role of checkpoint kinase 1 in preventing …...role of checkpoint kinase 1 in preventing premature...

Role of Checkpoint Kinase 1 in Preventing Premature Mitosis

in Response to Gemcitabine

Meredith A. Morgan,1Leslie A. Parsels,

2Joshua D. Parsels,

2Alefiyah K. Mesiwala,

1

Jonathan Maybaum,2and Theodore S. Lawrence

1

Departments of 1Radiation Oncology and 2Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan

Abstract

The deoxycytidine analogue 2V,2V-difluoro-2V-deoxycytidine(gemcitabine) is a potent radiation sensitizer in a varietyof solid tumors and tumor cell lines. Previous studies haveshown that radiosensitization by gemcitabine is accompaniedby simultaneous depletion of dATP pools (through ribonucle-otide reductase inhibition) and accumulation in the S-phase ofthe cell cycle. Because of the importance of cell cycleredistribution in gemcitabine-mediated radiosensitization,we investigated the role of checkpoint kinase (Chk) 1 andChk2 in gemcitabine-induced cell cycle arrest. We hypothe-sized that gemcitabine might induce Chk1 or Chk2 signaltransduction pathways that mediate S-phase arrest. We foundthat radiosensitizing concentrations of gemcitabine inducedaccumulation of phosphorylated Chk1 and Chk2 and down-regulation of Cdc25A in BxPC-3 (10 nmol/L), Panc-1(100 nmol/L), A549 (30 nmol/L), RKO (30 nmol/L), andSW620 (30 nmol/L) cells. Depletion of Chk1 from Panc-1 cellsprevented the down-regulation of Cdc25A in response togemcitabine. Furthermore, Chk1 depletion permitted Panc-1and SW620 cells treated with gemcitabine to enter mitosisdespite incomplete DNA synthesis. However, depletion ofneither Chk1 nor Chk2 abrogated the inhibition of DNAsynthesis in response to gemcitabine. These results provideevidence that Chk1 negatively regulates entry into mitosis inresponse to gemcitabine. Furthermore, these data imply thatChk1 acts to coordinate the cell cycle with DNA synthesis, thuspreventing premature mitotic entry in gemcitabine-treatedcells. (Cancer Res 2005; 65(15): 6835-42)

Introduction

2V,2V-Difluoro-2V-deoxycytidine (gemcitabine), a deoxycytidineanalogue, is a potent radiation sensitizer both in vitro andclinically (1–3). Gemcitabine requires phosphorylation to produceits radiosensitizing and cytotoxic activity. Deoxycytidine kinasecatalyzes a series of sequential phosphorylations convertinggemcitabine to the monophosphorylated, diphosphorylated, andtriphosphorylated metabolites (dFdCMP, dFdCDP, and dFdCTP,respectively). dFdCTP can interfere with DNA synthesis bycompetition with endogenous dCTP and incorporation intoreplicating DNA. dFdCDP is a potent inhibitor of ribonucleotidereductase, reducing the synthesis of deoxynucleotide triphos-phates, primarily dATP (1). This causes cells to redistribute intothe early S-phase of the cell cycle. Correlative studies have

suggested that simultaneous depletion of dATP pools (throughribonucleotide reductase inhibition) and accumulation in theS-phase of the cell cycle are required to achieve radiosensitiza-tion by gemcitabine (1, 4, 5).Given the critical role of cell cycle arrest in radiosensitization, it

