Biomedical subjects

J Doniger

Publications and source records attributed to J Doniger.

At least 19 recordsLinked to original sources

PubMed1995

Human herpesvirus 6A ts suppresses both transformation by H-ras and transcription by the H-ras and human immunodeficiency virus type 1 promoters.

Human herpesvirus 6 strain U1102 (HHV-6A) was shown to contain a 1,473-bp functional transformation suppressor gene (ts). ts exhibited 24% identity and 51% similarity to adeno-associated virus type 2 Rep68/78. Like adeno-associated virus type 2 Rep68/78, HHV-6A ts suppressed H-ras transformation of NIH 3T3 cells. Suppression of H-ras transformation was eliminated by translation termination linker mutation at amino acid 25, 125, or 245. These data indicated the importance of the C-terminal portion of the ts protein. H-ras transformation was suppressed by ts only when H-ras was expressed by its endogenous H-ras promoter and not when it was expressed by the heterologous murine osteosarcoma virus long terminal repeat (LTR). Furthermore, ts suppressed chloramphenicol acetyltransferase (CAT) activity when the CAT gene was expressed from the H-ras promoter but not the murine osteosarcoma virus LTR promoter. Taken together, the data showed that ts suppressed H-ras transformation at the level of the H-ras promoter. To further identify the interaction of ts with transcriptional regulatory elements, the human immunodeficiency virus type 1 (HIV-1) LTR was used. This promoter was selected because it has well-defined transcriptional regulatory elements for both basal and activated transcription, because its activity is inhibited by the Rep68/78 gene, and because both HHV-6 and HIV-1 naturally infect CD4+ T cells in vivo and have been shown to infect the same cell in vitro. ts suppressed expression from both wild-type and upstream mutant HIV-1 LTR-CAT constructs. However, downstream HIV-1 TAR mutations reversed ts suppression, indicating that TAR is one of the critical elements involved. The data presented demonstrated that HHV-6A ts functionally suppressed H-ras transformation and HIV-1 LTR expression and thus that it may be useful in future gene therapy.

3T3 Cells↗

PubMed1994

Nucleic acid binding and intracellular localization of unr, a protein with five cold shock domains.

The unr gene was identified as a transcription unit located immediately upstream of N-ras in the genome of several mammalian species. While this genetic organization could be important for the transcriptional regulation of unr and N-ras, the function of the protein product of unr is unknown. unr is ubiquitously expressed and codes for an 85 kDa protein which is not closely related to previously characterized proteins. Nevertheless, a search for protein motifs has indicated the presence of five 'cold shock domains' within unr, a motif present in procaryotic cold shock proteins and in the vertebrate Y box factors. As these proteins have been reported to interact with nucleic acids, we investigated whether unr could bind to some classes of nucleic acids. We report here that unr has a high affinity for single-stranded DNA or RNA and a low affinity for double-stranded nucleic acids. Its low affinity for double-stranded DNA clearly distinguishes unr from the Y box factors. The binding of unr to RNA does not appear to depend upon extended sequence motifs but requires some level of sequence complexity as unr has only a low affinity for most simple polymers including polyA stretches. unr is also characterized by its low affinity for double-stranded and structured RNAs. We further determined that unr is mostly localized in the cytoplasm, and is in part associated with the endoplasmic reticulum. These studies indicate that unr is a novel single-stranded nucleic acid binding protein which is likely to be associated with cytoplasmic mRNA in vivo.

Cloning, Molecular↗

PubMed1994

Transcriptional activation of minimal HIV-1 promoter by ORF-1 protein expressed from the SalI-L fragment of human herpesvirus 6.

