The cell-coded polypeptide U90 increased by herpes simplex virus type 2 infection induces Fos and DNA synthesis.

Lytic infection with herpes simplex virus type 2 (HSV-2) induces synthesis of a cell-coded protein of molecular mass 90 kDa, termed U90. U90, whose expression is specific in tumour cells is, in addition, excreted from he cell. To determine if the function of U90 could be advantageous for the virus the protein was purified from the medium of HSV-2 infected cells, shown to be similar to the internalized form and its effect on cell growth studied. Addition of U90 to quiescent fibroblast cells stimulated teh expression of Fos, the product of the cellular transcription factor c-fos and increased the incorporation of [3H]thymidine into DNA, factors which may facilitate HSV-2 replication. However, U90 might also induce abnormal cells such as those in precancerous lesions to proliferate.

HSV-2 increases the mitochondrial aspartate amino transaminase characteristic of tumor cells.

A set of polypeptides has previously been described as being immunoprecipitated from tumor cell lysates by the sera of tumor-bearing animals (TBS) or by antisera raised to herpes simplex virus type 2 (HSV-2)-infected cells. These polypeptides were not immunoprecipitated from control cells. The principal polypeptides detected of MW 200, 90 (U90 and L90), 40, and 32 kDa were increased by HSV-2 infection. This communication describes the purification of the 40-kDa protein and its identification by amino acid sequence analysis as being homologous to the mature form of mitochondrial aspartate amino transaminase (mAAT). Two different forms of mAAT were purified and sequenced. One form, of low abundance, was immunoprecipitated by TBS and corresponded to the 40-kDa protein immunoprecipitated from tumor, but not control, cell lysates. Western blotting of the 40-kDa protein immunoprecipitated by TBS showed that it was a form of mAAT found only in tumor cells. The other abundant form reacted in Western blots with antibody to mAAT and was clearly the constitutive enzyme, present in all cells. In general, mAAT was increased up to fourfold after infection with HSV-2, HSV-2 infection also increased mAAT enzyme activity. Northern blotting and quantitative PCR showed that mAAT was induced by HSV-2 at the level of transcription. The specific form of mAAT immunoprecipitated from tumor, but not control, cell lysates could have a role in tumorigenesis, could be a putative tumor cell marker, or could be a target for antitumor drugs. HSV-2 probably induces enzymes of glutamine metabolism because HSV-2 requires glutamine for growth, thus revealing potential new antiviral targets.

S-adenosylmethionine metabolism in herpes simplex virus type 2-infected cells.

Infection of primary rat embryo cells with herpes simplex virus type 2 has previously been reported to produce a dramatic and rapid inhibition of cellular DNA methylation. However, it has neither an immediate effect on S-adenosylmethionine breakdown nor on the relative pool sizes of S-adenosylmethionine and S-adenosylhomocysteine.

The characteristic which makes the cell-coded HSV-inducible U90 distinctive in transformed cells is its greatly increased half-life.

A set of polypeptides detected in transformed cells of M(r) values 90,000 (a doublet) 40,000 and 32,000 that are recognised by immunoprecipitation of L-[35S]methionine-labelled tumour cell lysates, with sera from tumour-bearing rats and with antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, were previously reported (Macnab et al., 1985, 1992; Hewitt et al., 1991). These proteins are cell-coded and cannot be precipitated from similarly radiolabelled control cells. Two of these polypeptides are significantly induced by HSV-2 infection (Hewitt et al., 1991; Macnab et al., 1992). This paper further characterises one of these polypeptides, U90, and addresses which properties distinguish the behaviour of U90 in tumour cells, whether there is an equivalent in control cells and whether U90 can be induced without HSV replication. U90 can be immunoprecipitated from radiolabelled human cells which are capable of extended passage in culture, as well as from human tumour cells, rodent tumour cells and cells of different lineages, e.g. epithelial, fibroblastic and lymphoid. Purification of U90 and the subsequent production of monospecific antibodies revealed, by western blotting, a homologue of 90 kDa in control cells. Western blotting shows that HSV can increase the amount of the U90 homologue in these normal cells. The absence of an immunoprecipitate of the U90 homologue from control cells is a result of the very short half-life (i.e. high turnover) of the protein in these cells (32.9 minutes) as opposed to tumour cells (about 13 hours).(ABSTRACT TRUNCATED AT 250 WORDS)

Oestrogen receptor message in premalignant and normal cervical cells: a methodological study.

