Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

Characterization of p53 in Chinese hamster cell lines CHO-K1, CHO-WBL, and CHL: implications for genotoxicity testing.

Since the p53 gene function is critical to how a cell responds to DNA damage, we investigated the p53 status in Chinese hamster cell lines commonly used in genotoxicity tests for cytogenetic damage around the world. These included: Chinese hamster ovary K1 (CHO-K1), Chinese hamster ovary WBL (CHO-WBL), and Chinese hamster lung (CHL) cells. The results of DNA sequencing, protein analysis, and cell cycle analysis demonstrate that the CHO-K1 and CHO-WBL cell lines have mutant p53 sequence [a mutation in codon 211 in exon 6 resulting in a change from Thr (ACA) to Lys (AAA)], mutant protein (high spontaneous levels that are non-inducible after X-irradiation), and mutant function (lack of G1 checkpoint). Interestingly, the CHL cell line has a completely wild-type p53 DNA sequence. However, the CHL cells have an abnormally high spontaneous level of wild-type p53 protein expression that is not inducible after X-irradiation, yet there is some evidence of G1 delay after irradiation. The protein data suggests that p53 in CHL cells is not being regulated normally, and thus is probably not functioning normally. The mechanism leading to this abnormal regulation of p53 in CHL cells clearly does not involve mutation in the p53 gene. Overall, the CHL cell line may be similar to the CHO cell lines, in that they all appear to have abnormal p53 function. Further work is needed to determine whether the presence of spontaneously high levels of wild-type p53 in CHL cells results in a difference in response to DNA damage (quantitatively or qualitatively) compared to the p53 mutant CHO cell lines.     

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G₁/S checkpoint, ATM and p53 signaling pathways in p53-wild-type cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G₁/S checkpoint pathway in p53 wild-type lines and not in p53-mutant cells. These responses are coupled with G₂/G₁ checkpoint effectors p21^CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G₂ cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wild-type and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G₂ arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wild-type and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.Key words: DNA damaging agent, G₂ arrest, microarray, PARP inhibition, p53, topotecan, veliparib (ABT-888)     

Characterization of the p53-Dependent Postmitotic Checkpoint following Spindle Disruption

The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G₁ following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53^+/+ and p53^−/− murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G₁ cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21^−/− fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.     

An effect of cell-cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells

Using synchronous populations obtained by selectively detaching mitotic cells from cultures grown in monolayer, we demonstrate here that Chinese hamster ovary (CHO) cells exhibit a differential sensitivity to mutation induction by UV as a function of position in the cell cycle. When mutation induction to 6-thioguanine (TG) resistance is monitored, several maxima and minima are displayed during cell-cycle traverse, with a major maximum occurring in early S phase. Although cells in S phase are more sensitive to UV-mediated cell lethality than those in G₁ or G₂/M phases, there is not a strict correlation with induced mutation frequency. Fluence-response curves obtained at several times during the cell cycle yield D_q values approximating 6 J/m². The primary survival characteristic which varies with cell cycle position is D₀, ranging from 2.5 J/m² at 6 h after mitotic selection to 5.5 J/m² at 11 h afterward. Based on studies with asynchronous, logarithmically growing populations, as well as those mitotically selected to be synchronous, the optimum phenotypic expression time for induced TG resistance is 7–9 days and is essentially independent of both UV fluence and position in the cell cycle. All isolated mutants have altered hypozanthine—guanine phosphoribosyl transferase (HGPRT) activity, and no difference in the residual level of activity was detected among isolated clones receiving UV radiation during G₁, S, or late S/G₂ phases of the cell cycle. Changes in cellular morphology during cell-cycle traverse do not contribute to the differential susceptibility to UV-induced mutagenesis.     

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21^WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21^WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21^WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G₁ arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G₁ arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21^WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21^WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.     

Dyrk1A Phosphorylates p53 and Inhibits Proliferation of Embryonic Neuronal Cells

Down syndrome (DS) is associated with many neural defects, including reduced brain size and impaired neuronal proliferation, highly contributing to the mental retardation. Those typical characteristics of DS are closely associated with a specific gene group “Down syndrome critical region” (DSCR) on human chromosome 21. Here we investigated the molecular mechanisms underlying impaired neuronal proliferation in DS and, more specifically, a regulatory role for dual-specificity tyrosine-(Y) phosphorylation-regulated kinase 1A (Dyrk1A), a DSCR gene product, in embryonic neuronal cell proliferation. We found that Dyrk1A phosphorylates p53 at Ser-15 in vitro and in immortalized rat embryonic hippocampal progenitor H19-7 cells. In addition, Dyrk1A-induced p53 phosphorylation at Ser-15 led to a robust induction of p53 target genes (e.g. p21^CIP1) and impaired G₁/G₀-S phase transition, resulting in attenuated proliferation of H19-7 cells and human embryonic stem cell-derived neural precursor cells. Moreover, the point mutation of p53-Ser-15 to alanine rescued the inhibitory effect of Dyrk1A on neuronal proliferation. Accordingly, brains from embryonic DYRK1A transgenic mice exhibited elevated levels of Dyrk1A, Ser-15 (mouse Ser-18)-phosphorylated p53, and p21^CIP1 as well as impaired neuronal proliferation. These findings suggest that up-regulation of Dyrk1A contributes to altered neuronal proliferation in DS through specific phosphorylation of p53 at Ser-15 and subsequent p21^CIP1 induction.     

