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THE JOURNAL OF BIOLOGICAL CHEMISTRY

© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 50, Issue of December 15, pp. 39702–39709, 2000 Printed in U.S.A.

Apoptosis Induced by Cadmium in Human Lymphoma U937 Cells through Ca 2 -calpain and Caspase-Mitochondria- dependent Pathways*

Received for publication, August 14, 2000 Published, JBC Papers in Press, September 1, 2000, DOI 10.1074/jbc.M007369200

Min Li‡§, Takashi Kondo , Qing-Li Zhao, Fu-Jun Li**, Kiyoshi Tanabe, Yoko Arai‡, Zong-Can Zhou§, and Minoru Kasuya‡

From the Department of Public Health, the Department of Radiological Sciences, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, 930-0194, Japan, the §Department of Toxicology, School of Public Health, Beijing Medical University, Beijing, 100083, China, and the **Department of Industrial Hygiene and Occupational Disease, School of Public Health, China Medical University, Shenyang, 110001, China

Apoptosis induced by cadmium has been shown in many tissues in vivo and in cultured cells in vitro. How- ever, its molecular mechanism is not fully understood. When the human histiocytic lymphoma cell line U937 was treated with cadmium for 12 h, evidence of apo- ptotic features, including change in nuclear morphol- ogy, DNA fragmentation, formation of DNA ladder in agarose gel electrophoresis, and phosphatidylserine ex- ternalization, were obtained. Moreover, loss of the mito- chondrial membrane potential ( m ) was observed in the cadmium-treated cells and was inhibited by a broad caspase inhibitor (Z-VAD-FMK). Caspase inhibitors sup- pressed the DNA fragmentation in the order of Z-VAD- FMK > caspase-8 inhibitor > caspase-3 inhibitor. Ex- pression of Bcl-x L and Bid decreased significantly in the cadmium-treated cells, although no apparent change in Bcl-2 and Bax expression was found. Tetrakis-(2-pyri- dylmethyl) ethylendiamine, a cell-permeable heavy metal chelator, partially reversed the increase of fluo- rescence of Fura-2 in the cadmium-treated cells. In ad- dition, verapamil (70 M), a voltage-dependent Ca 2 channel blocker, inhibited the DNA fragmentation in- duced by cadmium less than 100 M and decreased the fluorescence of Fura-2. Cadmium up-regulated the ex- pression of type 1 inositol 1,4,5-trisphosphate receptor (IP 3 R) but not type 2 or type 3 IP 3 R. Calpain inhibitors I and II partially prevented DNA fragmentation. No ef- fects of Z-VAD-FMK on the expression of type 1 IP 3 R or of calpain inhibitors on the loss of m were observed. These results suggest that cadmium possibly induced apoptosis in U937 cells through two independent path- ways, the Ca 2 -calpain-dependent pathway and the caspase-mitochondria-dependent pathway.

Cadmium, a potent toxic metal, is very harmful to the envi- ronment and to human beings because of its long lifetime. The toxicity of cadmium as an industrial pollutant and a food con- taminant, and as one of the major components in cigarette smoke is well established (1). Cadmium can cause a number of lesions in many organs, such as the kidney, the testis, the lung, the liver, the brain, the bone, the blood system, etc. (2). How-

* This work was supported by the Nishiyama Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed. Tel.: 81-76-434-7265; Fax: 81-76-434-5190; E-mail: kondot@ms.toyama-mpu.ac.jp.

ever, the mechanism of toxicity of cadmium is not yet clear. Recently, several reports have shown that cadmium can induce apoptosis of many tissues and cells both in vivo and in vitro, such as the cells of the respiratory system (3), the testis (4–6), the kidney (7, 8), the liver (9), and the immune system (10, 11), etc. This evidence indicates that apoptosis probably plays a very important role in acute and chronic intoxication with cadmium. Further research on this aspect of cadmium would have significance in the prevention and cure of the diseases induced by cadmium. Apoptosis is a fundamental form of cell death, and it plays an essential role in the development and homeostasis of multicel- lular organisms. Apoptosis disorders are associated with many diseases, such as cancer, autoimmune disorders, neurodegen- erative disease, toxin-induced disease, etc. (12). In the apo- ptotic process, caspases, a family of asparate proteases, lie in a pivotal position (13). Two independent pathways of apical caspases activation, receptor-intermediated caspase 8 and mi- tochondria-cytochrome c intermediated caspase 9 activation, converge on the activation of executing caspases, key substrate cleavage, and apoptotic death (14). Intracellular Ca 2 homeostasis is very important in main- taining the normal function of the cell. Increases and decreases

in calcium ion are possible causes of apoptosis (15, 16). The

radius of Cd 2 , a common form of free cadmium in the body, is

very similar to that of Ca 2 (0.099 and 0.097 nm, respectively).

