Leukemia Research 24 (2000) 1033–1039

Granulocyte-colony stimulating factor induced intranuclearendonuclease in murine leukemia cell line

Atsushi Handa *, Takuya Kashimura, Akiko Yamamoto, Ikuo Murohashi,Masami Bessho, Kunitake Hirashima

Di6ision of Hematology and Infectious Diseases, Department of Internal Medicine, Saitama Medical School, Saitama 350-0451, Japan

Received 9 February 2000; accepted 22 May 2000

Abstract

We have reported that murine leukemia cell line (C2M-A5) induced apoptosis by G-CSF. To clarify the mechanism, mRNAexpression of apoptosis-related genes was studied. It revealed transient over-expression of c-myc, H-ras and p53 and down-expres-sion of bcl-2. These changes were known as triggers of endonuclease induction. After 96 h culture with G-CSF, apoptosis wasoccurred simultaneously with endonuclease (37 kd) activation. This endonuclease induced the digestion of double-strand DNAand might be associated with caspase3. Although G-CSF accelerates cell growth and prevents apoptosis in general, it is acontradictory effect. We concluded that G-CSF induced endogenous endonuclease activity in C2M-A5. © 2000 Elsevier ScienceLtd. All rights reserved.

Keywords: G-CSF; Apoptosis; Endonuclease; Myeloid leukemia cells; c-myc; H-ras; p53; bcl-2; Caspase3

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1. Introduction

There are two distinct patterns of cell death, necrosisand apoptosis, which have been recognized on the basisof morphological and biochemical analyses [1,2]. Todate evidence has been found that various intracellularmolecular pathways, including proto-oncogenes and tu-mor suppressor genes, are related to apoptosis andconverged to induce apoptosis in tumor cells [3,4].Morphologically, apoptosis is characterized by cellshrinkage and chromatin condensation. These morpho-logical changes are thought to be associated with en-donuclease activity. Recent reports have shown thatleukemia cells have several endonucleases and their

metal dependencies are lineage specific [5,6]. There isalso evidence that apoptosis in leukemia cells was in-duced by some cytokines, TGF-b and IFN-b [7].

We have established a radiation-induced murinemyeloid leukemia cell line (C2M-A5) [8]. Intravenousinoculation of C2M-A5 cells into C3H/He mice resultedin the development of myeloid leukemia. However,death of the mice due to leukemia was suppressed bythe subcutaneous injection of recombinant humangranulocyte-colony stimulating factor (rh-G-CSF), pos-sibly because of the induction of apoptosis to theleukemic cells [9]. G-CSF is one of the hematopoieticgrowth factors which stimulate the growth and differen-tiation of hematopoietic cells, and it is capable ofpreventing the apoptosis [10,11]. This factor regulatesthe proliferation and promotes the survival of somemyeloid leukemic cells [12–15], as well as normal gran-ulocytic progenitor cells. In this study, to clarify thesecontradictory effects of G-CSF on apoptosis, we ana-lyzed the changes of mRNA expression of apoptosis-re-lated genes first, and then on endonuclease during theapoptosis induced by rh-G-CSF.

Abbre6iations: CAD, caspase3-activated DNase; rh-G-CSF, recom-binant human granulocyte-colony stimulation factor.

* Corresponding author. Present address: Hematology Branch, Na-tional Heart, Lung and Blood Institute, National Institutes of HealthBldg 10/Rm 7C 218, 9000 Rockville Pike, Bethesda, MD 20892-1652,USA. Tel.: +1-301-4962480; fax: +1-301-4968396.

E-mail address: handaa@nih.gov (A. Handa).

0145-2126/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 5 -2126 (00 )00074 -6

A. Handa et al. / Leukemia Research 24 (2000) 1033–10391034

2. Materials and methods

2.1. G-CSF

Purified CHO cell-derived rh-G-CSF, a special giftfrom Chugai Pharmaceutical Co. Ltd (Tokyo, Japan),with a specific activity of 108 U/mg was dissolved insaline and added to the cultures at a final concentrationof 100 ng/ml.

