Background
One of the inherent properties of high-risk human papillomavirus (HR-HPV) types belonging to the α-genus of the
Papillomaviridae is their ability to immortalize primary human epithelial cells. In pioneering experiments conducted in the late eighties it was shown that transfection of the native HPV16 or HPV18 genomes could extend the proliferative life-span of foreskin keratinocyte cultures. After a phase of crisis, immortal cell clones would emerge from most of these cultures [
1,
2]. In subsequent experiments the viral proteins driving the immortalization process were identified [
3,
4]. The oncoproteins E6 and E7 interact with numerous cell regulatory proteins, most notably with p53 and pRb respectively, thereby deregulating the cell cycle, DNA repair and apoptosis which inevitably leads to genetic instability [
5,
6]. Indeed, the inactivation of the p16
INK4A-Rb pathway has more recently been identified to play a critical role in cellular senescence [
7,
8]. However, bypassing one of the regulatory pathways for senescence is not sufficient for immortal cell growth. Several additional genetic alterations including the activation of telomerase are indispensable for immortality [
9]. Somatic cell fusion experiments suggest that the immortal phenotype can be complemented and thereby implies loss of gene function as a characteristic step in this process [
10,
11]. In our previous studies using microcell-mediated chromosome transfer into HPV-immortalized cells we provided functional evidence for senescence associated genes located on chromosomal regions 10p14-15 and 4q35→qter [
12,
13]. Importantly, these regions are more frequently affected by allele loss in cervical carcinomas than precancers [
12‐
14] and thus warranted a more detailed investigation. In order to identify candidate genes which contribute to senescence we had in earlier experiments performed microarray expression analyses with a particular focus on the above chromosomal regions. Two genes were found to be significantly down-regulated in cervical cancers as compared to precancers (CIN3). However, ectopic expression of neither gene could induce senescence in HPV-transformed cells [
15]. A possible weakness of that study may have been the use of pooled RNA for the array experiments which may have masked differences in the expression levels of several genes. In the present study we have therefore modified our strategy. Instead of pooled RNA, RNA from 12 microdissected CIN3 and 11 cervical carcinomas were used for individual expression analyses. Ten out of 15 genes which were found to be significantly down-regulated in cervical cancers versus CIN3 could be validated by quantitative RT-PCR and showed highly significant differences in expression between the two groups. Of seven genes cDNAs were overexpressed in HPV-immortalized cell lines, primary fibroblasts or primary keratinocytes to test their potential to induce senescence.
Methods
Tissue specimens
Biopsies of histopathologically confirmed CIN3 and squamous cell carcinoma (CxCa) from patients at the Department of Gynaecology at the Jena University Hospital were available for this study. This study was approved by the Ethics Committee of the Friedrich-Schiller-University Jena (reference number: 1402-09/04). Informed consent for the use of residual tissue for research was given. All cases were HPV-16 positive. Biopsies were embedded in Tissue-Tek (Sakura Finetek Germany GmbH, Staufen, Germany) and stored at -80°C. Sectioning was performed using a cryo-microtome (Shandon cryotome SME, Thermo Fisher Scientific, Wilmington, USA). Sections of 12–15 μm thickness were prepared. To protect the RNA from degradation, all instruments used for RNA isolation were treated with RNaseZap (Ambion/Applied Biosystems, Darmstadt, Germany) and rinsed with DEPC-water. Sections were placed on pre-cooled PALM MembraneSlides NF (RNase- and DNase-free; PALM Microlaser Technology, Bernried, Germany) which were treated with UV-light for 30 min thereby allowing better adhesion. The slides were quickly dried on a thermoplate at 40°C to protect tissue and RNA from humidity. Tissue-Tek medium interferes with the laser beam during microdissection and was therefore removed by washing the slides in 70% ethanol followed by drying on a thermoplate.
Laser capture microdissection
Immediately before microdissection the tissue sections were stained with a 1%-solution of Cresyl Violet acetate (minimum dye content 70%, Sigma-Aldrich, Seelze, Germany) in 96% ethanol for 1 min, differentiated with 70% ethanol and dried on a heating plate. Microdissection and pressure catapulting of tumor or dysplastic areas were performed on a PALM MicroBeam (Zeiss, Jena, Germany). The specimens were collected in PALM AdhesiveCaps 500 opaque (PALM Microlaser Technologies, Bernried, Germany).