Cd 2 not only competitively inhibits the influx of Ca 2 (17) but

also causes the increase of [Ca 2 ] i through inhibiting Ca 2 -

ATPase on the membrane of the depot of calcium (18). In addition, cadmium can activate or inhibit some calcium-related

enzymes instead of Ca 2 (19). Thus, investigation on the role of interactions of calcium ions in cadmium-induced apoptosis is important for understanding the mechanism of toxicity of cadmium. In this study, the molecular mechanism of apoptosis induced

by cadmium in human histiocytic lymphoma U937 cells, which

possess the Ca 2 /Mg 2 -dependent endonuclease (20), was in- vestigated. We herein show that two independent pathways,

the Ca 2 -calpain pathway and the caspase-mitochondria path- way, are probably involved in the regulation of cadmium-in- duced apoptosis.

MATERIALS AND METHODS

Reagent—Cadmium chloride, verapamil, 3,3 -dihexyloxacarbocya- nine iodide (DiOC 6 (3)), 1 dichlorodihydrofluorescein diacetate, and dihy-

1 The abbreviations used are: DiOC 6 (3), 3,3 -dihexyloxacarbocyanine iodide; TPEN, tetrakis- (2-pyridylmethyl) ethylendiamine; Annexin

39702 This paper is available on line at http://www.jbc.org

Cadmium-induced Apoptosis in U937 Cells

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droethidium were purchased from Wako Pure Chemical industries Ltd. (Tokyo, Japan). An annexin V/FITC (fluorescein isothiocyanate-labeled annexin V) kit was obtained from Immunotech, France. Fura-2/AM was obtained from Dojindo Laboratory (Kumamoto, Japan). Cell-Tak was purchased from Collaborative Research (Bedford, MA). TPEN were purchased from Sigma-Aldrich. A set of caspase inhibitors and calpain inhibitor I and II were purchased from Calbiochem-Novabiochem Corp.

Bcl-2, Bcl-x L , Bax, Bid, Bad, IP 3 Rs 1, 2, and 3, and -actin monoclonal or polyclonal antibodies were purchased from Santa Cruz Biotechnol-

ogy, Inc.

Cell Culture and Treatment—U937 cells obtained from the Japanese Cancer Resource Bank were grown in RPMI 1640 culture medium containing 10% heat-inactivated fetal bovine serum. Apoptosis in U937 cells was induced with cadmium chloride. Some cells were pretreated

with caspase inhibitors (100 M) or calpain inhibitor I and II (20 and 50 M) for 2 h prior to exposure to cadmium. Assessment of Apoptosis—Quantitative DNA fragmentation assay

was carried out according to the method of Sellins and Cohen (21). The

formation of DNA ladder was examined in 1.2% agarose gel electro- phoresis (22). Cell morphology was examined by Giemsa staining. Phos- phatidylserine (PS) externalization of apoptosis was determined by

two-color analysis of Annexin V/FITC binding and propidium iodide (PI) uptake using flow cytometry (EPICS XL TM Coulter, Beckman) accord-

ing to the instructions of the manufacturer.

Change in Ca 2 /Cd 2 Intracellular Concentration—After treatment for 6 h, cells were collected by centrifugation and washed with Ca 2 -free HEPES-buffered Ringer solution (HR; pH 7.4, 118 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl 2 , 1.0 mM Na 2 HPO 4 , 5.5 mM glucose, and 10 mM HEPES). The buffer was supplemented with 0.2% bovine serum albu-

min Fraction V, 2% minimal Eagle’s essential amino acids, and 2 mM

L-glutamine. About 3 10 5 cells in 1 ml of HR were loaded with Fura-2/AM for 30 min at 37 °C. Then an aliquot of cell suspension (10 l) was transferred to a glass bottom dish coated with Cell-Tak and left

