Biomedical subjects

M M Pater

Publications and source records attributed to M M Pater.

At least 37 records · Page 2Linked to original sources

PubMed1996

Malignant transformation of human ectocervical cells immortalized by HPV 18: in vitro model of carcinogenesis by cigarette smoke.

In addition to the established role of human papillomaviruses (HPVs) in cervical cancer, smoking has been suggested to be an important cofactor. Previously, primary human ectocervical cells immortalized by HPV types 16 and 18 DNA did not form tumors on nude mice. Here, we derived a new line of HPV 18-immortalized ectocervical cells (HEC-18-1), which was also non-tumorigenic. To examine the role of cigarette smoking in the progression of cervical cancer initiated by HPV 18, we adapted these cells to growth in serum and high calcium and treated the cells with cigarette smoke condensate until tumorigenic cells (HEC-18-1C) were produced. Moderate and late passage serum-adapted untreated HEC-18-1 (HEC-18-1S) remained non-tumorigenic. A typical HEC-18-1C tumor was an invasive squamous cell carcinoma, from which we established a clonal line of cells (HEC-18-1CT). Although the physical state of HPV 18 was not affected by malignant transformation and the gene expression of HPV 18 was not affected by malignant transformation and the gene expression of HPV 18 was affected little, the differentiation of the epithelium derived in organotypic (raft) culture from HEC-18-1CT was altered dramatically. Moderate and late passage HEC-18-1 and HEC-18-1S were reconstructed into mild dysplasia in organotypic (raft) culture. On the other hand, the moderate passage malignantly transformed HEC-18-1CT displayed severe dysplasia/carcinoma in situ in raft culture. We describe here the first direct evidence of the role of cigarette smoke in the progression of HPV-initiated carcinogenesis using an in vitro model system.

Animals↗

PubMed1995

Splice sites of human papillomavirus type 16 E6 gene or heterologous gene required for transformation by E7 and accumulation of E7 RNA.

Transformation of primary baby rat kidney cells by the human papillomavirus type 16 (HPV 16) E7 gene and efficient accumulation of E7 RNA have been shown by this laboratory to depend on the integrity of the nucleotide position (nt) 880 splice donor site. Here, the splice sites within the HPV 16 E6 open reading frame (ORF) and the sites of the SV40 splicing unit were examined for an ability to provide this requirement. Constructs containing the HPV 16 E6 sites and the SV40 splice site sequences were used for transformation and RNase protection assays. E6 splice sites supported a low level of transformation, in assays for complete HPV 16 early region constructs containing loss-of-function mutations of the nt 880 site. Using constructs with wild-type E6 or SV40 splice sites showed that both splice sites could substitute similarly for the requirement in cis of the nt 880 site for transformation. HPV 16 E6 mutated splice site and SV40 splice site in reverse, nonfunctional orientation relative to the promoter, were not transformation competent. The HPV 16 E7 RNA levels for the E6 splice site constructs correlated closely with the transformation frequency. The SV40 splice sites were required for E7 transcript accumulation. The results showed E6 splice site function and evidence for enhanced exon skipping from E6 splice donor site to acceptor sites 3' of the E7 ORF. This was shown with constructs containing loss-of-function mutations of the nt 880 site. These results confirmed the function of the splice sites by the transformation competent constructs and suggested lower transformation frequency than for wild type was due to skipping of the E7 exon. These patterns of transcripts may have a role in the regulation of gene expression during progression to malignancy. The combined results revealed that the general presence of a functional splice donor site was absolutely required for transformation by HPV 16 E7 and accumulation of E7 RNA.

Animals↗

PubMed1995

Prevention of carcinoma in situ of human papillomavirus type 16-immortalized human endocervical cells by retinoic acid in organotypic raft culture.

OBJECTIVE: To determine the effect of retinoic acid on the development of severe dysplasia or carcinoma in situ from endocervical cells containing human papillomavirus (HPV) type 16. METHODS: Two independent lines of HPV 16-immortalized endocervical cells were reconstructed into two squamous epithelial tissues using the organotypic raft culture system to examine the differentiated phenotype. The effect of retinoic acid on dysplastic morphology of differentiation of the epithelia was examined by light microscopy of stained sections and electron microscopy. The endocervical cell type cytokeratin expression pattern was determined by indirect immunofluorescence using specific monoclonal antibodies. Ribonucleic acid expression of the HPV 16 E7 oncogene was examined by in situ hybridization. RESULTS: Untreated HPV 16-immortalized endocervical cells were reconstructed into squamous dysplastic lesions resembling carcinoma in situ observed in women. Retinoic acid-treated rafts formed epithelia composed of two to three cell layers of columnar-like cells resembling simple epithelium of the endocervix. Electron microscopy and cytokeratin expression patterns confirmed the histology of a differentiated endocervical phenotype after treatment with retinoic acid. Expression of HPV 16 E7 was modestly lower in treated epithelia, preferentially in basal cells. CONCLUSION: Retinoic acid prevents the histology and cytokeratin differentiation markers of carcinoma in situ of HPV 16-immortalized endocervical cells. Because the epithelia closely mimic HPV 16-containing severe dysplasias and native endocervical epithelium in women, this immortalized endocervical cell-raft system may be useful as a model to assess the efficacy of agents such as retinoic acid for preventing progression of these lesions to malignant cervical carcinoma.

