 (IFN-AR1) and receptor2 (IFN-AR2) showed no significant difference among RCC cell lines, and no difference was found between isoforms 1 and 2.

3. Phosphorylation of Stat1 and Stat3 by IFN-α in RCC using Western blotting. Stat1 were expressed at high levels and significantly phosphorylated in ACHN compared toNC 65 when they were treated with 50 or 100 IU/mL IFN-α for 30 min. However, no significant difference was found in Stat3 or phosphorylated STAT3 between ACHN and NC 65.

well-absorbance of blank)/(absorbance of untreated controlwell-absorbance of blank)] × 100%.

Flow cytometric analysisIFN-α-treated RCC cells were collected and fixed with 70%ethanol at −20◦C overnight, washed three times with PBS,and incubated at 37◦C for 30 min in 7-AAD staining so-lution (BD Biosciences Pharmingen, San Diego, CA, USA).Cell counts at each phase of the cell cycle were estimated us-ing a FACSCalibur flow cytometer (BD Biosciences, San Jose,CA, USA) and Cellquest 3.0 software.

Detection of caspase activityThe activities of caspase 3 and 9 were measured using anAPOPCYTO Caspase Colorimetric Assay kit (Medical & Bi-ological Laboratories Co., Nagoya, Japan) according to themanufacturer’s instructions. The absorbance representingthe formation of p-nitroanilide was measured with a micro-culture plate reader at 405 nm.

Western blottingProtein was extracted and the concentration was assessedusing the Bradford dye-binding protein assay (Bio-Rad,

Richmond, CA, USA), and SDS polyacrylamide gel elec-trophoresis was performed. Phospho-Jak1 (Tyr1022/1023)and Jak1, phospho-Tyk2 (Tyr1054/1055) and Tyk2, and thephospho-Stat1 (Tyr701) and Stat1 (9H2) antibodies werepurchased from Cell Signaling Technology (Danvers, MA,USA). The proliferation cell nuclear antigen (PCNA) mon-oclonal antibody (PC10) was purchased from Sigma-Aldrich(St. Louis, MO, USA), and the anti-beta–actin monoclonalantibody (Abcam, Cambridge, UK) was used as an internalcontrol. The immune complexes were detected using the ECLplus Western blotting detection system (Amersham, Ayles-bury, UK).

Construction of vectors and transfectionThe full-length protein coding sequence of Stat1 (GenBankaccession number NM 007315) was synthesized by RT-PCRusing cDNA from the ACHN cell line as a substrate. PurifiedPCR products were inserted into the pcDEF3 vector and con-firmed by sequence analysis. NC 65 was transfected with thisvector or with pcDEF3 alone (without an insert) using Lipo-fectamine 2,000 (Invitrogen, Carlsbad, CA, USA) accordingto the manufacturer’s instructions. Selection of stable clones

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Growth Suppression of RCC by IFN-α

Figure 1. Growth suppression following treatment with IFN-α in RCCcell lines, ∗p < .01. IFN-α caused dose-dependent cell-growth inhibi-tion in the RCC cell lines, ACHN, Caki-1, and A498 were determinedto be IFN-α sensitive and NC 65; KY/RC-17 were resistant cell lines.All experiments were performed in triplicate, and error bars representthe standard deviation (SD).

was carried out using G418 and confirmed by Western blot-ting.

Statistical analysisAll determinations were repeated in triplicate, and the resultswere expressed as the mean ± standard deviation (SD). Sta-tistical significance was determined using the Student’s t-test,and a p value of .05 or less was considered significant.

RESULTS

Susceptibility of RCC cell lines to IFN-αIFN-α caused dose-dependent cell-growth inhibition in theRCC cell lines used in this study (Figure 1). IFN-α exertedits growth inhibitory activity at a concentration as low as100 IU/mL, which is around the therapeutic concentration.The IC50 of IFN-α in the ACHN, Caki-1, A498, NC 65, andKY/RC-17 cell lines was 2423 ± 232, 2553 ± 246, 2170 ± 259,5569 ± 379, and 4988 ± 335 IU/mL, respectively. Althoughno statistical difference in IC50 was observed for ACHN,Caki-1, and A498, these were all significantly lower than thatobserved for NC 65 and KY/RC-17. Based on the suscepti-bility of these cell lines to IFN-α, cell lines ACHN, Caki-1,and A498 were determined to be IFN-α sensitive and NC 65,KY/RC-17 were considered a resistant cell line in this study.