seemed important to understand the mechanism underlyinggemcitabine-induced S-phase arrest. In response to DNA damage,the cell cycle halts, which prevents the propagation of cells withdamaged DNA. The two predominant signaling pathways regulat-ing cell cycle progression following DNA damage are the ataxiatelangiectasia mutated (ATM)/checkpoint kinase (Chk) 2 andataxia telangiectasia related (ATR)/Chk1 signal transduction path-ways. The ATM/Chk2 and ATR/Chk1 pathways elicit control at theG1, S, and G2 cell cycle checkpoints (6, 7). In response to ionizingradiation–induced DNA double-strand breaks, the ATM/Chk2pathway prevents DNA synthesis and cell cycle progression (8).The ATR/Chk1 pathway is induced in response to agents thatinhibit DNA replication either directly (aphidicolin; hydroxyurea) orindirectly (ultraviolet radiation; refs. 9, 10). The ATM/Chk2 andATR/Chk1 signaling pathways converge on the Cdc25 phosphatasefamily. Within the Cdc25 family, Cdc25A promotes G1-S transitionthrough stimulation of cyclin E/A-cyclin-dependent kinase (Cdk) 2complexes (11) and promotes G2-M transition through cyclinB-Cdk1 activation (12, 13). Cdc25A is degraded after phosphory-lation by either Chk1 or Chk2 in a ubiquitin- and proteosome-dependent manner in response to DNA damage. Althoughradiation-induced Cdc25A degradation tends to depend on Chk2(8) and hydroxyurea- or ultraviolet radiation-induced degradationof Cdc25A on Chk1 (14), crossover pathways do exist (15, 16).Because of the importance of cell cycle redistribution in

gemcitabine-mediated radiosensitization, we designed a study toassess the role of Chk1 and Chk2 in gemcitabine-induced cell cyclearrest. We hypothesized that gemcitabine, as an inhibitor ofribonucleotide reductase or as DNA chain terminator, might induceboth Chk1 and Chk2 signal transduction pathways. When we foundthat both Chk1 and Chk2 were phosphorylated and Cdc25A down-regulated in response to gemcitabine, we used small interferingRNA (siRNA) techniques to directly assess the roles of Chk1 andChk2 in gemcitabine-induced cell cycle arrest. We hypothesizedthat Chk1 and/or Chk2 might directly mediate DNA synthesisinhibition by gemcitabine or, alternatively, that Chk1 and/or Chk2might act to coordinate DNA synthesis with the rest of the cellcycle machinery. We tested this hypothesis by simultaneouslymonitoring DNA content and entry into mitosis of cells treatedwith gemcitabine and depleted of Chk1 or Chk2.

Materials and Methods

Cell culture. We chose a panel of cell lines with varying abilities to be

radiosensitized by gemcitabine. A549, BxPC-3, Panc-1, RKO, and SW620

cells were grown in DMEM (Panc-1 and SW620) or RPMI supplementedwith 10% fetal bovine serum (FBS) and 2 mmol/L glutamine. In addition,

Requests for reprints: Theodore S. Lawrence, Department of Radiation Oncology,University of Michigan Medical Center, UH-B2C490, Box 0010, 1500 East MedicalCenter Drive, Ann Arbor, MI 49109-0010. Phone: 734-647-9955; Fax: 734-763-7371;E-mail: [email protected].

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RKO cells were supplemented with 1.5 g/L sodium bicarbonate, 0.1 mmol/Lnonessential amino acids, and 1.0 mmol/L sodium pyruvate. Cells were

passaged weekly. Cells were routinely screened for Mycoplasma contam-

ination.

Gemcitabine. Gemcitabine was obtained from Eli Lilly (Indianapolis,IN). Gemcitabine was dissolved in PBS and stored at �20jC. Cells were

treated with 10 to 300 nmol/L gemcitabine for 24 hours.

Irradiation. Cells were irradiated at room temperature using a Pantak

DXT300 orthovoltage unit with 6 Gy at a dose rate of f3 Gy/min.Dosimetry was carried out using an ionization chamber connected to an

electrometer system that was directly traceable to a National Institute of

Standards and Technology calibration.

Clonogenic survival. Cells were subcultured into 100 mm2 dishes and

treated for 24 hours with gemcitabine. Cells were then irradiated and

processed for clonogenic survival according to the method described

previously (17). Cell survival curves were fitted using the linear quadratic

equation, and the mean inactivation dose was calculated according to the

method of Fertil et al. (18). The radiation enhancement ratio was calculated

by dividing themean inactivation dose under control conditions by themean

inactivation of gemcitabine-treated cells. Cytotoxicity was measured by the

surviving fraction of gemcitabine-treated cells to untreated control cells.

