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CELLULAR AND MOLECULAR
Departments of Environmental Medicine (G.M., M.J.M.) and Radiation Oncology (P.C.K.), School of Medicine and Dentistry, University of Rochester, Rochester, New York; and Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky (J.C.S.)
Received November 12, 2004; accepted February 17, 2005.
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). Treatment with arsenic trioxide, a source of trivalent, inorganic arsenic, otherwise known as arsenite, produces complete remission in more than 50% of APL patients (Miller et al., 2002). Chemotherapeutic blood plasma levels of arsenic trioxide corresponding to 1 to 4 µM arsenite lead to apoptosis of leukemic cells, whereas lower levels (0.2–1 µM arsenite) lead to differentiation (Chen et al., 2001). The large body of research directed at understanding the mechanism through which arsenic acts to eliminate APL cells is summarized by Miller et al. (2002). Various mechanisms have been proposed, but no unified agreement has been reached. Some examples of proposed mechanisms include: degradation of the PML-RAR
fusion oncoprotein characteristic of APL cells, influence on signaling pathways (such as activation of mitogen-activated protein kinase), and induction of oxidative stress (Huang et al., 1999; Jing et al., 1999; Cai et al., 2000; Zhu et al., 2001). Arsenite reacts with biomolecules through binding to vicinal thiol groups, resulting in disruption of protein activity (Reglinski, 1998; Miller et al., 2002). It has been shown to inhibit enzymes such as succinic dehydrogenase and acetylcholine esterase (Reglinski, 1998; Chou et al., 2000). These targets are affected by arsenite at very high concentrations and may be more relevant to overt arsenite toxicity than to chemotherapeutic effects. The specific proteins targeted by more clinically relevant concentrations of arsenite are unknown.
To date, most study of the chemotherapeutic mechanism of arsenite has concentrated on experiments with the NB4 cell line (Wang et al., 1998; Jing et al., 1999; Cai et al., 2000). NB4 cells were derived from an APL patient, and they contain the PML-RAR fusion oncoprotein (de The and Chelbi-Alix, 2001). The U937 cell line is another model system for studying the effects of arsenite (McCabe et al., 2000). U937 was derived from a non-Hodgkin's lymphoma associated with a block in differentiation of monocyte progenitors (Sundstrom and Nilsson, 1976). U937 does not contain the PML-RAR fusion protein, yet arsenite inhibits its growth and enhances its differentiation (McCabe et al., 2000). Therefore, U937 is a good model system for the study of arsenite's effects on myeloid leukemia cells outside of its effects on PML-RAR. Because U937 cells respond to arsenite similarly to APL cells, arsenite's mechanism of action in this context will shed light on its chemotherapeutic mechanism.
Sodium arsenite, another source of trivalent, inorganic arsenic, can cause a growth delay in U937 cells at concentrations below 10 µM (McCabe et al., 2000). However, the nature of this delay is unknown. Cell growth is dependent on the efficient passage of cells through all phases of the cell cycle (Murray and Hunt, 1993). During times of stress, it is commonly held that cells may pause in their cell cycle progression at one of two major checkpoints. One checkpoint lies at the end of G1 as a cell prepares to enter S phase. The other can be triggered during G2, before a cell commits to mitosis. However, cell cycle progression can be altered outside of these two best studied checkpoints. In response to DNA damage, not only do cells arrest in G2 phase, but S phase transit is also slowed to allow for DNA repair to occur in parallel with its replication (Abraham, 2001). Agents that interfere with mitotic spindle function induce a metaphase arrest, preventing asymmetrical distribution of chromosomes (Howell et al., 2000). Many proteins are involved in the regulation of each of these cell cycle transitions. To narrow the field of possible molecular targets of arsenite, it is desirable to define the cell cycle phase during which cells are most sensitive. Here, we present evidence that supports previous findings that G2/M cells are most sensitive to arsenite-induced cell cycle arrest leading to apoptosis (Li and Broome, 1999; Park et al., 2001; Halicka et al., 2002; Ling et al., 2002; States et al., 2002; Cai et al., 2003). However, another dramatic effect of arsenite is an induction of nonphase-specific cell cycle prolongation (i.e., arsenite increases the length of time it takes a cell to transit each phase of the cell cycle). The prolongation of cell cycle transit time has the potential to contribute greatly to growth suppression by arsenite, because at any given time, the percentage of cells susceptible to G2/M arrest and apoptosis is small compared with the percentage of cells susceptible to G1 or S phase delay.
