To determine whether integration of human papillomavirus (HPV) DNA sequences could lead to the deregulation of genes implied in oncogenesis, we analysed the HPV integration sites in a series of nine cell lines derived from invasive genital carcinomas. Using in situ hybridization, HPV16 or 18 sequences were found at chromosome band 8q24, the localization of MYC, in IC1, IC2, IC3, IC6 and CAC-1 cells and at other sites in IC4, IC5, IC7 and IC8 cells. We then localized viral sequences at the molecular level and searched for alterations of MYC structure and expression in these cells. MYC genomic status and viral integration sites were also analysed in primary tumors from which IC1, IC2, IC3 and IC6 cells were derived. In IC1, IC2 and CAC-1 cells, HPV DNA was located within 58 kb of MYC, downstream, upstream, or within MYC. In IC3 and IC6 cells, HPV DNA was located 400–500 kb upstream of MYC. Amplification studies showed that, in IC1, IC2 and IC3, viral and MYC sequences were co-amplified in an amplicon between less than 50 and 800 kb in size. MYC amplification was also observed in primary tumors, indicating that this genetic alteration, together with viral insertion at the MYC locus, had already taken place in vivo. MYC was not amplified in the other cell lines. MYC mRNA and protein overexpression was observed in the five cell lines in which the HPV DNA was inserted close to the MYC locus, but in none of the lines where the insertion had occurred at other sites. MYC activation, triggered by the insertion of HPV DNA sequences, can be an important genetic event in cervical oncogenesis.
Specific types of human papillomaviruses (HPVs) are associated with a large majority of cervical neoplasia (Bosch et al., 1995; Walboomers et al., 1999). High-risk HPV E6 and E7 viral oncoproteins have been shown to immortalize cells in vitro (Münger et al., 1989; Halbert et al., 1991) and their ability to inactivate p53 and pRB, respectively, leads to genomic instability and increased cell proliferation (Munger et al., 2004). The additional cooperation of activated oncogenes such as KRAS (Crook et al., 1988) or FOS (Pei et al., 1993), however, is necessary for malignant conversion. Although persistent HPV infection is a critical event leading to cervical intraepithelial neoplasia (CIN) (Walboomers et al., 1999), additional host and environmental cofactors also play a major role in the long-lasting evolution of CIN to invasive cancer. Little data have been published concerning differences in genetic abnormalities between CIN and invasive carcinoma. Activation of FGFR3 (Cappellen et al., 1999; Rosty et al., 2005a), KRAS (Crook et al., 1988; Jiko et al., 1994), HRAS (Riou et al., 1988), MYB (Nurnberg et al., 1995) and MYC (Riou et al., 1987; Zhang et al., 2002) has been reported in invasive carcinoma of the cervix. The integration of viral DNA into the host genome is seen in a minority of cervical intraepithelial neoplasia compared to a majority of invasive cancers (Cullen et al., 1991; Klaes et al., 1999; Kalantari et al., 2001) and corresponds thus to a crucial step in oncogenesis. Viral genome disruption and integration lead to the deregulated expression (Schwarz et al., 1985) and increased stability (Jeon and Lambert, 1995) of the RNA messengers derived from viral oncogenes that act in the tumor process. Integration may also lead to the activation, or to the inactivation of cellular genes implied in oncogenesis.
