Sa1821 Abnormal Expression of Epigenetic Regulators in Mesenchymal Stem Cells Explains Helicobacter Induced Gastric Carcinogenesis

Abstract

Background Epigenetic changes are heritable changes in gene expression without alteration of the coding sequence of the gene. Epigenetic regulators are key players in stem cell biology and carcinogenesis. We have shown that bone marrow derived MSC home to the gastric mucosa during Helicobacter infection and differentiate abnormally as dysplastic cells, can initiate, contribute to and become gastric adenocarcinoma. The time can be dramatically decreased by introducing MSC which have been repeatedly passaged in culture and allowed to spontaneously transform. The aim of the present study was to investigating the abnormal expression of the regulatory elements of polycomb (PcG) and trithorax (TrX) complexes over time in MSC and correlate these changes with tumor forming potential. Materials and MethodsMSC isolated from C57BL/6 mice bone marrow were placed in culture and grown for up to 1 year. Cells at each passage were tested for their ability to contribute to tumor formation in C57BL/6 mice, and frozen for later use and analysis. 84 genes representing mouse polycomb and trithorax complexes were studied using quantitative PCR analysis. RNA was isolated quantified and reverse transcribed into cDNA followed by quantitative PCR using Mouse Polycomb & Trithorax Complexes PCR Array (SAbioscience). Sequence analysis for the DNA binding domain of TP53 gene was performed at the core facility of the UMass. Results Differences in gene expression were detected beginning prior to the third passage. The fold change for each gene was calculated using 2(-ΔΔCT) method. The most significantly regulated genes were kdm5d, Rbp2, RnaseL, Zbtb16, cbx5, cbx7, Dnmt3I, phf19 and smarca1. P53 mutations were detected as early as passage 3. Early in culture, substitution mutations were common while at later passages insertions and deletion were detected. Despite dramatic genetic dysregulation, only cells grown for a year or greater in culture formed tumors in the C57BL/6 mouse. Conclusion and Discussion: Abnormal expression of these epigenetic regulatory elements may be involved in induction of p53 mutation. Tautomeres (C T substitution) are types of mutation that appear after DNA replication. This type of mutation is repaired through DNA proofreading mechanism occur directly after DNA synthesis. The presence of this type of mutation in passages 6, 9 and 12 indicate a defect in the DNA proofreading mechanism which may be linked to abnormal expression of these epigenetic regulatory elements. Also the presence of these tautomeres show that the mutations likely occur randomly along the DNA, not only in the P53 gene. The persistent accumulation of these mutations along the DNA over time, in parallel with the abnormal expression of the epigenetic regulatory elements results intomalignant transformation ofMSC.