Znt. J. Cancer: 50,356-364 (1992) 0 1992 Wiley-Liss, Inc.
Publication of the InternationalUnion Against Cancer Publication de I'Union InternationaleContre le Cancer
EXPRESSION AND SPLICING PATTERNS OF HUMAN PAPILLOMAVIRUS TYPE-16 mRNAs IN PRE-CANCEROUS LESIONS AND CARCINOMAS OF THE CERVIX, IN HUMAN KERATINOCYTES IMMORTALIZED BY HPV 16, AND IN CELL LINES ESTABLISHED FROM CERVICAL CANCERS L. SHERMAN I, N. ALLOUL', I. GOLAN',M. DURST'and A. BAR AM^ 'Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel; 'Institutfur Virusforschung, Deutsches Krebsforschungszentrum,Heidelbetg, Germany; and 'Department of Obstetrics and Gynecology,A . Serlin Hospital, Hakea, Tel-Aviv, Israel. We have analysed the splicing patterns of human papillomavirus (HPV) type-I6 mRNAs in a human epithelial cell line immortalized by HPV 16 (HPKII), in cell lines established from cervical carcinomas (SiHa and CaSki) and in pre-invasive and invasive carcinomas of the cervix. The presence of mRNA species previously described, which could encode the E6, E61, E611, E6111, E7, E2, E X , E4, E5 and LI proteins, was determined, using the RNA polymerase chain reaction (PCR) technique with primers that flank unique splice sites. The state of the viral DNA in the tumor biopsies was established by Southern blot analysis. The various HPV 16 transcripts could be detected in cell lines and in tumor biopsies. The size of the RNA PCR products were in agreement with the previously mapped splice sites. The full range of transcripts was revealed in the HPKll cell line and in a number of pre-invasive carcinomas. Messenger RNAs which could encode the E6111, E4 and E5 proteins were most prevalent in all types of tumor. The overall results of DNA and RNA analyses in cell lines and tumor specimens indicate that (I) expressionof either of the early or late transcripts studied is not specifically related to (a) tumor stage or (b) the physical state of the viral genome; and (2) alterations in the splicing patterns of HPV 16 transcripts may not be involved in tumor progression.
Approximately 22 distinct types of human papillomavirus (HPV) preferentially infect the anogenital mucosa and are found in a spectrum of lesions ranging from benign condylomas, through cervical intra-epithelial neoplasms (CINs) to invasive carcinomas (De Villiers, 1989). HPV type-16 predominates in high-grade CINs and invasive carcinomas, implying an important role in the development of cancer (zur Hausen, 1989). Although HPV-16 DNA and RNA are consistently detected in tumor specimens, at present it is not clear whether alterations in the transcription pattern of HPV 16 occur during stages in the progression of cervical neoplasia. The genomes of HPV 16 in pre-malignant lesions exist generally in an episomal state, whereas in carcinoma integrated viral DNA is frequently observed (Lehn et al., 1985, 1988; Durst et al., 1985; Cullen et al., 1991). The integration break in the viral genome often occurs within the El-EZ gene region and this event is presumed to result in de-regulation of the expression of the E6 and E7 genes and promotion of neoplasia (zur Hausen, 1989). Active transcription was consistently observed in HPV-16-positive carcinomas and carcinoma cell lines (Yee et al., 1985; Smotkin and Wettstein, 1986; Baker et al., 1987; Shirasawa et al., 1988). The transcription pattern was extensively studied in carcinoma cell lines and only in some tumor biopsies. By Northern blot hybridization with gene-specific probes (Baker et al., 1987; Shirasawa et al., 1988) primer extension and S1 analyses (Smotkin and Wettstein, 1986; Smotkin efal., 1989) the presence of transcripts representing the E6 and E7 open reading frames (orfs) was revealed. The transcripts were fused at their 3' end with cellular sequences. Three types of E6-E7 mRNA were identified (Smotkin et al., 1989); 2 mRNAs termed E61 and E6II are spliced with the E6 orf. The E61 transcript is presumed to encode the E7 protein which is abundantly expressed in carcinomas and the derived cell lines (Smotkin et al., 1989).
