Requirement of the human chromosome 11 long arm for replication of herpes simplex virus type 1 in nonpermissive Chinese hamster x human diploid fibroblast hybrids

Somatic cell hybrids between Chinese hamster (CH) lung cells (V79/380-6), nonpermissive for productive infection by herpes simplex type 1 (HSV-1), and permissive human diploid cells support productive HSV-1 infection as long as they retain human chromosome 11. Human chromosome 3 has been reported to complement nonpermissivity in (CH) Don cells (1). Intraspecies hybrids between Don/a3 and V79/380-6 cells, however, did not support HSV-1 replication, indicating lack of complementation. The block in both nonpermissive CH cell lines was determined to involve a step beyond replication of the parental viral DNA. In cell hybrids between nonpermissive Don/a23 cells and human fibroblasts containing a t(11;15) (p11;p12) translocation, HSV-1 production was dependent solely on the presence of either human chromosome 11 or the der(11) (p11 leads to qter) translocation product containing the long arm of chromosome 11. Chromosome 3 was excluded by a discordancy rate of 59%. We conclude that the long arm of human chromosome 11 carries one or more genes coding for host functions necessary for the production of progeny HSV-1 DNA.     

Identification of human papillomavirus type 18 transforming genes in immortalized and primary cells.

The selective retention and expression of the E6-E7 region of human papillomavirus (HPV) types 16 and 18 in cervical carcinomas suggests that these viral sequences play a role in the development of genital neoplasia. Each of three possible gene products, E6, E6*, and E7, from this region of HPV-18 were examined for transforming properties in several types of rodent cells. We have found that in immortalized fibroblasts, both E6 and E7 (but not E6*) are capable of inducing anchorage-independent growth. In rat embryo cells, the HPV-18 E7 open reading frame was an effective immortalizing agent and complemented an activated ras oncogene for transformation. In both immortalized and primary cells, transformation was observed when the HPV-18 sequences were expressed from either the HPV-18 promoter or a heterologous promoter. The E6-E7 region is not, however, the sole transforming domain of HPV-18, since another portion of the early region, possibly E5, also exhibited transforming capability in immortalized fibroblasts. The development of human cervical carcinomas may therefore involve a series of steps involving multiple viral and cellular gene products.     

Regional assignment of the human gene for platelet-type phosphofructokinase (PFKP) to chromosome 10p: novel use of polyspecific rodent antisera to localize human enzyme genes

Human phosphofructokinase (PFK; EC 2.7.1.11) is under the control of three structural loci which encode muscle-type (M), liver-type (L), and platelet or fibroblast-type (P) subunits; human diploid fibroblasts and leukocytes express all three loci. In order to assign the human PFKP locus to a specific human chromosome, in this study, we have examined ten human X rodent somatic cell hybrids for the expression of human P subunits using a mouse anti-human P subunit-specific antiserum in an active-enzyme-immunoprecipitation technique. In nine of ten hybrids studied, the expression of the PFKP locus segregated concordantly with chromosome 10 and none other, indicating that PFKP is located on chromosome 10; the discordancy rates for all the other chromosomes were 0.2 or greater. In the one discordant hybrid, only the long arm of chromosome 10 was retained and PFKP was not expressed. Human fibroblasts from a patient with duplication of the short arm of chromosome 10 consistently exhibited PFK activity values 180% of normal. These data indicate that human PFKP is located on the short arm of chromosome 10, and that a gene dosage effect is demonstrable in fibroblasts with a duplication of 10p. The use of rodent antihuman antibody combined with immunoprecipitation aided by staphylococci-bearing protein A may find general application in mapping human enzyme genes, when human and rodent gene-products are not distinguishable by other means.     

The 55 kDa regulatory subunit of protein phosphatase 2A plays a role in the activation of the HPV16 long control region in human cells with a deletion in the short arm of chromosome 11.

Previous results indicated that SV40 small t is essential for SV40-induced transformation of diploid cells but dispensable for the transformation of cells with a deletion on the short arm of chromosome 11 (del-11 cells). From these results we concluded that del-11 cells contain a cellular 'SV40 small t-like' factor, which is able to transactivate the HPV16 long control region (LCR) and to complement SV40 large T in transformation. Since SV40 small t and the regulatory 55 kDa subunit (PR55) of protein phosphatase 2A (PP2A), have been shown to inhibit the enzyme activity of PP2A, the PR55 beta subunit could be the putative 'small t-like' factor. In accordance with this hypothesis, we show that the PR55 beta subunit is highly expressed in del-11 but not in diploid cells and is able to trans-activate the HPV16 LCR in diploid cells. Moreover, inhibition of PP2A by okadaic acid resulted in trans-activation of the HPV16 LCR in diploid cells. Alignment of PR55 and SV40 small t showed a common four amino acid motif DKGG. We present evidence that the integrity of this motif is necessary for the PP2A-mediated ability of SV40 small t to trans-activate the HPV16 LCR.     

