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J Doniger

Publications and source records attributed to J Doniger.

At least 37 records · Page 2Linked to original sources
PubMed1992

Identification of two promoters within human cytomegalovirus morphologic transforming region II.

A 980-bp subfragment of human cytomegalovirus (HCMV) strain Towne has been previously identified as morphologic transforming region II (mtrII) because of its ability to induce focal transformation of NIH 3T3 cells. Transcripts from this region, which could encode the three open reading frames (ORFs), 79, 83, and 34 amino acids (aa), detected by DNA sequence analysis, are expressed early during HCMV infection. In this report, the mRNA start sites for promoters (P1 and P2) were mapped within Towne mtrII by primer extension using RNAs isolated from transformed NIH 3T3 cells. The Towne mtrII promoters exhibited similar activities to the SV40 enhancerless early promoter. Equivalent promoter activities were detected within the mtrII colinear nontransforming region from HCMV strain Tanaka. Two subclones of Towne mtrII (5' 440-bp and 3' 540-bp), each containing one promoter, were generated utilizing a unique BgII site which also interrupted the 79-aa ORF. In transfection assays, neither the 5' 440-bp promoter subclone containing a truncated 79-aa ORF nor the 3' 540-bp subclone containing intact 83- and 34-aa ORFs exhibited transforming activity. These data indicated that transformation by HCMV mtrII did not occur by promoter insertion. The identification of these early promoters will allow further studies on the regulation of important HCMV early genes known to be involved in viral/host interactions.

3T3 Cells↗
PubMed1992

The product of unr, the highly conserved gene upstream of N-ras, contains multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif.

We show that the open reading frame transcribed from the unr gene (immediately upstream of N-ras) in mammals consists of multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif found in prokaryotic cold-shock proteins, and eukaryotic DNA-binding proteins. Alignment of the CSD sequences of unr with those from other proteins reveals a core of similarity for which a consistent secondary structure prediction can be derived. This prediction suggests that the CSD consists primarily of beta-sheet, in contrast to most known eukaryotic DNA-binding proteins. Sequence analysis of the 3' end of the guinea pig unr gene shows that the core of one CSD repeat is encoded in a single exon, consistent with the modular assembly of the gene from ancestral CSD-coding units.

Amino Acid Sequence↗
PubMed1991

Immortalization of normal human oral keratinocytes with type 16 human papillomavirus.

Primary human oral keratinocytes were transformed by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA, and two transformed cell lines named human oral keratinocytes-16A and -16B (HOK-16A and HOK-16B) were established. While normal cells and cells transfected with vector only exhibited a limited lifespan, the HOK-16A and HOK-16B lines demonstrated immortality and altered morphology from their normal counterpart. The HOK-16A and HOK-16B lines contained approximately 40 and approximately 25 copies of intact HPV-16 DNA as integrated form per cell respectively, and both cell lines expressed several viral specific poly(A+) RNAs. Notably these cell lines also overexpressed cellular myc proto-oncogene in comparison with the normal counterpart. However, the immortalized cell lines were not able to produce tumors in nude mice, indicating that the cells are partially transformed. The HOK-16A and HOK-16B lines are, therefore, useful for investigating the multistep molecular events of oral carcinogenesis.

Blotting, Northern↗
PubMed1991

HPLC purification of polymerase chain reaction products for direct sequencing.

Same day PCR amplification and sequencing is desired in situations where one needs to sequence a number of PCR products. The rapid, high-yield purification of PCR products via the use of high performance, anion-exchange chromatography yields sequencing results comparable to those obtained from techniques requiring subcloning of the PCR product. This can be achieved by standard dideoxynucleotide sequencing technology without the need to prepare prelabeled primers and additional internal primers or to gel purify the PCR product. In addition, this chromatographic technique offers the potential of isolating several PCR products from the same amplification mixture.

Base Sequence↗
PubMed1990

HSV-2-induced tumorigenicity in HPV16-immortalized human genital keratinocytes.

A subgenomic region of HSV-2, BglII N, is capable of converting immortal genital epithelial cells containing integrated HPV16 sequences into tumorigenic squamous cell carcinoma cells. Moreover, tumor-derived cultured cells and immortal cells that had been transfected with HSV-2/Bg/II N and kept in continuous culture subsequently lost the HSV-2 sequences. The HSV-2/Bg/II N sequence was ineffective on normal cells. Thus, HSV-2/Bg/II N may act as a cofactor in the genesis of a carcinoma but is not required to maintain the transformed phenotype. Although papillomaviruses (HPVs) are currently receiving much attention because of their association with cervical squamous carcinomas, ample reasons exist to suggest a multifactorial etiology in which additional factors are necessary to convert dysplastic lesions to carcinomas. The hypothesis that specific HPV types may be necessary but not sufficient to cause cancer is reinforced. Thus, HSV-2-transforming sequences have a potential role in the etiology of human cervical cancer.

