plos one 2012

Interferon-b Induces Cellular Senescence in CutaneousHuman Papilloma Virus-Transformed HumanKeratinocytes by Affecting p53 Transactivating ActivityMaria V. Chiantore1, Serena Vannucchi1, Rosita Accardi2, Massimo Tommasino2, Zulema A. Percario3,

Gabriele Vaccari4, Elisabetta Affabris3, Gianna Fiorucci1,5, Giovanna Romeo1,6*

1 Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanita, Rome, Italy, 2 Infections and Cancer Biology Group, International

Agency for Research on Cancer-WHO, Lyon, France, 3 Department of Biology, University of Rome 3, Rome, Italy, 4 Department of Veterinary Public Health and Food Safety,

Istituto Superiore di Sanita, Rome, Italy, 5 Institute of Molecular Biology and Pathology, CNR, Rome, Italy, 6 Department of Medico-Surgical Sciences and Biotechnologies,

Sapienza University, Rome, Italy

Abstract

Interferon (IFN)-b inhibits cell proliferation and affects cell cycle in keratinocytes transformed by both mucosal high riskHuman Papilloma Virus (HPV) and cutaneous HPV E6 and E7 proteins. In particular, upon longer IFN-b treatments, cutaneousHPV38 expressing cells undergo senescence. IFN-b appears to induce senescence by upregulating the expression of thetumor suppressor PML, a well known IFN-induced gene. Indeed, experiments in gene silencing via specific siRNAs haveshown that PML is essential in the execution of the senescence programme and that both p53 and p21 pathways areinvolved. IFN-b treatment leads to a modulation of p53 phosphorylation and acetylation status and a reduction in theexpression of the p53 dominant negative DNp73. These effects allow the recovery of p53 transactivating activity of targetgenes involved in the control of cell proliferation. Taken together, these studies suggest that signaling through the IFNpathway might play an important role in cellular senescence. This additional understanding of IFN antitumor action andmechanisms influencing tumor responsiveness or resistance appears useful in aiding further promising development ofbiomolecular strategies in the IFN therapy of cancer.

Citation: Chiantore MV, Vannucchi S, Accardi R, Tommasino M, Percario ZA, et al. (2012) Interferon-b Induces Cellular Senescence in Cutaneous Human PapillomaVirus-Transformed Human Keratinocytes by Affecting p53 Transactivating Activity. PLoS ONE 7(5): e36909. doi:10.1371/journal.pone.0036909

Editor: Alfredo Fusco, Consiglio Nazionale delle Ricerche (CNR), Italy

Received October 19, 2011; Accepted April 10, 2012; Published May 16, 2012

Copyright: � 2012 Chiantore et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Research in our laboratory is currently funded in part by ‘‘Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (PRIN 2008) del Ministerodell’Istruzione, dell’Universita e della Ricerca’’ and Progetti Ateneo, Sapienza University, 2009 and 2011. The funders had no role in study design, data collectionand analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

The group of cellular proteins known as Interferons (IFNs)

appeared to be expressed in infected cells as an early response to

viral infection but, in addition to their antiviral activity, IFNs also

have a profound effect on cell growth [1].

IFN-a2 was the first human protein shown to be effective for

cancer treatment and the first economically viable clinical product

developed from recombinant DNA technology in cancer therapy.

Antitumor activity of IFNs is probably exerted through direct

and indirect mechanisms. It is conceivable that numerous direct

effects play a central role in the overall antitumor response, such as

down-regulation of oncogene expression, induction of differenti-

ation, inhibition of cell cycle progression and induction of tumor

suppressor genes, and programmed cell death [2]. However,

additional understanding of IFN antitumor action and mecha-

nisms influencing tumor responsiveness or resistance appears

necessary to aid further promising development of biomolecular

strategies in IFN therapy of cancer.

The most extensively studied anticancer treatment-induced

mechanism is apoptotic programmed cell death. Nevertheless, the

correlation between the induction of apoptosis and drug response

cannot explain the overall tumor cell sensitivity [3]. Numerous

recent studies have shown that in cells where apoptosis is blocked,

non-apoptotic cell death or irreversible cell growth arrest, namely

senescence, can be activated as potential tumor-suppressor

mechanisms [4]. The concept of senescence is applied in general

to the irreversible proliferative arrest of cells caused by various

stresses [5] including oxidative damage, telomere dysfunction, and

DNA damage. One particularly relevant source of stress in tumor

cells is derived from the aberrant proliferative signals of oncogenes

which may trigger senescence through a process known as

oncogene-induced senescence, functioning as a potential tumor

suppressor mechanism.

