Jan ter Schegget

Novel Short-Fragment PCR Assay for Highly Sensitive Broad-Spectrum Detection of Anogenital Human Papillomaviruses

A novel set of polymerase chain reaction (PCR) primers, designated SPF1 and SPF2 and located in the L1 region, was developed for universal detection of human papillomavirus (HPV). A short PCR fragment (SPF) of only 65 pb was synthesized. SPF amplimers were detected in a microtiter-based hybridization system, using a mixture of oligonucleotide probes. The SPF system allowed detection of at least 43 different HPV genotypes. The clinical performance of the novel SPF system was assessed in three different patient groups. 1) Analysis of 534 cervical scrapes, obtained from treated patients, showed that the detection rate in 447 (83.7%) scrapes with normal cytology was significantly higher using the SPF system as compared with the universal primer set GP5+/6+ (P < 0.001). 2) The SPF assay detected HPV DNA in 299 (98.4%) of 304 scrapes with cytological dyskaryosis. 3) The SPF system detected HPV DNA in 100% of 184 formalin-fixed, paraffin-embedded cervical carcinoma specimens. In conclusion, the novel SPF system permitted universal and highly sensitive detection of HPV DNA in diverse clinical materials and may improve the molecular diagnosis and epidemiology of this important virus.

A new method for the isolation of herpes simplex virus type 2 DNA

Abstract Sodium-iodide density gradient centrifugation of lysates of BSC cells infected with herpes simplex virus type 2 was carried out in the presence of ethidium bromide. Two DNA bands could be visualized. The lower DNA band was further identified as herpes simplex virus type 2 DNA by its plaque-forming capacity and sedimentation behavior.

Multifocal vulvar intraepithelial neoplasia grade III and multicentric lower genital tract neoplasia is associated with transcriptionally active human papillomavirus

Background. The incidence of vulvar intraepithelial neoplasia Grade III (VIN III) is increasing and is diagnosed at a younger age than previously. VIN III is often multifocal and frequently coexists with multicentric dysplastic lesions in the cervix and vagina. Warty‐type VIN III more often has been found to contain human papillomavirus (HPV) DNA than basaloid‐type VIN III. The authors performed HPV DNA polymerase chain reaction (PCR) analysis in 48 VIN III biopsies and reverse transcriptase (RT)‐PCR in 8 HPV‐16 DNA‐positive multifocal VIN III biopsies to detect E6/E7 transcripts.

Application of the NASBA nucleic acid amplification method for the detection of human papillomavirus type 16 E6-E7 transcripts

Using a human papillomavirus type 16 (HPV-16) E6-E7 specific primer set in a nucleic acid sequence-based amplification (NASBA) reaction, detection of HPV-16 transcripts was accomplished in a single enzymatic reaction at 41 degrees C. The NASBA reaction product was visualized either by Northern bolt analysis with an HPV-16 E6-E7-specific 32P-labelled oligonucleotide probe or by a non-radioactive enzyme-linked gel assay (ELGA). In combination with a rapid nucleic acid extraction procedure this method appears to be very suitable for the sensitive and specific detection of HPV-16 transcripts on small amounts of HPV-16-expressing cells of various sources, including cervical smears.

Human papillomavirus DNA after treatment of cervical dysplasia: Low prevalence in normal cytologic smears

The presence of human papillomavirus (HPV) DNA in relation to cervical cytology was evaluated after treatment of cervical dysplasia.

Cutaneous squamous cell carcinoma and p53 codon 72 polymorphism: a need for screening?

The association between human papillomavirus (HPV)–associated cervical cancer and cutaneous squamous cell carcinoma and codon 72 polymorphism in the p53 gene is not unequivocal. Especially, it is not known whether carriers of the arginine form have an increased risk of cancer that necessitates screening. The alternative is that the polymorphism is a tumor marker instead of a risk factor. We set out a case‐control study to determine the risk of squamous cell carcinoma of the skin in individuals with the p53 codon 72 arginine genotype in order to establish the possible need for screening. The distribution of the different p53 codon 72 genotypes was examined in 86 subjects with a history of cutaneous squamous cell carcinoma and in 168 controls. Additionally, 121 subjects who had had histologically proven basal cell carcinoma and 108 subjects who had had non‐familial malignant melanoma were tested. p53 polymorphism was evaluated by polymerase chain reaction (PCR) using DNA samples from peripheral blood lymphocytes. In a subgroup of patients with squamous cell carcinoma and controls, the presence of epidermodyplasia verruciformis human papillomavirus (EV‐HPV) DNA was determined in plucked eyebrow hair. Differences in the distributions of the genotypes among cases and controls were calculated, and univariate and multivariate analyses were performed to assess the risk to develop cutaneous squamous cell carcinoma in the presence of the p53 codon 72 arginine genotype. Frequency distributions of the three different genotypes (homozygous for the arginine allele, heterozygous for the two alleles, and homozygous for the proline allele) were similar among the squamous cell carcinoma group and the control group: 47.1%, 46.0% and 6.9% versus 47.8%, 45.8% and 6.4%, respectively. Statistical analysis showed no significant differences between these groups. In patients with squamous cell carcinoma and controls who harbored EV‐HPV DNA in their plucked eyebrow hair, similar results were obtained. The distributions of the p53 codon 72 genotypes in the basal cell carcinoma and malignant melanoma group were also not significantly different from the control group. p53 codon 72 arginine homozygosity does not appear to represent a significant risk factor for cutaneous squamous cell carcinoma and screening seems not to be indicated. Mol. Carcinog. 30:56–61, 2001.

