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Published as doi: 10.1096/fj.07-8389com.
 (The FASEB Journal. 2008;22:622-632.)
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(The FASEB Journal. 2008;22:622-632.)
© 2008 FASEB

Impairment of the telomere/telomerase system and genomic instability are associated with keratinocyte immortalization induced by the skin human papillomavirus type 38

Anne-Sophie Gabet*, Rosita Accardi*, Angelica Bellopede*, Susanne Popp{dagger}, Petra Boukamp{dagger}, Bakary S. Sylla*, J. Arturo Londoño-Vallejo{ddagger} and Massimo Tommasino*,1

* International Agency for Research on Cancer, Lyon, France;

{dagger} Deutsches Krebsforschungszentrum, Heidelberg, Germany; and

{ddagger} CNRS-Institut Curie-UPMC, UMR7147, Paris, France

1Correspondence: Infections and Cancer Biology Group, International Agency for Research on Cancer, World Health Organization, 150 Cours Albert Thomas, 69372 Lyon, France. E-mail: tommasino{at}iarc.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The skin human papillomavirus (HPV) types belonging to the genus beta of the HPV phylogenetic tree appear to be associated with nonmelanoma skin cancer. We previously showed that the beta HPV type 38 E6 and E7 oncoproteins are able to inactivate the tumor suppressors p53 and retinoblastoma. Here, both viral proteins were expressed in primary human skin keratinocytes in order to study their effects on the telomere/telomerase system. We show that immortalization of skin keratinocytes induced by HPV38 E6/E7 is associated with hTERT gene overexpression. This event is, in part, explained by the accumulation of the p53-related protein, {Delta}Np73. Despite elevated levels of hTERT mRNA, the telomerase activity detected in HPV38 E6/E7 keratinocytes was lower than that observed in HPV16 E6/E7 keratinocytes. The low telomerase activation in highly proliferative HPV38 E6/E7 keratinocytes resulted in the presence of extremely short and unstable telomeres. In addition, we observed anaphase bridges, mitotic multipolarity, and dramatic genomic aberrations. Interestingly, the ectopic expression of hTERT prevents both telomere erosion and genomic instability. Thus, we showed that in HPV38 E6/E7 keratinocytes characterized by unscheduled proliferation, suboptimal activation of telomerase and subsequent extensive telomere shortening result in genomic instability facilitating cellular immortalization. Gabet, A.-S., Accardi, R., Bellopede, A., Popp, S., Boukamp, P., Sylla, B. S., Londoño-Vallejo, J. A., Tommasino, M. Impairment of the telomere/telomerase system and genomic instability are associated with keratinocyte immortalization induced by the skin human papillomavirus type 38.


Key Words: HPV38 E6 and E7 • telomere dysfunctions • chromosomal abnormalities


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OVER 90 DIFFERENT HUMAN papillomaviruses (HPVs) have been characterized and recently classified by genera, based on their DNA sequences (1) . The different HPV types can have different tissue tropism and can infect mucosa of the genital or upper respiratory tract or the skin. It has been conclusively proven that a subgroup of the mucosal HPVs, referred to as high-risk types, is the etiological cause of cervical cancer (2) . These HPV types, e.g., HPV16 and HPV18, are able to immortalize human keratinocytes, their natural host cells in vivo. This event is mainly mediated by two viral oncoproteins, E6 and E7, which alter cell cycle control and apoptosis via their interaction with and degradation of p53 and the retinoblastoma gene product (pRb), respectively (3) .

In addition to the mucosal high-risk HPV types, emerging lines of evidence suggest that cutaneous HPVs of the genus beta of the HPV phylogenetic tree (1) may also be involved in human carcinogenesis. These cutaneous HPV types have been initially detected in nonmelanoma skin cancer (NMSC) among patients suffering from a rare autosomal recessive cancer-prone genetic disorder termed epidermodysplasia verruciformis (EV) (reviewed in ref. 4 ). The development of highly sensitive polymerase chain reaction (PCR) -based assays for HPV DNA detection has shown that the presence of beta HPV DNA is not exclusively restricted to skin lesions of EV patients but can also be frequently found in NMSCs of immunocompromised and immunocompetent individuals (4) .

Initial studies focused on characterizing the biological properties of different EV HPV types, e.g., HPV5 and 8, showed that their E6 protein can interfere with the regulation of UV-induced apoptosis (5) . In addition, we have previously demonstrated that the EV HPV38 E6 and E7 proteins, like HPV16 E6/E7, are able to alter the functions of p53 and pRb (6 , 7) . However, the two viruses inactivate p53 by distinct mechanisms. Unlike HPV16, HPV38 does not induce p53 degradation but rather promotes its phosphorylation and stabilization, leading to the accumulation of {Delta}Np73, a potent inhibitor of p53 transcriptional functions (7) . In agreement with these data, HPV38 E6 and E7 considerably increase the life span of primary human skin keratinocytes (HSKs), the natural target cells of the virus (6) .

