Student Research: Mark J. McMillan
MS, , 1998
Faculty Advisor: Elaine M. Faustman
Establishment of a Novel Method for Analyzing Cell Cycle-specific Changes in Gene Expression
Methylmercury (MeHg) is an environmentally-relevant metal and potent developmental neurotoxicant in animals and humans. Evaluation of children exposed in utero in Japan and Ira has demonstrated a range of developmental effects including mental retardation, cerebral palsy, growth retardation, blindness, and death. Histological examination of brain tissue from exposed children and animals reveals inhibition of cell migration and cell proliferation. To evaluate molecular mechanisms underlying MeHg-induced cell cycle inhibition, we have previously characterized alterations in gene expression of brain tissue from in vitro exposed rat CNS cells. Those investigations demonstrated MeHg-induced changes in gene expression at exposure concentrations associated with cell cycle inhibition in both G0/G1 and G2/M, and demonstrated induction of the genes GADD 45 and GADD 153. The purpose of the experiments presented here was to develop methods for examining cell cycle-specific induction of cell cycle regulatory genes. Methods included isolating micromass CNS cell cultures from gestational day 12 rat embryos and exposing them to 0 and 1 mM MeHg over 24 hr. Following exposure, cells were harvested, stained with a DNA fluorochrome, Hoechst 33342, and sorted into G0, G1, S, and G2/M by flow cytometry. Messenger RNA from sorted cells was reverse transcribed in situ and amplified due to the limiting number of cells attained following cell sorting and mRNA expression was analyzed on reverse Northern blots. We evaluated 2% paraformaldehyde, ethanol/acetic acid (95:5 v/v), and non-fixative methods to determine that non-fixed micromass CNS samples were the most useful for our application. Using fresh, non-fixed cells we were able to demonstrate that cell cycle-specific gene expression might be ascertained following MeHg treatment in sorted CNS micromass cells. Consistent with previous investigations, low-level MeHg exposure (1mM) altered normal cell cycle phase distribution as indicated by changes in cell cycle phase distribution in CNS micromass cells. Together, these data provide a basis for additional investigation of the role of cell cycle-specific gene expression following MeHg exposure.