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M Dürst

Publications and source records attributed to M Dürst.

At least 37 records · Page 2Linked to original sources
PubMed1997

Specific chromosomal imbalances in human papillomavirus-transfected cells during progression toward immortality.

High risk human papillomaviruses (HPVs) known to be closely associated with cervical cancer, such as HPV16 and HPV18, have the potential to immortalize human epithelial cells in culture. Four lines of HPV-transfected keratinocytes were analyzed by comparative genomic hybridization at different time points after transfection. A number of chromosomal imbalances was found to be highly characteristic for the cultures progressing toward immortality. Whereas several of these were new and previously not found as recurrent aberrations in cervical tumors, some were identical to chromosomal changes observed during cervical carcinogenesis. The data put new emphasis on the studied cell system as a relevant model for HPV-induced pathogenesis.

Cell Line↗
PubMed1997

Detection and characterization of human papillomavirus type 45 DNA in the cervical carcinoma cell line MS751.

The cervical carcinoma-derived cell line MS751 was examined for human papillomavirus (HPV) DNA and RNA. A genomic fragment containing both viral and cellular sequences was cloned. Sequence analysis showed that MS751 cells contain a partially deleted HPV-45 genome integrated at a single chromosomal site. HPV sequences from the E6-E7 region are expressed as poly(A)+ RNA.

Base Sequence↗
PubMed1997

Interaction of human papillomavirus type 16 and adeno-associated virus type 2 co-infecting human cervical epithelium.

Recently, we hypothesized that the tumour-suppressive, human helper-virus-dependent, adeno-associated parvoviruses (AAV) may interfere with transforming functions of human papillomaviruses (HPV) in the development of cervical carcinoma. Here, we demonstrate that in cervical epithelium containing papillomavirus DNA, AAV DNA can be detected in a replication-competent form and that AAV proteins are expressed. In cultured cells containing integrated AAV-2 DNA, transfection of HPV-16 DNA induced rescue of infectious AAV-2, revealing helper functions of HPV-16. Similarly, cotransfection of HPV-16 and AAV-2 DNAs into human epithelial cell lines led to replication of AAV-2, and, in keratinocytes, to a cytopathic effect. These data suggest an interaction of the two viruses, possibly influencing the development of HPV-related lesions.

Animals↗
PubMed1996

Promoter usage in the E7 ORF of HPV16 correlates with epithelial differentiation and is largely confined to low-grade genital neoplasia.

Human papillomavirus type 16 (HPV16) transcription in HPV16-positive vulvar intraepithelial neoplasia (VIN), cervical intraepithelial neoplasia (CIN) and cervical carcinomas was analyzed using RNA-RNA in situ hybridization. Subgenomic probes were constructed which specifically detected individual spliced E6/E7 transcripts as well as transcripts initiated within the E7 open reading frame (ORF). In most biopsies, viral RNA was predominantly initiated in the E6 ORF at promoter P97 and contained the E6*I splice. Three of 7 VIN, 13 of 37 CIN and I of 13 cervical carcinomas expressed significant amounts of mRNA that were initiated within the E7 ORF. Promoter activity in the E7 ORF correlated with epithelial differentiation and viral late gene (LI) expression. Our data therefore do not support the finding of Böhm et al. (1993) which suggested that the predominant transcript(s) in HPV16-associated high-grade neoplasms and genital carcinomas is initiated within the E7 ORF. Rather, our data suggest that the major HPV16 transcript in high-grade cervical neoplasms and carcinomas is initiated in the E6 ORF and encodes the E7 oncoprotein.

Animals↗
PubMed1995

Malignant progression of an HPV16-immortalized human keratinocyte cell line (HPKIA) in vitro.

