Biomedical subjects

M M Pater

Publications and source records attributed to M M Pater.

At least 55 records · Page 3Linked to original sources

PubMed1992

Human papillomavirus 16 DNA immortalizes two types of normal human epithelial cells of the uterine cervix.

Premalignant cervical lesions occur at the squamo-columnar junction and in endocervical epithelium and squamous ectocervical epithelium, in descending order of frequency. However, previously only ectocervical cells have been clearly shown to be immortalized in vitro by the oncogenic human papillomaviruses (HPVs). This report describes the immortalization of normal human ecto- and endocervical epithelial cells by the intact HPV 16 genome. Ectocervical epithelial cells (HEC) became immortalized (HEC-16) without crisis while endocervical cells (HEN) were immortalized (HEN-16) after undergoing crisis. HEN-16 and HEC-16 contained integrated HPV 16 DNA, expressed E6 and E7 mRNA, and were aneuploid and nontumorigenic. They also expressed cytokeratins in a pattern similar to their distinct normal parental cells. These results suggest that both squamous and simple epithelial cells of uterine cervix are targets for immortalization by HPV 16.

Adult↗

PubMed1991

Activity and enhancer binding factors for BK virus regulatory elements in differentiating embryonal carcinoma cells.

We have studied cell type specificity of expression of the human papovavirus BK regulatory elements in undifferentiated and differentiated embryonal carcinoma (EC) cells as a model system. While the activity of the regulatory elements of this virus was marginal in undifferentiated cells, differentiation by retinoic acid and DMSO resulted in a dramatic increase in the activity. To correlate in vivo activity of the regulatory elements with interaction with cellular transcription factors, we performed DNase I footprinting experiments. A GC-rich region was protected in both undifferentiated and differentiated cells. An additional four protected sites were detected in retinoic acid-differentiated cells and at least one of these additional sites was weakly protected in DMSO-differentiated cells. The sequences of the differentiated cell type-specific protected regions showed homology to a nuclear factor 1 (NF-1) binding motif and to a muscle creatine kinase gene enhancer motif. The intensity, competition, and pattern of protection of these sites were different in the two differentiated cell types, suggesting the involvement of different transcription factors regulating the activity of BKV regulatory elements in the two cell types.

BK Virus↗

PubMed1991

RU486 inhibits glucocorticoid hormone-dependent oncogenesis by human papillomavirus type 16 DNA.

We have previously reported (Pater et al., Nature 335, 832-835, 1988) the glucocorticoid hormone-dependent oncogenic transformation of primary baby rat kidney (BRK) cells by a combination of human papillomavirus (HPV) type 16 DNA and the activated form of the human Ha-ras-1 (ras) oncogene. In this study we provide evidence for the inhibition of such hormone-dependent transformation by the hormone antagonist, RU486. The level of inhibition was dependent on the ratio of hormone to antagonist and severe inhibition was observed at a 1:5 ratio. The effect of RU486 on the growth of HPV16-transformed cells was also studied. Severe growth inhibition and cell death occurred when cells transformed in the absence of RU486 were subsequently grown in the presence of either a 1:2.5 or 1:5 ratio of hormone to hormone antagonist. RU486 did not alter the level or electrophoretic mobility pattern of HPV16 mRNA expressed in transformed cells, when antihormone was applied either continuously or subsequent to transformation by HPV. These results indicate the importance of continuous viral gene expression for initiation and maintenance of the transformed phenotype.

Animals↗

PubMed1990

Oncogenic transformation by human papillomavirus type 16 deoxyribonucleic acid in the presence of progesterone or progestins from oral contraceptives.

Compelling evidence supports a role of certain types of human papillomaviruses as the cause of cervical cancer. In addition to human papillomaviruses, other agents, such as hormones, have been implicated as cofactors in this type of neoplasia. In this study we provide evidence for oncogenic transformation of primary baby rat kidney cells by human papillomavirus type 16 deoxyribonucleic acid plus ras oncogene in the presence of progesterone but not estrogen. Integrated and intact human papillomavirus type 16 deoxyribonucleic acid is present and expressed in all the five progesterone-transformed colonies that we examined. Moreover, all these cell lines are capable of anchorage-independent growth and induce tumors in syngeneic animals. We also observed oncogenic transformation with human papillomavirus type 16 deoxyribonucleic acid plus ras in the presence of ethanol-soluble extracts from two brands of commonly used oral contraceptive tablets. No transformation is achieved in the presence of ethanol-soluble extracts from the inert tablets, provided in packages of each brand of oral contraceptive. These results may have implications for a papillomavirus-hormone link to cervical neoplasia.