The SalI-L fragment of human herpesvirus 6 (HHV-6) strain U1102 transformed rodent cells and transactivated the HIV-1 LTR 10- to 15-fold in both monkey fibroblasts and human T-lymphocytes. In this report, the SalI-L transactivator of the HIV-1 LTR was localized to ORF-1 which codes for a protein of 357 amino acids. To determine if ORF-1 required functional Sp1 binding sites or the TATA box element of HIV-1 LTR for transactivation, 5'-deletion mutants of the HIV-1 LTR were employed. Plasmids pBS/SalI-L, pBS/SalI-L-SH, and pC6/ORF-1(S), a mammalian expression vector containing ORF-1, all transactivated a deletion mutant of HIV-1 LTR lacking functional Sp1 binding sites (CD-54). These studies demonstrate that transactivation occurred in the absence of Sp1 binding sites and required only a minimal HIV-1 promoter which contains the TATA box element. The specificity of the SalI-L transactivator for HIV-1 LTR was demonstrated by its inability to transactivate the human papillomavirus type 16 or 18 early promoters. The ORF-1 gene was cloned into and expressed from the pET17b bacterial expression vector. Purified ORF-1 protein was obtained by ammonium sulfate precipitation, Mono-S chromatography, and anti-T7. Tag immunoaffinity chromatography. Transactivation of the HIV-1 LTR by ORF-1 protein was demonstrated by electroporation studies in vivo and by transcription studies in vitro. To substantiate the putative biological role of ORF-1, pBS/SalI-L, pBS/SalI-L-SH, and pC6/ORF-1 all reactivated tat-defective HIV-1 provirus from latently infected cells expressing CD4. Thus, the data presented suggest that HHV-6 infection could have a cofactor role in the progression of AIDS.

Amino Acid Sequence↗

PubMed1994

A 79 amino acid oncogene is responsible for human cytomegalovirus mtrII induced malignant transformation.

Human cytomegalovirus (HCMV) morphological transforming region (mtr)II is the only HCMV mtr that was retained and expressed in transformed mouse or rat cells. The minimal transforming region has previously been shown to be within a 980-bp BanII/XhoI subfragment which encodes three open reading frames (ORF) of 34, 79, and 83 amino acids. This report provides definitive evidence that the 79-aa ORF is responsible for mtrII mediated tumorigenic transformation. The 79-aa ORF, subcloned into a mammalian expression vector, pCHC79orf, induced morphologic transformation of NIH 3T3 cells. These transformed cells expressed 79-aa ORF specific transcripts and were tumorigenic when injected into nude mice. A construct containing a triple termination linker inserted after codon 24 failed to transform NIH 3T3 cells to tumorigenicity even though 79-aa ORF specific transcripts were expressed. Furthermore, when the triple termination linker was inserted after codon 49, tumorigenic transformation still occurred. These results demonstrate that the 79-aa ORF is the oncogene within HCMV mtrII and that the first 49-aa are sufficient.

3T3 Cells↗

PubMed1994

A transforming fragment within the direct repeat region of human herpesvirus type 6 that transactivates HIV-1.

HHV-6 infection has been associated with several malignancies including non-Hodgkin's lymphoma and Hodgkin's disease by the presence of high antibody titer and/or the presence of HHV-6 DNA. To understand their oncogenic potential, SalI restriction fragments from HHV-6 strain U1102 were transfected into NIH3T3 cells to assess transforming ability. A 3.9-kbp SalI-L DNA fragment spanning the junction of the direct repeat left (DRL) and unique long segment (UL) regions of HHV-6 induced foci of morphologically altered cells. The SalI-L transformed NIH3T3 focal lines induced tumors in nude mice within 2 weeks. The retention of HHV-6 specific DNA observed in SalI-L transformed cells and their tumor-derived lines suggest a possible maintenance function. Since both HHV-6 infection as well as transforming fragments from other DNA viruses have been shown to transactivate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR), SalI-L was examined for transactivation activity. SalI-L up-regulated HIV-1 LTR CAT 10-15 fold in both monkey CV-1 and human T Jurkat cells. The further study of the SalI-L transforming fragment exhibiting transactivation of HIV-1 LTR will elucidate whether these two activities are encoded by a single gene and will aid in the understanding of the interaction between HHV-6 and HIV-1 as it relates to progression of AIDS and/or AIDS-related malignancies.