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-bromodeoxyuridine, fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse IgG1 specific monoclonal antibody, 1x cell lysis buffer, and Ac-DEVD-7-amino-4-methylcoumarin substrate were purchased from BD Biosciences PharMingen (San Diego, CA). Allophycocyanin (APC)-conjugated goat anti-mouse IgM antibody was purchased from Caltag Laboratories (Burlingame, CA). The TG-3 antibody (in hybridoma culture supernatant) was a generous gift from Dr. Peter Davies (Albert Einstein College of Medicine, New York, NY). HEPES solution (1 M) and 1x PBS were purchased from Invitrogen (Carlsbad, CA). Glycerol and dithiothreitol were purchased from J. T. Baker (Phillipsburg, NJ). Cell Culture. U937 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 supplemented with 5% fetal bovine serum (FBS), 1% L-glutamine, and 0.1% gentamicin. RPMI 1640, FBS, glutamine, and gentamicin were obtained from Invitrogen. Cells were maintained in logarithmic growth at a density between 0.1 and 1 x 106 cells/ml at 37°C in a humidified atmosphere consisting of 5% CO2. For most experiments, the seeding cell density was 2.0 x 105 cells/ml.
PI Exclusion Assay. U937 cells (2.7 x 106 cells/10 ml) were cultured for up to 72 h with or without arsenite. Cells were washed twice with 1x PBS, then 1 x 106 cells in 1 ml of PBS were stained with 1.67 µg of propidium iodide. PI fluorescence was determined by flow cytometry on a FACScalibur (BD Biosciences, San Jose, CA). Cells with intact membranes excluded PI and were counted as viable. The percentage of PI-excluding cells in each sample was normalized to the percentage of such cells in the control sample. A minimum of 10,000 cells/sample were analyzed. Data were collected and analyzed using CellQuest software.
Flow Cytometric Analysis of Cell Cycle Progression. U937 cells were enriched for cells in various cell cycle phases via centrifugal elutriation as described by Donaldson et al. (1997). Cells (2.0 x 106 cells/10 ml) were exposed to 0 to 5 µM arsenite and/or 0 to 50 µM Z-VAD-FMK for 0 to 24 h and pulse-labeled by incubation with 10 µM BrdU for 15 min at 37°C in a humidified atmosphere with 5% CO2. After being washed with 10 ml of 1x PBS, the cells were fixed with 70% ethanol overnight at 4°C. Fixed cells were rinsed with PBS and incubated with 0.2 mg/ml pepsin/2 N HCl/1x PBS for 20 min at 37°C. Following two washes with 1x PBS/0.5% BSA/0.5% Tween 20, cells were treated with 1x PBS/2% FBS for 20 min at room temperature. Cells were incubated with 0.5 µg of purified mouse -bromodeoxyuridine and 1 µl of TG-3 hybridoma culture supernatant/10 µl in the dark overnight at 4°C and washed in 1x PBS/0.5% BSA/0.5% Tween 20. Cells were incubated with 0.5 µg of FITC-conjugated rat anti-mouse IgG1 monoclonal antibody and 0.1 µg of APC-conjugated goat anti-mouse IgM antibody/10 µl in the dark for 2 h at 4°C and washed in 1x PBS/0.5% BSA/0.5% Tween 20. They were then incubated in 500 µl of PBS containing 10 µg/ml PI in the presence of RNase A (100 U/ml final) for at least 30 min at room temperature. PI fluorescence (i.e., DNA content), FITC fluorescence (BrdU incorporation), and APC fluorescence (TG-3 labeling) were determined by flow cytometry on a FACScalibur (BD Biosciences). A minimum of 20,000 cells/sample were analyzed. Data were collected and analyzed using CellQuest software.