2.2. Cell line and culture

Myeloid murine leukemia cell line C2M-A5 was es-tablished by limiting dilution from the cell line C2Mwhich was originated from a radiation-induced myeloidleukemia in a C3H/He mouse [8]. C2M-A5 cells weremaintained in McCoy’s medium (Gibco BRL, Gaithers-burg, MD) supplemented with 10% horse serum (Hy-clone, Logan, UT) and 50 mg/ml gentamicin (GM;Schering-Plough, Osaka, Japan). Then the cellswere subcultured with the same culture media by re-plenishing the fresh McCoy’s medium every 4 days.Cell viability was measured by trypan blue dye exclu-sion.

2.3. RNA preparation and Northern blot analysis

Total RNA was prepared in guanidium isothio-cyanate and extracted in phenol/chloroform [16].Northern blot analysis was carried out as described byThomas with a slight modification [17]. Total RNA(20–50 mg) was electrophoresed on a 1.0% agarosegel containing 2.2 M formaldehyde and transferred tonylon membrane (Hybond-N, Amersham PharmaciaBiotech, Piscataway, NJ) followed by hybridizationwith nick-translated 32P-labeled probes. The followingcDNA fragments were used as probe; c-myc,0.467 kb and 6-H-ras, 0.56 kb (Takara, Otsu, Japan)p53, 1.3 kb and bcl-2, 2.7 kb, (Oncogene Science,Cambridge, MA). Autoradiogrames were analyzed byscanning densitometry with Densitoron 20-HR (Joko,Tokyo, Japan). To quantitate relative gene expressionrates, the hybridization signals for each gene werecompared to the expression rates for 28S ribosomalRNA, which were determined by the ethidiumbromidestain.

2.4. Preparation of nuclei and induction of nuclearcondensation in 6itro

The cells were maintained at 1×106/ml and mediumwas exchanged after 2 days of culture. After 96 h thecells were cultured with or without rh-G-CSF (100ng/ml), then centrifuged at 600×g for 10 min andwashed twice with PBS. The pellets were re-suspendedin an ice-cold hypotonic buffer (10 mM Hepes pH

7.4/10 mM KCl/2 mM MgCl2/1 mM DTT/1 mMPMSF), incubated on ice for 20 s, and then homoge-nized 20 strokes in a Dounce homogenizer. After cen-trifugation and removal of cell supernatant, nuclei werewashed once and resuspended in hypotonic buffer. Nu-clei were incubated at 37°C with the supernatant (cyto-plasmic fraction) of rh-G-CSF-treated or untreatedcells. Nuclear condensation was determined morpho-logically or by TUNEL method which was based onTdT activity using commercial kit (Amersham Pharma-cia Biotech, Piscataway, NJ) [18]. In addition, DNAwas extracted in the nuclei and DNA fragmentation ongel electrophoresis were performed as described below[9].

2.5. Determination of apoptosis

The percentage of apoptotic cells was determinedwith Wright–Giemsa-stained cells after cyto-centrifuga-tion (1500 rpm, 5 min) onto a glass slide using aCytospin (Shandon, London, UK). Counts of 500 cellsor isolated nuclei were evaluated. Apoptotic cells weresmall in size and had fragmented nuclei compared withthe intact C2M-A5 cells [1,2] and also determined byTUNEL method [18]. The DNA was electrophoresedthrough 1.0% agarose and examined for DNA fragmen-tation as described previously [9].

2.6. Detection of the endonuclease acti6ities

C2M-A5 cells were cultured with or without rh-G-CSF (100 ng/ml) for 96 h. The cells were washed threetimes with PBS and re-suspended in 200 ml of samplebuffer (0.1 M Tris–HCl pH 7.5/1% Nonidet-P40/2 mMPMSF/2.5 mM DTT). After a 5 min incubation on ice,the samples were centrifuged at 12 000 rpm for 15 min.The resulting supernatant of the samples was separated.The pellets were re-suspended in 200 ml of the samplebuffer, sonicated for 2 min, then the DNA fragmentswere removed using glass milk (BIO 101 Inc, Tokyo,Japan). For determination of endonuclease or nucleaseactivity, the gel system was used as originally describedby Rosenthal [19] with minor modifications. The sam-ples were concentrated four times by using a Ultra free5000 (Nihon Millipore Ltd., Tokyo, Japan), and elec-trophoresed in 15% polyacrylamide gel with 160 mg/mlsalmon sperm DNA (Research Genetics, Huntsville,AL) incorporated. After electrophoresis, the gel wasrinsed and incubated at 37°C in the reaction buffer (0.1M Tris–HCl pH 6.8, pH 7.0 or pH 8.0/1 mM EDTA/2mM MgCl2) with or without addition of 3 mM CaCl2and/or MgCl2 and brought to a final concentration of 5mM. The gel was stained with ethidiumbromide andexamined for the presence of DNA digestion using aUV trans-illuminator.