for 10 min. After addition of HR to the chamber, digital imaging by

Fura-2 fluorescence (ratio of 340 nm/380 nm at 510 nm) was carried out with an inverted microscope (TE300; Nikon) and a digital image proc- essor (Argus 50 CA; Hamamatsu Photonics, Japan) (23). Mitochondrial Trans-membrane Potential ( m ) Assay—After treat-

ment with cadmium, cells were stained with 40 nM DiOC 6 (3) in 1 ml of phosphate-buffered saline and 1% fetal bovine serum for 15 min at 37 °C, then washed twice with phosphate-buffered saline, and har- vested for flow cytometry (24). Western Blot Analyses of Proteins—Control and Cd 2 -treated cells were collected and washed with cold phosphate-buffered saline. Cells

were lysed at a density of 10 6 cells/50 l of lysis buffer (1 M Tris-HCl, 5 M NaCl, 1% Nonidet P-40 (v/v), 1% sodium deoxycholate, 0.05% SDS, 1

mM

phenylmethylsulfonyl fluoride) for 20 min. After brief sonication,

the

lysates were centrifuged at 12,000 rpm for 10 min at 4 °C, and

protein content in the supernatant was measured using a Bio-Rad protein system. Western blot analyses of Bcl-2, Bcl-x L , Bax, Bid, Bad, IP 3 Rs 1, 2, and 3, and -actin were performed using specific polyclonal or monoclonal antibodies (see above), as described previously (25).

RESULTS

Induction of Apoptosis by Cadmium—The results of exami- nations of DNA fragmentation, DNA ladder formation, obser- vation of cell morphology, and PS externalization consistently revealed that cadmium induced apoptosis in U937 cells. Non- random DNA fragmentation has been regarded as one of the typical end points of apoptosis (26). Fig. 1A illustrates that

V/FITC, fluorescein isothiocyanate-labeled annexin V; PS, phosphati- dylserine; PI, propidium iodide; HR, HEPES-buffered Ringer solution; IP 3 R, inositol 1,4,5-trisphosphate receptor; calpain inhibitor I, N-acetyl- Leu-Leu-norleucine-aldehyde; Calpain inhibitor II, N-acetyl-Leu-Leu- normethionine-aldehyde; Boc-D-FMK, Boc-Asp(OMe)-fluoromethyl- ketone; Z-VAD-FMK, benzyloxycarbonyl-Val- Ala-Asp(OMe)-fluoro- methylketone; Z-YVAD-FMK, benzyloxycarbonyl-Tyr-Val-Ala-Asp (OMe)-fluoromethylketone; Z-VDVAD-FMK, benzyloxycarbonyl-Val- Asp(OMe)-Val-Ala-Asp(OMe)-fluoromethylketone; Z-DEVD-FMK, ben- zyloxycarbonyl-Asp(OCH 3 )-Glu(OCH 3 )-Val-Asp(OCH 3 )-fluoromethyl- ketone;Z-WEHD-FMK, benzyloxycarbonyl-Trp-Glu(OMe)-His-As- p(OMe)-fluoromethylketone; Z-VEID-FMK, benzyloxycarbonyl-Val- Glu(OMe)-lle-Asp(OMe)-fluoromethylketone; Z-IETD-FMK, benzyloxy- carbonyl-lle-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone; Z-LEHD- FMK, benzyloxycarbonyl-Leu-Glu(OMe)-His-Asp(OMe)-fluoro- methylketone; AM, acetoxy methyl.

methylketone; AM, acetoxy methyl. F IG . 1. Cadmium-induced DNA fragmentation in U937 cells.

FIG. 1. Cadmium-induced DNA fragmentation in U937 cells. A, U937 cells were treated with cadmium at final concentrations indicated for 3, 6, 12, and 24 h. Then DNA fragmentation assay was carried out according to the method of Sellins and Cohen (21). Data are presented as the means S.D. (n 4). B, U937 cells were treated with 100 M cadmium for 12 h, and DNA was extracted from control and cadmium- treated cells. Then the formation of DNA ladder was examined in 1.2% agarose gel electrophoresis.