In addition to cell cycle checkpoint and apoptosis alteration, impairment of the telomere/telomerase system plays a key role in the high-risk HPV-induced immortalization of keratinocytes. Telomeres consist of TTAGGG repetitions located at chromosome extremities and are synthesized by the telomerase complex. In a somatic cell characterized by very little or no telomerase activity, telomeres shorten as a function of cellular division to finally reach a critical size, leading to replicative senescence. By contrast, almost all tumor cells harbor a very high level of telomerase activity, allowing telomere maintenance and indefinite proliferation (reviewed in ref. 8 ). However, suboptimal telomerase activity in a context of rapid cellular growth has been shown to lead to telomere attrition and dysfunctions that, together with p53 and p16INK4a/Rb pathway inactivation, result in dramatic genomic rearrangements promoting cellular immortalization (8 9 10) . A similar scenario has been observed during the immortalization process driven by HPV16. In fact, recent reports indicate that HPV16 E6 is able, through its association with the E3 ubiquitin ligase E6AP, to activate the transcription of the hTERT (human telomerase reverse transcriptase) gene encoding the catalytic subunit of the telomerase complex (11 , 12) . This effect directly results in telomerase activity up-regulation (13) . Nevertheless, a continuous telomere shortening is observed in increasing population doublings (PDs) of HPV16 E6 expressing keratinocytes (14) . This event, together with inactivation of p53 and p16INK4a/Rb pathway by the HPV16 E6/E7 oncoproteins, is thought to be responsible for the accumulation of chromosomal rearrangements that in turn favors cellular immortalization (15 , 16) .

So far, nothing is known about the effect of skin HPV types on the regulation of the telomere/telomerase system. Therefore, we have determined whether the HPV38 E6 and E7 oncoproteins are able to alter this system and whether this plays a role in the immortalization of HSKs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Phoenix cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum. Three types of primary human keratinocytes (HSK) were used in independent experiments, as specified in the Results section and grown as described previously (6 , 17) , namely: 1) foreskin keratinocytes, 2) female neonatal keratinocytes (Cambrex, Walkersville, MD, USA), and 3) adult skin keratinocytes.

Retroviral expression vectors
Two retroviral vectors, pLXSN (Clontech, Palo Alto, CA, USA) and pBabe-puro (18) , were used in the study. The pLXSN HPV38 E6/E7 construct was previously described (6) , and pBabe-puromycin hTERT construct was kindly provided by Dr Jerry Shay, Simmons Comprehensive Cancer Center (Dallas, TX, USA).

Gene silencing
Silencing of {Delta}Np73 expression was achieved using antisense oligonucleotide, while HPV38 E6/E7 gene expression was down-regulated using the pRetroSuper construct (pRS), expressing small hairpin RNAs (shRNAs) for the polycistronic HPV38 E6 and E7 mRNA (pRS 38E6/E7), as previously described (7) .

Retroviral infections
High-titer retroviral supernatants (>5.106 IU/ml) were generated by transient transfection of Phoenix cells and used to infect primary HSKs as described by Caldeira et al. (6) . After infection, cells were selected in 100 µg of G418/ml (8–10 days) or 0.4 µg of puromycin/ml (4 or 5 days).

RT-PCR and real-time PCR analyses
Total RNA was isolated from keratinocytes using the Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA, USA), adding a DNase I treatment to prevent cellular DNA contamination in the PCR reaction. cDNAs were synthesized from 1 µg of total RNA samples by reverse transcription using the first-strand cDNA synthesis kit (MBI Fermentas GmbH, St. Leon-Rot, Germany). PCRs were performed using primers specific for all forms of hTERT (TOT-hTERT-F: 5'-cgagctgctcaggtctttcttttatg-3'; TOT-hTERT-R: 5'-ccacgacgtagtccatgttcacaatc-3'; TERT2109-F: 5'-gcctgagctgtactttgtcaa-3'; TERT2531-R: 5'-aggctgcagagcagcgtggagagg-3' (19) ; HPV38 E7 (HPV38-E7-F: 5'-attgacctgcattgccac-3'; HPV38-E7-R: 5'-cggtggcccacacgtatagtt-3') or HPV16 E7 (HPV16-E7-F: 5'-cagctcagaggaggaggatg-3'; HPV16-E7-R: 5'-gcccattaacaggtcttcca-3') or GAPDH (GAPDH-F: 5'-gccaaaagggtcatcatc-3'; GAPDH-R: 5'-tgccagtgagcttcccgttc-3'). The HotStarTaq DNA Polymerase (Qiagen, Valencia, CA, USA) was used for hTERT amplifications. For semiquantitative RT-PCR analysis, PCRs were performed using serial dilutions of cDNA and the FL hTERT primers (FL-hTERT-F: 5'-tacgacaccatcccccag-3'; 5'-FL-hTERT-R: 5'-aagcgtaggaagacgtcgaa-3'). PCR products were separated by electrophoresis through 2% agarose gels and the intensity of PCR bands was quantified by the Quantity 1 4.1.0 program (Bio-Rad Laboratories, Hercules, CA, USA) and normalized to the levels of GAPDH.

Real-time PCR was performed using the LightCycler® FastStart DNA MasterPLUS SYBR Green I kit (Roche, Mannheim, Germany) with the following primers: hTERT-total-F (5'-tgtttctggatttgcaggtg-3') and hTERT-total-R (5'-gttcttggctttcaggatgg-3') that amplify all forms of hTERT. The hTERT signals were normalized to the GAPDH values obtained using the primers described above (GAPDH-F and GAPDH-R).

Transfection
A plasmid containing the hTERT promoter cloned in front of the luciferase reporter gene (20) was cotransfected with increasing amounts of pRS 38E6/E7 in HPV38 E6/E7 HSKs using the lipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA). As an internal control for transfection efficiency, a plasmid expressing the Renilla luciferase gene was also included in each transfection. Similarly, {Delta}Np73 antisense oligonucleotide was cotransfected with the hTERT-luciferase construct using the oligofectamineTM reagent (Invitrogen). Total DNA levels were equilibrated with empty pRS construct or {Delta}Np73 sense oligonucleotide. After 48 h, cells were collected and Firefly and Renilla luciferase activities were measured with the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA).