Flow Cytometric Analysis of Cell Cycle Kinetics. U937 cells (2.0 x 106 cells/10 ml) were pulse labeled by incubation with 10 µM BrdU for 15 min at 37°C in a humidified atmosphere with 5% CO2. They were then washed once with 1x PBS and cultured for 4 h with or without arsenite. After being washed with 10 ml of PBS, the cells were fixed with 70% ethanol overnight at 4°C. Fixed cells were labeled with anti-BrdU and TG-3 antibody, stained with PI, and analyzed as described above.
Caspase Activity Assay. Elutriated U937 cells were exposed to 0to5 µM arsenite and/or 0 to 50 µM Z-VAD-FMK for 8 or 12 h. Cells were harvested and washed in ice-cold PBS. Cell pellets were snapfrozen in dry ice bath to preserve protein integrity and stored at –80°C. Pellets were lysed with 1x cell lysis buffer (10 mM Tris-HCl/10 mM NaH2PO4/NaHPO4, pH 7.5/130 mM NaCl/1% Triton X-100/10 mM sodium pyrophosphate). Cell lysate was added to 1x HEPES buffer (20 mM HEPES, pH 7.5, 10% glycerol, and 2 mM dithiothreitol) containing Ac-DEVD-7-amino-4-methylcoumarin substrate. The fluorescence emission yielded by 7-amino-4-methylcoumarin release was monitored every 45 s for 1 h at a temperature of 37°C in a SPECTRAmax GEMINI XS Dual-Scanning Microplate Spectrofluorometer from Molecular Devices (Sunnyvale, CA). The protein concentration of each lysate was determined by bicinchoninic acid protein assay using a kit from Pierce Chemical (Rockford, IL). Caspase activity was expressed in terms of arbitrary fluorescence units released per mg protein per second.
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5 µM) inhibit cell growth but do not immediately induce cell death. Arsenic trioxide (As2O3), rather than sodium arsenite (NaAsO2), is the chemical form of the drug approved by the Food and Drug Administration for the treatment of APL. Both are sources of trivalent inorganic arsenic, differing mainly in their stoichiometry. The experiments shown in Fig. 1 were repeated using 0.5 to 5 µM arsenic trioxide to establish that the effect is similar to that seen with sodium arsenite. The results were virtually identical to those shown in Fig. 1 except that arsenic trioxide inhibits growth and reduces viability at lower concentrations (data not shown), a difference predicted by the fact that arsenic trioxide contains more arsenic per mole than does sodium arsenite. We chose to use sodium arsenite in the remaining experiments because it is far more soluble in aqueous solution than is arsenic trioxide, which must be solubilized in 1 M sodium hydroxide prior to use.
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Arsenite Induces Mitotic Arrest in U937 Myeloid Leukemia Cells. Centrifugal elutriation was used to obtain a cell fraction containing an enriched population of cells within the G2/M phase of the cell cycle. Following elutriation and collection of the enriched G2/M fraction, BrdU-pulsed cells were labeled with anti-BrdU and monoclonal TG-3 antibody and stained with PI to verify their cell cycle phase distribution. TG-3 antibody recognizes mitosis-specific phosphorylation of nucleolin, making TG-3 labeling a useful marker to discriminate mitotic cells from G2 phase cells (Anderson et al., 1998). As shown in Fig. 2, A and B, G2/M cells were highly enriched following elutriation (compare top and bottom right quadrants of Fig. 2, A and B), and TG-3 staining revealed that 
12% of the elutriated cells were mitotic. Cells from this elutriated fraction were treated for up to 8 h with sodium arsenite. Cell cycle analysis at the end of 8 h of treatment showed that the mitotic population (top right quadrant) decreased in untreated samples (Fig. 2C) but increased in the presence of arsenite (Fig. 2D). The accumulation of mitotic cells suggests that arsenite arrested cells in mitosis. This was confirmed by a BrdU pulse-chase experiment to track the progression of S phase cells through mitosis. Asynchronous cells were pulsed with BrdU prior to treatment with sodium arsenite for up to 12 h. Before and after treatment, cell cycle distribution was analyzed with anti-BrdU, TG-3 antibody, and PI (Fig. 3; data not shown). In a representative experiment, before treatment, 1.7% of the cells were shown to be mitotic by TG-3 labeling (Fig. 3A, leftmost panel, total of top and bottom right quadrants). As expected, all of the mitotic cells were BrdU-negative (bottom right quadrant). After 4 h, in untreated samples, most of the cells that were in G2/M at the beginning of the experiment have moved out of mitosis as shown by a lack of TG-3 labeling. Few cells remain in the BrdU-negative mitotic population (Fig. 3A, middle panel, bottom right quadrant). With arsenite treatment, the population of BrdU-negative mitotic cells remains constant over 4 h (Fig. 3A, compare bottom right quadrants of leftmost and rightmost panels), indicating that cells in mitosis upon arsenite treatment are arrested there. Additionally, cells are also slow to transit from S phase into mitosis, as evidenced by the decreased percentage of BrdU-positive mitotic cells in the arsenite-treated samples after 4 h (Fig. 3A, compare top right quadrants of middle and rightmost panels). In an asynchronous population, the percentage of cells in mitosis is small (2%), but we have observed consistent, statistically significant differences between untreated and arsenite-treated cells (Fig. 3B), suggesting that there is a real difference in the kinetics of cell cycle transit between these two populations.
