E1A functions as a coactivator of retinoic acid-dependent retinoic acid receptor-beta 2 promoter activation

The retinoic acid (RA) receptor (RAR) beta 2 promoter is strongly activated by RA in embryonal carcinoma (EC) cells. We examined this activation in the P19 EC-derived END-2 cell line and in E1A-expressing counterparts and found strong RA-dependent RAR beta 2 promoter activation in the E1A-expressing cells, which was not observed in the parental cell line, indicating a possible role for E1A in RAR beta 2 activation. In transient transfection assays, E1A functioned as a coactivator of RA-dependent RAR beta 2 promoter activation and, moreover, was able to restore this activation in cells lacking RAR beta 2 activation. By deletion analysis, two regions in the RAR beta 2 promoter were identified that mediate the stimulatory effect of E1A: the RA response element and TATA box-containing region and a more up-stream region between -180 and -63, in which a cAMP response element-related motif was identified as a target element for E1A. In addition, determination of endogenous E1A-like activity by measuring E2A promoter activity in transient transfection assays in EC and differentiated cells revealed a correlation between RA-dependent RAR beta 2 promoter activation and the presence of this activity, suggesting an important role for the cellular equivalent of E1A in regulation of the RAR beta 2 promoter

International Pig-a gene mutation assay trial: Evaluation of transferability across fourteen laboratories

Experiments described herein were designed to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE and SYTO 13 dye were used to label leukocyte-depleted blood samples, and the frequency of CD59-negative erythrocytes (RBCCD59-) and CD59-negative reticulocytes (RETCD59-) were determined via flow cytometric analysis. To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to the prototypical mutagen N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20 and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: pre-dosing and on Days 15 and 30. Blood samples were processed according to standardized sample processing and data acquisition protocols, and three endpoints were measured: %reticulocytes, frequency of RETCD59-, and frequency of RBCCD59-. As illustrated by the analysis of technical replicates, the methodology was found to be highly reproducible, as experimental coefficients of variation approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the responses that were observed among different laboratories. Dose-related increases in the frequency of RETCD59- and RBCCD59- were consistently observed on Day 15. Whereas maximal RETCD59- responses tended to occur by Day 15, peak RBCCD59- responses occurred at approximately Day 45. Elevated mutant phenotype cell frequencies were maintained through the latest time-point studied (Day 90). High concordance correlation coefficients show a remarkable level of agreement between the reference site and the test sites. Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories