Abstract
Abstract 4711
Promyelocytic leukemia protein (PML) was found in discrete, punctuate subnuclear structures known as PML nuclear bodies (PML NBs) in normal somatic cells and is an important factor acting downstream of wnt/β-catenin in the regulation of cell proliferation and differentiation. It is well-known that PML can bind cbp/p300 to promote TCF/LEF dependent transcription of downstream genes in 293T cells. However, its function in human mesenchymal stem cells (hMSCs) remains unclear. In our former studies, we found PML NBs in mitosis metaphase of hMSCs were more abundant than in Hela cells and 293T cells, which indicates PML may have special functions in hMSCs compared with tumor cells and normal somatic cells. The aim of our study is to investigate PML's function during proliferation and differentiation in hMSCs in contrast to Hela cells and 293T cells. hMSCs were isolated from bone marrow of healthy volunteers by density gradient centrifugation using Ficoll-paque and cultured (LG-DMEM, 10%FBS) with their characteristic adherence and morphology. hMSC immunophenotype was analyzed by flow cytometry and demonstrated uniform positivity for CD29, CD44, CD166 and CD105, and negativity for CD34, CD45 and HLA-DR. MTT assay and flow cytometry were used to evaluate proliferation of hMSCs. Osteogenic differentiation of hMSCs was perfoprmed and differentiated cells were identified with biochemical and morphological approaches. The levels of PML mRNA and protein expression during proliferation and differentiation were detected by RT-PCR, western blot and immunofluorescence while Hela cells and 293T cells were used as controls. Results showed the mean levels of mRNA and protein expression significantly increased during proliferation of hMSCs and 293T cells and positively correlated with the proliferation status of hMSCs. However, the expression of PML decreased in proliferating Hela cells (Fig. 1A, B). Intranuclear NBs of MSCs immunostained with PML monoclonal antibody and showed increased levels during proliferation, but remained very low in Hela cells throughout their proliferation. (Fig2). The above results indicate PML may take part in regulating the proliferation of hMSCs, and the mechanism involved is different from that in tumor cells. In tumor cells, the main function of PML maybe a suppressor of malignant cell growth. And PML protein is reduced or almost completely lost by post-transcriptional mechanisms. This loss is associated with tumor cell proliferation while regulation of cell proliferation and differentiation is the more important function of PML in hMSCs. During osteogenic differentiation of MSCs, ALP which is an early marker of osteogenic differentiation increased while PML's expression accordingly increased as detected by RT-PCR. (Fig 1C). Intranuclear NBs during differentiation are bigger but unequal in size compared with controls. (Fig3) These changes indicated that PML may interact with other molecules in NBs and act as a complex to regulate the process of osteogneic differentiation, but the exact mechanism involved is unclear and needs further study.
Disclosures:
No relevant conflicts of interest to declare.
NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs
