Biomedical subjects

M Dürst

Publications and source records attributed to M Dürst.

51 records · Page 3Linked to original sources

PubMed1991

Failure to detect human papillomavirus sequences at the 3p21 rearrangement site in pleomorphic adenomas.

Eighteen salivary gland pleomorphic adenomas, including nine with a t(3;8)(p21;q12) as the sole chromosome abnormality, were tested for the presence of human papillomavirus (HPV) DNA and for rearrangements of the cellular flanking regions of a HPV 16 integration site mapping to 3p21, as previously described in a cervical carcinoma. In none of the tumors could evidence for either HPV DNA or related sequences, or a rearranged allele of the integration site, be found.

Adenoma↗

PubMed1991

Inverse relationship between human papillomavirus (HPV) type 16 early gene expression and cell differentiation in nude mouse epithelial cysts and tumors induced by HPV-positive human cell lines.

Two human papillomavirus type 16 (HPV 16)-immortalized human keratinocyte cell lines (HPK) were shown to have retained the ability for differentiation after subcutaneous injection into nude mice. These properties were maintained even at late passage. HPK cells gave rise to transiently growing cysts which exhibited an epitheliumlike architecture. Moreover, differentiation-specific markers such as cytokeratin 10, involucrin, and filaggrin were shown to be expressed in an ordered succession. RNA-RNA in situ hybridization revealed heterogeneous and low levels of HPV 16 E6-E7 RNA in the basal layer of the cysts. In contrast, in progressively growing tumors induced by HPK cells containing an activated ras oncogene (EJ-ras) or in tumors induced by the cervical carcinoma cell line CaSki, high levels of E6-E7-specific RNA could be detected. Irrespective of the growth potential of these cell lines in nude mice, viral transcription was always more evident in the basal layer and in proliferatively active cells rather than in differentiated cells. This contrasts with viral gene expression in HPV 16 positive low-grade cervical dysplasia, in which abundant viral transcriptional activity was mapped to the upper third of the epithelium. It is suggested that the physical state of the viral DNA, i.e., integrated viral DNA in the cell lines as opposed to extrachromosomal DNA in low-grade cervical dysplasia, may influence viral gene regulation.

Animals↗

PubMed1991

The early genes E6 and E7 of cancer associated human papilloma viruses as targets of tumor suppression?

We have transplanted HPV 18 positive nontumorigenic hybrid cells and tumorigenic hybrid segregants as well as nontumorigenic and tumorigenic human keratinocytes immortalized by HPV 16-DNA transfection (HPK cells and HPK-ras cells) into nude mice and grown them under in vivo conditions for different periods of time. By analyzing gene expression at the mRNA level of the early viral genes E6/E7 and a number of cytoskeletal cellular genes we wanted to test the hypothesis that the nontumorigenic phenotype of these cells in vivo may be determined by the specific downregulation of expression of the oncogenic E6/E7 genes by the product(s) of tumor suppressor gene(s). The results obtained showed that: 1) The nontumorigenic hybrid cells (in contrast to tumorigenic segregants) stopped to proliferate about 3 days after transplantation. At this time E6/E7 gene expression was already drastically reduced, whereas at day 2 expression was still high. This suppression specifically affected the HPV 18 E6/E7 genes and preceded cell death by at least 10 days. 2) The same specific suppression of HPV E6/E7 gene expression occurred during in vivo growth of the nontumorigenic HPK cells. These cells gave rise to transiently growing cysts, but had retained the in vivo differentiation potential of normal keratinocytes (orderly expression of cytokeratins 1 and 10, involucrin and filaggrin). HPV 16 E6/E7 gene expression was very low and clearly restricted to a small subset of basal cells, thus showing an inverse relationship to terminal differentiation. In contrast, in the tumors induced by HPK-ras cells E6/E7 gene expression extended from the basal cells into suprabasal cells and terminal differentiation was retarded. These results support the hypothesis that during in vivo growth tumor suppressor gene(s) negatively regulated HPV E6/E7 gene expression resulting in the stop of proliferation of the nontumorigenic cells.

Animals↗

PubMed1990

Differential response of nontumorigenic and tumorigenic human papillomavirus type 16-positive epithelial cells to transforming growth factor beta 1.

