JNK is Associated with Bcl-2 and PP1 in Mitochondria: Paclitaxel Induces Its Activation and Its Association with the Phosphorylated Form of Bcl-2

It has been shown that the activation of JNK after paclitaxel-inducedmicrotubule damage is parallel to Bcl-2 phosphorylation, cell cycle arrest in mitosis andapoptosis. Using subcellular fractionation and immunocytochemistry, we found herethat a pool of activated JNK is located in mitochondria of HeLa cells treated withpaclitaxel. Furthermore, whereas the JNK protein is present in a tripartite complex withthe anti-apoptotic Bcl-2 protein and the PP1 phosphatase in mitochondria isolated fromcontrol cells, the activated form of JNK was associated with the phosphorylated form ofBcl-2, but devoid of PP1, in mitochondria isolated from paclitaxel-treated cells.Moreover, using an original cell-free system, we evidenced a direct involvement of JNKas the kinase responsible for the phosphorylation of mitochondrial Bcl-2 in mitoticarrested cells. Indeed, cytosols prepared from mitotic arrested cells led to a dosedependentphosphorylation of mitochondrial Bcl-2. Bcl-2 phosphorylation was inhibitedby CEP 11004, a JNK pathway inhibitor and by immunodepletion of JNK. Takentogether, these data show that JNK activation provides a molecular linkage frommicrotubule damages to the mitochondrial apoptotic machinery and also point to apivotal role for the JNK/Bcl-2/PP1 complex in the control of apoptosis followingpaclitaxel treatment.     

The phosphorylation status and anti-apoptotic activity of Bcl-2 are regulated by ERK and protein phosphatase 2A on the mitochondria

Bcl-2 protein play important roles in the regulation of apoptosis. We previously reported that the phosphorylation of Bcl-2 was augmented by treatment with protein phosphatase 2A (PP2A) inhibitor; however, the kinase responsible for Bcl-2 phosphorylation had not yet been identified. In this study, we identified extracellular-signal-regulated kinase (ERK) as the responsible kinase for the phosphorylation of Bcl-2. We also found that the transmembrane region (TM) deleted form of Bcl-2 (Bcl-2DeltaTM), which was unable to localize on the mitochondria was constitutively phosphorylated, whereas wild-type Bcl-2 that localized on the mitochondria, was present in its hypophosphorylated form. The phosphorylation of Bcl-2DeltaTM was retarded by treatment with MAP kinase ERK kinase (MEK) inhibitor and PP2A did not bind to Bcl-2DeltaTM. These observations suggest that Bcl-2DeltaTM is constitutively phosphorylated by ERK, but is not dephosphorylated by PP2A in human tumor cell lines. The phosphorylation of Bcl-2 resulted in a reduction in anti-apoptotic function, implying that dephosphorylation promoted the anti-apoptotic activity of Bcl-2 protein in human tumor cell lines. Thus, the present findings suggest that ERK and PP2A are physiological regulators of Bcl-2 phosphorylation, and these enzymes exert an influence on the anti-apoptotic function of Bcl-2.     

Bcl-2 phosphorylation and proteasome-dependent degradation induced by paclitaxel treatment: consequences on sensitivity of isolated mitochondria to Bid

Several studies have suggested that Bcl-2 phosphorylation, which occurs during mitotic arrest induced by paclitaxel, inhibits its antiapoptotic function. In the present study, we demonstrated that the level of phosphorylated Bcl-2 was threefold higher in mitochondria than in the nuclear membrane or endoplasmic reticulum. Our results show, in isolated mitochondria, that phosphorylation of Bcl-2 in mitosis does not modify either its integration into the mitochondrial membrane or the ability to release cytochrome c in response to Bid, a cytochrome c releasing agent. In HeLa cells, in which paclitaxel induces apoptosis, the nonphosphorylated form of Bcl-2 is degraded by a proteasome-dependent degradation pathway, whereas the phosphorylated forms of mitochondrial Bcl-2 appear to be resistant to proteasome-induced degradation. We found that low concentrations of recombinant Bid triggered a greater release of cytochrome c from mitochondria isolated from paclitaxel-treated HeLa cells than from mitochondria isolated from control HeLa cells. Taken together, these results show that Bcl-2 phosphorylation does not inhibit its function. On the contrary, Bcl-2 phosphorylation indirectly regulated its antiapoptotic action via protection against degradation. Indeed, in response to paclitaxel treatment, the level of Bcl-2 expression in mitochondria rather than its phosphorylation state could regulate the sensitivity of mitochondria to cytochrome c releasing agents in vitro.     

