Biomedical subjects

Thomas Iftner

Publications and source records attributed to Thomas Iftner.

15 recordsLinked to original sources

PubMed2007

Changes of epidermodysplasia verruciformis in benign skin lesions: the EV acanthoma.

Foci of histological changes of epidermodysplasia verruciformis (EV) were noted in five benign skin lesions. These skin lesions included an intradermal nevus, a pigmented seborrheic keratosis, an isolated papule on the forearm, a perianal lesion, and an acantholytic acanthoma. Because the changes resembled true EV so strongly despite the absence of clinical EV in these patients, we searched for EV-human papilloma virus (HPV) types in these skin lesions. Polymerase chain reaction (PCR) analysis on the formalin-fixed, paraffin-embedded blocks was performed. As a positive control, we included tissue from two HIV-positive patients with clinical EV proven by biopsy. Studies were also performed on five other archived biopsies that did not show changes of EV on multiple tissue sections. A nested PCR method detected EV-HPV types in three of the five benign skin lesions showing EV changes as well as in the positive controls. EV changes and EV-HPV can be found incidentally on biopsy in the absence of clinical EV; when such changes are the major histopathological finding in an isolated skin lesion, the lesion should be termed an EV acanthoma.

Acanthoma↗

PubMed2006

The absolute risk of cervical abnormalities in high-risk human papillomavirus-positive, cytologically normal women over a 10-year period.

In spite of the success of cervical cytology as a cancer-screening tool, it has important limitations, and human papillomavirus (HPV) testing may be valuable in future screening. The majority of women in screened populations, who test HPV positive, will have a concurrent normal smear, and we need more information about the risk for subsequent high-grade cervical lesions in these women. We examined 8,656 younger women (22-32 years old) and 1,578 older women (40-50 years old) who were followed for development of cervical neoplasia (cytology and/or histology) through the Danish Pathology Data Bank. We estimated the proportion of women developing cervical lesions of different types before a given time point as a function of time. Among women with normal cytology and positive high-risk Hybrid Capture 2 (HC2) test, 17.7% and 24.5% of younger and older women, respectively, had a subsequent abnormal Pap smear within 5 years. The risk of CIN3 or cancer within 10 years among younger women with positive HC2 test was 13.6% (10.9-16.2) and 21.2% (2.7-36.1) among older women. An analysis among younger women also being HC2-positive 2 years before baseline showed a subsequent 10-year risk of > or =CIN3 of 18% (14.6-21.5). Among older women where HPV may be added to general screening, the estimated absolute risk of > or =CIN3 in HC2-positive women was more than 20% within 10 years. These results indicate that even a single positive HPV test in cytologically negative women is substantially predictive of high-grade CIN and suggest that HC2 testing can help stratify women into different risk categories.

Adult↗

PubMed2006

Overview of the European and North American studies on HPV testing in primary cervical cancer screening.

Several studies suggest that HPV testing is more sensitive than cytology in primary cervical screening. These studies had different designs and were reported in different ways. Individual patient data were collected for all European and North American studies in which cytology was routinely performed and HPV testing was included as an additional parallel test. More than 60,000 women were included. The sensitivity and specificity of HPV testing were compared with routine cytology, both overall and for ages <35, 35-49 and 50+. The age-specific prevalence of high risk HPV (hr-HPV) was also analysed. HPV testing was substantially more sensitive in detecting CIN2+ than cytology (96.1% vs. 53.0%) but less specific (90.7% vs. 96.3%). The sensitivity of HPV testing was similar in all studies carried out in different areas of Europe and North America, whereas the sensitivity of cytology was highly variable. HPV sensitivity was uniformly high at all ages, whereas the sensitivity of cytology was substantially better in women over the age of 50 than in younger women (79.3% vs. 59.6%). The specificity of both tests increased with age. Positivity rates for HPV testing in women without high-grade CIN were region dependent. These results support the use of HPV testing as the sole primary screening test, with cytology reserved for women who test HPV positive. Large demonstration projects are needed to fully evaluate this strategy.

Adolescent↗

PubMed2006

Gene expression profiles reveal an upregulation of E2F and downregulation of interferon targets by HPV18 but no changes between keratinocytes with integrated or episomal viral genomes.

