Originul Contributions Human Papillomavirus Type 16 and 18 Gene Expression in Cervical Neoplasias MARK H. STOLER, MD, CHERYL R. RHODES, BS, APRIL WHITBECK, MS, STEVEN M. WOLINSKY, MD, LOUISE T. CHOW, PHD, AND THOMAS R. BROKER, PHD Human papillomavirus


cated in the generation

of progressive

ruses produce


cervical neoplasms. The vi-

families of overlapping

that are linked to differentiation,

HPV type 16 and type 18 subgenomic specific to individual



making it necessary to analyze

gene expresslion in the context of morphology. riboprobes

(condylomata acuminata) and are only rarely associated with high-grade squamous intraepithelial lesions (SILs) or invasive squamous cancers. In contrast, HPV-16 is the most prevalent virus to infect the uterine cervix and is closely associated with the entire range of intraepithelial and invasive squamous neoplasia and sometimes with glandular neoplasia.’ While accounting for fewer cervical infections, HPV-18 is most consistently associated with aclenocarcinomas and small cell neuroendocrine cancers of the cervix and less frequently with invasive squamous cancer.“-’ Despite the inability to propagate HPVs productively in cultured cells, molecular analysis of recombinant HPV DNAs in vitro has yielded a wealth of information on their genomic organization, protein functions, and transcriptional regulation (Fig 1). Several lines of evidence have shed light 011 the probable mechanisms of HPV-induced neoplasms. Active transcription and translation of the E6-E7 region of high-risk virus type HPV-16 or HPV-18 have been demonstrated in cervical cancers and in cell lines derived from cervical cancers, such as HeLa, SiHa. and CaSki.“.xe’”The EtiE7 regions of the high-risk viruses, but not those of the low-risk types alone, are able to immortalize primary human keratinocytes’7-“5 and to transform primary baby rat kidney cells in collaboration with an activated HaR4s or fos gene.9”-‘7 This multifactorial requirement is consistent with experimental carcinogenesis.?s Persistent E6 and E7 expression is essential to maintain the transformed phenotype. 2q-31Mechanistic support for the roles of the E6 and E7 proteins of high-risk viruses in cervical neoplasms comes from recent evidence that they form complexes with the proteins encoded by the host tumor suppressor genes, r 53 and retinoblastoma susceptibility (RB), respectIvely.-2-34 Bmdmg of E6 protein facilitates the degradation of ~53.“’ Presumably, binding of the viral oncoproteins disrupts normal p53 ;and pRB controls of the cell cycle and generates a phenocopy of p53 and RB mutations (see review, ref 36). Thus, regulation of E6 and E7 expression is anticipated to be central to viral carcinogenesis. Unique to the mucosotrophic HPVs, the E6 promoter can be repressed in transient transfection assays following binding of proteins encoded by the homologous viral E2 open reading frame (ORF) to DNA sequences, ACCNGGGT, adjacent to the E6 promoter TATA motif.“‘-‘” Viral DNA is often found integrated in cancers and in cervical carcinoma cell lines, sometimes accompanied by deletion of the E2 ORF and

types 16 and 18 are strongly impli-

We have developed

clones from which ‘H-labeled


families can be generated

in vitro. Using these probes for in situ hybridization,

we examined

serial sections of archival biopsy specimens of the spectrum of genital lesions. In low-grade frames



squamous lesions, all viral open reading and

the most



spanned the lE4 and E5 open reading frames at the 3’ end of the E region. L region transcription

coding for the capsid proteins was

restricted to terminally differentiated

keratinocytes. As the grade

of neoplasia






and, with few exceptions,

and overall

transcripts ceased to exist. The E6-E7 transforming variably derepressed.



the coexistence

viral DNAs.


