Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines.

A series of human carcinoma cell lines was examined for human papillomavirus (HPV) DNA sequences with the use of HPV-6, HPV-11, HPV-16, and HPV-18 DNA probes. Six of eight cell lines derived from human cervical carcinomas were shown to contain integrated HPV DNA sequences. In five of these six lines, HPV-specific polyadenylated RNA species could also be identified. The expression of HPV sequences was detected in three lines with a HPV-18 DNA probe and in two lines with a HPV-16 DNA probe. Of the two lines which contained HPV-16 specific RNA, one contained HPV DNA sequences which hybridized only to an HPV-16 probe, and the other contained HPV DNA sequences which hybridized to both HPV-16 and HPV-18 DNA probes. Six cell lines established from human squamous-cell carcinomas of the bladder, pharynx, lung, esophagus, and vulva were negative for HPV-6, HPV-11, HPV-16, and HPV-18 DNA sequences under stringent hybridization conditions.     

Human papillomavirus DNA is present in a subset of unselected breast cancers

OBJECTIVE: The major molecular events in the genesis of most breast cancers are unknown. However, human papillomaviruses (HPV) have been reported to be found in a significant portion of breast cancers of women with concomitant cervical intraepithelial neoplasia III. To investigate a potential HPV-breast cancer link, we carried out a small survey to identify HPV in unselected, general breast cancer tissues. STUDY DESIGN/METHODS: Deoxyribonucleic acid (DNA) was isolated from 17 breast cancer tissues (and one cervical swab) taken from our local, randomly selected patient population. Two different previously characterized broad-spectrum primer sets (targeting the E6/E7 or L1 regions) were used to amplify HPV DNA, and another primer set was used to amplify the ColE1/pBR322 origin of replication by polymerase chain reaction amplification. The polymerase chain reaction product DNA was analyzed by dot blot hybridization with HPV-16, -18, -31, or pRB322 DNA probes. Total cellular DNA was also analyzed by one- and two-dimensional Southern blot analysis. Finally, the E6/E7 polymerase chain reaction products were cloned, sequenced, and compared to previously cloned HPV types. RESULTS: Polymerase chain reaction/dot blot analysis by both the HPV E6-E7 and L1 primer sets identified the same 6 out of 17 (35%) breast cancers as being HPV positive. ColE1/pBR322 origin targeted polymerase chain reaction/dot blot analysis failed to identify plasmid contamination. One- and two-dimensional Southern blot analysis showed that the breast cancers specimens contained significant levels of HPV DNA and that the viral DNA was largely episomal. The sequences of the HPV clones demonstrated that HPV-16, -18, and possibly type 11 were present within the breast cancer specimens. Furthermore, the HPV sequences cloned from the cervical swab and breast cancer of the same patient were found to be identical. CONCLUSIONS: These data suggest that HPV may be associated with a significant subset of breast cancers, and further suggest that additional studies are warranted.     

Human papillomavirus-16 and -18 replication in esophagus squamous cancer cell lines does not require sheterologous E1 and E2 proteins

Human papillomaviruses (HPVs) are doublestranded DNA viruses that replicate in the nuclei of squamous epithelial cells. HPVs can be classified into high-risk (e.g., types 16, 18, 31, and 33) or low-risk (e.g., types 6, 11, and 30), depending on their association with benign or malignant tumors. We recently described the association of HPV-16 and -18 with esophagus squamous cell cancer. HPV replication was studied in representative cell lines derived from esophagus cancers. HPV-16 and -18 genomes were independently transiently transfected into HCE-4 and HCE-7 cell lines with and without E1 and E2 genes under heterologous promoters. Southern blot analysis demonstrated that these cell lines support viral replication. However, heterologous E1 and E2 are not required for HPV replication. These findings suggest that specific host nuclear factors in esophageal squamous epithelial cells may support HPV replication. © 1995 Wiley-Liss, Inc.     