Administration, Oral↗

PubMed1990

Ubiquitous and cell-type-specific protein interactions with human papillomavirus type 16 and type 18 enhancers.

We have studied the protein-DNA interactions of human papillomavirus types 16 and 18 constitutive enhancer elements using DNasel footprinting experiments with nuclear extracts from four cervical carcinoma cell lines (C33A, HeLa, SiHa, and CaSki) and one fibroblast cell line (143B). Among nine footprints for the HPV 16 enhancer region, six footprints contain nuclear factor 1 (NF1) binding GCCAA motif. In vitro competition experiments suggest that the same factors are shared by all six of these motifs. Two other sequence motifs have consensus sequences for transcription factor AP1. Another sequence motif, for which uv crosslinking studies reveal interaction with four protein molecules, is a strong positive modulator of HPV 16 enhancer function in vivo and shares 100% homology to a sequence motif, GTTTTAA, in the tissue-specific enhancer of the c-mos oncogene. Footprints on the HPV 18 enhancer show five protected regions with homologies to NF1, AP1 and EFII transcription factor binding motifs. One sequence motif of the HPV 18 enhancer has three repeats of a TTTTA sequence contained within the c-mos sequence motif and interacts with at least four different individual polypeptides, as judged by uv crosslinking experiments.

Base Sequence↗

PubMed1990

Activity and enhancer binding factors for JC virus regulatory elements in differentiating embryonal carcinoma cells.

We have studied cell-type-specific expression by JC virus (JCV) DNA regulatory sequences using embryonal carcinoma (EC) cells as a model system. In transient transfection assays, JCV enhancer demonstrated activity in retinoic acid-differentiated neuronal type cells but not in undifferentiated or DMSO-differentiated muscle type cells. To correlate in vivo activity with the binding of transcription factors, we performed DNasel footprinting experiments. Retinoic acid-treated EC cell extracts provided three completely protected regions, each containing sequences with homology to nuclear factor 1 (NF1) binding motifs and the partially protected TATA box. Oligonucleotide competition studies suggest that all three NF1 binding motifs are bound by the same factors but with different affinities and that there are cooperative interactions between NF1 proteins binding to adjacent regions. No protected region other than the partially protected TATA box was detected in undifferentiated and DMSO-differentiated EC cells in which JC regulatory sequences were not expressed.

Animals↗

PubMed1989

Trans-regulation and differential cell specificity of human papillomavirus types 16, 18, and 11 cis-acting elements.

The noncoding region (ncr) of human papillomavirus (HPV) types 16, 18, and 11 contains promoter and/or enhancer function. We have localized the sequence containing the constitutive enhancers of HPV types 16, 18, and 11 to 315, 230, and 213 bp fragments, respectively, for comparative studies. The region of homology shared between the enhancers of the three viruses is limited to the sequence ATTTTTGGCTT, which is also present in the ncr of HPV 6b and 33. We have also examined the enhancer activity of the HPV ncrs in three human cervical carcinoma cell lines, one noncervical human carcinoma cell line, and one monkey kidney established cell line. We observed cell-specific differences in the constitutive expression of the enhancers in the various cell lines. The conditional enhancer activity of the ncr of the viruses is increased in trans by the E2 gene product of HPV 16. Transactivation by E2 is mediated through the E2 binding motif on HPV enhancer plasmids with a heterologous but not with a homologous promoter. Our preliminary studies also indicate a repressor function for the E7 gene of HPV 16.

Base Sequence↗

PubMed1988

Glucocorticoid-dependent oncogenic transformation by type 16 but not type 11 human papilloma virus DNA.

Squamous cell carcinoma of the uterine cervix is one of the most common cancers among women. Correlation between human papilloma virus (HPV) infection of the uterine cervix and the development of cervical neoplasia has been established. More recent studies have shown the presence and expression of integrated HPV types 16 and 18 DNA sequences in 70-80% of cervical tumours and tumour cell lines. It has been suggested that, in addition to HPVs, other agents such as hormones and tobacco products act as cofactors in cervical neoplasia (for review see ref. 15). The presence and expression of a glucocorticoid-responsive element in HPV-16 has been reported. Here we provide evidence for the oncogenic transformation of primary cells with a combination of HPV-16 DNA, but not HPV-11 DNA, and the activated form of the human Ha-ras oncogene only in the presence of the glucocorticoid hormone dexamethasone.