Small interfering RNA. SMARTpool Chk1, Chk2, and nonspecific

control pool (�pool) siRNAs were purchased from Dharmacon (Lafayette,

CO). Panc-1 cells were transfected with siRNA using Oligofectaminetransfection reagent (Invitrogen, Carlsbad, CA) according to the

manufacturer’s protocol. Cells in Opti-MEM reduced serum medium

(Invitrogen) were treated with 100 nmol/L siRNA and Oligofectamine.After 4 hours, medium was adjusted to contain 10% FBS. On the next day

after transfection, cells were replated and then exposed to drug 24 hours

later. SW620 cells were transfected with siRNA using LipofectAMINE 2000

(Invitrogen) according to the manufacturer’s protocol. Cells weretransfected in the presence of 10% FBS. Twenty-four hours after

transfection, medium was exchanged with normal growth medium. On

the next day, SW620 cells were replated and then exposed to drug 48

hours later.Flow cytometry. Cells were harvested and fixed in 70% ethanol. For DNA

content flow cytometry, cells were stained with a solution of 0.018 mg/mL

propidium iodide (PI) and 0.04 mg/mL RNase A. For flow cytometry with

P-histone H3, cells were permeabilized with 0.25% Triton-X 100 and thenresuspended in P-histone H3 antibody (Upstate Biotechnology, Lake Placid,

NY) followed by a FITC-conjugated anti-rabbit secondary antibody as

described previously (19). Cells were then stained with PI (0.033 mg/mL).Premature and normal mitoses were separated by first defining normal

mitosis under control conditions as P-histone H3–positive cells with 4N

DNA content and then applying these parameters to treated samples.

Premature mitosis was defined as the P-histone H3–positive cells with <4NDNA content. For bromodeoxyuridine (BrdUrd) flow cytometry, cells were

exposed to 30 Amol/L BrdUrd for 15 minutes and processed as described

previously (20) using an antibody recognizing BrdUrd (PharMingen, San

Diego, CA) followed by a FITC-conjugated goat anti-mouse secondaryantibody (Sigma Chemical Co., St. Louis, MO). In each experiment, a control

sample without BrdUrd was processed to determine the background signal.

Human lymphocytes or trout erythrocyte nuclei (BioSure, Grass Valley, CA)were included as internal standards. Cells were analyzed by counting 10,000

events on a Beckman Coulter (Fullerton, CA) Epics Elite (University of

Michigan Flow Cytometry Core) or 40,000 events on a Becton Dickinson

FACScan (San Jose, CA).Immunoblotting. Whole-cell lysates were prepared in 10 mmol/L Tris

(pH 7.4), 2% SDS, 1� Complete protease inhibitor cocktail (Roche,

Mannheim, Germany), 1 mmol/L NaF, 2 mmol/L Na3VO4, and 1 mmol/L

Na2PO7. Protein concentration was determined with the BCA Protein AssayReagent (Pierce, Rockford, IL). Samples were diluted in 1� loading buffer

[0.32 mol/L Tris-HCl, 10% glycerol, 2% SDS, 0.2% bromophenol blue, 4% 2-

mercaptoethanol (pH 6.8)] before loading onto 7.5% or 10% polyacrylamide

gels. Separated proteins were transferred to polyvinylidene difluoridemembranes and hybridized overnight at 4jC with antibodies recognizing

P-Chk1 (S317 or S345), P-Chk2 (T68; Cell Signaling Technology, Beverly,

MA), Chk2 (N-17), Cdc25A (F-6; Santa Cruz Biotechnology, Santa Cruz, CA),or Chk2 (clone 7; Upstate Biotechnology). Membranes were probed with

secondary antibodies, incubated with Enhanced Chemiluminescence Plus

(Amersham Biosciences, Little Chalfont, United Kingdom) and then

exposed to film.