Senescent cells are identified in vitro by distinctive morphological

changes, such as large cell size, flat vacuolated morphology, the

inability to synthesize DNA, the formation of domains of

heterochromatin called Senescence-Associated Heterochromatin

Foci (SAHF) and the expression of a Senescence-Associated b-

galactosidase activity (SA-b-gal) [6,7]. One of the earliest steps in

the senescence programme is the translocation of histone

chaperone HIRA (Histone Repression factor A) into promyelo-

cytic leukemia (PML) nuclear bodies (NBs), but the role played by

HIRA localization into PML bodies has not yet been identified.

PML bodies are nuclear structures known to serve as sites of

PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e36909

protein modification and the assembly of macromolecular

regulatory complexes, and have been extensively implicated in

the induction of senescence and apoptosis [8,9].

Consistent with its role in tumor suppression, the critical

senescence pathways converge on the two major tumor suppressor

genes p53 and pRb, whose mutations or inactivation are most

common in all cancers [10].

Abrogation of senescence can be achieved by SV40 large T, a

combination of HPV oncoproteins E6 and E7, E1A and MDM2

coexpression or small interfering RNA against pRb and p53 [11].

Tumors initiated by loss of p53 can be eliminated by senescence

induced by p53 restoration, tumor regression being achieved

through an innate immune response that leads to the clearance of

senescent cells [12,13]. Cellular senescence results in altered gene

expression including IFNs and their related genes [14]. PML is

known to be regulated by the interferon pathway via the STAT

transcription factors [15]. IFN also regulates many other

components of PML nuclear bodies, suggesting that in conjunction

they mediate the antiviral and antiproliferative activities of this

cytokine [9]. Cellular senescence is induced in human fibroblasts

by prolonged IFN-b treatment through DNA damage signaling

and a p53-dependent pathway [16]. IFN-a also induces replicative

senescence in endothelial cells after continuous stimulation [17].

Taken together, these studies suggest that signaling through the

IFN pathway might play an important role in cellular senescence.

Human Papilloma Viruses (HPVs) are small DNA viruses

involved in the development of both benign and malignant lesions

localised in different anatomic districts, that are able to replicate

exclusively in the stratified squamous cutaneous and mucosal

epithelium. More than 100 different HPV types have been isolated

so far, and they can be sub-grouped into cutaneous or mucosal

according to their ability to infect the skin or the mucosa of the

genital or upper-respiratory tracts [18]. To date, the causative

association between mucosal high risk-HPV and cervical carcino-

ma has been clearly demonstrated. It is well known that the

expression of E6 and E7 viral oncoproteins is a common feature of

cervical cancer cells and is strongly implicated in the process of

cancer development, E6 and E7 principally targeting and

inhibiting p53 and pRb tumor suppressor proteins, respectively

[19]. Emerging lines of evidence support the involvement of the

cutaneous HPV types belonging to the beta genus in non

melanoma skin cancer (NMSC). However, although the role of

beta HPV types in NMSC in Epidermodysplasia verruciformis

(EV) patients is well accepted, their involvement in skin

carcinogenesis in the general population is not entirely proven.

The transforming properties of the majority of cutaneous HPV

types have been poorly investigated. Tommasino and co-workers

[20] have shown that HPV38 E7 appears to act similarly to

HPV16 E7, by binding to pRB and promoting its degradation via

the proteasome pathway. On the contrary, HPV38 E6 oncopro-

tein differs from mucosal high risk HPV E6 proteins in the

mechanism by which it counteracts p53 activity. The expression of

HPV38 E6 and E7 in human keratinocytes induces the

stabilization of p53, which in turn selectively activates transcrip-

tion of DNp73, a p53 inhibitor [21]. High DNp73 levels have been

found in a number of human malignancies including cancers of

the breast, prostate, liver, lung and thyroid [22]. Recently, it has

been shown that IFN-a reduces DNp73 levels in Huh7 hepatoma

cells, and this effect correlates to increased susceptibility to IFN-atriggered apoptosis [23].

Here, we show that prolonged treatment with IFN-b induces

senescence in cutaneous HPV38-transformed keratinocytes. PML

is essential in IFN-b induction of senescence in HPV38-

transformed keratinocytes, and both p53 and p21 pathways

contribute to the execution of the phenomenon. p53 colocalyzes

with IFN-b-induced PML into PML Nuclear Bodies. By

recruitment of p53 into NBs, IFN-b can modulate p53 post-

translational modification at specific phosphorylation and acety-

lation sites and downregulate DNp73 expression, leading to the

recovery of p53 transactivating activity of selected target genes

involved in cell proliferation control.

Results

IFN-b Inhibits Cell Proliferation of K38 and K16 CellsKeratinocytes expressing E6 and E7 proteins of HPV-38 and

HPV-16, referred to as K38 and K16 respectively, were obtained

as described [20].