Telomeric repeat amplification protocol (TRAP) assays
A telomerase activity assay was performed on 0.5 µg cellular extracts using a PCR-based commercially available kit (TeloTAGGG Telomerase PCR ELISAPLUS kit, Roche). The levels of the PCR products were determined by a colorimetric reaction in an ELISA.

Immunoblot analysis
Total protein extracts were prepared by lysing cells in lysis buffer (20 mM Tris-HCl pH 8, 200 mM NaCl, 0.5% Nonident P40, 1 mM EDTA, 10 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin) for 20 min at 4°C. After centrifugation (12000 g, 5 min), the supernatant was collected and used for immunoblot analysis. Cell extracts were fractionated by electrophoresis on a sodium dodecyl sulfate (SDS) -polyacrylamide gel. Proteins were transferred onto a Polyscreen PVDF membrane (NEN Life Sciences, Boston, MA, USA) in a Trans-Blot semidry electrophoretic transfer cell (Bio-Rad) (130 mA, 1 h 30 min). {Delta}Np73 levels were determined by immunoblotting using an anti p73 (Antip73 Ab-1; Calbiochem, Novachem, Nottingham, UK, dilution 1:1000) since no other p73 isoforms were expressed in HPV38 E6/E7 HSK (7) . β-actin levels were determined using an anti β-actin (C4; MP Biomedicals, Orangeburg, NY, USA, dilution 1:5000). The intensity of the protein bands was quantified and the {Delta}NP73 levels were normalized according to the β-actin signal.

Quantitative fluorescence in situ hybridization analysis
Metaphase chromosome spreads were prepared from normal or HPV38 E6/E7 HSKs after treatment with colchicine (0.2 µg/ml, 3h), hypotonic (KCl, 0.56 M) and methanol:acetic acid (3:1 v:v). Fixed cells were hybridized with a telomeric (C3TA2)3-Cy3 PNA probe and counterstained with 4'-6-diamidino-2-phenylindole (DAPI). Fluorescent signals were visualized under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) with the appropriate filters and captured using a charge-coupled device (CCD) camera (Photometrics-Sensys, Tucson, AZ, USA). Original black-and-white Cy3 images were used for quantitative analysis using the Iplab Spectrum P Software (Scanalytics, Rockville, MD, USA). Distribution of fluorescence intensities was obtained by averaging the pixel intensities of individual telomeric signals and subtracting background levels measured at interstitial chromosome segments.

Anaphase bridge index determination
Monolayer cultured keratinocytes were fixed with formaldehyde 3.7% and stained with DAPI. Slides were examined under a Zeiss Axioplan 2 microscope (Carl Zeiss, Thornwood, NY, USA) using x100 magnification. The anaphase bridge index corresponds to the percentage of anaphases harboring at least one bridge.

Immunostaining
Immunofluorescence staining of nocodazole synchronized keratinocytes was carried out on paraformaldehyde-fixed cells. Cells were first permeabilized with a solution containing Triton X-100 0.5% and incubated O/N at 4°C with the mouse anti-{alpha} tubulin antibody (A11126; Molecular Probes, Eugene, OR, USA) diluted 1:400 in PBS/BSA. After extensive washing, cells were incubated with FITC-conjugated anti-mouse antibody (F2883; Sigma-Aldrich, St. Louis, MO, USA) diluted 1:400 in PBS/BSA and mounted in Vectashield® Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Data shown correspond to the percentage of dividing cells harboring more than two mitotic poles.

Multiplex fluorescence in situ hybridization analysis
Metaphase spreads were prepared as described previously (21) . Combinatorial labeling of whole chromosome painting probes and multicolor-hybridization were performed according to the method described by Popp et al. (22) . Hybridized metaphase spreads were evaluated under a fluorescence microscope (Leica Microsystems, Milton Keynes, Buckinghamshire, UK). Under control of the Leica quantitative fluorescence in situ hybridization software, images were acquired separately for each fluorochrome using a Sensys CCD camera (Photometrics-Sensys) with a Kodak KAF 400 chip. Images were processed using the Leica Multicolor Karyotyping (MCK) software package for spectral image analysis. On average, 10 metaphase spreads were evaluated per cell line.

Statistical analyses
The two-sample t test (Student’s t test) was used for statistical analysis of significance. A value of P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immortalization of human skin keratinocytes by HPV38 E6 and E7 is associated with up-regulation of hTERT mRNA expression
HPV16 E6 and E7, when expressed in primary human keratinocytes, are able to induce unscheduled cellular proliferation through the inhibition of the p53 and pRb pathways and bypass of the replicative senescence checkpoint, leading ultimately to immortalization (3) . We have previously shown that expression of E6 and E7 of the cutaneous HPV38 significantly increases the life span of HSKs (6) . To further assess the carcinogenic potential of HPV38, keratinocytes from different anatomic regions and donors were independently transduced with HPV38 E6 and E7 recombinant retroviruses, and their long-term proliferative capability was followed. We obtained three different immortal HSK lines that have now reached ~60 (from adult skin keratinocytes, line 1), 200 (from female neonatal keratinocytes, line 2), and 370 (from foreskin keratinocytes, line 3) PDs. Unlike what has been observed in HPV16 E6/E7 HSKs, immortalization of HPV38 E6/E7 HSKs was always preceded by an apparent lag phase independently of the type of keratinocyte used in the experiment. Figure 1 A shows the growth curves of lines 1, 2, and 3 HSKs up to DP 60. Immediately after infection, all HPV38 E6/E7 HSK lines grew for a few DPs and then stopped proliferating, enlarged, and remained growth-arrested for a period of 3–6 wk. Thereafter, islands of small proliferating cells appeared and continued to grow without further interruption. Two representative images of line 2 HPV38 E6/E7 HSKs and HPV16 E6/E7 HSKs are shown in Fig. 1B . The lag phase and associated phenotype were never observed during keratinocyte immortalization promoted by HPV16 E6 and E7 proteins (Fig. 1A, B ). These findings confirmed our initial data and showed that like E6 and E7 of HPV16, HPV38 E6 and E7 promote the immortalization of skin keratinocytes.