Transient replication of human papillomavirus DNAs.

Information on papillomavirus DNA replication has primarily derived from studies with bovine papillomavirus type 1 (BPV-1). Our knowledge of DNA replication of the human papillomaviruses (HPVs) is quite limited, in part because of the lack of a cell culture system capable of supporting the stable replication of HPV DNA. This study demonstrates that the full-length genomic DNAs of HPV types 11 and 18 (HPV-11 and HPV-18), but not HPV-16, are able to replicate transiently after transfection into several different human squamous cell carcinoma cell lines. This system was used to identify the viral cis and trans elements required for DNA replication. The viral origins of replication were localized to a region of the viral long control region. Like BPV-1, E1 and E2 were the only viral factors required in trans for the replication of plasmids containing the origin. Cotransfection of a plasmid expressing the E1 open reading frame (ORF) from HPV-11 with a plasmid that expresses the E2 ORF from HPV-6, HPV-11, HPV-16, or HPV-18 supported the replication of plasmid DNAs containing the origin regions of HPV-11, HPV-16, or HPV-18, indicating that there are functions shared among the corresponding E1 and E2 proteins and origins of these viruses. Although HPV-16 genomic DNA did not replicate by itself under experimental conditions that supported the replication of HPV-11 and HPV-18 genomic DNAs, expression of the HPV-16 early region functions from a strong heterologous promoter supported the replication of a cotransfected plasmid containing the HPV-16 origin of replication. This finding suggests that the inability of the HPV-16 genomic DNA to replicate transiently in the cell lines tested was most likely due to insufficient expression of the viral E1 and/or E2 genes required for DNA replication.     

E5 proteins of human papillomavirus types 11 and 16 transactivate the c-fos promoter through the NF1 binding element.

Human papillomavirus type 11 (HPV-11) and HPV-16 contain an E5 gene that can induce c-fos gene expression in mouse fibroblasts. This study investigated the human c-fos promoter characteristics by mapping the c-fos promoter sequence with several deletion and point mutants that confer responsiveness to E5 of HPV-11 or HPV-16. The mutant studies show that NF1 binding sequences within the c-fos promoter were crucial for the induction of the c-fos gene by E5, and the gel shift assay study suggested that E5 of both HPV-11 and HPV-16 is associated, perhaps indirectly, with this NF1 element in the transactivation of the human c-fos promoter. Using an inducible system, we demonstrate that increased induction of the HPV-11 E5 gene in cells led to increased transactivation of the NF1 element. In addition, the transactivating activity of a series of HPV-11 E5 mutants on the NF1 element had a strong correlation with their respective transforming activities.     

Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells.

Integration of human papillomavirus type 16 (HPV-16) DNA into a host chromosome has been hypothesized to result in altered expression of two viral transforming genes, E6 and E7, in cervical cancers. In order to investigate the role that changes in viral genomic state and gene expression play in cervical carcinogenesis, we have derived clonal populations of human cervical epithelial cells which harbor multiple copies of either extrachromosomal or integrated viral DNA. The clonal populations harboring extrachromosomal HPV-16 DNA stably maintained approximately 1,000 viral copies for at least 15 passages (approximately 100 cell doublings), which contrasted with the unstable HPV-16 replicons in the parental counterpart. In the clonal populations harboring integrated viral DNA, 3 to 60 copies of HPV-16 DNA were found integrated in either of two forms: type 1, in which all the copies of HPV-16 DNA were disrupted in the E2 open reading frame upon integration, and type 2, in which intact viral copies were flanked by disrupted viral copies and cellular sequences. Despite the lower HPV-16 DNA copy number, the clonal populations with integrated viral DNA had levels of E7 protein that were in most cases higher than those found in the clonal populations harboring extrachromosomal viral DNA. Irrespective of viral genomic state, the clonal populations were capable of undergoing terminal differentiation and unable to form colonies in soft agar, which is indicative of the nontumorigenic nature of these cells. Importantly, a cell population with integrated viral DNA was found to outgrow another with extrachromosomal DNA when these cells were cocultured over a period of time. Thus, integration of human papillomaviral DNA correlates with increased viral gene expression and cellular growth advantage. These observations are consistent with the hypothesis that integration provides a selective advantage to cervical epithelial precursors of cervical carcinoma.     

Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines.

