Purpose: The purpose of our study was to characterize a human papillomavirus (HPV) 16 DNA integration in the genome of a rapidly progressive, lethal cervical cancer in a 39-year-old woman.

Experimental Design: An HPV 16 integration site from cervical cancer tissue was cloned and analyzed using Southern blot hybridization, nucleotide sequencing, fluorescence in situ hybridization analysis for chromosomal localization and comparison with the draft human genome sequence.

Results: HPV 16 DNA (3826 bp) was integrated into the genome of the tumor sample and contained an intact upstream regulatory region and E6 and E7 open reading frames. Both 5′ and 3′ viral-cell junction regions contained direct repeat and palindrome sequences. The chromosomal location of the viral integration and cellular deletion was mapped to chromosome 14q32.3 using both a somatic cell hybrid panel and fluorescence in situ hybridization. Search of the draft human genome sequence confirmed the chromosomal location and revealed a disruption of the TNFAIP2 cytokine/retinoic acid-inducible gene.

Conclusions: On the basis of the lack of sequence homology between the viral and cellular site of integration and the structure of the viral-cell junctions, it seems that HPV 16 DNA integrates into the host genome by a mechanism of nonhomologous recombination. We suggest that, taken together, maintenance of E6 and E7 expression, loss of the E2 gene and disruption of the TNFAIP2 gene through viral integration contributed to the rapid progression of cervical cancer in this patient. Availability of the human genome sequence will facilitate identification of cellular genes involved in cervical cancer by high-throughput analysis of viral integration sites.

Cervical cancer is the third most common cancer in women, accounting for ∼10% of all new cancers worldwide (1). Integration of HPV2 DNA into the host genome represents an important event in cervical carcinogenesis. This event increases the transcript stability and protein expression of the viral E6 and E7 genes (2). HPV integration has been found to occur at many sites in the human genome, with a greater frequency in or near fragile sites, oncogenes, or translocation breakpoints (3). Characterization of these integration sites, to date, have not identified a highly prevalent chromosome or HPV type-specific integration site or region. This report describes the cloning and analysis of an HPV 16 DNA sequence integrated into, and disrupting, the TNFAIP2 gene of a cervical cancer tumor from a patient who died of a rapid-onset cervical cancer.

Tumor tissue was obtained from a 39-year-old female who presented with pelvic pain and vaginal bleeding. Her past medical history and HIV status were unremarkable. A Pap smear taken 12 years before this visit was normal. Cervical biopsies and clinical evaluation revealed Stage IB invasive squamous cell carcinoma. A radical hysterectomy and bilateral radical pelvic lymphadenectomy were performed. Histological analysis revealed a deeply infiltrating, well-differentiated squamous cell carcinoma with involvement of the lower uterine segment. Although the surgical margins were free of tumor, there was microscopic tumor in four of nine iliac lymph nodes. The patient declined adjuvant radiation therapy. She returned 18 months later with widespread metastatic cancer.

High-molecular-weight DNA was isolated from the patient’s tumor and 2normal endometrial tissue and analyzed by restriction enzyme digestion and Southern blot hybridization as described previously (4). A partial genomic library was constructed by ligating BglII-digested DNA into the BamHI site of EMBL3 as described (4). Recombinants containing the HPV 16 integration region were identified by filter hybridization using radiolabeled HPV 16 DNA; plaque-purified and recombinant-phage DNA was isolated from large-scale liquid lysate. A partial restriction map of the integration region was constructed from single- and multiple-restriction digestions. The genomic fragment, containing the integrated HPV 16 and both viral-cell junction regions, was subcloned into pUC19, and the DNA sequence of the junction regions was determined by dideoxy-chain-termination method (5). To identify the specific chromosome containing the integrated HPV 16 DNA, a 3.3-kb 5′ single-copy BglII/BamHI fragment flanking the integrated viral sequences was used to screen a panel of 43 rodent × human somatic cell hybrids by Southern blot hybridization (6). These hybrids retained the entire rodent genome but segregated human chromosomes in different combinations. Presence of the single-copy DNA fragment was correlated with the concordance of human chromosomes to establish its location (7).