Figure 1
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  		Figure 1. Immortalization of HPV38 E6/E7 HSKs is preceded by an apparent lag phase. A) Primary keratinocytes (HSKs) from different donors were infected with control, HPV16, or HPV38 E6/E7 recombinant retroviruses, and their short-term proliferative capability was followed. The graph represents the number of population doublings (PDs) at the indicated times (days). PD 0 refers to the point at which drug selection was complete after retroviral infection. The gray arrow indicates the time when primary keratinocytes infected with empty retrovirus (pLXSN) entered senescence. B) Morphology of HPV16 E6/E7 or line 2 HPV38 E6/E7 HSKs at indicated PDs.

hTERT overexpression is considered to be a key event during the immortalization of human keratinocytes induced by HPV16 E6 and E7 proteins (3) . Therefore, we next determined the status of hTERT transcription in line 2 HPV38 E6/E7 HSKs. After retroviral infection, keratinocytes grown at different PDs were collected for RT-PCR and real-time PCR analyses. We found that like HPV16 E6/E7 HSKs, line 2 HPV38 E6/E7 HSKs displayed higher levels of hTERT mRNA than did mock cells (pLXSN HSKs) (Fig. 2 A, B). However, HPV16 appeared to be more efficient at early PDs than HPV38 in up-regulating hTERT as shown by real-time PCR analysis (Fig. 2B ). We also observed that hTERT mRNA levels gradually increased in line 2 HPV38 E6/E7 HSKs with the increase of PDs (Fig. 2B ). The hTERT up-regulation induced by HPV38 E6 and E7 was also observed in adult skin keratinocytes from different donor (line 1) by RT-PCR (supplementary Fig. 1A) and real-time PCR (data not shown). Increased expression of hTERT mRNA was directly linked to the presence of the viral proteins. In fact, silencing E6 and E7 expression in line 2 HPV38 E6/E7 HSKs by small hairpin RNAs (shRNAs) led to a significant down-regulation of the activity of the hTERT promoter cloned in front of luciferase reporter gene (Fig. 2C ).


Figure 2
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  		Figure 2. HPV38 E6/E7 up-regulate hTERT gene expression. A) After transduction with the indicated retroviruses, cells were grown and RNAs were extracted at indicated PDs. After reverse transcription, the same amounts of cDNAs were subjected to PCR analysis using specific primers for hTERT, HPV16 E7, HPV38 E7, and GAPDH genes. B). hTERT mRNA levels were determined by real-time PCR. The data are the means of three experiments. The differences in hTERT mRNA levels between 1) pLXSN HSKs PD1 and HPV16 E6/E7 HSKs PD15; and 2) pLXSN HSKs PD1 and line 2 HPV38 E6/E7 HSKs PD5, 14, 22, or 32 were statistically significant, P ≤ 0.0006. C) HPV38 E6/E7 HSKs were transiently transfected with the hTERT promoter-driven Firefly luciferase reporter plasmid and increasing amounts (from 0.25 to 1 µg) of a retroviral vector expressing shRNA for HPV38 E6 and E7 transcript (pRS 38E6/E7). Results are expressed as induction of luciferase activity (%) over the pRS control after normalization with the Renilla luciferase activity. Shown are the means ± SD of one representative experiment performed in triplicate. Similar results were obtained in three independent experiments. The differences in luciferase activity in line 2 HPV38 E6/E7 HSKs transfected with empty pRS vector or with different concentration of pRS 38E6/E7 construct (0.25, 0.5, or 1.0 µg) were statistically significant, P ≤ 0.028. D). {Delta}Np73 was silenced using antisense oligonucleotide. Antisense (AS) and sense (S) oligonucleotides directed against {Delta}Np73 were transfected in HPV38 keratinocytes by oligoligofectamine. Forty-eight hours after transfection, cells were collected and protein extracts were prepared. The levels of {Delta}Np73{alpha}- and β-actin as loading control were detected by immunoblot analysis (left). Signals of immunoblotting of three independent experiments were quantified (right). E) hTERT mRNA levels were determined by real-time PCR in cells transfected with S and AS {Delta}Np73 oligonucleotides. The data are the means of four independent experiments. The difference in hTERT mRNA levels in line 2 HPV38 E6/E7 HSKs transfected with S or AS {Delta}Np73 oligonucleotide is statistically significant, P = 0.024. F) HPV38 E6/E7 HSKs were transiently transfected with the hTERT promoter-driven Firefly luciferase reporter plasmid and {Delta}Np73 S or AS oligonucleotide (500 ng). Results are expressed as induction of luciferase activity (%) over the control ({Delta}Np73 S oligonucleotide) after normalization with the Renilla luciferase activity. Shown are the means ± SD of one representative experiment performed in triplicate. Similar results were obtained in three independent experiments.