Carcinoma, Squamous Cell↗
PubMed1990

Polyomavirus-based shuttle vectors for studying mechanisms of mutagenesis in rodent cells.

We have constructed a series of polyomavirus-based shuttle vectors for analyzing mechanisms of mutagenesis in rodent cell systems. These vectors contain the supF suppressor tRNA gene which serves as the mutagenesis target; the pBR327 replication functions and ampr gene for replication and selection in bacteria; and the polyomavirus genome which permits replication in rodent cells. The polyoma genomes used in these vectors vary in their enhancer regions, causing varying efficiencies of replication in different types of rodent cells. One of the vectors (pPySLPT-2) which replicates particularly well in several different rodent cell types (i.e., Chinese hamster ovary, mouse hepatoma and mouse lymphoma) was used to compare mutation induction by UV radiation in UV repair-deficient mouse lymphoma L5178Y-R cells with mutagenesis in the related UV repair-proficient line, L5178Y-S. In both cell types, UV-induced mutants could be recovered at frequencies up to 50-fold higher than that of the spontaneous background. At a given UV fluence the L5178Y-R cells were more highly mutable than the L5178Y-S cells. Our results indicate that these new polyomavirus-based vectors should be useful for analysis of the molecular mechanisms of mutation induction in rodent cell systems, and in particular should allow detailed analysis of mutagenesis in the well characterized rodent somatic cell mutants.

Animals↗
PubMed1990

Frequent activation of non-ras transforming sequences in neoplastic Syrian hamster cells initiated with chemical carcinogens.

Carcinogen-caused transformation of Syrian hamster embryo cells has been widely used as a model for experimental carcinogenesis. However, analysis of the molecular mechanisms of hamster cell transformation has been limited. To expand the understanding of the molecular basis of this system, 22 independently derived Syrian hamster neoplastic cell lines initiated with chemical carcinogens were screened for the presence of dominant transforming sequences by DNA transfection into mouse NIH3T3 cells. High molecular weight DNAs from 12 (55%) of these cell lines transformed NIH3T3 cells through serial transfection cycles. NIH3T3 transformants contained hamster-specific repetitive sequences, which co-segregated with the transformed phenotype in successive transfection rounds. Results from Southern hybridization analyses and p21ras mobility assays indicated the presence of N-ras oncogenes, presumably activated by point mutations at codon 61, in 3 of the 12 (25%) transfection positive lines, all initiated with sodium bisulfite; non-ras transforming sequences were apparently activated in the remaining 9 (75%) lines. DNA prepared from NIH3T3 transformants derived from cell line 81C39 was analysed by Southern hybridization with a battery of 38 probes including non-ras oncogenes known to score as positive in the NIH3T3 assay as well as other retroviral and mammalian oncogenes. Each probe hybridized to DNA fragments showing the mobility characteristic of NIH3T3 protooncogenes, but failed to detect homolog sequences of hamster origin, even under hybridization conditions which allowed their detection in hamster DNA. Results show that ras activation occurs at a low frequency in hamster neoplastic transformation and strongly suggest that novel transforming sequences are activated, thus validating the use of this system for investigating the role of non-ras transforming sequences in neoplasia.

Animals↗
PubMed1989

Transient appearance of photolyase-induced break-sensitive sites in the DNA of ultraviolet light-irradiated Syrian hamster fetal cells.

Syrian hamster fetal fibroblasts (HFC) were examined for photolyase-induced break-sensitive sites after ultraviolet light (UV) exposure and growth. These sites, observed in excision-defective human xeroderma pigmentosum (XP) cells, are due to cleavage of the internal phosphodiester bond of UV-induced pyrimidine dimers. Excision-inefficient HFC acquired photolyase-induced break-sensitive sites during incubation after UV (10 J/m2). However, these were observed transiently, with a maximum of 5% of the pyrimidine dimers at 9 h post UV; by 18 h they were undetectable. Caffeine (1 mM) delayed the peak of photolyase-induced break-sensitive sites by 2 h. In human XP cells photolyase-induced break-sensitive sites accumulate to a plateau level of about 20% of the pyrimidine dimers. The present results extend to rodent cells the observation that cleavage of the internal phosphodiester bond of pyrimidine dimers may be an early step in their excision repair. Furthermore, the data suggest that photolyase-induced break-sensitive sites might be necessary for replication bypass at pyrimidine dimers.

Animals↗
PubMed1989

Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma.