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Arsenite Induces Apoptosis in G2/M Phase U937 Cells. Arsenite (5 µM) is known to induce apoptosis in U937 cells (McCabe et al., 2000). Many studies have been published in support of a G2/M phase-specific sensitivity to arsenite-induced apoptosis in this and other cell types (Li and Broome, 1999; Park et al., 2001; Halicka et al., 2002; Ling et al., 2002; States et al., 2002; Cai et al., 2003). Given the induction of mitotic arrest seen with arsenite treatment, we further investigated the cell cycle phase specificity of the apoptotic response. Centrifugal elutriation was used to obtain cell fractions enriched for cells with G1, S, or G2/M phase DNA content. Cell cycle phase distribution for each fraction was determined by analysis with BrdU and PI (Fig. 4; data not shown). Cells from these elutriated fractions were treated for 8 or 12 h with sodium arsenite. Treatment duration was chosen to maximize the detection of caspase activity because the relatively small number of cells obtained in each fraction of a centrifugal elutriation limits the number of samples that can be generated. Caspase activity was detected only in arsenite-treated samples with G2/M phase populations larger than those in the corresponding untreated samples (Fig. 4). An untreated population enriched for G2/M phase cells largely progressed into G1 phase over 8 h. When treated with 5 µM arsenite, the same population was delayed in G2/M phase and contained activated caspases (Fig. 4B). Caspase activation was also detected in arsenite-treated S phase-enriched cells that were delayed in G2/M following incubation for 12 h (Fig. 4C). On the other hand, arsenite treatment delays G1 phase-enriched cells in G1 phase, but caspase activation is largely absent (Fig. 4A). This suggests, in agreement with previous studies, that arsenite induces apoptosis specifically in delayed G2/M phase cell populations (Li and Broome, 1999; Park et al., 2001; Halicka et al., 2002; Ling et al., 2002; States et al., 2002; Cai et al., 2003). Experiments using the general caspase inhibitor, Z-VAD-FMK, further supported that G2/M phase is the specific cell cycle phase at which cells are vulnerable to apoptosis induction by arsenite. A cell fraction enriched for cells with S phase DNA content was obtained by centrifugal elutriation, and its cell cycle distribution was confirmed by analysis with BrdU and PI (Fig. 5A). Cells from this S phase-enriched fraction were treated for 16 h with sodium arsenite, with Z-VAD-FMK, or with both arsenite and the inhibitor. Once again, the treatment time was chosen to maximize the effect to most efficiently use the elutriated cells. Z-VAD-FMK treatment, alone, had no effect on cell cycle distribution. Over the course of 16 h, most of the cells in the control sample move from S phase to G1 or S phase of the next cell cycle (Fig. 5B). These results are not unexpected because cell cycle kinetics analyses have shown that untreated U937 cells complete S phase in approximately 10 to 12 h and G2/M phase in approximately 4 to 8 h (data not shown). In the experiment depicted in Fig. 5, many of the cells are already near the end of S phase at elutriation, allowing plenty of time for them to enter a subsequent S phase by 16 h. With arsenite treatment, however, 16.9% of cells are arrested in mitosis, and a population of BrdU-negative cells with S phase DNA content appears (Fig. 5, A and C). The addition of Z-VAD-FMK to the culture results in the complete inhibition of arsenite-induced caspase activity (Fig. 4). Also, more cells are arrested in mitosis in response to arsenite (24.5%), and fewer cells appear in the BrdU-negative population with S phase DNA content (Fig. 5, A and D). Because a portion of this population depends on caspase activity, it is likely that this portion represents G2/M phase cells in which caspase-dependent DNA degradation is occurring. It is appropriate to say that this population has sub-G2 DNA content because one would say that cells undergoing apoptosis out of G1 phase develop sub-G1 DNA content (Nicoletti et al., 1991).