3T3 Cells↗

PubMed1993

Localization and sequence analysis of morphological transforming region III within human cytomegalovirus strain Towne.

A 7.6-kbp BamHI/XbaI EJ subfragment of the Towne XbaI-E fragment of human cytomegalovirus (HCMV) strain Towne has been previously designated as morphological transforming region III (mtrIII) because it induced focal and tumorigenic transformation of rodent fibroblasts. However, in two separate cell systems, mtrIII sequences were not retained because they were not detected in either the focal, tumor or tumor-derived cell lines. In this report, mtrIII was localized to a 2.1-kbp SalI/XbaI DNA fragment. The sequence of the 2,085-bp region was determined and compared to the colinear DNA from HCMV strain AD169. DNA sequence analysis revealed the presence of five open reading frames in Towne mtrIII, two of which are conserved in strain AD169. The localization and sequence analysis of mtrIII will allow further investigation as to the mechanism(s) by which HCMV may play a role in human cancer.

Base Sequence↗

PubMed1992

Down-regulation of keratin 14 gene expression after v-Ha-ras transfection of human papillomavirus-immortalized human cervical epithelial cells.

Keratin expression in human cervical squamous cell carcinoma (SCC) lines differed significantly from both normal and human papillomavirus (HPV) immortalized exocervical cells. Keratin 14 (K14) expression, determined by protein synthesis and mRNA levels, was dramatically down-regulated in the cervical SCC lines while keratin 5 (K5) expression was not. K14 expression was similarly down-regulated in an HPV-16 immortalized cervical cell line after tumorigenic transformation with recombinant v-Ha-ras DNA. Cultures derived from nude mouse tumor explants also exhibited an altered keratin profile and the levels of K14 protein synthesis, as well as K14 mRNA, were not detectable. In both cases K5 protein synthesis was not significantly down-regulated. In addition, neoplastic cervical SCC lines exhibited up-regulation of keratins 7, 8, 13, and 19, combined with slight down-regulation of keratins 6 and 16. Epidermal keratinocytes responded in a different manner to exocervical cells. Transfection of human papillomavirus-immortalized epidermal keratinocytes with the BglII N fragment of herpes simplex virus 2 produced a neoplastic cell line, but K5 and K14 expression remained unchanged. Thus, neoplastic transformation of human exocervical cells, both in vivo (spontaneous cervical SCC) and in vitro (HPV-16- and v-Ha-ras-induced cervical SCC), is accompanied by characteristic changes in keratin expression. The specific down-regulation of K14 in these tumorigenic cervical cells, in the absence of significant changes in the expression of K5, implies that the normal coordinate regulation of K5 and K14 gene expression has been uncoupled.

Blotting, Northern↗

PubMed1992

Prognostic significance of polymerase chain reaction detected human papillomavirus of tumors and lymph nodes in surgically treated stage IB cervical cancer.

This study describes the prognostic role of polymerase chain reaction detected human papillomavirus (HPV) in Stage IB cervical cancer patients treated with radical hysterectomy and pelvic and paraaortic node dissection. All tumors were confined to the cervix and all margins and nodes were disease free. Twenty-one patients were analyzed: 6 patients recurred within 20 months of initial therapy, while 15 had no evidence of disease with a minimum follow-up of 36 months. Polymerase chain reaction (PCR) was performed on paraffin-block tissue of the hysterectomy specimen cervical tumor and lymph nodes. Oligonucleotide probes for HPV types 6, 11, 16, 18, 31, 33, and 35 were used with consensus primers for uncharacterized HPV types created from an L1 constant region. Control tissues were run with each tumor sample to assure against contamination. HPV type confirmation was performed using diagnostic restriction sites. HPV was detected in all cervical tumors. Recurring tumors were infected with multiple types of HPV in all 6 tumors versus only 5 of 15 nonrecurring tumors being multiply infected (P = 0.023). No tumor had HPV 6 or 11, and the incidence of HPV 16, 31, 33, and 35 was not significantly different for recurrent versus nonrecurrent groups. HPV 18 was found in 5 of 6 recurring cancers versus 1 of 15 nonrecurring tumors (P = 0.0029). PCR typing of the histologically negative nodes that had been obtained at radical hysterectomy was done in all 6 recurring patients and in 6 nonrecurring patients. The recurrent patients had a significantly higher incidence of lymph nodes positive for HPV DNA (71%) than the nonrecurring patients (35%) (P = 0.0047). These observations suggest that HPV 18 cervical cancer patients, those with infections of multiple types, and those with HPV DNA in histologically negative lymph nodes may be at increased risk for recurrence.

