FANCD2 monoubiquitination and activation by hexavalent chromium [Cr(VI)] exposure: activation is not required for repair of Cr(VI)-induced DSBs.
Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA crosslinking agents. FA is a genetically heterogeneous disease with at least 11 complementation groups. The eight cloned FA proteins interact in a common pathway with established DNA-damage-response proteins, including BRCA1 and ATM. Six FA proteins (A, C, E, F, G, and L) regulate the monoubiquitination of FANCD2 after DNA damage by crosslinking agents, which targets FANCD2 to BRCA1 nuclear foci containing BRCA2 (FANCD1) and RAD51. Some forms of hexavalent chromium [Cr(VI)] are implicated as respiratory carcinogens and induce several types of DNA lesions, including DNA interstrand crosslinks. We have shown that FA-A fibroblasts are hypersensitive to both Cr(VI)-induced apoptosis and clonogenic lethality. Here we show that Cr(VI) treatment induced monoubiquitination of FANCD2 in normal human fibroblasts, providing the first molecular evidence of Cr(VI)-induced activation of the FA pathway. FA-A fibroblasts demonstrated no FANCD2 monoubiquitination, in keeping with the requirement of FA-A for this modification. We also found that Cr(VI) treatment induced significantly more S-phase-dependent DNA double strand breaks (DSBs), as measured by gamma-H2AX expression, in FA-A fibroblasts compared to normal cells. However, and notably, DSBs were repaired equally in both normal and FA-A fibroblasts during recovery from Cr(VI) treatment. While previous research on FA has defined the genetic causes of this disease, it is critical in terms of individual risk assessment to address how cells from FA patients respond to genotoxic insult.
Vilcheck
Susan K
SK
Department of Pharmacology and Physiology, The George Washington University Medical Center, 2300 Eye Street, NW, Washington, DC 20037, United States.
Ceryak
Susan
S
O'Brien
Travis J
TJ
Patierno
Steven R
SR
D015153:Blotting, Western; D002478:Cells, Cultured; D002648:Child; D002857:Chromium / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects; D005199:Fanconi Anemia / Q000378:metabolism / Q000473:pathology; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism*; D051856:Fanconi Anemia Complementation Group Proteins / Q000378:metabolism; D005434:Flow Cytometry; D006657:Histones / Q000378:metabolism; D006801:Humans; D008297:Male; D016196:S Phase / Q000187:drug effects; D025801:Ubiquitin / Q000378:metabolism*
C499905:FANCD2 protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D051856:Fanconi Anemia Complementation Group Proteins; C468783:H2AX protein, human; D006657:Histones; D025801:Ubiquitin; D002857:Chromium; C074702:chromium hexavalent ion