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Arsenite Inhibits Cell Cycle Progression through All Phases of the Cell Cycle. It has been established that arsenite induces mitotic arrest and apoptosis of mitotic cells (Li and Broome, 1999; Ling et al., 2002). However, it was important to examine the effect of arsenite on other phases of the cell cycle because 98% of an asynchronous population is made up of nonmitotic cells, making it likely that effects on these cells have an important impact on overall growth inhibition. We have looked at progression of arsenite-treated cells from each cell cycle stage to the next and have found that arsenite slows cell growth in every phase of the cycle. Cells elutriated in G1 phase enter S phase more slowly in the presence of arsenite (Fig. 6B, compare left panels). Once in early S phase, arsenite-treated cells are slow to progress to late S phase (Fig. 6B, compare right panels). Analysis of U937 cell cycle kinetics using the method described by Begg et al. (1985) reveals that the DNA synthesis time in asynchronous populations of untreated cells is between 10 and 12 h (data not shown). When cells are treated with 5 µM sodium arsenite, the DNA synthesis time increases to 16 h, indicating that transit through S phase is affected. We noted earlier that cell movement from S phase to mitosis is slowed in the presence of arsenite (Fig. 3, right panel, compare top right quadrants). We wanted to determine whether it was the S to G2 phase or the G2 to M phase transition that was more sensitive to arsenite. We used centrifugal elutriation to collect fractions of cells excluding those in G1 phase to ensure that the cell populations under study contained only actively cycling cells. These fractions were allowed to grow in the presence or absence of arsenite for 8 h. An 8-h treatment allowed enough time for most cells in the untreated samples to progress out of S phase but not enough time for mitotic cells to enter a subsequent cell cycle. After treatment, the samples were analyzed by BrdU, TG-3, and PI staining. The results clearly show that cell passage from any cell cycle phase to the next is inhibited by arsenite (Fig. 7). At elutriation, 49% of cells are in late S phase (Fig. 7A). In control samples after 8 h, 44% of cells have moved on from S phase to later phases of the cell cycle (Fig. 7A, compare left and top right panels). In arsenite-treated samples, however, only 38% are able to exit S phase during the 8 h treatment (Fig. 7A, compare left and bottom right panels). Similar cell cycle phase transition inhibition is seen for movement from G2 to M phase and, as established earlier, for movement out of mitosis (Fig. 7B).
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Since an important action of arsenite in U937 cells seems to be slowing of S phase progression, we thought that perhaps arsenite as a chemotherapeutic agent may be more effective if administered in combination with a drug that targets S phase cells. Such a drug is the topoisomerase inhibitor camptothecin. U937 cells were cultured in the presence of 40 nM camptothecin together with 1 µM sodium arsenite, and cell recovery at 24 or 48 h was determined using a Coulter counter. As shown in Fig. 8, the decrease in cell recovery from camptothecin-treated cultures was enhanced by the addition of only 1 µM sodium arsenite. It is possible that both arsenite and camptothecin may be effective chemotherapeutic agents at lower concentrations if used in combination.