DNA fragmentation induced by cadmium was concentration- dependent and time-dependent; DNA fragmentation increased with increasing concentrations of cadmium up to 100 M after 6, 12, and 24 h of exposure to cadmium but decreased beyond 100 M. However, no DNA fragmentation increased after 3 h of treatment. Furthermore, DNA fragmentation culminated 12 h after treatment with cadmium. Thus, 100 M cadmium and 12 h of treatment were often used in this study. After cadmium treatment for 12 h, observation of DNA ladder formation and morphological changes supported the evidence of DNA frag- mentation (Figs. 1B and 2, C and D). The observation of cell morphology illustrated that cadmium-treated cells underwent prominent cytoplasmic aggregation, nucleatic condensation, and fragmentation, which were typical signs of apoptotic fea- tures. Morphological change (including PI staining) showed

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FIG. 2. Phosphatidylserine exter- nalization and morphological change induced by cadmium in U937 cells. A and B, U937 cells were treated with cad- mium at final concentrations indicated for 3, 6, 12, and 24 h. Then the percent- ages of early apoptotic (A) and secondary necrotic (B) cells were determined by flow cytometry with Annexin V/FITC and PI double staining. These data represent three experiments. C and D, after expo- sure to 0 (C) or 100 M (D) cadmium for 12 h, cells were collected for Giemsa staining and then examined under a mi- croscope at a magnification of 10 100.

Cadmium-induced Apoptosis in U937 Cells

of 10 100. Cadmium-induced Apoptosis in U937 Cells F IG . 3. Effects of verapamil on
of 10 100. Cadmium-induced Apoptosis in U937 Cells F IG . 3. Effects of verapamil on

FIG. 3. Effects of verapamil on the increase of fluorescence of Fura-2 in U937 cells treated with cadmium and cadmium-induced DNA fragmenta- tion. A, after treatment with 100 M cad- mium for 6 h with or without verapamil (70 M), control and treated cells were loaded with Fura-2/AM for 30 min, and the fluorescent image of a single cell was obtained as described under “Materials and Methods.” Then 100 cells were ran- domly selected, and the fluorescence ra- tios of 340 and 380 nm (F340/380) of a single cell were analyzed. The histogram shows the relationships between the number of cells and the ratio of F340/380. B, U937 cells were treated with cadmium at indicated concentrations for 6 h with or without verapamil (70 M) and analyzed as shown in A. To simplify the analysis results, the histogram was changed to a lineal graph at the point where the F340/ 380 ratio was equal to 1.5. C, when U937 cells were treated with cadmium at indi- cated concentrations for 12 h in the pres- ence of verapamil (70 M), DNA fragmen- tation was measured. The bars in the figure indicate the means S.D. (n 3).

that at concentrations of more than 100 M, the number of necrotic cells apparently increased (data not shown). PS externalization is an early symbol of apoptosis (27). Flow cytometry using Annexin V/FITC and PI double staining re- vealed that after exposure to cadmium, cells with externalized PS significantly increased depending on cadmium concentra- tion and exposure time. At 6 h, early apoptotic cells (only

Annexin V/FITC staining) were predominantly observed; as time progressed or as the concentration increased, secondary necrotic cells in a later stage of apoptosis (double Annexin/ FITC V and PI staining) were in the main position. These results indicate that at later stages of apoptosis, the plasma membrane was damaged by cadmium (Fig. 2, A and B). Elevation of Ca 2 /Cd 2 Concentration in Cells—To eluci-

Cadmium-induced Apoptosis in U937 Cells

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date the effect of the interaction between Ca 2 and Cd 2 in apoptosis induced by cadmium, we used Fura-2, which is de- rived from Fura-2/AM through esterase in cells, to determine the change in ion concentration of the cells. Fluorescence in cells treated for 6 h with cadmium became much stronger than that in control cells, and this change showed a dose-effect

relationship. Unfortunately, the excitation response of Fura-2

to Cd 2 is almost an exact match to that of Fura-2 to Ca 2 .

Therefore, it is difficult to distinguish Ca 2 from Cd 2 (28).