G1 arrest induced by IFN-αWe analyzed the induction of apoptosis or cell-cycle arrest byIFN-α in RCC cells. Flow cytometric analysis revealed thatIFN-α caused a dose-dependent cell-cycle arrest at G1 in allfive RCC cell lines (Figure 2). In contrast, IFN-α did not sig-nificantly induce apoptosis in any of the cell lines, even at5,000 IU/mL (data not shown).

Caspase and antiproliferative assaysCaspase activity was detected in RCC cells treated with IFN-α, though IFN-α did not affect the activity of caspase 3 orcaspase 9 [Figure 3(A), results for 50, 100, 500, 1,000 IU/mLin ACHN and NC65 are shown). We also evaluated the pro-liferative ability of RCC cell lines treated with IFN-α, and the

Figure 2. Detection of apoptotic cells in RCC cell lines treated withIFN-α. Determined by flow cytometric analysis, IFN-α induced G1cell-cycle arrest in all five RCC cell lines, however, it did not induceapoptosis in any of the cell lines. All experiments were performed intriplicate, and error bars represent the standard deviation (SD).

proliferating cell nuclear antigen (PCNA) protein was used asa marker of proliferative ability as assessed by Western blot-ting. In all five RCC cell lines, the expression of PCNA wasdecreased by IFN-α treatment in a dose-dependent manner[Figure 3(B), results for ACHN and NC65 are shown)].

Phosphorylation of Jak-Stat pathway components by IFN-αWe evaluated the induction of phosphorylation of Jak-Statpathway components by IFN-α using Western blot analysis.Jak1, Tyk2, and Stat1 were expressed at high levels and signif-icantly phosphorylated in the ACHN, Caki-1, and A498 celllines following treatment with IFN-α. In NC 65 and KY/RC-17, although expression and phosphorylation of Jak1 and

Copyright C© 2011 Informa Healthcare USA, Inc.

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Figure 3. Caspase activity and PCNA protein expression induced byIFN-α. (A) IFN-α did not increase the activity of caspase 3 or 9 inall five RCC cell lines, the results for ACHN and NC 65 were shown.(B) PCNA protein expression in RCC cell lines treated with IFN-α,as assessed by Western blot analysis. PCNA expression decreased in adose-dependent manner following IFN-α treatment. All experimentswere performed in triplicate, and error bars represent the SD.

Tyk2 was observed at a level similar to that in IFN-α-sensitiveRCC cell lines; Stat1 was expressed and activated at a lowerlevel following IFN-α treatment (Figure 4, results for ACHN,Caki-1, NC 65, and KY/RC-17 are shown). These results sug-gested that deficiency in Stat1 might contribute to the resis-tance of RCC cells to IFN-α treatment.

Effect of restoring Stat1 on the susceptibility of RCC cells toIFN-αThe susceptibility of NC 65 cells to IFN-α treatment follow-ing transfection with a Stat1 vector was evaluated. In trans-fected NC 65 cells, although the expression and phosphory-lation of Jak1 and Tyk2 by IFN-α was observed at a level sim-ilar to that in the mock-transfected cell line, high expressionof Stat1 was found; and, Stat1 phosphorylation was signifi-cantly increased when the cells were treated with IFN-α com-pared to the phosphorylation levels in the mock-transfectedcell line [Figure 5(A)]. Moreover, resistance to IFN-α wasalso reversed and dose-dependent growth suppression wasobserved in the transfected NC 65 cells [Figure 5(B)].

DISCUSSION

For metastatic RCC, IFN-α therapy is the most common op-tion at present (6). However, the mechanism of IFN-α in-duced cell-growth suppression and resistance remains ob-scure. Immunotherapy by IFN-α was reported to mediateimmune modulation by increasing the infiltration of acti-vated, mature dendritic cells and functionally active CD8+ Tcells in renal tumors (7). IFN-α also markedly increases theamount of cell-surface CD317 and augments the antibody-

Figure 4. Tyrosine phosphorylation of Jak–Stat pathway componentsin RCC cell lines treated with IFN-α. In NC 65 and KY/RC-17, Stat1was expressed and activated at a lower level following IFN-α treatmentcompared to that of ACHN and Caki-1.

dependent cellular cytotoxic activity of RCC cells (8). Sev-eral groups have also indicated that IFN-α induces apoptosisand/or cell cycle arrest in vitro, depending on the IFN typeand tumor cell properties (9–11).