It has been recently reported that the p53-related protein, {Delta}Np73, positively regulates the hTERT transcription (23) . Because HPV38 E6/E7 led to {Delta}Np73 accumulation in keratinocytes (7) , we transfected {Delta}Np73 antisense oligonucleotide in line 2 HPV38 E6/E7 HSKs and assessed hTERT expression by real-time PCR. hTERT mRNA levels were approximately decreased by 50% in cells transfected with the {Delta}Np73 antisense oligonucleotide (Fig. 2D, E ). In agreement with these findings, transfection of line 2 HPV38 E6/E7 HSKs with {Delta}Np73 antisense oligonucleotide resulted in a decrease of the activity of the hTERT promoter cloned in front the luciferase reporter (Fig. 2F ). Thus, our results indicate that {Delta}Np73 is a downstream effector of the HPV38 E6/E7-mediated hTERT up-regulation, although we do not exclude the possibility that HPV38 can up-regulate hTERT expression with additional mechanisms.

HPV38 E6 and E7 up-regulate the telomerase activity
Since hTERT gene regulation is the limiting factor for telomerase activity (24) , we next performed TRAP assays in order to determine whether the accumulation of hTERT mRNA in line 2 HPV38 E6/E7 HSKs resulted in an increased activity of the enzyme. The basal level of telomerase activity detected in mock cells (pLXSN HSKs PD1) was extremely low and corresponded to the lower detection threshold of our TRAP assay. HPV38 E6/E7 HSKs displayed greater telomerase activity than did mock cells even few PDs after retroviral transduction (Fig. 3 A). Interestingly, continuous growth of the HPV38 E6/E7 HSKs was associated with a further increase in telomerase activity (Fig. 3A ). However, this telomerase activation in HPV38 E6/E7 HSKs was clearly lower than the one observed in HPV16 E6/E7 HSKs at similar PDs (Fig. 3A ).


Figure 3
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  		Figure 3. HPV38 E6/E7 HSKs displayed a moderate up-regulation of telomerase activity compared with HPV16 E6/E7 HSKs. A) The same amounts (0.5 µg) of cellular extracts of indicated cells were submitted to a TRAP-ELISA assay. The telomerase activity detected in HPV16 and 38 E6/E7 HSKs is expressed as fold induction respect to telomerase activity detected in HSKs pLXSN PD1. The data are the means of three experiments. The differences in telomerase activity between the following HSK cultures are statistically significant: 1) pLXSN HSKs PD1 vs. line 2 HPV38 E6/E7 HSKs PDs 5, 14, 22, or 32, P ≤ 0.0002; 2) pLXSN HSKs PD1 vs. HPV16 E6/E7 HSKs PDs 1, 15, or 30 P < 0.0001; 3) line 2 HPV38 E6/E7 HSKs PDs 5 or 14 vs. HPV16 E6/E7 HSKs PDs 1 or 15, P ≤ 0.0002; 4) line 2 HPV38 E6/E7 HSKs PDs 5 or 14 vs. line 2 HPV38 E6/E7 HSKs PDs 22 or 32, P ≤ 0.0002. B) After transduction with the indicated retroviruses, cells were grown and RNAs were extracted at indicated PDs. After reverse transcription, cDNAs were subjected to PCR analysis using primers, allowing the detection of the FL, {alpha}, β, {alpha}/β forms of the hTERT mRNA. HSKs expressing ectopic levels of FL hTERT (hTERT HSKs) were used as positive control. C) Total RNA was extracted from the indicated cells and subjected to reverse transcription. Then, semiquantitative PCR analysis was performed on three different dilutions (1:1, 1:5, 1:10) of cDNA using specific primers for the FL form of hTERT and the GAPDH gene (top). Intensity of the FL hTERT PCR bands was quantified using the lower dilutions (1:5 and 1:10) and normalized to the signal of GAPDH. The data are the means of three independent experiments (bottom). The differences in hTERT FL mRNA levels in the following HSK cultures are statistically significant: 1) line 2 HPV38 E6/E7 HSKs PDs 5 or 32 vs. HPV16 E6/E7 HSKs PD 30, P ≤ 0.0013; 2) line 2 HPV38 E6/E7 HSKs PD 5 vs. line 2 HPV38 E6/E7 HSKs PD 32, P = 0.007.

It has been reported that, in addition to the full-length (FL) mRNA, alternative splicing of the hTERT transcript can occur within the region encoding the conserved reverse transcriptase motifs of hTERT, giving rise to the production of nonfunctional spliced variants ({alpha}, β, and {alpha}/β forms) (25 , 26) . The primers used in the RT-PCR experiments illustrated in Fig. 2A did not allow the discrimination between the FL form responsible for telomerase activity and the alternative spliced forms (ALS). To determine whether telomerase activity in all HSK lines correlated with FL form, we characterized the alternative splicing of hTERT transcript in HPV38 E6/E7 HSKs, performing RT-PCR using a set of primers allowing the amplification of all four hTERT mRNAs (19) . As shown in Fig. 3B , all hTERT mRNA forms could be detected in HPV16 E6/E7 and HPV38 E6/E7 HSKs. The β hTERT mRNA was the predominant form in HPV38 E6/E7 HSKs, while the FL appeared to be the most abundant form in HPV16 E6/E7 HSKs (Fig. 3B ). Semiquantitative RT-PCR using FL-specific primers showed that the expression of the FL transcript was higher in HPV16 E6/E7 HSKs than in HPV38 E6/E7 HSKs (Fig. 3C ). Moreover, in accordance with the TRAP assay results, we observed that FL mRNA expression increased together with telomerase activity in the latter PDs of HPV38 E6/E7 HSKs (PDs 32) (Fig. 3C ).