Results

To determine whether radiosensitization by gemcitabine isaccompanied by perturbation of the cell cycle, we examinedBrdUrd incorporation and cell cycle distribution in BxPC-3, Panc-1,A549, RKO, and SW620 cells in response to gemcitabine. Weinvestigated both noncytotoxic and cytotoxic conditions thatwould produce radiosensitization (Table 1) and found that S-phasearrest accompanied radiosensitization. In these experiments,S-phase arrest was characterized by the presence of BrdUrd-positivecells with a cessation of cell doubling. In BxPC-3 and Panc-1 cells,treatment with gemcitabine under radiosensitizing conditions(10-30 and 100-300 nmol/L, respectively) resulted in arrest of thecells in the early S-phase of the cell cycle (Fig. 1; Table 2). In BxPC-3and Panc-1 cells, S-phase arrest and radiosensitization wereproduced by noncytotoxic concentrations of gemcitabine (10 and100 nmol/L, respectively). Under conditions of moderate radio-sensitization (10 nmol/L), A549 cells accumulated in both early andmiddle S-phases. A subpopulation of A549 cells arrested in earlyS-phase was visible in the histogram of propidium iodide–stainedcells (data not shown). However, greater sensitization was causedby a higher concentration (30 nmol/L) that produced S-phasearrest. In RKO cells, a lower, nonradiosensitizing concentration ofgemcitabine (10 nmol/L) did not arrest cell cycle progression,whereas a higher cytotoxic concentration of gemcitabine (20-30nmol/L) caused arrest and radiosensitization. In SW620 cells,conditions that produced radiosensitization and cytotoxicity(30-100 nmol/L) also caused early S-phase arrest. These resultsprovide a correlation between radiosensitization and redistributionof cells into S-phase by gemcitabine.To begin to understand the mechanism of the S-phase arrest by

gemcitabine, we examined the DNA damage–induced cell cyclecheckpoint proteins, Chk1 and Chk2. We measured the levels ofChk1 and Chk2 phosphorylated at sites required for enzymatic

Table 1. Radiosensitization by gemcitabine

Cell type

(nmol/L gemcitabine)

Radiation

enhancement ratio

Surviving

fraction

BxPC-3 (10)* 1.77 F 0.10 1.07 F 0.07

BxPC-3 (30) 2.38 F 0.24 0.60 F 0.10Panc-1 (30)* 1.19 F 0.10 1.06 F 0.03

Panc-1 (100) 1.76 F 0.02 0.94 F 0.07

A549 (10)c 1.31 F 0.04 0.96 F 0.03

A549 (30) 1.70 F 0.08 0.52 F 0.11RKO (10)b 1.03 F 0.06 0.98 F 0.03

RKO (20) 1.36 F 0.16 0.55 F 0.14

SW620 (1) 0.95 F 0.11 0.93 F 0.11SW620 (10) 1.31 F 0.07 0.73 F 0.27

SW620 (100) 1.74 F 0.11 0.13 F 0.10

*Lawrence et al. (1).cLawrence et al. (29).bChen et al. (28).

Cancer Research

Cancer Res 2005; 65: (15). August 1, 2005 6836 www.aacrjournals.org



activity. Cells treated with gemcitabine under radiosensitizingconditions showed an accumulation of S317 and S345 phosphor-ylated Chk1 (P-Chk1 S317 and P-Chk1 S345, respectively) in all fivecell lines (Fig. 2A and B) with no change in the total level of Chk1protein (Fig. 2C). In BxPC-3 and Panc-1 cells, P-Chk1 accumulationwas observed as early as 8 hours after the start of gemcitabinetreatment and persisted during the 24-hour gemcitabine treatment,whereas in A549 and RKO cells the induction occurred later (16-24hours; data not shown). We also measured the levels of T68phosphorylated Chk2 (P-Chk2 T68) in gemcitabine-treated cells.Radiosensitizing concentrations of gemcitabine also produced anaccumulation of P-Chk2 (T68) in BxPC-3, Panc-1, A549, RKO, andSW620 cells (Fig. 2D) without change in total Chk2 protein levels(Fig. 2E). These results suggest that both Chk1 and Chk2 areactivated by the conditions associated with S-phase arrest andradiosensitization.In addition, we wished to assess the interaction between