The DNA of human papillomavirus (HPV) types found in cervical carcinomas can immortalize primary human keratinocytes. However, in analogy to tumor progression in vivo, HPV-immortalized keratinocytes require secondary events for malignant conversion. Here, we report on an HPV16-immortalized keratinocyte cell line (HPKIA) which after gamma-irradiation and long term culturing in vitro has acquired the ability to form squamous cell carcinomas in nude mice. The HPV16 integration locus and the viral transcript pattern of HPKIA cells at different passages have remained unaltered. A difference in cytokeratin expression was noted for HPKIA-induced cysts and HPKIA-induced carcinomas. In addition to the expression of suprabasal markers such as cytokeratin 10 and involucrin, carcinomas also express cytokeratin 8 and 18. The latter cytokeratin pair is often expressed in high-grade cervical neoplasia and cervical squamous cell carcinomas. Extensive cytogenetic analyses of nontumorigenic HPKIA cells and their tumorigenic segregants has revealed no single chromosomal abnormality which is confined to all tumorigenic cells. A consistent net loss of chromosomes 3, 5, 9, 12, and 22 was evident for all malignant cells. HPKIA cells represent all stages of transformation and are thus useful for defining secondary genetic events that potentially mark malignant progression in human cells in vivo.

Animals↗
PubMed1995

Epithelial differentiation fails to support replication of cloned human papillomavirus type 16 DNA in transfected keratinocytes.

Research on the life cycle of human papillomaviruses (HPVs) has suffered from the lack of a model system that allows the use of molecularly cloned HPV DNA. In this study, we analyzed replication of molecularly cloned HPV-16 genomes after transfection into cells of two human keratinocyte cell lines. Transfected cells were grown in cell culture (in vitro) or in transplantation chambers on the flanks of nude mice (in vivo). When DNA was extracted after different time intervals, replication of HPV-16 DNA could not be detected. Even the formation of a stratified epithelium under in vivo conditions failed to support vegetative replication. In contrast, transfection of molecularly cloned HPV-11 DNA resulted in replication of viral DNA in vitro. It seems likely that besides epithelial cell differentiation, a number of other factors influence HPV-16 replication.

Animals↗
PubMed1995

Expression of CD44 splice variants in normal, dysplastic, and neoplastic cervical epithelium.

Expression of splice variants of the CD44 adhesion molecule has been implicated in metastatic spread of various human tumor cells, including malignant lymphomas and colon, mammary, and gastric carcinomas, and has been correlated to a poor prognosis for the respective patients. To determine whether variant CD44 molecules might also contribute to the metastatic spread of cervical cancer, we analyzed the CD44 expression pattern in normal cervical epithelium and in low- and high-grade cervical dysplasia and compared it with invasive and metastasizing cervical cancers, including cell lines derived thereof by immunofluorescence and exon-specific PCR amplification of reverse-transcribed CD44 transcripts. We observed that normal cervical epithelium and dysplastic lesions express high levels of standard CD44 in the basal and spinous epithelial layers. CD44 molecules encompassing variant exons v5 and v6 are strongly expressed throughout the epithelium. Low levels of variants encompassing exon v7 are expressed in basal and spinous layers, with particular strength in the suprabasal layer. Low levels of epitopes encoded by v8 and v10 are expressed in the basal and spinous layers. In cervical cancers, including lymph node metastasis, we observed strong expression of v5 and v6, but almost no expression of v7, v8, and v10. Expression of the CD44 standard form appeared to be down-regulated in some cancers when compared to normal cervical epithelium. Thus, expression of variant CD44 molecules is related to distinct differentiation of mucosal squamous epithelia in the female genital tract. No correlation of the expression of variant CD44 isoforms, including the v7-v8 fusion epitope with tumor progression or lymphatic spread, was observed.

Alternative Splicing↗
PubMed1994

Genetic analysis of an in vitro model system for human papillomavirus type 16-associated tumorigenesis.

We have investigated the genetic basis for malignant progression of an human papillomavirus type 16 immortalized human keratinocyte cell line (HPKIA) by somatic cell fusion. In this cell culture model system for human papillomavirus type 16-associated tumorigenesis, early passage cells (HPKIA-nt) are nontumorigenic, whereas gamma-irradiated late passage cells (HPKIA-t) are tumorigenic when tested in nude mice. Fusion of HPKIA-t cells with normal human keratinocytes or with HPKIA-nt gave rise to hybrid clones in which the tumorigenic phenotype is suppressed. This observation is in line with complementation studies conducted for other tumor cell systems and indicates that, also in the case of HPKIA-t cells, the tumorigenic phenotype is recessive. Hybrid clones derived by fusion of HPKIA-t with cells of the cervical carcinoma cell lines C4-I, HeLa, or C33A were also nontumorigenic. However, the tumorigenic phenotype was retained in all of the HPKIA-t x SiHa and HPKIA-t x MS751 hybrids tested. The cervical carcinoma cell lines SiHa and MS751, therefore, cannot complement the gene defect(s) related to tumor growth of HPKIA-t cells. This may suggest that HPKIA-t, SiHa, and MS751 cells share the same defect(s) in regulatory pathway(s) involved in tumor suppression. Differences in the steady state levels of viral E6-E7 transcripts were noted for most of the hybrid cells. The observations of this study underline the potential use of nontumorigenic and tumorigenic HPKIA cells to define genetic differences that may also correlate with tumor progression in vivo.