Similar data were obtained in independent experiments using line 1 HPV38 E6/E7 HSKs, in which FL and ALS forms of hTERT were detected. (Supplemental Fig. 1B ). In addition, line 1 HPV38 E6/E7 HSKs displayed lower telomerase activity than HPV16 E6/E7 HSKs (Supplemental Fig. 1C ), as observed in the experiments with line 2 HPV38 E6/E7 HSKs shown in Fig. 3A, B .

Together, these results demonstrate that up-regulation of FL hTERT mRNA expression and telomerase activation are lower in HPV38 E6/E7 HSKs than in HPV16 E6/E7 HSKs.

HPV38 E6/E7 HSKs display short and unstable telomeres
Despite a strong telomerase activity up-regulation, continuous telomere shortening occurs in HPV16 E6 expressing keratinocytes over increasing PDs (14) . Since HPV38 E6 and E7 proteins considerably induce proliferation of keratinocytes, we next assessed the telomere length by Q-FISH after 9 and 25 PDs (line 2 HPV38 E6/E7 HSKs). As shown in Fig. 4 A, line 2 HPV38 E6/E7 HSKs displayed shorter telomeres than did normal HSKs. In addition, with increased PDs, short telomeres accumulate, as did abnormal chromosome extremities without any detectable telomeric signals corresponding to signal-free ends (SFE) (Fig. 4A, B ). At the same time, end-to-end chromosome fusions and ring chromosomes were visible (Fig. 4C, D ). Together, these observations suggest that the telomerase activity up-regulation in HPV38 E6/E7 HSKs is not sufficient to compensate for telomere erosion and to allow telomere stability.


Figure 4
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  		Figure 4. HPV38 E6/E7 HSKs harbor short and unstable telomeres. Metaphase spreads were stained using a telomeric (C3TA2)3-Cy3 PNA probe and counterstained with DAPI. A) The graph shows the distribution of single fluorescence intensities within the indicated cell population. SFE indicates extremities with no detectable telomeric signals. B) Representative Q-FISH pictures showing signal-free chromosome ends (indicated by arrows) in HPV38 E6/E7 HSKs. Positive telomeric signals (in red) can be observed at the ends of the chromatids (in blue). C) Representative Q-FISH pictures showing end-to-end fusion events (indicated by arrows) in HPV38 E6/E7 HSKs, sometimes involving extremities with still-visible telomeres. D) Representative Q-FISH picture showing a ring chromosome in HPV38 E6/E7 HSKs.

In this context, artificial increase of telomerase activity in early PDs of HPV38 HSKs should attenuate telomere erosion and instability. Therefore, we next transduced these cells before the lag phase with a recombinant retrovirus expressing the FL hTERT mRNA, leading to a twofold increase of telomerase activity immediately after retroviral infection (data not shown). Q-FISH analyses were performed after 25 PDs, and as expected, we found that telomeres were greatly elongated in HPV38 E6/E7 HSKs expressing the ectopic hTERT (Fig. 4A ) and that neither SFE nor end-to-end fusions were detected (Fig. 4A and data not shown). Similar results were obtained using the line 1 HPV38 E6/E7 HSKs (Supplemental Fig. 1D and data not shown), excluding the possibility that the phenomena observed were due to HPV38 independent event, e.g., insertion of the retroviral vector in the host genome. Thus, ectopic hTERT expression prevents telomere erosion and rearrangements in HPV38 E6/E7 HSKs.

Telomere instability in HPV38 E6/E7 HSKs leads to anaphase bridge formation and mitotic defects
Chromosomes harboring very short telomeres are often involved in end-to-end fusions and can form bridges during anaphase (9 , 10 , 27 , 28) . Therefore, we next determined the anaphase bridge index (ABI) in normal and HPV38 E6/E7 HSKs. The ABI was increased more than 10-fold in line 2 HPV38 E6/E7 HSKs in comparison to mock cells (23.1% vs. 1.9%, respectively, P<0.0001) (Fig. 5 A). A similar difference in ABI was observed in line 1 HPV38 E6/E7 HSKs and the primary cells (27.5% vs. 1.9%, respectively, P<0.0001). Figure 5B shows representative images of anaphase bridges detected in line 2 HPV38 E6/E7 HSKs. Interestingly, ectopic expression of hTERT in line 2 HPV38 E6/E7 HSKs and subsequent telomere stabilization led to an approximate six-fold reduction of the ABI in comparison to keratinocytes expressing the viral genes only (4% vs. 23.1%, respectively, P<0.0001) (Fig. 5A ). Also, overexpression of hTERT in line 1 HPV38 E6/E7 HSKs led to a decrease of ABI (27.5% in HPV38 E6/E7 HSKs and 7.4% in hTERT HPV38 E6/E7 HSKs, P=0.004). These results indicate the involvement of dysfunctional telomeres in anaphase bridge formation.


