Unité Mixte Institut Pasteur/INSERM (U.190), Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris cedex 15, France1
Laboratoire de Pathologie, Section Médicale et Hospitalière, Institut Curie, Paris, France2
Author for correspondence: Gérard Orth. Fax +33 1 45 68 89 66. e-mail gorth{at}pasteur.fr
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Abstract |
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Introduction |
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In order to investigate the relationships between integration of HPV sequences and alterations of the structure and expression of a myc protooncogene, we have further analysed two genital invasive carcinomas (IC2 and IC4) previously found to harbour HPV-16- and HPV-18-related sequences, respectively, integrated at the chromosomal sites of c-myc (IC2) and N-myc (IC4) (Couturier et al., 1991 ). The IC2 tumour had been found to harbour rearranged and amplified c-myc sequences without any evidence of c-myc overexpression, whereas a 10- to 20-fold amplification of the N-myc gene and high levels of N-myc transcripts had been observed in the IC4 tumour (Couturier et al., 1991
).
Our data show that DNA sequences of HPV-45 are integrated in the 3' untranslated part of the N-myc gene in IC4 cells, that a major N-myc transcript terminates upstream of the viruscell junction and that high levels of N-myc protein are expressed. In contrast, we found no evidence for integration of HPV-16 within or in the vicinity of the c-myc gene in IC2 cells and we show that the major c-myc rearrangement corresponds to the insertion of a non-coding cellular sequence into the second intron of the gene.
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Methods |
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Dual-colour-fluorescent in situ hybridization.
Chromosome preparations of IC2 and IC4 cells obtained at the 12th subculture were hybridized with a mixture of probes specific for HPV-16 and c-myc (IC2) or for HPV-45 and N-myc (IC4). HPV DNA probes were biotin-labelled by using a nick translation kit (Boehringer Mannheim). Digoxigenin-labelled c-myc and N-myc DNA probes were purchased from Oncor. After incubation in 2x SSC at 37 °C for 30 min and denaturation in 70% formamide/2x SSC at 70 °C for 2 min, chromosome preparations were hybridized with the denatured probes (5 ng/µl of each) in 50% formamide/2x SSC at 37 °C for 15 h. Slides were washed at 37 °C in 50% formamide/2xSSC and then 2x SSC. Detection was achieved by using an FITCavidin/anti-digoxigenin rhodamine solution (Oncor). Slides were mounted in Vectashield-DAPI (Vector Laboratories). Fluorescence observation and image capture were performed on a Leica DMRB microscope fitted with a Quantix digital camera (Photometrics). The three-colour images (DAPI, FITC and rhodamine) were processed on a Quips Smart Capture workstation (Vysis).
DNA, RNA and protein preparation.
Total DNA was prepared from tumour biopsy specimens and human placenta by standard methods of phenolchloroform extraction followed by ethanol precipitation (Sambrook et al., 1989 ). DNA, RNA and proteins were extracted simultaneously as described by Coombs et al. (1990)
. Briefly, frozen samples corresponding to 107 IC2 cells (12th subculture) and tumours (100 mg) obtained after grafting 106 IC4 cells (12th subculture) or 106 SKNBE cells to athymic mice were homogenized in 4 M guanidinium thiocyanate by using an Ultraturrax T25 homogenizer (Janke & Kunkel, IKA-Labortechnik). The homogenates were layered onto a 5·7 M caesium chloride cushion and centrifuged for 18 h at 130000 g at 20 °C. RNA was recovered from the pellet by dissolution in 0·3 M sodium acetate (pH 6) and ethanol precipitation. DNA was extracted by phenolchloroform and ethanol precipitation from the caesium chloride layer after dialysis and incubation for 24 h with proteinase K (10 µg/ml). The protein-containing guanidine phase was dialysed against 100 mM ammonium bicarbonate and lyophilized. DNA and RNA concentrations were measured by spectrophotometry at 260 nm and proteins were quantified by using the Bio-Rad DC protein assay.
Southern blot hybridization` experiments.
Total cell DNA preparations (10 µg) obtained from tumour biopsy specimens and from human placenta were digested with BamHI, EcoRI, HindIII or PstI restriction endonucleases, electrophoresed in 0·8% agarose gels, blotted onto nitrocellulose filters (Hybond-C, Amersham International) and hybridized with random-primed, 32P-labelled DNA probes (Couturier et al., 1991 ). Probes corresponded to cloned HPV-16 and HPV-45 genomes excised from plasmid sequences, as well as to PCR amplification products obtained by using primers specific for c-myc exon 1 (nt 306325 and 884865), exon 2 (nt 47284747 and 50665047) and exon 3 (nt 67316750 and 77777758) (Gazin et al., 1984
) and from the N-myc exon 3 (nt 50035022 and 56125593) (Stanton et al., 1986
). Membranes were exposed to X-ray films for 1648 h.