In this study, we evaluated the growth suppression andsusceptibility of RCC cells to IFN-α. IFN-α did not induceapoptosis in any cell line or at any concentration tested anddid not affect the activity of caspase 3 or caspase 9. In-stead, IFN-α treatment suppressed tumor growth by induc-ing G1 cell-cycle arrest in a dose-dependent manner. More-over, IFN-α suppressed the proliferative ability of RCC cells,which was confirmed by examining PCNA expression. Wealso examined the expression and phosphorylation of Jak-Stat components induced by IFN-α in RCC to reveal themechanism of growth suppression and susceptibility. Jak1and Tyk2 were expressed and activated by IFN-α at a highlevel in both IFN-α sensitive (ACHN, Caki-1, and A498) andIFN-α resistant (NC 65 and KY/RC-17) cell lines, whereasStat1 was expressed at a lower level and could not be phos-phorylated following IFN-α treatment in NC 65 and KY/RC-17. By restoring the expression of Stat1, the resistance of NC65 to IFN-α was reversed. These results suggested that IFN-αsuppressed the growth of RCC cells by activating the Jak-Statpathway.

We further analyzed other possible factors affecting IFN-α signaling, such as the IFN-α receptor. The resistance ofRCC lines to the antiproliferative action of IFN-α was re-ported not affected by defects in ligand binding or in IFN-receptor structure (12). In our study, despite the different

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Growth Suppression of RCC by IFN-α

Figure 5. Tyrosine phosphorylation of Stat1 is induced by IFN-α inNC 65 cells transfected with a Stat1 vector (A), and the susceptibilityof NC 65 to IFN-α is also increased following transfection with thisvector (B), ∗p <.01. All experiments were performed in triplicate, anderror bars represent the SD.

susceptibilities of the RCC cell lines to IFN-α, the expres-sion levels of the IFN-α receptor did not show any signifi-cant differences and no mutation was detected via RT-PCRand cDNA sequencing (shown in supplemental data). Stat3 isanother transcription factor that can be activated by IFN-α.Although Stat3 is reported to have antiproliferative and anti-oncogenic effects (13), it mainly appears to play a critical rolein cell proliferation, differentiation, and survival (14, 15). Inthe present study, Stat3 was expressed and activated by IFN-α in all four RCC cell lines assayed (shown in supplementaldata), and we speculate that different ratios of Stat1 versusStat3 could account for their different responses to IFN-α.Furthermore, blocking Stat3 might increase the susceptibilityof RCC to IFN-α. Another study indicated that Caki-2 cells,in which neither protein expression nor phosphorylation ofJak1 and Tyk2 was detected, have a low response to IFN-α

(16). Several studies have also demonstrated that suppressorof cytokine signaling (SOCSs) inhibits IFN-α-induced acti-vation of the Jak-Stat pathway (17, 18). One study showedthat an IFN-α-resistant cell line exhibited enhanced SOCS3mRNA expression in response to IFN-α stimulation and thatblocking SOCS3 partially restored IFN sensitivity (19). Inbrief, the processes affecting susceptibility of RCC to IFN-αare complex and elusive. IFN-α signal transduction and IFN-α-stimulated genes are commonly analyzed, but no singlemechanism can explain the total resistance of RCC to IFN-αat present. Although Shimazui et al. suggested that the pro-tein level of Jak-Stat is independent of IFN-α sensitivity (20),the expression of Stat1 was clearly associated with IFN-α sen-sitivity in our study. Because the in vitro response may not berelevant to in vivo efficacy, a study with clinical RCC samplesshould be encouraged to clarify the role of Jak-Stat in IFN-αresistance.

In summary, we investigated the susceptibility of RCCcells to IFN-α and the mechanisms of growth suppressionand resistance. We concluded that IFN-α could induce cell-cycle arrest at G1 by activating the Jak-Stat pathway, and thedefective Stat1 expression is associated with the resistance ofRCC cells to IFN-α. Our results also suggested that the Jak-Stat pathway could be an appropriate target for improving theresponse of RCC to IFN-α treatment.

ACKNOWLEDGMENTS

This study was supported by a grant from National Natu-ral Science Foundation of China and the Scientific ResearchFoundation for the Returned Overseas Chinese Scholars,State Education Ministry.

DECLARATION OF INTEREST

The authors report no conflict of interest. The authors aloneare responsible for the content and writing of this paper.

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