The regular preservation and expression of the E6-E7 gene region in cancerous tissues lead to the assumption that these genes are required for the establishment and maintenance of the neoplastic state (Schlegel et al., 1990). This assumption is also supported by numerous in vitro assays which have demonstrated the transforming capacity of the E6 and E7 orfs of HPV 16 (Schlegel et aL, 1990) and the evidence showing the direct correlation between HPV E6/E7 gene transcription and the growth rate of human cervical cancer cells (von KnebelDoeberitz et al., 1988). Information regarding the expression of HPVs in precancerous lesions is limited. Technical limitations, including the small size of biopsies and the paucity of certain RNA transcripts, have prevented precise analysis of transcripts from being performed. In situ hybridization studies in CIN biopsies demonstrated that the most abundant hybridization signals occurred from the E4-ES orfs, with weaker signals from the E6-E7 orfs and the 5' portion of the E2 orf (Crum et al., 1988). Northern blot analyses with subgenomic DNA probes revealed 4 types of mRNA which represented all the orfs of the early region (Shirasawa et al., 1988). The exact structure of these mRNAs was not determined. Analyses of the splicing pattern of the E6-E7 transcripts in CINs by RNA polymerase chain reaction (PCR) revealed the same pattern of splicing as previously described in carcinoma cell lines (Cornelissen et al., 1990; Johnson et al., 1990). Data on the structure of HPV 16 mRNAs present in pre-cancerous lesions were recently obtained from analyses of cell lines established from CINs (Schneider-Maunoury et al., 1990; Doorbar et al., 1990). The W12 cell line derived from a CLN I lesion (Stanley et al., 1989) contains the HPV-16 genome in an episomal state and this seemed to dictate a different program of gene transcription. Analyses of cDNAs obtained from these cells (Doorbar et al., 1990), revealed the presence of early mRNAs with novel splicing patterns, ending at the viral Poly(A) site. These were an E6 mRNA (E6III) which could encode E6 protein, whose terminus is derived from an out of frame region of the E2/E4 coding sequence; an E2 mRNA which could encode the E2C protein, derived from the C terminus of the E2 orf; and a transcript which could encode the E4 and E5 proteins. Two novel late mRNAs were also identified. It is not known whether these mRNA species are unique to the W12 keratinocytes or are commonly expressed in HPV-16-containing lesions. To understand the relation between the expression of individual transcripts and tumor development, and to determine whether alterations in the splicing patterns of specific mRNAs are related to tumor stage, or the physical state of the viral genome, we analysed the transcription pattern and the state of HPV-16 DNA in a wide range of naturally occurring tumors and in cell lines containing HPV-16 DNA. For this Received: June 27,1991 and in revised form August 30,1991.
TRANSCRIPTION O F HPV 16 IN TUMORS AND CELL LINES
purpose we used the RNA PCR procedure which enabled us to detect and determine the splicing patterns of a variety of HPV-16 transcripts, in small tissue specimens. MATERIAL AND M E T H O D S
Cell lines, tissue specimens and probes The CaSki and SiHa cell lines were obtained from the ATCC (Rockville, MD). HPKII cells were obtained from Dr. M. Durst (Heidelberg, Germany). Cells were grown in DulbecCO'S Modified Eagle's Medium (DMEM) supplemented with 10% FCS. Tissue specimens taken from cervical lesions and tumors were obtained from the Gynecology Departments of Serlin (Hakirya) and Sheba Hospitals, Tel-Aviv. Upon excision, biopsies were immediately frozen in liquid nitrogen and then stored at -80°C. Biopsies, from tissues adjacent to those analysed for HPV, were cut for histology. Extraction of RNA and DNA High-molecular-weightDNA and RNA were extracted simultaneously from tissue specimens by the guanidinium cesium chloride procedure as described by Meese and Blin (1987). Restriction enzyme cleavage and blot hybridization Between 10 and 20 pg of DNA were digested with the appropriate restriction endonuclease and fractionated on a 0.8% agarose gel in TAE buffer (40 mM Tris-acetate, 2 mM EDTA) containing 1 pg/ml ethidium bromide. The DNA was denatured and transferred to nitrocellulose filter as described by Southern (1975). Filters were hybridized with HPV-16 DNA, "P-labelled by the random priming method to specific activity of lo9cpm/pg DNA (Maniatis et al., 1989). Hybridization were carried out at 42°C in 50% formamide 