Preferential in vivo DNA repair of melphalan-induced damage in human genes is greatly affected by the local chromatin structure.
To investigate the molecular mechanisms of action of the nitrogen mustard melphalan in patients treated for multiple myeloma, the in vivo induction and repair of melphalan-induced DNA damage was measured in genes with different transcriptional activity (b-actin>p53>N-ras>d-globin) from leukocytes of 20 multiple myeloma patients following chemotherapeutic administration of high-dose melphalan (200mg/m(2)) and autologous blood stem cell transplantation. Heterogeneous repair was found among the studied genes. The extent of repair was always in the order: b-actin>p53>N-ras>d-globin, correlating with the gene transcriptional state. Similar findings were obtained using peripheral blood mononuclear cells (PBMC) from healthy volunteers following in vitro treatment with melphalan, indicating that these results are not malignant disease-specific. Following in vitro treatment of PBMC from healthy volunteers with alpha-amanitin, an inhibitor of RNA polymerase II that can also induce condensation of chromatin structure, a significant inhibition of the removal of melphalan-induced damage in the three active genes but not in the silent d-globin gene was found, suggesting that transcription and/or chromatin structure may play important roles in the preferential DNA repair. When the in vivo DNA damage formation and repair in multiple myeloma patients following chemotherapeutic administration of melphalan was measured in the two strands of the active genes, no strand bias was found, indicating that the global genome repair subpathway of nucleotide excision repair may play a crucial role in the repair of these adducts. These results were also confirmed in PBMC from healthy volunteers following in vitro treatment with melphalan. Using micrococcal nuclease digestion of nuclei isolated from PBMC of multiple myeloma patients before the chemotherapeutic treatment, as well as from PBMC of healthy volunteers, we probed the chromatin structure in each gene and found that the "looseness" of the chromatin structure correlated with the levels of the gene-specific repair, being again in the order: b-actin>p53>N-ras>d-globin. To conclude, the in vivo gene-specific repair of melphalan-induced damage in humans is greatly affected by the local chromatin structure.
Souliotis
Vassilis L
VL
National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48 Vassileos Constantinou Ave., Athens 11635, Greece. vls@eie.gr
Dimopoulos
Meletios A
MA
Episkopou
Hara G
HG
Kyrtopoulos
Soterios A
SA
Sfikakis
Petros P
PP
D000199:Actins / Q000235:genetics; D000546:Amanitins; D018906:Antineoplastic Agents, Alkylating / Q000009:adverse effects*; D002843:Chromatin / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics*; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D016158:Genes, p53 / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D008558:Melphalan / Q000009:adverse effects*; D015336:Molecular Probe Techniques; D009101:Multiple Myeloma / Q000188:drug therapy / Q000235:genetics*; D010957:Plasmids / Q000235:genetics
D000199:Actins; D000546:Amanitins; D018906:Antineoplastic Agents, Alkylating; D002843:Chromatin; D017931:DNA Primers; D008558:Melphalan