Cultured Rodent Cells Research Articles

Alternative aspects of adipocyte function

This symposium was conceived, organized, and presented by members of NCR-97—Regulation of Adipose Accretion in Meat Animals. This group was organized over 25 yr ago and has been continuously involved in investigation of the biology of the adipocyte, the regulation of adipocyte hyperplasia and hypertrophy, and modulation of adipose tissue accretion in animals raised for meat production. Cattle, pigs, and sheep have been the major subjects, with some investigators using poultry. Rodent models and cells in culture have added to the scope of the research, particularly in regard to cellular and molecular mechanisms. In recent years, the adipocyte has become a more popular target for investigation, partly because of the interest in problems associated with human obesity, and partly because it has become known that adipose tissue is an endocrine organ secreting a number of compounds that can act at other sites in the body. This symposium presented concepts of the adipocyte that deviate from the usual discussions of endocrine and pharmacological regulation of anabolic and catabolic lipid metabolism, and hyperplasia and hypertrophy of the cells. The symposium was sponsored by Elanco Animal Health, Monsanto, Pharmacia Animal Health, and the USDA/CSREES. The publication costs were funded by USDA/ARS.

Production of antibodies in plants and their use for global health

Recombinant antibodies can be used to diagnose, treat and prevent disease by exploiting their specific antigen-binding activities. A large number of drugs currently in development are recombinant antibodies and most of these are produced in cultured rodent cells. Although such cells produce authentic functional products, they are expensive, difficult to scale-up and may contain human pathogens. Plants represent a cost-effective, convenient and safe alternative production system and are slowly gaining acceptance. Five plant-derived therapeutic recombinant antibodies (plantibodies) are undergoing clinical evaluation, three of which can be used as prophylactics.

A rapid switch in sympathetic neurotransmitter release properties mediated by the p75 receptor

Cardiac function is modulated by norepinephrine release from innervating sympathetic neurons. These neurons also form excitatory connections onto cardiac myocytes in culture. Here we report that brain-derived neurotrophic factor (BDNF) altered the neurotransmitter release properties of these sympathetic neuron-myocyte connections in rodent cell culture, leading to a rapid shift from excitatory to inhibitory cholinergic transmission in response to neuronal stimulation. Fifteen minutes of BDNF perfusion was sufficient to cause this shift to inhibitory transmission, indicating that BDNF promotes preferential release of acetylcholine in response to neuronal stimulation. We found that p75(-/-) neurons did not release acetylcholine in response to BDNF and that neurons overexpressing p75 showed increased cholinergic transmission, indicating that the actions of BDNF are mediated through the p75 neurotrophin receptor. Our findings indicate that p75 is involved in modulating the release of distinct neurotransmitter pools, resulting in a functional switch between excitatory and inhibitory neurotransmission in individual neurons.

Evaluation of a Quantitative Product-enhanced Reverse Transcriptase Assay to Monitor Retrovirus in mAb Cell-culture

Murine hybridoma cells used in the production of monoclonal antibodies (mAbs) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAbs intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by evaluation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In a previous report, we demonstrated the utility of TaqMan fluorogenic 5′-nuclease product-enhanced reverse transcriptase (TM-PERT) assays for measuring reverse transcriptase (RT) activity in laboratory-scale cell-culture samples and RT removal by laboratory-scale models of processing steps. In this report, we evaluate the specificity, accuracy, range, precision and robustness of TM-PERT for this purpose. We find that this assay detects RT activity contained in xenotropic murine leukemia virus (X-MuLV) and CHO cell type C particles and quantifies particle numbers comparably to other assays (e.g. transmission electron microscopy, viral sequence specific TaqMan). Cell derived DNA polymerases appear to contribute only modestly to the assay background and RT activity in clarified cell culture harvests is contained largely in Type C particles. TM-PERT is linear and precise between 107and 1013pU/ml, establishing the assay range. The assay is robust in that test article storage condition and DNA/protein content had little impact on assay performance. Thus, TM-PERT appears to be an acceptable assay to measure type C particles in rodent cell culture samples.