Animals↗
PubMed1993

Major histocompatibility complex and human papillomavirus type 16 E7 expression in high-grade vulvar lesions.

To determine whether expression of the human papillomavirus (HPV) type 16 E7 open reading frame influences expression of major histocompatibility complex (MHC) antigens on the surface of squamous epithelial cells, serial frozen sections from seven HPV type 16-positive, high-grade vulvar intraepithelial neoplasia (VIN 2-3) lesions were tested for viral transcription by RNA-RNA in situ hybridization, for MHC expression by immunohistochemical staining with antibodies to MHC class I and II molecules, and for keratinocyte differentiation by immunohistochemical staining with anti-filaggrin and cytokeratin 10 antibodies. Despite the histologic appearance of high-grade VIN lesions, expression patterns of cytokeratin 10 and filaggrin suggested a certain degree of keratinocyte differentiation in all specimens. These differentiation markers were especially prominent in parakeratotic and hyperkeratotic superficial areas, which did not express MHC antigens or contain E7 mRNA. Expression of MHC class I molecules within dysplastic tissues was greater than within HPV type 16-negative, normal vulvar epithelium from the same patients. In five of the VIN 2-3 specimens anti-MHC class I antibodies reacted more strongly with cells of the basal and suprabasal layers than with cells of the epithelial surface. In one lesion basal cells stained less intensively than surface cells, whereas in another specimen all epithelial layers were equally MHC class I positive. Staining with anti-MHC class II antibodies was generally restricted to isolated foci, representing invading lymphocytes, tissue macrophages, and Langerhans cells. In two lesions, however, there was heterogeneous keratinocyte expression of MHC class II proteins, perhaps due to inflammation. Major histocompatibility complex antigen detection was independent of the presence or distribution pattern of E7-specific transcripts. Hence, a correlation between MHC and E7 expression appears unlikely in warty VIN lesions.

Adult↗
PubMed1993

Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.

The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection.

Animals↗
PubMed1992

Regulation of growth and gene expression in human papillomavirus-transformed keratinocytes by transforming growth factor-beta: implications for the control of papillomavirus infection.

Cervical carcinogenesis is a multistep process that appears to be initiated by infection of squamous epithelial cells in the cervix with one of a limited number of human papillomavirus (HPV) types. However, the mechanisms involved in the evolution of benign, HPV-induced lesions to malignancy have not yet been fully elucidated. Transforming growth factor-beta (TGF-beta), a multifunctional growth factor produced by cells in the skin, inhibits the proliferation of foreskin and cervical keratinocytes in vitro. We examined the effects of TGF-beta on growth and virus early-gene expression in cell lines immortalized by two HPV types associated with cervical carcinogenesis as well as the expression of TGF-beta 1 mRNA transcripts in normal and HPV-positive cells in vivo and in vitro. We found that normal and HPV-positive cells expressed similar levels of TGF-beta 1 mRNAs and exhibited similar patterns of responsiveness to three isoforms of TGF-beta in both monolayer and modified organotypic cultures. Of particular interest is our finding that the expression of the E6 and E7 early viral transforming regions of both HPV16 and HPV18 was reversibly and rapidly inhibited by TGF-beta. In one HPV16-positive cell line examined in detail, inhibition of HPV expression required protein synthesis and occurred at the level of transcription. HPV-immortalized cells selected for resistance to in vitro differentiation signals remained sensitive to TGF-beta-mediated growth inhibition. These results, showing that both growth and virus gene expression in HPV-transformed cells were responsive to TGF-beta, suggest that endogenous growth factors produced by different cell types in squamous epithelium may play a role in the progression of cervical neoplasia.