Persistent infections with human papillomaviruses type 18 can result in the development of cervical cancer. HPV18 genomes persist extrachromosomally in low-grade and precancerous lesions but are always integrated in cervical cancers, and this might contribute to the progression of HPV18-induced lesions. To address whether integration induces additional changes in host cells, several keratinocyte lines with wild type and replication-deficient E1 mutant HPV18 (E1C-TTL) genomes were analyzed with high density oligonucleotide arrays. In comparison to normal keratinocytes, wild type and integrated E1C-TTL HPV18 genomes deregulate the expression of 280 annotated genes. However, the comparison of wild type with E1C-TTL cell lines did not reveal any significant differences, indicating that neither the loss of E1 nor viral integration induces additional gene expression changes in low passage HPV18-positive keratinocytes. Half of the deregulated genes have been described as targets of the p16/Rb/E2F, p53, interferon or NFkappaB pathways consistent with the functions ascribed to the viral E6 and E7 oncoproteins, but the other half can currently not be ascribed to certain pathways.

Cell Line↗

PubMed2006

Disruption of the Y-box binding protein-1 results in suppression of the epidermal growth factor receptor and HER-2.

The overexpression of the epidermal growth factor receptor (EGFR) and HER-2 underpin the growth of aggressive breast cancer; still, it is unclear what governs the regulation of these receptors. Our laboratories recently determined that the Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, induced breast tumor cell growth in monolayer and in soft agar. Importantly, mutating YB-1 at Ser(102), which resides in the DNA-binding domain, prevented growth induction. We reasoned that the underlying cause for growth attenuation by YB-1(Ser(102)) is through the regulation of EGFR and/or HER-2. The initial link between YB-1 and these receptors was sought by screening primary tumor tissue microarrays. We determined that YB-1 (n = 389 cases) was positively associated with EGFR (P < 0.001, r = 0.213), HER-2 (P = 0.008, r = 0.157), and Ki67 (P < 0.0002, r = 0.219). It was inversely linked to the estrogen receptor (P < 0.001, r = -0.291). Overexpression of YB-1 in a breast cancer cell line increased HER-2 and EGFR. Alternatively, mutation of YB-1 at Ser(102) > Ala(102) prevented the induction of these receptors and rendered the cells less responsive to EGF. The mutant YB-1 protein was also unable to optimally bind to the EGFR and HER-2 promoters based on chromatin immunoprecipitation. Furthermore, knocking down YB-1 with small interfering RNA suppressed the expression of EGFR and HER-2. This was coupled with a decrease in tumor cell growth. In conclusion, YB-1(Ser(102)) is a point of molecular vulnerability for maintaining the expression of EGFR and HER-2. Targeting YB-1 or more specifically YB-1(Ser(102)) are novel approaches to inhibiting the expression of these receptors to ultimately suppress tumor cell growth.

Breast Neoplasms↗

PubMed2006

Comparative analysis of 19 genital human papillomavirus types with regard to p53 degradation, immortalization, phylogeny, and epidemiologic risk classification.

We have analyzed E6 proteins of 19 papillomaviruses able to infect genital tissue with regard to their ability to degrade p53 and the thus far unknown immortalization potential of the genomes of human papillomaviruses (HPV) 53, 56, 58, 61, 66, and 82 in primary human keratinocytes. E6 proteins of HPV types 16, 18, 33, 35, 39, 45, 51, 52, 56, 58, and 66, defined as high-risk types, were able to induce p53 degradation in vitro, and HPV18-, HPV56-, and HPV58-immortalized keratinocytes revealed markedly reduced levels of p53. In contrast, the E6 proteins of HPV6 and 11 and HPV44, 54, and 61, regarded as possible carcinogenic or low-risk HPV types, respectively, did not degrade p53. Interestingly, the E6 proteins of HPV 53, 70, and 82 inconsistently risk classified in the literature were also found to induce p53 degradation. The genomes of HPV53 and 82 immortalized primary human keratinocytes that revealed almost absent nuclear levels of p53. These data suggest a strict correlation between the biological properties of certain HPV types with conserved nucleotide sequence (phylogeny), which is largely coherent with epidemiologic risk classification. HPV types 16, 18, 33, 35, 39, 45, 51, 52, 56, 58, and 66, generally accepted as high-risk types, behaved in our assays biologically different from HPV types 6, 11, 44, 54, and 61. In contrast, HPV70, regarded as low-risk type, and HPV53 or HPV82, with inconsistent described risk status, were indistinguishable with respect to p53 degradation and immortalization from prototype high-risk HPV types. This could imply that other important functional differences exist between phylogenetically highly related viruses displaying similar biological properties in tissue culture that may affect their carcinogenicity in vivo.