IIn contrast, in HPV-18

region was in-

the patterns of HPV-16 of episomal


the L2 and Ll gene

and integrated

lesions, all the viral template

DNA appeared to have integrated. Integration was deduced to have occurred near the boundary of the El and E2 open reading frames. Viral transcription

patterns were similar in carcinomas in situ and

in invasive c;arcinomas, regardless

of the histologic

cell types or

the associated virus types, consistent with the notion that additional host gene alterations were necessary for progression.

On the basis

of viral gene expression

in vivo and the E6 promoter


in vitro, we discuss a molecular


nism for HP’V-associated 128. Copyright





0 1992 by W.B. Saunders Company

The human papillomaviruses (HPVs) induce diverse manifestations of epithelial neoplasia in vertebrates. Over 70 types of HPV have been molecularly cloned, some two dlozen of which are trophic for the anogenital tract.’ Approximately two thirds of the HPV-associated anogenital neoplasms involve HPV types 6, 11, 16, and 18. Types 6 and 11 cause benign exophytic genital warts Front the Department of Pathology, The Cleveland Clinic Foundation, Cleveland, OH; and the Departments of Pathology, Medicine/ Ini’ectious Diseases Unit. and Biochemistry, and The Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester. NI’. Accepted for publication October 3, 1991. Supported by US Public Health Service grants no. CA43629 to M.H.S. and CA36200 to l~.‘I‘.C KP~ wwdrr RNA expression. in situ hybridization, epithelial differentiation. >viral pathogenesis. cervical cancers. Address t:cwrespondence and reprint requests to Mark H. Staler. Ml), Department of Pathology--125, The Cleveland Clinic Fowldarion. 9500 Euclid Ave, Cleveland, OH 44195. Copyright 0 1992 by W.B. Saunders Company 0046.81’i7/(12~302-0006$5,00/O 117


Volume 23, No. 2 (February


viruses have been revealed in serial sections of natural or ex erimental condylomata using in situ hybridizaF The results demonstrate tion.“,,“’ that high levels of DNA replication and mRNA transcription depend on squamous cell differentiation and that the relative abundances of individual messages can vary over 1 ,OOOfold in the same cells. This report describes studies of a spectrum of cervical neoplasms and contrasts the viral gene expression patterns of different HPV types and histopathologies. MATERIALS


Probe Strategy and Construction of Message-Specific Probes FIGURE 1. Human papillomavirus type 16 DNA sequence organization, mRNAs, and protein coding potentials. The viral genome of 7,906 base pairs is a revision of the published sequence.83 Open reading frames in the three possible translation phases are represented by open boxes, and the first AUG protein initiation codon in each is marked with a vertical dashed line. Bent arrows denote the confirmed positions of the RNA splice donor and acceptor sites. The mRNAs or splices determined (species d, e, f, g, h, i, j, and l)50,56,51or inferred (species a, b. c. k, and m) by a highly conserved sequence homology to the HPV-I 1 messages48~5’~52are displayed as continuous or interrupted arrows; beads symbolize the transcription initiation sites, the arrowheads mark the 3’cleavage and poly(A) addition sites, and gaps in the arrows represent introns removed during RNA splicing. Only one of three predicted upstream exons of the E2 mRNA is shown. Additional possibilities are as in species g or i. Messenger RNA exon-specific subgenomic clones are depicted with shaded boxes, and their precise restriction site boundaries are listed in Table I. The proteins encoded are predicted on the basis of mRNA sequences. All E region transcripts can conceptually encode E5 protein. The promoter for the L2 mRNA is not certain.