The transforming growth factor (TGF) beta s are multifunctional polypeptide growth factors with diverse biological effects, including inhibition of epithelial cell proliferation both in vitro and in vivo. To investigate the possible role of TGF beta 1 in the regulation of papillomavirus infection and papillomavirus-associated transformation, we compared the response to TGF beta 1 of normal keratinocytes, human papillomavirus, type 16 (HPV 16)-positive-immortalized keratinocytes (nontumorigenic), and HPV 16-positive cervical carcinoma cells (tumorigenic) with respect to DNA synthesis and protooncogene expression. All HPV 16-immortalized cell lines were nearly as inhibited by TGF beta 1 as normal keratinocytes, whereas two cervical carcinoma cell lines (Caski and Siha) were refractory to growth inhibition by TGF beta 1. Cell surface receptors for TGF beta 1 were present on both normal and carcinoma cell lines. In all cases, growth inhibition by TGF beta 1 was accompanied by suppression of Steady-state levels of c-myc mRNA. In contrast, TGF beta 1 induced the expression of c-jun mRNA transcripts in normal, immortalized, and tumorigenic cells. We also studied the effect of TGF beta 1 on HPV 16 mRNA expression. Steady-state levels of HPV 16 mRNA transcripts were suppressed by TGF beta 1 in the nontumorigenic HPK cells but were unaffected in the tumorigenic lines. These findings suggest that TGF beta 1 may be an in vivo modulator of HPV infection and that loss of responsiveness to this growth inhibitory signal may be involved in HPV-associated malignant transformation.

Cell Division↗

PubMed1989

Glucocorticoid-enhanced neoplastic transformation of human keratinocytes by human papillomavirus type 16 and an activated ras oncogene.

Human papillomaviruses (HPV) are known etiological agents of benign proliferation of the skin and mucosa (papillomas and warts). They have also been implicated in the development of cervical dysplasia and anogenital carcinoma. The close association of HPV type 16 DNA with the majority of cervical carcinomas suggests the involvement of the virus in this type of cancer. We have developed an in vitro multistep model for human epithelial cell carcinogenesis. Primary human epidermal keratinocytes acquired an indefinite lifespan in response to transfection with HPV 16 DNA but did not undergo malignant conversion. Addition of Kirsten murine sarcoma virus (Ki-MSV), which contains an activated K-ras oncogene, to these cells induced morphological alterations associated with the acquisition of neoplastic properties. The frequency of transformation by Ki-MSV was markedly enhanced by the inclusion of glucocorticoid. At optimal conditions, a 125-fold stimulation was observed. These findings demonstrate the malignant conversion of human primary epithelial cells in culture by the cooperation of HPV type 16 and an activated ras oncogene and support a multistep process for neoplastic conversion. The availability of a human epithelial cell transformation model should facilitate studies of the interaction between HPV and human epithelial cells.

Blotting, Western↗

PubMed1988

Regional chromosome localization of human papillomavirus integration sites near fragile sites, oncogenes, and cancer chromosome breakpoints.

The integration sites of human papillomavirus (HPV) DNA within the cervical carcinoma cell line C4-I and a primary cervical tumor were mapped by in situ hybridization. Cloned cellular sequences flanking the integrated viral DNA were used as probes. For the cell line, the viral integration site was mapped to chromosome region 8q21-q22.3, while in the primary tumor chromosome band 3p21 was the target for integration. The HPV DNA integration appears to occur in the vicinity of fragile sites, oncogenes, and chromosome breakpoints that are characteristic of hematologic malignancies and solid tumors. The integration of HPV may thus promote chromosome changes in cancer cells.

Cells, Cultured↗

PubMed1988

Selective suppression of human papillomavirus transcription in non-tumorigenic cells by 5-azacytidine.

The transcription of human papillomavirus type 18 (HPV 18) is selectively suppressed in non-tumorigenic HeLa x fibroblast or HeLa x keratinocyte cell hybrids by 5-azacytidine. In contrast, viral gene expression is not influenced by 5-azacytidine in both tumorigenic hybrid segregants and in the parental HeLa cells. The suppression mechanism seems to operate at the level of initiation of transcription since nuclear run-on experiments show the absence of elongated nascent viral RNA, whereas the transcription of cellular reference genes remains unaffected. Down-regulation of HPV 18 mRNA correlates directly with cessation of cellular growth and can be abolished using the protein synthesis inhibitor cycloheximide. Furthermore human keratinocytes immortalized by HPV 16 but still retaining the non-tumorigenic phenotype reveal the same inhibitory effect on viral transcription after treatment with 5-azacytidine. These results support a model of a postulated intracellular control mechanism, directed against papillomavirus transcription, which can be induced by 5-azacytidine and appears to correlate with the presence of specific chromosomes in non-tumorigenic cells.