Blotting, Northern↗
PubMed1992

Human papillomavirus type 16 (HPV 16) gene expression and DNA replication in cervical neoplasia: analysis by in situ hybridization.

We have analyzed human papillomavirus (HPV) type 16 RNA expression in premalignant cervical lesions of different severity and in squamous cervical cancers by RNA-RNA in situ hybridization in order to find differences in the topographic distribution of viral RNA, which might correlate with the severity of disease. In the basal layer of low-grade squamous intraepithelial lesions (SIL) only weak transcription of viral early genes was observed. Signal intensity increased strongly in the more differentiated cells accompanied by high levels of HPV DNA replication. This pattern of viral gene expression, together with the onset of viral late transcription in the upper differentiated layer of the epithelium, most likely reflects the productive phase of viral infection. In contrast, in high-grade SIL viral transcription was comparatively strong in basal cells and evenly distributed throughout the undifferentiated epithelium. This difference of viral transcription in the basal layer of the respective lesions points to an altered regulation of viral gene expression which may be causally linked to the progression of precursor lesions. Evidence for disrupted expression of 3' early genes (E2, E4, and E5), analogous to the situation in HPV-DNA containing cervical carcinoma-derived cell lines, was not found in any of the HPV-16-positive premalignant lesions nor in the majority of cancers. The similarity of the viral transcription pattern of high-grade SIL and cancers suggests that additional host gene alterations are necessary for malignant progression.

Biopsy↗
PubMed1992

Geographical dependence of sequence variation in the E7 gene of human papillomavirus type 16.

We have determined nucleotide sequences of the E7 open reading frame (ORF) of human papillomavirus type 16 (HPV-16) isolates obtained from 32 genital tumours and two HPV-16-transformed human keratinocyte cell lines. In comparison to the prototype HPV-16 isolated from a German cervical cancer biopsy, no sequence variations were noticed in either the two cell lines or the 10 biopsies that were obtained from German patients. In contrast only three of 22 (13.6%) of Tanzanian isolates showed the prototype sequence. In 18 of these biopsies two alterations (T to C and T to G) not affecting the amino acid sequence were found within the HPV-16 E7 ORF (nucleotide positions 789 and 795) but eight of these isolates contained an additional change (nucleotide position 647) coding for serine instead of asparagine (amino acid position 29). One tumour harbours HPV-16 DNA with a mutation (C to T) at nucleotide 790 changing the E7 amino acid sequence (arginine to cysteine) at position 76. Our findings suggest that clustering of E7 sequence variants may occur in different geographical regions of the world.

Amino Acid Sequence↗
PubMed1992

Sense and antisense transcripts of human papillomavirus type 16 in cervical cancers.

Human papillomavirus type 16 (HPV-16) transcription was analysed in one squamous cervical carcinoma by cDNA cloning and DNA sequencing, and in eight additional squamous cervical carcinomas and 11 precancerous lesions by RNA-RNA in situ hybridization. The nucleotide sequences of the cDNA clones revealed structures of early HPV-16 mRNAs (E6*-E7-E1 E4-E5) in agreement with data reported for other premalignant and malignant tumours. cDNA clones possibly representing viral RNA of antisense orientation were also detected. These RNAs included sequences of the upstream regulatory region, part of the early and the late region of the genome. In three of eight squamous cervical carcinomas examined by in situ hybridization, signals specific for viral antisense RNA were also found. The antisense RNAs had a predominantly nuclear localization. Viral antisense RNA could not be detected in any of 11 HPV-16-positive premalignant lesions. The expression of HPV antisense RNA is likely to be linked to viral integration into the host genome. The possible effects of viral antisense transcription with regard to tumour progression remain to be determined.

Female↗
PubMed1992

Molecular variants of human papillomavirus type 16 from four continents suggest ancient pandemic spread of the virus and its coevolution with humankind.

