Differentiation of human epidermal cells transformed by SV40.

Human epidermal cells were transformed with DNA from wild-type SV40 virus or with DNA from a temperature-sensitive A mutant (tsA209). The SV40-transformed cells differed from nontransformed cells in their morphologic appearance, growth properties, and expression of certain characteristics associated with differentiation. The transformed cells were more variable in size and shape than their nontransformed counterparts and were less stratified and less keratinized. While the growth properties of the cells were similar under optimal growth conditions, the transformed cells could be propagated under stringent growth conditions that did not support the growth of nontransformed human epidermal cells. The transformants still required a 3T3 feeder layer for growth, remained anchorage dependent as assayed in soft agar, and were not tumorigenic in athymic nude mice. The expression of certain differentiated functions of the human epidermal cell, the presence of keratins and cross-linked envelopes, was decreased in the transformed cells, and these functions could be restored at the nonpermissive temperature in the tsA209 transformed cells.

Purification of intact microtubules from brain.

Hundred-fold purification of intact microtubules from homogenates of rat brain is reported. The success of purification depends on stabilizing the microtubule structure by the combined effects of hexylene glycol, acidic Ph, and low temperature. A practical, negative stain, electron microscopic assay is used to study purity and stability of microtubule fractions. The purified fractions show a major band which migrates like purified tubulin in the SDS gel electrophoresis system.

Association of human papillomavirus types 16 and 18 E6 proteins with p53.

Human papillomavirus type 16 (HPV-16) is a DNA tumor virus that is associated with human anogenital cancers and encodes two transforming proteins, E6 and E7. The E7 protein has been shown to bind to the retinoblastoma tumor suppressor gene product, pRB. This study shows that the E6 protein of HPV-16 is capable of binding to the cellular p53 protein. The ability of the E6 proteins from different human papillomaviruses to form complexes with p53 was assayed and found to correlate with the in vivo clinical behavior and the in vitro transforming activity of these different papillomaviruses. The wild-type p53 protein has tumor suppressor properties and has also been found in association with large T antigen and the E1B 55-kilodalton protein in cells transformed by SV40 and by adenovirus type 5, respectively, providing further evidence that the human papillomaviruses, the adenoviruses, and SV40 may effect similar cellular pathways in transformation.

The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product.

Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.

Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade.

The E6AP ubiquitin-protein ligase (E3) mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The crystal structure of the catalytic hect domain of E6AP reveals a bilobal structure with a broad catalytic cleft at the junction of the two lobes. The cleft consists of conserved residues whose mutation interferes with ubiquitin-thioester bond formation and is the site of Angelman syndrome mutations. The crystal structure of the E6AP hect domain bound to the UbcH7 ubiquitin-conjugating enzyme (E2) reveals the determinants of E2-E3 specificity and provides insights into the transfer of ubiquitin from the E2 to the E3.

Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation.

Levels of the tumor suppressor protein p53 are normally quite low due in part to its short half-life. p53 levels increase in cells exposed to DNA-damaging agents, such as radiation, and this increase is thought to be responsible for the radiation-induced G1 cell cycle arrest or delay. The mechanisms by which radiation causes an increase in p53 are currently unknown. The purpose of this study was to compare the effects of gamma and UV radiation on the stability and ubiquitination of p53 in vivo. Ubiquitin-p53 conjugates could be detected in nonirradiated and gamma-irradiated cells but not in cells which were UV treated, despite the fact that both treatments resulted in the stabilization of the p53 protein. These results demonstrate that UV and gamma radiation have different effects on ubiquitinated p53 and suggest that the UV-induced stabilization of p53 results from a loss of p53 ubiquitination. Ubiquitinated forms of p21, an inhibitor of cyclin-dependent kinases, were detected in vivo, demonstrating that p21 is also a target for degradation by the ubiquitin-dependent proteolytic pathway. However, UV and gamma radiation had no effect on the stability or in vivo ubiquitination of p21, indicating that the radiation effects on p53 are specific.

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53.

The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro.

Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins.

E6-AP is a 100-kDa cellular protein that mediates the interaction of the human papillomavirus type 16 and 18 E6 proteins with p53. The association of p53 with E6 and E6-AP promotes the specific ubiquitination and subsequent proteolytic degradation of p53 in vitro. We recently isolated a cDNA encoding E6-AP and have now mapped functional domains of E6-AP involved in binding E6, association with p53, and ubiquitination of p53. The E6 binding domain consists of an 18-amino-acid region within the central portion of the molecule. Deletion of these 18 amino acids from E6-AP results in loss of both E6 and p53 binding activities. The region that directs p53 binding spans the E6 binding domain and consists of approximately 500 amino acids. E6-AP sequences in addition to those required for formation of a stable ternary complex with E6 and p53 are necessary to stimulate the ubiquitination of p53. These sequences lie within the C-terminal 84 amino acids of E6-AP. The entire region required for E6-dependent ubiquitination of p53 is also required for the ubiquitination of an artificial E6 fusion protein.

Cell-heritable stages of tumor progression in transgenic mice harboring the bovine papillomavirus type 1 genome.