Figure 5
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  		Figure 5. Anaphase bridges and multipolar mitosis occurred during HPV38 E6/E7 HSK division. A) The indicated cells were grown on cover slides and formaldehyde-fixed, and DNA was stained with DAPI. For ABI determination, a total of 105 to 220 anaphases from 6–8 cover slides were examined for each cell type. B) Representative pictures of anaphase bridges detected in HPV38 E6/E7 HSKs. C) Keratinocytes were synchronized with nocodazole and subjected to immunofluorescence staining using anti-{alpha}-tubulin antibody and anti-mouse FITC-conjugated antibodies. Cellular DNA was counterstained with DAPI. To determine the percentage of dividing cells harboring more than two poles, a total of 190 to 330 mitosis from 3 or 4 cover slides were examined for each cell type. D) Representative pictures of multipolar mitosis in HPV38 E6/E7 HSKs with cellular DNA stained in blue and {alpha}-tubulin stained in green.

It has been recently demonstrated that dysfunctional telomeres can also lead to mitotic multipolarity (27 , 28) . Therefore, we determined the degree of mitotic abnormalities in normal and line 2 HPV38 E6/E7 HSKs by {alpha}-tubulin immunofluorescence staining. Approximately 31% of dividing keratinocytes expressing the viral proteins were involved in multipolar mitosis (i.e., ≥3 poles) (Fig. 5C, D ), while multipolar metaphases were rare (0.8%) in mock cells (P=0.0009) (Fig. 5C ). As observed in the case of anaphase bridge formation, ectopic hTERT expression prevented significantly the accumulation of mitotic abnormalities in HPV38 E6/E7 HSKs (9.1% in HPV38 E6/E7 HSKs+hTERT vs. 31% in HPV38 E6/E7 HSKs, P=0.013) (Fig. 5C ). Similarly, line 1 HPV38 E6/E7 HSKs showed a high percentage of multipolar mitosis (23.2%) in comparison to the primary cells (0.85%) (P=0.0004), which was strongly reduced by hTERT overexpression (8%, P=0.008), confirming the involvement of short and unstable telomeres in such an event.

Together, these results demonstrate that telomere shortening in HPV38 E6/E7 HSKs promotes the occurrence of mitotic defects like anaphase bridges and multipolar mitosis.

HPV38 E6/E7 HSKs display polyploidy and chromosomal rearrangements
Telomere dysfunctions, anaphase bridge events and mitotic multipolarity promote both numerical and structural chromosome aberrations that facilitate, together with the inhibition of cell cycle checkpoints, cellular immortalization (9 , 10 , 27 28 29) . To determine whether the events described above could result in the formation of stable genetic rearrangements in HPV38 E6/E7 HSKs, we performed multiplex fluorescence in situ hybridization (M-FISH) analyses, which allow the identification of all chromosomes in a metaphase (22) (Fig. 6 ). Our data showed that the line 2 HPV38 E6/E7 HSKs are predominantly polyploid, with a chromosome number ranging from 62 to 110. Irrespective of the chromosome number, all metaphases are characterized by two stable translocations, t(1;5), t(9;10), emphasizing their clonal origin (Fig. 6A ). In addition to these stable translocations, we detected an average of 5 sporadic aberrations per metaphase, with a maximum of 10 (present in only one metaphase). A quite different situation was observed in the HPV38 E6/E7 HSKs expressing ectopic hTERT. This population was also clonal as it was characterized by two stable translocations, i(8q), t(17;17) (Fig. 6B ). Moreover, from this population, two subpopulations had emerged as being defined by a del(Xq) or a translocation involving the chromosome 9 (Fig. 6B ). However, in opposition to what was observed in HPV38 E6/E7 HSKs, all metaphases analyzed were diploid or pseudodiploid (range: 41 to 47 chromosomes), and only a few extra aberrations were seen (~1 additional aberration per metaphase). These results indicate that the introduction of ectopic hTERT in HPV38 E6/E7 HSKs and subsequent telomere lengthening, make less probable the occurrence of genomic rearrangements.


Figure 6
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  		Figure 6. Polyploidy and a high rate of chromosomal rearrangements characterized HPV38 E6/E7 HSKs. Metaphase spreads were prepared, and combinatorial labeling of whole chromosome painting probes and multicolor fluorescence in situ hybridization (M-FISH) were performed. Representative pictures of the M-FISH data for HPV38 E6/E7 HSKs (A) and HPV38 E6/E7 HSKs expressing ectopic hTERT cell lines (B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of EV HPV types in carcinogenesis has not been completely proven. Recent studies from our and other laboratories have shown that E6 and E7 oncoproteins of certain EV HPV types, e.g., HPV8 and 38, display transforming activities in in vitro and in vivo models (6 , 7 , 30 , 31) . Here, we show that, analogously to HPV16, EV HPV types can also lead to activation of telomerase, a key event in cellular transformation. In fact, expression of HPV38 E6 and E7 in HSKs led to activation of hTERT transcription and telomerase activity. HPV16-induced hTERT expression is mediated by E6 and depends on its ability to bind the cellular proteins E6AP. Indeed, it has been shown that HPV16 E6 together with E6AP promote the degradation of the hTERT transcriptional repressor NFX-91 through the proteasome pathway (11) . Moreover, a recent study demonstrated that E6AP plays an important role in HPV16 E6-induced hTERT promoter acetylation (12) . It is not yet known whether E6AP is involved in HPV38-induced hTERT expression. However, our results show that the p53-related protein {Delta}Np73 is, at least in part, responsible for hTERT up-regulation in HPV38 E6/E7 HSKs. {Delta}Np73 is known to act as a dominant-negative inhibitor of p53 and its related proteins (i.e., p63 and p73), which are strong repressors of hTERT expression (23 , 32) . Moreover, {Delta}Np73 also interferes with the E2F/pRb-mediated repression of hTERT transcription (23) . In HPV38 E6/E7 HSKs, the {Delta}Np73 level is considerably elevated (7) , and we show here that its down-regulation by antisense oligonucleotide leads to hTERT transcriptional down-regulation. Therefore, {Delta}Np73 plays a role in HPV38-mediated immortalization not only by leading to alteration of the p53 transcriptional functions, preventing activation of proapoptotic genes (7) but also by inducing hTERT overexpression.