Normal and abnormal biopsies of the uterine cervix, to a total of 124 samples, have been analysed for the detection of oestrogen receptor (ER) mRNA. The tough fibrous nature of the cervix proved very resistant to disaggregation in the first instance and subsequently, it was difficult to extract good high molecular weight message. This necessitated a systematic study of methodological technique, including two methods of tissue disaggregation and five techniques of extraction of ER mRNA, which in total involved the use of 124 cervical biopsies. The most successful method involved chopping the tissue, then digesting the cells with proteinase K and extracting the nucleic acids in salt and sodium dodecyl sulphate. Using the perfected technique, 16 cervical biopsies obtained at serial intervals from four women did not show any differences in ER mRNA in cervical biopsies either in the presence of oral contraception or histological abnormality. The successful method described will prove valuable for the detection of ER message in human tumours and other tissues of similar nature.

Patients with cervical cancer produce an antibody response to an HSV-inducible tumour-specific cell polypeptide.

Anti-sera raised against HSV-2-infected cells (WI) and the sera of animals bearing tumours (TBS) to HSV-2 transformed cells contain antibodies to a set of tumour-specific cell-coded polypeptides. The specificity of these polypeptides for tumour cells is monitored by the ability of [35S]-L-methionine labelled proteins to be immunoprecipitated by these anti-sera, in contrast to control cells from which the polypeptides are not precipitated. The polypeptides which share an epitope and are co-precipitated are of MWs 90,000 (a doublet), 40,000 and 32,000. The upper 90,000-MW polypeptide (U90) is induced by HSV-2 infection. This communication deals with the 40,000-MW polypeptide which was shown to be immunoprecipitated by TBS and a monoclonal antibody (MAb) raised to the DNA-binding proteins of HSV-2-infected cells. Immunological and biochemical studies reveal that the 40,000-MW protein which is immunoprecipitated comprises more than one polypeptide, and that the proteins may need to interact to produce the peptide pattern specific for the tumour form of the immunoprecipitated 40,000-MW protein. WI antisera and TBS both recognise antigens specific for tumour cells in sections of cervical-carcinoma tissue. Sera from patients with cancer of the cervix contain antibodies to a cell-coded polypeptide of MW 40,000, which by peptide analysis is indistinguishable from the 40,000-MW polypeptide induced by HSV-2 infection and immunoprecipitated by WI and TBS.

A transformation-specific polypeptide distinct from heat shock proteins is induced by herpes simplex virus type 2 infection.

A tumour-specific polypeptide designated U90 is one of a set of polypeptides which are encoded by the host cell and are specific for the transformed cell state, being immunoprecipitated by the sera of tumour-bearing animals. The interest in these tumour-specific polypeptides centres on the finding that they are also recognized by antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, implying some role for HSV-2 in tumorigenesis. The peptide map of HSV-2-induced U90 is indistinguishable from that of U90 present in uninfected tumour cells, including mouse cells transformed by human papillomavirus type 16. In tumour cells, U90 is located principally in the plasma membrane fraction and cannot be induced by heat shock, glucose starvation, or treatment with tunicamycin or calcium ionophore. U90 is not related to either the heat shock protein of Mr 90,000 (HSP90) or the glucose-related polypeptide of Mr 94,000 (GRP94) as determined by peptide mapping and the use of monospecific, monoclonal and antipeptide antibodies. This suggests that U90 is a novel transformation-specific protein which can be induced by infection with HSV-2.

Prospective serial study of viral change in the cervix and correlation with human papillomavirus genome status.

OBJECTIVE: To observe in serial fashion the histological changes of human papilloma virus (HPV) infection of the cervix and to correlate these with the detection of HPV genomes. DESIGN: A prospective longitudinal study to perform colposcopy and at 0, 9 and 18 months to obtain a smear, a biopsy for histology and HPV-16 genome detection by DNA/DNA hybridization. SETTING: Glasgow Family Planning Centre. SUBJECTS: Eighty-two women recruited on the basis of a routine smear showing viral change without dyskaryosis. INTERVENTIONS: Women found to have CIN 3 were treated with laser. MAIN OUTCOME MEASURES: Cytology and cervical biopsy results and HPV-16 DNA detection by Southern blotting obtained serially from individual subjects. RESULTS: Of 82 women recruited, 10% were positive for HPV-16 detection by Southern blotting. Detection of HPV-16 genomes did not correlate with either CIN or viral infection assessed histologically and serial hybridizations in a given individual showed fluctuation in HPV-16 detection. Polymerase chain reaction in a subset of samples revealed 46% positive for HPV DNA. CONCLUSION: No clear correlation exists between HPV genome detection and the histological appearance. HPV-16 genome detection alone may not be a useful predictor of precancerous progression.