In a previous work (Couturier et al., 1991) we have shown that chromosome band 8q24, to which MYC maps, was a common site for integration of HPV DNA in IC1, IC2 and IC3 cells, derived from genital carcinomas. Northern blot experiments showed that MYC was overexpressed in IC1 cells. More recently, using DNA stretching (Herrick et al., 2005), we found that MYC was co-amplified together with HPV18 DNA sequences in IC1 cells. These data suggest that MYC activation could be secondary to the insertion of HPV DNA sequences at the MYC locus. Although a large diversity of integration sites has been reported (Wentzensen et al., 2004), MYC chromosome region is the most recurrent localization for HPV insertion, found in about 10% of genital neoplasias, observed in chromosome band 8q24 by fluorescence in situ hybridization (FISH) (Dürst et al., 1987; Couturier et al., 1991; Hori et al., 1991; Brink et al., 2002) or at the MYC locus by DNA/RNA analysis (Wentzensen et al., 2002; Ferber et al., 2003b; Ziegert et al., 2003; Kraus et al., 2005). However, despite this recurrence, no evidence of MYC overexpression directly associated with the insertion of HPV DNA at the MYC locus has been shown so far. This prompted us to further study HPV integration sites at the molecular level and MYC status/expression by quantitative polymerase chain reaction (PCR) and Western blot in the IC1, IC2 and IC3 cell lines previously analysed, as well as in IC4 cells (Couturier et al., 1991; Sastre-Garau et al., 2000) and in five other cell lines newly established from cervical carcinoma. In five of the nine cell lines, HPV DNA was found at the MYC locus. MYC overexpression, with or without amplification, was seen in all five cell lines with HPV sequences inserted close to MYC but not in those with viral sequences inserted at other sites. We also show here that MYC amplification was already present in the original tumors from which the three cell lines with MYC amplification were derived.
Localization of integrated human papillomavirus DNA sequences in cell lines and in IC2, IC3, IC5, IC6 and IC8 primary genital carcinoma
The localization of HPV DNA sequences was first determined by FISH analysis of early-passage cultures using HPV probes on metaphase chromosomes. A single integration site was observed in seven of the nine cell lines analysed, whereas two sites were found in the two remaining cases (IC6 and CAC-1). Human papillomavirus DNA sequences were found in 8q24.21 in five of the nine cell lines (IC1, IC2, IC3, IC6, CAC-1) (Table 1). The other insertion sites were 2p24.3 (IC4), 7q31 (IC5), 13q22.1 (IC6), 1q32.2 (IC7), 19p13.2 (IC8) and 15q21.3 (CAC-1) (Table 1). Since MYC maps to 8q24.21, we performed FISH analysis using both HPV and MYC DNA probes in the five cell lines with HPV DNA sequences in this chromosomal band, which showed the superposition of MYC and HPV signals. The MYC signal associated with viral sequences was stronger than that of isolated MYC signals, suggesting that MYC was amplified at the insertion sites (Figure 1). In CAC-1 cells, MYC and HPV18 DNA sequences colocalized on a rearranged chromosome 8 and on a marker chromosome derived from a translocation between chromosomes 8 and 15 (R-banding data not shown) (Figure 1).
Detection of integrated papillomavirus sequence (DIPS) and Amplification of papillomavirus oncogene transcripts (APOT) techniques were used to characterize the viral integration sites at the molecular level. Detection of integrated papillomavirus sequence, based on DNA analysis, was informative in all cell lines and showed two integration sites in IC6 and CAC-1 cells, and a single site in the others. Human papillomavirus DNA sequences were always found in sites corresponding to the chromosome band identified by FISH (Table 1). Amplification of papillomavirus oncogene transcripts, based on RNA analysis, was informative in six of the nine cases. Five showed localization identical to that determined by FISH and DIPS, whereas an additional site was seen at 2p25 in CAC-1 cells (Table 1).
We identified genes that map in the vicinity of the integrated sequences by submitting the host genomic DNA sequences flanking the inserted viral DNA to the Working Draft Genome database. In cells with HPV DNA at 8q24.21, the viral sequences were found close to MYC, at a distance varying from 0 to 513 kb (Table 1). Two clusters of integration sites were seen, one at less than 60 kb from MYC (IC1, CAC-1) and the other at 400–500 kb upstream of MYC (IC3, IC6). For IC2 cells, two viral integration sites were found at the MYC locus in each of these two clusters: one at 58 kb from MYC and the other at 514 kb upstream of MYC (Table 1 and Figure 3). Other genes located near the inserted viral sequences were MYCN (75 kb) in IC4, ZNF277 (0 kb) in IC5, KLF12 (250 kb) in IC6, CAMK1G (230 kb) in IC7, NFIX (0 kb) in IC8 and DYX1C1 (43 kb) in CAC-1 (Table 1). Variability in the number of integration sites at the molecular level was observed in some lines. Two sites were seen at the MYC locus in IC1, IC6 and CAC-1, and four sites at the KLF12 locus in IC6, spanning in an interval of 22–78 kb (data not shown). This variability may be related to genetic rearrangements associated with the co-amplification of HPV and flanking DNA sequences. In all cases, an amplification of human DNA flanking integrated viral sequences was observed, ranging from 6 to 95 copies (Table 2).
