ORIGINAL ARTICLE

EFFECTS OF IFI16 OVEREXPRESSION ON THE GROWTHAND DOXORUBICIN SENSITIVITY OF HEAD AND NECKSQUAMOUS CELL CARCINOMA–DERIVED CELL LINES

Marco De Andrea, PhD,1,2* Daniela Gioia, PhD,2* Michele Mondini, PhD,1,2 Barbara Azzimonti, PhD,2

Filippo Reno, PhD,2 Giancarlo Pecorari, MD,3 Vincenzo Landolfo, MD,3 Massimo Tommasino, PhD,4

Rosita Accardi, PhD,4 Christel Herold-Mende, MD,5 Santo Landolfo, MD,1 Marisa Gariglio, PhD, MD2

1 Department of Public Health and Microbiology, Medical School of Torino, Via Santena 9,10126 Torino, Italy2 Department of Clinical and Experimental Medicine, Medical School of Novara,Via Solaroli 17, 28100 Novara, Italy. E-mail: gariglio@med.unipmn.it3 Department of Clinical Physiopathology, Medical School of Torino,Corso Bramante 88/90-10126 Torino, Italy4 Unit of Infection and Cancer, IARC, 150 Cours Albert Thomas, 69372 Lyon, France5 Department of Neurosurgery, University of Heidelberg, Im Neuenheimer Feld 400,69120 Heidelberg, Germany

Accepted 5 December 2006Published online 17 May 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/hed.20611

Abstract: Background. In a previous analysis of head and

neck squamous cell carcinomas (HNSCCs), we showed that the

levels of the interferon-inducible protein IFI16 inversely correlate

with cancer grade. In this study, we further evaluate the molecu-

lar role of IFI16 in the development of HNSCCs.

Methods. The effect of IFI16 expression was evaluated by its

retroviral restoration in an IFI16-negative HNSCC-derived cell

line, HNO136. Growth rate and soft agar colony formation were

evaluated. The effect of IFI16 restoration in cells exposed to dox-

orubicin was also analyzed.

Results. IFI16 restoration resulted in the inhibition of both cell

growth and in vitro transforming activity and increased doxorubicin-

induced cell death by accumulating the cells at the G2/M phase.

Conclusion. In agreement with our previous in vivo data,

IFI16 appears to be involved in maintaining the normal growth of

epithelial cells, whereas its downregulation may contribute to

uncontrolled cell proliferation and tumorigenesis. VVC 2007 Wiley

Periodicals, Inc. Head Neck 29: 835–844, 2007

Keywords: head and neck squamous cell carcinoma; IFI16;

doxorubicin; growth arrest; interferon

The interferon (IFN)-inducible HIN-200 familyincludes IFI16, IFIX, MNDA (myeloid nuclear dif-ferentiation antigen), and AIM2 (absent in mela-noma 2) genes in humans, and Ifi202a, Ifi202b,Ifi203, Ifi204, and D3 genes in mice.1,2 Theencoded proteins share a 200-amino acid signa-ture motif of type a and/or b and are primarily nu-clear phosphoproteins involved in the transcrip-tional regulation of genes that are important forcell-cycle control, differentiation, and apoptosis.3,4

The antitumor activity of HIN-200 proteins hasbeen demonstrated. Overexpression of the mouse

*Marco De Andrea and Daniela Gioia contributed equally to this work.

Correspondence to: M. Gariglio

VVC 2007 Wiley Periodicals, Inc.

IFI16 Activity in HNSCC-Derived Cell Lines HEAD & NECK—DOI 10.1002/hed September 2007 835

family proteins p202 and p204 retards prolifera-tion of various cell lines, delays G1 progressioninto S-phase, and accumulates cells with a DNAcontent equivalent to cells arrested in late G1.5,6

