title: Methylation damage response in hematopoietic progenitor cells.
abstract: The cellular response to methylation DNA damage was compared in multipotent CD34(+) hematopoietic stem cells and mature CD34(-) cells isolated from cord blood of the same donor. Cytofluorimetric analysis of freshly isolated cord blood cells indicated that both cell types were in the G0/G1 phase of the cell cycle. Quantitative RT-PCR identified a general trend towards high expression of several DNA repair genes in CD34(+) cells compared to their terminally differentiated CD34(-) counterparts. The overexpressed genes included members of the mismatch repair (MMR) (MSH2, MSH6, MLH1, PMS2), base excision repair (AAG, APEX), DNA damage reversal (O(6)-methylguanine DNA methyltransferase) (MGMT), and DNA double strand breaks repair pathways. These differences in gene expression were not apparent in CD34(+) and CD34(-) cells obtained following expansion of CD34(+) cells in a medium containing early acting cytokines. Early progenitor CD34(+) and early precursor CD34(-) cells form the two populations isolated under these experimental conditions, and both contain a significant proportion of cycling cells. The methylating agent N-methyl-N-nitrosourea (MNU) induced similar levels of apoptosis in these cycling CD34(+) and CD34(-) cells. Cytotoxicity required the presence of the MGMT inhibitor O(6)-benzylguanine and the timing of MNU cell death (48 and 72h) was similar in CD34(+) and CD34(-) cells. These data indicate that cycling CD34(+) and CD34(-) cells are equally sensitive to methylation damage. MGMT provides significant protection against MNU toxicity and MGMT and MMR play the expected roles in the MNU sensitivity of these cells.
authors.affiliation: Section of Experimental Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Ida
authors.initials: I
authors.lastname: Casorelli
authors.forename: Elvira
authors.initials: E
authors.lastname: Pelosi
authors.forename: Mauro
authors.initials: M
authors.lastname: Biffoni
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Cerio
authors.forename: Cesare
authors.initials: C
authors.lastname: Peschle
authors.forename: Ugo
authors.initials: U
authors.lastname: Testa
authors.forename: Margherita
authors.initials: M
authors.lastname: Bignami
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle; D004249:DNA Damage; D019175:DNA Methylation; D053843:DNA Mismatch Repair; D004260:DNA Repair / Q000235:genetics; D005312:Fetal Blood / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007231:Infant, Newborn; D008770:Methylnitrosourea / Q000633:toxicity; D011995:Recombination, Genetic
chemical_list: D018952:Antigens, CD34; D008770:Methylnitrosourea