Human Rad17 (hRad17) is centrally involved in the activation of cell-cycle checkpoints by genotoxic agents or replication stress. Here we identify hMCM7, a core component of the DNA replication apparatus, as a novel hRad17-interacting protein. In HeLa cells, depletion of either hRad17 or hMCM7 with small-interfering RNA suppressed ultraviolet (UV) light- or aphidicolin-induced hChk1 phosphorylation, and abolished UV-induced S-phase checkpoint activation. Similar results were obtained after transfection of these cells with a fusion protein containing the hMCM7-binding region of hRad17. The hMCM7-depleted cells were also defective for the formation of ATR-containing nuclear foci after UV irradiation, suggesting that hMCM7 is required for stable recruitment of ATR to damaged DNA. These results demonstrate that hMCM7 plays a direct role in the transmission of DNA damage signals from active replication forks to the S-phase checkpoint machinery in human cells.

Cancer cells constantly adapt to oxidative phosphorylation (OXPHOS) suppression resulting from hypoxia or mitochondria defects. Under the OXPHOS suppression, AMP-activated protein kinase (AMPK) regulates global metabolism adjustments, but its activation has been found to be transient. Whether cells can maintain cellular ATP homeostasis and survive beyond the transient AMPK activation is not known. Here, we study the bioenergetic adaptation to the OXPHOS inhibitor oligomycin in a group of cancer cells. We found that oligomycin at 100 ng/ml completely inhibits OXPHOS activity in 1 h and induces various levels of glycolysis gains by 6 h, from which we calculate the bioenergetic organizations of cancer cells. In glycolysis-dominant cells, oligomycin does not induce much energy stress as measured by glycolysis acceleration, ATP imbalance, AMPK activation, AMPK substrate acetyl-CoA carboxylase phosphorylation at Ser 79 , and cell growth inhibition. In OXPHOS-dependent LKB1 wild type cells, oligomycin induces 5-8% ATP drops and transient AMPK activation during the initial 1-2 h. After AMPK activation is completed, oligomycininduced increase of acetyl-CoA carboxylase phosphorylation at Ser 79 is still detected, and cellular ATP is back at preoligomycin treatment levels by sustained elevation of glycolysis. Cell growth, however, is inhibited without an increase in cell death and alteration in cell cycle distribution. In OXPHOS-dependent LKB1-null cells, no AMPK activation by oligomycin is detected, yet cells still show a similar adaptation. We also demonstrate that the adaptation to oligomycin does not invoke activation of hypoxia-induced factor. Our data suggest that cancer cells may grow and survive persistent OXPHOS suppression through an as yet unidentified regulatory mechanism.The bioenergetic organization, the fraction of cellular ATP produced by glycolysis and mitochondrial OXPHOS, 2 determines the bioenergetic homeostasis in cells. Tumors have a bioenergetic organization distinct from that of normal cells, in which the burden of ATP production increasingly shifts from OXPHOS to glycolysis, the so-called Warburg effect (1). Deregulation of multiple oncogenes and tumor suppressors during tumorigenesis contributes to this distinct neoplastic metabolism alteration because glycolysis is the downstream target of the altered pathways (2, 3). In addition, mitochondrial defects can also drive up the aerobic glycolysis to bioenergetically compensate for the loss of OXPHOS ATP production. The reduction in OXPHOS activity has been identified in widely spread cancer cells (4 -9). The switch of bioenergetic dependence from OXPHOS to glycolysis is proposed for cancer cells to cope with intermittent and chronicle hypoxia microenvironments, reduce mitochondrial-initiated cell death (3, 10), and promote invasion and metastasis (10 -12).Suppression of OXPHOS activity activates master energy stress sensor AMPK that reprograms the global cellular metabolism for the stress adaptation (13,14). In compensating for the loss of OXPHOS ATP...

The human CYP2Cs have been studied extensively with respect to the metabolism of clinically important drugs and endogenous chemicals such as arachidonic acid (AA). Five members of the mouse CYP2C family have previously been described that metabolize arachidonic acid into regio-and stereospecific epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids, which have many important physiological roles. Herein, we describe the cloning and characterization of a new mouse cytochrome P450 (P450), CYP2C44, which has the lowest homology with other known mouse CYP2Cs. Western blotting and real-time polymerase chain reaction detected CYP2C44 mRNA and protein in liver Ͼ Ͼ kidney Ͼ adrenals. Kidney contained approximately 10% of the CYP2C44 mRNA content of liver. CYP2C44 metabolized AA to unique stereospecific products, 11R,12S-EET and 8R, 9S-EET, which are similar to those produced by rat CYP2C23. CY2C23 is highly expressed in rat kidney and has been suggested to be important in producing compensatory renal artery vasodilation in response to salt-loading in this species. Immunohistochemistry showed the presence of CYP2C44 in hepatocytes, biliary cells of the liver, and the proximal tubules of the kidney. Unlike mouse CYP2C29, CYP2C38, and CYP2C39, CYP2C44 did not metabolize the common CYP2C substrate tolbutamide. CYP2C44 was not induced by phenobarbital or pregnenolone-16␣-carbonitrile, two prototypical inducers of hepatic P450s. The presence of CYP2C44 in mouse liver, kidney, and adrenals and the unique stereospecificity of its arachidonic acid metabolites are consistent with the possibility that it may have unique physiological roles within these tissues, such as modulation of electrolyte transport or vascular tone.