To test whether IFN-b could affect proliferation of both

transformed keratinocytes, K-38 and K-16 cells were treated with

IFN-b for several time points and the amounts of viable cells were

revealed. As shown in Fig. 1A, proliferation of both cell lines was

strongly affected by IFN-b.

To exclude that the antiproliferative effect of IFN-b could be

due to downregulation of E6/E7 expression, we checked HPV-38

and HPV-16 E6 and E7 mRNA levels by RT-PCR. No significant

variations were observed in either E6 or E7 expression in K38 and

K16 cells upon treatment with IFN-b for different time points

(data not shown).

We previously demonstrated that IFN-b exerts its antiprolifer-

ative effect on high-risk HPV-positive cell lines by lengthening cell

cycle S-phase progression [24]. We analyzed cell cycle distribution

of K38 and K16 cells after treatment with IFN-b for several time

points. Both cell lines showed a significant augment of S-phase cell

amount starting from 48 h of treatment. Interestingly, in K16 cells

S-phase cell accumulation increased with time whereas in K-38

cells the S-phase increase was followed by an augment of G1

population (Fig.1B).

K16 and K38 cells were pulse-labelled with BrdU for 5 h and

analysed for BrdU incorporation. As with what was observed in

SiHa and other mucosal high-risk HPV-positive cell lines [24], an

increased number of BrdU-positive cells reflecting an S-phase cell

accumulation was revealed in IFN-b treated K16 populations. On

the contrary, in K-38 samples the number of cells incorporating

BrdU upon IFN-b treatment appeared clearly reduced (Fig. 1C).

IFN-b Induces Cellular Senescence in K38 but Not in K16Cells

To study apoptosis and senescence induction, specific assays

were performed. Annexin-V externalization assay showed no

significant increase of apoptosis in either cell types after IFN-btreatment (data not shown). Senescent cells were quantified by

counting cells displaying b-galactosidase activity at pH 6.0 (SA-

bgal). This lysosomal hydrolase is elevated in senescent cells as a

result of lysosomal activity at suboptimal pH, which is detectable

only in senescent cells due to an increase in lysosomal content.

Interestingly, increasingly high percentages of senescent cells were

observed exclusively in K38 cells transformed by E6 and E7

proteins of cutaneous HPV genotype, starting from 4 days of IFN-

b treatment, compared to control keratinocytes (LXSN), K16 cells

and mucosal high risk HPV-positive cell lines (Fig. 2A, B, and C).

Involvement of PML, p53 and p21 in Cell SenescenceInduced by IFN-b in K-38 Cells

It is known that important senescence regulators are found in

IFN-inducible genes. In addition, it has been reported that

prolonged IFN-b stimulation can induce senescence in normal

cells through the activation of a DNA damage response triggered

IFNb-Induced Senescence and p53 Activity


by an ATM-chk2-p53 pathway [16]. We asked whether IFN-bcould induce the senescence phenotype in K38 cells through the

involvement of PML and the activation of p53, thus counteracting

the inhibitory action exerted on p53 by HPV-38 E6/E7

expression. We analysed the protein levels of PML, p53 and

p21, upon IFN-b treatment, and the respective involvement in

senescence through RNA silencing (siRNA) technique. Three

different siRNAs were used for PML, p53 and p21 genes. Upon

IFN-b treatment, PML was up-regulated as well as p21 (Fig. 3).

On the other hand in K16 cells, even if IFN-b treatment induces

PML expression, it is not detectable any increase in p21 protein

levels (data not shown).

PML seems to be an essential component of senescence

response in K38 cells, since, when PML expression is inhibited

by specific siRNAs (Fig. 4A), IFN-b-induced senescence is strongly

reduced (Fig. 4D). p21 silencing (Fig. 4C) partially affects IFN-b-

induced senescence (Fig. 4D), while p53 silencing (Fig. 4B) appears

much more effective (Fig. 4D), suggesting that different p53 targets

may be involved in IFN-b-induced senescence in K38 cells and

different pathways may cooperate towards this phenomenon.

PML Target Proteins Colocalyze in PML Nuclear BodiesIt is known that PML recruits into NBs p53 and different

proteins involved in p53 post-translational modifications that are

critical for the activation of p53 and for the selection of target

genes [25]. We studied colocalization of PML with p53 and

DNp73 through confocal microscopy analyses of K38 cells treated

with IFN-b for different time points. Fig. 5 shows that p53 (A) and

DNp73 (B) colocalyze with IFN-b-induced PML into PML NBs.

On the other hand, colocalization is not detectable after PML

silencing (data not shown).