gemcitabine and radiation on Chk1 and Chk2. In four of five cell

lines (BxPC-3, Panc-1, RKO, and SW620), radiation treatment (6 Gy)alone induced P-Chk1 protein levels at 30 minutes after irradiation(Fig. 2A and B). Treatment with gemcitabine before irradiationproduced an increase in P-Chk1 protein levels in BxPC-3, Panc-1,and SW620 cells compared with treatment with radiation orgemcitabine alone. As anticipated, radiation treatment resulted ininduction of P-Chk2 protein in all of the cell lines (Fig. 2D). Thiseffect was similar to that observed in cells treated with gemcitabinealone or with gemcitabine before irradiation. These findingssuggest that gemcitabine and radiation might have overlappingabilities to activate Chk1 and Chk2 in some cell lines but not inothers.Checkpoint kinase 1 regulates Cdc25A protein levels in

gemcitabine-treated cells. To further explore the mechanism ofS-phase arrest by gemcitabine, we investigated whether Chk1 orChk2 activation could produce Cdc25A degradation. Treatmentof cells with gemcitabine under radiosensitizing conditions causeda reduction in Cdc25A protein in all five cell lines (Fig. 3A).

Figure 1. Effect of gemcitabine on cell cycle distribution.BxPC-3, Panc-1, A549, RKO, and SW620 cells weretreated for 24 hours with low-dose or high-dosegemcitabine (Gem ). BrdUrd incorporation and PI stainingwere analyzed by flow cytometry. Representative of threeindependent experiments.

Checkpoint Kinase 1, Premature Mitosis, and Gemcitabine




In addition, radiation also resulted in a reduction of Cdc25Aprotein levels in both the presence and the absence of gemcitabinepretreatment. These results suggest that the induction of Chk1and/or Chk2 in response to gemcitabine or radiation produces adownstream response, which initiates a reduction in Cdc25Aprotein levels.To determine if the reduction in Cdc25A protein in response to

gemcitabine was mediated by Chk1 or Chk2, we used siRNA todeplete Chk1 or Chk2 from Panc-1 cells that were well sensitizedunder noncytotoxic conditions. In initial experiments, weconfirmed that cells depleted of Chk1 were not able to induceP-Chk1 protein in response to gemcitabine. Likewise, cellsdepleted of Chk2 were no longer able to induce P-Chk2 protein.Following treatment with Chk1 or Chk2 siRNA, Panc-1 cells weretreated with gemcitabine and Cdc25A protein levels wereassessed. Under control conditions (�pool), gemcitabine pro-duced the expected reduction in Cdc25A protein (Fig. 3B).However, in cells depleted of Chk1, Cdc25A protein levelspersisted despite treatment with gemcitabine. It is of interest tonote that the basal levels of Cdc25A were increased in cellsdepleted of Chk1. In addition, we found that gemcitabine stillproduced a reduction in Cdc25A protein levels in cells depleted ofChk2. These results provide evidence that Chk1, but not Chk2,regulates Cdc25A protein in both untreated and gemcitabine-treated cells.Checkpoint kinase 1 prevents premature mitosis in gemci-

tabine-treated cells. Next, we wished to understand how Chk1and Chk2 were involved in the cell cycle redistribution produced bygemcitabine. We postulated two means by which Chk1 or Chk2might regulate the cell cycle. We hypothesized that Chk1 and/orChk2 could be the cause of DNA synthesis inhibition bygemcitabine or, alternatively, that Chk1 and/or Chk2 might beactivated by DNA synthesis inhibition and thus act to coordinateDNA synthesis with the rest of the cell cycle machinery. Weexpected that if Chk1 or Chk2 were causative in DNA synthesisinhibition by gemcitabine, then depleting Chk1 or Chk2 wouldresult in new DNA synthesis; however, if induction of Chk1 or Chk2were the effect of DNA synthesis inhibition, then depleting Chk1 orChk2 would permit cell cycle progression despite incompletelyreplicated DNA. To address these two hypotheses, in cells depleted