Verapamil, a voltage-dependent Ca 2 channel inhibitor (10), reduced fluorescence of Fura-2 in cells; moreover, 70 M vera-

pamil delayed DNA fragmentation induced by cadmium. These results indicated that cadmium probably entered into U937 cell via voltage-dependent calcium channels, and verapamil inhib- ited this passage, resulting in delayed DNA fragmentation (Fig. 3). After combined treatment of cadmium and verapamil, the increased fluorescence (cells indicating a ratio higher than 1.6) appeared to indicate intracellular Ca 2 release induced by cadmium, because verapamil blocks both Ca 2 and Cd 2 entry into the cells. To further determine the contribution of [Ca 2 ] i to the in- crease in ion concentration, we used a cell-permeable, specific

K a 10 4.47 ),

Cd 2 chelator (TPEN, Cd 2 K a 10 16.33 ; Ca 2

and fluorescence in cells significantly decreased, but it did not

completely reverse the fluorescence of Fura-2 in cells, even though the concentration of TPEN increased to 100 M. These results suggest that in addition to augmentation of [Cd 2 ] i , [Ca 2 ] i was probably elevated (Fig. 4A). IP 3 R Expression and Effects of Calpain Inhibitor—Because cadmium competitively inhibits Ca 2 influx (18), the mecha-

nism of [Ca 2 ] i increase in cells exposed to cadmium has not been elucidated. In some apoptotic systems, elevation of [Ca 2 ] i was mediated through IP 3 Rs (25, 29). In our study, therefore, the expression of IP 3 R subtypes was investigated at 12 h after cadmium treatment (Fig. 4B). The expression of IP 3 R1 was apparently enhanced in a dose-dependent manner, but IP 3 R2 and IP 3 R3 were not detected. These results demonstrated that, probably via the IP 3 R1 pathway, cadmium induced the release

of

calcium into the cytoplasm from its intracellular depot, such

as

endoplasmic reticulum. In addition, caspase inhibitor (Z-

VAD-FMK) did not affect the expression of IP 3 R1. Calcium ion can act on multiple targets to trigger apoptosis (14). Recently, calpain, calcium-dependent protease has been considered as a possible target through which elevated calcium triggers apoptosis (30). Calpain inhibitor I significantly re- duced DNA fragmentation by 40% at low concentrations, and there was little difference between 20 and 50 M. However, calpain inhibitor II was not effective until its concentration

reached 50 M. The results, therefore, indicate that calpain may take part in the Ca 2 -dependent pathway of apoptosis induced by cadmium (see Fig. 6A). Loss of Mitochondrial Membrane Potential Induced by Cad- mium—In many systems, apoptosis is associated with the loss

of

mitochondrial inner membrane potential ( m ), which may

be

regarded as a limiting factor in the apoptotic pathway (31).

To observe the change in m in cells exposed to cadmium, DiOC 6 (3), a mitochondria-specific and voltage-dependent dye, was employed. After U937 cells were exposed to cadmium for 12 h, m was significantly reduced in a dose-dependent man- ner (Fig. 5C). However, at 3 and 6 h, the loss of m was indiscernible. Although it has been reported that cadmium was able to cause the loss of m via oxidant injury (32, 33), no significant increase in intracellular oxidants, such as hydrogen peroxide or superoxide, in the cadmium-treated cells was ob- served by flow cytometry with dichlorodihydrofluorescein diac- etate and dihydroethidium in our study (data not shown).

etate and dihydroethidium in our study (data not shown). F IG . 4. The effect of

FIG. 4. The effect of TPEN on the increase in fluorescence of cells induced by cadmium and the up-regulation of IP 3 R1 ex- pression caused by cadmium. After treatment with 100 M cadmium for 6 h, cells were loaded with Fura-2/AM for 30 min, and fluorescent images were obtained as described under “Materials and Methods.” Then TPEN (50 or 100 M) was added into the same sample, and fluorescence images were taken again. Then 100 cells were randomly selected, and the fluorescence ratios of 340 and 380 nm (F340/380) of a single cell were analyzed. The histogram shows the relationships be- tween the number of cells and the ratio of F340/380. B, U937 cells were treated with cadmium at indicated concentrations for 12 h, and protein extracts were prepared. When caspase inhibitors were used, cells were preincubated with 100 M Z-VAD-FMK for 2 h and then treated with 100 M cadmium. Protein extracts were separated by SDS-PAGE and immunoblotted as described under “Materials and Methods.” -Actin was used as a control for loading of protein.