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; Lu et al., 1999; Rousselot et al., 1999; Seol et al., 1999; Zhang et al., 1999; Zhu et al., 1999; Rego et al., 2000; Antman, 2001; Bachleitner-Hofmann et al., 2001; Chen et al., 2001; de The and Chelbi-Alix, 2001; Wang, 2001; Miller et al., 2002). Arsenite's chemotherapeutic mechanism is not well understood. Arsenic trioxide is Food and Drug Administration-approved for the treatment of APL, which is most often characterized by the presence of the oncogenic fusion protein PML-RAR within the leukemic cells (Chen et al., 2001; de The and Chelbi-Alix, 2001; Soignet, 2001). PML-RAR exerts a double dominant-negative effect on the function of both PML and RAR proteins, blocking both apoptosis and differentiation. It is thought that arsenite exerts its apoptotic and differentiative effects on these cells by inducing PML-RAR degradation (Zhu et al., 2001). However, degradation of the oncoprotein is not the sole mechanism of arsenite action because arsenite does induce apoptosis and differentiation in cell lines that lack the fusion protein (Wang et al., 1998; Lu et al., 1999; Rousselot et al., 1999; Seol et al., 1999; Zhang et al., 1999; Zhu et al., 1999; McCabe et al., 2000; Rego et al., 2000; Bachleitner-Hofmann et al., 2001). Therefore, arsenite may be useful in the treatment of cancers other than APL.
Arsenite's effects on cell cycle progression have been studied in many cell types. The identification of the most arsenite-sensitive cell cycle phase has been controversial. Some studies have found that certain cells arrest in G1 phase in response to arsenite (Zhang et al., 1998; Park et al., 2000). In U937 cells, we have found that arsenite treatment induces growth arrest and apoptosis to the greatest extent in G2/M phase (Figs. 2, 3, 4, 5). Also, G2/M phase cells specifically accumulate in response to combined arsenite and caspase inhibitor treatment (Fig. 5A), suggesting that it is cells in the G2/M compartment that are specifically susceptible to arsenite-induced apoptosis. However, by looking at cell cycle progression in synchronized cells, we were also able to observe that arsenite slows progression through the G1 checkpoint and through S phase (Figs. 6 and 7). These results may begin to explain why it has been difficult to pinpoint the cell cycle phase most affected by arsenite. Our results best agree with past studies that have found that trivalent arsenic species induce accumulation and apoptosis of mitotic cells in several cell types (Li and Broome, 1999; Park et al., 2001; Halicka et al., 2002; Ling et al., 2002; States et al., 2002; Cai et al., 2003). Park et al. (2001) reported that U937 cells accumulate in G2/M phase and undergo apoptosis in response to treatment with arsenic trioxide. They found that cells arrested in S phase by aphidicolin were resistant to apoptosis induced by trivalent arsenic. Their conclusion was that arsenite-induced apoptosis occurs specifically in cells arrested in G2/M phase. Our research takes their findings further in that we have followed the cell cycle progression of synchronized U937 cells in the absence of chemical manipulation, allowing us to eliminate any unexpected effects that may result from treating cells with two different cell cycle inhibitory agents simultaneously (i.e., trivalent arsenic and aphidicolin). Also, we were able to separate effects on G2 phase cells from those on mitotic cells by use of the TG-3 antibody. As a result, we found that sodium arsenite induces arrest followed by apoptosis in both of these distinct cell cycle compartments. Ling et al. (2002) used a panel of various cancer cell lines and found that all of them underwent mitotic arrest in response to arsenite. Along with mitotic arrest, the cells also displayed characteristics of apoptosis [activation of caspases, poly-(ADP-ribose) polymerase cleavage] that were specific to mitotic cells. Similarly, we found that arsenite-induced caspase activation occurs primarily in cells outside of G1 phase (Fig. 4) and that caspase inhibition exacerbates arsenite-induced G2/M phase arrest (Fig. 5). We used a terminal deoxynucleotidyltransferase dUTP nick end labeling assay to detect caspase-induced DNA strand breaks in elutriated cell populations and found that with 8 h of arsenite treatment, such breaks could only be detected in cells with S or G2/M phase DNA content (data not shown). It is important to understand the mechanism by which arsenite induces arrest and apoptosis in M phase because this may be an important way in which arsenic can target cancer cells.
