Methylation damage response in hematopoietic progenitor cells.
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.
Casorelli
Ida
I
Section of Experimental Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.
Pelosi
Elvira
E
Biffoni
Mauro
M
Cerio
Anna Maria
AM
Peschle
Cesare
C
Testa
Ugo
U
Bignami
Margherita
M
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
D018952:Antigens, CD34; D008770:Methylnitrosourea