For IC2, IC3, IC5, IC6 and IC8 cells, frozen histological sections from the original tumors from which these cells derived were available, allowing DNA extraction and viral integration sites analysis in these five primary carcinomas. In IC2, IC5, IC6 and IC8, the integration sites in the primaries were identical to those found in the respective derived cell lines, including the two sites (on chromosomes 8 and 13) observed in IC6. In IC3 primary tumor, HPV sequences were found integrated at the MYC locus at a distance of 85 bp from the site observed in IC3 cells. In IC1 primary tumor, the quality of the DNA extracted from formalin-fixed tumor tissue was not appropriate to determine the integration site.
Molecular pattern of human papillomavirus DNA sequences integrated in 8q24.21
In order to define the pattern of viral sequences integrated in 8q24.21, the HPV DNA in IC1, IC2, IC3, IC6 and CAC-1 cells was further analysed by DIPS-PCR using additional primers. In most cases, the viral genome was found interrupted in the L1 and E1/E2 ORFs, whereas the E6 and E7 ORF and the LCR were conserved in all cell lines (Figure 2). Rearrangements of the viral DNA sequences located at the viral/cellular junctions were observed, as exemplified by insertion of cellular sequences in E1 in IC3 cells, and in the L1/L2 sequences in IC1 (Figure 2).
MYC genomic status in cell lines and in IC1, IC2, IC3 and IC6 primary tumors
To determine whether the insertion of viral sequences was associated with alteration of MYC, we analysed MYC genomic status in all the cell lines using quantitative PCR. The MYC status could also be assessed in the IC1, IC2, IC3 and IC6 primary tumors, using DNA extracted from frozen (IC2, IC3, IC6) or formalin-fixed (IC1) histological sections.
In three (IC1, IC2, IC3) of the five cell lines with HPV sequences at the MYC locus, MYC was found to be amplified (12–17 copies) (Table 2). No evidence of MYC amplification was found in IC6 and in CAC-1 cells, which also contain HPV DNA at the MYC locus, nor in any of the other cells negative for viral DNA at this site (Table 2). The analysis of primary tumors from which IC1, IC2 and IC3 cell lines were derived also showed MYC amplification, indicating that the amplification had already taken place in vivo. In agreement with the MYC status seen in IC6 cells, no evidence of MYC amplification was found in the primary tumor from which these cells were derived.
In order to specify the boundaries of the amplified sequences in cells with HPV sequences near MYC (IC1, IC2, IC3, IC6 and CAC-1 cells), we performed quantitative PCR using a series of 12 primers pairs at about 100 kb distances flanking MYC (Figure 3). In all these cells, DNA sequences adjacent to the integrated HPV DNA were found to be amplified. The amplicon size varied from less than 50 to 800 kb (Figure 3). In IC3 cells with HPV16 sequences 513 kb upstream of MYC, a 530–786 kb-sized amplicon encompassed both viral and MYC sequences. In contrast, in CAC-1 cells with HPV18 sequences within MYC, the amplicon (0.3–200 kb) was just downstream of MYC, and MYC was not amplified.
MYC expression level analysis
MYC expression level was assessed by quantitative reverse transcription (RT)–PCR and western blot experiments. Using quantitative RT–PCR, the three cases with MYC amplification (IC1, IC2, IC3) showed MYC overexpression (3–10-fold that of normal cervix). Overexpression was also seen in CAC-1 cells (threefold) and in IC6 cells (2.5-fold), which show viral DNA insertion at the MYC locus, but without MYC amplification. None of the other cells (IC4, IC5, IC7, IC8) showed MYC overexpression (Table 2).