A. Handa et al. / Leukemia Research 24 (2000) 1033–1039 1035

2.7. Caspase3 and Bcl-2 acti6ities in G-CSF treatedcells

To determine the apoptosis related genes expressionsafter rh-G-CSF treatment, caspase3 and Bcl-2 activitieswere measured after rh-G-CSF treatment. Caspase3and Bcl-2 were determined by ELISA system followingattached manuals (caspase3: MBL, Nagoya, Japan; Bcl-2: Amersham Pharmacia Biotech, Piscataway, NJ). Thesamples were triplicated and these activities were mea-sured by microplate reader (405 nm) (BioRad, Her-cules, CA).

3. Results

3.1. Apoptosis associated genes re6ealed transientchanges

Although Northern blot analysis demonstrated theexpression of c-myc, H-ras, bcl-2 and p53 in the C2M-A5 stationary cells, genes expressions were changed bystimulation with rh-G-CSF (Fig. 1). Expression of Fas,which was detected in stationary cells, did not changewith the used assay conditions (data not shown). Afterthe rh-G-CSF-stimulation, over-expression of c-myc

was observed from 3 to 24 h peaking at 12 h, when theexpression rate was 10 times control (Fig. 1A). WhereasH-ras was over-expressed earlier than c-myc peaking at6 h when it was 10 times the control, but it went downto the normal level 48 h later with rh-G-CSF supple-ment (Fig. 1B). The expression rate of p53 started toelevate at 24 h after the addition of rh-G-CSF, peakingat 24 h (12 times the control) (Fig. 1D). In contrast tothe results on c-myc, H-ras, and p53, bcl-2 expressionwas down-regulated after 6 h of rh-G-CSF stimulationand continued for at least 48 h (0.1 times the control)(Fig. 1C).

3.2. Nuclear condensation was induced by theincubation of cell nuclei with rh-G-CSF treated cellsupernatant

The cytoplasmic fraction cultured with rh-G-CSF for96 h induced nuclear condensation and apoptotic bodyformation after 5 h incubation (Fig. 2). The condensednuclei were approximately 30% of the total nuclei ob-served under a microscope with the Wright–Giemsastain, however, TUNEL positive cells which indicatedapoptotic cells were 72.493.2% (n=3). In contrast,only 5% of the nuclei were condensed after incubationwith cytoplasm cultured without rh-G-CSF. As the

Fig. 1. (a) Expression of genes related to the apoptosis of C2M-A5 cells after incubation. Data from Northern analysis were calculated bydensitometry (n=5). The relative amount of mRNA is shown in OD units (mRNA/rRNA, 28S) and plotted against time. A: c-myc, B: H-ras,C: bcl-2, D: p53. (b) Northern blot analysis of (A) c-myc, (B) H-ras, (C) bcl-2, and (D) p53.

A. Handa et al. / Leukemia Research 24 (2000) 1033–10391036

Fig. 2. Morphological change of the nuclei from C2M-A5 after 5 h incubation with rh-G-CSF treated (96 h) (left) or untreated (right) cytoplasmicfraction. Wright–Giemsa stain was done to clarify the condensation of nuclei.

same as rh-G-CSF untreated cytoplasm induced only7.290.4% (n=3) TUNEL positives (Fig. 3). DNAfragmentation was occurring at the same time as theapoptotic body appeared morphologically (Fig. 4).