5 x SSC, 2 x Denhardt, 10% dextran sulfate and 100 pg/ml salmon sperm DNA, for 24 hr. The filters were washed twice with 2 x SSC, 0.1% SDS at room temperature and twice in 0.1 x SSC, 0.1% SDS, at 50°C. Autoradiography was performed for 4- 10 days at 80°C with Kodak X-omat film. cDNA synthesis and PCR ampliJication Synthesis of each cDNA and its subsequent amplification was performed with 1 pg cell or biopsy RNA and the appropriate primers (Table I). RNA was mixed in a total volume of 20 pl in a reaction mix containing reverse transcriptase buffer (BRL), 25 units RNasin (Promega, Madison, WI), 1.0 mM of each of the dNTPs, 35 pmol of the reverse (anti-sense) primer, 200 units of cloned MuLV-1 reverse transcriptase (RT) (BRL, Gaithersburg, MD). The reaction
was allowed to proceed for 30 min at 42°C and then the RT was inactivated at 95°C for 5 min. Amplification of cDNA was followed by adding 35 pmol of the 5' (sense) primer in 80 p1 amplification buffer and 2 U Taq polymerase (bioexcellence, Anglian Biotec Limited, Colchester, UK). The reaction was subjected to 30 PCR amplification cycles, beginning with DNA denaturation at 94°C for 1 min, followed by a primer annealing step at 55°C for 1min and an elongation step at 72°C for 2 min. The final elongation step was prolonged for 8 min. In cases where a second round of amplification was performed, 2% of the first mix was amplified using the same reaction parameters. Analysis of RNAIPCR products One-fifth of the amplified product was separated by electrophoresis through 1.4% agarose gel. The DNA was transferred by blotting onto nylon membrane (Gene Screen Plus, NEN, Boston, MA) in 0.5 N NaOH, 1.5 M NaCl and hybridized with the appropriate oligonucleotide probe (see Table I), endlabelled with ("P) ATP (3000 Ci/mmol) and T4polynucleotide kinase (Maniatis et al., 1989). Hybridizations were carried out at 42°C in 6 x SSC, 1 x Denhardt, 0.5% SDS, 200 pg/ml salmon sperm DNA 500 pg/ml tRNA, for 16 hr. Filters were washed twice in 2 x SSC, 0.5% SDS at room temperature and then twice in 1 x SSC, 1% SDS, 0.5% sodium PPi at 50°C. Autoradiography was performed for 3-12 hr at -80°C. RESULTS
1. RNA PCR analysis of HPV 16 transcripts in immortalized keratinocytes and carcinoma cell lines We applied the RNA PCR method (Kawasaki et al., 1988) to analyse the transcription pattern of HPV 16 in naturally occurring tumors and cell lines associated with HPV 16. Initial experiments were conducted with RNA obtained from 3 cell lines, all containing integrated viral genomes. The SiHa and CaSki cell lines, established from cervical carcinomas, contain 1 and 500 HPV 16 genome copies per cell respectively (Yee et al., 1985; Baker et al., 1987), whereas the immortalized non-malignant keratinocyte cell line HPKII contains about 10 viral copies of HPV 16 DNA (Durst et al., 1987, 1991). To establish the validity and sensitivity of the RNA PCR assay, we first attempted to detect viral transcripts known to be expressed in these cell lines, and later we examined the expression of other transcripts, recently revealed in the W12 cell line derived from a CIN lesion (Doorbar et al., 1990). The oligonucleotide primers were designed to selectively amplify mRNA species that utilize previously mapped unique
TABLE I - HPV-16 mRNA SPECIES AND OLIGONUCLEOTIDEPRIMERS USED IN THE GENERATION AND AMPLIFICATION OF cDNAs AND TARGET
PROBES USED IN THE DETECTION OF THE PCR PRODUCTS
mRNA species Coding capacity
Primers Splice site
E6,E7,E1/E4,E5 E6I,E7,El /E4,E5 E6II,E7,E1/E4,E5 E6III.ES ElIE4,ES
E6"E6 I) E6"E6[11) E6"U El"E4
16E6S-101 16E6~-101 16E6~-101 16E6~-101 16E7~-8 18 16E7~-818 16E7~-818 16Els-1242 16Els-1242 16E7~-571 16E7s-818a 16E4~-3383 16E1S- 1242
PCR product Expected size
16E7as-570 16E7as-570 16E7as-570 16E4~-3383 16E4~-3383 16E4~-3383 16E4~-3383 16E4~-3383 16E4~-3383 16E2as-2880 16E2as-2880 16Llas-5673 16Llas-5673
676 494 377 200 137 592 795 134 589 533 286 313 124
16E7as-776 16E7as-776 16E7as-776 16E4as-3430 16E4as-3430 16E5as-3885a 16E4as-4088b 16E5as-3430 16E5as-3885a 16E2as-2930 16E2as-2930 16Llas-5700 16Llas-5700
The targeted mRNA species and splice sites refer to those depicted in Figure 1. Oligonucleotide rimers and probes (20 mer) were synthesized according to the sequence determined by Seedorf efal. (1985). The orientation of the primers, sense(s) or antisense (as?, and the nucleofide,numberof the 5' nucleotide, relative to the HPV 16 genome, is included in the name of each primer. Alternative primers used to detect identical splice sites were indicated as a and b.
SHERMAN ET AL.
