Lipid peroxidation product 4-hydroxy-2-nonenal modulates base excision repair in human cells

Oxidative-stress-driven lipid peroxidation (LPO) is involved in the pathogenesis of several human diseases, including cancer. LPO products react with cellular proteins changing their properties, and with DNA bases to form mutagenic etheno-DNA adducts, removed from DNA mainly by the base excision repair (BER) pathway.One of the major reactive aldehydes generated by LPO is 4-hydroxy-2-nonenal (HNE). We investigated the effect of HNE on BER enzymes in human cells and in vitro. K21 cells pretreated with physiological HNE concentrations were more sensitive to oxidative and alkylating agents, H₂O₂ and MMS, than were untreated cells. Detailed examination of the effects of HNE on particular stages of BER in K21 cells revealed that HNE decreases the rate of excision of 1,N⁶-ethenoadenine (ɛA) and 3,N⁴-ethenocytosine (ɛC), but not of 8-oxoguanine. Simultaneously HNE increased the rate of AP-site incision and blocked the re-ligation step after the gap-filling by DNA polymerases. This suggested that HNE increases the number of unrepaired single-strand breaks (SSBs) in cells treated with oxidizing or methylating agents. Indeed, preincubation of cells with HNE and their subsequent treatment with H₂O₂ or MMS increased the number of nuclear poly(ADP-ribose) foci, known to appear in cells in response to SSBs. However, when purified BER enzymes were exposed to HNE, only ANPG and TDG glycosylases excising ɛA and ɛC from DNA were inhibited, and only at high HNE concentrations. APE1 endonuclease and 8-oxoG-DNA glycosylase 1 (OGG1) were not inhibited. These results indicate that LPO products exert their promutagenic action not only by forming DNA adducts, but in part also by compromising the BER pathway.     

ATP7B activity is stimulated by PKCɛ in porcine liver

Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)–ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10⁻⁸ M), a PKC activator, increased Cu(I)–ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 μM Ca²⁺, whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced V_max without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.