Animals↗

PubMed1988

Isolation and characterization of a variant of human papillomavirus type 11 from a nasal inverting (Schneiderian) papilloma.

We have previously reported the presence of a variant of human papillomavirus (HPV) type 11 in a nasal inverting papilloma [Respler et al., 1987]. In the present study, we have cloned molecularly the DNA of this variant at its unique restriction enzyme Bam HI site into lambda BF101 phage. Restriction enzyme mapping and DNA sequencing revealed that the genome of this virus contained an extra 531 base pair (bp) which was the repeat of most of the noncoding region (ncr) of HPV 11. Insertion of transcriptional control elements, including the repeated sequence, in front of the chloramphenicol acetyltransferase (CAT) gene resulted in a 5- to 30-fold increase in expression in transfected cells, as compared to the constructs containing a single ncr of HPV 11. This increased expression was due to enhanced levels of CAT RNA the synthesis of which is initiated by the viral promoter element.

Acetyltransferases↗

PubMed1988

Expression of human papillomavirus types 16 and 18 DNA sequences in cervical carcinoma cell lines.

A total of six cervical carcinoma cell lines, of which two contained human papillomavirus (HPV) type 16 and four contained HPV type 18 DNA, were examined for viral RNA expression by spot-blot hybridization. All six cell lines contained HPV-specific cytoplasmic poly(A)-containing RNA. The same RNA species were detected in both cytoplasmic and polysomal fractions, indicating nonselective translation of these messages into protein products. Primer extension analysis of RNA from the cell lines revealed the presence of three major start sites at the 5' end of the E6 gene for RNA from HPV 16-containing cell lines and four major start sites at the 5' end of the E6 gene for RNA from HPV 18-containing cell lines. One cell line, ME 180, had a distinct pattern with only one minor HPV 18 start site. Northern-blot hybridization using the E6-E7 region of HPV 16 and 18 DNAs as probes revealed the presence of E6-E7 transcripts in all the cell lines except ME 180. Southern-blot analysis revealed that while sequences hybridizing to the E6-E7 coding region were present, the 5' end region containing the enhancer-promoter region of viral DNA is deleted in ME 180. Subsequent Northern-blot hybridization using the E1 region of HPV 18 DNA as probe revealed that the viral RNA in ME 180 is transcribed from this region.

Blotting, Northern↗

PubMed1988

Expression of the control elements of BK and SV40 viruses in human cells exhibiting different transformed phenotypes.

Transformation of primary human embryonic kidney (HEK) cells as selected by either focus assay or growth in soft agar after their transfection with BK virus (BKV) DNA alone or BKV DNA plus the activated form of human Ha-ras oncogene has been previously reported (A. Pater and M. M. Pater, J. Virol. 58, 680-683 (1986]. In order to examine the expression of the regulatory elements of papovaviruses in each of the transformed phenotypes, chloramphenical acetyltransferase (CAT) plasmids under the control of BK or SV40 early promotor were used in transient assays. The expression of CAT, as driven by either SV40 or BK early promoters, was approximately 100-fold higher in HEK cells transformed by BKV DNA plus Ha-ras oncogene than in cells transformed only by BKV DNA. The higher CAT expression was not due to high levels of plasmid replication in these cells. Time course studies revealed that the higher CAT activity could be explained largely by greater plasmid uptake and stability in BK plus ras-transformed cells.

BK Virus↗

PubMed1988

Functional role of BK virus tumor antigens in transformation.

We have examined the role of the human papovavirus BK virus (BKV) tumor (T) antigen(s) in the maintenance of transformation and have identified the domain of T antigen essential for transformation. BKV-transformed BHK 21 and NIH 3T3 cells expressing antisense T-antigen RNA lose their ability to grow in soft agar, indicating the need for the continued expression of T antigen for the maintenance of the transformed phenotype. Experiments using translation termination linker insertion and deletion mutagenesis of BKV T antigen demonstrate that amino acids 356 to 384 are essential for transformation. Although BKV T antigen shares 100, 95, and 82% amino acid homology with that of simian virus 40 (SV40) for the nuclear localization signal, p53-binding domain, and DNA-binding domain, respectively, the transformation domains of BKV and SV40 T antigens share only 54% homology. Also, BKV T antigen lacks a substantial portion of the ATPase domain of SV40, and our results indicate the dispensability of the remaining portion for transformation by this protein. We suggest that the differences in the amino acids in the identified transformation domains together with the differences in the ATPase domains may account for the differences in the transformation potentials of the two proteins.