3.3. Endonuclease acti6ity was induced after the cellincubation with rh-G-CSF

Endonuclease activity was detected in the nuclearfraction of rh-G-CSF-treated C2M-A5 cells with thenuclease activity gel system (Fig. 5). After 5 h ofincubation in a reaction buffer, pH 8.0 containing 3mM Ca2+ and 5 mM Mg2+, the electrophoresed geldemonstrated that the only nuclear fraction treatedwith 100 ng/ml of rh-G-CSF for 96 h showed DNAdigestion activity as a black spot on white background.From the size marker, the endonuclease was estimatedto be about 37 kd. These conditions (pH 8.0 andpresence of calcium/magnesium) and its size were com-patible to DNase I. In the samples which were incu-bated up to 7 days in the reaction buffer pH 5.0, noDNA digestion was detected with or without additionof 10 mM EDTA (data not shown).

3.4. Caspase3 and Bcl-2 acti6ities

Caspase3 and Bcl-2 were measured in cell lysateswith or without 96 h rh-G-CSF treatment. Caspase3were significantly increased by rh-G-CSF stimulationand Bcl-2 were decreased as undetectable level (Fig. 6).On these stage, more than 70% of C2M-A5 cells werekilled by apoptosis.

4. Discussion

Generally G-CSF is recognized as a cytokine thatstimulates myeloid precursor growth and differentiationbut does not lead to apoptosis. Previously we havefound that in a radiation-induced murine myeloidleukemia cell line (C2M-A5), apoptosis is induced byadministration of rh-G-CSF to culture media, and thatthe anti-leukemic effect of this cytokine is also ascribedto apoptosis induction [9,14]. We have already reportedthe rh-G-CSF induced apoptosis of C2M-A5, and thatthe apoptosis could be demonstrated 48 h after admin-istration of rh-G-CSF, suggesting relatively slow induc-tion of apoptosis [14]. In another paper, rh-G-CSFup-regulated expression of cyclin D1 and cdk4 proteinsand reciprocally down-regulated expression of cyclin

Fig. 3. The rates of condensate nuclei or TUNEL positive nuclei weredetermined by 500 nuclei observation under microscopy (n=3).C2M-A5 cells lysates (cytoplasmic fraction) with or without rhG-CSFtreated were incubated with C2M-A5 nuclei for 5 h at 37°C.

A. Handa et al. / Leukemia Research 24 (2000) 1033–1039 1037

Fig. 4. Electrophoresis of DNA extracted from C2M-A5 nuclei after5 h incubation with rh-G-CSF treated or untreated cytoplasmicfraction. Lane 1: incubated with rh-G-CSF untreated cytoplasmicfraction, Lane 2: incubated with rh-G- CSF treated (2 h) cytoplasmicfraction, Lane 3: incubated with rh-G-CSF treated (96 h) cytoplasmicfraction.

activation of cell growth and cell cycles were revealed(data not shown). Although the mechanism of thechanges in c-myc and H-ras expression is not docu-mented in tumor cells, the changes in expressions ofthese genes are so widespread as to suggest a criticalrole in apoptosis [21–24]. Reports indicate that c-mycis the major oncogene whose expression is raised duringthe period of apoptosis [21,22] and that the down-ex-pression of H-ras is a trigger for apoptosis [23,24]. Theelevated expressions of c-myc and rapid return to thestationary level of H-ras supported the role in apopto-sis. After the early stage of growth-stimulation, we alsodemonstrated increased expression of p53 and down-ex-pression of bcl-2, which are apoptosis-inducing [25] andapoptosis-suppressing genes [26], respectively.

The up-regulation of c-myc expression leads to theactivation of Ca2+ and Mg2+ dependent endonuclease,and also the down expression of H-ras induces theendonuclease [23,24]. Because of our results on theexpression change of c-myc and H-ras, we focused onendonuclease activity, which is thought to be the keyenzyme later in the process of apoptosis. Some reportsdescribe that the elevation of endonuclease activityduring the apoptosis of non-adherent marrow mononu-clear cells from myelodysplastic syndrome or acutemyelogenous leukemia [27,28]. These endonucleases areendogenous and not inducible. On the other hand,another report shows that apoptosis sensitivity is deter-mined by endogenous endonuclease content in chroniclymphocytic leukemia cells [29]. In other case, the en-donuclease which is Mg2+ dependent with an optimalpH of 7.5–8.5, was related with myelogenous leukemiaand not lymphoid leukemia [30] and that the cells from