Post-translational Modification of p53 by IFN-bThe expression of HPV38 E6 and E7 in human keratinocytes

induces the stabilization of p53, as shown by WB analysis of p53 in

K38 cells compared with control keratinocytes (LXSN), K16 cells

and high risk HPV-positive cell lines SiHA and ME-180 (Fig. 6A).

This p53 stabilization can be related to increased phosphorylation

[21] and acetylation (Fig. 6C).

In this respect, we may hypothesize that IFN-b, through PML

up-regulation, can lead to the recovery of p53 transactivating

activity of target genes involved in cell proliferation control.

Therefore p53 phosphorylation and acetylation status was

analyzed in K38 cells treated with IFN-b. IFN-b modulates p53

phosphorylation status at different phosphorylation sites (Ser-6,

Ser-15, Ser-46, Ser-392, Fig. 6B) while acetylation is mainly

downregulated in Lys-320 (Fig. 6C). Ser-6, Ser-392, and Lys-320

seem to be the most important p53 post-translational modifica-

tions involved in IFN-b-induced senescence in K38 cells. In fact,

when PML expression is silenced, IFN-b is not able to modulate

p53 Ser-6, Ser-392, and Lys-320 status (Fig. 6D).

Accardi et al. [21] reported that p53 stabilization in K38 cells

leads to transcriptional activation of DNp73, a p53 inhibitor, able

to inhibit p53 transactivation of genes involved in cell growth

suppression. It has been shown that IFN-a reduces DNp73 levels

in Huh7 hepatoma cells and this effect correlates to increased

susceptibility to IFN-a triggered apoptosis [23]. We observed that

in K38 cells, IFN-b treatment downregulates DNp73 mRNA levels

(data not shown). The DNp73 protein expression appears to be

reduced upon IFN-b treatment (Fig. 6E), probably as a result of

the p53 post-translational modifications induced by IFN-b. In fact,

when PML expression is silenced, DNp73 protein levels are not

downregulated by IFN-b (Fig. 6D).

Real time PCR array results indicate that some genes involved

in senescence and growth control are IFN-b-upregulated (Fig.6F).

In particular, the observed induction of p53 target genes Bax and

Pig3 indicates that IFN-b treatment leads to the recovery of p53

transactivating activity of selected target genes involved in the

control of cell proliferation. In fact, it has been reported that

modification of specific p53 phosphorylation and acetylation sites

may correlate to the transactivation of growth related genes,

suggesting a tissue and promoter-specific p53 activity regulation

[26]. PML depletion reduces IFN-b induction of Bax and Pig3 in

K-38 cells (Fig.6F), indicating the role of PML in the ability of

IFN-b to recover p53 transactivation activity of specific target

genes.

Discussion

IFNs were the first successful biological therapy for human

malignancy and currently there are several approved IFN cancer

therapies. Clinical effectiveness of different IFN subtypes in

treatment of various forms of cancer has been extensively reviewed

[1]. Better definition of therapeutic molecular targets appears to be

critical to fully realize the potential of IFNs in oncology and

further understand the mechanisms of antitumor action of the IFN

family.