In vitro transformation and molecular characterization of Colobus monkey venereal papillomavirus DNA

The DNA of a monkey papillomavirus (CgPV-1), originally isolated from a penile lesion on a Colobus monkey was cloned into the EcoRI site of the pUC18 vector and characterized. Using a variety of restriction enzymes a physical map of the DNA was constructed. Cross-hybridization with a variety of animal and human papillomaviruses under high (Tm-22 degrees C) and low (Tm-40 degrees C) stringency conditions indicated various degrees of homology. CgPV-1 showed higher homology with HPVs than it did with any other animal papillomaviruses tested. DNA similarities with the human papillomaviruses HPV-16 and HPV-18 that are frequently associated with cervical cancer, were manifested by extensive cross-hybridization under stringent conditions. Functional alignment of the genomic map of CgPV-1 with that of HPV-16 was carried out by determination of homology between specific restriction fragments of the two viral genomes in cross-hybridization analyses. This alignment was refined by sequencing two regions of approximately 200 bp of the CgPV-1 DNA, and aligning them by computer with their homologous HPV-16 counterparts. CgPV-1 DNA in its pUC18 vector, transformed NIH 3T3 cells with roughly the same efficiency as BPV-1, as determined by the number of transformed foci generated per ug of DNA. The data presented indicate that the state of the CgPV-1 viral DNA in these transformed cells is integrated and partially deleted, not unlike the genomes of HPV-16 and HPV-18 characterized in cell lines derived from cervical cancers.     

Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.     

Sequence variants of human papillomavirus type 16 in clinical samples permit verification and extension of epidemiological studies and construction of a phylogenetic tree.

Genomic variability between different viral isolates provides a powerful epidemiological tool for verifying ultrasensitive diagnostic procedures, understanding infectious pathways in individuals and human populations, and studying viral evolution. The potential of this approach has not yet been exploited for the diagnosis of human papillomaviruses (HPVs) like HPV type 16 (HPV-16), which are involved in genital cancer. Toward this end, we amplified by polymerase chain reaction, cloned, and sequenced a 364-bp noncoding segment of the HPV-16 genome from cell lines, cervical biopsy specimens, and cervical smears. The HPV-16 genomes in the cell lines SiHa and CaSki showed an identical point mutation, and in the SiHa cell line it had an additional 38-bp deletion. Only 4 of 22 cervical lesions biopsied from patients at several hospitals in Singapore contained HPV-16 DNA with the prototype sequence, while the DNAs of the other 18 cervical lesions differed by 1 to 10 mutations. This excludes contaminations with cloned HPV-16 DNA as the source of this DNA. To test whether this diversity was a geographic idiosyncrasy, we analyzed 25 cervical biopsy specimens from Brazil. Eight of these contained the prototype sequence, while 17 were mutated. Altogether, 11 genomic variants were found in the Singaporean samples and 12 genomic variants were found in the Brazilian samples, and only 5 of these occurred identically in both cohorts. All variants could be connected to form a phylogenetic tree, with some branches being specific for each cohort. This suggests that the variants did not originate over a short period in the individual patient but, rather, evolved consecutively while spreading throughout humankind.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of vector contamination in detection of human papillomavirus DNA using full-length genomic DNA probes.

Nucleic acid hybridization techniques are currently the most specific and sensitive procedures available for diagnosing and typing human papillomavirus (HPV) infection. HPV genomic DNA cloned into vector pBR322 or related vectors are commonly used as probes for detection of HPV. When isolating HPV insert DNA however, it is difficult to remove all pBR322 DNA. This can result in false positive readings when clinical specimens harbouring sequences homologous to pBR322 are screened. It was found that up to 200 ng of vector-like sequences in a clinical sample could be blocked by the addition of non-labelled, digested pBR322 sequences to the hybridization reaction.     

The presence of inhibitory RNA elements in the late 3'-untranslated region is a conserved property of human papillomaviruses

Here we have tested the inhibitory activity of the late untranslated region (UTR) of nine different human papillomavirus (HPV) types representing three different genera and six different species. These HPVs include both low-risk and high-risk types. We found that the late UTR of the various HPVs all displayed inhibitory activity, although they inhibited gene expression to various extent. The late UTR from the two distantly related HPV types 1 and 16, which are two different species that belong to different genera, each interacted with a 55 kDa protein. This protein cross-linked specifically to both HPV-1 and HPV-16 late UTR, although it bound more strongly to HPV-16 than to HPV-1, which correlated with the higher inhibitory activity of the HPV-16 late UTR. Mutagenesis experiments revealed that inactivation of two UGUUUGU motifs in the HPV-16 late UTR or two UAUUUAU motifs in the HPV-1 late UTR resulted in loss of binding of p55. In summary, these results demonstrate that the presence inhibitory elements encoding PuU(3-5)Pu-motifs in the HPV late UTR is a conserved property of different HPV types, species and genera, and suggest that these elements play an important role in the viral life cycle.     