Dephosphorylation of Plk1 occurs through PP2A-B55/ENSA/Greatwall pathway during mitotic DNA damage recovery

Recovery from DNA damage is critical for cell survival. However, serious damage cannot be repaired, leading to cell death for prevention of abnormal cell growth. Previously, we demonstrated that 4N-DNA accumulates via the initiation of an abnormal interphase without cytokinesis and that re-replication occurs during a prolonged recovery period in the presence of severe DNA damage in mitotic cells. Mitotic phosphorylated Plk1 is typically degraded during mitotic exit. However, Plk1 has unusually found to be dephosphorylated in mitotic slippage without cytokinesis during recovery from mitotic DNA damage. Here, we investigated how Plk1 dephosphorylation is established during recovery from mitotic DNA damage. Mitotic DNA damage activated ATM and Chk1/2 and repressed Cdk1 and Greatwall protein kinase, followed by PP2A activation through the dissociation of ENSA and PP2A-B55. Interaction between Plk1 and PP2A-B55α or PP2A-B55δ was strongly induced during recovery from mitotic DNA damage. Moreover, the depletion of PP2A-B55α and/or PP2A-B55δ by siRNA transfection led to the recovery of Plk1 phosphorylation and progression of the cell cycle into the G₁ phase. Therefore, to adapt to severe DNA damage, the activated Greatwall/ENSA signaling pathway was repressed by ATM/Chk1/2, even in mitotic cells. Activation of the PP2A-B55 holoenzyme complex induced the dephosphorylation of Plk1 and Cdk1, and finally, mitotic slippage occurred without normal chromosome segregation and cytokinesis.     

Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis

Tyrosine phosphorylation of cdc2 is regulated in the cell cycle of mouse 3T3 fibroblasts. Phosphotyrosine in cdc2 is detectable at the onset of DNA synthesis and becomes maximal in the G2 phase of the cell cycle. Quantitative tyrosine dephosphorylation of cdc2 occurs during entry into mitosis and no phosphotyrosine is detected during the G1 phase of the cell cycle. While increasing tyrosine phosphorylation of cdc2 correlates with the formation of a cdc2/p62 complex, the tyrosine phosphorylated cdc2 is inactive as a histone H1 kinase. cdc2 is fully dephosphorylated in its most active mitotic form, yet specific tyrosine dephosphorylation of interphase cdc2 in vitro is insufficient to activate the kinase. In vivo inhibition of tyrosine dephosphorylation by exposure of cells to a phosphatase inhibitor is associated with G2 arrest, which is reversible upon the removal of the phosphatase inhibitor. Tyrosine dephosphorylation of cdc2 may be one of a number of obligatory steps in the mitotic activation of the kinase.     

Activation of protein kinase C delta following cerebral ischemia leads to release of cytochrome C from the mitochondria via bad pathway

Background

The release of cytochrome c from the mitochondria following cerebral ischemia is a key event leading to cell death. The goal of the present study was to determine the mechanisms involved in post-ischemic activation of protein kinase c delta (δPKC) that lead to cytochrome c release.