CYP2C19 is selective for the 4'-hydroxylation of S-mephenytoin while the highly similar CYP2C9 has little activity toward this substrate. To identify critical amino acids determining the specificity of human CYP2C19 for S-mephenytoin 4'-hydroxylation, we constructed chimeras by replacing portions of CYP2C9 containing various proposed substrate recognition sites (SRSs) with those of CYP2C19 and mutating individual residues by site-directed mutagenesis. Only a chimera containing regions encompassing SRSs 1--4 was active (30% of wild-type CYP2C19), indicating that multiple regions are necessary to confer specificity for S-mephenytoin. Mutagenesis studies identified six residues in three topological components of the proteins required to convert CYP2C9 to an S-mephenytoin 4'-hydroxylase (6% of the activity of wild-type CYP2C19). Of these, only the I99H difference located in SRS 1 between helices B and C reflects a change in a side chain that is predicted to be in the substrate-binding cavity formed above the heme prosthetic group. Two additional substitutions, S220P and P221T residing between helices F and G but not in close proximity to the substrate binding site together with five differences in the N-terminal portion of helix I conferred S-mephenytoin 4'-hydroxylation activity with a K(M) similar to that of CYP2C19 but a 3-fold lower K(cat). Three residues in helix I, S286N, V292A, and F295L, were essential for S-mephenytoin 4'-hydroxylation activity. On the basis of the structure of the closely related enzyme CYP2C5, these residues are unlikely to directly contact the substrate during catalysis but are positioned to influence the packing of substrate binding site residues and likely substrate access channels in the enzyme.

A cDNA encoding a new cytochrome P450 was isolated from a mouse brain library. Sequence analysis reveals that this 1,958-base pair cDNA encodes a 57-58-kDa 502-amino acid polypeptide that is 70 -91% identical to CYP2J subfamily P450s and is designated CYP2J9. Recombinant CYP2J9 was co-expressed with NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9 cells using a baculovirus system. Microsomes of CYP2J9/CYPORtransfected cells metabolize arachidonic acid to 19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically distinct from other P450s. Northern analysis reveals that CYP2J9 transcripts are present at high levels in mouse brain. Mouse brain microsomes biosynthesize 19-HETE. RNA polymerase chain reaction analysis demonstrates that CYP2J9 mRNAs are widely distributed in brain and most abundant in the cerebellum. Immunoblotting using an antibody raised against human CYP2J2 that cross-reacts with CYP2J9 detects a 56-kDa protein band that is expressed in cerebellum and other brain segments and is regulated during postnatal development. In situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2 IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje cells. Importantly, 19-HETE inhibits the activity of recombinant P/Q-type Ca 2؉ channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release. Based on these data, we conclude that CYP2J9 is a developmentally regulated P450 that is abundant in brain, localized to cerebellar Purkinje cells, and active in the biosynthesis of 19-HETE, an eicosanoid that inhibits activity of P/Q-type Ca 2؉ channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.Cytochromes P450 catalyze the NADPH-dependent oxidation of arachidonic acid to cis-epoxyeicosatrienoic acids (5,8,11,and 14,, 1 mid-chain cis-trans-conjugated dienols , and/or -terminal alcohols of arachidonic acid

We recently identified five different murine CYP2C cDNAs from a murine cDNA library. When expressed in a bacterial cDNA expression system, all five recombinant proteins metabolized arachidonic acid but produced distinctly different profiles. In addition, some CYP2C mRNAs were found in extrahepatic tissues, as well as in liver. Immunoblots with an antibody raised against recombinant CYP2C38, which recognizes all five murine CYP2Cs, demonstrated that among extrahepatic tissues, colon and cecum contained the highest amount of CYP2Cs. The highest concentration of CYP2Cs occurred in cecum and colon (cecum >/= proximal colon >> distal colon), with lower levels in duodenum, jejunum, and ileum. Immunohistochemical studies revealed that CYP2Cs were localized principally in epithelial cells and autonomic ganglia in gut and colon. Polymerase chain reaction amplification of reverse-transcribed mRNA using murine CYP2C-specific primers followed by cloning and sequencing identified CYP2C40 as the major CYP2C isoform expressed in murine intestinal tract. Recombinant CYP2C40 metabolized arachidonic acid in a regio- and stereospecific manner to 16(R)-HETE (hydroxyeicosatetraenoic acid) as the major product. To our knowledge, CYP2C40 is the first enzyme known to produce primarily 16-HETE. We conclude that CYP2C40 is one of the major cytochrome P450 proteins in the mouse intestinal tract. In the light of vasoactive and anti-neutrophilic effects of 16-HETE, we hypothesize that CYP2C40 may play an important role in endogenous biological functions in intestine.

Many anticancer agents activate NF-kappaB, which plays an important role in the survival of cancer cells. Inhibition of NF-kappaB activity may therefore potentiate the efficacy of anticancer agents. We found that a previously used anticancer agent Streptonigrin (SN) was also a potent NF-kappaB inducer. Using a specific IKKbeta inhibitor IV (Podolin et al., J Pharmacol Exp Ther 2005; 312: 373-381), we revealed that the activation of NF-kappaB was mediated through DNA damage-induced activation of IKK complex. Furthermore, we demonstrated that SN-induced DNA damage was unrelated to reactive oxygen species but to the hydroquinone form of SN converted by the NAD(P)H:quinine oxidoreductase (NQO1). The study suggests that the combination of SN with IKK inhibitor may improve efficacy over the use of single agent.