Senescence is a permanent cell cycle arrest that is resistant to

growth factors and other signals that induce cell proliferation. It

has been proposed that senescence prevents cancer in the early

stages of its development [6]. Tumor suppressors such as p53, pRb

and PML are critical regulators of senescent programme [10,25],

and genes required for senescence are often found to be mutated

in human cancers. Cellular senescence is induced in human

fibroblasts by prolonged IFN-b treatment through DNA damage

signaling and a p53-dependent pathway [16]. IFN-a also induces

replicative senescence in endothelial cells after continuous

stimulation [17], and treatment with IFN-c induces cellular

senescence in young human umbilical vascular endothelial cells

[27]. However, whether induction of senescence is sufficient to

repress tumor in vivo is controversial. Recent reports showed that

conditional restoration of p53 in mice with hepatocarcinomas,

sarcoma or lymphoma is able to promote tumor regression

[13,12]. In addition, it has been reported that HeLa cells cease

proliferation and undergo senescence by introduction of the

bovine papillomavirus E2 gene that inhibits the expression of the

Figure 1. IFN-b affects cell proliferation in K16 and K38 cells. (A) IFN-b inhibits cell proliferation in K16 and K38 cells. Cells were seeded intriplicate at 105 cells per 35 mm dish and, after 24 h, IFN-b was added to the cultures for the indicated times. IFN-b treated and control cells werecounted in a hemocytometer and viability was evaluated by trypan blue exclusion. Data represent means 6 s.d. of three independent experiments.** = p,0.01; *** = p,0.001. (B) IFN-b treatment differently affects cell cycle progression of K16 and K38 cells. K16 and K38 cells were treated with IFN-b for the indicated time points. DNA staining was performed by incubating cells in PBS containing 0.18 mg/ml propidium iodide and 0.4 mg/mlDNase-free RNase. Cells were analysed on a FACScan flow cytometer. DNA profiles, derived from one representative experiment of three performed,are shown. (C) IFN-b treatment differently affects DNA synthesis in K16 and K38 cells. To determine the number of S-phase nuclei, cells were plated intriplicate at 105 cells per 35 mm dish, treated with IFN-b for different time points and incubated with 50 mM BrdU for the last 5 hours. BrdU treatedsamples were then fixed and stained with an anti-BrdU monoclonal antibody followed by a rhodamine conjugated goat anti-mouse antibody. BrdU-positive cells were counted under a fluorescence microscope. Data represent means 6 s.d. of three independent experiments.doi:10.1371/journal.pone.0036909.g001



HPV18 E7 gene [28]. Antisense sequences directed against

HPV16 E6 and E7 genes transfected in SiHa cells contributed

to apoptosis and senescence [29].

In contrast to mucosal high-risk HPV types, the involvement of

cutaneous HPV types in human carcinogenesis is still unclear.

Cutaneous HPV types that belong to the beta genus of the HPV

phylogenetic tree were first isolated in patients suffering from EV,

a rare autosomal recessive cancer-prone genetic disorder, and are

consistently detected in NMSC from EV, immunocompromised

and normal individuals [30]. The transforming properties of the

majority of the cutaneous HPV types have been poorly

investigated. It has been reported that cutaneous HPV5 E6

protein targets and abrogates Bak function by promoting its

proteolitic degradation both in vitro and in regenerated epithelium

[31]; however, regulation of Bax has also been reported [32]. The

E6 protein of HPV5 compromises the repair of UV-induced

thymine dimers [33] and E6 of HPV7 forces keratinocytes into the

S1-phase by inhibiting p53-activated, pro-apoptotic genes [34].

HPV8 E6 is able to bind XCRR1 that functions in a single strand

DNA repair [35] and it has been shown that UV-irradiated

cutaneous HPV8 E2-transgenic mice develop invasive carcinoma-

tous lesions more rapidly than non-irradiated counterparts [36].

Moreover, E6/E7 expression of HPV20 influences proliferation

and differentiation of the skin of UV-irradiated transgenic mice

[37]. The anti-apoptotic activity and the delay of the DNA repair

mechanism may lead to the persistence of UV-damaged kerati-

nocytes, suggesting that cutaneous HPV types may be involved in

the early stages of carcinogenesis.

A different mechanism behind the lack of cell cycle arrest in

cutaneous HPV expressing cells is the up-regulation of DNp73 as a

result of p53 accumulation observed in HPV38 E6 and E7

expressing human keratinocytes. DNp73 in turn inhibits the

capacity of p53 to induce the transcription of genes involved in

growth suppression [20,21]. This observation, together with the

efficiency of pRb binding and degradation by HPV38 E7, the

HPV38 E6/E7-induced suboptimal activation of telomerase and

the HPV38 E6/E7 transforming properties in vivo [38], seems to

indicate that HPV38 E6 and E7, differently from proteins of other

cutaneous HPV types, may be involved in the maintenance of

oncogenic transformation.

We have previously reported that type I IFNs inhibit cell

proliferation in high risk mucosal HPV-positive Squamous

Carcinoma Cell (SCC) lines by inducing a significant accumula-

tion of cells in S-phase [24]. The S-phase deregulation triggers

apoptotic cell death specifically mediated by the pro-apoptotic

factor TRAIL [39].

The present study shows that IFN-b affects cell proliferation in

keratinocytes expressing E6 and E7 proteins of cutaneous HPV-38

to a greater extent than in E6 and E7 mucosal HPV-16

transformed cells. In particular, K38 cells undergo senescence

upon prolonged IFN-b treatment. IFN-b appears to induce

senescence by up-regulating the expression of the tumor suppres-

sor PML. Indeed, experiments of gene silencing via specific

siRNAs have shown that PML is essential in the execution of

senescence programme and that both p53 and p21 pathways are

involved in senescence induction by IFN-b in K38 keratinocytes.

P53 and PML are critical mediators of senescence. PML is

essential for the formation of discrete protein assemblages in the

nucleus known as Nuclear Bodies (NBs) [40]. PML recruits into

NBs p53 and proteins involved in p53 post-translational modifi-

cations that are essential for the activation of p53 and for the

selection of target genes, such as the DNA damage responsive

kinases ATM and ATR [25]. ATM kinase phosphorylates p53 at

Ser-15, a senescence-inducible modification [41], in IFN-b-

induced cellular senescence in human fibroblasts [16]. Over-

expression of PML is capable of inducing premature senescence by

stabilizing p53 via p53 acetylation on Lys-382 and phosphoryla-

tion on Ser-15 and Ser-46 [42]. In contrast, deacetylation of p53

antagonizes PML-induced premature senescence [43].

