The molecular pathogenesis of the myeloid leukemias that frequently occur in patients with Fanconi anemia (FA) is not well defined. Hematopoietic stem cells bearing inactivating mutations of FA complementation group C (FANCC) are genetically unstable and hypersensitive to apoptotic cytokine cues including IFN-gamma and TNF-alpha, but neoplastic stem cell clones that arise frequently in vivo are resistant to these cytokines. Reasoning that the combination of genetic instability and cytokine hypersensitivity might create an environment supporting the emergence of leukemic stem cells, we tested the leukemia-promoting effects of TNF-alpha in murine stem cells. TNF-alpha exposure initially profoundly inhibited the growth of Fancc-/- stem cells. However, longer-term exposure of these cells promoted the outgrowth of cytogenetically abnormal clones that, upon transplantation into congenic WT mice, led to acute myelogenous leukemia. TNF-alpha induced ROS-dependent genetic instability in Fancc-/- but not in WT cells. The leukemic clones were TNF-alpha resistant but retained their characteristic hypersensitivity to mitomycin C and exhibited high levels of chromosomal instability. Expression of FANCC cDNA in Fancc-/- stem cells protected them from TNF-alpha-induced clonal evolution. We conclude that TNF-alpha exposure creates an environment in which somatically mutated preleukemic stem cell clones are selected and from which unaltered TNF-alpha-hypersensitive Fancc-/- stem cells are purged.

Patients with the genomic instability syndrome Fanconi anemia (FA) commonly develop progressive bone marrow (BM) failure and have a high risk of cancer. Certain manifestations of the disease suggest that the FA immune system is dysfunctional and may contribute to the pathogenesis of both BM failure and malignancies. In this study, we have investigated inflammation and innate immunity in FA hemopoietic cells using mice deficient in Fanconi complementation group C gene (Fancc). We demonstrate that Fancc-deficient mice exhibit enhanced inflammatory response and are hypersensitive to LPS-induced septic shock as a result of hemopoietic suppression. This exacerbated inflammatory phenotype is intrinsic to the hemopoietic system and can be corrected by the re-expression of a wild-type FANCC gene, suggesting a potential role of the FANCC protein in innate immunity. LPS-mediated hemopoietic suppression requires two major inflammatory agents, TNF-α and reactive oxygen species. In addition, LPS-induced excessive accumulation of reactive oxygen species in Fancc−/− BM cells overactivates the stress kinase p38 and requires prolonged activation of the JNK. Our data implicate a role of inflammation in pathogenesis of FA and BM failure diseases in general.

The proinflammatory cytokine tumor necrosis factor α (TNFα) inhibits hematopoietic stem cell (HSC) expansion, interferes with HSC self-renewal and compromises the ability of HSC to reconstitute hematopoiesis. We have investigated mechanisms by which TNFα suppresses hematopoiesis using the genomic instability syndrome Fanconi anemia mouse model deficient for the complementation-group-C gene (Fancc). Examination of senescence makers, such as senescence-associated β-galactosidase, HP1-γ, p53 and p16INK4A shows that TNFα induces premature senescence in bone marrow HSCs and progenitor cells as well as other tissues of Fancc–/– mice. TNFα-induced senescence correlates with the accumulation of reactive oxygen species (ROS) and oxidative DNA damage. Neutralization of TNFα or deletion of the TNF receptor in Fancc–/– mice (Fancc–/–;Tnfr1–/–) prevents excessive ROS production and hematopoietic senescence. Pretreatment of TNFα-injected Fancc–/– mice with a ROS scavenger significantly reduces oxidative base damage, DNA strand breaks and senescence. Furthermore, HSCs and progenitor cells from TNFα-treated Fancc–/– mice show increased chromosomal aberrations and have an impaired oxidative DNA-damage repair. These results indicate an intimate link between inflammatory reactive oxygen species and DNA-damage-induced premature senescence in HSCs and progenitor cells, which may play an important role in aging and anemia.

Oridonin, the main active component of Rabdosia rubescens, has antitumor activities in experimental and clinical settings. The aims of the current study were to explore the anticancer abilities of oridonin in hepatoblastoma (HB) HuH-6 cells and to investigate the underlying mechanisms. We found that oridonin inhibited HuH-6 cell in vitro growth in a dose- and time-dependent manner. Further, oridonin induced HuH-6 cell apoptosis and G2/M cell cycle arrest. Upon studying the mechanism, we found that oridonin treatment caused endoplasmic reticulum (ER) stress activation. Meanwhile, ER stress inhibitor salubrinal- or inositol-requiring enzyme 1 (IRE-1) shRNA silencing inhibited oridonin's anti-HuH-6 effects, while ER stress inducers thapsigargin (Tg) and tunicamycin (Tm) mimicked oridonin's actions on HuH-6 cells. Oridonin also activated apoptosis signal regulating kinase 1 (ASK1)-c-Jun N-terminal kinase 1 (JNK1) signaling in cultured HuH-6 cells, which was inhibited by IRE-1 silencing. Importantly, the JNK inhibitors suppressed oridonin-induced growth inhibition and apoptosis in HuH-6 cells. In conclusion, our results suggest that oridonin induces growth inhibition and apoptosis in cultured HuH-6 cells involving ER stress and ASK1/JNK signaling pathways, which enhances our understanding of the molecular mechanisms of oridonin in HB management.