Expression of AIM2 is lost by frame-shift muta-tions in colorectal tumors, and loss of MNDAexpression in prostate carcinoma is linked withthe progression of more aggressive metastaticprostate cancer.7,8 Consistent with these findings,increased levels of IFI16 in prostate epithelialcells contribute to senescence-associated irrevers-ible cell growth arrest and inhibit the colony for-mation capability of prostate cancer cell lineslacking IFI16.9 Sustained IFI16 expression inhuman umbilical vein endothelial cells suppressestheir migration and cell-cycle progression, alongwith tube morphogenesis activity.10 Moreover, arole of IFI16 in commitment to growth arrest or ap-optosis under condition of DNA damage induced byionizing radiation or neocarcinostatin treatmenthas also been recently demonstrated.11 Finally,expression of IFIX in human breast cancer celllines reduces their anchorage-dependent and -in-dependent growth in vitro, and tumorigenicity inmice.12

Immunohistochemical analysis of IFI16expression in a series of primary head and necksquamous cell carcinomas (HNSCCs) revealed astrong correlation between IFI16 expression lev-els and tumor histopathologic grade. SustainedIFI16 immunoreactivity was present in low-grade, less aggressive tumors, whereas IFI16 wasbarely detectable or negative in high-grade, moreaggressive tumors.13,14 Thus, the lower prolifera-tion rate of IFI16 strongly positive tumors may berelated to the antiproliferative activity of theHIN200 proteins.

To further understand the functional rela-tionship between IFI16 expression levels andgrowth regulation of HNSCC, IFI16 expressionlevels were analyzed in a series of freshly estab-lished HNSCC tumor cultures.15 In 1 cell line,designated HNO136, expression of IFI16 wasnot detectable. Ectopic expression of IFI16 inthis cell line demonstrated that its restorationreduces both growth rate and in vitro transform-ing activity and increased doxorubicin-inducedcell death by accumulating the cells at the G2/Mphase. As already reported for prostate cancer,our observations support the idea that loss ofIFI16 function in head and neck squamous epi-thelia contributes to the development of HNSCCand increases their sensitivity to cytotoxicdrugs, such as doxorubicin.

MATERIALS AND METHODS

Cell Cultures, Treatment, Retroviral Infection, Cell

Growth, and Soft Agar Assay. HNSCC-derivedprimary cell cultures, obtained as previouslydescribed,15 were cultured in Dulbecco’s modifiedEagle’s medium (DMEM; Sigma-Aldrich, Milan,Italy) supplementedwith 10% heat inactivated fe-tal bovine serum (FBS; Sigma-Aldrich). HaCaTcells were cultured in DMEM supplemented with10% heat inactivated FBS. Cells were incubatedat 378C in a humidified atmosphere containing5% CO2 and 95% air. Cells were split every 3 to 4days to ensure logarithmic growth. Normalhuman epidermal keratinocytes (NHEK) fromneonatal foreskin were obtained from CambrexBio Science (Milan, Italy). They were maintainedin serum-free keratinocytes growth medium(KGM; Cambrex Bio Science). p53 mutationalanalysis was performed as previously described.16

When indicated, cells were treated with 0.3 lMdoxorubicin (Sigma-Aldrich) for 18 hours, washedout to remove the drug and cultured for an addi-tional 24, 48, and 72 hours to perform FACS anal-ysis or immunoblotting. Treatment with IFN-b(Sigma-Aldrich) was performed with 1000 U/ml.

The 2700 bp blunt-ended Cla I/BamH I IFI16cDNA from pBKS-IFI16 plasmid (kindly providedby JA Trapani), was introduced into the pCLXSNvector (Imgenex, San Diego, CA) following diges-tion with Xho I to generate the pCL-IFI16 plas-mid. The empty pCLXSN vector, carrying a neo-mycin resistance gene, was used as a control.High-titer retrovirus-containing supernatantswere generated by transient transfection of sec-ond-generation retrovirus producer Phoenix cellsand used to infect the cells as previously describedby Pear et al.17 After infection, HNO cells wereselected in 800 lg/mL neomycin for 10 days anddesignated HNO-neo or HNO-IFI16 (carryingpCLXSN and pCL-IFI16, respectively).

Cell growth in monolayer was evaluatedby MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide) assay as previously des-cribed.18 Cells were plated in triplicate at a densityof 15,000 cells/well in a 24-well cell culture plates.Cells were allowed to adhere overnight and thentreated with IFN-b (1000 U/ml). At the indicatedtime points, cell growth was measured by MTTassay. Briefly, 60 lL of MTT (5 mg/mL in PBS)were added to the medium for 4 hours. Mediumand MTT were then removed, dimethyl sulfoxidewas added, and absorbance was measured at570 nm.

