The ability to analyze the molecular events involved in adenovirus transformation of mammalian cells critically has been impeded by the lack of a highly transformable target cell. The majority of investigations have used heterogeneous primary or secondary rat embryo cells that are semipermissive for group C adenovirus-i.e., serotypes 1, 2, or 5 (Adl, Ad2, and AdS, respectively) (1-6). With primary rat embryo cells, a certain proportion ofthe infected population is permissive for viral replication and produces infectious virus, whereas a small fraction of infected cells integrate viral DNA and become morphologically transformed (usually <10-5 transformants per infected cell) (3, 6). The frequency of Ad5 transformation of rat embryo cells can be enhanced 7-to 8-fold by treating the cells with chemical carcinogens prior to viral infection and by growing infected cells in the presence of the tumor promoting agent phorbol 12-myristate 13-acetate (6-8). However, even with these modifications, the frequency ofadenovirus transformation is still too low to allow accurate appraisal ofthe crucial virus-cell interactions required for stable transformation. In addition, because the majority of transformation assays have used genetically diverse animals as sources of embryo cultures, the quantitation and reproducibility oftransformation in different assays and laboratories have been major problems.By systematically screening >100 cloned populations derived from five established rat embryo cell lines, a specific clone of Fischer rat embryo cells (CREF cells) was isolated. The cells derived from this clone are transformed by Ad5 at least 150-fold more frequently than primary or secondary rat embryo cells. This report describes the CREF transformation system and the phenotypic properties of CREF clones transformed with wildtype Ad5 and the temperature-sensitive mutant H5ts125. Since previous studies have shown that wild-types Ad5 and Ad2 transformed rat embryo cells usually contain only part of the viral genome, including approximately the left hand 15% (9-11), whereas H5tsl25-transformed rat embryo cells transformed and cultured at 36 or 39.5°C usually contain the complete viral genome (10, 11), the patterns of viral DNA integration in wildtype-and H5tsl25-transformed CREF clones have also been determined. MATERIALS AND METHODSCell Cultures. A series of five early passage and established rat embryo cell cultures isolated from 10-to 14-day gestation Sprague-Dawley and Fischer rats were cloned and analyzed for stability at confluence, anchorage dependence, and transformability by adenovirus (6,12). The cell cultures studied included NRK, a normal rat kidney cell line, provided by David K. Howard; 3Y1, an established Fischer rat embryo cell line supplied by Nada Ledinko; SDRE-1 and SDRE-2, two independently isolated early passage Sprague-Dawley rat embryo fibroblast cell lines established in this laboratory as described (13); and an early passage F2408 Fischer rat embryo fibroblast cell line provided by Claudio Basilico. The CREF ...
Research Summary:Frederica Perera, DrPH, PhD, is a Professor of Environmental Health Sciences and serves as the Director of the Columbia Center for Children 's Environmental Health (CCCEH). Dr. Perera is internationally recognized for pioneering the field of molecular epidemiology, beginning with studies of cancer and then applying molecular techniques within studies of pregnant women and their children. Since 1998, she and her colleagues at CCCEH have tracked the health of more than 700 NYC pregnant women and their children. Exposures being studied include the combustion pollutants polycyclic aromatic hydrocarbons (PAH), pesticides, secondhand smoke, and chemicals in plastics and flame retardants. Outcomes include asthma, neurobehavioral development, cancer, and obesity.Her areas of specialization include prevention of environmental risks to children, molecular epidemiology, cancer prevention, environment-susceptibility interactions in cancer, developmental damage, asthma, and risk assessment. She is the author of over 300 publications and has received numerous honors, including the first Irving
The tumor-promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) caused a 2-to 3-fold enhancement of transformation of secondary rat embryo cells that had been infected with a temperature sensitive mutant of adenovirus type 5 (Hts125). In addition, transformed foci appeared earlier and were larger in cultures grown in the presence of TPA. The addition of TPA could be delayed until up to 7 days after viral infection and still enhancement was observed. Exposure It is now well recognized that the carcinogenic process is often multifactor in terms of etiology and multistep in its evolution (1). One of the earliest demonstrations of this was the study by Rous and Kidd (2) demonstrating that wounding or chemical treatment led to the appearance of skin tumors on the ears of rabbits that had been infected with Shope papilloma virus. One of the most extensively studied multistep animal models is that of two-stage mouse skin carcinogenesis (3) in which application of a subcarcinogenic dose of an "initiating carcinogen" followed by repeated application of a "promoting agent" results in the induction of multiple skin tumors. The most potent promoting agents for mouse skin are 12-0-tetradecanoyl-phorbol-13-acetate (TPA) and related plant diterpenes (4-6). Recent studies indicate that TPA enhances the in vitro transformation of fibroblast cell cultures previously exposed to either polycyclic aromatic hydrocarbons (7,8) or ultraviolet light (9).The purpose of the present study was to determine whether TPA might also enhance the in vitro transformation of cells previously infected with a transforming virus. The virus chosen for our studies is a temperature-sensitive mutant of adenovirus type 5 (H1ts125), which has a higher transforming efficiency than wild-type adenovirus type 5 (10-12). Since initiating carcinogens are known to act synergistically with certain viruses in tumor induction in the whole animal (13,14) and during cell transformation in vitro (13-16), we have also examined the effects of exposing our cell cultures to polycyclic aromatic hydrocarbons prior to the addition of H5ts125 and TPA. Viral Transformation Assays. Transformation assays were performed on secondary rat embryo cultures as described (18). Approximately 2 X 106 rat embryo cells were pretreated with 0.1-0.0125 jug of Me2BzA per ml or 0.5-0.05 jug of BzP per ml for 18 hr. The carcinogen-containing medium was removed, the cells were washed twice with Ca2+-and Mg2+ free phosphate-buffered saline, and the cells were then infected with 30 plaque-forming units of H5ts125 per cell (10-12) in 0.2 ml of phosphate-buffered saline for 3 hr at 370. Control cultures were treated with either 0.5% acetone (carcinogen solvent) or growth medium (modified Eagle's medium) for 18 hr and then infected with H5ts125. The cultures were then trypsinized and 1 X 103 cells were plated in each of five 50-mm tissue culture plates for survival assays or 2 X 105 cells were plated in each of ten 50-mm tissue culture plates for transformation assays. Plates for surviv...
Dysregulation of cyclin expression has been reported for several human malignancies, including breast cancer. To further investigate the role of cyclin genes in mammary tumorigenesis we analyzed the expression of cyclins D1, E and A and other cell cycle-related proteins in a series of nine N-methyl-N-nitrosourea-induced primary rat mammary tumors. Western blot analysis revealed a 10- to 15-fold increase in the level of cyclin D1 protein in most (7/9) of the tumors, when compared with normal rat mammary gland. The two tumors that did not show this increase also displayed negligible levels of the retinoblastoma protein. A moderate increase, 1.5- to 2-fold, in the level of cyclin E was observed in four tumors and three tumors displayed abnormal low molecular weight cyclin E-related proteins. None of the tumors showed amplification of the cyclin D1 or E genes when studied by Southern blot analysis. All nine tumors showed a 2- to 6-fold increase in the level of cyclin A protein. Most of the tumors also displayed a marked increase in levels of the CDK2 and CDK4 proteins. These changes did not appear to be simply a consequence of increased cell proliferation, as assessed by proliferating cell nuclear antigen analysis. Thus, aberrant expression of cyclins and other cyclin-related genes occurs frequently in mammary tumorigenesis in both rodents and humans.
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