RNA isolation, RNA quality and integrity assessment
The microdissected samples were dissolved in 400-500 μl TRIzol reagent (Invitrogen, Karlsruhe, Germany) and mixed with 0.33 volumes of chloroform. For a clear separation of the aqueous and organic phase the TRIzol-Chloroform solution was centrifuged at 14000 rpm for 20 min at 4°C in Phase Lock Gel Heavy tubes (Eppendorf, Hamburg, Germany). The aqueous phase was collected and mixed with 2 volumes of 70% ethanol. Total RNA was extracted with the RNeasy Micro Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions (which include DNase digestion) and was eluted in 14 μl RNase-free water. To assess the concentration of the total RNA, 1 μl was directly measured on a NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies, Thermo Fisher Scientific, Wilmington, USA). The integrity of the RNA was determined using a 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) thus enabling quality comparison to be made between the samples. The median RIN (RNA integrity number) of the RNA samples, which were hybridised to microarrays, was 6.2 (+/- 1.12).
Microarray hybridization and data analysis
Finally, RNA from 12 microdissected CIN3 and 11 CxCa were used for Agilent Whole Human Genome Oligo Microarray (4 × 44 K slide) hybridization in one-colour experiments with Cy3-labeled samples. Reverse transcription into cDNA, labeling of cDNA, hybridization and scanning of the microarrays were done according to the manufacturers’ protocols. Quality control was performed with the Feature Extraction Software by Agilent. Background signals were eliminated and all gene expression values were normalized to the median. Genes with expression values over 20 were set as expressed. Further gene analyses were performed with the software GeneSpring from Agilent. For group comparison the Welch t-test with a p-value cut-off of 0.05 was applied, followed by a multiple testing correction according to Benjamini and Hochberg [
16].
Validation with real-time PCR (qPCR)
The microarray data of all candidate genes was validated by reverse transcription qPCR. For this purpose the remaining RNA from the array analyses and from further microdissected biopsies, altogether 20 CIN3 and 20 CxCa, were used. All cases were HPV16-positive.
200 ng RNA of each sample was reverse transcribed in a total volume of 40 μl using SuperScript II Reverse Transcriptase (Invitrogen, Karlsruhe, Germany) according to the manufacturers’ protocol. Quantitative PCR was done with an ABI 7300 SDS system (Applied Biosystems, Darmstadt, Germany) using the PowerSybrGreen Master Mix (Applied Biosystems, Darmstadt, Germany). Reactions were performed with cDNA equivalent to 5 ng RNA in a volume of 25 μl volume comprising gene-specific forward and reverse primers (10pmol each) (Additional file
1: Table S1). To determine the normalisation factor (NF) we used the three most stable housekeeping genes (HKGs)
GAPDH,
HPRT and
ACTB, which were identified by the method of Vandesompele and colleagues [
17]. PCR data were analysed using REST (relative expression software tool) [
18].
Amplification, cloning and sequencing of cDNA
To generate expression-constructs of the genes of interest we used cDNA-clones available from ImaGenes (Berlin, Germany) (Additional file
2: Table S2). The coding sequences were amplified with the Long Expand Template PCR-System (Roche, Mannheim, Germany). The PCR comprised dNTP’s (350 μM), forward and reverse primers (300 nM), 1× buffer, plasmid-DNA (10 ng), enzyme-mix (3.75 U). The following PCR cycling parameters were employed: 94°C for 2 min initial denaturation, followed by 10 cycles at 94°C for 10 s, annealing temperature according to Table S3 (Additional file
3: Table S3) for 30 s, elongation at 68°C for 2 min. The next 20 cycles consisted of 94°C for 15 s, 65°C for 30 s, 68°C for 2 min with an additional increase of 20 s at 68°C for every cycle. The PCR products were separated on 2% agarose gels and the bands of predicted size were excised. DNA was eluted with Zymoclean Gel DNA Recovery Kit (Zymo research, Orange, USA) following the manufacturers protocol. PCR-products were cloned into pJet using the CloneJET PCR Cloning Kit (Fermentas, St. Leon-Rot, Germany) following the manufacturer’s instructions and transformed into E.coli XL1-Blue. Subcloning into the lentiviral vector pCDH-CMV-MCS-EF1-Puro (#CD510B-1, BioCat, Heidelberg, Germany) was done via the restriction enzymes shown in Table S4 (Additional file
4: Table S4). The ligation reaction consisted of: 50 ng pCDH-vector; 3U Ligase (Fermentas, St. Leon-Rot, Germany); 2 μl 10× buffer and 10 ng DNA-fragment in a total volume of 20 μl. The mix was incubated at 14°C for 16 h and then cloned into E.coli Stbl3 (Invitrogen, Karlsruhe, Germany). To ensure functionality all constructs were sequenced at Seqlab (Göttingen, Germany) (Additional file
5: Table S5).