Development of human cell lines from multiple organs.

While the majority of carcinogenesis studies have relied on the use of rodent cells in culture, experimental models to define the role of carcinogenic agents in the development of cancers must be established by using a variety of human cells. Unlike rodent cells, normal human cells in culture rarely undergo spontaneous transformation and have generally proven to be resistant to neoplastic transformation by carcinogens. Remarkable progress has been made during the past decade in human cell transformation systems. Malignant transformation of human cells in culture has been achieved by a stepwise process: immortalization and conversion of the immortalized cells to tumorigenic cells. One of the critical initial events in the progression of normal human cells to tumor cells is the escape from cellular senescence, with few exceptions; normal human cells require immortalization to provide a practical system for carcinogenesis studies. Different cell types require different conditions and transforming agents to achieve a useful cell line. The current state of the art in immortalization of human cells will be presented.

Internalization of Carcinogenic Lead Chromate Particles by Cultured Normal Human Lung Epithelial Cells: Formation of Intracellular Lead-Inclusion Bodies and Induction of Apoptosis

Occupational exposure to certain particulate hexavalent chromium [Cr(VI)] compounds, such as lead chromate, has been associated with lung cancer and respiratory tract toxicity. We have previously shown that apoptosis is a major mode of death in cultured rodent cells treated with soluble sodium chromate and particulate lead chromate. Here we report the cellular and molecular effects of lead chromate and sodium chromate in normal human lung small airway epithelial (HSAE) cells, which may be one of the targets for Cr(VI)-induced lung cancer and respiratory tract toxicity. Phagocytosed lead chromate particles and intracellular lead-inclusion bodies (LIB) were observed by transmission electron microscopy and confirmed by X-ray analysis. HSAE cells exposed to lead chromate and sodium chromate underwent dose-dependent apoptosis. The cellular uptake and genomic interactions of both Cr and lead (Pb) were examined by inductively coupled plasma mass spectrometry (ICPMS) coupled with a novel, direct-injection high-efficiency nebulizer (DIHEN). Using this approach, we have quantitated a dose-dependent formation of Cr-DNA adducts and DNA-associated Pb in lead chromate–treated HSAE cells. The formation of LIB in normal human lung cells exposed to lead chromate indicates that ionic Pb is released from the particles and thus might contribute to the cell toxicity caused by lead chromate. Internalization and dissolution of lead chromate particles and the interaction of ionic Cr and Pb with DNA, may be components of the mechanism of lead chromate carcinogenesis. Lead chromate–induced apoptosis may be a mechanism to eliminate cells with chromium- and/or lead-damaged DNA.

Concepts of immortalization in human mammary epithelial cells.

Cellular immortality is a hallmark of neoplastic transformation, the detection of which in vitro may be only limited by providing stringent culture conditions for neoplastic cells. Unlike cultured rodent cells, spontaneously immortal human mammary epithelial cell lines cultured from presumably normal cells have rarely been identified. Only two reports described cell lines that arose in mammary tissue explants obtained from women with fibrocystic disease or intraductal hyperplasia (Briand et al. 1987; Soule et al. 1990). For clarity, and by definition, a normal human mammary epithelial cell (HMEC) population or clone that eventually senesces in culture is termed a “strain”, while a clone or population that has unlimited replicative potential is termed a “line”. The principles and players that confer unlimited in vitro replicative potential upon HMEC strains overlap partially with findings from cultured human diploid lineages, e.g., fibroblast-like cells and keratinocytes. Similarities between immortalized fibroblast-like cultures and HMECs include the functional repression of cell cycle- and DNA damage responsive-proteins p53, pRB, p16, and p21 (reviewed in Campisi et al. 1996; Garkavtsev et al. 1998). Nonetheless, human HMECs have unique phenotypes and require stringent culture conditions. This chapter will be limited to a discussion of human-derived mammary epithelial cells.

Effect of 3-aminotriazole on anchorage independence and mutagenicity in cadmium- and lead-treated diploid human fibroblasts.

Cadmium and lead have been shown to induce cellular transformations and gene mutations in cultured rodent cells, as well as tumours in live animals. However, the mechanisms by which these metals cause cellular transformations and mutations in human cells have not been explored. In this study, we investigated the abilities of cadmium and lead to induce anchorage-independent transformations and hprt gene mutations in diploid human fibroblasts. Human fibroblasts were exposed to either cadmium acetate (0-60 microM) or lead acetate (0-2 mM) for 24 h. After removal of the metals, the cells were kept in exponential growth for 7 and 9 days before mutation and anchorage-independence assays were taken, respectively. Both cadmium and lead significantly induced anchorage-independent colonies in dose-dependent manners; the frequencies of anchorage-independent colonies induced by these metals were similar to those induced by N-methyl-N'-nitro-N-nitrosoguanidine at approximately equal cytotoxic dose ranges (30-10% survival). 3-Aminotriazole at non-cytotoxic dosages decreased catalase activity by >80%, and markedly enhanced cadmium-induced cytotoxicity and anchorage-independent colonies. Cadmium uptake by human fibroblasts was not affected by 3-aminotriazole co-administered with 10 microM of cadmium; whereas cadmium uptake and accumulation were enhanced 1.5-fold by 3-aminotriazole co-administered with 1-2.5 microM of cadmium. Lead-induced anchorage-independence or cytotoxicity was not affected by 3-aminotriazole co-treatment; however, 3-aminotriazole did significantly enhance lead uptake and accumulation in human fibroblasts. Neither cadmium- nor lead-induced 6-thioguanine-resistant mutation frequency in human fibroblasts. Co-administering these metals with 3-aminotriazole did not enhance mutations in human fibroblasts. These results suggest that cadmium and lead may both act as tumour promoters in diploid human fibroblasts, and that reactive oxygen species is more important in cadmium- than lead-induced cytotoxicity and anchorage-independence.

Immunofluorescence analysis of diazepam-induced mitotic apparatus anomalies and chromosome loss in Chinese hamster cells.

To study the mechanisms leading to diazepam (DZ)-induced chromosome loss, we evaluated the effect of the drug on the distribution of cytoplasmic and mitotic apparatus proteins using specific antibodies. The use of antibodies directed against dynein and kinetochores (CREST staining) suggested that chromosomes arranged in monopolar spindles were interacting with short fibers originating from the monopole. Interestingly, nearly 50% of DZ-induced monopolar mitoses showed a punctate staining of centrosomes when evaluated with an anti-gamma-tubulin antibody. The extent of phosphorylation of mitotic proteins was not affected by drug treatment, as shown by staining the cells with an antibody against mitotic phosphorylated proteins (MPM-2). After recovery of DZ, nearly 20% of anaphases were abnormal and mainly consisted of multipolar anaphases and lagging chromosomes; this was consistent with a high frequency of kinetochore-containing micronuclei as evaluated by CREST antibody staining in cells that had divided only once after drug removal, i.e. binucleate cells obtained by cytochalasin-B treatment. Our data confirmed that DZ is a powerful inducer of chromosome loss in cultured rodent cells. Moreover, our results indicate that DZ interfered with the correct assembly of centrosomes.

A Conserved Element in the Protein-Coding Sequence Is Required for Normal Expression of Replication-Dependent Histone Genes in Developing XenopusEmbryos