Human cervical carcinoma cell lines that harbor human papillomavirus (HPV) have been reported to retain selectively and express HPV sequences which could encode viral E6 and E7 proteins. The potential importance of HPV E6 to tumors is suggested further by the observation that bovine papillomavirus (BPV) E6 can induce morphologic transformation of mouse cells in vitro. To identify HPV E6 protein, a polypeptide encoded by HPV-16 E6 was produced in a bacterial expression vector and used to raise antisera. The antisera specifically immunoprecipitated the predicted 18-kd protein in two human carcinoma cell lines known to express HPV-16 RNA and in mouse cells morphologically transformed by HPV-16 DNA. The 18-kd E6 protein was distinct from a previously identified HPV-16 E7 protein. The HPV-16 E6 antibodies were found to be type specific in that they did not recognize E6 protein in cells containing HPV-18 sequences and reacted weakly, if at all, to BPV E6 protein. The results demonstrate that human tumors containing HPV-16 DNA can express an E6 protein product. They are consistent with the hypothesis that E6 may contribute to the transformed phenotype in human cervical cancers that express this protein.     

Chromosome 7 suppresses indefinite division of nontumorigenic immortalized human fibroblast cell lines KMST-6 and SUSM-1.

Using nontumorigenic immortalized human cell lines KMST-6 (KMST) and SUSM-1 (SUSM), we attempted to identify the chromosome that carries a putative senescence-related gene(s). These cell lines are the only ones that have been established independently from normal human diploid fibroblasts following in vitro mutagenesis. We first examined restriction fragment length polymorphisms on each chromosome of these immortalized cell lines and their parental cell lines and found specific chromosomal alterations common to these cell lines (a loss of heterozygosity in KMST and a deletion in SUSM) on the long arm of chromosome 7. In addition to these, we also found that introduction of chromosome 7 into these cell lines by means of microcell fusion resulted in the cessation of cell division, giving rise to cells resembling cells in senescence. Introduction of other chromosomes, such as chromosomes 1 and 11, on which losses of heterozygosity were also detected in one of the cell lines (KMST), to either KMST or SUSM cells or of chromosome 7 to several tumor-derived cell lines had no effect on their division potential. These results strongly suggest that a gene(s) affecting limited-division potential or senescence of normal human fibroblasts is located on chromosome 7, probably at the long arm of the chromosome, representing the first case in which a specific chromosome reverses the immortal phenotype of otherwise normal human cell lines.     

Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system.

Integrated human papillomavirus type 16 (HPV-16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki by fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV-16 DNA were quantified using a microscope equipped with a cooled-CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotinylated centromere probe specific for chromosomes 13 and 21. Ratios of the distance from 13pter to the HPV-16 signals to the entire chromosome length were approximately 0.63 +/- 0.05 in both SiHa and CaSki cells, indicating the possibility of a common integration domain on chromosome 13. In SiHa cells, no additional signals were observed on other chromosomes. This observation, taken together with literature reports that SiHa cells contain 1 to 2 copies of the HPV-16 genome in this region of chromosome 13, suggests that each fluorescein signal on chromosome 13 represents one equivalent of the HPV-16 genome. The total integrated fluorescence intensity in isolated CaSki metaphase chromosome spreads was approximately two orders of magnitude greater than that of a single copy of HPV-16 DNA in SiHa cells, indicating an increase in HPV-16 copy number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human papillomavirus type 16 (HPV-16) genomes integrated in head and neck cancers and in HPV-16-immortalized human keratinocyte clones express chimeric virus-cell mRNAs similar to those found in cervical cancers

Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.     

The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes.

To determine the function of the E5 open reading frame (ORF) of the human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts were transformed to colony formation in soft agar, and the transformation frequency was increased by epidermal growth factor (EGF) but not by platelet-derived growth factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-16 were mitogenic in primary human foreskin epithelial cells (keratinocytes) and acted synergistically with EGF. Investigation of keratinocytes expressing HPV-16 E5 showed that the number of endogenous EGF receptors (EGFRs) per cell was increased two- to fivefold. Immunofluorescence microscopy of HPV-16 E5-expressing keratinocytes indicated that there was an apparent delay in the internalization and degradation of EGFRs compared with controls. Kinetic studies with [125I]EGF showed that the ligand underwent normal internalization and degradation in both HPV-16 E5-expressing and control keratinocytes, but in E5-expressing cells, a greater number of receptors recycled back to the cell surface within 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphorylation of the EGFR on tyrosine, an indication of receptor kinase activity, was of greater magnitude in the HPV-16 E5-expressing keratinocytes than in control cells, although the basal level of receptor phosphorylation was similar.     

DNA sequences of human papillomavirus types 11, 16, and 18 in lesions of the uterine cervix in the west of Scotland.