Carcinoma in Situ↗

PubMed1995

Alterations in physical state and expression of human papillomavirus type 18 DNA following crisis and establishment of immortalized ectocervical cells.

Integration of episomal human papillomavirus (HPV) DNA in infected cervical lesions during malignant progression is frequently observed, but the importance of integration is poorly understood. We have studied immortalization by HPV-18 of human cervical cells as an in vitro model system. Here, the status and expression of HPV-18 DNA in precrisis ectocervical keratinocytes was compared with that in the same cells after crisis and establishment of immortalization. Southern blots revealed, and two-dimensional gel analysis confirmed, that the precrisis culture contained more than 100 copies/cell of episomal HPV-18 DNA and no detectable integrated viral DNA. In contrast, the postcrisis cells contained a low copy number of only integrated viral genome. The Northern blot patterns of E6-E7 and E2/E4 RNA expression were also different. Analysis of RNA by RT-PCR indicated that neither culture expressed the unspliced HPV-18 E6 oncogene present in tumor cell lines and that the precrisis, but not postcrisis, culture expressed the full-length E2 repressor. The two cultures displayed a similar keratinocyte morphology in vitro and a similar low grade dysplasia in vivo and both were non-tumorigenic. These results suggest that, although insufficient for complete malignant conversion, viral DNA integration during crisis is associated with the establishment of an immortalized phenotype in which HPV-18 DNA is integrated and HPV-18 RNA expression is altered.

Adult↗

PubMed1994

Role of steroid hormones in potentiating transformation of cervical cells by human papillomaviruses.

Human papillomaviruses (HPVs) are etiologically involved in cervical neoplasia, and epidemiological evidence suggests that steroid hormones can increase the risk of this cancer in HPV-infected women. Steroids can interact with hormone-response elements in the viral long control region, enhancing HPV transcription and resulting in transformation of cervical cells. Subsequent malignant progression may involve virus-induced chromosomal instability, facilitating viral DNA integration and deregulation of gene expression.

Animals↗

PubMed1994

Differential regulation by c-jun and c-fos protooncogenes of hormone response from composite glucocorticoid response element in human papilloma virus type 16 regulatory region.

Glucocorticoid hormones positively regulate human papilloma virus (HPV) type 16 gene expression, and we have previously shown that this regulation is through three glucocorticoid response elements (GREs). The GRE at nucleotide 7640 is a composite GRE (cGRE) containing an overlapping activator protein-1 (AP-1) motif for the c-jun homodimer and c-jun/c-fos heterodimer. This report examined the effects of c-jun and/or c-fos AP-1 protooncogenes and the glucocorticoid hormone dexamethasone on expression of the HPV 16 cGRE in AP-1-deficient P19 embryonal carcinoma cells. The activity of the full-length HPV 16 enhancer was progressively increased with increasing levels of c-jun. The hormone induced an additional response. For the c-jun/c-fos heterodimer, the response to hormone was progressively diminished. Site-specific mutations of the cGRE revealed that the regulation by AP-1 and hormone required both GRE and the AP-1 motif. An enhancer fragment containing the cGRE and excluding the two simple GREs gave similar results. Two disruption mutations of the AP-1 site confirmed the requirement of this site for hormone response. A cGRE oligonucleotide construct substantiated the effect of c-jun for response to hormone. For heterodimer, activity and hormone response were both also progressively increased. The results reveal a unique cross-talk between the distinct AP-1- and hormone-signaling pathways, suggesting the involvement of a complex interaction of c-jun and c-fos and glucocorticoid hormone receptor with the HPV 16 cGRE, resulting in novel control patterns for regulating viral expression.

Animals↗

PubMed1994

Human papillomavirus 18-immortalized endocervical cells with in vitro cytokeratin expression characteristics of adenocarcinoma.

OBJECTIVE: To determine whether human papillomavirus (HPV) 18 has a role in the development of adenocarcinoma from human endo- or ectocervical cells. METHODS: Secondary cultures of human endo- and ectocervical cells were assayed for immortalization by HPV 18 DNA using lipofection. The effects of immortalization on the patterns of cytokeratin expression were determined by indirect immunofluorescence using monoclonal antibodies. The differentiation phenotype of the immortalized cells was investigated by a modified in vivo implantation system. RESULTS: Both endo- and ectocervical cells were immortalized by HPV 18. The immortalized cells contained integrated HPV 18 DNA and expressed E6-E7 RNA. The immortalized endocervical cells had a cytokeratin phenotype characteristic of adenocarcinoma, whereas the immortalized ectocervical cells retained a distinct cytokeratin expression pattern of normal parental cells. In an in vivo implantation system, endocervical cells formed a lesion resembling severe dysplasia or carcinoma in situ, whereas ectocervical cells developed into a lesion resembling mild dysplasia. Both cell lines were nontumorigenic in nude mice. CONCLUSION: Both endo- and ectocervical cells are targets for immortalization by HPV 18. Based on cytokeratin expression patterns, immortalized endocervical cells, but not ectocervical cells, may be useful as a model for premalignant lesions that progress into adenocarcinoma.

Adenocarcinoma↗

PubMed1993

Glucocorticoid requirement for growth of human papillomavirus 16-transformed primary rat kidney epithelial cells: correlation of development of hormone resistance with viral RNA expression and processing.

The establishment of transformation of primary baby rat kidney epithelial cells by human papillomavirus type 16 DNA requires glucocorticoid hormones (Pater et al., Nature (Lond.), 335: 832-835, 1988). In this report we provide evidence that growth of transformed baby rat kidney cells in culture also requires glucocorticoids. However, transformed cells for which growth does not require hormone readily arise after a brief period of crisis, if cultured without added hormone. No reduction of glucocorticoid receptor was evident in non-hormone-requiring cells. The expression of human papillomavirus 16 RNA in these cells was analyzed by Northern blot, primer extension, and RNase protection analysis. Cells that do not require hormone had greatly reduced levels of transcripts initiated from the viral P97 promoter. However, there is evidence for compensating alterations to allow more efficient expression of E7 mRNA, since the growth of these cells is correlated with altered patterns of viral RNA expression and processing.

Animals↗

PubMed1993

In vivo cytokeratin-expression pattern of stratified squamous epithelium from human papillomavirus-type-16-immortalized ectocervical and foreskin keratinocytes.

The association between human papillomavirus (HPV) type 16 and cervical squamous-cell carcinoma has been well documented, and HPV 16 DNA has been shown to immortalize human genital keratinocytes in vitro. Using a panel of cytokeratin(CK)-specific antibodies, we examined the CK expression pattern, an important characteristic of epithelia, of the stratified squamous epithelium reconstructed in vivo from monolayer cultures of 2 human ectocervical and 3 human foreskin keratinocyte cell lines immortalized by HPV 16 DNA. Whereas the abnormal epithelium formed from these grafts presented certain aspects of mature terminal differentiation, such as morphology and expression of CK10/11, the expression patterns for CK19, and especially CK13, were more obviously abnormal. CK18 expression was not detectable in any of the epithelia formed from the 5 cell lines. In contrast, for an HPV-16-immortalized endocervical cell line and the CaSki cervical-carcinoma cell line there was expression of CK18. Our results indicate that HPV-16-induced immortalization of keratinocytes is associated with disruption of the normal CK expression pattern in stratified squamous epithelium and that expression of particular CKs can be differentially disrupted.

Animals↗

PubMed1993

Human JC virus perfect palindromic nuclear factor 1-binding sequences important for glial cell-specific expression in differentiating embryonal carcinoma cells.

The brain cell specificity of the human papovavirus JC virus was examined by site-directed mutagenesis of the nuclear factor 1 (NF1) motifs within the viral regulatory region. The NF1 motif sites, located within the 98-bp tandem repeats that contain 6-bp perfect inverted palindromic sequences, were important for glial cell-specific expression of JC virus in differentiated embryonal carcinoma cells in vivo. The NF1 site on the late side of the repeats was not important, a fact confirmed by in vitro transcription studies. These observations were correlated with in vitro DNase I footprinting and mobility shift assays, which demonstrated specific interactions of factors in glial cell nuclear extracts with NF1 sites.

Base Sequence↗

PubMed1993

Multiple human papillomavirus type 16 glucocorticoid response elements functional for transformation, transient expression, and DNA-protein interactions.