Replication-dependent histone genes in the mouse and Xenopusshare a common regulatory element within the protein-encoding sequence called the CRASα element (coding region activating sequence α) which has been shown to mediate normal expression in vivoand to interact with nuclear factors in vitroin a cell cycle-dependent manner. Thus far, the α element has only been studied in rodent cells in culture, and its effect on histone gene expression during development has not been determined. Here we examine the role of the α element in histone gene expression during Xenopusdevelopment which features a switch in histone gene expression from a replication-independent mode in oocytes to a replication-dependent mode in embryos after midblastula stage. In vivoexpression experiments involving wild-type or α-mutant mouse H3.2 genes show that mutation of the CRASα element results in a fourfold decline of expression in embryos, but does not affect expression in oocytes. Two distinct α sequence-specific binding activities were detected in both oocyte and embryonic extracts. A slowly migrating DNA-binding complex was present at relatively constant levels throughout development from the earliest stages of oogenesis through larval stages. In contrast, levels of a rapidly migrating complex were high in stage I and II oocytes, declined in stage II–VI oocytes, remained low in unfertilized eggs and cleavage stage embryos, and rose dramatically after the midblastula transition. The molecular masses of the factors forming the slow and rapidly migrating complexes were estimated to be approximately 110 and 85 kDa, respectively. DNA-binding activity of the 85 kDa α-binding factor was affected by phosphorylation, binding with higher affinity in the dephosphorylated state. The abrupt increase in DNA-binding activity of the 85-kDa α-binding factor at late blastula coincides with the switch to the replication-dependent mode of histone gene expression. We propose that the conserved α element present in the coding sequence of mouse and Xenopuscore histone genes is required for normal replication-dependent histone expression in the developing Xenopusembryo.

Extreme N terminus of E1A oncoprotein specifically associates with a new set of cellular proteins.

By interacting with key regulatory proteins such as the pRb family, cyclins, cyclin-dependent kinases and p300/CBP of host cells, adenoviral E1A interferes with various cellular processes to provide a suitable environment for the replication of viruses. E1A may promote DNA synthesis and cell cycle progression, immortalize rodent cells in culture and transform cultured cells in cooperation with E1B, Ras, or other oncoproteins. Both extreme N terminus and conserved region 1 of E1A are required for the immortalization and the transformation of rodent cells, transcriptional repression and specific induction of the expression of cellular genes such as the proliferating cell nuclear antigen (PCNA) and heat shock protein 70 (HSP70). Although the molecular mechanisms of these functions of E1A are not fully understood, it is believed that protein-protein interactions may play essential roles. In this communication, we report that a new set of cellular proteins with apparent molecular weight of 200, 90, 45, 30, and 28 specifically associate with the extreme N terminus of E1A. Further analysis demonstrate that these associations do not depend on E1A's association with p300 or pRB. Neither the 30 kDa nor the 28 kDa polypeptide is identical to Cdc2 or Cdk2. The region of E1A required for the protein interaction is also required for the recently identified N-terminal transactivation activity of E1A. Our observations suggest that in addition to p300/CBP, the new set of cellular proteins may be involved in the functional complexity of the N terminus of E1A, thus predicting a p300/CBP independent pathway.

Genotoxic Effects Induced by Airborne Particulates on Tracheobronchial Epithelial Cells <italic>In Vitro</italic>

Airborne particulate matter adsorbs chemicals, forming a locus for chemical interactions and a vehicle for transmission. Chemical analyses have shown that airborne particulate matter from heavily industrialised areas contains more than 500 chemical substances, among them putative and known carcinogens, particularly polycyclic aromatic hydrocarbons, acridines, traces of heavy elements etc. (Goldstein, 1983). It has been recognised for many years that high levels of particulate air pollution can lead to increased mortality and morbidity. Dockery and Schwartz (1995) reported that daily mortality was associated with particulate air pollution measured as total suspended particulates (TSP). Similar analysis have shown positive correlations between particulate air pollution and daily mortality in six communities in the United States (Dockery etal., 1993; Schwartz, 1994). Other epidemiologic studies have highlighted an association between inhalable particulates with an aerodynamic diameter of less than 10 |im and especially fineparticulates below 2.5 [xm incriminated to contribute to excess morbidity and mortality in certain U.S. cities (Pope et al., 1995). The respiratory tract is the major site of exposure to airborne particulates (Heyder, 1993) and genotoxic activity of airborne particulates leading to mutation and cancer has been well documented (Epstein et al., 1977; Chrisp and Fisher, 1980). An important target of aerosols, especially of inhalable particulate matter with a particle size smaller than 10 (j,m are the tracheobronchial epithelial cells. The most common cancer in man, the bronchogenic carcinoma, originates from these cells (Tomatis, 1990). Several in vitro models have been developed for the study of respiratory toxicology, carcinogenesis and mutagenesis utilising rodent and human tracheal epithelial cells in culture (Nettesheim and Barrett, 1984; Harris 1987). In previous studies it has been reported that organic extracts of airborne particulates are cytotoxic, mutagenic and carcinogenic in a number of short-term bioassays using rodent and human tissue culture cells (Seemayer et al., 1988; Hornberg et al., 1993; Hornberg and Seemayer, 1995a, b; Motykiewicz etal., 1996). In this study we present data of genotoxic effects of a sample of airborne particulates collected in Duisburg, a city in the highly industrialised Rhine-Ruhr district, Germany. As target cells we utilised tracheal epithelial cells of the golden Syrian hamster, the rat and human bronchial epithelial cells of the line BEAS-2B (Reddel et al., 1988).