Normal human foreskin keratinocytes cotransfected with the neomycin resistance gene and recombinant human papillomavirus (HPV) DNAs (types 16, 18, 31, and 33) that have a high or moderate association with cervical malignancy acquired immortality and contained integrated and transcriptionally active viral genomes. Only transcripts from the intact E6 and E7 genes were detected in at least one cell line, suggesting that one or both of these genes are responsible for immortalization. Recombinant HPV DNAs with low or no oncogenic potential for cervical cancer (HPV1a, -5, -6b, and -11) induced small G418-resistant colonies that senesced as did the nontransfected cells. These colonies contained only episomal virus DNA; therefore, integration of HPV sequences is important for immortalization of keratinocytes. This study suggests that the virus-encoded immortalization function contributes to the pathogenesis of cervical carcinoma.

Carcinoma↗
PubMed1989

Induction of human cervical squamous cell carcinoma by sequential transfection with human papillomavirus 16 DNA and viral Harvey ras.

Clinical and epidemiological data are consistent with the hypothesis that human papillomaviruses (HPVs) are a factor in genital, particularly cervical cancer. Although HPV16 and 18 are found primarily in cervical malignancy, the transfection of HPV16 or 18 DNA into cervical cells results in immortalization but not tumorigenicity. The addition of activated Ha-ras, an oncogene found in some cervical cancers expressing HPV16 or 18, to HPV16-immortalized human cervical cells results in malignancy as proven by the formation of cystic squamous cell carcinomas by HPV16-Ha-ras cells in nude mice. This two-stage model utilizing relevant human cells demonstrates that HPVs play a critical role in cervical malignancy and provides a system for elucidating critical cellular changes associated with progression to malignancy.

Animals↗
PubMed1988

Characterization of normal human exocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18 DNA.

An in vitro system for studying the interaction between human papillomavirus (HPV) 16 and 18 recombinant DNA and normal human exocervical epithelial cells is described. Eight HPV-immortalized human exocervical epithelial cell lines were established; all the lines contained either integrated HPV16 or 18 sequences and expressed HPV mRNAs. Thus, integration and expression appear to be required for immortalization. Immortalized cells (greater than 200 population doublings to date) divided rapidly (doubling time of 30 to 46 h) and morphologically resembled primary cultures of normal human exocervical epithelial cells. They expressed a keratin pattern consistent with their origin from exocervical epithelium. When cultured at high density or in the presence of serum they terminally differentiated. Sublines resistant to terminal differentiation were selected by growth in serum-supplemented medium. Keratin pattern changes suggest they have some properties in common with cervical squamous carcinoma cells. However, HPV-immortalized cell lines were not tumorgenic in nude mice. Thus, HPV16/18 is not carcinogenic by itself. These cell lines represent an appropriate model for studying factors that regulate HPV gene expression in normal cervical epithelial cells and examining the influence of cocarcinogens on neoplastic progression.

Animals↗
PubMed1988

Coordinate N-ras mRNA up-regulation with mutational activation in tumorigenic guinea pig cells.

Tumorigenic guinea pig cell lines with mutationally activated N-ras alleles also exhibited up-regulated N-ras mRNA. Mutational activation and mRNA up-regulation were limited to tumorigenic cells; preneoplastic progenitors were unaffected. Therefore, up-regulation occurred at a late stage of carcinogenesis closely associated with acquisition of tumorigenicity. cDNA and S1 protection analysis demonstrated that polyadenylation site of the short N-ras message and the mRNA start sites were different from that reported for human. The promoter region contained no canonical TATA or CCAAT boxes, but exhibited GGGCGG and CCGCCC SPl binding motifs characteristic of growth control genes. Moreover, both mutant and wild-type alleles were up-regulated in a guinea pig line heterozygous for N-ras codon 61. Coordinate N-ras mutational activation and up-regulation in five independent tumorigenic lines with unique chromosome constitutions suggests that both events are required for expression of the neoplastic phenotype.

Alleles↗
PubMed1988

Continuous cell lines with altered growth and differentiation properties originate after transfection of human keratinocytes with human papillomavirus type 16 DNA.

Immortalization of human keratinocytes (HKc) by human papillomavirus type 16 (HPV16) is reproducible at a high frequency, is due directly to the presence of the viral sequences in the cells, and occurs independently from the genetic characteristics of the host cells. Ten human keratinocyte strains, each derived from a different individual, were transfected with pMHPV16d and selected with G418. Eight became established lines. Two strains, which failed to grow shortly after successful G418 selection, were negative for HPV16 DNA. No lines were established following transfection of the same HKc strains with vector sequences only. The immortalized lines maintained a constant number of copies of the viral genome integrated into the cellular DNA. Each line showed a unique integration pattern of HPV16 sequences into the cellular genome, but expressed similar patterns of viral messages. Sublines able to grow in the absence of growth factors (epidermal growth factor and bovine pituitary extract), and others which became resistant to differentiation stimuli (serum and calcium) were obtained by selection in growth factor-free medium and serum-supplemented medium, respectively. The establishment of continuous cell lines is a direct consequence of the presence of viral sequences; however, because none of these lines formed tumors in nude mice, additional events must be necessary for progression of malignancy. HPV16-immortalized human keratinocyte lines can be used to investigate and identify the viral factors involved with the modification of growth and differentiation control by HPV16.