Animals↗

PubMed2005

Papillomavirus E2 protein induces expression of the matrix metalloproteinase-9 via the extracellular signal-regulated kinase/activator protein-1 signaling pathway.

Papillomaviruses are involved in the development of cancers of the female cervix, head and neck, and skin. An excellent model to study papillomavirus-induced tumor induction and progression is the New Zealand White rabbit, where the skin is infected with the cottontail rabbit papillomavirus (CRPV). This leads to the formation of benign tumors that progress into invasive and metastasizing carcinomas without the need for cofactors. We have shown previously that specific mutations in the transactivation domain of the transcription/replication factor E2 cause a dramatic loss in the tumor induction efficiency of the viral genome and a major deficiency in tumor progression as we show now. By comparing wild-type (WT) and mutant E2-induced skin tumors, we found high levels of matrix metalloproteinase-9 (MMP-9) protein and transcripts in WT CRPV-E2-induced tumors in contrast to certain mutant CRPV-E2-induced papillomas and normal uninfected skin. Stable cell lines and reporter assays revealed that E2 from different papillomavirus types is able to transactivate the MMP-9 promoter via the promoter-proximal activator protein-1 (AP-1) site as shown in reporter gene assays with mutant MMP-9 promoter constructs. Furthermore, WT E2 but not mutant E2 strongly transactivated a minimal promoter reporter construct with multiple AP-1 sites. The MMP-9 protein induced in cells expressing E2 degrades collagen matrices as measured in Matrigel-based invasion/mobility assays. E2-induced MMP-9 expression can be blocked by a chemical inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (PD 098059), suggesting that E2 activates the MAPK/ERK signaling pathway, which is further supported by the induction of ERK1 in CRPV-E2-transfected cells.

Animals↗

PubMed2004

Gene profiling of cottontail rabbit papillomavirus-induced carcinomas identifies upregulated genes directly Involved in stroma invasion as shown by small interfering RNA-mediated gene silencing.

To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 zeta and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin beta, which are involved in cell motility and tissue invasion, as well as casein kinase 1 alpha, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin beta in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.

Animals↗

PubMed2003

The prevalence of human papillomavirus genotypes in nonmelanoma skin cancers of nonimmunosuppressed individuals identifies high-risk genital types as possible risk factors.

Nonmelanoma skin cancer is the most commonly diagnosed malignant disease in Caucasians. Known risk factors include fair skin, sun exposure, male gender, advancing age, and the presence of solar keratosis. No viral risk factors have been established thus far. To examine the association between nonmelanoma skin cancer and infection with human papilloma virus (HPV) types, we performed a retrospective study in which skin biopsies were collected from 496 nonimmunosuppressed patients attending dermatologic clinics during a defined period and for whom a biopsy or resection of a tumor was indicated for medical reasons. A total of 390 patients with histologically confirmed diagnosis of warts (n = 209), solar keratosis or Bowen's disease (n = 91), squamous cell carcinoma (n = 72), or basal cell carcinoma (n = 18), as well as 106 control patients with normal skin was analyzed for infection with HPV and, if positive, HPV typed by sequencing. Logistic regression was performed to separately investigate association of certain HPV types with the occurrence of warts, precancerous lesions, and skin cancer compared with normal skin. For all three histological groups, both crude risk and risk adjusted for age, sex, and sun exposure were calculated. HPV DNA was detected in only 4.7% of controls, in 90.9% of benign warts, in 60.4% of precancerous lesions, in 59.7% of squamous cell carcinoma, and in 27.8% of basal cell carcinoma, which demonstrates that viral infection is specifically linked to skin disorders. The distribution of viral types found is distinctly different between warts and precancers or cancers, supporting an etiologic role of specific HPV types. This is supported by statistical analysis, where after adjusting for age, gender, and sun exposure, the odds ratio for nonmelanoma skin cancer in patients who were DNA positive for the high-risk mucosal HPV types, 16, 31, 35, and 51 was 59 (95% confidence interval, 5.4-645) with normal skin as controls. These findings suggest that persistent infections of the skin with high risk genital HPV types recently identified as significant risk factors for cervical cancer may also represent a risk factor for nonmelanoma skin cancer in a nonimmunosuppressed population.