Adult↗

PubMed1992

Sequential combined tumorigenic effect of HPV-16 and chemical carcinogens.

We immortalized oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines, human oral keratinocytes-16A (HOK-16A) and -16B (HOK-16B). These cell lines were morphologically different from the normal counterpart, contained HPV-16 DNA as integrated form and expressed numerous viral genes. However, these cells proliferated only in culture medium containing low calcium (0.15 mM) and are not tumorigenic in nude mice. To test the hypothesis that tumors can be developed by sequential combined effect of human papillomavirus and chemical carcinogens in the oral cavity, these immortalized cell lines were chemically transformed by exposure to either benzo[a]pyrene or methanesulfonic acid ethyl ester. Such transformants proliferated in medium containing physiological calcium levels (1.5 mM) and demonstrated enhanced growth potential in nude mice, whereas primary human oral keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. Chemically transformed cells contained integrated, intact HPV-16 sequences and transcribed significantly higher amount of HPV-16 E6/E7 messages and transforming growth factor-alpha (TGF-alpha) compared with the immortalized oral keratinocytes. Like the HPV-immortalized cell lines, the chemically transformed oral keratinocytes contained lower levels of newly synthesized, wild-type p53 proteins compared to normal cells, and expressed wild-type c-Ha-ras. These results indicate that this in vitro system is useful for investigating the mechanisms of multistep oral carcinogenesis.

Base Sequence↗

PubMed1992

High frequency of latent and clinical human papillomavirus cervical infections in immunocompromised human immunodeficiency virus-infected women.

In 32 human immunodeficiency virus (HIV)-infected women, routine gynecologic examination was performed with colposcopy and Papanicolaou smear; cervical swabs were collected for human papillomavirus (HPV) DNA screening and typing; and immune status was assessed by CD4 T-cell count. Dot blot analysis was specifically chosen for HPV DNA screening to detect only relatively substantial HPV DNA infections. Polymerase chain reaction analysis was used for precise DNA typing of dot blot-positive samples. The HPV data were assessed for immune status; a subject with a CD4 T-cell count below 200/microL was considered functionally immunosuppressed. The frequency of dot blot positivity was fivefold higher among immunocompromised (nine of ten) than relatively immunocompetent (four of 22) HIV-infected women. Moreover, four immunosuppressed women, compared with no immunocompetent subjects, had evidence of HPV DNA without signs of HPV-associated lesions by cytology or histology (ie, latent HPV infection). Furthermore, four of nine of the immunocompromised, compared with four of 21 immunocompetent, subjects had cervical intraepithelial neoplasia. These frequencies are high compared with those reported in the general population. Finally, HPV 18 was detected in five of the ten women with CD4 T-cell counts below 200/microL and in only one of the 22 with CD4 T-cell counts above that level. These results suggest that the normal immune system suppresses latent and clinical HPV cervical infections and that the efficiency of suppression may be HPV type-specific. Furthermore, impaired immune status, as reflected by CD4 T-cell count, is an important factor increasing the severity of HPV-induced cervical infections in this population.

Adult↗

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↗