Molecular cloning of DNA sequences from cervical intraepithelial neoplasia that hybridize to human cytomegalovirus DNA.

We have prepared EMBL3 libraries of DNA extracted from the cervix of a patient with cervical intraepithelial neoplasia (CIN) and isolated seven recombinant clones containing sequences that hybridize to human cytomegalovirus (HCMV) DNA. Restriction analysis of one clone with BamHI and SalI endonucleases revealed that the insert DNA showed a high degree of homology to the HCMV Ad169 genome over the region between the HindIII K/E site and the SalI site located within the BamHI P fragment. The HCMV insert in the CIN clone is integrated and flanked by cellular sequences. The major immediate early gene that encodes a polypeptide of approximately 69 kD was found to be conserved in the CIN clone. Transfection of clones encoding the immediate early region of HCMV resulted in cells that were positive in immunofluorescence studies with two monoclonal antibodies directed against the HCMV 69 kD immediate early polypeptide. Infection of human ectocervical cells with HCMV Ad169 revealed that they could express the 69 kD polypeptide encoded by the immediate early gene but could not replicate the virus, whereas HCMV was able to replicate productively in cultured endocervical cells. HCMV has been shown to activate endogenous retroviruses and also to transcriptionally activate the long terminal repeat of human immunodeficiency virus. Activation of virus and cellular genes by HCMV may be a means by which this virus is involved in the multistage process of oncogenesis and/or the activation of latent infections.

Stimulation of estrogen receptor mRNA levels in MCF-7 cells by herpes simplex virus infection.

Infection of estrogen-responsive cells (MCF-7) with herpes simplex virus type 1 or 2 stimulates expression of the estrogen receptor message. Experiments on infection with the mutant virus, tsK, together with transfection studies implicate the virion protein, Vmw65, in the response. Cellular protein synthesis is essential for estrogen receptor mRNA expression.

Cervical carcinoma and human cytomegalovirus.

Human cytomegalovirus (HCMV) is a sexually transmitted virus which has been implicated in both cervical cancer and its precancerous state, cervical intraepithelial neoplasia (CIN). The evidence for this and the current possible mechanisms by which HCMV could be involved are discussed at the molecular level.

Sexually transmitted cancers and viruses.

Both cervical cancer and its precancerous state cervical intraepithelial neoplasia (CIN) have the characteristics of being sexually transmitted. Formerly herpes simplex virus (HSV) but more recently human papillomavirus (HPV) which both infect the cervix have been implicated in causation. The role of these viruses as possible initiators of cancer or as potential cofactors of cocarcinogens is discussed at the molecular level within the context of the disease process.

Human papillomavirus in paired normal and abnormal cervical biopsies--implications for treatment.

A total of 143 consecutive patients with abnormal cervical cytology was examined at a large colposcopy clinic in Glasgow. Each patient had paired biopsies from normal and abnormal cervical epithelium examined both histologically and by immunoperoxidase staining for human papillomavirus (HPV) infection. More than 71% of the abnormal biopsies and 39% of the normal paired biopsies had histological evidence of HPV infection. Cytological evidence of HPV infection was seen in 38.5% of cervical smears. Immunocytochemistry revealed HPV antigen in 22% of the abnormal biopsies and in 4.2% of the 'normal' biopsies. The presence of HPV infection in colposcopically normal cervical tissue both inside and outside the transformation zone may help to explain why current methods for treatment of cervical HPV infection are often unsuccessful.

Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus.

Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.

Histological and cytological evidence of viral infection and human papillomavirus type 16 DNA sequences in cervical intraepithelial neoplasia and normal tissue in the west of Scotland: evaluation of treatment policy.