Tumorigenesis of dermal fibroblasts in a line of transgenic mice carrying the BPV-1 genome was found to involve distinct proliferative stages. Cell cultures derived from normal skin, from benign proliferative fibromatoses, and from malignant fibrosarcomas each evidenced distinguishable, cell-heritable characteristics. The latent viral genome was transcriptionally inactive in normal-appearing skin and was activated in the dermal fibromatoses. Fibrosarcoma cells grew continuously in culture, formed domelike foci, and had a more rounded, anaplastic appearance. Independent cultures derived from the fibromatoses varied in their proliferative characteristics, which correlated well with the levels of viral gene expression. In contrast, progression to malignancy was not accompanied by a further increase in transgene activity, which strongly implicated cellular genetic changes in the later stages of tumorigenesis.

Functional characterization of interferon regulatory factor 3a (IRF-3a), an alternative splice isoform of IRF-3.

Virus infection of numerous cell types results in the transcriptional induction of a subset of virus- and interferon (IFN)-stimulated genes. The beta IFN (IFN-beta) gene is one of these rapidly induced genes; it serves as a fundamental component of the cellular defense response in eliciting potent antiviral, immunomodulatory, and antiproliferative effects. One of the transcription factors involved in the stringent regulation of IFN-beta production following virus infection is interferon regulatory factor (IRF) 3 (IRF-3). We have characterized an alternatively spliced isoform of IRF-3 that we have called IRF-3a. IRF-3a can selectively and potently inhibit virus-induced activation of the IFN-beta promoter. IRF-3a lacks half of the DNA binding domain found in IRF-3 and is unable to bind to the classical IRF binding elements, IFN-stimulated response elements. These studies suggest that IRF-3a may act as a modulator of IRF-3.

A stable bovine papillomavirus hybrid plasmid that expresses a dominant selective trait.

We describe a bovine papillomavirus hybrid plasmid containing the neomycin resistance gene from Tn5 inserted into a mammalian cell transcriptional unit. This plasmid is maintained as a stable extrachromosomal element (20 to 100 copies per diploid genome) in mouse cells selected either for the transformed phenotype or for resistance to the aminoglycoside G418. Cells selected for G418 resistance initially display a flat, nontransformed phenotype before exhibiting the gross morphological characteristics of transformation. The delay in the appearance of the transformed phenotype indicated that some intracellular event or series of events subsequent to the establishment of transcriptionally active bovine papillomavirus 1 hybrid plasmid is required for the manifestation of the transformed phenotype.

Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression.

The wild-type (wt) p53 protein has transcriptional activation functions which may be linked to its tumor suppressor activity. Many mutant p53 proteins expressed in cancers have lost the ability to function as transcriptional activators and furthermore may inhibit wt p53 function. To study the mechanisms by which mutant forms of p53 have lost their transactivation function and can act in a dominant negative manner, a structure-function analysis of both mutant and engineered truncated forms of p53 was carried out. We show that different mutant p53 proteins found in cancers vary in the ability to inhibit the transcriptional transactivation and specific DNA binding activities of wt human p53. This transdominant effect was mediated through the carboxy-terminal oligomerization region. The role of the transactivation activity in transformation suppression by wt p53 was also examined by constructing an N-terminal deletion mutant lacking the transactivation domain. This mutant was unable to transactivate but could bind specifically to DNA. Although it was impaired in its ability to suppress transformation of primary rat embryo fibroblasts by adenovirus E1A plus activated ras, the N-terminal deletion mutant still had some suppression activity, suggesting that additional functions of p53 may contribute to transformation suppression.

Enhancer-dependent expression of the rat preproinsulin gene in bovine papillomavirus type 1 vectors.

The effect of position in a bovine papillomavirus type 1 (BPV-1) vector on foreign gene expression was assessed with the rat preproinsulin (rI1) gene. The rI1 gene was inserted at each of the BPV-1/pML2d junctions in either transcriptional orientation in derivatives of the pdBPV-1(142-6) vector which consists of the BamHI linear genome of BPV-1 DNA cloned into pML2d. Transformed lines of C127 cells were established and assayed for rI1 gene expression. Cells containing the rI1 gene at the 3' end of the BPV-1 transforming region expressed rat proinsulin, whereas cells with the gene at the 5' end of the nontransforming region did not. Variability in the plasmid copy number or in the extent of DNA rearrangement could not account for this difference. We conclude that the expression of the rat preproinsulin gene (which is normally tissue specific for pancreatic islet cells) in C127 cells depends on the transcriptional activation afforded by viral enhancer sequences located at the 3' end of the transforming region. Intervening BPV-1 or pML2d sequences appear to block this enhancer-mediated gene activation. In agreement with enhancer-dependent activation, a rat preproinsulin gene located in a blocked position (i.e., not adjacent to the BPV-1 enhancer) could be activated by the insertion of a DNA fragment containing the simian virus 40, Moloney murine sarcoma virus, or BPV-1 enhancer element adjacent to the rI1 gene. Thus, a gene which is normally not expressed in a particular cell may be activated when placed adjacent to a viral enhancer in a BPV-1 vector.

