Fanconi D2 Research Articles

Influence of PJ34 on the genotoxicity induced by melphalan in human multiple myeloma cells.

IntroductionThe aim of this study was to evaluate the potential biological activity of N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acetamide hydrochloride (PJ34) on the genotoxicity induced by melphalan in human multiple myeloma cells.Material and methodsThe inhibitory effects of the drugs on the growth of RPMI8226 cells were determined by Cell Counting Kit-8 (CCK-8) assay. The expression of Fanconi anemia/breast cancer (FA/BRCA) pathway related genes was determined by western blot analysis. Cell cycle phase and apoptosis were analyzed by flow cytometry. Coadministration of PJ34 and melphalan had additional effects on cell cycle distribution and enhanced apoptosis of RPMI8226 cells. PJ34 plus melphalan inhibited cell-cycle progression, as evidenced by the increased proportion of cells in the G2/M phase with the decreasing proportion of cells in the G0/1 and S phases.ResultsHowever, no significant synergistic effect of PJ34 and melphalan on cell proliferation was observed. These effects were accompanied by inhibition of the FA/BRCA pathway by downregulation of Fanconi D2 (FANCD2) protein expression. The results showed that treatment with 60 µmol/l of PJ34 previously to melphalan administration increased cell apoptosis. Pretreatment also caused cell cycle arrest.ConclusionsThis study suggests that enhancement of melphalan efficacy may be best achieved by the poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor PJ34. The effects of PJ34 are associated with inhibition of the FA/BRCA pathway, increased apoptosis percentage, and G2/M cell cycle arrest. Administration of PJ34 has been shown to protect DNA from damage induced by melphalan. This corroborates the biological activities of PJ34 and points to the need for further studies.

Monoubiquitinated Fanconi anemia D2 (FANCD2-Ub) is required for BCR-ABL1 kinase-induced leukemogenesis

Fanconi D2 (FANCD2) is monoubiquitinated on K561 (FANCD2-Ub) in response to DNA double-strand breaks (DSBs) to stimulate repair of these potentially lethal DNA lesions. FANCD2-Ub was upregulated in CD34+ chronic myeloid leukemia (CML) cells and in BCR-ABL1 kinase –positive cell lines in response to elevated levels of reactive oxygen species (ROS) and DNA cross-linking agent mitomycin C. Downregulation of FANCD2 and inhibition of FANCD2-Ub reduced the clonogenic potential of CD34+ CML cells and delayed BCR-ABL1 leukemogenesis in mice. Retarded proliferation of BCR-ABL1 -positive FANCD2−/− leukemia cells could be rescued by FANCD2 expression. BCR-ABL1 –positive FANCD2−/− cells accumulated more ROS-induced DSBs in comparison to BCR-ABL1 –positive FANCD2+/+ cells. Antioxidants diminished the number of DSBs and enhanced proliferation of BCR-ABL1 –positive FANCD2−/− cells. Expression of wild-type FANCD2 and FANCD2(S222A) phosphorylation-defective mutant (deficient in stimulation of intra-S phase checkpoint but proficient in DSB repair), but not FANCD2(K561R) monoubiquitination-defective mutant (proficient in stimulation of intra-S phase checkpoint but deficient in DSB repair) reduced the number of DSBs and facilitated proliferation of BCR-ABL1 –positive FANCD2−/− cells. We hypothesize that FANCD2-Ub plays an important role in BCR-ABL1 leukemogenesis due to its ability to facilitate the repair of numerous ROS-induced DSBs.

Monoubiquitination of the Fanconi Anemia D2 (FANCD2) Protein Regulates the Transforming Potential of BCR/ABL

We showed before that BCR/ABL-mediated leukemogenesis is associated with the elevation of reactive oxygen species (ROS), which in addition to enhancing signaling pathways may increase the number of oxidative DNA lesions including DNA double-strand breaks (DSBs) [Blood, 2005, 2006]. CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain higher number of DSBs induced by ROS in comparison to CD34+ cells from healthy donors [Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cell-enriched populations display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Thus, BCR/ABL-positive leukemia cells appear to accumulate an excess of ROS-induced DSBs, which may cause apoptosis if not repaired. We reported before that homologous recombination repair (HRR), involving RAD51 protein, plays a pivotal role in the response of BCR/ABL-positive leukemia cells to numerous DSBs induced by ROS [Blood, 2005]. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 by FANCL ubiquitinase and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 (FANCD2-Ub) interacts with RAD51 to facilitate HRR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint. We detected an increased amount of FANCD2-Ub in BCR/ABL-positive leukemia cell line and CD34+ CML-CP and CML-BC cells in comparison to normal counterparts. This effect was not associated with up-regulation of FANCD2 ubiquitinase FANCL or down-regulation of FANCD2 deubiquitinase USP1, but was reversed after inhibition of BCR/ABL kinase with imatinib and reduction of ROS with antioxidant vitamin E (VE) or N-acetylcysteine (NAC). Therefore we postulate that BCR/ABL kinase-dependent ROS-induced FANCD2- Ub may play a role in leukemic transformation. This hypothesis is supported by impaired transformation potential of BCR/ABL kinase in FANCD2−/− murine bone marrow cells in comparison to +/+ counterparts. Restoration of the expression of FANCD2 protein in FANCD2−/− cells rescued the transforming potential of BCR/ABL kinase. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2−/− human lymphoblast cell line. Again BCR/ABL kinase did not exert a negative effect on the proliferation of FANCD2-reconstituted cells. The growth defect of BCR/ABL-positive FANCD2−/− cells was accompanied by delayed leukemogenesis in SCID mice. Growth potential of BCR/ABL-positive FANCD2−/− could be rescued by co-expression of FANCD2 wild-type and S222A mutant, but not the K561R mutant. This observation supports our hypothesis that K561 monoubiquitination, but not S222 phosphorylation of FANCD2 might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL employs RAD51-dependent HRR to repair numerous DSBs induced by ROS, elevated expression of FANCD2-Ub may facilitate this process. This speculation is supported by the observation that although BCR/ABL-positive FANCD2−/− cells and +/+ counterparts display similar levels of ROS and oxidized DNA bases, the former cells accumulate more DSBs evaluated by neutral comet assay and immunostained gamma-H2AX nuclear foci. This effect could be reversed by the expression of FANCD2 S222A mutant, but not K561R mutant, again implicating FANCD2-Ub in reparation of these DSBs. Elevated levels of ROS-mediated DSBs in BCR/ABL-positive FANCD2−/− cells did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging ROS by VE and NAC reduced the number of DSBs and eliminated the growth defect in BCR/ABL-positive FANCD2−/− cells without affecting their +/+ counterparts. In conclusion we hypothesize that FANCD2-Ub, but not FANCD2-phosphoS222 may play an important role in BCR/ABL-dependent leukemogenesis probably due to its ability to interact with RAD51 and facilitate HRR of the numerous ROS-induced DSBs.