of Chk1 or Chk2, we monitored DNA content by PI staining andprogression of the cell cycle by P-histone H3 staining. We foundthat the depletion of Chk1 and Chk2 from cells did not prevent theaccumulation of cells with a S-phase DNA content induced bygemcitabine (Fig. 4A). These results suggest that neither Chk1 norChk2 is the cause of DNA synthesis arrest in gemcitabine-treatedcells.To test the alternative hypothesis of whether Chk1 and/or Chk2

act to coordinate DNA synthesis with the cell cycle, we monitoredentry into mitosis by staining for P-histone H3 (19). As anticipated,treatment with gemcitabine markedly reduced the number of cellsentering mitosis (Fig. 5A and B). The percentage of cells in mitosiswas reduced from 1.9% to 0.5% in Panc-1 cells and from 2.5% to0.7% in SW620 cells by gemcitabine. Depletion of Chk2 ingemcitabine-treated cells had no effect on the fraction of cellsentering mitosis. However, depletion of Chk1 in gemcitabine-treated Panc-1 cells resulted in the entry of cells into mitosisdespite incomplete DNA synthesis (4.2%). In SW620 cells, a smallpercentage of cells were observed in premature mitosis (0.4%) inresponse to gemcitabine and nonspecific siRNA (�pool +gemcitabine). However, depletion of Chk1 in gemcitabine-treatedSW620 cells produced a marked increase in the percentage of cellsprematurely entering mitosis (1.7%). Depletion of Chk1 or Chk2from cells in the absence of gemcitabine treatment did notproduce any consistent alterations in the fraction of mitotic cells.Although Chk2 was substantially reduced by siRNA (Figs. 4B and5C), it was not completely suppressed. Therefore, we cannotexclude the possibility that Chk2 is involved in cell cycleprogression following gemcitabine. Nonetheless, we conclude thatChk1, but not Chk2, prevents premature mitosis in gemcitabine-treated cells and acts to coordinate DNA synthesis with the rest ofcell cycle machinery.

Discussion

In this study, we have found that treatment with gemcitabineunder radiosensitizing conditions produces arrest in early S-phasethat is associated with accumulation of the phosphorylated formsof Chk1 and Chk2. Although both Chk1 and Chk2 werephosphorylated, the subsequent degradation of Cdc25A in responseto gemcitabine was mediated by Chk1. Neither Chk1 nor Chk2 weredirectly responsible for the inhibition of DNA synthesis induced bygemcitabine. However, Chk1 negatively regulated entry into mitosisin gemcitabine-treated cells. These findings suggest that gemcita-bine stimulates Chk1 to initiate a G2-M cell cycle checkpoint.Furthermore, these data imply that Chk1 acts to coordinate the cellcycle with DNA synthesis, thus preventing premature mitotic entryin gemcitabine-treated cells (Fig. 6).There are two possible scenarios for how DNA synthesis might

be arrested in response to DNA damage. Firstly, arrest of DNAsynthesis could be directly mediated through checkpoint activa-tion. Alternatively, arrest might occur for other reasons, such aschain termination, which activates checkpoints. Others havefound that Chk1 and Chk2 cause the S-phase arrest in responseto topoisomerase I poisons. For example, Xiao et al. (13) foundthat Chk1 mediates the S-phase checkpoint induced by campto-thecin, whereas Yu et al. (21) observed that Chk2 mediates thecamptothecin-induced S-phase checkpoint. These studies showedthat depletion of Chk1 or Chk2 abrogated the camptothecin-induced S-phase arrest. In contrast to camptothecin, gemcitabinecould directly arrest DNA synthesis through the depletion of

Table 2. Percentage of cells in S, G1, or G2-M phase

Cell type

(nmol/L gemcitabine)