836 IFI16 Activity in HNSCC-Derived Cell Lines HEAD & NECK—DOI 10.1002/hed September 2007

Anchorage-independent cell growth was deter-mined by analyzing the formation of colonies insoft agar. Pools of stable cell lines were harvestedand aliquots were added to 60-mm dishes contain-ing 0.5% (w/v) SeaPlaque agarose (Cambrex BioScience) in complete medium on top of a layer of1% (w/v) SeaPlaque agarose. Aliquots of 1000;10,000; and 100,000 cells were plated in duplicate.After 16 days of culture, 25 squares of 1 cm2 werecounted and photographed.

Western Blot Analysis. For crude extract prepa-ration, cells were lysed in 3% sodium dodecyl sul-fate (SDS)-lysis buffer containing 125 mM Tris–HCl pH 6.8, 3% SDS, 10 mM dithiothreitol (DTT),10% glycerol with the addition of protease inhibi-tors (0.2 mM phenylmethanesulfonyl fluoride—PMSF, 1 mg/mL pepstatin, 0.1 mM benzamidine, 2mg/mL aprotinin) and briefly sonicated. Insolublematerial was removed by centrifugation at 13,000rpm for 5minutes. Protein concentrationwas deter-mined by the Bio-Rad Dc Protein Assay (Bio-RadLaboratories, Milan, Italy). Proteins were sepa-rated on 8.5%, 10%, or 12.5% SDS-polyacrylamidegels and transferred onto a polyvinyl difluoride(PVDF) membrane (GE Healthcare Europe GmbH,Milan, Italy) according to the instruction manual.Themembraneswere blocked in a blocking solution(10 mM Tris–HCl pH 7.5, 0.1MNaCl, 0.1% Tween-20, and 5% [w/v] nonfat dry milk) overnight at 48C,and incubated with the following primary antibod-ies: affinity-purified anticarboxy-terminal IFI16rabbit polyclonal (diluted 1:2000), antiactin mousemonoclonal (1:5000; Chemicon International,Temecula, CA), anti-14-3-3r (clone 3B9) mousemonoclonal (1:500; Sigma-Aldrich), anti-p53 (DO-1)mouse monoclonal (1:1000), anti-p16 (F-12) mousemonoclonal (1:300), anti-p21waf1/cip1 (C-19) rabbitpolyclonal (1:2000), anticyclin A (H-432) rabbit pol-yclonal (1:1000), anticyclin B1(H-433) rabbit poly-clonal (1:1000), and anticyclin D1(HD11) mousemonoclonal (1:2000) all from Santa Cruz Biotech-nology (Santa Cruz, CA). Appropriate secondaryantibodies conjugated with horseradish peroxidasewere used (GE Healthcare Europe GmbH) and thereaction was visualized by enhanced chemilumi-nescence (SuperSignal West Pico Substrate; PierceBiotechnology, Rockford, IL), according to the man-ufacturer’s instructions. Densitometry analysiswas performed with Quantity One software (Bio-Rad Laboratories).

Cell-Cycle Analysis. Cell-cycle analysis was per-formed measuring the propidium iodide staining

of DNA in flow cytometry. Briefly, adherent cellswere trypsinized, harvested, and fixed in 70%cold ethanol for 60 minutes at 48C. Cells werethen washed twice with PBS, incubated at 378C incitrate buffer (0.05MNa2HPO4, 25 mM sodium ci-trate, and 0.1% Triton X-100 pH 7.8), and stainedwith 200 lg/mL PI for 30 minutes in the presenceof RNase (100 ng/mL). Cellular DNA content wasthen assessed with a FACScan flow cytometer(Becton-Dickinson, Milan, IT) equipped with 15mW air-cooled argon ion laser operating at 488nm. PI red fluorescence was measured through along pass 620 nm filter. Red fluorescence wasamplified in linear mode to identify cell-cyclephases. A minimum of 15,000 events per samplewere collected at a low flow rate setting (12 mL/min) to improve the coefficient of variation on theDNA histograms. DNA data analysis was per-formed with the ModFit LT software program(Becton-Dickinson).