Using Western blots, MYC protein overexpression was observed in the IC1, IC2, IC3, IC6 and CAC-1 cells with HPV DNA at the MYC locus, but in none of the other cell lines (Figure 4a). This is in agreement with the mRNA expression data (Figure 4b), and shows that MYC activation is associated with HPV DNA sequences at the MYC locus in these carcinoma-derived cell lines.
A great diversity in the localization of integrated HPV DNA sequences has been found in genital tumors or tumor-derived cell lines. Up to 181 different sites corresponding to 243 cases have been observed (Brink et al., 2002; Klimov et al., 2002; Wentzensen et al., 2004; Kraus et al., 2005; and our unpublished results). In spite of this scattering, it is remarkable that the chromosome band 8q24, to which MYC maps, is the most recurrent site for insertion of HPV DNA, as it is found in 10.7% (26/243) of the all cases (Dürst et al., 1987; Popescu et al., 1987; Couturier et al., 1991; Hori et al., 1991; Brink et al., 2002; Wentzensen et al., 2002; Ferber et al., 2003b; Ziegert et al., 2003; Kraus et al., 2005). A low level of MYC overexpression has been found in HeLa cells harboring HPV18 sequences in chromosome band 8q24 (Dürst et al., 1987; Lazo et al., 1989), but no evidence of MYC activation directly related to HPV insertion at the MYC locus has been published.
Here we report the overexpression of MYC in five cell lines derived from genital carcinomas with HPV16 or 18 DNA sequences inserted within 500 kb of the MYC gene, and in none of four lines with HPV DNA at other sites. We show that MYC overexpression could be related to the co-amplification of MYC and HPV sequences, a genetic event already present in the primary tumor, together with the insertion of viral DNA sequences at the MYC locus. We show also that, in two cases, MYC overexpression occurred without gene amplification. In addition to the data concerning the new cell lines and primary tumors analysed, quantitative PCR and Western blot experiments used in the present work permitted to reveal MYC amplification in IC1 and IC3 cells and MYC overexpression in IC2 and IC3, not previously observed (Couturier et al., 1991).
MYC activation has been found to play a role in different types of human tumors. In Burkitt lymphoma, MYC overexpression is secondary to the translocation of heavy or light immunoglobulin chains genes at a distance of 100–150 kb upstream (Joos et al., 1992) or 300 kb downstream (Henglein et al., 1989; Zeidler et al., 1994) of MYC. MYC activation was also observed in invasive carcinomas that develop in various organs such as breast, prostate, stomach or colon (Pelengaris et al., 2002). This activation, which is generally related to a moderate level of amplification, is often found in poorly differentiated tumors (Nesbit et al., 1999). The present observation that HPV DNA sequences are usually located at the median part of the amplicon, together with the pattern of the co-amplified MYC and viral sequences on DNA stretched molecules previously reported (Herrick et al., 2005), clearly indicate that, in the genital tumors analysed, integration of viral DNA occurred prior to gene amplification. In two cases, both associated with HPV18, MYC was overexpressed but not amplified. The Long Control Region of the HPV18 genome harbors enhancing sequences, the activity of which is restricted to epithelial cells, and is dependent upon the assembly of an enhanceosome containing multiple cellular factors (Bouallaga et al., 2000). Viral sequences could therefore increase MYC transcription level. In animal models, viral sequences were found to be responsible for the expression of the nearby MYC or MYCN gene in murine leukemia virus-induced T-cell lymphoma (Selten et al., 1984) or the woodchuck hepatitis virus-induced hepatocarcinoma (Fourel et al., 1994), respectively.