splice sites (Smotkin et al., 1989; Doorbar et al., 1990; Rohlfs et al., 1991). Primers positioned at both sides of the splice junction were selected, based on the known DNA sequence of HPV 16 (Seedorf et al., 1985). Single-stranded cDNA was made from total RNA by priming reverse transcriptase with the down-stream (anti-sense) primer, and subsequently amplified by PCR. The RNA PCR product was analysed by agarose gel electrophoresis and Southern blot hybridization with 32Plabelled target probe, contained within the expected PCR fragment. The various mRNA species investigated are depicted in Figure 1. Oligonucleotides used for the generation and amplification of cDNAs, and target probes used to detect the RNA-specific PCR products, are listed in Table I. From the E6-E7 gene region, 3 RNA-specific PCR products were generated with the primer pair E6s-lOl/E7as-776, identical for HPKII, CaSki, and SiHa cells (Fig. 2, E6'E6). The size of the products 377, 494, 676 bp, corresponds with the expected size of the E6II and E61 transcripts alternatively spliced within the E6 orf and the unspliced E6 RNA (Smotkin et al., 1989). The latter PCR product could, however, be derived from DNA present in the RNA samples, as suggested
by the presence of a similar band in the control reactions with no reverse-transcriptase added (data not shown). The expression of the non-spliced E6 transcript in HPKII and CaSki cells was conclusively proved using primers targeting the E6AE4 splice (described below). The major E6-E7 RNA species detected in HPKII, CaSki and SiHa cells was the E61 RNA, as found in previous analyses (Srnotkin and Wettstein, 1986; Smotkin et al., 1989; Rohlfs et al., 1991). The recently reported E6III mRNA species (Doorbar et al., 1990) could be detected with the primer pair E6~-101/E4as3430 (Table I). RNA/PCR product, with the expected size of 200 bp, was generated with RNA from HPKII and CaSki cells (Fig. 2, E6"E4). Inability to obtain the respective product with RNA from SiHa cells was expected from the known integration site of the single genome present in SiHa cells, which interrupts the E2 and the E4 orfs (Baker et al., 1987). In addition to the E6III product, 3 additional RNA PCR products were generated with the E6/E4 primer pair. The sizes of these products correspond with the expected sizes, 555, 672, 854 bp, for the E6II,E6I and the non-spliced E6 mRNAs, if these were spliced to the E4-E5 orfs via the splice site between
HPV-16 GENOME (E71
FIGURE 1 - HPV 16 mRNA species and coding potentials. Top: diagram of the genome of HPV 16 represented in a linear fashion, and positions of open reading frames. Bottom: RNA species described previously and positions of splice sites (Smotkin et aL, 1989; Doorbar eta/., 1990; Rohlfs et al., 1991). The nucleotide numbers are according to the sequence determined by Seedorf et al. (1985). Numbers of common splice sites are shown only once. The open boxes represent potential coding regions; coding capacity of each species is indicated on the right. The positions of primers used to generate and amplify the correspondingcDNAs are denoted by arrowheads, above (sense) and below (anti-sense), each mRNA. The positions of the target probes used to detect the corresponding RNA PCR products are indicated by empty arrowheads.
TRANSCRIPTION OF HPV 16 IN TUMORS AND CELL LINES
FIGURE 2 - Profiles of RNA PCR products from CaSki, SiHa, and HPKII cells. For detection of each RNA species, 1 pg of RNA was subjected to RNA PCR with the appropriate primers (Table I). cDNA synthesis and PCRs were carried out as described in "Material and Methods". Products were revealed by electrophoresison ethidium-bromide-stained gels and Southern blot hybridization, each with the specific target probe. Also applied to the gels were the 123-bpmarker fragments (BRL) (M). Autoradiograms of Southern blots are presented. The RNA targeted splice is indicated at the top of each lane. The sizes of the RNA PCR products reacting with the target probes are denoted only once (for HPKII). The bars on the right show the positions of the 123-bp marker fragments. The position of the first fragment (123 bp) is also indicated by an arrow (right). nucleotides 880/3357. These PCR results suggest that the E6 and E6 I1 transcripts join the 5' orfs to the 3' orfs as was previously described for the E6 I transcript (Tanaka et al., 1989; Schneider-Maunoury et al., 1990; Doorbar et al., 1990; Rohlfs et al., 1991). Messenger RNA species sharing the El'E4 splice were detected with the primer pair E7s-818/E4as-3430 which border the El"E4 splice site (Fig. 1, Table 11). The expected El'E4 137 bp RNA PCR product was obtained with RNA from HPKII and CaSki cells (Fig. 2, El"E4). This product could represent, in addition to the 3 E6-E7 transcripts, also a shorter transcript which is initiated at the E7 orf (Doorbar et al., 1990). The E2 gene of HPV 16 encodes 2 different proteins, the E2 protein implicated in trans-repression of the expression of the E6-E7 genes, and the E2C repressor protein derived from the c terminus of the E2 orf (Sousa et al., 1990). A putative E2 mRNA was recently revealed in HPKII cells and its splice site was mapped within the El orf (Rohlfs et al., 1991). With the primer pair E7s-818/Elas-2930, the expected 286-bp RNA PCR product was generated, with RNA from HPKII cells and also CaSki and SiHa cells (Fig. 2, El'Ela). cDNAs representing the El"E1 splice junction could be also produced with the sense primer positioned in the E7 orf (E7s-571) (Fig. 2, El'E1). Cloning and nucleotide sequence analysis of the 533-bp product from HPKII cells confirmed the