Inhibition of Caspase Inhibitors in Cadmium-induced Apo- ptosis—Because of the loss of mitochondrial membrane poten- tial in the cadmium-treated cells, it was speculated that caspases may play an essential role in the process of cadmium- triggered apoptosis. First, Z-VAD-FMK (caspase inhibitor I) and Boc-D-FMK (caspase inhibitor III), two kinds of broad spectrum, irreversible caspase inhibitors, were used. Z-VAD- FMK almost completely inhibited cadmium-induced DNA frag- mentation (Fig. 6B); Boc-D-FMK also showed a similar result (data not shown). Moreover, Z-VAD-FMK also inhibited the loss of m . These results suggest that cadmium probably triggers apoptosis of U937 cells via a caspase-dependent pathway. To determine which kinds of caspases take effect in apoptosis induced by cadmium, a series of inhibitors, caspase-1 inhibitor VI (Z-YVAD-FMK), caspase-2 inhibitor I (Z-VDVAD-FMK), caspase-3 inhibitor II (Z-DEVD-FMK), caspase-5 inhibitor

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FIG. 5. Loss of m induced by cad- mium. U937 cells were treated with 100 M cadmium for 12 h, cells were stained with 40 nM DiOC 6 (3) as described under “Materials and Methods,” and then m was measured by flow cytometry. When caspase inhibitor (Z-VAD-FMK (100 M)) and calpain inhibitors (calpain inhibitors I (20 M) and II (50 M)) were used, cells were preincubated for 2 h and then treated with 100 M cadmium. A, control. B, cadmium (100 M). D, cadmium (100 M) calpain inhibitors I (20 M). E, cad- mium (100 M) calpain inhibitors II (50 M). F, cadmium (100 M) Z-VAD-FMK (100 M). C, after treatment with cad- mium at indicated concentrations for 12 h, the percentage of cells with low m was measured, as shown in Fig. 5A. The results were presented as the means S.D. (n 5).

Cadmium-induced Apoptosis in U937 Cells

S.D. ( n 5). Cadmium-induced Apoptosis in U937 Cells I (Z-WEHD-FMK), caspase-6 inhibitor I (Z-VEID-FMK),

I (Z-WEHD-FMK), caspase-6 inhibitor I (Z-VEID-FMK), caspase-8 inhibitor II (Z-IETD-FMK), and caspase-9 inhibitor I (Z-LEHD-FMK), were employed for preventing DNA fragmen- tation in apoptosis. The results revealed that both caspase-8 inhibitor and caspase-3 inhibitor were able to prevent cadmi- um-induced DNA fragmentation. Moreover, caspase-8 inhibitor was more effective than caspase-3 inhibitor in this respect (Fig. 6). Expression of Apoptosis-related Protein—Cleavage of Bid is important for the release of cytochrome c from mitochondria in CD95-induced apoptosis (34). Bid was present as a 26-kDa protein in U937 cells (Fig. 7A). The proform of Bid slowly decreased depending on the cadmium concentration. At the same time, caspase inhibitor (Z-VAD-FMK) and caspase-8 in-

hibitor (Z-IETD-FMK) both inhibited the cleavage of Bid in- duced by 100 M cadmium. A member of the Bcl-2 family, Bcl-x L , significantly decreased in a concentration-dependent manner, and at the same time, Bcl-x S , which is the cleaving product of Bcl-x L, slowly appeared as Bcl-x L was reduced . It has been reported that Bcl-x L and Bcl-x S have completely contrary effects; the former inhibits but the latter promotes apoptosis (35) (Fig. 7B). This evidence is consistent with our results. Caspase inhibitor (Z-VAD-FMK) and caspase-8 inhibitor (Z-IETD-FMK) did not affect the ex- pression of Bcl-x L . In contrast, no change in the expression of Bcl-2/Bax proteins was apparent in cadmium-induced apopto- sis in U937 cells (Fig. 7C), although Bcl-2/Bax has been re- garded as the regulator of the release of cytochrome c in apo-

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Cadmium-induced Apoptosis in U937 Cells 39707 F IG . 6. Effects of caspase inhibitors and calpain

FIG. 6. Effects of caspase inhibitors and calpain inhibitors on DNA fragmentation induced by cadmium. A, after preincubation with various caspase inhibitors (100 M), U937 cells were treated with 100 M cadmium. Then DNA fragmentation assay was carried out according to the method of Sellins and Cohen (21). The data are pre- sented as the means S.D. (n 3). B, U937 cells were preincubated with calpain inhibitor I (20 and 50 M) and II (20 and 50 M) and then treated with 100 M cadmium. Measurement of DNA fragmentation is shown in A.

ptosis (36). Bad expression was not detectable in U937 cells treated with cadmium, although Bcl-x L can counteract the effect of Bax (37) and be sequestered by Bad in cytosol (38).