Through its numerous targets, MYC acts as a key regulator of major cell functions such as proliferation, apoptosis, differentiation and DNA metabolism (Dang, 1999). In cooperation with E6 in HPV-infected keratinocytes, MYC may also activate the telomerase reverse transcription promoter (Veldman et al., 2003). Recent data have underlined the important role of MYC in the differentiation of skin epithelium (Pelengaris et al., 2002; Alonso and Fuchs, 2003). Through the depletion of integrin β (Waikel et al., 2001), MYC was found to act selectively on epidermal stem cells (Gandarillas and Watt, 1997), driving them into the transit amplification compartment and stimulating differentiation into interfollicular epidermis and sebocytes (Frye et al., 2003). In an experimental model, MYC activation was sufficient to induce a neoplastic phenotype in the skin (Pelengaris et al., 1999).
It is important to underline that, comparing the histological type of cervical carcinomas, when it was documented, to the cartography of viral integration sites (present study and (Thorland et al., 2000; Klimov et al., 2002; Ferber et al., 2003b), 9/10 cases with HPV DNA sequences at the MYC locus corresponded to adenocarcinomas, whereas 22/29 of cases with viral sequences at other sites were squamous cell carcinoma (P=4.10−4). HPV18 is frequently associated with adenocarcinoma (Lombard et al., 1998) and preferentially integrates at the MYC locus (Ferber et al., 2003b). However, we and others (Brink et al., 2002; Wentzensen et al., 2002) have also found HPV16 DNA at this locus. It is well established that cervical neoplasia start at the squamous columnar junction of the cervix epithelium, a part of the mucosa in which the basal cells, exposed to infections, have the potential to differentiate into either squamous or glandular cells. MYC activation, driven by HPV integration into epithelial stem cells, might play a role in the glandular differentiation of transformed keratinocytes, in cooperation with microenvironmental interactions, and thus account in part for the high rate of adenocarcinoma associated with HPV DNA inserted at the MYC locus.
Besides MYC, a number of genes, potentially involved in carcinogenesis, such as CEACAM5, FANCC (Wentzensen et al., 2002; Ferber et al., 2003b), TP63 (Wentzensen et al., 2002), TERT (Ferber et al., 2003a), MYOF, Ribosomal protein gene RPS27 (Klimov et al., 2002), MYCN (Sastre-Garau et al., 2000), NFIB (Ziegert et al., 2003), have also been found to correspond to targets for the insertion of HPV DNA. In spite of this diversity, only a few cases with an integration pattern compatible with a mechanism of insertional mutagenesis have been observed (Wentzensen et al., 2002). Cellular genome deletion, observed at the site of integrated HPV DNA (Wilke et al., 1996), may also lead to the inactivation of cellular genes with tumor suppressive properties. A well-documented example of inhibition of the APM1 gene, occurring secondary to the insertion of HPV68 DNA sequences in ME180 cells, has been reported (Reuter et al., 1998).
Biological parameters that condition the integration of foreign DNA into the cellular genome are poorly documented. In a large series of HPV-associated tumors, viral integration sites were found preferentially distributed in transcribed cellular sequences (Klimov et al., 2002; Wentzensen et al., 2002; Ziegert et al., 2003). Extensive analyses of retrovirus integration sites have shown that active genes (Schroder et al., 2002) or transcription start regions (Wu et al., 2003) are preferential integration targets. Fragile sites have also been reported to facilitate HPV DNA integration (Thorland et al., 2003), but other factors may influence the nature of the genetic rearrangements that implicate insertion of foreign DNA and illegitimate recombination. In Burkitt lymphoma, the frequency of the different types of translocation observed was highly correlated with the probability that the two partners were in physical proximity in the cell nucleus (Roix et al., 2003). DNA repair mechanisms have also been found involved in the process of viral integration. During retroviral integration, the initial event is detected as DNA damage by the host cell and completion of the integration process requires the DNA protein kinase-mediated pathway (Daniel et al., 1999). In another model, adenovirus oncoproteins were found to target cellular proteins involved in the DNA repair process that prevent normal cells from malignancy and regulate genome stability (Stracker et al., 2002). These data, associated with the fact that integration of HPV DNA occurs at a single site in most cases of invasive cervical cancer, indicate that integration is not an incidental event but rather corresponds to an active process. This process, involving the cell machinery, probably participates in viral maintenance and also constitutes an irreversible step in cell transformation. Although viral integration into the host genome is probably a random event, cell clones with biological advantages secondary to this rearrangement must become selected for during tumor development. Indeed, as a consequence of integration, the deregulated expression of the viral E6 and E7 oncoproteins leads to the overexpression of genes involved in cell proliferation and/or division (Rosty et al., 2005b). The data we have presented here, concerning the most frequently found integration site of HPV DNA in cervical carcinoma, in addition to the few examples reported so far (Reuter et al., 1998; Sastre-Garau et al., 2000), indicate that the alteration of cellular gene, secondary to the integration of viral HPV DNA, can also participate in the tumor process.