previously mapped splice site between nucleotides 880/2708 (data not shown). This result indicates that El spliced transcripts could be initiated upstream of the E7 orf, possibly at p97. The splice site of the E2C mRNA was mapped between nucleotides 1301 and 3357 (El*'E4) (Doorbar et al., 1990). Using the Els-l241/E4as3430 set of primers, RNA PCR product with the expected size of 134 bp was generated, with RNA from CaSki and HPKII cells (Fig. 2, El*'E4). Since the latter RNA PCR product could also be derived from the doubly spliced E2M (Ll) mRNA described (Doorbar et al., 1990), we used another primer combination to distinguish between these 2 mRNAs. With the down-stream primer positioned beyond the second splice donor of the putative E2M (Ll) (El'E4"Ll) mRNA, so as to exclude amplification of the latter, we could selectively amplify the E2C RNA. With the primers Els-1241iE5as-3885, RNA PCR product of 586 bp was generated with RNA from HPKII and CaSki cells (data not shown). To examine whether RNAs detected in CaSki and HPKII cells with primers that span the specific splice site contain the entire E4 and E5 region, RNA PCR assays were conducted with anti-sense primers positioned in the E5 orf. Targeting the El"E4 splice with the primer pair Els-818/ E5as-3885 or Els-818/E5as-4088, the expected RNA PCR products of 592 and 791 bp were enerated using RNA from HPKII and CaSki cells (Fig. 2, E l /?E4a,b), thus suggesting the
SHERMAN ET AL. TABLE I1 - PHYSICAL STATE OF IIPV 16 DNA AND VIRAL mRNAs DETECTED IN TUMOR BIOPSIES
mRNA speciesisize of PCR products 177
B-20 B-32 B-58 B-73 B-78 B-84 v-7 B-90 B-91 B-92 B-93 Total positive TS-2 TS-3 s-7 s-12
S-13 s-20 Total positive
CIN-2 CIN-2-3 CIN-3 CIN-3 CIN-2 CIN-3 CIN-3 CIN-3 CIN-3 CIN-3 CIN-2 Adenocarcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma Metastasis Squamous carcinoma
E+I E+I E+I E+I E+I E E+I E I E E I I I I I I
+ + + + + + +
+ 9 + -
+ + + + + + + + 9
+ ++ * +
+ + +
+ + + +
+ + + + 11 + + +* + +
+ + + + + + + + f
+ 11 + + ++ * +*
+ + + + + + + + + + +*
++* +* + 6
+ +* + + +-
+ + + + +
+ + 8
The physical state of the viral DNA, either episomal (E) or integrated (I), as interpreted from Southern blot analyses, is indicated. The presence of each RNA species was assayed in 2 separate reactions. Positive hybridization signals obtained after a second round of amplification are denoted with an asterix. (-) lack of hybridization signals after 2 separate rounds of PCR and re-amplification.
existence of full-length transcripts from the early gene region in both cell lines. High-molecular-weight bands the size of the uninterrupted genome, which hybridized with the specific target probes, were observed with some of the primer combinations. These could be derived from unspliced pre-mRNA sequences or DNA sequences co-amplified with the RNA-specific products. In summary, from the early gene region we could detect in CaSki and HPKII cells the entire range of transcripts analysed (Fig. 1, Table I) whereas in SiHa cells we could detect those transcripts whose splice sites are positioned within the E G E 7 and El orfs, located upstream of the viral integration site (Baker et al., 1987). In addition to 7 spliced mRNAs from the early gene region, we examined the expression of 2 mRNA species from the late region. With the primer pair E4~-3383/Llas-5700RNA PCR product, the size of 313 bp was generated, which correspond with the expected size for the second splice of the doubly spliced E2M (L1) transcript described (Doorbar et al., 1990). This product was obtained with RNA from HPKII cells but not with RNA from CaSki and SiHa (Fig. 2, E4AL1).Primer pairs ElS-1241/Llas-5700 or E7s-818/Llas-5700 repeatedly failed to produce bands of 124 bp and 548 bf respectively, which could be expected if an L1 mRNA ( E l L1) spliced between nucleotides 1301/5637 was present in these cell lines. Both primer combinations also failed to produce RNA PCR products sized 399 or 823 bp respectively, which would be expected if the first exon of the doubly spliced E2M (Ll) mRNA was linked to the second exon via the splice between nucleotides 1301 and 3357 as described (Doorbar et al., 1990). These results suggest that L1 mRNA species other than those described in W12 cells exist in HPKII cells.