Biopsy samples from 27 patients referred to a colposcopy clinic in Glasgow for cervical abnormalities were assessed for the relations among colposcopic appearances, cytological and histological diagnosis, expression of papillomavirus antigen, and the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 deoxyribonucleic acid (DNA) sequences. Specimens were from colposcopically abnormal areas of the transformation zone and from colposcopically apparently normal areas of the zone in the same patients (paired matched internal control tissue). All 27 women referred for abnormal smears had colposcopic abnormalities. HPV-16 or 18 DNA sequences were detected in 20 of the 27 colposcopically abnormal biopsy samples and 13 of the 27 paired normal samples. Twelve samples of colposcopically normal tissue contained histological evidence of viral infection but only four of these contained HPV DNA sequences. The other nine samples of colposcopically normal tissue which contained HPV DNA sequences were, however, histologically apparently normal. HPV-6 and 11 were not detected. Integration of the HPV-16 genome into the host chromosome was indicated in both cervical intraepithelial neoplasia and control tissues. In two thirds of the HPV DNA positive samples the histological grade was classed as normal, viral atypia, or cervical intraepithelial neoplasia grade 1. Papillomavirus antigen was detected in only six of the abnormal and three of the normal biopsy samples, and HPV DNA was detected in all of these. The detection of HPV DNA correlates well with a combination of histological and cytological evidence of viral infection (20 of 22 cases in this series). A poor correlation between the site on the cervix of histologically confirmed colposcopic abnormality and the presence of HPV DNA sequences implies that a cofactor other than HPV is required for preneoplastic disease to develop. A separate study in two further sets of biopsy samples examined the state of HPV DNA alone. The sets were (a) 43 samples from cervical intraepithelial neoplasia and nine external controls and (b) 155 samples from cervical intraepithelial neoplasia, cervical cancer, vulval intraepithelial neoplasia, and vulval cancer and external controls. HPV-11 was found in only two (4.7%) of the 43 specimens from cervical intraepithelial neoplasia, whereas HPV-16 was found in 90 (58%) of the other 155 specimens. These results also suggest that HPV subtype is subject to geographical location rather than being an indicator of severity of the lesion or of prognosis.

Herpes simplex virus and human cytomegalovirus: their role in morphological transformation and genital cancers.

Herpes simplex virus (HSV) and human cytomegalovirus (HCMV) are candidates for the induction of premalignant or malignant disease. Morphological transformation studies have failed to demonstrate a viral oncogene, a virus-coded transforming protein or any sequence of DNA that uniquely transforms cells according to one-hit kinetics. Thus the mechanism of transformation is complex. The transformed cells are, however, all oncogenic in the host animal and in immunocompetent mice. Direct evidence for the presence of these viruses in human genital tumours is the finding that a small proportion (about 10%) retain fragments of virus DNA from different regions of the virus genomes. In contrast human papillomavirus (HPV) is strongly associated with genital neoplasia, being present in over 80% of tumours. However, HPV can also be detected in histologically normal tissue. The most persuasive roles for HSV and HCMV in human tumourigenesis are as mutagens, as activators of cellular transcription or in switching on the synthesis of host cell proteins not normally expressed in untransformed cells. In these roles the prospects of further defining roles for HSV and HCMV in the multistage process of oncogenic transformation are good.

Human papillomavirus type 16 DNA from a vulvar carcinoma in situ is present as head-to-tail dimeric episomes with a deletion in the non-coding region.

A number of genital cancer biopsy samples were screened for the presence of human papillomavirus type 16 (HPV-16) DNA sequences. One of these samples (a vulvar carcinoma in situ) was found to contain more than 100 copies of HPV-16 DNA sequences per cell. Using this tumour DNA, a genomic library was constructed in bacteriophage lambda and the library was screened for recombinant phage containing HPV-16 sequences. Five recombinant phage clones were isolated and their DNA was analysed by restriction endonuclease digestion and blot hybridization. All five recombinants contained two copies of the HPV-16 genome present in a head-to-tail arrangement. The data are consistent with the presence of HPV-16 sequences in the tumour DNA arranged as genomic dimers in a circular episomal configuration. The HPV-16 genomes contained a deletion within the non-coding region, a region which includes the viral origin of DNA replication and transcriptional control sequences. Possible consequences of this deletion for viral replication and transcription are discussed.