We have amplified by the polymerase chain reaction, cloned, and sequenced genomic segments of 118 human papillomavirus type 16 (HPV-16) isolates from 76 cervical biopsy, 14 cervical smear, 3 vulval biopsy, 2 penile biopsy, 2 anal biopsy, and 1 vaginal biopsy sample and two cell lines. The specimens were taken from patients in four countries--Singapore, Brazil, Tanzania, and Germany. The sequence of a 364-bp fragment of the long control region of the virus revealed 38 variants, most of which differed by one or several point mutations. Phylogenetic trees were constructed by distance matrix methods and a transformation series approach. The trees based on the long control region were supported by another set based on the complete E5 protein-coding region. Both sets had two main branches. Nearly all of the variants from Tanzania were assigned to one (African) branch, and all of the German and most of the Singaporean variants were assigned to the other (Eurasian) branch. While some German and Singaporean variants were identical, each group also contained variants that formed unique branches. In contrast to the group-internal homogeneity of the Singaporean, German, and Tanzanian variants, the Brazilian variants were clearly divided between the two branches. Exceptions to this were the seven Singaporean isolates with mutational patterns typical of the Tanzanian isolates. The data suggest that HPV-16 evolved separately for a long period in Africa and Eurasia. Representatives of both branches may have been transferred to Brazil via past colonial immigration. The comparable efficiencies of transfer of the African and the Eurasian variants to the New World suggest pandemic spread of HPV-16 in past centuries. Representatives of the African branch were possibly transferred to the Far East along old Arab and Indonesian sailing routes. Our data also support the view that HPV-16 is a well-defined virus type, since the variants show only a maximal genomic divergence of about 5%. The small amount of divergence in any one geographic location and the lack of marked divergence between the Tanzanian and Brazilian African genome variants two centuries after their likely introduction into the New World suggest a very slow rate of viral evolution. The phylogenetic tree therefore probably represents a minimum of several centuries of evolution, if not an age equal to that of the respective human races.

Base Sequence↗
PubMed1992

Binding of the human E2F transcription factor to the retinoblastoma protein but not to cyclin A is abolished in HPV-16-immortalized cells.

The adenovirus E1A, SV40 large T and papillomavirus E7 proteins immortalize primary cells by virtue of their ability to bind the retinoblastoma gene product (pRB) and other cellular proteins, including cyclin A and the prRB-related protein, p107. It has been demonstrated that these viral oncogene products will prevent the inhibition of positive growth regulators by pRB, one of them being the E2F transcription factor. Here we show that the interactions of pRB and cyclin A with E2F are present also in normal keratinocytes and in primary human fibroblasts. In human keratinocytes immortalized by human papillomavirus 16 (HPV-16), expressing high levels of HPV-16 E7 protein, complexes between E2F and pRB are disrupted. In this cell line, as well as in HeLa cells which express HPV-18 E7, complexes containing E2F and cyclin A are maintained, indicating that this interaction is not sensitive to the viral oncoprotein and that cyclin A can associate with E2F independently of pRB. In vitro binding experiments suggest that the E7 gene product is able to preferentially abolish the interaction of pRB with E2F, leaving the cyclin A complexes intact. Our findings suggest that E7-dependent immortalization of human cells is associated with modifications of E2F multiprotein complexes.

Base Sequence↗
PubMed1991

Viral transcription in human keratinocyte cell lines immortalized by human papillomavirus type-16.

Human papillomavirus type-16 (HPV-16) transcription in two human keratinocyte cell lines (HPK) immortalized by transfection of viral DNA in vitro was analyzed by nucleotide sequencing of cDNA clones, and in addition by primer extension analysis and S1 nuclease and exonuclease VII digestion of poly(A)+ RNA. A novel mRNA species which probably initiates in the E7 ORF and in which the 5'-part of the E1 ORF (splice donor at position (pos.) 880) is joined to an exon comprising the entire E2 ORF (splice acceptor at pos. 2708) was found in both cell lines. This mRNA has the potential to encode a full-length E2 protein, which is known to function as a repressor of transcription initiated at P97. cDNAs derived from the late region of the viral genome and the use of a late polyadenylation signal at pos. 7320-7325 are described. In agreement with RNA data published by others the major promoter for HPV-16 transcription is located at pos. 97. mRNA species encoding full-length or truncated forms of the E6 protein, and species characterized by an E1i [symbol see text] E4 splice junction (which provided the E4 open reading frame (ORF) with an ATG triplet) were identified.

Base Sequence↗