2. Detection and establishment of the physical state of HPV I 6 DNA in tumor specimens To establish the correlation between HPV 16 gene transcription and the state of the viral DNA, both were analysed in the same tissue specimens. The presence of HPV 16 DNA in genital specimens, and the physical state of the genomes, were determined by Southern blot analysis. The hybridization pattern of undigested tumor DNA was compared with DNA that has been digcstcd with a "no-cut'' restriction endonuelease (Hind111 or BglII), with a "single-
cut" enzyme (BamHI), and a multi-cut enzyme (PstI or EcoRI). Integration of viral DNA was suggested by the appearance of hybridization signals which co-migrated with the high-molecular-weight DNA in the uncut DNA, an alteration in the position of the high-molecular-weight DNA signals after digestion with a "no-cut'' enzyme, and observation of "off-size'' bands after digestion with the appropriate restriction enzymes. Figure 3 shows hybridization profiles of DNAs isolated from 5 representative tumor specimens. Analyses of biopsies obtained from cervical carcinomas and one metastasis revealed the presence of integrated DNA, without episomal forms (eg. specimen Ts-2). The hybridization profiles of DNAs from a primary cervical carcinoma (specimen S-12) and lung metastasis (S-13), both from the same patient, were identical, indicating the common origin of these tumors. Most of the specimens diagnosed as CINs showed the presence of episomal forms (e.g. specimen B-84). In some specimens (e.g. specimen B-32), the hybridization signals appeared in the positions of the episomal forms, but in addition within the high-molecular-weight DNA. Treatment with BamHI or PstI yielded, in addition to the genomic bands, novel bands. This pattern of hybridization suggests the existence of integrated viral DNA, although the presence of rearranged multimers and deleted molecules of HPV DNA cannot be excluded. In one CIN specimen (B-91) there was no evidence for episomal HPV DNA. In summary, 95 biopsies from cervical diseases, including flat and exophytic condylomas (39 specimens), CINs stages 1-3 (44 specimens), carcinomas (1 1 specimens) and one metastasis, were screened for the presence of HPV 16 DNA. Seventeen biopsies were positive (Table 11). In 4 CINs, only episomal forms were detected. Six of 11 high-grade pre-cancerous lesions of the cervix (CIN 2-3) contained episomal and possibly also integrated forms of HPV DNA (Table 11). One CIN3 biopsy appears to contain only integrated DNA. In 5 carcinomas and 1 metastasis, only integrated viral DNA was observed.
3. R N A PCR analysis of HPV 16 transcripts in biopv specimens from CINs and carcinomas Tumor biopsies examined for HPV 16 DNA were analysed for the transcription phenotype. The presence of 7 early
TRANSCRIPTION OF HPV 16 IN TUMORS AND CELL LINES
FIGURE3 - Hybridization analysis of DNA from cervical specimens. High-molecular-weightDNAs obtained from intra-epithelial neoplasms (CINs) specimens B32 and B84, adenocarcinoma (CAC) specimen TS2, squamous-cell carcinoma (CSC) specimen S12, and lung metastasis specimen S13 (M), either undigested (U), or after digestion with BamHI (B), HindIII (H), EcoRI (R), PstI (P), were electrophoresed on 0.8% agarose els Also applied to the gels were 1 ng of cloned HPV 6 and HPV 16 DNA digested with BamHl (6, 16) and DNA size markers (M). T i e DNA fragments were blotted onto nitrocellulose membrane and probed with 3ZP-labelledHPV 16 DNA. Arrowheads (left) indicate the positions of BamHI and PstI fragments of the prototype HPV 16 DNA (7.9 and 2.8,1.8,1.5, 1.1,0.5 kb). The migration of supercoiled (I) and open circular (11) viral DNA is also indicated. Bars (right) show the positions of DNA fragments of lambda phage digested with HindIII and phk174 DNA digested with Hae 111. PstI digests of DNAs from specimens S12 and S13 were blotted on a separate filter. transcripts and 2 late transcripts was determined for each biopsy by RNA PCR. When RNA PCR products could not be detected by Southern blot hybridization, another 30 cycles of amplification were performed with 2% of the first PCR mix. Samples were considered negative for the expression of the respective mRNA, after 2 separate RNA PCR assays and reamplifications. Figure 4 shows Southern blot analyses of RNA PCR products obtained from a representative set of tumors. The results of transcript analyses of all tumor biopsies are summarized in Table 11. HPV transcripts were found in all the tumor biopsies. From the E6-E7 gene region we could detect the 3 RNA PCR products (Fig. 2, E6"E6). These were produced with RNA from most of the biopsies, but not all. RNAs from specimens B-32 and B-58 obtained from pre-malignant lesions (CIN 2-3) and RNA from specimen TS-3 obtained from cervical squamous carcinoma, repeatedly failed to produce the expected E61 and E6II RNA PCR products, although products from other mRNA species were generated with the same RNA preparations (Fig. 4). Lack of expression of the E61 and E6II mRNA was not the result of interruption of the E6-E7 gene region, as deduced from Southern analyses of the HPV DNA, and the ability to produce the third PCR product corresponding to the entire E6-E7 region. The pattern of splicing of the E61 and E6II transcripts, judged by the size of the RNA PCR products, did not differ between CINs, carcinomas and the metastasis analysed. The third RNA PCR product of 676 bp which corresponds to the entire E6-E7 region was observed in all the tumors; however, we could not exclude the possibility that this was the result of amplification of DNA present in the RNA samples. The presence of the mRNA discussed above could be conclusively demonstrated in 5 biopsies (specimens B-20, B-90, B-91, B-92,