Cloned HPV-16 and HPV- 18 DNAs were gifts of Drs 1.. Gissmann and H. zur Hausen. Whole genomic clones in pGEM vectors have been described previously.55 On the basis of DNA sequence homology and the mRNA structures of HPV types 6 and 11, a number of spliced HPV-16 and HPV-18 mRNA species were anticipated and subsequently confirmed by cDNA On the basis of known or predicted mRNA analysis. 47.5”.5’~5’.57 structures, nonoverlapping restriction fragments from HPV16 and HPV-18 unique to messages encoding the E6 plus Eli. the El, the E2, the E4 plus E5, rhe L’L. and the I,1 proteins were separately subcloned into pCEM1, pGEM? (Promega. Madison, WI), or pBRLl9 (Life Technologies, Gaitlersburg, MD), vectors for in vitro transcription (Fig 1 and Table 1). Each fragment and its orientation in the vector was confirmed by diagnostic restriction digestions. The probe for the putative Eli”E4 and E5 messages detects all RNAs polyadenylated at the E region poly(A) site. Their relative abundance can be inferred from differences between the cytoplasmic signals generated by the E4-E5 probe and those from the IS-E7 and E? probes combined.

Clinical Material and In Situ Hybridization of Serial Sections


sequences.“.“-” In sum, these observations suggest that inopportune overexpression of the E6 and E7 viral-transforming proteins following disruption of synthesis of the E2 repressor proteins plays a critical role in the initiation and maintenance of HPV carcinogenesis (for reviews, see refs 46 and 47). To corroborate these hypotheses, it is necessary to examine viral gene expression in detail in the full range of lesions associated with papillomaviruses. The more common and accessible HPV-6and HPV-1 l-associated condylomata have provided materials for detailed studies of the viral RNA structures.4”-52 The spatial and temporal patterns of genetic activities of these low-risk

TABLE ORFs Whole EG-E7 El E2 E4-E5 1.2 1.1

Patient biopsies were fixed in neutral bufftred 10% formalin. Following routine processing through ,gTaded ethanols and xylene, tissues were embedded in paraffin, sectioned at 3 to 4 pm, and stained with hematoxylin-eosin for initial diagnosis. Additional unstained serial sections were mounted on 3-aminopropyl triethoxysilane-coated glass microscope slides for in situ hybridization. The final section was stained with hematoxylin-eosin to verify the lesional morphology. In vitro transcription to generate whole genomic or CXOIIin both polarities was carried specific, ‘H-labeled riboprobes out as described previously.“’ Sense-strand probes were in the same polarity as viral mRNAs and hybridized only to the viral

1. Human Papillomavirus HPV-I 6 Restriction


Probes HPV-18


Note. Whole genomic DNA or restriction fragments Methods. Nucleotide positions are given in parentheses.

of HPV-16

and HPV-I8




EcoRI (2441-7857/l-2470) BamHI (lPO)-Him11 (658) FspI (1132)-EcoRI (2440) SfanI (2806)-SfanI (3242) HincII (3606)-HgaI (4275) EcoRV (4670)-HincII (5 144) XbaI (5731)-BamHI (6929)

BamHI (6153-7906/l-6152) SphI (7470)-KpnI (884) KpnI (885)-AvaII (2714) AvaII (2715)-HincIl (3211) Fnu4HI (3415).Fnu4HI (3943) MstII (4338)-KpnI (5383) BamHI (6153)-SphI (7469) were cloned

into vectors

as described

in Materials





DN.4 if the DNA was predenatured by heating in 95% formanlide at 65°C for 15 minutes. Antisense-strand probes were in the opposite orientation and hybridized uniquely to viral transcripts when the DNA in the specimens was not heat denatured. III vitro transcription from the proper promoter was separately carried out for each of the clones in the presence of “H-UTP and “H-CT1 (approximately 30 or 60 Ci/mmol, respectiveI> I, yielding probes with a specific activity of 1.1 X IO” dpm/~g. WC chose to use “H- rather than ‘“S-labeled prt)brs because of the tower energy and shol-ter track path of the, beta particles emitted by the decay of the radioisotope. (:onscquentl~, rhc d

Human papillomavirus type 16 and 18 gene expression in cervical neoplasias.

Human papillomavirus (HPV) types 16 and 18 are strongly implicated in the generation of progressive cervical neoplasms. The viruses produce complex fa...
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