It has been shown that PML interacts with CBP/p300

acetyltransferase and stabilizes p53 through Lys-382 acetylation

[42]. PML also recruits the tumor suppressor homeodomain-

interacting protein kinase-2 (HIPK2) which induces p53Ser46

phosphorylation [44]. It has been reported that HIPK2-mediated

phosphorylation of p53Ser46 is required for the CBP-induced p53

acetylation at Lys-382 [45]. PML has also been recently identified

as a direct target of p53 revealing a regulatory positive feedback

loop between p53 and PML [46].

Our results indicate that in K38 cells p53 colocalyzes with IFN-

b-induced PML into PML NBs. IFN-b can significantly modulate

Figure 2. IFN-b induces senescence in K38 cells. (A) Control keratinocytes (LXSN), K16, K38 cells and high risk HPV-positive squamous carcinomacell lines ME-180, Caski, HeLa and SiHa were treated with IFN-b for 4 days and senescent cells were quantified by counting cells displaying senescent-associated b-galactosidase (SA-bgal) activity at pH 6.0. (B) Percentage of senescent cells in K16 and K38 cells treated with IFN-b for different timepoints. (C) SA-bgal-positive K38 blue cells observed under a light microscope after 4 days of IFN-b treatment. * = p,0.05; *** = p,0.001.doi:10.1371/journal.pone.0036909.g002

Figure 3. IFN-b affects the expression of proteins involved in senescence in K38 cells. Western blot analysis of PML, p53 and p21expression in K38 treated with IFN-b for different time points. Whole cell extracts were resolved on SDS-PAGE and transferred onto PVDF membrane.Immunoblotting was performed as reported in M&M.doi:10.1371/journal.pone.0036909.g003



p53 phosphorylation at Ser-6,-15,-46, and -392 and acetylation

status mainly at Lys-320. It has been also shown that p53

acetylation in Lys-320 is the first step in IFN-b-induced senescence

in human fibroblasts [16].

DNA damage response is required for the activation of p53 in

response to oncogenes. Oncogene induced senescence is accom-

pained by DNA replicative stress, including prematurely termi-

nated DNA replication forks and DNA double-strand breaks

caused by hyper-DNA replication [47]. Consistent with this, Ras-

induced senescence is associated with activation of DNA damage

response effectors, such as ATM/ATR and Chk2/Chk1, and

inactivation of these DNA damage effectors by RNA interference

attenuates oncogene induced senescence [48,49]. We observed

that the inhibition of ATM and ATR prevents IFN-b induction of

senescence in K38 keratinocytes, suggesting that IFN-b might

induce senescence through a p53-dependent DNA damage

pathway (data not shown).

It has been reported that HPV16 E6 mediates resistance to IFN-

induced senescence through inhibition of p53 acetylation by

binding to CBP/p300. Conversely, treatment of HPV16 E7-

expressing cells with IFN ultimately resulted in cellular senescence

through a process that is dependent upon acetylation of p53 by

CBP/P300 [50]. Moreover, HPV16 E7 up-regulates SIRT1, thus

attenuating p53 activity via its deacetylation [51]. It has been

shown that HPV16 E6 can induce multiple site phosphorylation of

p53 [52]. HPV38 E6 and E7 expression in human keratinocytes

induces phosphorylation of p53, which leads to the up-regulation

of DNp73 and the inhibition of p53 transcriptional induction of

genes involved in growth suppression [21]. All together these

observations indicate that p53 post-translational modifications are

critical for p53 involvement in senescence programme induced by

IFN-b and that modulation of p53 activity could be a common

strategy utilized by both mucosal and cutaneous HPV to inhibit

p53 function.

Figure 4. Effect of PML, p53 and p21 silencing on senescence induction by IFN-b in K38 cells. PML (A), p53 (B) and p21 (C) were silencedby specific small interfering RNAs and protein expression was analyzed by Western blot in cells treated with IFN-b for 4 days. (D) Senescenceinduction by IFN-b (4 days treatment) was evaluated by SA-bgal staining. * = p,0.05; ** = p,0.01.doi:10.1371/journal.pone.0036909.g004



Figure 5. p53 and DNp73 co-localyze with IFN-b-induced PML into PML Nuclear Bodies. (A,B) For confocal microscopy analysis, K38 cellswere cultured on glass bottom dishes (MatTek Corporation) and treated with IFN-b for 4 days. Cells were then fixed in PBS 4% paraformaldehyde for30 min on ice, immuno-fluorescence labelling was performed as described in Materials and Methods and sample were analyzed using confocalmicroscope (Leica TCS SP5).doi:10.1371/journal.pone.0036909.g005



We report that by recruitment of p53 into NBs via PML

induction, IFN-b may modulate p53 phosphorylation and

acetylation status and downregulate DNp73 expression in K38

keratinocytes, leading to the recovery of p53 transactivating

activity of selected target genes involved in cell proliferation

control. Real time PCR array confirms that some genes involved

in senescence and growth control are IFN-b-upregulated, in

agreement with the reported observations [25] that modification of

specific p53 phosphorylation and acetylation sites may correlate to

the transactivation of growth related genes, suggesting a tissue and

promoter-specific p53 activity regulation.