Plasmids and Luciferase Assay. p53RELuc andmutp53RELuc plasmids contain 2 copies of a wildtype (50-CGAATTCGAACATGTCCGAACATGTT-GAGATCTGCC-30) or mutated p53 binding seq-uence (50-CCGAATTCGAAAATTTCC GAATCCT-TTGAGATCTGCC-30), respectively, subcloned inthe pGL3-Promoter plasmid, harboring SV40minimal promoter (Promega,Milan, IT).

Equal amounts (4 lg) of each reporter plasmid(harboring firefly Luc cDNA) along with 0.15 lg ofpRLSV40 plasmid (harboring Renilla Luc cDNA),included as a control for transfection efficiency,were introduced into HNO136-neo and -IFI16cells by Lipofectamine Plus (Invitrogen, Milan,IT). After 24 hours, cells were treated with 0.3 lMdoxorubicin. After 36 hours incubation, cells wereassayed for chemiluminescence using the dual lu-ciferase reporter system as suggested by the manu-facturer’s instructions (Promega). Chemilumines-cence wasmeasured using a PlateLumino Luminom-eter (Stratec Biomedical System, Birkenfeld, D). Forall studies, valueswerenormalized bydividing valuesfor firefly Luc expressed as relative light units (RLU)on values obtained from Renilla Luc. The resultingluciferase activity is expressed as fold induction rela-tive to basal levels measured in HNO136-neo cellstransfectedwith p53RELuc and left untreated.

RESULTS

Loss of IFI16 Expression in a HNSCC-Derived Cell

Line. We have previously shown that the IFI16

IFI16 Activity in HNSCC-Derived Cell Lines HEAD & NECK—DOI 10.1002/hed September 2007 837

expression in HNSCC tightly correlates withlower histopathological grading and proliferationindex of the tumor.13 To further investigate therole of IFI16 in epithelial cell growth, its basal aswell as IFN-induced levels were evaluated in a se-ries of freshly cultures of tumor fragments, grownas adherent monolayers with epithelioid morphol-

ogy that was retained through as many as 50 to 80subcultures.15 As shown in Figure 1, panel A,IFI16 protein was detected, although at differentlevels, in the majority of the cell lines tested. Onecell line, designated HNO136, showed no IFI16expression, even after 24 hours IFN-b treatment.As already reported with the in vivo observations,these results indicate that expression of IFI16 israrely lost in head and neck cancer cells. DNAsequence analysis of p53 exon 4–9 in HNO136revealed no mutation, thus offering the possibilityto investigate IFI16 antiproliferative activity, whichis at least in part dependent on functional p53.

By contrast, HNO150 cells expressed detecta-ble levels of IFI16 protein, and IFN treatmentresulted in a significant increase. DNA sequenceanalysis of p53 exon 4–9 in HNO150 revealed amutation in exon 5with a 135 Cys-to-Tyr substitu-tion. This cell line was therefore chosen to assessIFI16 activity in a cellular background bearing amutated p53. To investigate the potential role ofIFI16 in IFN-induced tumor suppression inHNSCC, we showed that IFN-b treatment sup-pressed the growth of the 2 cell lines harboringwild-type p53, namely HNO124 and HNO41 (�51and �54%, respectively) (Figure 1, panel B),which correlated with the induction of IFI16(Figure 1, panel A). As expected, IFN-b treatmentwas less effective in growth inhibition of HNO136(�33%), which lacks IFI16, and HNO150, whichharbors mutated p53 (�37%).