Animals↗

PubMed1986

Human papillomavirus types 16 and 18 sequences in early cervical neoplasia.

A total of 100 colposcopic biopsies from patients with abnormal Papanicolau's tests were surveyed for the presence of human papillomavirus (HPV) types 16 and 18 sequences by spot-blot hybridization. HPV 16 and 18 DNA sequences were detected in 58% of the biopsies. None of the cervical intraepithelial neoplasia grade I (CIN I) contained HPV 16 while 50% of the CIN III lesions (carcinoma in situ, CIS) contained HPV 16. HPV 18-related sequences were equally represented in CIN I, II, and III. Southern-blot hybridization of total undigested cellular DNA revealed the presence of HPV DNA sequences only in an episomal form. While the restriction enzyme patterns in HPV 16-positive samples were mostly identical to the originally cloned sequence, the restriction enzyme pattern for HPV 18-positive samples were different from that of HPV 18 but identical to each other. Furthermore, this DNA hybridized more strongly to HPV 18 under nonstringent conditions, suggesting a new type.

Colposcopy↗

PubMed1986

Transformation of primary human embryonic kidney cells to anchorage independence by a combination of BK virus DNA and the Harvey-ras oncogene.

Primary human embryonic kidney (HEK) cells were transformed by a focus assay with BK virus (BKV) DNA molecularly cloned at its unique EcoRI site. Both viral DNA sequences and viral tumor antigens were present and expressed in all the foci that we examined. However, cells isolated from foci were incapable of growth in soft agar. We then examined the transformation of HEK cells after their transfection with a combination of BKV DNA and either the normal or the activated form of the human Ha-ras oncogene (EJ c-Ha-ras-1). Only the cells transfected with a combination of BKV DNA and the activated form of Ha-ras were capable of growth in soft agar. Both BKV and Ha-ras DNAs were present in the transformed colonies. BKV tumor antigens and the Ha-ras p21 protein were also expressed.

Antigens, Viral, Tumor↗

PubMed1985

Differential requirement for SV40 early genes in immortalization and transformation of primary rat and human embryonic cells.

A series of recombinant plasmids carrying various DNA fragments of SV40 early region were used to test for their ability to immortalize primary cultures of rat embryo (RE) and human embryonic kidney (HEK) cells. When primary RE cells were transfected with plasmids containing an entire early region of wild-type SV40- or a deletion mutant in the small tumor (t) antigen, dl 1410-DNA, they were all immortalized. The immortalized cells could grow in soft-agar medium and produced large tumor (T)-antigen. Cultured RE cells transfected with pW2-t, which contains a deletion in the large-T-specific coding region, also gave rise to continuous cell lines. Interestingly, two of nine RE lines immortalized by pW2-t could also grow in soft-agar medium. The plasmid pW-t8 carrying a similar fragment of SV40 DNA as pW2-t, but lacking the processing and polyadenylation signal sequences, also immortalized RE cells. Surprisingly, the plasmid pD-t1 which contains neither the intact large-T nor the small-t function also immortalized RE cells. However, the RE lines immortalized by pW-t8 or pD-t1 were unable to grow in soft-agar medium and displayed a wide range of growth phenotypes. On the contrary, when primary HEK cells were used for immortalization experiments, only those SV40 plasmids carrying the intact large-T function were able to generate immortalized lines. The growth properties of these immortalized HEK lines can be categorized into two groups. Those HEK lines immortalized by the large T alone grew slightly denser and rounder than their parental normal HEK cells, while those immortalized by both the large-T and small-t antigens grew extremely fast, reached higher density, piled up on each other, and were anchorage independent. In addition, when these SV40 plasmids were used to directly transform primary HEK cells by the focus assay, the large-T clone, pD3-05, only transformed HEK cells to form light foci. Transfection by the large-T plus the small-t sequences either in cis or in trans, did increase the frequency of focus formation, and gave rise to dense foci which could grow in soft-agar medium.

Animals↗

PubMed1985

Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix.