Total S G1 G2-M

BxPC-3 (control) 38 F 6 50 F 9 8 F 3

BxPC-3 (10) 75 F 4 18 F 5 3 F 2BxPC-3 (30) 77 F 3 16 F 3 3 F 1

Panc-1 (control) 37 F 7 44 F 7 12 F 2

Panc-1 (100) 65 F 3 23 F 4 4 F 1

Panc-1 (300) 62 F 6 25 F 6 4 F 1A549 (control) 32 F 5 55 F 7 7 F 3

A549 (10) 65 F 12 20 F 4 3 F 2

A549 (30) 69 F 6 18 F 1 2 F 1RKO (control) 53 F 2 27 F 2 16 F 1

RKO (10) 64 F 0 22 F 1 8 F 4

RKO (30) 91 F 1 3 F 1 1 F 1

SW620 (control) 52 F 4 38 F 5 7 F 1SW620 (30) 88 F 4 7 F 2 3 F 1

SW620 (100) 83 F 11 8 F 4 2 F 1

Cancer Research




deoxynucleotide triphosphate pools and incorporation into DNA.In turn, arrested DNA synthesis might then stimulate cell cyclecheckpoint activation. For camptothecin, inhibition of DNAsynthesis might not be mediated by the topoisomerase I cleavagecomplexes alone but is likely dependent on induction of a cellcycle checkpoint (22, 23). Taken together, these findings suggest

that drugs that directly affect DNA synthesis (such as theantimetabolites) may show a different pattern of checkpointresponses from drugs that affect other cellular functions, such asDNA unwinding. In addition, although our studies showed thatneither Panc-1 nor SW620 cells treated with gemcitabine resumeDNA synthesis in response to Chk1 depletion, Pan et al. (24) have

Figure 2. Effect of gemcitabine on Chk1 and Chk2. A-E, cells were treated for 24 hours with gemcitabine (Gem 1, BxPC-3, A549, and RKO, 10 nmol/L; Panc-1,100 nmol/L; SW620, 30 nmol/L; Gem 2, BxPC-3, A549, and RKO, 30 nmol/L; Panc-1, 300 nmol/L; SW620, 100 nmol/L) or for 8 hours with hydroxyurea (HU ) or leftuntreated (C ). Additionally, cells were treated with 6 Gy irradiation alone (6Gy ) or following 24 hours of gemcitabine treatment (Gem 1 + 6Gy ). Irradiated cells wereincubated for an additional 30 minutes after irradiation. Levels of Chk1 or Chk2 in whole-cell lysates were detected by immunoblotting with antibodies that specificallyrecognize S317 (A ) or S345 (B) phosphorylated Chk1, total Chk1 (C ), T68 phosphorylated Chk2 (D ), or total Chk2 (E ). h-actin is shown as a loading control.Representative of at least three independent experiments.





found that Chk1 depletion allows gemcitabine-treated U2OS cellsto resume DNA synthesis. The difference between our findingsand theirs might be due to inherent differences among Panc-1,SW620, and U2OS cells or differences in their metabolism ofgemcitabine.Our finding that depletion of Chk1 results in the premature entry

of gemcitabine-treated cells into mitosis could be a result of the

elevated expression of Cdc25A in Chk1-depleted cells. Over-expression of Cdc25A has been shown to drive cells into mitosiseven in the presence of incompletely synthesized DNA (12, 25).Furthermore, Molinari et al. (26) showed that Cdc25A over-expression caused cells with arrested DNA synthesis (by hydroxy-urea) to enter mitosis. These findings suggest that Chk1 depletionor Cdc25A overexpression can drive the cell cycle machinery

Figure 3. Effect of gemcitabine on Cdc25A protein levels. A, cells were treated as described in Fig. 2. Levels of Cdc25A were measured by immunoblotting.B, cells were either mock treated (�) or treated with �pool, Chk1, or Chk2 siRNA as indicated. After 24 hours, gemcitabine (100 nmol/L) was added to cells for anadditional 24 hours. Levels of Cdc25A, Chk1, Chk2, and h-actin proteins were measured in the cell lysates. Representative of three independent experiments.