Azacitidine↗

PubMed1987

Transcriptional regulation of the human papillomavirus-16 E6-E7 promoter by a keratinocyte-dependent enhancer, and by viral E2 trans-activator and repressor gene products: implications for cervical carcinogenesis.

The transcriptional promoter of the candidate E6-E7 transforming gene region of human papillomavirus (HPV)-16 (P97) was active in transiently transfected cervical carcinoma cells when linked to the HSV-1 tk or bacterial cat genes. Sequences 5' to P97 contain a short enhancer element responding to cellular factor(s) in uninfected human foreskin keratinocytes and in cervical carcinoma cells, but not in human or animal fibroblasts. The E2 trans-activator products of HPV-16 or of the related bovine papillomavirus (BPV)-1 further elevated HPV-16-driven transcripts in co-transfections, and required the presence of E2-binding ACC(N)6GGT cores in cis. A 'short E2' C-terminal repressor gene product (sE2) of HPV-16 or the BPV-1 sE2 repressor not only inhibited viral E2 trans-activation, but also suppressed enhancer response to keratinocytic factors. Suppression by the sE2 products was abolished by deletion of the E2-binding cores in cis or by a mutation in the sE2 DNA binding domain. The keratinocyte-dependent enhancer is likely to contribute to the epithelial cell tropism of HPV-16, and may direct persistent E6-E7 gene transcription in response to cellular factors in cervical carcinoma cells in which the viral E2 genes are inactive.

Animals↗

PubMed1987

Papillomavirus sequences integrate near cellular oncogenes in some cervical carcinomas.

The chromosomal locations of cellular sequences flanking integrated papillomavirus DNA in four cervical carcinoma cell lines and a primary cervical carcinoma have been determined. The two human papillomavirus (HPV) 16 flanking sequences derived from the tumor were localized to chromosome regions 20pter----20q13 and 3p25----3qter, regions that also contain the protooncogenes c-src-1 and c-raf-1, respectively. The HPV 16 integration site in the SiHa cervical carcinoma-derived cell line is in chromosome region 13q14----13q32. The HPV 18 integration site in SW756 cervical carcinoma cells is in chromosome 12 but is not closely linked to the Ki-ras2 gene. Finally, in two cervical carcinoma cell lines, HeLa and C4-I, HPV 18 DNA is integrated in chromosome 8, 5' of the c-myc gene. The HeLa HPV 18 integration site is within 40 kilobases 5' of the c-myc gene, inside the HL60 amplification unit surrounding and including the c-myc gene. Additionally, steady-state levels of c-myc mRNA are elevated in HeLa and C4-I cells relative to other cervical carcinoma cell lines. Thus, in at least some genital tumors, cis-activation of cellular oncogenes by HPV may be involved in malignant transformation of cervical cells.

Attachment Sites, Microbiological↗

PubMed1987

Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA.

A proliferating population of human foreskin keratinocytes (presently in the sixtieth passage) has been obtained after transfection with human papillomavirus (HPV) type 16 DNA. In contrast, the control cultures did not survive beyond the sixth passage. Cytogenetic analysis of cells taken from the twelfth passage revealed a heteroploid male karyotype. In approximately 50% of the cells a common marker chromosome was found, suggesting a clonal origin for at least part of the population. This is further substantiated by Southern blot analysis of cellular DNA which revealed oligomeric HPV 16 genomes integrated at a single site within the host DNA. RNA transcribed from the early region of the HPV 16 genome was identified in the cytoplasm. The immortalizing effect of HPV 16 DNA on human keratinocytes could be reproduced in a second experiment. Such cell lines represent an unique system to study the interaction of HPV with its natural target cell in vitro.

Cell Division↗

PubMed1985

Human papillomavirus type 16 DNA sequence.

The complete nucleotide sequence of HPV16 DNA (7904 bp) cloned from an invasive cervical carcinoma was determined. Homology comparisons allowed us to align the major open reading frames with the other published papilloma virus DNA sequences. The general organization of the open reading frames is similar to that of the other four papillomavirus (BPV1, HPV1a, HPV6b, CRPV) already sequenced. The sequence reveals an interruption of the reading frame coding for a suspected E1 protein.