Our results also indicate that as a consequence of hTERT transactivation, the telomerase activity is up-regulated in HPV38 E6/E7 HSKs. However, this activation remains moderate compared to that observed in HPV16 E6/E7 HSKs at similar PDs. By investigating the expression pattern of the ALS hTERT mRNA, we found that both HPV16 and 38 E6/E7 HSKs express the ALS forms. However, lower amounts of FL hTERT mRNA were detected in HPV38 E6/E7 HSKs compared with HPV16 E6/E7 HSKs. Because only the FL hTERT transcript is responsible for telomerase activity, this result could explain the different levels of telomerase activity measured in HPV16 or HPV38 E6/E7 cell lines. In addition, although the precise function of the ALS hTERT transcripts remains unclear, it is possible that they interfere with translation efficiency of the FL form and/or with the activity of the FL product. Interestingly, our data obtained in in vitro experimental models are in agreement with the in vivo situation, since alternative splicing of the hTERT mRNA and telomerase activation occur in a large proportion of NMSC lesions (33 , 34) , supporting a possible link between cutaneous HPV type infection and skin cancer development.

We also show that despite increased hTERT transcription and telomerase activation, telomere erosion occurs in HPV38 E6/E7 HSKs with increasing PDs. Our Q-FISH results clearly demonstrate that these cells harbor extremely short and unstable telomeres. Several reports indicate that such telomeres are often enrolled in breakage-fusion-bridge (BFB) cycles and promote the occurrence of mitotic abnormalities (reviewed in ref. 35 ). These events, together with the absence of functional cell cycle checkpoint, trigger increasing chromosomal instability, ultimately resulting in structural aberrations and polyploidy (9 , 10 , 27 28 29) . Here, we describe a similar scenario in HPV38 E6/E7 HSKs. Indeed, we observed in these cells that dysfunctional telomeres are involved in fusion events resulting in dicentric or ring chromosome formation. These chromosomes are unable to separate properly during mitosis, and at anaphase, form chromatin bridges that are susceptible to breakage. As observed here, chromatin bridges can also be responsible for cytokinetic failure resulting in multipolar mitosis. These events in the majority of cases may lead to cellular death. However, because of the inactivation of p53 and pRb pathways by E6 and E7 (6 , 7) , HPV38 E6/E7 HSKs continue to proliferate despite telomere dysfunctions and mitotic abnormalities. In this context, we observed that these cells accumulate genetic defects, resulting in a polyploid cell population harboring chromosomal aberrations. The occurrence of particular chromosomal rearrangements could confer growth advantages to few clones, allowing them to survive and acquire a transformed phenotype. This hypothesis is supported by the clonal origin of the HPV38 E6/E7 cell lines. Studies on HPV16 suggest that among other, loss of chromosome 11, deletions in chromosomes 3p, 6p, and 10p or gain of 3q are clearly associated with immortalization (36) . Preliminary data indicate that a nonrandom rearrangement of chromosome 9 occurs during immortalization of HSKs by HPV38 E6/E7 (data not shown). Interestingly, polyploidization and alteration of chromosome 9 have already been observed in NMSC cells (37 , 38) .

Similarly to the early reactivation of telomerase during precrisis period of SV40-ER transformed cells (39) , the ectopic expression of FL hTERT form before the lag phase strongly prevents the telomere erosion and the occurrence of mitotic defects and chromosomal aberrations described above.

In summary, our data indicate that similar to what has been observed in HPV16 E6/E7-expressing cells, HPV38 E6/E7 HSKs are characterized by unscheduled proliferation, suboptimal activation of the telomerase and subsequent telomere shortening that are at least partially responsible for their genomic instability (14 15 16 , 40) . Thus, although HPV16 and 38 appear to differ in some extent in the mechanisms used for targeting cellular proteins, e.g., p53, they induce similar events and both results in cellular immortalization. Together, our results provide additional lines of evidence for the carcinogenic potential of HPV38.


   ACKNOWLEDGMENTS
 
We are grateful to all the members of our laboratory for their cooperation; Dr. Jerry Shay (Simmons Comprehensive Cancer Center, Dallas, TX) for providing the pBabe-puro hTERT construct; Heidi Holtgreve for her excellent technical assistance in the M-FISH analysis; Franck Mortreux and Perrine Galia for the real-time PCR hTERT primers; and Uzma Hasan, John Daniel, and Veronique Bouvard for critical reading of the manuscript. The study was supported by grants from La Ligue Contre le Cancer (Comité du Rhône), the Association pour la Recherche sur le Cancer, the European Union (LSHC-2005–018704), and the Association for International Cancer Research to M.T.; a grant from La Ligue Contre le Cancer (Comité du Rhône) to B.S.S.; a grant from the European Union (LSH-CT-2004–502943) to P.B.; and a grant from the Association pour la Recherche sur le Cancer (3803) to J.A.L.V.

Received for publication March 6, 2007. Accepted for publication August 30, 2007.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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