Basic cell culture.

This article will describe the basic techniques required for successful cell culture. It will also act to introduce some of the other chapters in this volume. It is not intended, as this volume is not, to describe the establishment of a tissue culture laboratory, nor to provide a historical or theoretical survey of cell culture. There are several books that adequately cover these areas, including the now somewhat dated but still valuable volume by Paul (1), the multi-authored Methods in Enzymology volume edited by Jakoby and Pastan (2), and the new edition of Freshney (3). Instead, this chapter's focus will be on the techniques for establishing primary rodent cell cultures from embryos and adult skin, maintaining and subculturing these fibro-blasts and their transformed derivatives, and the isolation of genetically pure strains. The cells described are all derived from Chinese hamsters since, to date, these cells, have proved to be the most useful for somatic cell genetics (4,5). The techniques, however, are generally applicable to most fibroblastic cell types.

Sister chromatid exchanges in rodent tracheal epithelium exposed in vitro to environmental pollutants

In our highly industrialized world, air pollution has become a major topic. The human respiratory tract is constantly exposed to air pollutants by inhalation. Besides gaseous pollutants airborne particulates are of great importance, containing a complex mixture of several hundred substances. The tracheobronchial epithelium is the major target site of airborne particulates as well as the origin of the most common cancer in man, the bronchogenic carcinoma. In our study we collected samples of airborne particulates in winter 1991 in the highly industrialized Rhine-Ruhr area (Germany) with a high-volume sampler on glass fiber filters. Airborne particulates were extracted with di-chloromethane and quantitatively transferred to dimethyl sulfoxide (DMSO) for tissue culture experiments. As target cells for genotoxicity testing we used cultures of rodent tracheal epithelial cells from the Syrian golden hamster and from the rat. Induction of “sister chromatid exchanges” (SCE) was utilized as a sensitive cytogenetic endpoint for evaluation of the genotoxic activity of extracts of airborne particulates. In presence of global extracts (GEX) we observed a dose-dependent, highly significant increase of SCE in tracheal epithelial cells of the Syrian golden hamster and of the rat. It is remarkable that even quantities of chemical substances equivalent to airborne particulates from less than 1 m 3 of air were genotoxic. Results of this study and earlier reports demonstrate that rodent tracheal epithelial cells offer a reliable and sensitive in vitro model for genotoxicity testing of airborne particulates. Therefore, tracheal epithelial cells in vitro appear a meaningful alternative to other human and rodent cell culture systems which have been used for genotoxicity testing of air pollutants.

Abundance and subcellular localisation of cyclin D3 in human tumours.

The D-type cyclins are positive regulators of the G1 phase of the mammalian cell cycle. Cyclins D1 or D2 are over-expressed in several types of cancer, transform rodent cells in culture and therefore harbor hallmarks of cellular proto-oncogenes. In contrast, no data on expression of cyclin D3 in tissues and tumours are presently available. We have raised monoclonal antibodies (MAbs) specific for cyclin D3 and examined abundance and subcellular localisation of this G1 cyclin in a series of human cultured cell types and in 180 primary tumours of diverse histogenesis. Cyclin D3 localised predominantly in nuclei of normal and tumour cells both in culture and in situ, and a pronounced cell-to-cell variation of its abundance was reminiscent of cyclins D1 and D2. Immunohistochemical analysis of tumour and corresponding normal tissues showed strong aberrant accumulation of cyclin D3 in a subset (about 10%) of breast carcinomas, whereas only weak-to-moderate expression was found in colorectal, head and neck and uterine carcinomas, melanomas and soft tissue sarcomas. The specificity of the immunohistochemical data was confirmed by immunoblotting analysis of tissue and tumour lysates. Our results indicate that over-abundance of cyclin D3 is considerably less frequent than that of cyclin D1, yet we identify subsets of breast tumours, and potentially lymphomas, as candidate tumour types with elevated cyclin D3 expression.