Methods/Findings

We used a rat model of cardiac arrest as an in vivo model, and an in vitro analog, oxygen glucose deprivation (OGD) in rat hippocampal synaptosomes. Cardiac arrest triggered translocation of δPKC to the mitochondrial fraction at 1 h reperfusion. In synaptosomes, the peptide inhibitor of δPKC blocked OGD-induced translocation to the mitochondria. We tested two potential pathways by which δPKC activation could lead to cytochrome c release: phosphorylation of phospholipid scramblase-3 (PLSCR3) and/or protein phosphatase 2A (PP2A). Cardiac arrest increased levels of phosphorlyated PLSCR3; however, inhibition of δPKC translocation failed to affect the OGD-induced increase in PLSCR3 in synaptosomal mitochondria suggesting the post-ischemic phosphorylation of PLSCR3 is not mediated by δPKC. Inhibition of either δPKC or PP2A decreased cytochrome c release from synaptosomal mitochondria. Cardiac arrest results in the dephosphorylation of Bad and Bax, both downstream targets of PP2A promoting apoptosis. Inhibition of δPKC or PP2A prevented OGD-induced Bad, but not Bax, dephosphorylation. To complement these studies, we used proteomics to identify novel mitochondrial substrates of δPKC.

Conclusions

We conclude that δPKC initiates cytochrome c release via phosphorylation of PP2A and subsequent dephosphorylation of Bad and identified δPKC, PP2A and additional mitochondrial proteins as potential therapeutic targets for ischemic neuroprotection.     

PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF

FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex. CyclinD/cdk4-expressing RCS cells became resistant to FGF-induced p107 dephosphorylation and growth arrest, and maintained significantly high levels of cyclin E/cdk2 activity and of phosphorylated p130 at later times of FGF treatment. We explored the involvement of a phosphatase in p107 dephosphorylation. Expression of the SV40 small T-Ag, which inhibits the activity of the PP2A phosphatase, or knockdown of the expression of the PP2A catalytic subunit by RNA interference prevented p107 dephosphorylation and FGF-induced growth arrest of RCS cells. Furthermore, an association between p107 and PP2A was induced by FGF treatment. Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation.     

Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis through blockage of caspase-2 activation

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.     

p34cdc2 phosphorylation sites in histone H1 are dephosphorylated by protein phosphatase 2A1.

The protein phosphatase activity in rat liver cytosol or nuclear extracts that dephosphorylates histone H1 which has been phosphorylated by p34cdc2 is inhibited completely by okadaic acid, but unaffected by inhibitor-2 or magnesium ions, demonstrating that the only enzyme in this tissue capable of dephosphorylating this substrate is a type 2A phosphatase. Fractionation of the cytosol by anion-exchange chromatography and gel filtration demonstrated that histone H1 phosphatase activity coeluted with the major species of protein phosphatase 2A, termed PP2A1 and PP2A2. PP2A1 was the most active histone H1 phosphatase, its histone phosphatase phosphorylase phosphatase activity ratio being 6-fold higher than PP2A2 and 30-fold higher than the free catalytic subunit PP2AC. It is concluded that PP2A1 is likely to be the enzyme which dephosphorylates p34cdc2-labelled histone H1 in vivo and that the A and B subunits which interact with PP2AC in this species each play a key role in facilitating dephosphorylation of this substrate. The results demonstrate that PP2A, in addition to being involved in suppressing the activation of p34cdc2 in vivo, can also function to reverse at least one of its actions.     

Apoptosis and mitotic arrest are two independent effects of the protein phosphatases inhibitor okadaic acid in K562 leukemia cells.