DISCUSSION

The present study shows that cadmium is able to induce apoptosis in U937 cells, and in this process, there may be two different and independent pathways for inducing apoptosis by cadmium. One is the Ca 2 -calpain-dependent pathway, and the other is the caspase-dependent pathway. The Ca 2 -Calpain-dependent Pathway—In recent years, the effect of IP 3 R in apoptosis has been the focus of attention. The elevation of and selective expression of IP 3 R subtype involved in apoptosis are due to many stimuli, such as anti-immuno- globulin M, dexamethasone (29), irradiation (25), B-cell anti- gen receptor (39), etc. On the other hand, cells with failure in Ca 2 elevation owing to deficient IP 3 R1 or IP 3 R2 are resistant

to apoptosis by dexamethasone, irradiation, or Fas ligand in

U937 cells (40). Our study reveals that cadmium is able to up-regulate the expression of IP 3 R1 but not IP 3 R2 or IP 3 R3 in

U937 cells. It has been reported that a majority of IP 3 R1 was distributed on the surface of endoplasmic reticulum (41). Hence, because of competitive inhibition of Ca 2 influx via the

calcium channel and the expression of IP 3 R1 in U937 cells, the elevation of calcium ion caused by cadmium possibly derives from endoplasmic reticulum. Verapamil delayed cadmium-in- duced DNA fragmentation, probably because it inhibited the

Cd 2 influx via the Ca 2 channel so that the IP 3 R1 up-regula-

tion was extended. Of course, the possibility should not be excluded that cadmium in cells may directly mobilize Ca 2 or

enhance Ca 2 mobilization, because cadmium can inhibit all types of Ca 2 -ATPase (19, 42, 43). Calpain is a family of Ca 2 -dependent cystein proteases whose members are expressed ubiquitously (44). Calpain has

been reported to be involved in several models of apoptosis and

to take effect as a target of Ca 2 -dependent activation (45, 46).

Presumably, calpain plays an essential role in apoptosis be- cause of cadmium, because calpain inhibitors inhibited the DNA fragmentation induced by cadmium in our study. Fur-

thermore, calpain inhibitor I was more potent than calpain inhibitor II. In addition, in addition to calpain, calcium ions are able to activate other targets to trigger apoptosis, such as

Ca 2 /Mg 2 -dependent endonuclease, whose involvement has

been shown in UV-induced apoptosis in U937 cells. On the other hand, the other effects of cadmium in cells should be considered. Cd 2 not only causes Ca 2 elevation but also activates some calcium-related enzymes, for example, pro- tein kinase C (47), mitogen-activated protein kinase (48), cal- modulin-dependent kinase (49), etc. Hence, further research is necessary to determine whether cadmium can directly activate apoptotic proteases (calpain, calcium-dependent endonuclease, etc.) instead of calcium. The Caspase-dependent Pathway—The caspase family is di-

vided into two groups: one group, derived from procaspase with long predomains (caspase-2, -8, -9, and -10), is called “initiator”

or “upstream” caspases, and the other, which is derived from

precursor with short predomains is called “effector” or “down- stream” caspases (caspase-3, -6, -7, and -14) (50). In our study, Z-VAD-FMK (broad spectrum inhibitor) inhibited DNA frag- mentation and PS externalization induced by cadmium, sug- gesting that caspase may be involved in the process of cadmi- um-triggered apoptosis. Furthermore, caspase-8 and -3 inhibitors also inhibit cadmium-induced DNA fragmentation, but caspase-2, -6, -7, and -9 inhibitors do not. Therefore, we hypothesize that probably caspase-8 is the most apical caspase

in cadmium-induced apoptosis, and finally signal converges to

caspase-3. According to previous reports, caspase-8 not only directly cleaves and activates caspase-3 (51) but also indirectly activates caspase-3 by inducing cytochrome c release (52). Ac- cording to observations, the loss of m because of cadmium seems to support this evidence. Bid, a proapoptotic Bcl-2 family member containing BH 3 domain, can be cleaved by caspase-8, and the cleaved Bid, the carboxyl-terminal fragment, translocates to mitochondria to induce the release of cytochrome c, which is 500 times more numerous than Bax (53). In our study, the decrease in the proform of Bid means that it is cleaved in cadmium-induced apoptosis, and Z-VAD-FMK and caspase-8 inhibitor block this cleavage. This suggests that the cleavage of Bid is

caspase-8-dependent.