Activation of ATM/ATR signaling in human embryonic stem cells after DNA damage

Embryonic stem cells (ESCs) are the progenitors of all adult cells; consequently, genomic abnormalities in them may be catastrophic for the developing organism. ESCs are characterized by high proliferation activity and do not stop in checkpoints upon DNA-damage executing only G₂/M delay after DNA damage. ATM and ATR kinases are key sensors of double-strand DNA breaks and activate downstream signaling pathways involving checkpoints, DNA repair, and apoptosis. We examined activation of ATM/ATR signaling in human ESCs and revealed that irradiation induced ATM, ATR, and Chk2 phosphorylation, and γH2AX foci formation and their colocalization with 53BP1 and Rad51 proteins. Interestingly, human ESCs exhibit noninduced γH2AX foci colocalized with Rad51 and marking single-strand DNA breaks. Next, we revealed the significant contribution of ATM, Chk1, and Chk2 kinases to G₂/M block after irradiation and ATM-dependent activation (phosphorylation) of p53 in human ESCs. However, p53 activation and subsequent induction of p21 ^Waf1 gene expression after DNA damage do not result in p21^Waf1 protein accumulation due to its proteasomal degradation.     

p16INK4A Participates in a G1 Arrest Checkpoint in Response to DNA Damage

Members of the INK4 protein family specifically inhibit cyclin-dependent kinase 4 (cdk4) and cdk6-mediated phosphorylation of the retinoblastoma susceptibility gene product (Rb). p16^INK4A, a prototypic INK4 protein, has been identified as a tumor suppressor in many human cancers. Inactivation of p16^INK4A in tumors expressing wild-type Rb is thought to be required in order for many malignant cell types to enter S phase efficiently or to escape senescence. Here, we demonstrate another mechanism of tumor suppression by implicating p16^INK4A in a G₁ arrest checkpoint in response to DNA damage. Calu-1 non-small cell lung cancer cells, which retain Rb and lack p53, do not arrest in G₁ following DNA damage. However, engineered expression of p16^INK4A at levels compatible with cell proliferation restores a G₁ arrest checkpoint in response to treatment with γ-irradiation, topoisomerase I and II inhibitors, and cisplatin. A similar checkpoint can be demonstrated in p53^−/− fibroblasts that express p16^INK4A. DNA damage-induced G₁ arrest, which requires the expression of pocket proteins such as Rb, can be abrogated by overexpression of cdk4, kinase-inactive cdk4 variants capable of sequestering p16^INK4A, or a cdk4 variant incapable of binding p16^INK4A. After exposure to DNA-damaging agents, there was no change either in overall levels of p16^INK4A or in amounts of p16^INK4A found in complex with cdks 4 and 6. Nonetheless, p16^INK4A expression is required for the reduction in cdk4- and cdk6-mediated Rb kinase activity observed in response to DNA damage. During tumor progression, loss of p16^INK4A expression may be necessary for cells with wild-type Rb to bypass this G₁ arrest checkpoint and attain a fully transformed phenotype.     

Effects of p21Cip1/Waf1 at Both the G1/S and the G2/M Cell Cycle Transitions: pRb Is a Critical Determinant in Blocking DNA Replication and in Preventing Endoreduplication

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21^Cip1/Waf1 and p27^Kip1 are limited to cell cycle control at the G₁/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G₁/S transition, contributes to regulation of the G₂/M transition. Both G₁- and G₂-arrested cells were observed in all cell types, with different preponderances. Preponderant G₁ arrest in response to p21 expression correlated with the presence of functional pRb. G₂ arrest was more prominent in pRb-negative cells. The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest Bs. In addition, DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G₂-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G₁ arrest as well as to blockade of DNA replication from either G₁ or G₂ phase is the pRb regulatory system. Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Effects of cis-platinum(II) diamminedichloride on survival and the rate of DNA synthesis in synchronously growing HeLa cells in the absence and presence of caffeine