Treatment of human myeloid leukemia K562 cells with the serine/threonine protein phosphatases inhibitor okadaic acid induced mitotic arrest followed by apoptosis in a synchronized manner. The effect was observed at drug concentrations that inhibited the protein phosphatase type 2A but not type 1. We investigated whether apoptosis was a consequence of the preceding mitosis arrest or was induced independently by okadaic acid. We found that (1) apoptosis, but not mitotic arrest, was inhibited in cells with constitutive expression of Bcl-2; (2) pretreatment of cells with the DNA synthesis inhibitor hydroxyurea blocked the mitotic arrest but not the apoptosis mediated by okadaic acid; (3) down-regulation of c-myc gene was associated with apoptosis, but not with mitotic arrest; and (4) inhibition of protein synthesis abrogated mitotic arrest, but not apoptosis. The results suggest that inhibition of protein phosphatase 2A by okadaic acid provokes mitotic arrest and apoptosis of leukemia cells by independent mechanisms.     

Doublecortin induces mitotic microtubule catastrophe and inhibits glioma cell invasion

Doublecortin (DCX) is a microtubule (MT) binding protein that induces growth arrest at the G2–M phase of cell cycle in glioma and suppresses tumor xenograft in immunocompromised hosts. DCX expression was found in neuronal cells, but lacking in glioma cells. We tested the hypothesis that DCX inhibits glioma U87 cell mitosis and invasion. Our data showed that DCX synthesizing U87 cells underwent mitotic MT spindle catastrophe in a neurabin II dependent pathway. Synthesis of both DCX and neurabin II were required to induce apoptosis in U87 and human embryonic kidney 293T cells. In DCX expressing U87 cells, association of phosphorylated DCX with protein phosphatase-1 (PP1) in the cytosol disrupted the interaction between kinesin-13 and PP1 in the nucleus and yielded spontaneously active kinesin-13. The activated kinesin-13 caused mitotic MT catastrophe in spindle checkpoint. Phosphorylated-DCX induced depolymerization of actin filaments in U87 cells, down-regulated matrix metalloproteinases-2 and -9, and inhibited glioma U87 cell invasion in a neurabin II dependent pathway. Thus, localization of the DCX–neurabin II–PP1 complex in the cytosol of U87 tumor cells inhibited PP1 phosphatase activities leading to anti-glioma effects via (1) mitotic MT spindle catastrophe that blocks mitosis and (2) depolymerization of actin that inhibits glioma cell invasion.     

Regulation of Polo-like kinase 1 by DNA damage in mitosis. Inhibition of mitotic PLK-1 by protein phosphatase 2A

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Polo-like kinase 1 (Plk1) is an important regulator of several events during mitosis. In addition to Plk1 functions in cell cycle, Plk1 is involved in DNA damage check-point in G2 phase. Normally, ataxia telangiectasia-mutated kinase (ATM) is a key enzyme involved in G2 phase cell cycle arrest following DNA damage, and inhibition of Plk1 by DNA damage during G2 occurs in a ATM/ATR-dependent manner. However, it is still unclear how Plk1 is regulated in response to DNA damage in mitosis in which Plk1 is already activated. Here, we show that treatment of mitotic cells with doxorubicin and gamma-irradiation inhibits Plk1 activity through dephosphorylation of Plk1, and cells were arrested in G2 phase. Treatments of the phosphatase inhibitors and siRNA experiments suggested that PP2A pathway might be involved in regulating mitotic Plk1 activity in mitotic DNA damage. Finally, we propose a novel pathway, which is connected between ATM/ATR/Chk and protein phosphatase-Plk1 in DNA damage response in mitosis.     

Inhibitory role of TRIP-Br1 oncoprotein in anticancer drug-mediated programmed cell death via mitophagy activation