Cell culture
For functional studies, the following cell lines were used: HPKIA and HPKII (both HPV16-immortalized human keratinocytes at passages p83 and p289, respectively) [
19,
20], CaSki and SiHa (both HPV16-positive cervical carcinoma), HeLa, SW756 and C4.I (all HPV18-positive cervical carcinoma). All cervical carcinoma cell lines were obtained from the ATCC. Primary human keratinocytes and fibroblasts were isolated from foreskins of four donors. Primary keratinocytes were cultured in EpiLife (Cascade Biologics, Portland, USA) with human keratinocyte growth supplement (HKGS, Cascade Biologics, Portland, USA). All other cell lines were grown in Dulbecco’s modified Eagle medium (D-MEM; Gibco Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C with 5% CO
2.
Lentivirus production
For the generation of recombinant lentivirus particles HEK 293 T cells were transfected using the calcium phosphate-method. One day before transfection 1.8×106 cells were seeded into a 6 cm-plate to achieve 80-90% confluence by the next day. The transfection mix comprising CaCl2 and BES-buffered saline, 2 μg pCDH expression plasmid, 2 μg pMDL, 1 μg pRSV and 0.4 μg pVSV-g was dropped onto the cells into 3 ml D-MEM (+10% FCS) and cultured over night at 35°C and in 3%CO2. The next day the cells were washed 2× with PBS and cultured over night with 3 ml medium at 32°C and in 5% CO2. About 48 h after transfection the virus-containing medium could be harvested. A 3rd generation packaging system with plasmid pMDLg/pRRE (included HIV-1 gag/pol genes; gag, coding for the virion main structural proteins; pol, responsible for the retrovirus-specific enzymes; RRE, a binding site for the Rev protein), plasmid pRSV-Rev (included parts of HIV-1 rev gene, controlling the export rate of mRNAs) and plasmid pCMV-VSVG (envelope plasmid) were used to produce lentiviral particles.
Lentiviral gene transduction
One day before transduction the target cells were seeded into 12-well-plates at a density of 1×106 cells/well. Virus-containing medium was harvested and filtered through a 0.45 μm filter. For each well 1 ml of virus-containing medium was mixed with 2 μg Polybrene (stock 5 mg/ml; Sigma-Aldrich, Steinheim, Germany) to replace the culture medium. Transduction was performed twice on the first day, temporally separated by 6 h, and a third time on the next day. Polybrene was used only for the first transduction. After adding the virus-containing medium the plates were centrifuged at 1500 rpm for 45 min at RT and subsequently cultured at 32°C and in 5% CO2 in the incubator. Six hours after the third transduction the virus-containing medium was removed and replaced with unmodified culture medium and the cells were cultured under normal conditions. For establishing stable clones selection was performed with 0.5 μg - 1 μg/ml puromycin hydrochlorid (Sigma-Aldrich, Steinheim, Germany) for 5–7 days. Assays were done in triplicate and repeated four times.
β-galactosidase assay
After removing the culture medium, cells were washed in PBS and fixed in a 3% formaldehyde solution for 5 min at RT. Cells were washed again with PBS and incubated with fresh senescence-associated β-galactosidase (SA-β-gal) solution as described by Dimri and colleagues [
21]. Incubation was performed at 37°C over night.
Northern blot
Northern blotting was done according to a standard protocol. Approximately 4 μg of total RNA were separated in a 1% MOPS- agarose gel and blotted onto a nylon membrane. Radioactively labeled DNA probes were generated by random priming (Roche Applied Science, Mannheim, Germany) with [32P]-ATP (Hartmann Analytic GmbH, Braunschweig, Germany). For removal of non-integrated nucleotides G-50 Sephadex-columns (Roche Applied Science, Mannheim, Germany) were used. Hybridization was performed for 2–3 days at 42°C followed by stringent washing and exposition to X-ray films for 3-14d at -80°C.
Immunofluorescence
1×105 cells suspended in 150 μl PBS were centrifuged at 500 rpm for 2 min at RT onto a glass slides. After fixation with 4% paraformaldehyde for 10 min, slides were washed with Tris-buffered solution (50 mM Tris, 150 mM NaCL) with 0,1% Tween-20 (TBST). Slides were then blocked with 50 μl normal serum (donkey-serum for SORBS2- and goat-serum for p16INK4a- antibody, respectively)(each 1:5 dilution in TBST) for 20 min at RT. Primary antibodies specific for SORBS2 (ArgBP2 H-15, Santa Cruz Biotechnologies, Heidelberg, Germany) (dilution 1:200) or p16INK4A (Roche mtm Laboratories, Heidelberg, Germany) (dilution 1:2) were then applied and incubated over night at 4°C followed by 3 washing steps with 1× TBST for 5 min each. Incubation with the secondary antibody for SORBS2 staining (donkey anti-goat IgG-FITC sc-2024, 1:100; Santa Cruz Biotechnologies, Heidelberg, Germany) was done for 45 min at RT. Afterwards slides were washed again 3 times for 5 min with washing buffer, rinsed with aqua bidest and covered with antifade/DAPI-solution under a coverslip. P16INK4a -detection was performed using EnVision™Detection System Rabbit/Mouse (Dako Deutschland GmbH, Hamburg, Germany) according to the manufacturers protocol. Slides were then counterstained with hematoxylin and covered with coverslips in gelatine. Fluorescence or brightfield images were obtained with a Zeiss Axioplan 2 microscope and a Axio Cam HRc using 20× and 40× objectives using AxioVision Application Rel.4.5.2, (Zeiss, Jena, Germany).
Western blot
Western blotting was done according to a standard protocol for semi dry procedure. Cells growing in a 6 cm dish were lysed in 300 μl of 1% SDS solution with 1:100 protein inhibitor (Serva Electrophoresis, Heidelberg, Germany). Approximately 10-15 μg protein was separated by 10% SDS-PAGE and transferred to an Immobilon-P membrane (Millipore, Bedford, USA). For immunostaining antibodies specific for SORBS2 (ArgBP2 H-15 goat, 100 ng/ml, Santa Cruz Biotechnologies, Heidelberg, Germany), TLR3 (TLR3 L-13, N-14 and Q-18, each goat IgG, 200 ng/ml, Santa Cruz Biotechnologies, Heidelberg, Germany) and ACTB (ACTB mouse, 125 ng/ml, BD Bioscience, Heidelberg, Germany) were used. The secondary antibodies were labelled with horseradish peroxidase (HRP) (anti-rabbit 0.04 ng/ml, anti-goat 0.08 ng/ml, anti-mouse 0.04 ng/ml; Dianova, Hamburg, Germany). The first antibodies were incubated over night at 4°C and the second antibodies for 1 h at RT. A 5% milk solution was used for blocking. All antibodies were diluted in 1% BSA solution. Staining was visualized using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Fisher Scientific, Dreieich, Germany).
Discussion
Escape from senescence is a prerequisite for carcinogenesis. Our previous functional studies involving somatic cell fusions and micro-cell mediated chromosome transfers have linked recessive gene alterations with the acquisition of immortality [
12,
13,
22]. In this study we have identified and validated 10 genes located within the chromosomal regions 4q35 → qter and 10p14-p15 that were down-regulated in cervical cancer as compared to CIN3. Down-regulation of SORBS2, TLR3 and GATA-3 has already been described in the context of cervical carcinogenesis. SORBS2 was shown to be expressed in normal cervical epithelium and CIN3 but at significantly reduced levels in CxCa [
15]. Immunhistochemical analysis for GATA-3 showed a clear nuclear staining in normal cervical squamous epithelium, CIN1 and CIN2 lesions, but 11% of CIN3 and 67% of CxCa revealed a complete absence of GATA-3 immunostaining [
23]. TLR3, a toll-like receptor eliciting an antiviral response in the presence of HPV oncogene expression, is increased in CIN3 followed by a decrease in CxCa [
24]. The remaining 7 genes with exception of PFKFB3 are not linked to cancer when reviewing the literature. PFKFB3, in particular the splice variant UBI2K4 is considered to be a tumor suppressor protein in glioblastomas [
25].