Base Sequence↗

PubMed1985

The physical state of human papillomavirus type 16 DNA in benign and malignant genital tumours.

Cloned DNA from human papillomavirus (HPV) type 16 was subjected to restriction enzyme analysis. A genome size of 7.8 +/- 0.1 kb was determined and restriction maps were prepared. Fragments of HPV 16 DNA were nick-translated and hybridized with fragments of HPV 6b DNA. The two genomes appeared to be colinear. The physical state of HPV 16 DNA in genital tumours was analysed. In each of six benign tumours the viral DNA was detected exclusively as 8 kb circles. In four malignant tumours the viral DNA appeared to be integrated within the host genome but one cervical carcinoma and one case of Bowen's disease also contained oligomeric episomal molecules of viral DNA. One cervical carcinoma (WV 2965), containing only integrated viral DNA, was examined in detail. HPV 16 DNA was integrated as head-to-tail tandem repeats at more than one site. Three virus/cell junction fragments from this tumour were cloned. Two contained lengths of repetitive cellular DNA and one a length of apparently single copy cellular DNA.

Animals↗

PubMed1984

Presence of human papillomavirus in genital tumors.

Human papillomavirus (HPV) types 16 and 18 have been identified in two different human cervical carcinomas. The viral DNAs were molecularly cloned and used as probes to screen a large number of genital tumors by Southern blot analysis. HPV-16 or HPV-18 sequences, respectively, were found in a high percentage of cervical carcinomas, but only in a small number of condylomata acuminata or flat condylomas. The majority of the latter lesions, however, contained HPV-6 or HPV-11 sequences, respectively, which in contrast were detected only rarely in carcinomas in situ or invasively growing carcinomas. A similar distribution of the different papillomaviruses was observed when cell swabs taken from the cervix were tested by in situ hybridization.

Animals↗

PubMed1983

DNA sequence and genome organization of genital human papillomavirus type 6b.

The complete nucleotide sequence of the circular double-stranded DNA of the genital human papillomavirus type 6b (HPV6b) comprising 7902 bp was determined and compared with the DNA sequences of human papillomavirus type 1a (HPV1a) and bovine papillomavirus type 1 (BPV1). All major open reading frames are located on one DNA strand only. Their arrangement reveals that the genomic organization of HPV6b is similar to that of HPV1a and BPV1. The putative early region includes two large open reading frames E1 and E2 with marked amino acid sequence homologies to HPV1a and BPV1 which are flanked by several smaller frames. The internal part of E2 completely overlaps with another open reading frame E4. The putative late region contains two large open reading frames L1 and L2. The L1 amino acid sequences are highly conserved among analyzed papillomavirus types. By sequence comparison, potential promoter, splicing and polyadenylation signals can be localized in HPV6b DNA suggesting possible mechanisms of genital papillomavirus gene expression.

Amino Acid Sequence↗

PubMed1983

A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions.

DNA from one biopsy sample of invasive cancer of the cervix contained sequences hybridizing with human papillomavirus (HPV) type 11 DNA only under nonstringent conditions. This DNA was molecularly cloned in lambda phage. Under stringent conditions of hybridization it cross-hybridized to a minor extent (less than 0.1%) with HPV types 10, 14, and 15 and showed no homology with DNA of other human HPV types. We therefore propose to designate it tentatively as HPV 16. HPV 16 DNA was used as a probe to test additional cancer biopsy samples from cervical, vulval, and penile cancer, as well as benign genital warts (condylomata acuminata) and cervical dysplasias for the presence of homologous sequences. In 61.1% (11/18) of cervical cancer samples from German patients sequences were found hybridizing with HPV 16 DNA under conditions of high stringency. In contrast, only 34.8% (8/23) of cancer biopsy samples from Kenya and Brazil revealed this DNA. Vulval and penile cancer biopsy samples hybridized to 28.6% (2/7) or 25% (1/4), respectively. Only 2 out of 33 condylomata acuminata contained HPV 16 DNA. Both positive tumors harbored in addition HPV 6 or HPV 11 DNA. The data thus indicate that HPV 16 DNA prevails in malignant tumors, rendering an accidental contamination with papillomavirus DNA from adjacent papillomas rather unlikely. The rare presence in benign genital papillomas in addition to common genital papillomaviruses suggests a dependence of HPV 16 replication on helper virus.

Bacteriophage lambda↗