Faculty Bibliography

The response to exposure of all-trans-retinoic acid (RA) during development varies from physiologic to severe teratogenic outcomes and is dependent upon the dose and the stage of development in all species. Effects of RA-mediated teratogenesis may be due to its ability to cause apoptosis. We have recently reported the modulation of p53 in murine stem cells by RA. The aim of this study was to characterize the temporal and spatial pattern of p53 expression in Swiss Webster mouse fetuses following maternal treatment with a single oral dose of 100 mg/kg body weight of RA during organogenesis. RA treatment resulted in a decreased p53 mRNA level in fetuses 24, 48, and 72 h after maternal treatment as detected by semiquantitative reverse transcriptase polymerase chain reaction. Western blot analysis showed a decrease in p53 protein at 24 and 48 h. Immunohistochemistry revealed decreased localization of p53 in the neuroepithelium of fetuses exposed to RA in utero. RA treatment also resulted in decreased nuclear p21 and decreased expression of cytosolic as well as nuclear p27 at 72 h in the fetuses. These results demonstrated that RA-mediated teratogenesis is accompanied by a reduction in the temporal and spatial pattern of p53 gene and protein expression in addition to the disruption of the cell cycle by modulation of p21 and p27.

The Myc family of genes regulates proliferation, differentiation, and apoptosis. Temporal expression of Myc family genes and several pro-apoptotic genes were investigated during Swiss Webster mice organogenesis after maternal treatment with an oral dose of 100 mg/kg trans-retinoic acid (RA) or vehicle on day 10 post-coitum. Reverse transcriptase-polymerase chain reaction and ribonuclease protection assay revealed decreased c-myc expression at 48 h followed by an increase at 72 h in fetuses from RA-treated dams. Increased c-Myc protein was detected at 72 h in the RA-treated group. In utero RA-treatment resulted in decreased expression of max, mad, caspases, bax, and bad genes at 48 h. Terminal uridinetriphosphate nick end-labeling (TUNEL) analysis revealed increased apoptosis at 24-48 h, followed by decreased apoptosis 72 h after in utero RA-exposure, which correlated with the decreased expression of pro-apoptotic genes noted at 48 h. Further investigations are needed to understand the role of Myc family genes during RA-mediated teratogenesis.

Apoptosis plays an important role during embryonic development. Apoptotic cell death is executed by caspases and can be regulated by the Bcl-2 family of genes. Ribonuclease protection assay was used to investigate the expression of selected apoptosis-related genes of the Bcl-2 family, pro-apoptotic Bax, Bad and anti-apoptotic Bcl-2, during differentiation of murine embryonic stem cells (ES) mediated by all-trans-retinoic acid. The mRNA expression of caspase 3, caspase 6 and certain pro-inflammatory cytokines was also investigated simultaneously. ES cells exposed to 1 microM all-trans-retinoic acid on day 8, 9 and 10 of differentiation revealed increased expression of Bax and Bad compared to the vehicle-treated cells. No effect on Bcl-2 mRNA was noted after all-trans-retinoic acid treatment. Increased mRNA expression of caspase 3 and caspase 6 in all-trans-retinoic acid-exposed ES cells suggested that caspases play an important role in retinoic acid-mediated apoptosis during ES differentiation. Increase in the expression of TNF alpha and macrophage migration inhibitory factor (MIF) was noted in retinoic acid-treated cells on day 14. Significant increase observed in interferon gamma inducing factor (IGIF/IL-18) mRNA expression in all-trans-retinoic acid-treated cells on day 14 and 17 did not translate to increased INF gamma expression. No change in the expression of other pro-inflammatory cytokines was noted with all-trans-retinoic acid treatment. The function of TNF alpha, IGIF/IL-18 and MIF in all-trans-retinoic acid-treated cells during ES differentiation and apoptosis is still speculatory. Results suggested that RA-mediated apoptosis during neural differentiation of ES cells involves up-regulation of caspase 3, caspase 6, Bad, and Bax.

c-Myc regulates cellular proliferation, differentiation, and apoptosis. Temporal expression of c-Myc during all-trans-retinoic acid (RA)-mediated neural differentiation in murine embryonic stem cell (ES) was investigated. Correlation to the modulation of dimerizing partners Max and Mad that may influence c-Myc signaling and transcription regulation was elucidated for the first time in these cells. In RA-treated cells, increase in c-myc mRNA was detected by reverse transcriptase polymerase chain reaction on days 11 and 14 of differentiation compared with the vehicle-treated controls. The results were further corroborated by ribonuclease protection assay (RPA). Western blots revealed an increase in c-Myc protein only on day 14 of differentiation in RA-treated cells. Increases in max and mad gene transcription detected by RPA at times of elevated c-Myc in RA-treated ES cells suggest that a transient increase in c-Myc protein expression may influence differential dimerization of Myc partners needed for signaling in the neural differentiation of ES cells.