Biochemical transformation of LM(TK-) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene

Recombinant plasmid pAGO codes for herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) and consists of a 2-kbp HSV-1 DNA fragment inserted at the unique PvuII cleavage site of plasmid pBR322. A hybrid plasmid, designated pMH110, has been derived from plasmid pAGO by deleting the 1689-bp pBR322 nucleotide sequence of pAGO, which extends from the BamHI to the PvuII cleavage site, and the 250-bp HSV-1 nucleotide sequence of pAGO, which extends from the PvuII to the BglII cleavage site. Plasmid pMH110 biochemically transformed LM(TK^?)cells to the TK⁺ phenotype. The biochemically transformed cell lines had the following properties: (i) they were resistant to the growth-inhibiting effects of 1 mM thymidine; and (ii) they expressed an HSV-1-specific TK activity. This HSV-1 TK activity was purified after labeling biochemically transformed cell lines [LM(TK^?)/TF pMH110 E2 and LM(TK^?)/TF pMH110 Hc2] with [³⁵S]methionine. Immunoprecipitation experiments revealed that the TK polypeptides made in the biochemically transformed cells had molecular weights of about 39,000 to 40,000, which are about the same as the molecular weights of the TK polypeptides previously purified from HSV-1-infected LM(TK^?) cells and other biochemically transformed cell lines. The experiments support the hypothesis that the functional coding region of the HSV-1 TK gene is 3′ to the BglII cleavage site, and they also suggest that the HSV-1 TK messenger RNA may have been initiated in cells transformed by HincII- and EcoRI-cleaved pMH110 DNA at a site in cellular (or plasmid) DNA upstream from the HSV-1 DNA BglII cleavage site.     

Induction of productive human papillomavirus type 11 life cycle in epithelial cells grown in organotypic raft cultures

The study of the human papillomavirus (HPV) life cycle was hampered for more than 50 years by the lack of a conventional cell culture system for propagating HPV. Considerable progress has been made in the production of several HPV types using either organotypic rafts or human epithelial xenografts in immunocompromised mice. In this study, we demonstrated episomal maintenance of HPV-11 DNA in N-Tert cells. HPV-11 episomal DNA containing cell populations grown in raft culture showed induction of the productive viral life cycle. HPV-11 DNA amplification and viral capsid antigen synthesis were detected in differentiated layers of epithelia. The viruses generated were able to infect keratinocytes in vitro, which indicate that viruses generated were infectious. The demonstration of the productive HPV-11 life cycle in raft culture from cloned HPV-11 DNA will facilitate genetic analyses of viral gene functions that was not possible using the human xenograft athymic mouse model.     

Cloning of hepatitis A virus genome

Hepatitis A virus (HAV) was highly purified from faeces. The genomic RNA was transcribed to cDNA and this DNA was then cloned into plasmid pBR 322 at the Pst I site, and clones were selected in presence of tetracycline. Most clones contained inserts which hybridized to HAV-specific RNA isolated from HAV-infected cell cultures derived from a human hepatocellular carcinoma. Two clones expressed low amounts of viral antigens.     

Molecular cloning of DNA complementary to tobacco vein mottling virus RNA

RNA isolated from tobacco vein mottling virus (TVMV) was used as a template for avian myeloblastosis virus RNA-dependent DNA polymerase, primed with oligo(dT). The largest single-stranded cDNA synthesized was 10 kb, the same as the viral RNA. This material was converted to double-stranded cDNA with Escherichia coli DNA polymerase I and digested with restriction endonuclease HindIII. The cDNA fragments were ligated to HindIII-digested plasmid pBR322 and the product used to transform E. coli strain DG-75. Clones containing recombinant plasmids were selected by ampicillin resistance, and those containing TVMV RNA sequences were selected by colony hybridization with a single-stranded cDNA probe. Four different sizes of recombinant plasmid were reproducibly observed. The inserted DNA portion could be excised in each case with HindIII. The lengths of inserted DNA were 3.0, 1.85, 1.1, and 0.72 kb. A similar procedure was used with PstI-digested cDNA and pBR322. A single type of recombinant plasmid, containing a DNA insertion of 1.85 kb, was reproducibly observed. Hybridization with TVMV RNA confirmed that the five inserted DNA segments were derived from the viral RNA. Hybridization of each recombinant plasmid with the others established that each of the cloned HindIII fragments was unique and that one of them overlapped the cloned PstI fragment. The cloned cDNA fragments were ordered by establishing a restriction map of the cDNA. Together the cloned cDNA fragments account for over 80% of the viral genome.     