Synchronously growing HeLa cells demonstrated a different profile of DNA synthesis to that observed for Chinese hamster V79-379A cells after treatment with cis-Platinum(II) diamminedichloride (cis-Pt(II)) in the G₁ phase of the cell cycle. The progression of G₁ phase treated cells into the DNA synthetic phase was not affected. The peak rate of DNA synthesis in the first cycle was decreased in a dose dependent manner. However, no displacement in the time of appearance of this peak rate of DNA synthesis was observed in the first cycle as had been observed in Chinese hamster V79-379A cells. The timing of mitosis after the first cycle was delayed in a dose dependent manner and resulted in a concomitant delay in the appearance of the peak rate of DNA synthesis in the second cycle. The peak rate of DNA synthesis in the second cycle was reduced in a dose dependent manner. The ability of cells to divide after the first cycle was not related to their eventual ability to survive. Incubation of HeLa cells with caffeine after treatment with cis-Pt(II) did not increase the toxicity of cis-Pt(II). This was consistent with the lack of effect of caffeine posttreatment on the rate of DNA synthesis in cis-Pt(II) treated synchronously growing HeLa cells. HeLa cells did not show the characteristics of caffeine sensitive replication repair, nor did they show evidence for the presence of an inducible repair system. The rate of DNA synthesis, cell number and survival data were discussed in relation to a mechanism of cell death proposed for Chinese hamster cells.     

Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G₀/G₁ cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G₀/G₁ cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.     

The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation,leading to G1 arrest

Cellular stresses, including growth factor deprivation, inflammatory cytokines or viral infection promote RAX/PACT-dependent activation of the double-stranded RNA-dependent protein kinase, PKR, to phosphorylate eIF2α, resulting in translation inhibition and apoptosis. In addition, PKR has been reported to regulate p53, STAT1 and NFκB. Here, we report that RAX/PACT interacts with the SUMO E2 ligase Ubc9 to stimulate p53-Ubc9 association and reversible p53 sumoylation on lysine 386. In addition, expression of RAX/PACT in a variety of cell lines promotes p53 stability and activity to increase p53 target gene expression. Significantly, while the expression of RAX/PACT, PKR or p53 alone has little effect on the cell cycle of p53-null H1299 cells, co-expression of p53 with either RAX/PACT or PKR promotes a 25–35% increase of cells in G₁. In contrast, co-expression of RAX/PACT with the sumoylation-deficient p53(K386R) mutant or with the desumoylase SENP1 fails to induce such a G₁ arrest. Furthermore, co-expression of p53, RAX/PACT and the dominant-negative PKR(K296R) mutant inhibits RAX/PACT-induced, p53-dependent G₁ growth arrest and expression of RAX/PACT in pkr^+/+ but not pkr^−/− MEF cells promotes p53 and p21 expression following gamma irradiation. Significantly, p53 stability is decreased in cells with reduced RAX/PACT or PKR following doxorubicin treatment, and expression of exogenous RAX/PACT promotes phosphorylation of wild-type but not p53(K386R) on serine 392. Collectively, results indicate that, in response to stress, the RAX/PACT-PKR signaling pathway may inhibit p53 protein turnover by a sumoylation-dependent mechanism with promotion of p53 phosphorylation and translational activation leading to G₁ cell cycle arrest.Key words: p53, PKR, RAX, PACT, Ubc9, sumoylation     

Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G₁-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G₁-phase. The role of ste9⁺ in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G₁-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G₁/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10^ts, cells arrested in G₁-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G₁-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G₁ progression possibly by controlling the amount of the mitotic cyclin in the G₁-phase.     

p53 signalling controls cell cycle arrest and caspase‐independent apoptosis in macrophages infected with pathogenic Leptospira species

Pathogenic Leptospira species, the causative agents of leptospirosis, have been shown to induce macrophage apoptosis through caspase‐independent, mitochondrion‐related apoptosis inducing factor (AIF) and endonuclease G (EndoG), but the signalling pathway leading to AIF/EndoG‐based macrophage apoptosis remains unknown. Here we show that infection of Leptospira interrogans caused a rapid increase in reactive oxygen species (ROS), DNA damage, and intranuclear foci of 53BP1 and phosphorylation of H2AX (two DNAdamage indicators) in wild‐type p53‐containing mouse macrophages and p53‐deficient human macrophages. Most leptospire‐infected cells stayed at the G₁ phase, whereas depletion or inhibition of p53 caused a decrease of the G₁‐phase cells and the early apoptotic ratios. Infection with spirochaetes stimulated a persistent activation of p53 and an early activation of Akt through phosphorylation. The intranuclear translocation of p53, increased expression of p53‐dependent p21^Cip1/WAF1 and pro‐apoptotic Bcl‐2 family proteins (Bax, Noxa and Puma), release of AIF and EndoG from mitochondria, and membrane translocation of Fas occurred during leptospire‐induced macrophage apoptosis. Thus, our study demonstrated that ROS production and DNA damage‐dependent p53‐Bax/Noxa/Puma‐AIF/EndoG signalling mediates the leptospire‐induced cell cycle arrest and caspase‐independent apoptosis of macrophages.     

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G₀/G₁ cell cycle arrest and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G₀/G₁ cell cycle phase arrest and increased levels of p27^kip1 in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G₀ state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.