Materials and methods
Cell lines and culture
IC1, IC3, IC4 (Couturier et al., 1991), CAC-1 cells (Hayakawa et al., 1988) and IC5, IC6, IC7, IC8 were derived from invasive carcinoma of the uterine cervix, and IC2 from a penile cancer (Couturier et al., 1991). For IC2, IC3 and IC6 cells, frozen histological sections corresponding to the initial tumor were retrieved from the Institut Curie tumor bank and could be used for DNA extraction. For IC1 tumor, only DNA extracted from formalin-fixed tumor tissue could be obtained for molecular analysis. Human papillomavirus characterization showed that these cell lines were associated with HPV16 (IC2, IC3 and IC7), HPV18 (IC1, IC5, IC6, IC8 and CAC-1) or HPV45 (IC4). Cells were cultured in RPMI 1640 supplemented with 5% fetal calf serum.
Fluorescence in situ hybridization experiments
Human papillomavirus DNA sequences have been found previously localized by FISH at chromosome bands 8q24.1 in IC1, IC2, IC3 and 2p24 in IC4 (Couturier et al., 1991) and colocalized with MYC and MYCN in IC2 and IC4, respectively (Sastre-Garau et al., 2000). In the present study, FISH experiments were performed for IC5, IC6, IC7, IC8 and CAC-1 cells, according to the procedure previously described (Sastre-Garau et al., 2000). After chromosomal localization of HPV DNA sequences, metaphase preparations of IC1, IC3, IC6 and CAC-1 were further analysed by dual-color FISH, using HPV16 or 18 probe and a SpectrumOrange labeled MYC probe (Vysis, Downers Grove, IL, USA) in the hybridization mixture.
Viral integration sites at molecular level
After DNA and RNA extraction, human genomic sequences adjacent to integrated HPV16, 18 or 45 DNA have been identified with the APOT (amplication of papillomavirus oncogenes transcripts) (Klaes et al., 1999) and/or the DIPS-PCR (detection of integrated papillomavirus sequences by ligation-mediated PCR) (Luft et al., 2001) techniques.
The APOT technique allows to distinguish mRNAs derived from episomal versus integrated HPV DNA. Briefly, total RNAs were reverse transcribed using an oligo(dT) primer coupled to a linker sequence [(dT)17-p3]. cDNAs were then amplified by PCR, using a HPV E7-specific oligonucleotide as forward primer and an oligonucleotide specific for the linker as reverse primer. One-tenth of the PCR product was used as a template for a second round of amplification using an internal primer specific for the HPV E7 and the [(dT)17-p3]. Final PCR products were submitted to electrophoresis and PCR products whose size was different from that of the major transcript derived from episomal DNA sequences (around 1000 bp) were cloned with a TA cloning kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Positive clones were sequenced using Big Dye Kit V1.1 (Applied Biosystems, Foster City, CA, USA), and sequences were submitted to databases (Working draft April 2003).
Detection of integrated papillomavirus sequences by ligation-mediated PCR was used to amplify genomic viral-cellular junctions. DNA was digested with either Taq1 or Sau3AI. Enzyme-specific adapters were ligated to the restriction fragments, and the ligation products obtained were subjected to PCR amplification. This amplification consisted of a first round of linear PCR with a viral-specific primer, followed by a second round of exponential PCR with a viral-specific primer internal to the previous one and a second primer specific for the adapter. Polymerase chain reaction products the size of whose was different from the predicted episomal segment were excised from an agarose gel, purified and sequenced (Luft et al., 2001) (see Supplementary Information for additional primers).