Virus-specific transcription in bovine papillomavirus-transformed mouse cells

We have characterized the viral-specific RNAs present in C127 mouse cells transformed by the BPV-1 virus or by cloned DNA. Five distinct BPV-1 polyadenylated RNA species can be detected by size fractionation on denaturing formaldehyde agarose gels, measuring 1050, 1150, 1700, 3800, and 4050 bases. These RNA species are all transcribed from the same DNA strand and map to a region between 0.34 and 0.85 map units of the 8-kb BPV1 DNA. All five viral RNA species share a common 3′ terminus at 0.34 map units and the 5′ ends map at approximately 0.47, 0.485, 0.55, 0.785, and 0.85 map units. Analysis of the three most abundant viral RNA species (1050, 1150, and 1700 bases) did not reveal any internal splicing; but the presence of a remote 5′ leader sequence could not be ruled out.     

Molecular cloning and characterization of human papillomavirus type 7 DNA

Human papillomavirus type 7 (HPV-7) was first described in 1981 but so far could not be molecularly cloned. It has been found almost exclusively in hand warts of butchers. We have cloned the complete genome in pBR 322, established its physical map, demonstrated the colinear genome organization with HPV-18 and analyzed the degree of homology with other HPV types and bovine papillomavirus (BPV) types. In order to investigate whether HPV-7 might be a so far unidentified bovine virus, we screened 37 bovine tumor DNAs using Southern blot analysis for its presence, with exclusively negative results. From our data we conclude that the HPV-7 genome shows all characteristics of a papillomavirus genome and that its origin is most likely human.     

A new type of papillomavirus associated with cancer of the uterine cervix

A new human papillomavirus (HPV) type was detected by low-stringency Southern blot hybridization analysis of DNA from an endocervical adenocarcinoma. The genomic DNA of the virus, which was obtained as two BamHI fragments of 3.75 and 4.1 kb, was molecularly cloned into lambda L47 and subsequently subcloned into pBR322 for further characterization. Hybridization studies demonstrated that these viral DNA isolates were only distantly related to other HPV and thus represented a new type of HPV, called HPV 35. A restriction enzyme map was prepared which allowed a comparison of the genetic organization of this HPV with that of HPV 6b; the results demonstrated collinearity of the HPV 35 genome with that of HPV 6b. Prevalence studies revealed that HPV 35 was present in 2 of 158 (1%) anogenital intraepithelial neoplasia and in 3 of 69 (4%) anogenital cancers. Thus HPV 35 is a low-prevalence human papillomavirus associated with anogenital intraepithelial neoplasia and cancer.     

Human papillomavirus (HPV) and Merkel cell polyomavirus (MCPyV) in non small cell lung cancer

Certain types of human papillomavirus (HPV) induce cancers, especially cervical cancers in women. A meta-analysis of the literature suggests that HPV is also associated with 20%–25% of non small cell lung carcinoma (NSCLC). Merkel cell Polyomavirus (MCPyV) causes most Merkel cell carcinomas in immunocompromised hosts, and is associated with some squamous carcinomas of skin in immunocompetent individuals. Since both oncogenic viruses appear to involve the tonsils and, therefore, have clear access to the lungs, we examined that the possible association of HPV and MCPyV infections with lung cancers, especially, NSCLC. DNAs were extracted from 51 frozen tissues from 30 lung cancer patients, and examined for the presence of HPV and MCPyV by PCR and DNA sequencing analysis. Clinical data was correlated with the viral status. HPVs were only detected in 5 adenocarcinomas (16.7% of all lung cancers examined). Three were positive for HPV-16, 1 for HPV-11 and 1 had an unknown HPV type DNA. None was identified in benign tissue. MCPyV DNA was detected in 5 NSCLCs (16.7%). Three of the 5 were identified in squamous carcinomas, 1 in adenocarcinoma, and 1 in an unspecified NSCLC. Two additional samples were positive for MCPyV DNA within benign adjacent lung tissue only. In one adenocarcinoma, HPV-11 was identified in an adenocarcinoma, and MCPyV DNA was detected in the adjacent “benign” tissue. HPV and MCPyV were directly associated with 33.3% of NSCLC. Further studies are necessary to determine if polyomavirus and papillomavirus are necessary risk factors for some cases of NSCLC.