Unconjugated bilirubin is considered a potent antioxidant when present at moderate levels. However, at high concentrations, it produces severe neurological damage and death associated with kernicterus due to oxidative stress and other mechanisms. While it is widely recognized that oxidative stress by different toxic insults results in severe damage to cellular macromolecules, especially to DNA, no data are available either on DNA damage in the brain triggered by hyperbilirubinemia during the neonatal period or on the activation of DNA repair mechanisms. Here, using a mouse model of neonatal hyperbilirubinemia, we demonstrated that DNA damage occurs in vivo in the cerebellum, the brain region most affected by bilirubin toxicity. We studied the mechanisms associated with potential toxic action of bilirubin on DNA in in vitro models, which showed significant increases in DNA damage when neuronal and nonneuronal cells were treated with 140 nM of free bilirubin (Bf), as determined by γH2AX Western blot and immunofluorescence analyses. Cotreatment of cells with N-acetyl-cysteine, a potent oxidative-stress inhibitor, prevented DNA damage by bilirubin, supporting the concept that DNA damage was caused by bilirubin-induced oxidative stress. Bilirubin treatment also activated the main DNA repair pathways through homologous recombination (HR) and nonhomologous end joining (NHEJ), which may be adaptive responses to repair bilirubin-induced DNA damage. Since DNA damage may be another important factor contributing to neuronal death and bilirubin encephalopathy, these results contribute to the understanding of the mechanisms associated with bilirubin toxicity and may be of relevance in neonates affected with severe hyperbilirubinemia.
Metabolic deregulation, a hallmark of cancer, fuels cancer cell growth and metastasis. Here, we show that phosphoserine phosphatase (PSPH), part of the serine metabolism pathway, is upregulated in patient-derived melanoma samples.
PSPH
knockdown using short hairpin RNAs (shRNAs) blocks melanoma tumor growth and metastasis in both cell culture and mice. To elucidate the mechanism underlying PSPH action, we evaluated
PSPH
shRNA-expressing melanoma cells using global metabolomics and targeted mRNA expression profiling. Metabolomics analysis showed increase in 2-hydroxyglutarate (2-HG) levels in
PSPH
knockdown cells. 2-HG inhibits the TET family of DNA demethylases and the Jumonji family of histone demethylases (KDM and JMJD), which is known to impact gene expression. Consistent with these data,
PSPH
knockdown in melanoma cells showed reduced DNA 5-hydroxymethylcytosine (5hmC) and increased histone H3K4me3 modifications. 2-HG treatment also inhibited melanoma growth. The nCounter PanCancer Pathways Panel–based mRNA expression profiling revealed attenuation of a number of cancer-promoting pathways upon
PSPH
knockdown. In particular, PSPH was necessary for nuclear receptor NR4A1 expression. Ectopic NR4A1 expression partly rescued growth of melanoma cells expressing
PSPH
shRNA. Collectively, these results link PSPH to facilitation of melanoma growth and metastasis through suppression of 2-HG and thus activation of pro-oncogenic gene expression.
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