Chromosomes from lymphocytes for in situ hybridization were obtained from methotrexate-synchronized cultures after incubation for 30 min with 0.1 μg/ml colcemid. The flanking single-copy cellular sequence was nick translated with biotin-11 dUTP (Enzo Biochemical, Farmingdale, NY) and hybridized to the slides as described previously (8). For the detection of the hybridization signal, 60 μl of fluorescent-avidin (Oncor, Rockville, Maryland) were applied to each slide. The intensity of biotin-linked fluorescence was amplified by adding 60 μl of biotinylated goat antiavidin antibody followed by a second layer of fluorescein-avidin (9, 10). After amplification, the slides were stained for 90 s with propidium iodine (2 μg/ml) and examined with an Olympus BH-2 microscope equipped with epifluorescence filters. One hundred metaphases with distinct hybridization signals were examined. In addition to identifying the single hybridized chromosomes, 10 duplicate photomicrographs of hybridized and G-banded chromosomes were analyzed.

Structure and Organization of HPV 16 in the Cervical Cancer Tumor Tissue.

The gross structure and organization of HPV 16 DNA were determined by digesting tumor DNA with restriction enzymes that either cleave the viral genome once or multiple times (e.g., EcoRI and PstI, respectively) or do not cleave (e.g., HindIII and BglII) HPV 16. The blot was probed with linearized HPV 16 DNA radiolabeled to high specific activity (data not shown). Digestion with EcoRI and BglII revealed fragments larger than anticipated for episomal HPV. Digestion of an intact HPV 16 genome with PstI should yield fragments 2.8, 1.8, 1.5, and 1.1 kb in size. However, after digestion with PstI, only the 2.8 and 1.8 kb fragments were detected indicating the presence of a partial HPV 16 genome. Taken together, these data indicated that a portion of the HPV 16 genome was integrated into the tumor DNA. To further characterize this region, the 12.7-kb BglII fragment containing the entire integrated HPV 16 genome with both viral-cell junctions was cloned. Analysis of the cloned integrated viral genome by restriction analyses and hybridization with the HPV 16 probe indicated that ∼3800 bp of HPV 16 sequence was present. The organization of the region containing the integrated viral genome based on a partial restriction map of the HPV 16 integration site is shown in Fig. 1.

To characterize the viral-cell junctions, DNA sequence determination was performed. The DNA sequence of the junction regions is shown in Fig. 1. Both direct repeats and palindrome sequences were present at both junctions. In addition, at both viral-cell junctions there was an addition of a short novel sequence corresponding to a duplication of 11 bp of viral sequence at the 5′ junction and 5 bp at the 3′ end, which created short direct repeats (see Fig. 1).

Chromosomal Location of the HPV Integration and Cellular Deletion.

Restriction endonuclease mapping of the cellular region containing the integrated HPV 16 DNA revealed a deletion of cellular sequences at the region of recombination. To identify the specific chromosome bearing the integrated HPV 16 DNA and deletion, a 5′ single-copy sequence was used to probe a panel of 43 rodent × human somatic cell hybrids. Hybrid cell DNA was digested with EcoRV and blotted with the radiolabeled single-copy genomic flanking probe (data not shown). Human DNA and those hybrids containing the appropriate chromosome had a 16-kb hybridizing fragment, whereas no signal was seen in rodent DNA (data not shown). The presence of the 16-kb hybridizing fragment was 97% concordant with human chromosome 14 (data not shown). On the basis of the high degree of discordance with the remaining human chromosomes, the HPV 16 DNA integration was assigned to human chromosome 14.

To identify the chromosomal sublocalization of the viral integration, in situ hybridization of chromosomes deriving from peripheral leukocytes with a biotinylated probe of cellular sequences flanking the HPV integration resulted in a specific fluorescence signal at the distal end of the long arm of an acrocentric chromosome consistent with chromosome 14. This site was labeled in >90% of the metaphases, and no other chromosome exhibited fluorescence spots on sister chromatids. This demonstrated homology of the cellular flanking probe to a single genomic site. After examination of hybridized and G-banded chromosomes, the labeled site was localized on chromosome 14 at band q32.3 (see Fig. 2).

In Silico Analysis of the Viral Integration Site.