We have previously shown that human papillomavirus type 16 (HPV-16) can efficiently transform primary baby rat kidney cells in the presence of the steroid hormones progesterone and the glucocorticoid dexamethasone. To study this effect of hormone, different combinations of the previously identified glucocorticoid response element (GRE) at nucleotide 7640 of HPV-16 and the other two GREs that we have recently identified, at nucleotides 7385 and 7474, were mutated. The previously described GRE and the other two GREs were shown to be functional for the induction of transformation by dexamethasone. In addition, transient assays in cervical HeLa cells demonstrated the functional importance of the three individual GREs. Assays for in vitro interaction demonstrated the specific binding of a 97-kDa protein, the glucocorticoid receptor, to both recently identified HPV-16 GREs.

Animals↗

PubMed1993

In vitro and in vivo analysis of cellular origin of cervical squamous metaplasia.

We have previously shown that cultured normal human endocervical cells (HENs) form epithelium resembling squamous metaplasia in vivo. To analyze the cellular origin of squamous metaplasia, the cytokeratin and mucin expression and morphological features of HENs in monolayer cultures and in implants beneath the skin of nude mice were examined. Primary HENs had two distinct morphological phenotypes in vitro pleomorphic epithelial cells and keratinocytelike cells. Using a panel of monoclonal antibodies for various cytokeratins (CKs), we observed that the pleomorphic cells, which were the primary outgrowths, expressed CK7 and CK18 and produced mucin, suggesting their origin to be the mucosecretory columnar cells (CCs) of the endocervix. Keratinocytelike cells were observed in proximity of the CC-like cells after a few days of HEN culture. Interestingly, these cells were homogeneously negative for CK7 expression, as for native reserve cells (RCs), and homogeneously positive for CK13 expression with the antibody that is specific for RCs. During early passages, the culture consisted mostly of the RC-like keratinocytelike cells, and in the late passages, the CC-like cells were predominant. HEN implants in nude mice morphologically formed epithelia similar to immature squamous metaplasia and showed variable CK18 expression. Moreover, they showed homogeneous CK13 expression throughout all layers and expressed mucin and CK7 in the suprabasal cells. The possibility that the HEN culture was originally a mixed population of CCs and RCs, that we failed to detect, cannot be eliminated. Our results support the more likely view that the endocervical simple epithelia, which form squamous metaplasia, are bipotential cells and undergo differentiation readily and reversibly to give rise to CC-like and RC-like cells in culture.

Adult↗

PubMed1993

Human papillomavirus type 16 expression in cervical keratinocytes: role of progesterone and glucocorticoid hormones.

OBJECTIVE: To determine the role of the steroid hormones, progesterone and glucocorticoids, and the viral hormone response elements, in the episomal expression of human papillomavirus (HPV) type 16 in primary human ectocervical cells. METHODS: In situ hybridization and mutagenesis were used to assess the requirements of these hormones and the HPV 16 glucocorticoid/progesterone response elements in the induction of HPV 16 expression in ectocervical cells. RESULTS: The assays detected a marked increase in viral messenger RNA only after treatment of the cells with either of the steroid hormones. This response was inhibited by the anti-progestin RU 486 in a concentration-dependent manner. Mutagenesis of the previously identified hormone response element in the regulatory region of the HPV 16 genome had no effect on hormone-induced HPV gene expression. We have now identified two additional hormone response elements. Different combinations of mutations in the three hormone response elements showed that all three were independently sufficient for the hormone-mediated induction of viral transcription. CONCLUSIONS: Steroid hormones induce HPV 16 gene expression in cervical keratinocytes directly through three hormone response elements in the regulatory region of the viral genome. The anti-progestin RU 486 inhibits this induction. Because the physical state of HPV DNA in this in vitro system and in premalignant cervical lesions is extrachromosomal, steroid hormones may have a critical role in modulating HPV expression in such lesions.

Cells, Cultured↗

PubMed1992

Squamous metaplasia of normal and carcinoma in situ of HPV 16-immortalized human endocervical cells.