Mutagenic specificity of solar UV light in nucleotide excision repair-deficient rodent cells.

To investigate the role of nucleotide excision repair (NER) in the cellular processing of carcinogenic DNA photoproducts induced by defined, environmentally relevant portions of the solar wavelength spectrum, we have determined the mutagenic specificity of simulated sunlight (310-1100 nm), UVA (350-400 nm), and UVB (290-320 nm), as well as of the "nonsolar" model mutagen 254-nm UVC, at the adenine phosphoribosyltransferase (aprt) locus in NER-deficient (ERCC1) Chinese hamster ovary (CHO) cells. The frequency distributions of mutational classes induced by UVB and by simulated sunlight in repair-deficient CHO cells were virtually identical, each showing a marked increase in tandem CC-->TT transitions relative to NER-proficient cells. A striking increase in CC-->TT events was also previously documented for mutated p53 tumor-suppressor genes from nonmelanoma tumors of NER-deficient, skin cancer-prone xeroderma pigmentosum patients, compared to normal individuals. The data therefore indicate that the aprt gene in NER-deficient cultured rodent cells irradiated with artificial solar light generates the same distinctive "fingerprint" for sunlight mutagenesis as the p53 locus in NER-deficient humans exposed to natural sunlight in vivo. Moreover, in strong contrast to the situation for repair-component CHO cells, where a significant role for UVA was previously noted, the mutagenic specificity of simulated sunlight in NER-deficient CHO cells and of natural sunlight in humans afflicted with xeroderma pigmentosum can be entirely accounted for by the UVB portion of the solar wavelength spectrum.

Origins and functions of cells essential for periodontal repair: the role of fibroblasts in tissue homeostasis.

A review is undertaken of rodent model systems and cell culture studies that address the role of periodontal fibroblasts in tissue homeostasis in both normal function and after wound healing. Fibroblasts are the predominant cells of the periodontal ligament (PL) and of healthy gingiva and have important roles in the development, function and regeneration of the tooth support apparatus. In normally functioning periodontal tissues cell turnover involves generation of new cells by proliferation which in turn is balanced by apoptopic cell death. Consequently PL fibroblasts comprise a renewal cell system in steady-state. PL cell progenitors can generate multiple types of more differentiated, specialized cells including large numbers of fibroblastic cells and more limited numbers of osteogenic or cementogenic cells. However PL fibroblasts constitutively block osteogenesis and thereby maintain the PL width. Proliferating progenitor cell populations of the PL are enriched in locations adjacent to blood vessels and in contiguous endosteal spaces from where they migrate to the body of the PL. Large increases of cell formation and cell differentiation occur after wounding but surprisingly, the cells that repopulate the PL adjacent to the root surface are largely post-mitotic. As PL cell populations comprise multiple lineages, it is likely that after wounding, the separate phenotypes repopulating the wound site will be selected by environmental factors. Further, the specific repopulating lineages will strongly influence the form and function of the nascent tissue. To illustrate the specificity of fibroblast functions, examples of migratory and contractile fibroblast phenotypes are provided which exhibit constitutively different levels of gelsolin and alpha-smooth muscle actin respectively, cytoskeletal proteins which are markers for these cell types. Fibroblasts contribute to PL homeostasis by their abilities to remodel tissues, to repopulate wounds, to influence the metabolism of other cell types and to create a new fibrous attachment.

The role of estrogen in mammary carcinogenesis.