None of the above genes have thus far been associated with the loss of senescence. We had expected that reconstitution of gene expression in HPV-immortalised keratinocyte cell lines such as HPKIA and HPKII or cervical carcinoma derived cell lines would induced senescence. However, this was not the case and leaves room for speculation. In previous analyses using micro-cell mediated transfer either of the entire chromosome 4 or 10 resulted in senescence of HPKII cells. In subsequent studies using derivative of chromosomes 4 and 10 the chromosomal region harbouring putative senescence genes were narrowed down by exclusion [
12,
13]. In other words none of the derivative chromosomes could induce senescence. It is therefore conceivable that a candidate senescence gene is indeed located within this region but to exert its effect on senescence the product of another gene located on the same chromosome is required. If this gene is lacking, ectopic expression of the senescence gene itself will have no effect. We have therefore ectopically expressed the candidate genes in primary human keratinocytes and fibroblasts which should not have any genetic aberrations. Of the seven candidate genes only SORBS2-2 and TLR3 could reproducibly induce senescence both in fibroblasts and keratinocytes. SORBS2-1, a shorter splice variant of SORBS2, could only induce senescence in fibroblasts. The reason for this phenomenon is unclear. Both isoforms encode relevant functional parts of the protein such as the SoHo (Sorbin homology) and the three SH3 (Src homology 3) domains at the amino and carboxyl terminal end, respectively.
Clearly, the type of senescence observed in this study is not to be confused with proliferative senescence which results when telomeres have reached a critical minimal length and thereby have lost their protective structure. Senescence can also be induced in the absence of telomere shortening or dysfunction by a variety of conditions. This phenomenon is referred to as premature senescence and is either induced by stress as a consequence of suboptimal culturing conditions [
26], or by elevated oncogene expression [
9]. Indeed, aberrant activation of signal transduction pathways and promoting cell cycle regulators can result in a DNA-damage response (DDR) and thereby induce premature senescence. In this context the role of c-Abl in DNA repair may be helpful to understand our finding that SORBS2 can induce senescence. SORBS2 interacts with poly-proline motifs of c-Arg and c-Abl kinases via its three SH3 domains. A complex of the ubiquitin ligase Cbl and SORBS2 mediates the ubiquitination and degradation of c-Abl [
27]. Within this complex, SORBS2 acts as a regulator of ubiquitination and degradation of c-Abl. It remains to be shown if this pathway is relevant for premature senescence. At a first glance the association between TLR3 and senescence may be interpreted as a lentivirus induced phenomenon. TLR3 is a member of the toll-like receptor family involved in the response of the innate immune system to pathogens such as double-stranded RNA viruses [
28]. TLR3 activation results in the induction of intracellular pathways mediated by NF-қB and MAPK and can lead to IFN-beta and TNF-alpha gene expression [
29,
30]. Furthermore, NF-қB activation can lead to the production of proinflammatory cytokines expression such as IL-1 and apoptosis [
28] or IL-1 and IFN-beta induced senescence [
31,
32]. We can therefore not completely exclude that the senescence observed in the TLR3 encoding lentivirus infected primary cells reflects a bias of the expression system. However, it should be considered that the very same recombinant virus could not induce senescence in HPV16-immortalized HPK cells. Moreover, the “empty” virus which served as negative control in all experiments, could not induced senescence above background in any of the cell lines or primary fibroblasts or keratinocytes analysed.
The observation that the endogenous expression of both SORBS2 and TLR3 is increased drastically in primary keratinocyte cultures at near senescent passage provides further support that these genes may contribute to senescence. In ongoing experiments we are investigating whether this increase in gene expression is also characteristic of replicative senescence. Moreover, modulation of both genes by shRNA-knockdown may result in a prolonged life-span of keratinocytes in culture which would be another indication for a physiological role of either protein in the senescence process.
Acknowledgements
We thank PD Dr. Jörg Müller (Institute for Molecular Cell Biology, Jena) for providing the lentiviral packaging plasmids and sharing his know-how.
Critical reading of the manuscript by Alfred Hansel and Norman Häfner is greatly appreciated.
Grant support
This study was supported by the Deutsche Forschungsgemeinschaft (DFG BA 2890/1-3 to C.B. and M.D.).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ML performed and analysed experiments and wrote the manuscript, LJ contributed to the experiments, SM performed microarray experiments, HF supervised the microarray experiments, IBR supervised the study, MD and CB designed and supervised the study and revised the manuscript. All authors read and approved the final manuscript.