In the U937 cell model, however, it seems that the effects of arsenite on mitotic cells, although severe, may be out-weighed by its ability to stall cell cycle progression in other phases of the cell cycle. For instance, it is interesting that no appreciable cell death is observed even at 72 h of arsenite treatment at a concentration known to induce apoptosis in the U937 cell line (Fig. 1B). Although apoptotic cells will maintain membrane integrity longer than necrotic cells, under cell culture conditions, at late stages, even apoptotic cell membrane integrity will be compromised. The conclusion we can draw from the information in Fig. 1 is that arsenite's inhibitory effects on cell recovery are less a result of the induction of cell death, be it apoptotic or necrotic, and more a result of growth inhibition. We went on to observe that arsenite inhibits cell cycle progression through all phases of the cell cycle (Figs. 6 and 7). The difference in the percentage of cells that transit from phase to phase between control and arsenite-treated samples, as shown in Fig. 7B, is partly a function of the decreased movement through the preceding cell cycle transitions. However, the difference increases with each subsequent transition, showing that arsenite exerts an inhibitive influence at each phase. Because cells in every phase of the cell cycle are affected, arsenite treatment profoundly decreases the rate of U937 cell growth (Fig. 1A). Cells that survive mitosis in the presence of arsenite (seen, for example, in the bottom left quadrant of Fig. 2D) remain susceptible to growth inhibition in other phases, further highlighting the importance of arsenite's effects on nonmitotic cells. Our conclusion is that although mitotic cells are most sensitive to arsenite-induced apoptosis, arsenite also inhibits growth in this cell line through the accumulation of its smaller effects on progression through each cell cycle phase.
We have observed that arsenite-treated cells are slow to move from G1 to S phase (Fig. 6). The G1 checkpoint is usually mediated via p53 activation, but the U937 cell line lacks p53 protein because of a 46-base deletion mutation in the p53 gene (Sugimoto et al., 1992). However, p21 activation has been observed in these cells, especially in the context of differentiation induction by 1, 25-dihydroxyvitamin D3 (Liu et al., 1996). We have used immunoblotting to look for increases in p21 levels in arsenite-treated U937 cells and have found none (data not shown). We have similarly found no changes in levels of other negative regulators of the G1 checkpoint: p27 and retinoblastoma protein (data not shown). These results were initially surprising, but it soon became clear that arsenite may not inhibit cell growth in this cell line by targeting a defined checkpoint. The uniformity of its effect on progression through G1, S, and G2 phases suggests that it does not activate a target with a negative influence at a distinct cell cycle phase transition, but rather that it inhibits something fundamental to the movement of cells into and through the cycle. Likely candidates are proteins involved in the process of DNA synthesis. A direct impairment of DNA synthesis would explain the apparent hesitation of cells to enter S phase and the increased S phase transit time (Fig. 6; data not shown). Impaired DNA synthesis may also cause subtle DNA damage that could trigger a G2 delay. Future studies will examine the effects of arsenite on the initiation of DNA synthesis and on the elongation of nascent DNA strands.
We have presented data that suggest that an overall slowing of cell cycle progression is a pronounced effect of arsenite in U937 cells. The most sensitive cells are those in mitosis because they undergo arsenite-induced apoptosis, but the growth-slowing effect of arsenite affects cells in every phase. In conclusion, arsenite treatment induces apoptosis in mitotic cells, as expected from the results of previous studies. Induction of apoptosis is an important chemotherapeutic mechanism that may potentially be used against many cancers. However, another important action of arsenite in U937 cells is to slow cell cycle progression of nonmitotic cells, prolonging the time spent in G1 and S phases. For this reason, arsenite as a chemotherapeutic agent may be more effective if administered in combination with a drug such as camptothecin that targets cells in S phase of the cell cycle, as suggested by the data in Fig. 8. Ultimately, a precise target of arsenic action remains to be identified, but knowledge of the specific cellular response to arsenic in a given cell type dramatically narrows the list of possible mediators of arsenic chemotherapy.
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Footnotes |
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ABBREVIATIONS: APL, acute promyelocytic leukemia; PML, promyelocytic leukemia protein; RAR, retinoic acid receptor-; BrdU, 5-bromo-2'-deoxyuridine; BSA, bovine serum albumin; PI, propidium iodide; Z-VAD-FMK, N-CBZ-Val-Ala-Asp(O-Me) fluoromethyl ketone; FITC, fluorescein isothiocyanate; APC, allophycocyanin; PBS, phosphate-buffered saline; FBS, fetal bovine serum.
Address correspondence to: Dr. Michael J. McCabe, Jr., Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, 575 Elmwood Ave., Rochester, NY 14642. E-mail: michael_mccabe{at}urmc.rochester.edu
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