Subsequent to the above experiments, a draft of the human genome sequence was published (11). The cellular sequences from each junction region were searched using the BLAST program against the available human genome sequence. Both 5′ and 3′ cellular junction sequences were identified within a single contig (gi:13647973) corresponding to chromosomal band region 14q32. Further analysis using the NCBI human genome software3 allowed us to identify the exact nucleotides corresponding to the human sequence at the viral-cell junctions. The 3′ end of the HPV 16 partial genome was integrated into the second intron of the TNFAIP2 gene (12), whereas the 5′ end of the viral sequence was contiguous with DNA 13,422 bp upstream of this site. This deleted region included the TNFAIP2 promoter, translation initiation start site and first 2 exons. Using the “map view” component of the NCBI software revealed that this was a gene-rich region including the CDC42-binding protein kinase β, eukaryotic translation initiation factor 5 and TNF receptor-associated factor-3 genes.

To determine whether other HPV integration sites occur in this region, a review of the literature was performed and a table listing all reported integration sites was assembled (see Table 1). A cell line transformed by HPV 16 was found to have an integration at 14q32. In addition, there are multiple HPV integrations localized to chromosomal bands that contain myc genes (e.g., 2p24, N-myc, and 8q24, c-myc). However, there are only two other reports of HPV DNA integrating into characterized genes (13, 14).

We have cloned and analyzed an HPV 16 DNA integrated into the genome of a cervical cancer specimen from a patient who died at 41 years of age from metastatic disease. The chromosomal location of the viral integration and cellular deletion were mapped to chromosome 14q32.3 using a somatic cell hybrid panel, fluorescence in situ hybridization and in silico analysis. Viral integration caused the deletion of 13,422 bp including the promoter and first two exons of the TNFAIP2 gene. This gene has been found to be inducible by TNF-α and RA and is a possible target implicated in promyelocytic anemia (12, 15).

On the basis of the lack of sequence homology between the viral and cellular site of integration and the structure of the viral-cell junctions, it seems that this HPV 16 DNA integrated into the host genome by a mechanism of nonhomologous recombination. Interestingly, sequences at the junctions of nonhomologous recombined fragments have been identified with short repeats as described in this HPV 16 integration (see Fig. 1; Ref. 16). Such features may represent a general dysfunction of DNA repair leading to the accumulation of additional genetic abnormalities and rapid progression. Both the 5′ and 3′ viral-cell junction regions contain direct-repeat and palindromic sequences at AT-rich regions. It has recently been suggested that double-strand breaks may occur in the center of a palindrome, leading to illegitimate recombination events between similar AT-rich sequences (17). Breakpoints in palindrome AT-rich repeats have been reported in other chromosomal translocations (18).

HPV integration in chromosome 14q32 has been reported in an HPV 16 immortalized keratinocyte line (19). This observation supports the notion that viral integration in this region may be a contributing factor to the selective growth advantage of HPV-containing cells (19). Other HPV integrations have been associated with deletion of cellular sequences and potential structural or functional proto-oncogene alterations (20, 21, 22). Chromosomal alterations associated with viral integrations include deletions, duplications, and translocations that can lead to loss of heterozygosity or to alterations of growth regulatory genes (19). Chromosome-14 band 32 has been implicated in many translocations associated with T- and B-cell lymphomas. In addition, the location of a potential proto-oncogene, TCLIA, which is associated with one type of T-cell lymphoma, is at 14q32.3 (23).

Integration of HPV 16 into the RA-inducible TNFAIP2 gene is interesting but of unknown significance. Retinoids play an important role in cellular differentiation, proliferation, and growth of cervical epithelium (24, 25). The mechanism of action of retinoids is not well understood. Molecular analysis has indicated that the RA-dependent growth suppression of cervical cancer cells could be mediated by inhibiting E6 and E7 (26). Whereas, other studies suggest that RA-dependent changes in HPV-immortalized cell proliferation are not correlated with E6 and E7 transcription products (27). In HeLa cells, the combination of RA with IFN-γ leads to an additive antiproliferative effect on cell growth (28). There are also different degrees of radiosensitization by RA in cervical cancer cell lines (29). Clinical responses to RA are minimal, despite activity in vitro(30, 31). The disparity between cell line and clinical response to RA may be attributable to up-regulation or down-regulation of the TNFAIF2 gene. However, functional data, such as reintroduction of the wild-type gene into cancer cells, will be required to evaluate the importance of TNFAIP2 disruption in cervical cancer. Nevertheless, in addition to chromosomal location 2p24 and 8q24, 14q32 should be considered an HPV-integration hot spot. Whether this region contains genes involved in the development of cervical cancer, such as c-myc, or fragile sites more susceptible to DNA recombination remains to be determined.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

                
2

The abbreviations used are: HPV, human papillomavirus; NCBI, National Center for Biotechnology Information; TNF, tumor necrosis factor; RA, retinoic acid.