Restoration of IFI16 Expression in the HNO136 Null

Cell Line Reduces Its Growth Rate. To gain moreinsight into the role of IFI16 in HNSCC cell lines,we chose to restore it in the HNO136 null cell lineand to force its expression in the HNO150 andHNO124 cell lines, harboring mutant and wild-type p53, respectively. To this end, cultures wereinfected with high-titer retroviruses coexpressingIFI16 with a neomycin resistance gene to isolatecells in which the virus was stably transduced. Aretroviral vector expressing the neomycin resist-ance gene alone was used as control. Infected cellpopulations were selected for 10 days in neomycinand then used as pooled populations for all subse-quent analyses. The levels and cellular localiza-tion of the product of the transduced gene wereanalyzed by western blotting and immunofluores-cence analysis. As shown in Figure 2, panel A, sta-bly transduced cell lines efficiently expressedIFI16. Moreover, immunofluorescence analysisconfirmed that IFI16 was properly localized in thenucleus (data not shown). Comparison of the

FIGURE 1. (A) Western-blot analysis of IFI16 protein levels in

HNSCC-derived cell lines. For each cell line, the p53 status is

indicated: where present, the mutations and their location in the

protein are listed (WT ¼ absence of mutation). Whole cell

extracts were extracted from both untreated (�) and interferon-

b (IFN-b)-treated (+) cells and then subjected to immunoblot-

ting. The actin protein levels serve as loading control. The data

shown are representative of 3 independent experiments. (B)

Suppression of the growth of HNSCC-derived cell lines by IFN

treatment. Cells were treated with IFN-b (full symbols) or left

untreated (open symbols) and the growth rate was determined

by MTT assay at the indicated time points. Each measurement

was made in triplicate. The relative absorbance at 570 nm was

determined by setting the absorbance on day 1 at 1.

838 IFI16 Activity in HNSCC-Derived Cell Lines HEAD & NECK—DOI 10.1002/hed September 2007

growth rates of the parental, neo-, and IFI16-transduced cells showed that IFI16 expressionsignificantly reduced the growth of transduced

HNO136 (�93%) and to a lower extent that ofHNO124, but not HNO150 (�33 vs �13%, respec-tively, when comparing IFI16-transduced cellswith the parental cell line) (Figure 2, panel B).Soft agar assay was used to determine the effect ofIFI16 on the in vitro transforming property. Con-sistent with the results of growth rate, coloniesfrom IFI16-transduced HNO136 and HNO124cells were significantly lower in number (Figure 2,panel C) and reduced in size when compared withparental cells (data not shown). As expected,empty vector-transduced cells displayed a numberof colonies and plating efficiency similar to that ofthe parental cells. By contrast, the number of colo-nies in IFI16-transduced HNO150 cells was simi-lar to that of empty vector-transduced cells. Takentogether, these results suggest that IFI16 restora-tion in HNO136 null cells and overexpression inHNO124 (harboring wild-type p53), suppressedtheir growth rate and in vitro transforming activ-ity. By contrast, IFI16 overexpression in HNO150(harboring mutated p53) did not exert antiproli-ferative activity, further indicating that IFI16-mediated inhibition of cell growth, in part,depends on the presence of functional p53.19,20

Restoration of IFI16 Expression in HNO136 Cells

Augments Doxorubicin-Induced G2 Arrest. Pre-vious studies demonstrated that ectopic expres-sion of IFI16 plays a role in commitment to growtharrest or apoptosis when associated with anti-cancer agents, such as neocarcinostatin or ionis-ing radiations.11 To examine the effect of IFI16expression in drug-induced cytotoxicity, theeffects of doxorubicin, a widely used anticanceragent that acts by stabilizing ‘‘cleavable com-plexes’’ of DNA with topoisomerase II, in theHNO136 cellular model was analyzed.21

FIGURE 2. Effects of IFI16 expression on the growth rates and

in vitro tumorigenicity of HNSCC-derived cell lines. (A) West-

ern-blot experiments showing IFI16 efficient expression upon

retroviral infection. HNO-IFI16, cells infected with pCL-IFI16 ret-

roviral vector; HNO-neo, cells infected with the empty retroviral

vector pCLXSN. Whole cell extracts from stably transduced

cells were subjected to immunoblotting. The actin protein levels

serve as loading control. (B) Growth rates of parental, neo- and

IFI16 transduced HNO136, HNO150, and HNO124 cells,

respectively. Cells were seeded and cell growth was measured

every 2 days by the Trypan blue exclusion method. Data are

expressed as mean 6 SD (n ¼ 3). (C) Soft-agar assay showing

reduction of colonies number following IFI16 transduction. The

cells were seeded in soft agar and the colony number was

scored at 3 weeks after seeding. Histograms show the number

of colonies formed and are means 6 SD of 3 independent

experiments.