Besides Bid, other Bcl-2 family members are associated with the release of cytochrome c. For example, Bax promotes cyto-

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Cadmium-induced Apoptosis in U937 Cells

39708 Cadmium-induced Apoptosis in U937 Cells F IG . 7. The expression of several members of

FIG. 7. The expression of several members of Bcl-2 family proteins. After treatment with cadmium at the indicated concentrations for 12 h, protein extracts were prepared. When caspase inhibitors were used, cells were preincubated with Z-VAD-FMK (100 M) and caspase-8 inhibitor (100 M) for 2 h and then treated with 100 M cadmium. The protein extracts were separated by SDS-PAGE and immunoblotted as described under “Materials and Methods.” -Actin was used as a control for loading of protein. A, Bid. B, Bcl-x L and Bcl-x S . C, Bcl-2 and Bax.

Bid. B , Bcl-x L and Bcl-x S . C , Bcl-2 and Bax. F IG

FIG. 8. Scheme of the cell signaling pathway mediating the Cd 2 -induced apoptosis. Cd 2 enters into U937 cells through the voltage-dependent Ca 2 channel and up-regulates IP 3 R1 expression, and then Ca 2 release from endoplasmic reticulum (ER) is induced. Ca 2 activates calpain and induces DNA fragmentation and apoptosis. On the other hand, cadmium can possibly activate caspase-8 to induce apoptosis. Caspase-8 may directly activate the effector caspases (caspase-3 and -7) responsible for many of the biochemical and morpho- logical changes in apoptosis. Alternatively, cadmium can cause loss of the m , cleavage of Bid and Bcl-x L , and increase of Bcl-x S . These factors are probably responsible for the release of cytochrome c and activation of caspase-9, with subsequent activation of effector caspases to induce apoptosis. Therefore, two independent pathways, the Ca 2 - calpain pathway and the caspase-mitochondria pathway, may be in- volved in the regulation of cadmium-induced apoptosis.

chrome c release, but Bcl-2 and Bcl-x L counteract the effect of Bax and inhibit the release of cytochrome c (37, 54). Moreover, Bcl-x L can itself bind to cytochrome c and Apaf-1 to prevent apoptosis (55, 56). In cadmium-induced apoptosis, the change in Bcl-2 and Bax is not apparent, but the level of Bcl-x L appar- ently decreases in a concentration-dependent manner, and Bcl- x S , which is the product of Bcl-x L cleavage and which promotes apoptosis (36), increases along with the decrease in Bcl-x L . Bad, another Bcl-2 family members that sequesters Bcl-x L , was not detected in U937 cells. Taken together, these results indi- cate that Bid and Bcl-x L are possibly involved in the caspase- dependent pathway of cadmium-induced apoptosis, resulting in cytochrome c release and enhancement of the apo- ptotic process. Although no reports have been published showing the acti- vation of caspase-8 induced by cadmium, it is speculated that cadmium may activate caspase-8 by elevating the expression of Fas ligand. This is not only because caspase-8 is a key compo- nent of the Fas/APO 1 death receptor-triggered apoptosis path- way (53), but also because many other apoptotic pathways initiated by distinct stimuli require Fas engagement. For in- stance, cell apoptosis triggered by anticancer drugs (57–59), irradiation (60) and ceramide (61) is mediated by up-regulation of Fas L and its interaction with Fas. Of course, we cannot exclude the possibility that cadmium activates caspase-8 via a pathway independent of Fas L, such as activated Lck (62). Further study is necessary to elucidate the mechanism. In summary, possibly via two different pathways, cadmium induces apoptosis in U937 cells (Fig. 8). In addition, because caspase degrades calpastatin, which is an endogenous inhibitor of calpain (63), and promotes the Ca 2 -calpain pathway and because the Bcl-2 family and Ca 2 act on each other (64), they may complement each other.

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