Fanconi Anemia D2 (FANCD2) Is Required To Overcome Reactive Oxygen Species (ROS) - Induced DNA Double-Strand Breaks in BCR/ABL-Positive Leukemias.

BCR/ABL-mediated transformation is associated with elevation of ROS which, in addition to enhancing the cytoplasmic signaling pathways, may increase the number of oxidative DNA lesions including DSBs. Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DSBs induced by ROS. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 interacts with RAD51 during HR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint in response to DNA damage. Our studies detected an enhanced interaction between RAD51 and FANCD2 in BCR/ABL-positive leukemia cells in comparison to normal counterparts implicating the role in repair of ROS-dependent DSBs. In addition, higher levels of monoubiquitinated FANCD2 protein was detectable in CML patient cells at chronic phase and in blast crisis, and in BCR/ABL-transformed cells in comparison to non-transformed cells. This effect was reversed after inhibition of BCR/ABL kinase with STI571. Therefore, FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. This speculation is supported by impaired transformation potential of the BCR/ABL kinase in FANCD2−/− murine bone marrow cells in comparison to +/+ counterparts. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2−/− human lymphoblast cell line. The growth defect of BCR/ABL-positive FANCD2−/− cells was accompanied with delayed leukemogenesis in SCID mice. Growth ability of BCR/ABL-positive FANCD2−/− cells could be rescued by co-expression of the wild-type and S222A mutant of FANCD2, but not the K561R mutant. This observation suggested that K561 monoubiquitination, but not S222 phosphorylation might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL cells employ RAD51-dependent HR to repair numerous DSBs induced by ROS, elevated expression of monoubiquitinated FANCD2 may facilitate this process. This hypothesis is supported by the observation that BCR/ABL-positive FANCD2−/− cells and +/+ counterparts display similar levels of ROS and oxidized DNA bases, however, the former cells accumulate more DSBs evaluated by neutral comet assay and detected by γ-H2AX foci immunostaining. This effect could be reversed by the expression of FANCD2 S222A, but not K561R mutant, again implicating HR in reparation of these DSBs. Elevated levels of ROS-mediated DSBs in BCR/ABL-positive FANCD2−/− cells did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging of ROS by vitamin E and N-acetylcysteine reduced the number of DSBs and eliminated the growth defect in BCR/ABL-positive FANCD2−/− cells without affecting their +/+ counterparts. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in BCR/ABL-dependent leukemogenesis, probably due to its ability to interact with RAD51 and facilitate HR repair of an excess of spontaneous DSBs induced by ROS.

Fanconi Anemia D2 Protein Contributes to BCR/ABL-Mediated Transformation of Hematopoietic Cells.

Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) or genotoxic treatment. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 interacts with RAD51 during HR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint in response to DNA damage. Our studies detected an enhanced interaction between RAD51 and FANCD2 in BCR/ABL-positive leukemia cells in comparison to normal counterparts. In addition, although the expression of FANCD2 was stimulated by BCR/ABL and growth factors, higher levels of FANCD2 monoubiquitination was detectable in CML patient cells at chronic phase and in blast crisis, and in BCR/ABL-transformed cells in comparison to non-transformed cells. This effect was reversed after inhibition of BCR/ABL kinase with STI571. Therefore, monoubiquitination of FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. BCR/ABL kinase displayed an impaired transformation potential in FANCD2-/- murine bone marrow cells in comparison to +/+ counterparts. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2-/- human lymphoblast cell line. Growth ability of BCR/ABL-positive FANCD2-/- cells could be rescued by co-expression of the wild-type and S222A mutant of FANCD2, but not the K561R mutant. This observation suggested that K561 monoubiquitination, but not S222 phosphorylation might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL cells employ RAD51-dependent HR to repair numerous DSBs induced by ROS, elevated expression of monoubiquitinated FANCD2 may facilitate this process. This hypothesis is supported by the observation that BCR/ABL-positive FANCD2-/- cells accumulate more DNA damage than +/+ counterparts as indicated by enzymatic assays converting oxidative DNA lesions into gaps detectable by comet assay. In addition, enhanced oxidative DNA damage in BCR/ABL-positive FANCD2-/- cells produced a variety of DNA lesions including abasic sites, and single- and double-strand breaks assessed by neutral comet assay. Moreover, BCR/ABL-positive FANCD2-/- cells accumulated higher numbers of DSBs detected by γ-H2AX immunostaining and displayed discrete apoptosis. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in the initial steps of BCR/ABL dependent leukemogenesis, probably due to its ability to interact with RAD51 and facilitate HR repair of an excess of spontaneous DSBs induced by ROS.