        
3

Internet address: http://www.ncbi.nlm.nih.gov/genome/guide/human.

Fig. 1.

Integration of HPV 16 DNA sequences into the TNFAIP2 gene. Top, a circular diagram of an HPV 16 genome integrating into and deleting the first and second exons of the TNFAIP2 gene. Bottom, the fine structure of the integrated HPV 16 viral genome and the viral-cell junction sequences. Capital letters, HPV 16 sequences; lower-case letters, cellular DNA; boxed sequences, duplicated viral sequences; small arrows, direct repeat sequences.

Fig. 1.

Integration of HPV 16 DNA sequences into the TNFAIP2 gene. Top, a circular diagram of an HPV 16 genome integrating into and deleting the first and second exons of the TNFAIP2 gene. Bottom, the fine structure of the integrated HPV 16 viral genome and the viral-cell junction sequences. Capital letters, HPV 16 sequences; lower-case letters, cellular DNA; boxed sequences, duplicated viral sequences; small arrows, direct repeat sequences.

Close modal
Fig. 2.

Subchromosomal localization of HPV integration by fluorescence in situ hybridization. a, human metaphase from normal peripheral leukocyte cultures after in situ hybridization with a biotinylated probe exhibiting fluorescent label at the terminus of the long arm of an acrocentric chromosome. In b, after G-banding, the labeled chromosome is identifiable as chromosome 14 and the labeled site is assigned at band q32.3.

Fig. 2.

Subchromosomal localization of HPV integration by fluorescence in situ hybridization. a, human metaphase from normal peripheral leukocyte cultures after in situ hybridization with a biotinylated probe exhibiting fluorescent label at the terminus of the long arm of an acrocentric chromosome. In b, after G-banding, the labeled chromosome is identifiable as chromosome 14 and the labeled site is assigned at band q32.3.