The short arm of chromosome 11 likely is involved in the regulation of the human papillomavirus type 16 early enhancer-promoter and in the suppression of the transforming activity of the viral DNA.

Loci on the short arm of chromosome 11 between 11p11 and 11p15 likely are involved directly or indirectly in the regulation of the HPV-16 enhancer-promoter strength and this may contribute to the control of the level of viral early gene expression. Transient expression assays have shown that the HPV-16 enhancer-promoter functions much stronger in fibroblasts in which this region of chromosome 11 (del-11 cells) is deleted than in normal diploid human embryonic fibroblasts. The differential regulation of the HPV-16 long control region may be due either to the presence or activity of a cellular transcription factor which up-regulates HPV-16 early gene expression in del-11 cells or to a factor which down-regulates expression in diploid cells. Since the HPV-16 enhancer containing fragment (nt 7003 to nt 57) in combination with the SV40 promoter functions equally well in del-11 cells as in diploid cells a target of this factor likely is located in the putative HPV-16 early promoter region. The relative resistance of diploid human embryonic fibroblasts to HPV-16 induced transformation could be explained by the inactivity of the HPV-16 early promoter as these cells could be transformed by HPV-16 DNA constructs in which the early gene expression was driven from a heterologous enhancer-promoter. These results may indicate that loci on the short arm could suppress HPV-16-induced transformation by down-regulating the activity of the viral early promoter.

Transcriptional regulation of human papillomavirus type 16 LCR by different C/EBPβ isoforms

During genital human papillomavirus (HPV) infection several cytokines are released, such as interleukin‐1 (IL‐1), tumor necrosis factorα (TNFα), IL‐6, and IL‐8. These cytokines may play a role in the immune surveillance against viral infection. Two of these cytokines, IL‐1 and TNFα, suppress the transcription of the HPV16 early genes. CAATT/enhancer binding proteinβ (C/EBPβ), which is activated by IL‐1 and TNFα, has been suggested to act as a mediator of this transcriptional downregulation. C/EBPβ contains three different translation initiation sites that can lead probably by leaky ribosome scanning to the generation of three isoforms of C/EBPβ, namely full‐length C/EBPβ, liver enriched transcriptional activator protein (LAP), and liver enriched inhibitory protein (LIP). When transiently expressed in C33A and HeLa cells, the first two C/EBPβ isoforms activate the HPV16 long control region (LCR). LIP, which acts as an antagonist of C/EBPβ, represses the HPV16 LCR activity. Our observation that treatment of HeLa cells with IL‐1 leads to induction of LIP supports the hypothesis that the LCR downregulation by IL‐1 is mediated by LIP. Mol. Carcinog. 28:42–50, 2000.

Human papillomavirus type 16 transcripts expressed from viral-cellular junctions and full-length viral copies in CaSki cells and in a cervical carcinoma

We have mapped using the RNA PCR the viral-cellular junctions of HPV16 viral-cellular cotranscripts expressed in CaSki cells and a cervical carcinoma to nt 3728 and 881, respectively. Both junctions were located within the E1-E2 region. Examination of the cellular sequences of the cotranscripts showed the presence of a polyadenylation signal in each of the transcripts. In CaSki cells and in the cervical carcinoma transcripts derived from the full-length early region including the E2 transcript were also detected. Our results suggest that the utilization of a cellular polyadenylation site could be important in the development of cancer by HPV.

Inhibition of human papillomavirus-16 long control region activity by interferon-gamma overcome by p300 overexpression.

Although interferons (IFNs) are currently used in the treatment of various human papillomavirus (HPV)‐associated lesions, their mechanisms of action are still unclear. In this study, we clearly demonstrated that IFN‐γ was a strong inhibitor of HPV‐16 long control region (LCR) activity in two human cervical carcinoma cell lines. The effect of IFN‐γ was dose dependent. We investigated whether the effect of IFN‐γ on HPV‐16 LCR could involve the inhibition of the CREB‐binding protein (CBP)/p300 family of transcriptional coactivators. In support of this model, we demonstrated by transfection experiments that a 12S E1A mutant (RG2), which interacts poorly with p300 and CBP in comparison to wild‐type E1A, was less able to repress human papillomavirus (HPV) 16 long control region (LCR) than wild‐type E1A. More important, overexpression of p300 was able to increase the HPV‐16 LCR activity and to overcome inhibition by IFN‐γ. Finally, we demonstrated that p300 could cooperate with c‐jun to activate HPV‐16 LCR. According to our results, IFN‐γ might inhibit HPV‐16 LCR transcription by activating the signal transducer and activator of transcription 1α, which in turn might compete for p300/CBP binding with specific transcription factors involved in LCR activation.