The importance of cervical squamous metaplasia and human papillomavirus 16 (HPV 16) infection for cervical carcinoma has been well established. Nearly 87% of the intraepithelial neoplasia of the cervix occur in the transformation zone, which is composed of squamous metaplastic cells with unclear origin. HPV DNA, mostly HPV 16, has been found in 90% of cervical carcinomas, but only limited experimental data are available to discern the role of HPV 16 in this tissue specific oncogenesis. We have initiated in vivo studies of cultured endocervical cells as an experimental model system for development of cervical neoplasia. Using a modified in vivo implantation system, cultured normal endocervical epithelial cells formed epithelium resembling squamous metaplasia, whereas those immortalized by HPV 16 developed into lesions resembling carcinoma in situ. In contrast, their ectocervical counterparts formed well differentiated stratified squamous epithelium and a lesion with mild dysplastic change, respectively. The HPV 16-immortalized cells showed in vivo cytokeratin expression patterns similar to their respective normal counterparts, confirming their different origins. Thus, this study provides direct experimental evidence for the transformation of simple epithelial cells of endocervical origin into stratified squamous metaplasia and indicates the differential susceptibility of endo- and ectocervical epithelial cells for conversion to cancer by HPV 16.

Animals↗

PubMed1992

Mitochondrial nucleic acids as internal standards for blot hybridization analyses.

A plasmid, designated p72, constructed from human lung carcinoma DNA inserted into the promoterless herpes simplex virus thymidine kinase gene pML-TK-Bgl II vector, hybridizes strongly to human nucleic acids on Southern and Northern blots. The portion of the DNA insert responsible for the strong signal following hybridization to human DNA or RNA is a 167-bp 3' terminal portion of the mitochondrial 16S ribosomal RNA gene. The expression of this gene is constitutive in the several human cell lines that were tested and is unaffected by exposure to cytotoxic chemicals that alter the expression of nuclear genes. This plasmid offers an excellent tool for studies of perturbations of gene expression and for controlling for the variations in sample preparation, loading, and transfer in Southern or Northern analysis of nucleic acids.

Blotting, Northern↗

PubMed1992

The first 124 nucleotides of the E7 coding sequences of HPV16 can render the HPV11 genome transformation competent.

The human papillomavirus (HPV) type 16 is associated with the majority of cervical tumors and is capable of oncogenically transforming cells in culture. HPV type 11 is rarely associated with malignant lesions and does not transform cells in vitro. While the E7 of HPV16 is necessary and sufficient for transformation of rodent cells, the E7 gene of HPV11 is not transforming. In the present report we demonstrate that the HPV11 genome, with the first 124 nucleotides of the HPV16 E7 open reading frame fused to the last 198 nucleotides of the HPV11 E7 open reading frame, becomes transformation competent.

Amino Acid Sequence↗

PubMed1992

Glucocorticoid-dependent transformation by human papillomavirus type 16 E7 coding and 3' noncoding sequences.

The establishment of transformation of primary rodent cells by human papillomavirus (HPV) type 16 DNA requires glucocorticoid hormones (Pater et al., Nature 335, 832-835, 1988). Here we provide evidence by mutational analysis that, in the context of the hormone-regulated HPV 16 promoter/enhancer, the only protein coding sequences of HPV 16 required are those of the E7 gene. Moreover, additional sequences adjacent to the 3' end of E7 coding sequences are also essential for the establishment of the transformed phenotype. Splice donor sites, especially an E7 ORF 3' proximal one, are implicated for this cis-acting function, since specific deletion mutations of these splice sites greatly or completely reduced the frequency of transformation and the level of E7 RNA.

Animals↗

PubMed1992

Nucleotide 880 splice donor site required for efficient transformation and RNA accumulation by human papillomavirus type 16 E7 gene.

Mutations within coding sequences of the various human papillomavirus type 16 (HPV-16) genes have been used to demonstrate that the HPV-16 E7 gene is necessary and sufficient for transformation of rodent cells. We now provide evidence that, in addition to E7 coding sequences, a small cis-acting region immediately flanking the 3' end of E7 coding sequences is also required for transformation. This was shown by translation termination linker insertion, progressive deletion analysis, and site-directed mutagenesis. Disruption of the nucleotide (nt) 880 splice donor site within the 3'-flanking region by deletion of as few as 4 nt or substitution of 3 nt totally abolished transformation. Regeneration of the wild-type sequence in a previously transformation-incompetent splice site mutant restored transformation. Mutating the wild-type splice donor site to the consensus splice site resulted in a stronger transformation phenotype, while mutating the +2 position of the consensus sequence significantly reduced the frequency of transformation. It was shown with RNase protection assays that the amount of E7 mRNA in transformation-deficient splice site mutants was much lower. Nuclear runoff experiments revealed that there was no change in the rate of synthesis of E7 message in the nt 880 splice site mutant. Furthermore, mutations of HPV-16 sequences indicated that the two other early region splice donor sites have no more than minor roles in transformation and efficient RNA accumulation. These results indicate that the specific integrity of the nt 880 splice donor site is essential for both accumulation of E7 RNA and efficient E7-mediated transformation.

Animals↗