Close modal
Table 1

Chromosomal regions containing integrated HPV sequences

Tumor or cell lineHistological typeViral typeChromosomal locationReferences
V18 Sqa HPV 16  (32)  
FEH18L Cl HPV 18 1p22-p31  (33)  
FEA Cl HPV 18 1q12-q21  (33)  
CC-10 Gc HPV 45 1q21-23  (34)  
CC192 Sq HPV 16 1q21  (14)  
Cervix tumor grown in vitro Sq HPV 18 2p24  (22)  
M15 Sq HPV 16 2q34-35  (32)  
H705 Sq HPV 16 3p14  (35, 36)  
Cervix CA cell line Arg HPV 18 3p21-22  (37)  
CC-8 As HPV 45 3q26-29  (34)  
CC-11 As HPV 67 t(3;13)(p23-26;q22-31)  (34)  
Hela (cervix CA cell line) Aden HPV 18 5p11-15 abn.  (8, 38, 39)  
CC171 Sq HPV 16 6p22  (14)  
CC5a Sq HPV 16  (32)  
C4-1 (metastatic cervix cancer cell line) Sq HPV 18 8q22  (40)  
Cervix tissue grown in vitro Aden HPV 18 8q24  (22)  
Cervix tissue grown in vitro Aden HPV 16 8q24  (22)  
Penile tissue grown in vitro Sq HPV 16 8q24  (22)  
YIK-1 (cervix CA cell line) Arg HPV 16 8q24  (37)  
Hela (cervix CA cell line) Aden HPV 18 8q24  (8, 21, 38, 39)  
V15 Sq HPV 16  (32)  
CK 11 and CK 12 Cl HPV 33 9p13  (8, 21, 38, 39)  
Hela Aden HPV 18 9q31-q34  (38, 39)  
Tonsillar CA Sq HPV 6a 10q24  (41)  
SK-v (vulvar CA cell line) Sq HPV 16 12q13  (42)  
   12q14-15  (43)  
SW 756 (cervix CA cell line) Sq HPV 18 12q13  (44)  
   12q14-15  (44, 45)  
TC146 Cl HPV 16 13q14  (46)  
SiHa (cervix cell line) Sq HPV 16 13q21-31  (4, 8)  
CK 1 to CK 10 Cl HPV 33 13q33-34  (47)  
CSCC-7 Sq HPV 16 t(3;14)(p14.1-14.3;14)  (34)  
CSCC-1 Sq HPV 16 14q  (34)  
HKcHPV16d-2 Cl HPV 16 14q32  (19)  
Cervix CA tissue Sq HPV 16 14q32.3 This study 
CC226 Sq HPV 16 17q23  (14)  
ME180 Cl HPV 68 18q21  (48)  
Hela Aden HPV 18 22q12-q13  (8, 38, 39)  
CC-10 Gc HPV 45 22q13  (34)  
Tumor or cell lineHistological typeViral typeChromosomal locationReferences
V18 Sqa HPV 16  (32)  
FEH18L Cl HPV 18 1p22-p31  (33)  
FEA Cl HPV 18 1q12-q21  (33)  
CC-10 Gc HPV 45 1q21-23  (34)  
CC192 Sq HPV 16 1q21  (14)  
Cervix tumor grown in vitro Sq HPV 18 2p24  (22)  
M15 Sq HPV 16 2q34-35  (32)  
H705 Sq HPV 16 3p14  (35, 36)  
Cervix CA cell line Arg HPV 18 3p21-22  (37)  
CC-8 As HPV 45 3q26-29  (34)  
CC-11 As HPV 67 t(3;13)(p23-26;q22-31)  (34)  
Hela (cervix CA cell line) Aden HPV 18 5p11-15 abn.  (8, 38, 39)  
CC171 Sq HPV 16 6p22  (14)  
CC5a Sq HPV 16  (32)  
C4-1 (metastatic cervix cancer cell line) Sq HPV 18 8q22  (40)  
Cervix tissue grown in vitro Aden HPV 18 8q24  (22)  
Cervix tissue grown in vitro Aden HPV 16 8q24  (22)  
Penile tissue grown in vitro Sq HPV 16 8q24  (22)  
YIK-1 (cervix CA cell line) Arg HPV 16 8q24  (37)  
Hela (cervix CA cell line) Aden HPV 18 8q24  (8, 21, 38, 39)  
V15 Sq HPV 16  (32)  
CK 11 and CK 12 Cl HPV 33 9p13  (8, 21, 38, 39)  
Hela Aden HPV 18 9q31-q34  (38, 39)  
Tonsillar CA Sq HPV 6a 10q24  (41)  
SK-v (vulvar CA cell line) Sq HPV 16 12q13  (42)  
   12q14-15  (43)  
SW 756 (cervix CA cell line) Sq HPV 18 12q13  (44)  
   12q14-15  (44, 45)  
TC146 Cl HPV 16 13q14  (46)  
SiHa (cervix cell line) Sq HPV 16 13q21-31  (4, 8)  
CK 1 to CK 10 Cl HPV 33 13q33-34  (47)  
CSCC-7 Sq HPV 16 t(3;14)(p14.1-14.3;14)  (34)  
CSCC-1 Sq HPV 16 14q  (34)  
HKcHPV16d-2 Cl HPV 16 14q32  (19)  
Cervix CA tissue Sq HPV 16 14q32.3 This study 
CC226 Sq HPV 16 17q23  (14)  
ME180 Cl HPV 68 18q21  (48)  
Hela Aden HPV 18 22q12-q13  (8, 38, 39)  
CC-10 Gc HPV 45 22q13  (34)  
a

Sq, squamous cell; Cl, transformed keritinocyte cell line; Gc, glassy cell; Arg, argyrophil small cell; As, adenosquamous; Aden, adenocarcinoma; CA, carcinoma.

We thank Steve O’Brien, National Cancer Institute (Frederick, MD) and his laboratory for assistance with the chromosomal localization using a rodent-human somatic cell hybrid panel.

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