Our results contribute to one of the most interesting current

research questions about the exact contribution of post-transla-

tional modification sites to the selectivity of the global transcrip-

tional programme of p53. Other important questions remain to be

answered to clarify the multitude and redundancy of p53 covalent

post-translational modifications required for p53-dependent se-

nescence. It is possible that no single specific post-translational

modification leads to specific p53 gene transactivation activity, but

each modification might help to regulate p53 function in a tissue

and promoter-specific manner.

Materials and Methods

Cell Cultures and TreatmentsPrimary human foreskin keratinocytes were transduced with

empty retrovirus pLXSN (control), or with pLXSN38E6E7 or

pLXSN16E6E7 as described by Caldeira et al., 2003 [20] and

were grown in KBM BulletKit (Lonza).

HPV16-positive cell line Caski and SiHa, HPV18-positive cell

line HeLa, and HPV68-positive cell line ME180, obtained from

the American Type culture Collection, were grown in Dulbecco’s

modified Eagle’s medium (DMEM) with 10% fetal bovine serum.

Cells were maintained in a humidified atmosphere of 5.5% CO2

at 37uC.

Human recombinant IFN-b (Rebif; 36108 IU/mg of protein;

Ares-Serono) was added to the medium at the concentration of

200 IU/ml.

Measurement of Cell ProliferationTransformed keratinocytes were seeded in triplicate at 105 cells

per 35 mm dish. After 24 h, IFN-b was added to the cultures for

the indicated times. Adherent cells were detached with 0.05%

trypsin-0.02% EDTA in PBS, suspended in growth medium, and

counted in a hemocytometer. Viability was evaluated by trypan

blue exclusion.

Flow CytometryFor cell cycle analysis, cells were fixed in 70% ice-cold ethanol

for at least 30 min. DNA staining was performed by incubating

cells in PBS containing 0.18 mg/ml propidium iodide and

0.4 mg/ml DNase-free RNase. Cells were analysed on a FACScan

flow cytometer (Becton and Dickinson).

BrdU IncorporationTo determine the number of S-phase nuclei, cells were plated in

triplicate at 105 cells per 35 mm dish, treated with IFN-b for

different time points and incubated with 50 mM BrdU for the last

5 hours. Samples were fixed with 95% ethanol, 5% acetic acid,

treated with 1.5 M HCl and stained with an anti-BrdU

monoclonal antibody (Amersham) followed by a rhodamine

conjugated goat anti-mouse antibody (Cappel).

Senescence-associated b-galactosidase StainingCells were plated in 12 multi-well, 0,56105 cells per well, and

treated with IFN-b at different time points. Senescent cells were

quantified by counting cells displaying senescent-associated b-

galactosidase activity at pH 6.0, assayed through Senescent

Detection Kit (Calbiochem) following manufacturer’s instruction.

Western Blot AnalysisTo analyse protein expression, control and IFN-b treated cells

were lysed in SDS reducing sample buffer. Total cell extracts were

clarified by centrifugation and boiled in the presence of 5% 2-

Mercaptoethanol and 0.01% bromophenol blue. Protein concen-

tration was determined (Bio-Rad Protein Assay) and 30 mg of total

proteins were resolved on SDS-PAGE and transferred onto PVDF

membrane (Amersham). The membranes were blocked with 5%

skim milk dissolved in PBS-T and incubated with primary

antibodies (mouse anti-p53; rabbit anti-p21 (Santa Cruz); rabbit

anti-PML (Bethyl); rabbit anti-phospho-p53 Ser6, anti-phospho-

p53 Ser15, anti-phospho-p53 Ser46, anti-phospho-p53 Ser392

(Cell Signaling); rabbit anti-acetyl-p53 Lys320, anti-acetyl-p53

Lys373/382 (Millipore), and anti-human b tubulin mouse IgG1

antibody (ICN), as an internal control. Immune complexes were

detected with horseradish peroxidase-conjugated goat anti-rabbit

and anti-mouse antiserums (ICN) followed by enhanced chemilu-

minescence reaction (Millipore).