Adolescent↗

PubMed2003

Chapter 12: Human papillomavirus technologies.

Currently, human papillomavirus (HPV) DNA tests validated in large trials and epidemiological studies are the hybrid capture second-generation (HC2) HPV DNA assay and a variety of polymerase chain reaction (PCR) protocols employing degenerate or consensus primers. This article describes the currently available technology for HPV detection and discusses novel technologies and their potential for large-scale screening. Ideally, an HPV test should allow detection of multiple HPV types, identify individual types, and provide quantitative information about the viral load of each individual type found. Moreover, it should be easy to perform, be highly reproducible, with a high specificity and sensitivity, and amenable for high throughput analysis and automation. Because we do not yet fully understand the true value of viral load and the biological relevance of the different HPV types, any HPV test should be able to detect the clinically relevant high-risk types with a sufficient sensitivity of at least 10 000 genome copies per sample. To validate the different current and future test systems and to compare inter-laboratory performance we urgently need reference samples, validated reagents, and standardized protocols.

Biotechnology↗

PubMed2003

Identification of the E9/E2C cDNA and functional characterization of the gene product reveal a new repressor of transcription and replication in cottontail rabbit papillomavirus.

Cottontail rabbit papillomavirus (CRPV) genomes mutated in the trans-activation domain of the E2 protein, which stimulates both viral DNA replication and transcription, are severely impaired in their ability to induce tumors in New Zealand White rabbits. A number of papillomaviruses encode, in addition to full-length E2, a shortened E2 protein or an E2 protein fused to a short stretch of amino acids derived from the small E8 open reading frame that counteract the activities of E2. We identified and cloned the novel cDNA E9/E2C of CRPV from papillomas of New Zealand White and cottontail rabbits and characterized the functions of the encoded gene product. E9/E2C was shown to be a bona fide repressor of minimal viral promoters, with the E9 domain being essential for this activity, and to repress E1/E2-dependent replication of a CRPV origin construct. In addition, E9/E2C counteracted the transactivation effect of the full-length E2 on minimal promoters containing several E2 binding sites. To investigate the role of E9/E2C in tumorigenesis, we constructed two CRPV genomes mutated in E9/E2C. One, designated CRPV-E9atgmut-pLAII, contained a mutation in the unique start codon in the E9 open reading frame, and the second E9/E2C mutant was constructed by the introduction of a stop codon close to the splice donor site at nucleotide 3714 that additionally prevented the correct splicing of the transcript. When we infected New Zealand White rabbits with these constructs, we surprisingly noted no differences in tumor induction efficiency, viral genome copy number, and viral transcription in comparison to wild-type CRPV.

Animals↗

PubMed2003

The papillomavirus E8-E2C protein represses DNA replication from extrachromosomal origins.

Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8-E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8-E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8-E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8-E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8-E2C (1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8-E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.

Base Sequence↗

PubMed2002

Interference of papillomavirus E6 protein with single-strand break repair by interaction with XRCC1.

XRCC1 protein is required for the repair of DNA single-strand breaks and genetic stability, and is essential for viability in mammals. XRCC1 functions as a scaffold protein by interacting and modulating polypeptide components of the single-strand break repair machinery, including AP endonuclease-1, DNA ligase IIIalpha, poly (ADP-ribose) polymerase, DNA polymerase beta and human polynucleotide kinase. We show here that the E6 protein of human papillomavirus type 1, 8 and 16 directly binds XRCC1. When tested in CHO derived XRCC1 'knock out' EM9 cells, co-expression of human papillomavirus 16 E6 with human XRCC1 reduced the ability of the latter protein to correct the methyl methane sulfate sensitivity of XRCC1 mutant CHO cell line EM9. These data identify a novel link between small DNA tumour viruses and DNA repair pathways, and suggest a novel explanation for the development of genomic instability in tissue cells persistently infected with papillomaviruses.