The in vivo and in vitro studies conducted to examine whether E2 functions as an initiator or a promoter in mammary carcinogenesis can be summarized as follows: (1) Clinical and animal studies in vivo have shown a positive correlation of up-regulation of E2 C16 alpha-hydroxylation with either the presence of or the risk for breast cancer, suggesting that this metabolic alteration may represent an early-occurring event in the multistep process of tumorigenesis. (2) The mammary tissue, target for carcinogenesis, exhibits cancer risk-dependent alteration in E2 metabolism in the rodent and human mammary explant culture model, indicating that E2 metabolites may directly influence the mammary epithelium. (3) The 16 alpha-hydroxylated metabolite of E2, 16 alpha-OHE1, induces genotoxic DNA damage and aberrant hyperproliferation similar to that induced by chemical carcinogens in the rodent cell culture model. In preinitiated or fully transformed rodent or human cells, 16 alpha-OHE1 promotes the expression of transformed phenotype. (4) The initiator-mediated perturbation of E2 C16 alpha-hydroxylation in rodent and human mammary explant cultures is modulated by naturally occurring dietary constituents that are known to modulate rodent mammary tumorigenesis. (5) The observed effect of E2 on mammary tumorigenesis may be due in part to the generation of 16 alpha-OHE1, which functions as a weak initiator or a potent promoter of tumorigenic transformation in mammary epithelial cells. (6) The reaction of 16 alpha-OHE1 with the transcription factor ER is unique in that it can be irreversible and leads to aberrant gene expression.

BS69, a novel adenovirus E1A-associated protein that inhibits E1A transactivation.

The adenovirus E1A gene products are nuclear phosphoproteins that can transactivate the other adenovirus early genes as well as several cellular genes, and can transform primary rodent cells in culture. Transformation and transactivation by E1A proteins is most likely to be mediated through binding to several cellular proteins, including the retinoblastoma gene product pRb, the pRb-related p107 and p130, and the TATA box binding protein TBP. We report here the cloning of BS69, a novel protein that specifically interacts with adenovirus 5 E1A. BS69 has no significant homology to known proteins and requires the region that is unique to the large (289R) E1A protein for high affinity binding. BS69 and E1A proteins coimmunoprecipitate in adenovirus-transformed 293 cells, indicating that these proteins also interact in vivo. BS69 specifically inhibits transactivation by the 289R E1A protein, but not by the 243R E1A protein. BS69 also suppressed the E1A-stimulated transcription of the retinoic acid receptor in COS cells, but did not affect the cellular E1A-like activity that is present in embryonic carcinoma cells. Our data indicate that BS69 is a novel and specific suppressor of E1A-activated transcription.

Regulation of viral and cellular gene expression by E1A proteins encoded by the oncogenic adenovirus type 12.

For more than 15 years human adenoviruses (Ad) have been a powerful tool for studying cellular processes such as regulation of gene expression, alternative splicing, polyadenylation, and replication. Especially the analysis of viral proteins encoded by early region 1A (E1A), which was shown to regulate transcription, has given many insights into how DNA viruses regulate their own and cellular gene expression. Moreover, E1A proteins have been the subject of extensive studies because of their ability to act as oncoproteins that cooperate with the adenovirus E1B gene products to transform rodent cells in culture and, in case of the oncogenic adenoviruses (e.g., Ad12), to induce tumors in animals (Gallimore et al. 1974; Graham et al. 1974a, b; Houweling 1980; Jochemsen et al. 1982). The ability to promote oncogenic transformation and transcriptional regulation appear to be distinct activities of the E1A polypeptides (for review see Moran and Mathews 1987). In oncogenic transformation, protein functions of region E1A are necessary to immortalize primary cells, whereas functions of region E1B are essential to obtain a fully transformed phenotype. The functions of region E1B can be substituted by specific cellular gene products, e.g., activated Ha-ras (Byrd et al. 1988; Ruley 1983). The reasons for the difference in oncogenicity of variant adenovirus serotypes are not yet understood.KeywordsAdenovirus TypeCellular Gene ExpressionCellular Transcription FactorHuman Adenovirus TypeRetinoblastoma Gene ProductThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.