Molecular cloning of rearranged myc sequences.
DNA libraries were constructed by inserting EcoRI-digested IC4 tumour DNA in a gt wes vector (Life Technologies) or BamHI-treated IC2 tumour DNA in a
GEM-11 vector (Promega). About 105 plaques were screened successively with N-myc exon 3 and HPV-45 DNA probes (IC4) or with c-myc exon 3 and HPV-16 DNA probes (IC2). Recombinant
phages were isolated as described previously (Sambrook et al., 1989
) from three plaques that hybridized with both N-myc and HPV-45 probes and were found to contain a 15 kb DNA fragment. An EcoRIXbaI fragment of 8 kb (IC4-EX-8kb) containing both N-myc and HPV-45 sequences was subcloned in pBluescript (Stratagene). Screening of the IC2 library yielded six plaques that hybridized with c-myc, five with HPV-16 and none with both probes. The c-myc-positive plaques were found to contain recombinant
phages with an 8 kb insert. A BamHIEcoRI DNA fragment of 3 kb (IC2-BE-3kb) containing the c-myc sequences was subcloned in pBluescript plasmid.
Nucleotide sequence analysis.
The nucleotide sequences of the cloned IC4-EX-8kb and IC2-BE-3kb DNA fragments were determined by using the ABI prism Bigdye terminator cycle sequencing kit with AmpliTaq FS DNA polymerase (Perkin-Elmer) as recommended by the supplier. Primers used corresponded to M13 oligonucleotide sequences complementary to sequences flanking the inserted fragment (M13 universal and reverse primers) and to internal oligonucleotides. Internal primers in both orientations were specific for N-myc intron 2 and exon 3 sequences (nt 37183737, 42114230, 47614780, 52655284, 57815800 and 62146233) (Stanton et al., 1986 ), HPV-45 sequences (nt 56325651, 61406169, 66096628, 71147133, 75737592, 264283, 574593 and 10961115) (Myers et al., 1994
) and c-myc intron 2 and exon 3 sequences (nt 57235742, 62696288, 67316750, 71927211 and 75577576) (Gazin et al., 1984
). Sequencing products were analysed with an ABI prism 377 DNA sequencer (Perkin-Elmer). Multiple alignments of DNA sequences were performed by using the CLUSTAL W program.
Northern blot hybridization experiments.
Samples (10 µg) of total RNA obtained from IC4 cells were electrophoresed in a denaturing (6% formaldehyde) 1% agarose gel and blotted onto a nitrocellulose membrane (Hybond-C, Amersham International) and strips were hybridized in parallel with different 32P-labelled DNA probes. Probes were prepared from genomic HPV-45 DNA or PCR amplification products obtained by using primers specific for N-myc exon 3 (nt 50035022 and 56125593) (Stanton et al., 1986 ), HPV-45 upstream regulatory region (URR) (nt 72717289 and 77057686) and HPV-45 ORFs E6 (nt 6382 and 593574), E7 (nt 574593 and 920901) and L1 (nt 52095228 and 70076988) (Myers et al., 1994
). Membranes were exposed to X-ray films for 1648 h.
Mapping of the 3' ends of N-myc transcripts in IC4 cells.
Total RNA (1 µg) isolated from IC4 cells was reverse-transcribed with the Superscript RT RNase H reverse transcription kit (Life Technologies), in the presence of an antisense adaptor primer [5' GACTCGAGTCGACCCGGG(dT)17 3'], according to the instructions of the manufacturer. After ethanol precipitation and centrifugation, the pellet was suspended in distilled water (20 µl) and 2µl of the cDNA mixture was used for PCR amplification (Frohman et al., 1988 ). Primers were specific for the untranslated part of N-myc exon 3 (nt 60776096) (Stanton et al., 1986
) and for the adaptor. PCR products were cloned in the SmaI site of pBluescript (Stratagene) and sequenced by using M13 universal and reverse primers.
Detection of N-myc protein.
Aliquots of protein extracts (50 µg) and prestained protein markers (New England BioLabs) were electrophoresed on a 10% SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell). The membrane was incubated with sheep anti-human N-myc IgG (Serotec) at a dilution of 1:200. After washing, the membrane was incubated with peroxidase-conjugated rabbit antibodies raised against sheep IgG (Dako) at a dilution of 1:1000. Proteins were visualized with the ECL chemiluminescent detection system as described by the manufacturer (Amersham International).