Aneuploidy↗
PubMed1988

The E7 open reading frame of human papillomavirus type 16 encodes a transforming gene.

Prior analysis of bovine papillomavirus has identified two genes, E5 and E6, which induce morphologic transformation of certain established cells. To study the transforming genes of human papillomavirus (HPV) type 16, the HPV type most commonly associated with cervical carcinoma, we examined subgenomic viral DNAs under control of a retroviral long terminal repeat for their capacity to induce cellular transformation of NIH 3T3 cells. Plasmids carrying the entire viral early region, the E6 and E7 (E6/E7) open reading frames (ORFs), or the E2,E4, and E5 (E2-E5) ORFs induced a very low frequency of focal transformation (0.4-1.7 ffu/micrograms DNA). In contrast, the plasmids with the entire early region of E6/E7, when selected by co-transfection with a transformation independent marker (neoR), induced anchorage independent growth with high efficiency. Cells selected after co-transfection with the marker gene and the E2-E5 plasmid grew much less efficiently in agar. Mutational analysis of the E6/E7 plasmid indicated that E7 was the gene primarily responsible for inducing anchorage independent growth. E7 gene therefore represents a third class of PV transforming gene. These results correlate with E7 (along with E6) being selectively retained and expressed in HPV associated cervical carcinomas and cell lines.

Animals↗
PubMed1987

Carcinogens with diverse mutagenic activities initiate neoplastic guinea pig cells that acquire the same N-ras point mutation.

N-ras has been identified by molecular cloning and DNA sequence analysis as the activated oncogene in carcinogen-induced guinea pig transformation. The deduced guinea pig amino acid sequence differs from that of human and mouse by 1 and 4 residues, respectively; the mismatches were in the C-terminal half of the fourth exon. The activated N-ras clone has an AT to TA transversion at the third position of codon 61 which results in the insertion of histidine instead of glutamine. The same activated N-ras gene with the identical mutation was found in all lines regardless of initiating carcinogen (aromatic aryl hydrocarbons or alkylating agents). These results suggest that the mutational event was independent of the mutagenic activity of the initiating carcinogen.

Amino Acid Sequence↗
PubMed1987

Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA.

Human keratinocytes and fibroblasts isolated from foreskin were transformed by transfection with recombinant human papillomavirus type 16 (HPV16) DNA. The transformed cells exhibited an extended (fibroblasts) or indefinite (keratinocytes) life-span compared with that of normal controls. In addition, HS27, a human fibroblast cell line previously transfected with origin-defective simian virus 40, was successfully transfected. HPV16 sequences were stably maintained in the cells, and extensive amplification and rearrangements occurred with continuous culturing. Moreover, both fibroblasts and keratinocytes expressed several specific HPV16 mRNAs. Because HPV16-transfected cells had viral transcripts and because transfection with the vector alone did not extend the life-span of the cells, it is likely that the virus was responsible for the indefinite life-span. Transfected fibroblast and keratinocyte lines will be useful for investigating the molecular biology of HPV16 and the interactions between the viral DNA and the human genome. Moreover, transfected keratinocytes provide a model for analyzing the effects of HPV16 on the differentiation properties of human epithelial cells.

Cell Transformation, Neoplastic↗
PubMed1987

Differential early viral gene expression in two stages of human papillomavirus type 16 DNA-induced malignant transformation.

Human papillomavirus (HPV) type 16 DNA induces progressive transformation in NIH 3T3 cells. Two types of cell lines, PM3T3G0 and PM3T3Fo, were isolated by G418 or focus selection, respectively, after transfection of cells by a recombinant HPV 16 DNA carrying the neo gene. These cell lines exhibited distinct phenotypes compared with controls. Saturation densities of PM3T3G0 and PM3T3Fo lines were two- to three- and five- to sevenfold greater than that of control NIH 3T3 cells, respectively. Neither cell type required high serum for growth, in contrast to NIH 3T3 cells. PM3T3G0 lines were premalignant, whereas PM3T3Fo lines manifested tumorigenicity within 2 weeks. Subpopulations of three PM3T3G0 lines underwent progressive transformation as reflected by focus formation. Analysis of HPV 16-specific mRNA species demonstrated that high levels of early and late gene expression were detected in premalignant PM3T3G0 lines, whereas relatively low quantities of selected gene messages were expressed in malignant transformants. Thus, high levels of viral gene expression are not crucial for malignant transformation.

Animals↗