IFI16 Activity in HNSCC-Derived Cell Lines HEAD & NECK—DOI 10.1002/hed September 2007 839

To elucidate the intrinsic mechanism by whichdoxorubicin inhibits cell growth of HNO136 cells,cell-cycle analysis was performed. Since it is well-

known that the actual mechanism of cell death af-ter doxorubicin treatment is strictly dependent onthe cellular model, we also analyzed its effect in

FIGURE 3. Effect of IFI16 restoration on cell-cycle distribution after doxorubicin treatment in HNO136-neo and HNO136-IFI16 cells

(panel A); normal human epidermal keratinocytes (NHEK) and HaCat cells (panel B). Cells were treated with doxorubicin for 18 hours

and the drug was washed out. The cells were cultured for an additional 24, 48, or 72 hours and then cell-cycle analysis was per-

formed. All histograms were plotted by using the same scale for both axes. Results are representative of 2 independent experiments.

840 IFI16 Activity in HNSCC-Derived Cell Lines HEAD & NECK—DOI 10.1002/hed September 2007

NHEK and in a nontransformed spontaneouslyimmortalized epithelial cell line (HaCaT).22 Cellswere treated with a moderate dose of doxorubicinfor 18 hours, washed out to remove the drug, andcultured for an additional 24, 48, and 72 hours toperform fluorescence-activated cell sorter (FACS)analysis. As evidenced by the flow cytometric his-togram profile depicted in Figure 3, panel A asrepresentative, treatment of HNO136 cellsresulted in a rapid accumulation of cells in the G2phase at 24 hours, whereas a minor fraction had aDNA content less than 2N, which is indicative ofapoptosis. Stably IFI16 transduced cells showed amore pronounced G2 arrest in comparison withneo, both at 24 and 48 hours (94.45% vs 82.17%,and 99.33% vs 86.48%, respectively). Interest-ingly, the percentage of cells in S phase was muchlower in IFI16-transduced cells in comparisonwith neo cells (5.55% vs 20.19%). At 72 hours,most of either neo- or IFI16-transduced cells died.By contrast, treatment of NHEKwith doxorubicinresulted in an initial weak accumulation of cellswith a 4N DNA content by 24 hours after drugwithdrawal, but most of the cells did not maintainthe cell-cycle arrest, lost viability, and detachedfrom the plate by 48 and 72 hours, thus account-ing for the diminished peaks in the histograms atthose times (Figure 3, panel B). HaCaTcells had amassive accumulation of cells with a 4NDNA con-tent 24 hours after treatment, still maintained at48 hours although to a lower extent, and then cellslost viability and detached from the plate. More-over, 72 hours after drug removal, a substantialproportion of nuclei had a sub-G1 DNA contentcharacteristic of apoptosis, indicating that in thiscell line the predominant way of doxorubicin-induced death is apoptosis.

To definitively exclude that in HNO136 cells,apoptosis is not the predominant mechanism ofdoxorubicin-induced death, caspase-3 activationhas been evaluated, since it is an importantupstream factor leading to apoptosis. Consistentwith FACS analysis, it was found that it caused asignificant increase in caspase-3 activation inHaCaTcells at 72 hours, which was not detectablein either NHEK or HNO136 cells (data notshown). Microscopic examination of doxorubicin-treated cells revealed that the most prominentfeature of cell death, observed at the time of drugremoval especially with HNO136-IFI16, was theappearance of enlarged cells containing multiple,completely, or partially separated, micronucleiwith evenly stained chromatin (data not shown).Such nuclear changes are characteristic of mitotic

catastrophe rather than apoptosis and indicatethat cells may continue to synthesize DNA andundergo aberrant nuclear division.23,24 Taken to-gether, these results clearly indicate that each cellline exhibited different modes of cell death afterdoxorubicin treatment as follows: apoptosis inHaCaT, mitotic cell death in HNO136, and verylikely necrosis in NHEK.

Altogether, these results demonstrate thatIFI16 restoration in HNO136 null cell line en-hanced doxorubicin-induced G2 arrest and its du-ration.