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Results |
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In order to map the 3' ends of the N-myc transcripts, poly(A)+ mRNAs of IC4 cells were reverse transcribed and the amplification products (about 200 bp) of the N-myc cDNA 3' ends were cloned and sequenced. Three polyadenylation sites were identified, at positions 6208, 6290 and 6293 of the N-myc sequence (Fig. 4); i.e. upstream of the bona fide AATAAA N-myc polyadenylation signal that is deleted upon integration of HPV-45 DNA sequences. This makes it likely that the cryptic polyadenylation signals AATAATA (nucleotide position 6149) and AACTAAA (nucleotide position 6264), located in the untranslated part of N-myc exon 3, were used for the 3'-end processing of the major 3·1 kb transcript. No cDNA corresponding to the minor 5·6 kb species was obtained. This transcript was found to hybridize with HPV-45 L1 and URR probes but not with the E6 and E7 probes (Fig. 2B
, lanes 36). It is thus likely that one of the putative polyadenylation signals (AATAAA) located at nucleotide positions 7310 and 7671 in the HPV-45 URR (Myers et al., 1994
) was used, resulting in the addition of about 2·2 or 2·5 kb of viral sequences to the 3·1 kb N-myc transcripts. A summary of the transcription data is given in Fig. 3(A)
.
N-myc protein overexpression in IC4 tumour cells
In human tumour cell lines expressing amplified N-myc sequences, the N-myc protein is detected as two doublets of closely migrating phosphorylated (p62 and p64) and non-phosphorylated (p58 and p60) polypeptides that are translated from two in-frame AUG codons located 25 bp apart in the second exon of the N-myc gene (Mäkelä et al., 1989 ). In order to determine whether high levels of N-myc mRNA in IC4 cells were associated with N-myc protein expression, Western blot experiments were performed using a protein extract from IC4 tumour cells. Neuroblastoma-derived SKNBE cells containing about 50 copies of the N-myc gene (de Crémoux et al., 1997
) and IC2 cells showing no N-myc transcript (Couturier et al., 1991
) were used as controls. Comparable amounts of N-myc protein were detected in IC4 cells and SKNBE cells, with apparent molecular masses ranging between 58 and 64 kDa. N-myc protein from IC4 cells migrated slightly ahead of that from SKNBE cells. No N-myc protein was found in IC2 cells (Fig. 5
).
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Insertion of cellular sequences within c-myc intron 2 in the IC2 tumour
Southern blot hybridization of IC2 DNA cleaved with the three non-cutting enzymes for the c-myc gene using c-myc exon 1, 2 or 3 probes showed extra bands with sizes of 15 (HindIII), 14 (BamHI), 7 or 6·5 kb (EcoRI) compared with the fragments detected in human placenta DNA (Fig. 6, lanes 24, 68 and 1012). Furthermore, an additional highly labelled band of 7 (HindIII), 8 (BamHI) or 3 kb (EcoRI) was detected with the exon 3 probe (Fig. 6
, lanes 4, 8 and 12). The DNA library constructed from BamHI-restricted IC2 DNA allowed further characterization of the rearranged and amplified sequences. Recombinant
phages isolated from six plaques hybridizing with the c-myc exon 3 probe were all found to contain an 8 kb BamHI fragment. None contained the abnormal 14 kb fragment detected by all c-myc probes. A 2·9 kb BamHIEcoRI fragment containing the c-myc sequences was subcloned and sequenced. This fragment was found to comprise a 399 bp non-coding sequence inserted upstream of c-myc sequences (2612 bp) encompassing most of intron 2, exon 3 and the downstream non-coding sequences (Fig. 7
). The 399 bp segment showed no significant similarity to the c-myc gene or to any of the sequences available in the EMBL database. The junction between the unknown sequences and the c-myc gene was mapped to nucleotide 5471 in intron 2. Only one nucleotide change was observed (C7775
T), located downstream of c-myc exon 3, compared with the published sequence (Gazin et al., 1984
).
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Discussion |
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In IC4 tumour cells, subgenomic HPV-45 DNA sequences were found to be integrated within the 3' untranslated part of N-myc exon 3, 15 nucleotides upstream of the bona fide polyadenylation signal, with a 10- to 20-fold amplification of both N-myc and viral sequences. The HPV-45 genome was interrupted in the 3' part of the L2 ORF and in the 5' part of the E1 ORF, a pattern similar to that described recently for HPV-45 in the cervical carcinoma-derived MS751 cell line (Geisbill et al., 1997 ). Despite the significant association of HPV-45 with invasive cervical carcinoma (Bosch et al., 1995
) and its worldwide distribution (Stewart et al., 1996
), data on the intratype variability of the E6 and E7 ORFs were available for two isolates only, the prototype (Myers et al., 1994
) and an isolate described by J. B. Kaplan and R. D. Burk (unpublished EMBL accession no. M38198). Alignment of the deduced amino acid sequences of IC4 HPV-45 oncoproteins with those of the two isolates disclosed a total of five variable amino acid positions in the E6 protein (residues 8, 10, 30, 53 and 118) and four in the E7 protein (residues 3, 5, 6 and 82). High levels of transcripts of the E6 and E7 region were detected in IC4 cells, supporting a role for the viral oncoproteins in the carcinogenesis process. In addition, a fusion ORF was created at the 3' viruscell junction that encodes a putative 153 amino acid protein including the 120 amino terminal residues of the viral E1 protein.