Chemotherapy has been widely used as a clinical treatment for cancer over the years. However, its effectiveness is limited because of resistance of cancer cells to programmed cell death (PCD) after treatment with anticancer drugs. To elucidate the resistance mechanism, we initially focused on cancer cell-specific mitophagy, an autophagic degradation of damaged mitochondria. This is because mitophagy has been reported to provide cancer cells with high resistance to anticancer drugs. Our data showed that TRIP-Br1 oncoprotein level was greatly increased in the mitochondria of breast cancer cells after treatment with various anticancer drugs including staurosporine (STS), the main focus of this study. STS treatment increased cellular ROS generation in cancer cells, which triggered mitochondrial translocation of TRIP-Br1 from the cytosol via dephosphorylation of TRIP-Br1 by protein phosphatase 2A (PP2A). Up-regulated mitochondrial TRIP-Br1 suppressed cellular ROS levels. In addition, TRIP-Br1 rapidly removed STS-mediated damaged mitochondria by activating mitophagy. It eventually suppressed STS-mediated PCD via degradation of VDACI, TOMM20, and TIMM23 mitochondrial membrane proteins. TRIP-Br1 enhanced mitophagy by increasing expression levels of two crucial lysosomal proteases, cathepsins B and D. In conclusion, TRIP-Br1 can suppress the sensitivity of breast cancer cells to anticancer drugs by activating autophagy/mitophagy, eventually promoting cancer cell survival.     

Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2.

Smac/DIABLO is a mitochondrial protein that potentiates some forms of apoptosis, possibly by neutralizing one or more members of the IAP family of apoptosis inhibitory proteins. Smac has been shown to exit mitochondria and enter the cytosol during apoptosis triggered by UV- or gamma-irradiation. Here, we report that Smac/DIABLO export from mitochondria into the cytosol is provoked by cytotoxic drugs and DNA damage, as well as by ligation of the CD95 death receptor. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bcl-2-overexpressing cells. Thus, in addition to modulating apoptosis-associated mitochondrial cytochrome c release, Bcl-2 also regulates Smac release, suggesting that both molecules may escape via the same route. However, whereas cell stress-associated mitochondrial cytochrome c release was largely caspase independent, release of Smac/DIABLO in response to the same stimuli was blocked by a broad-spectrum caspase inhibitor. This suggests that apoptosis-associated cytochrome c and Smac/DIABLO release from mitochondria do not occur via the same mechanism. Rather, Smac/DIABLO efflux from mitochondria is a caspase-catalysed event that occurs downstream of cytochrome c release.     

Potentiation of tumor necrosis factor-alpha-induced cell death by rottlerin through a cytochrome-C-independent pathway

The protein kinase C (PKC) signal transduction pathway negatively regulates receptor-initiated cell death. In HeLa cells, tumor necrosis factor-alpha (TNF)-mediated cell death involved mitochondria and was blocked by the overexpression of Bcl-2. The PKC-specific inhibitor bisindolylmaleimide and the PKCdelta inhibitor rottlerin enhanced TNF-induced cell death. We have investigated if potentiation of TNF-induced cell death by rottlerin involved amplification of the mitochondrial pathway. TNF induced cleavage of the proapoptotic protein Bid and release of mitochondrial cytochrome c. Rottlerin enhanced activation of caspase-8 and cleavage of Bid. It also enhanced activation of caspase-9 but it did not increase cytochrome c in the cytosol. It, however, increased release of mitochondrial apoptosis-inducing factor (AIF) to the cytosol. Overexpression of Bcl-2 prevented release of both cytochrome c and AIF to the cytosol. Prolonged exposure (> or =6 h) of HeLa cells to rottlerin and TNF decreased the level of cytochrome c but not of AIF in the cytosol. These results suggest that rottlerin activates a cytochrome-c-independent cell death pathway to potentiate cell death by TNF.     

Mitotic DNA damage response: Polo-like Kinase-1 is dephosphorylated through ATM-Chk1 pathway