Punch biopsy specimens of the cervix were examined both histologically and for the presence of human papillomavirus (HPV) DNA sequences. The presence of HPV DNA sequences was sought with the Southern blot technique using radioactively labelled HPV-6, 11, 16, and 18 DNA probes, both together and separately. Twenty six biopsy specimens were examined. Histological examination showed cervical intraepithelial neoplasia grade 2 or 3 in 16 specimens, viral changes (koilocytosis) in four, and inflammation or a normal appearance in three. Eleven specimens were negative for HPV DNA sequences, 10 contained HPV-16 DNA, four contained HPV-18 DNA, and one contained both HPV-18 and HPV-11 DNA. Episomal HPV-16 DNA was detected in one case of cervical intraepithelial neoplasia grade 3 and in five cases of cervical intraepithelial neoplasia grade 2/3 with koilocytosis; and episomal HPV-18 DNA was found in two specimens classed as cervical intraepithelial neoplasia grade 2/3, one of which also contained HPV-11 DNA, and in one specimen that showed viral changes alone. Integrated HPV DNA was found in six specimens (four with HPV-16 DNA and two with HPV-18 DNA), including two cases of chronically inflamed cervix with no histological evidence of viral infection or cervical intraepithelial neoplasia. Detection of viral DNA in early lesions may identify patients at risk of malignant progression. This is the first report of HPV-18 DNA in cervical intraepithelial neoplasia in Scotland.     

Mutational analysis of human papillomavirus type 11 E5a oncoprotein.

In this study, we investigated the structural basis of human papillomavirus type 11 (HPV-11) E5a transforming activity at the amino acid level. The effects of insertion, deletion , and substitution mutations on teh E5a transforming activity were determined by the assay of anchorage-independent growth. In the conserved Cys-X-Cys structure, substitution of Ser for Cys-73 resulted in indistinguishable transforming activity, whereas substitution of Ser for Cys-75 or Ser for both Cys-73 and Cys-75 retained 50 and 42% transformation, respectively. This suggests that Cys at position 75 may be important for transformation. Charge and structural changes at teh COOH termini of several mutants impaired transformation significantly, but those at the middle region did so only mildly. In addition, the 16,000-molecular-weight pore-forming protein (16K protein) is known to associate with BPV-1, HPV-6, and HPV-16 E5 proteins. In this study, we investigated the correlation between E5a-16K binding affinity and the transforming activity of E5a by the use of 11 E5a mutants. Results show that E5a and these 11 E5a mutants could bind to the 16K protein when these proteins were coexpressed in COS cells, suggesting that simple binding of the 16K protein by E5a may not be sufficient for cell transformation.     

Chromosomal insertion of human papillomavirus 18 sequences in HeLa cells detected by nonisotopic in situ hybridization and reflection contrast microscopy

Summary Genomic insertion of human papillomavirus (HPV) sequences is associated with the genesis of cervical carcinoma, and HPV-induced incipient cellular alterations may also present a requisite for the establishment of cell lines such as HeLa. Considering the theoretical importance of specific viral integration sites, we attempted to detect in HeLa cells the chromosomal location of DNA sequences homologous to HPV-16 and HPV-18 sequences by a nonisotopic high resolution in situ hybridization technique. Chromosome identification following in situ hybridization was possible by counterstaining of the same preparation with Chromomycin A₃, Distamycin A, and DAPI. Using this approach, we have assigned HPV-18 integration in HeLa cells to band 8q24 (a site including the locus of the myc-protooncogene), to an abnormal chromosome 22, and to a not yet identified marker chromosome possibly neighboring other oncogenic or activating sites. The sensitive detection technique described in this study presents a new approach involving in situ chromosome hybridization with biotinylated DNA probes in combination with reflection contrast microscopy and subsequent fluorescent R-and C-banding. The method allowed the assignment of a 7-kb HPV-18 DNA probe to human chromosomal sites important in growth regulation and cancerogenesis. It should prove useful in a number of similar studies using other viral and oncogenic DNA probes.     

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

Apoptotic HPV positive cancer cells exhibit transforming properties

Previous studies have shown that DNA can be transferred from dying engineered cells to neighboring cells through the phagocytosis of apoptotic bodies, which leads to cellular transformation. Here, we provide evidence of an uptake of apoptotic-derived cervical cancer cells by human mesenchymal cells. Interestingly, HeLa (HPV 18+) or Ca Ski (HPV16+) cells, harboring integrated high-risk HPV DNA but not C-33 A cells (HPV-), were able to transform the recipient cells. Human primary fibroblasts engulfed the apoptotic bodies effectively within 30 minutes after co-cultivation. This mechanism is active and involves the actin cytoskeleton. In situ hybridization of transformed fibroblasts revealed the presence of HPV DNA in the nucleus of a subset of phagocytosing cells. These cells expressed the HPV16/18 E6 gene, which contributes to the disruption of the p53/p21 pathway, and the cells exhibited a tumorigenic phenotype, including an increased proliferation rate, polyploidy and anchorage independence growth. Such horizontal transfer of viral oncogenes to surrounding cells that lack receptors for HPV could facilitate the persistence of the virus, the main risk factor for cervical cancer development. This process might contribute to HPV-associated disease progression in vivo.