B-93), using primers which could amplify both splice junctions of the E6-E7 transcripts (Fig. 4, E6"E4). With these primers we did not detect the E6-E7 RNA species in other biopsies, possibly because the E6III cDNA was preferentially amplified over the E6-E7 cDNAs, because of the selective advantage of its shorter length. RNA PCR products, representing the E6"E4 and the El"E4 splice sites, were generated with RNA obtained from all the tumors, including those which lacked expression of the E61 and E6II transcripts (Fig. 4, Table 11). These results indicate that the E6III and the E1/E4 E5 mRNAs are the most common species expressed in cervical lesions and tumors. From the E2 gene region we could obtain RNA PCR products corresponding to the E2C and the entire E2 mRNAs. Detection of the putative E2 mRNA with primers that detect the El"E1 splice (Rohlfs et al., 1991) was of interest. The expected product of 533 bp was produced with RNA from 8 CIN biopsies; however, with RNA from specimens B-20 and V-7 2 bands were repeatedly generated, both hybridizing with the specific target probe (Fig. 4, El"E1). The major band of 533 bp co-migrated with the RNA PCR product obtained with RNA from HPKII cells, and contained the authentic El"E1 splice as determined by nucleotide sequence analysis (data not shown). The second band of about 630 bp could be derived from an as yet unknown mRNA species, which is differently spliced within the El orf. RNA sequences corresponding to the major L1 capsid gene were detected in 6 biopsies, including 4 CINs and 2 invasive carcinomas (Fig. 4, Table 11). As was the case with RNA from HPKII cells, only RNA sequences corresponding to the El"L1 splice could be detected. The primer pair Els-1241/Llas-5700 failed to produce the RNA PCR products which would be
SHERMAN ET AL.
FIGURE 4 - RNA PCR products from tumor specimens revealed by Southern blot hybridization. RNA PCRs for detection of the various mRNA species and Southern analyses of products were carried out as described in "Material and Methods" and the legend to Figure 2. Autoradiograms of RNA PCR products from the indicated specimens are shown. The RNA splice targeted is shown at the top of each lane. Bars (right) show the positions of the 123-bpmarker fragments (BRL). expected if the putative E l / L l or the E2M (Ll) mRNAs (Doorbar et al., 1990) were present at detectable levels in one of the biopsies. The detection of late transcripts in HPKII cells and invasive carcinomas containing integrated genomes of HPV 16 implies that HPV 16 late gene transcription is not completely limited to terminally differentiated cells as previously suggested (Crum et al., 1988), and could occur from integrated genomes provided the respective region was preserved. DISCUSSION
The objective of our study was to determine whether alterations in the transcription and splicing patterns of HPV 16 mRNAs occur during the progression stages of cervical neoplasia and whether these are related to the physical state of the viral genome in the tumor cells. RNA PCR technology with primers selective for specific mRNAs has proved valuable for the detection of a wide range of HPV 16 transcripts in small tumor specimens, as well as in cells. Seven mRNA species from the early gene region and one from the late region were examined. The size of the RNA PCR products was in agreement with the previously mapped splice sites of the respective mRNA species. One difference between the data obtained from our RNA PCR analyses and from those carried out in the W12 cells (Doorbar et al., 1990) regards the structure of transcripts expressed from the L1 region. We could detect mRNA
sequences corresponding to the E4'"Ll splice previously mapped for the second splice site of the doubly spliced E2M (L1) mRNA. Analogous splice sites were mapped for the major L1 mRNA of HPV types 6 and 11 (species j) (Rotenberg et al., 1989) and for species e and j of HPV type 1 (PalermoDilts et al., 1990). However, we consistently failed to detect the presence of transcripts comparable to the L1 and the E2M (Ll) species, whose splice sites have been mapped between nucleotides 1301/5637 and 1301/3357, respectively (Doorbar et al., 1990). Whether alternatively spliced HPV 16 L1 mRNAs exist, different from those revealed in W12 cells, remains to be investigated. An important issue of this study was to determine whether other RNA species are expressed from circular extrachromosoma1 genomes and integrated viral DNA. Transcripts from the E6-E7 orfs, especially the E61, were previously identified in all HPV 16-containing cell lines analysed to date, irrespective of the state of the viral DNA (Smotkin and Wettstein, 1986; Smotkin et al., 1989; Tanaka et al., 1989; Doorbar et al., 1990; Schneider-Maunoury et al., 1990; Rohlfs et al., 1991). Our results show that the E6II1, E2C and L1 (E4AL1)transcripts, previously shown to be expressed from extrachromosomal genomes (Doorbar et al., 1990), were also expressed in the HPKII cell line, containing 10 copies of integrated viral DNA (Durst et al., 1987,1991). R6III and E2C transcripts were also expressed in CaSki cells containing multiple copies of integrated DNA (Baker et al., 1987). Moreover, we could demonstrate, in both cell lines, the presence of transcripts that