PML, p53 and p21 silencingSmall interfering RNAs (siRNAs) targeted to PML, p53 and p21

were designed and validated by Qiagen and a non-silencing

siRNA (Qiagen) served as control.

PML siRNAs were: 1) Sense:59-CGUCUUUUUCGAGAGU-

CUGtt-39;Antisense:59-CAGACUCUCGAAAAAGACGtt-39;

2)Sense:59-CCCGCAAGACCAACAACAUtt-39; Antisense: 59-

AUGUUGUUGGUCUUGCGGGtg-39; 3)Sense:59-GGGACC-

CUAUUGACGUUGAtt-39; Antisense: 59-UCAACGU-

CAAUAGGGUCCCtg-39.

p53 siRNA were: 1) Sense:59–GGAAAUUUGCGUGUGGA-

GUtt-39; Antisense: 59–CUCCACACGCAAAUUUCCtt-39; 2)

Sense: 59-GCAUCUUAUCCGAGUGGAAtt-39; Antisense: 59-

UUCCACUCGGAUAAGAUGCtg-39; 3) Sense: 59-GCA-

GUUAAGGGUUAGUUUAtt-39; Antisense: 59-UAAA-

CUAACCCUUAACUGCaa-39.

p21 siRNA (Dharmacon) was: Sense:5’-GAUGGAACUUC-

GACUUUGUUU-3’: Antisense:5’-PACAAAGUCGAAGUUC-

CAUCUU 3’.

Figure 6. IFN-b effect on p53 post-translational modification and expression of its target genes. (A) WB analysis of p53 in controlkeratinocytes (LXSN), K16 and K38 cells and in high risk HPV-positive squamous carcinoma cell lines SiHa and ME-180 treated with IFN-b for 48 h. (B)WB analysis of p53 phosphorylated at different phosphorylation sites. (C) WB analysis of acetylated p53. (D) WB analysis of phosphorylated andacetylated p53 and DNp73 in K38 cells silenced by PML siRNA and treated with IFN-b for 48 h. (E) WB analysis of DNp73 in K38 cells treated with IFN-bfor different time points. Whole cell extracts were resolved on SDS-PAGE and transferred onto PVDF membrane. Immunoblotting was performed asreported in M&M. (F) Real time PCR analysis of Bax and Pig3 was carried out on K38 cells treated with IFN-b for 48 h, also in the presence of PMLsiRNA. NT = not transfected. * = p,0.05; ** = p,0.01.doi:10.1371/journal.pone.0036909.g006



Shortly before transfection, 26105 cells per dish were seeded in

35 mm dishes in 1 ml of fully supplemented culture medium.

siRNA was diluted in 50 ml culture medium without supplements

to a final concentration of 10 nM. 3.5 ml of HiPerfect Transfection

Reagent (Qiagen) were added to the diluted siRNA and mixed by

vortexing. After an incubation of 10 min at room temperature, the

transfection complex was added drop-wise onto the cells. 24 hrs

post-transfection cells were treated with IFN-b for the indicated

time points.

ImmunofluorescenceImmunofluorescence staining of cells was performed on cells

grown on Glass Bottom Culture Dishes 14 mm Microwell poly-d-

lysine Coated from Mat Tek Corporation (Ashland, MA 01721

U.S.A.) and fixed with 4% formaldehyde, permeabilized with

PBS/0.1% Triton-X, and stained using the following primary

antibodies: anti-PML; ap53 (Santa Cruz), anti-DNp73 (Imgenex).

Anti-mouse-Fitc (Cappel), anti-mouse-Alexa 546 (Molecular Probe

# A11030), and anti-rabbit-Alexa 610 (Molecular Probe #A31551) were used as secondary antibodies. Sample were

analyzed using confocal microscope (Leica TCS SP5). Software:

LAS AF version 1.6.3 (Leica Microsystem).

Real-time PCRReal-time PCR was carried out by using the MESA GREEN

MasterMixes Plus, Low ROX (Eurogentec). The primer sequences

are: Bax F: 5’ TTT GCT TCA GGG TTT CAT CC 3’, R: 5’

ATC CTC TGC AGC TCC ATG TT 3’; Pig3 F: 5’ GCT TCA

AAT GGC AGA AAA GC 3’, R: 5’ AAC CCA TCG ACC ATC

AAG AG 3’.

Acknowledgments

We thank Roberto Gilardi for excellent editorial assistance.

Author Contributions

Conceived and designed the experiments: MVC GF GR. Performed the

experiments: MVC SV ZAP RA. Analyzed the data: MVC SV ZAP GV

EA GF GR. Contributed reagents/materials/analysis tools: RA MT.

Wrote the paper: MVC GF GR.

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