Fig. 5. Nuclease activity gel system using polyacrylamide gel. Nucleuslysates (10 mg protein) were prepared from C2M-A5 cells afterincubation with or without rh-G-CSF (100 ng/ml) and applied onSDS-polyacrylamide gel. Lane 1: without rh-G-CSF for 24 h, Lane 2:with rh-G-CSF for 24 h, Lane 3: without rh-G-CSF for 96 h, Lane 4:with rh-G-CSF for 96 h, Lane 5: positive control as DNase I. Afterelectrophoresis gel was incubated with the reaction buffer pH 8.0containing 5 mM Mg 2+ and 3 mM Ca 2+ for 5 h and stained withethidiumbromide. DNA digestion activity is visualized as black spotson white background using a UV trans-illuminator.

Fig. 6. The activities of Caspase3 (a) and Bcl-2 (b) with or withoutrh-G-CSF treatment for 96 h (n=3).

D2 and cdk2 proteins. This study showed that cell cycleactivation is crucial for this apoptosis [20].

After the administration of rh-G-CSF, we demon-strated over-expression of H-ras and c-myc in the earlystages, and that the expression level of H-ras was downmore rapidly than that of c-myc. In fact, in the earlystage of C2M-A5 cells cultured with rh-G-CSF, the

A. Handa et al. / Leukemia Research 24 (2000) 1033–10391038

acute myelogenous leukemia showed high levels of theendonuclease activity [6]. We have demonstrated theexistence of rh-G-CSF-induced endogenous endonucle-ase which has a molecular weight of 37 kd and is activeat pH 8.0 in the presence of Ca2+ and Mg2+, but isinactive below pH 5.0 or in the presence of EDTA. Thisendonuclease was not detected in rh-G-CSF untreatedcells. It digested salmon sperm DNA, which was adouble-stranded form. As shown in this study, cellfragmentation was induced by the incubation of nucleiwith the cytoplasmic fraction separated from the rh-G-CSF stimulated cells for 96 h. This evidence indicatedthat apoptosis inducible substance was existing in thecytoplasmic fraction. Based on these results, we as-sumed that this endonuclease is a DNase I-like enzyme.Although glucocorticoid induces endogenous endonu-clease in normal thymocytes [31,32], it is unusual thatcytokines induce these types of enzymes. As far as weknow there is no report that G-CSF induces anyendonuclease.

Recently caspase3-activated DNase (CAD) was dis-covered during apopotosis [33]. This DNase was associ-ated with caspase3 and induced by Fas-ligand or c-myc[33]. We also measured Caspase3 activity during apop-tosis and demonstrated the activation of caspase3 after96 h after G-CSF stimulation. In addition, the same asNorthern blot analysis, Bcl-2 was measured and de-tected the significant decrease after apoptosis. Thesedata supported that this enodonuclease is similar toCAD. However, we could not confirm this endonucle-ase as CAD because their molecular sizes were different(this endonuclease: 37 kd, and CAD: 40 kd) and noreport mentioned about the mice homolog [34].

From the current study, we conclude that G-CSFinduces a DNase I-like endonuclease or CAD-like en-donuclease in murine myelogenous leukemia cell line(C2M-A5). It is associated with expression changes inc-myc, H-ras, bcl-2, p53 and caspase3 which were com-bined with obvious controlling processes including cellcycles and/or apoptosis related proteins.

Acknowledgements

A. Handa provided the concept, design, collection ofdata, analysis and interpretation and drafted the paper.Dr. Handa also provided critical revision, gave finalapproval and gave statistical expertise. T. Kashimuraand A. Yamamoto assisted in data collection, providedtechnical support, study materials and helped withanalysis of the data and gave final approval. I. Muro-hashi provided technical support, helped with the draft-ing of the paper and gave final approval. M. Bessboprovided technical support and gave final approval. K.Hirashima helped to collect and assemble the data,assisted with obtaining funding and gave final approval.

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