MYC DNA copy number
MYC status was assessed by amplifying a DNA fragment in the MYC gene together with a DNA fragment within the PSA gene. Amplification was performed with 20–40 ng of DNA in a final volume of 25 μl, using the SYBR Green PCR Core Reagents Kit (Applied Biosystems) with 2 and 3 mM MgCl2 for MYC and PSA amplifications, respectively. Primers A221 (IndexTermCTGGCAAAAGGTCAGAGTCT) and A222 (IndexTermCTCTGACACTGTCCAACTTG), for MYC, and A92 (IndexTermAGGCTGGGGCAGCATTGAAC) and A93 (IndexTermCACCTTCTGAGGGTGAACTTG), for PSA, were used at 200 nM each, and the thermocycler conditions were 15 min, at 95°C, and 45 cycles (15 s at 95°C, 30 s at 62,5°C, 30 s at 72°C).
MYC DNA copy number was estimated by quantitative PCR using the 2−ΔΔct method with PSA as internal control sequence, and normal tissue as calibrator. MYC was considered to be amplified when the copy number was >4.
Characterization of the 8q24 amplicon and of human sequences flanking integrated HPV DNA
Characterization of the 8q24 amplicon was performed by quantitative PCR amplification of 12 DNA sequences distributed in an interval between 857 kb downstream and 212 kb upstream MYC (see Supplementary Information for primer sequences). All amplifications were performed with 3 mM MgCl2 in the conditions previously described for MYC DNA copy number assessment. DNA copy number of human sequences flanking virus insertion sites at 1q32.2, 2p24.3, 7q31 and 19p13.2 was determined by quantitative PCR with primers localized at less than 2 kb from viral DNA.
MYC mRNA and protein expression
Total RNAs were isolated using Trizol reagent (Invitrogen), as recommended by the supplier. Total RNA (1 μg) was reverse transcribed using the GeneAmp RNA PCR Core Kit (Applied Biosystems), as recommended by the manufacturer. One-hundredth of the cDNA was used for each PCR reaction in a final volume of 25 μl, in the presence of 600 nM of each specific primer and in the SYBR Green PCR master mix (Applied Biosystems). We used the primers A227 (IndexTermTGGTGCTCCATGAGGAGAC) and A228 (IndexTermCCACAGAAACAACATCGATTTC) for MYC amplification, and A12 (IndexTermAGTGAAGAACAGTCCAGACTG) and A13 (IndexTermCCAGGAAATAACTCTGGCTCAT) for TBP. Polymerase chain reaction amplifications were performed in an ABI PRISM 7900 (Applied Biosystem), with cycling conditions of 5 min at 50°C, 10 min at 95 °C and 40 cycles (15 s at 95°C, 1 min at 60°C).
MYC mRNA expression level was determined by relative quantitative PCR using the 2−ΔΔct method (Livak and Schmittgen, 2001), with TBP as internal control gene, and cervical normal tissue as calibrator. MYC was considered to be overexpressed when the RQ value was at least twice that found in the normal epithelium of the cervix.
MYC protein expression level was assessed using Western blot experiments. Total cellular extracts (20 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and electroblotted onto PVDF membrane for antibody detection. Antibodies against the c-myc protein (clone 9E10, Roche Applied Science, Meylan, France) and actin (clone AC-40, Sigma, St Louis, MO, USA) were used at dilutions of 1/5000 and 1/10 000, respectively, and incubated for 1 h, at room temperature.
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We thank Zofia Maciorowski and Edith Heard for their help in preparation of the manuscript.
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Peter, M., Rosty, C., Couturier, J. et al. MYC activation associated with the integration of HPV DNA at the MYC locus in genital tumors. Oncogene 25, 5985–5993 (2006) doi:10.1038/sj.onc.1209625
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