A total of eight human epithelial cell lines derived from the carcinoma of the cervix were examined for the presence of human papillomaviruses (HPVs) types 16 and 18 DNA sequences. Six out of eight cell lines contain sequences hybridizing to the DNA of these viruses. Two of the cell lines contain sequences hybridizing specifically to HPV 16. One of these two cell lines contains all of the HPV 16 sequences and the other cell line is missing fragments containing early regions E2 and E4 and some of the late regions. Four of the cell lines contain sequences hybridizing specifically to HPV 18. All these cell lines are missing fragments containing early regions E2, E4, and E5. Interestingly, all the cell lines contain sequences corresponding to early regions E1, E6, and E7.

Base Sequence↗

PubMed1985

Characterization of a new simian virus 40 mutant, tsA3900, isolated from deletion mutant tsA1499.

The simian virus 40 (SV40) mutant tsA1499 contains an 81-base-pair deletion in the region of A gene encoding the C-terminal portion of the large T antigen. This mutant is particularly interesting, since it is a temperature-sensitive mutant that is apparently able to separate the lytic growth and transforming functions of the SV40 large T antigen at 38.5 degrees C. We report the isolation of a tsA1499 revertant (tsA1499-Rev) which is no longer temperature sensitive for lytic growth but still contains the 81-base-pair deletion of tsA1499. Marker rescue experiments with tsA1499-Rev or wild-type strain 830 (wt830) DNAs revealed that the original tsA1499 mutant contained a second mutation within the HindIII-Fnu4HI restriction fragment between 0.425 and 0.484 map units. Sequencing of this DNA fragment from the tsA1499, tsA1499-Rev, and wt830 viruses revealed that tsA1499 contained a single-base transversion (C to G) at 0.455 map units (nucleotide 4261). This transversion resulted in the creation of a new RsaI cleavage site in the tsA1499 DNA and predicts an arginine-to-threonine substitution at amino acid position 186 in the mutant large T antigen. The DNA sequence of the tsA1499-Rev HindIII-Fnu4HI fragment was identical to that of wt830. To determine whether tsA1499 was temperature sensitive for lytic growth solely as a result of the newly discovered point mutation or because of a combination of the point and deletion mutations, a series of viruses were constructed which contained the point mutation, the deletion mutation, both mutations, or neither. This was done by ligating the PstI A and B DNA fragments from either tsA1499 or wt830 and transfecting the ligated DNA into BSC-1H monkey kidney cells. This experiment revealed that all viruses containing the point mutation (the tsA1499 PstI A DNA fragment) were temperature sensitive for lytic growth, regardless of the presence of the 81-base-pair deletion (the tsA1499 PstI B DNA fragment). This newly discovered point mutation, at nucleotide 4261, is therefore unique, since to our knowledge it is the first tsA mutation to be described in the 0.455-map-unit region of the SV40 genome. We then tested the effect of this unique mutation on the ability of the SV40 virus to transform cultured rat cells to anchorage independence.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1984

Transformation by purified early genes of simian virus 40.

A rapid soft-agar assay using baby hamster kidney (BHK21 cl.13) cells has been developed to establish the functional roles for the large T and small t antigens of SV40 in transformation. Plasmids expressing either large T or small t antigens of SV40 have also been constructed and these plasmids have been used separately or in combination for transformation. A large T clone, pD3-05, containing a deletion in the small t-specific coding region [0.584-0.54 map units (mu)], transformed a low-background subclone of baby hamster kidney (BHK21 cl.13) cell line and F111 rat fibroblasts to anchorage independence at a low level (10-20 and 1%, respectively, of an early region clone from wild type [WT], pW2). A WT-derived small t clone, pW2-t, containing a deletion in the large T-specific coding region (0.373-0.169 mu), did not transform F111 cells, but transformed BHK21 cells at a very low level (about 2% of pW2). Another WT-derived small t clone, pW2-t/B1, containing a larger deletion in the large T-specific coding region (0.512-0.169 mu), did not transform either BHK21 or F111 cells. However, cotransformation with pD3-05 clone and pW2-t or pW2-t/B1 clone increased the frequency of transformation to about the same level as that of pW2. The ability of the small t clones to enhance the transformation efficiency of the large T clone was not due to recombination between the two plasmids, since cotransformation with pD3-05 and a small t clone without the polyadenylation [poly(A)] signal sequence from WT, pW-t8, did not increase the frequency of transformation. When the frequency of transformation was determined by the focus assay using F111 cells, pD3-05 transformed as well as pW2. Also, cotransformation with pD3-05 and pW2-t/B1 did not increase the frequency of focus formation. Therefore, the small t antigen was not required for this morphological transformation.

Animals↗