Effect of Doxorubicin on Expression of Cell-Cycle

Regulatory Proteins. To elucidate the molecularbackground underlying the prolonged doxorubi-cin-induced G2 arrest observed in the cells con-taining IFI16, expression of cell-cycle regulatoryfactors in HNO136 was analyzed at differenttimes after drug removal. Protein lysates werealso prepared from NHEK, left untreated, or dox-orubicin-treated, as a normal counterpart. Asillustrated in Figure 4 (left part), expression ofp53 was markedly increased 24 hours after drugtreatment in NHEK, but it was reduced at latertimes. Accompanying the induction of p53, anincrease of p21waf1/cip1 levels was also observed at

FIGURE 4. Western-blot analysis of protein lysates prepared

from normal human epidermal keratinocytes (NHEK), HNO136-

neo and -IFI16 cells. To assess different aspects of regulation

contributing to cell-cycle arrest, cells were left untreated (�) or

treated with doxorubicin as described in materials and methods

section. Whole cell protein extracts were prepared and ana-

lyzed by immunoblotting for the indicated proteins. Results are

representative of 2 independent experiments.

IFI16 Activity in HNSCC-Derived Cell Lines HEAD & NECK—DOI 10.1002/hed September 2007 841

24 hours and then gradually decreased. The levelsof 14-3-3r (stratifin), a protein known for its abil-ity to prevent mitotic catastrophe in a colorectalcancer cell line, were slightly increased at 24hours after doxorubicin treatment.25 Cyclin B1 isa marker for G2/mitosis, and cyclin B1 levels werehigher in cycling untreated NHEK consistentwith the high proportion of cells in S and G2phase.26,27 Cyclin B1 disappeared at 24 hours af-ter doxorubicin treatment, indicating that thesecells had exited mitosis. No significant change inthe expression pattern of cyclin A, cyclin D, andp16 was observed. A different picture emergedwhen the protein expression in HNO136 (Figure4, right part) was analyzed. In this cell line p53,p21waf1/cip1 and 14-3-3r levels were unaffected ei-ther in neo- or IFI16-transduced cells after doxor-ubicin treatment. In surprising contrast toNHEK, sustained expression of IFI16 in HNO136cells caused an accumulation of cyclin B1 as earlyas 24 hours after treatment that was delayed to 48hours in IFI16-lacking cells. Since cyclin B1 is amarker of G2/M transition, these results are con-sistent with the FACS analysis showing G2/Maccumulation of HNO136-IFI16 and HNO136-neoat 24 and 48 hours, respectively.

Doxorubicin-Induced Cell Death in HNO136 is p53

Independent. Previous studies have shown thatdoxorubicin activates p53 DNA binding activity asmany other DNA-damaging agents.28,29 On thebasis of the crucial role of p53 in the execution ofsome forms of apoptosis, it has therefore beenspeculated that p53 could also play an importantfunction in antracycline cytoxicity. To verify thishypothesis, a luciferase assay using the p53RELucor mutp53RELuc reporter constructs, containingtandem repeats of the canonical or mutatedp53 binding site, respectively, was performed.IFI16- or empty vector-transduced HNO136 cellswere transiently transfected with the above-men-tioned plasmids, the day after they were treatedwith 0.3 lM doxorubicin and at 36 hours luciferaseactivity was evaluated. As shown in Figure 5, dox-orubicin treatment did not result in significantstimulation of the luciferase activity, indicatingthat this treatment did not increase p53 transcrip-tional activity.

To further confirm that p53 is not involved indoxorubicin-induced cell death, the pharmacologi-cal inhibitor pifithrin-alpha (PFT-a) was used toblock the p53-mediated transcriptional activa-tion. As expected, FACS analysis demonstrated

that pretreatment with the p53 inhibitor did notattenuate G2/M accumulation (data not shown).