Inducible expression of the human interferon beta 1 gene linked to a bovine papilloma virus DNA vector and maintained extrachromosomally in mouse cells.

A 1.6-kilobase DNA segment of the genomic human interferon beta 1 (IF-beta 1) gene was inserted into each of two possible orientations at the single HindIII site of a recombinant plasmid pBPV69T, consisting of the 69% transforming region of the bovine papilloma virus type 1 (BPV-1) and a modified SalI-SalI fragment of plasmid pBR322. After cleavage of the pBR322 sequences from this recombinant, BPV69T-IF-beta 1 hybrid DNAs were transfected onto C127 mouse cells by the standard calcium precipitation technique. Mouse cells transformed by this hybrid DNA produced low levels of human IF-beta 1 constitutively and responded to induction with either inactivated Newcastle disease virus or polyriboinosinic acid-polyribocytidylic acid. The BPV69T-IF-beta 1 hybrid DNA was nonintegrated in the transformed mouse cells but had acquired DNA sequences as a result of the transfection. Accurate transcripts of the IF-beta 1 mRNA were detected in cells only after induction. When the IF-beta 1 gene was oriented in the plasmid in the same direction of transcription as the BPV-1 genome, transcription was promoted from within the BPV-1 sequences. These results indicate that the regulatory sequences responsible for the inducible expression of the human IF-beta 1 gene are present in the 1.6-kilobase genomic segment and that these sequences can function in a free extrachromosomal state linked to BPV-1 sequences.

Bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector.

A novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. The foreign deoxyribonucleic acid (DNA) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. We have constructed a DNA hybrid molecule, BPV69T-rI1, containing the transforming region of bovine papilloma virus DNA and the rat preproinsulin gene I (rI1), and used it to transform susceptible mouse cells. DNA hybridization analysis has demonstrated the presence of multiple unintegrated copies of hybrid DNA molecules, with the bovine papilloma virus 1 DNA segment and the rI1 gene covalently linked in selected transformed cell lines. S1 nuclease analysis revealed the presence of a correctly spliced coding segment of the preproinsulin transcript similar or identical in its electrophoretic mobility to that of messenger ribonucleic acid produced in rat insulinoma cells. Significant levels of a protein immunoreactive with anti-insulin serum were detected by radioimmunoassay in the culture medium of transformed cells. Immunoprecipitation analysis in conjunction with competitive binding to bovine proinsulin established the identity of the protein as that of rat proinsulin.

The bovine papillomavirus distal "enhancer" is not cis essential for transformation or for plasmid maintenance.

We constructed a mutant of bovine papillomavirus type 1 (BPV-1) DNA that lacked a transcriptional enhancer located 3' to the polyadenylation site of the early viral RNAs expressed in transformed cells. This mutant DNA, when separated from the procaryotic sequences, transforms mouse cells with an efficiency comparable to that of the full BPV-1 genome, and it exists as a stable multicopy plasmid in transformed cells. The BPV-1 distal enhancer suppresses the effects of a cis-inhibitory element in pML2 sequences but is not essential for the expression of the viral genes involved in cellular transformation or plasmid maintenance.

Type 1 human T-cell leukemia virus small envelope protein expressed in mouse cells by using a bovine papilloma virus-derived shuttle vector.

In an attempt to express the small (transmembrane) envelope protein p21e of type 1 human T-cell leukemia (lymphotrophic) virus (HTLV-1) exclusive of other viral gene products, we have constructed a recombinant plasmid clone (pMBE-1) in a bovine papillomavirus-derived mammalian expression vector. Mouse C127 cells transfected with the pMBE-1 plasmid expressed the introduced HTLV-1 viral gene(s) as demonstrated by Northern blot and indirect immunofluorescence with natural human antisera. The transfected mouse cells were injected into BALB/c mice, and a monoclonal antibody was recovered which specifically recognizes a 21-kilodalton protein present in HTLV-1 virions, indicating that the pMBE-1 plasmid encodes the small envelope protein.

Oncogenicity of a nude mouse cell line transformed by a human papovavirus.

Primary cultures of NIH nude mouse (nu/nu) kidney cells were transformed with a human papovavirus (MMV). The transformed cell line expressed T-antigen, and MMV DNA was found to be associated with the cell DNA. When NIH nu/nu mice were inoculated with the transformed cells, they developed tumors at the injection site but failed to generate detectable levels of T-antibody. A control group of nu/+ littermates rejected the tumor inoculum but mounted an antibody response to T-antigen. It was proposed that nude mouse cells may be a suitable system to test oncogenicity of in vitro transformed cells.

Role of the human papillomaviruses in human cancer.

The papillomaviruses associated with human anogenital carcinomas encode two transforming genes, E6 and E7. The oncoprotein products of these two genes complex with the tumor suppressor gene products p53 and pRB, respectively. The loss of the normal function of these tumor suppressor gene products, either as a consequence of their association with E6 and E7 or by mutation, appears to be a common event in human cervical carcinogenesis.