IC4 cells express high levels of N-myc transcripts processed via cryptic polyadenylation signals located upstream of the 5' junction, as well as a minor N-myc/HPV-45 fusion transcript that terminates within the viral URR. It seems unlikely that these high levels of N-myc transcripts result from an increased half-life, since integration did not delete the U-rich region of the 3' untranslated part of the transcripts, a region found to be responsible for the rapid cytoplasmic turnover of c-myc transcripts (Jones & Cole, 1987 ). Since the N-myc protooncogene is not expressed in the normal cervical epithelium (Couturier et al., 1991
), its activation could result from insertional mutagenesis, possibly enhanced by the co-amplification of N-myc and of HPV-45 sequences, or from the amplification of the N-myc gene per se. Integration of viral sequences within the N-myc gene and the subsequent cis-activation of the protooncogene have commonly been observed in T cell lymphomas induced by the Moloney murine leukaemia virus (van Lohuizen et al., 1989
) and in hepatocarcinomas associated with woodchuck hepatitis virus (WHV) (Fourel et al., 1990
). The epithelium-specific enhancer identified in the HPV-18 URR (nt 75797738) (Thierry, 1993
) is highly conserved in the HPV-45 genome (nt 75787737) (Myers et al., 1994
). Insertion of this putative enhancer sequence about 9 kb downstream of the N-myc promoter in IC4 cells could possibly lead to its cis-activation. Transient transfection assays with the chimeric IC4 N-myc/HPV-45 sequence would demonstrate such a mechanism, as already shown for N-myc2WHV constructs (Wei et al., 1992
). On the other hand, the 10- to 20-fold amplification of the N-myc gene by itself could also account for the overexpression of the N-myc gene in IC4 cells, as observed in neuroblastoma (Brodeur et al., 1984
; Schwab et al., 1984
), retinoblastoma (Lee et al., 1984
) and small-cell carcinoma of the lung (Nau et al., 1986
). Comparable large amounts of N-myc protein were detected in IC4 cells and in SKNBE neuroblastoma-derived cells, which show a 50-fold amplification of the N-myc gene (de Crémoux et al., 1997
). The slightly faster migration of IC4 N-myc protein may indicate that this protein corresponds to the non-phosphorylated form (Mäkelä et al., 1989
). The effects of phosphorylation on the functions of myc proteins (transactivation, DNA binding, interaction with Max protein) are far from understood (Lüscher & Larsson, 1999
). It is worth stressing that mutations affecting two phosphorylation sites in the transactivation domain of the c-myc protein have been shown to result in a higher transforming potential (Nesbit et al., 1999
). Since myc protooncogenes encode transcription factors that regulate the expression of genes involved in cell proliferation and apoptosis (Nesbit et al., 1999
), it is likely that the activation of N-myc played a part in tumour progression.
Amplification, rearrangement and/or overexpression of the c-myc gene were reported to be frequent in invasive cervical carcinomas (Bourhis et al., 1990 ; Ocadiz et al., 1987
; Riou et al., 1985
). Whether these genetic alterations are related to the insertion of HPV sequences is still unknown. No evidence for the integration of HPV-16 sequences within or in the close vicinity of the c-myc gene was obtained for IC2 cells. A major c-myc rearrangement identified in IC2 tumour cells involved the insertion of non-coding cellular sequences within the 5' part of intron 2 and the amplification of the insert and the downstream c-myc sequences. In the absence of abnormal expression of the c-myc gene (Couturier et al., 1991
), it remains to be determined whether the integration of HPV-16 in band 8q24.1 in IC2 cells resulted in the abnormal expression of another as yet unidentified gene possibly involved in tumour progression. That c-myc amplification and/or overexpression can occur independently from virus integration in virus-associated cancers has been shown in Burkitts lymphoma cells associated with the EpsteinBarr virus (Magrath, 1990
) and in hepatocarcinomas induced by the ground squirrel hepatitis virus (Transy et al., 1992
). However, our data on IC4 cells provide evidence for a possible role of integration of HPV sequences in cervical carcinogenesis through a mechanism of insertional mutagenesis and/or gene amplification.
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Acknowledgments |
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Footnotes |
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Received 25 February 2000;
accepted 4 May 2000.