DISCUSSION

To investigate the role of IFI16 in head and necktumorigenesis, we first evaluated its expressionlevels in a series of HNSCC-derived cell lines. Im-munoblot analysis showed that the majority of thecell lines displayed variable basal levels of IFI16expression that could be further triggered by IFN-treatment, although to a different extents. By con-trast, 1 cell line, designated HNO136, expressedundetectable levels of endogenous IFI16, thatwere unaffected by IFN treatment. This cell linewas therefore chosen for reconstitution experi-ments to clarify the role of IFI16 in modulatingcell growth and differentiation. Analysis of the re-spective patient’s clinical data revealed a veryshort disease-free survival.15 The patient diedwithin 2months from the surgical treatment, sug-gesting a relevant role of the IFI16/p53 pathwayin controlling cell proliferation. This hypothesisis now proven by the finding that IFI16 reconstitu-tion by retroviral transduction, in the presenceof wild-type p53, downregulates HNO136 andHNO124 proliferation, and reverts at least inpart the malignant phenotype. By contrast, inHNO150, bearing a mutated p53, IFI16 overex-pression did not exert any antiproliferative activ-ity. Consistent with these observations, loss orreduction of IFI16 expression in vivo have beenshown to correlate with a more aggressive behav-

FIGURE 5. Effects of doxorubicin on p53 promoter activity in

luciferase assay. The p53RELuc or mutp53RELuc reporter con-

structs were transfected into HNO136-neo or HNO136-IFI16

cells. Cells were treated with doxorubicin or left untreated and

protein extracts were assessed for luciferase activity. The

resulting luciferase activity is expressed as fold of induction rel-

ative to basal levels measured in cells transfected with

p53RELuc and left untreated.

842 IFI16 Activity in HNSCC-Derived Cell Lines HEAD & NECK—DOI 10.1002/hed September 2007

ior of the tumor and worse follow-up. Moreover, asobserved in prostate and breast cancer cell lines,ectopic expression of IFI16 in HNO136 andHNO124 cells resulted in inhibition of cell growthand in vitro transforming activity, confirming thatit plays a role in maintaining the normal growthof epithelial cells.9,11

Proliferating cells respond to genotoxic stressby triggering a series of signaling events knownas cell-cycle checkpoints, which function to delaycell-cycle progression, thereby facilitating repairand preventing propagation of damaged geneticmaterials.25–27,29 Aberrant progression of cellsthrough the S andM phases of the cell cycle there-fore appears to be the lethal event triggered byDNA-damaging agents. In particular, it has beendemonstrated that doxorubicin treatment leads tocyclin B1 accumulation, probably because of bothan increase in protein synthesis and prevention ofits degradation.26 As with many other chemother-apeutic drugs, the ensuing induction of apoptosisis probably an important reason for doxorubicintherapeutic effect, at least inmyeloid cells.30 How-ever, despite its widespread clinical use and themany types of cellular damage doxorubicin hasbeen shown to cause, its cell death–inducing sig-nals are far from being well characterized, espe-cially in nonmyeloid cells. In HNO136 cells, thedoxorubicin treatment blocked G2/M progression,and the cells either progressed toward mitosis,and eventually died because of mitotic catastro-phe or started dying without entering mitosis. Incontrast to the myeloid cell and human ovarianteratocarcinoma cell results, p53 does not play animportant role in doxorubicin-induced cell death.Consistent with this finding, the lack of 14-3-3rinduction may be a consequence of the inability ofHNO136 cells to trigger p53 transcriptional activa-tion upon drug treatment, as demonstrated by theluciferase assay and by the absence of induction ofthe well-known p21waf1/cip1 responsive gene.

Altogether these results demonstrate thatthe definition of the physiological pathwaysinvolved in the control of cell proliferation mayprovide important information to understand theresponse of epithelial cells to DNA damagingagents, such as doxorubicin. Although additionalstudies are needed to better define the molecularpathway leading to this cooperation between dox-orubicin and IFI16, this report will serve to pro-vide the scientific community with a better under-standing of the molecular alterations that, byinactivating the p53-dependent pathways, lead tocarcinogenesis.

Acknowledgments. This workwas supportedby Lega italiana per la lotta contro i tumori, sectionof Novara, Special Project Oncology ‘‘Compagnia diSan Paolo’’, MIUR (‘‘Program 40%’’ to S.L. andM.G.) andTargetedProject fromRegionePiemonte.M.M. is a recipient of a Research Fellowship fromthe Fondazione Internazionale di Ricerca in Medic-ina Sperimentale, Turin.

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