Animals↗

PubMed2002

Factors associated with an increased risk of prevalent and incident grade III cervical intraepithelial neoplasia and invasive cervical cancer among women with Papanicolaou tests classified as grades I or II cervical intraepithelial neoplasia.

OBJECTIVE: Women with Papanicolaou tests classified as cervical intraepithelial neoplasia grade I or II are treated conservatively in many countries. However, these women are at an increased risk of having underlying prevalent and incident grade III cervical intraepithelial neoplasia and invasive cancer. This study was undertaken to identify factors that could predict these clinically important disease states. STUDY DESIGN: Five hundred women with Papanicolaou tests classified as persistent grade I or II cervical intraepithelial neoplasia underwent a repeat test, human papillomavirus testing with Hybrid Capture assay (Digene, Silver Spring, Md) and polymerase chain reaction, and colposcopy with histologic assessment. One hundred fifty-seven women with histologically proven grade I or II cervical intraepithelial neoplasia were monitored conservatively for a minimum of 9 months to assess predictors of incident grade III cervical intraepithelial neoplasia. RESULTS: One hundred fifty-one women with prevalent grade III cervical intraepithelial neoplasia and 5 women with prevalent invasive cancer were identified at the first colposcopy. A repeated Papanicolaou test classified as higher than grade II cervical intraepithelial neoplasia and detection of oncogenic human papillomavirus types were significant predictors of underlying grade III cervical intraepithelial neoplasia and cancer in the multivariate analysis. Seventeen of 157 women (10.8%) with grade I or II cervical intraepithelial neoplasia progressed to grade III cervical intraepithelial neoplasia. Age >30 years and detection of oncogenic human papillomavirus were significantly correlated with progression in the multivariate analysis. No progression was observed in women who were negative for human papillomavirus. CONCLUSION: The high rate of underlying prevalent grade III cervical intraepithelial neoplasia and cancer found in our study (31.2%) indicates that conservative management of women with persistent grade I or II cervical intraepithelial neoplasia should be discouraged. Colposcopy with histologic assessment should be recommended as the standard of care. However, for women with histologically proven grade I or II cervical intraepithelial neoplasia, subsequent conservative management was safe in our study for those who were negative for human papillomavirus by type-specific polymerase chain reaction.

Adolescent↗

PubMed2002

A transactivator function of cottontail rabbit papillomavirus e2 is essential for tumor induction in rabbits.

Infection of domestic rabbits with cottontail rabbit papillomavirus (CRPV) causes local papillomas which progress to carcinomas in more than 80% of cases. This animal model system therefore allows the identification of molecular mechanisms required for the induction and progression of epithelial tumors. The viral E2 protein stimulates both viral DNA replication and transcription, and these functions can be genetically separated. We introduced the respective mutations into CRPV E2 and found, in line with published data for other papillomavirus E2 proteins, that mutation of the highly conserved amino acid 37 or 73 resulted in replication-competent but transactivation-deficient E2 proteins, whereas E2 proteins with mutations at residue 39 were replication deficient and transactivation competent. The R37A, I73L, and I73A E2 mutants, showing a loss of transactivation function, and the R37K E2 mutant, which is still transactivation competent, were introduced into the whole genome of CRPV, which was then injected into the skin of rabbits. Strikingly, the ability to induce tumors within 6 weeks was abolished by each of the E2 mutations, in contrast to the tumor induction rate (93%) obtained with wild-type CRPV DNA. Two small papillomas induced by mutant E2 I73A CRPV DNA appeared as late as 12 or 24 weeks postinjection, were significantly smaller, and showed no further extension of growth. These data suggest that functionally conserved amino acids in the transactivation domain of E2 are also required for the induction and growth of epithelial tumors in rabbits infected with CRPV.

Animals↗