Figure 4. DNA content in response to gemcitabine in cells depleted of Chk1 or Chk2. A, Panc-1 cells were treated with �pool, Chk1, or Chk2 siRNA. After transfection,gemcitabine was added for an additional 24 hours. Cell cycle distribution was determined by the PI-stained DNA content. B, immunoblots of Chk1, Chk2, andh-actin proteins in Panc-1 cells treated with siRNA. Representative of three independent experiments.

Cancer Research




independent of DNA synthesis and lead cells into prematuremitosis.Although the focus of this study has been on assessing the role of

Chk1 and Chk2 in early S-phase arrest, it will be necessary to study

subsequent cellular events. For instance, it is unclear why Panc-1and BxPC-3 cells tolerate Chk1 and Chk2 activation and DNAsynthesis inhibition for up to 24 hours with minimal cytotoxicity,whereas A549, RKO, and SW620 cells show substantial cytotoxicity

Figure 5. Entry into mitosis in response to gemcitabine in cells depleted of Chk1 or Chk2. Panc-1 (A) and SW620 (B ) cells were treated as in Fig. 4. Entry into mitosiswas assessed by staining with an antibody recognizing P-histone H3. DNA content was assessed by PI staining. Immunoblots of Chk1, Chk2, and h-actin proteinsin SW620 cells treated with siRNA (C). Representative of three to eight independent experiments. The mean number F SE of cells in premature mitosis was 0.3 F 0.1%or 4.6 F 0.3% (n = 3) for Panc-1 cells treated with gemcitabine and �pool siRNA or gemcitabine and Chk1 siRNA, respectively, and 0.5 F 0.1% or 2.8 F 0.3%(n = 8), respectively, for SW620 cells.





under the same conditions. The most obvious factor that mightinfluence cytotoxicity is p53 status. For instance, p53 has beenshown to influence cell death in response to antimetabolitesthrough Fas-mediated apoptosis (27), and we have shown

previously that RKO cells with disrupted p53 function are moreresistant to gemcitabine-mediated cytotoxicity (28). Furthermore,in the current study, p53 mutant cells (BxPC-3 and Panc-1) tolerategemcitabine-induced S-phase arrest with minimal cytotoxicity,whereas p53 wild-type cells (A549 and RKO) do not (Table 1).However, in SW620 cells, which are p53 mutant, cell cycle arrest isaccompanied by cytotoxicity. Therefore, p53 is unlikely to be theonly factor regulating cytotoxicity in S-phase–arrested cells. It willbe important to distinguish the mechanism by which some cells arecapable of tolerating S-phase arrest in future studies. Furthermore,although we have begun to understand the roles of Chk1 and Chk2in gemcitabine-mediated cell cycle arrest, their influence oncytotoxicity and radiosensitization have not yet been investigated.It is possible that Chk1 depletion will enhance gemcitabine-mediated cytotoxicity and radiosensitization, thus providing atherapeutic potential. Studies of not only the initial activation ofcheckpoints but also the consequences of checkpoint activation insubsequent cellular events, such as survival, are required.

Acknowledgments

Received 6/24/2004; revised 5/4/2005; accepted 5/12/2005.Grant support: NIH grant CA78554 and University of Michigan Cancer Biology

Training Program.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Christine Canman for thoughtful review of this article and Dr. MaryDavis and Emily Ng for technical assistance.

Figure 6. Cell cycle effects of gemcitabine. Inhibition of DNA synthesis bygemcitabine, either through DNA incorporation or dATP pool depletion, leads tothe induction of P-Chk1. Chk1 in turn triggers the degradation of Cdc25A and thisultimately renders cyclin B-Cdk1 complexes inactive, thus preventing theprogression of cells into mitosis. In gemcitabine-treated cells depleted of Chk1Cdc25A protein persists, cyclin B-Cdk1 complexes are active and cells are driveninto mitosis despite incompletely replicated DNA.

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Cancer Research




2005;65:6835-6842. Cancer Res Meredith A. Morgan, Leslie A. Parsels, Joshua D. Parsels, et al. in Response to GemcitabineRole of Checkpoint Kinase 1 in Preventing Premature Mitosis

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