DNA damage during the cell division cycle can activate ATM/ATR and their downstream kinases that are involved in the checkpoint pathway, and cell growth is halted until damage is repaired. As a result of DNA damage induced in mitotic cells by doxorubicin treatment, cells accumulate in a G2-like phase, not in mitosis. Under these conditions, two mitosis-specific kinases, Cdk1 and Plk1, are inhibited by inhibitory phosphorylation and dephosphorylation, respectively. G2-specific phosphorylation of Cdc25 was increased during incubation after mitotic DNA damage. Inhibition of Plk1 through dephosphorylation was dependent on ATM/Chk1 activity. Depleted expression of ATM and Chk1 was achieved using small hairpin RNA (shRNA) plasmid constructs. In this condition, damaged mitotic cells did not accumulated in a G2-like stage, and entered into G1 phase without delay. Protein phosphatase 2A was responsible for dephosphorylation of mitotic Plk1 in response to DNA damage. In knockdown of PP2A catalytic subunits, Plk1 was not dephosphorylated, but rather degraded in response to DNA damage, and cells did not accumulate in G2-like phase. The effect of ATM/Chk1 inhibition was counteracted by overexpression of PP2A, indicated that PP2A may function as a downstream target of ATM/Chk1 at a mitotic DNA damage checkpoint, or may have a dominant effect on ATM/Chk1 function at this checkpoint. Finally, we have shown that negative regulation of Plk1 by dephosphorylation is important to cell accumulation in G2-like phase at the mitotic DNA damage checkpoint, and that this ATM/Chk1/PP2A pathway independent on p53 is a novel mechanism of cellular response to mitotic DNA damage.     

Prolonged mitotic arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction

Mitotic arrest induced by antimitotic drugs can cause apoptosis or p53-dependent cell cycle arrest. It can also cause DNA damage, but the relationship between these events has been unclear. Live, single-cell imaging in human cancer cells responding to an antimitotic kinesin-5 inhibitor and additional antimitotic drugs revealed strong induction of p53 after cells slipped from prolonged mitotic arrest into G1. We investigated the cause of this induction. We detected DNA damage late in mitotic arrest and also after slippage. This damage was inhibited by treatment with caspase inhibitors and by stable expression of mutant, noncleavable inhibitor of caspase-activated DNase, which prevents activation of the apoptosis-associated nuclease caspase-activated DNase (CAD). These treatments also inhibited induction of p53 after slippage from prolonged arrest. DNA damage was not due to full apoptosis, since most cytochrome C was still sequestered in mitochondria when damage occurred. We conclude that prolonged mitotic arrest partially activates the apoptotic pathway. This partly activates CAD, causing limited DNA damage and p53 induction after slippage. Increased DNA damage via caspases and CAD may be an important aspect of antimitotic drug action. More speculatively, partial activation of CAD may explain the DNA-damaging effects of diverse cellular stresses that do not immediately trigger apoptosis.     

S-phase arrest by reactive nitrogen species is bypassed by okadaic acid, an inhibitor of protein phosphatases PP1/PP2A

In mammalian cells DNA damage activates a checkpoint that halts progression through S phase. To determine the ability of nitrating agents to induce S-phase arrest, mouse C10 cells synchronized in S phase were treated with nitrogen dioxide (NO(2)) or SIN-1, a generator of reactive nitrogen species (RNS). SIN-1 or NO(2) induced S-phase arrest in a dose- and time-dependent manner. As for the positive controls adozelesin and cisplatin, arrest was accompanied by phosphorylation of ATM kinase; dephosphorylation of pRB; decreases in RF-C, cyclin D1, Cdc25A, and Cdc6; and increases in p21. Comet assays indicated that RNS induce minimal DNA damage. Moreover, in a cell-free replication system, nuclei from cells treated with RNS were able to support control levels of DNA synthesis when incubated in cytosolic extracts from untreated cells, whereas nuclei from cells treated with cisplatin were not. Induction of phosphatase activity may represent one mechanism of RNS-induced arrest, for the PP1/PP2A phosphatase inhibitor okadaic acid inhibited dephosphorylation of pRB; prevented decreases in the levels of RF-C, cyclin D1, Cdc6, and Cdc25A; and bypassed arrest by SIN-1 or NO(2), but not cisplatin or adozelesin. Our studies suggest that RNS may induce S-phase arrest through mechanisms that differ from those elicited by classical DNA-damaging agents.