TRANSCRIPTION OF HPV 16 IN TUMORS AND CELL LINES
encompass the entire early region and probably terminate the viral poly(A) site, consistent with mapping of mRNAs from the CaSki cells (Smits et al., 1991). Transcript analyses in tumor biopsies also failed to reveal differences in the variety of mRNAs or their splicing pattern between tumors carrying episomal or integrated forms of HPV 16 DNA, thus suggesting that other factors apart from the physical state of the genome must be important in determination of the transcriptional program of the respective HPV 16 mRNAs. Due to the small number of biopsies analysed in this study, accurate evaluation of transcript prevalence is difficult. In some cases, RNA PCR products were detected after a second round of amplification and this could reflect quantitative differences in the levels of specific mRNAs. However, since PCRs were carried out under standard conditions, without the modifications required for quantitative measurements, only qualitative estimation of HPV 16 gene expression was evaluated. It appears, from the overall data of transcript analyses in tumors at different stages of tumor progression and in nonmalignant keratinocytes versus carcinoma cell lines, that transcription of either of the early or late transcripts studied was not specifically related to the malignant stage, and in accordance no specific transcription phenotype could be assigned for pre-invasive vs. invasive tumors. Except for the E2 transcript, the entire set of mRNAs revealed in CIN biopsies (e.g. specimen B-20) could also be found in cervical carcinomas (eg. specimen 78). Although we were able to detect the putative E2 mRNA only in CIN biopsies, expression of this transcript could not be considered specific for the pre-invasive stage, since transcripts containing the El'El splice were also detected in CaSki and SiHa cells derived from cervical carcinomas. Products from the E2 gene region were implicated in the regulation of HPV gene transcription (Sousa et al., 1990). The E2C and the full-length E2 proteins are believed to act as trans-repressors, keeping the expression of the transforming E6 and E7 genes tightly regulated. We could not establish a transcriptional correlation between these gene classes. The E2 and the E2C mRNAs were detected in HPKII and CaSki cells and in 4 CIN biopsies, together with the 4 E6-E7 transcripts. Expression of the E2C mRNA alone was observed in 3 carcinomas, Ts-3, Ts-2, S-12, the last 2 of which also expressed the E61, the E6II and the E6III transcripts. It is possible that quantitative differences in the levels of the E6-E7 and the E2 transcripts exist, which could not be defined in our RNA PCR assays. A second possibility is that other E2
transcripts are involved in the regulatory functions. The existence of transcripts which are initiated by an intragenic promoter upstream of the E2 orf was suggested (Baker et al., 1987; Shirasawa et al., 1988) although these have not been recovered. The products of the E6 and E7 genes are believed to have a major role in HPV-induced neoplasia, and to be essential for both establishment and maintenance of the malignant cell phenotype (zur Hausen, 1989). We showed the presence of these transcripts in most of the tumors analysed, including CINs and carcinomas; however, some biopsies lacked the E61 and the E6II products. In these tumors we could detect the presence of the E6III species. The E6III mRNA, revealed as a unique species in the W12 cells (Doorbar et al., 1990), appears from our analyses to be commonly expressed in tumors and cell lines containing HPV 16. The exact structure of the putative E6III protein is not known, nor is its function. The observation that the E6III mRNA is consistently expressed implies an important role in tumor induction. Further studies are required to define its transforming activity. No alterations in the splicing pattern of the E6-E7 and E6III transcripts were observed between non-malignant and malignant cell lines or between pre-malignant and malignant tumors and the metastasis analysed. The splicing pattern of other mRNA species did not differ either, thus confirming and extending previous studies which have demonstrated the uniformity of splicing of the E6-E7 transcripts in premalignant and malignant carcinomas (Cornelissen et al., 1990; Johnson et al., 1990). The overall data of transcript analyses suggest that changes in the variety of transcripts, or their splicing pattern, are not involved in tumor progression. If other changes in HPV transcription play a role in tumor development, these might involve quantitative changes in the levels of the transcripts expressed, or regulation of initiation and termination of transcription. Further studies are required to clarify these points.
We thank Dr. De Villiers (German Cancer Research Center, Heidelberg) for providing the cloned HPV DNA, and Dr. G. Ben-Baruch (Gynecology Department, Sheba Hospital, Israel) for providing some carcinoma biopsies and for histological examinations. This research was supported by the Israel Cancer Association and the Chief Scientist Office of the Ministry of Health, Government of Israel.
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