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CELLULAR AND MOLECULAR
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina
Received May 15, 2006; accepted July 31, 2006.
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Abstract |
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-pifithrin (-PFT) or p53 small interference RNA (siRNA). In contrast, WI38 cell senescence induced by BU was associated with prolonged activation of extracellular signal-regulated kinase (Erk), p38 mitogen-activated protein kinase (p38), and c-Jun NH2-terminal kinase (JNK) and could be suppressed by the inhibition of Erk and/or p38 with PD98059 (2'-amino-3'-methoxyflavone) and/or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], respectively. However, inhibition of p53 with -PFT or p53 siRNA or JNK with SP600125 (1,9-pyrazoloanthrone) failed to protect WI38 cells from BU-induced senescence. These findings suggest that BU is a distinctive chemotherapeutic agent that can selectively induce normal human fibroblast senescence through the Erk and p38 pathways.
; Buggia et al., 1994). Unlike other commonly used chemotherapeutic agents, BU is unique because it primarily targets slowly proliferating or nonproliferating cells. As such, it causes various normal tissue injuries while killing leukemia cells. The common side effects associated with BU treatment include severe myelosuppression, hepatic venoocclusive diseases, pulmonary fibrosis, hemorrhagic cystitis, sterility, and cataracts, which limit its therapeutic efficacy as an antitumor agent (Bishop and Wassom, 1986; Morgan et al., 1991). However, the mechanism(s) of action by which BU injures normal tissues has not been well established.
As an alkylating agent, it is anticipated that BU can cause DNA alkylation leading to DNA-DNA and DNA-protein cross-links (Iwamoto et al., 2004; Mertins et al., 2004). Thereafter, these cross-links may trigger DNA damage responses and cause apoptosis or cell death through the p53 pathway. However, this mode of action was not supported by our recent studies (Meng et al., 2003a), where we found that, unlike other chemotherapeutic agents and ionizing radiation, BU does not induce significant apoptosis but senescence in one of its primary targets, e.g., hematopoietic stem cells (HSCs) independent of p53 activation (Meng et al., 2003b). This finding is consistent with the previous observation showing that no significant increase in apoptosis was detected in bone marrow biopsies from chronic myelogenous leukemia patients receiving BU chemotherapy (Thiele et al., 1997). However, it is not known whether induction of senescence is a specific cellular response of HSCs to BU or whether it can occur in other normal cells when the cells are treated with BU, nor have the molecular pathways mediating BU-induced cellular senescence been defined. Therefore, in the present study, we used the normal human diploid WI38 fibroblasts as a model system to determine whether BU treatment causes apoptosis or senescence in WI38 cells and to elucidate the molecular mechanisms by which BU induces cellular senescence because extensive knowledge about senescence pathways has been well delineated using this cell line (Serrano et al., 1997; Beausejour et al., 2003; Iwasa et al., 2003).
The results from our studies show that the response of WI38 cells to BU treatment is similar to that of HSCs. Both cell types undergo senescence but not apoptosis after exposure to BU in a dose-independent manner. In contrast, exposure of WI38 cells to a high dose of etoposide (Etop) induces apoptosis and to a low dose of Etop causes senescence, a typical cellular response to treatment with a cytotoxic chemotherapeutic agent (Robles and Adami, 1998; Robles et al., 1999; Roninson et al., 2001; Han et al., 2002; Roninson, 2002; Rebbaa et al., 2003; Zheng et al., 2004). Further investigations reveal that BU induces WI38 senescence independent of p53 as seen in HSCs (Meng et al., 2003b), whereas Etop relies on the p53 pathway to induce WI38 cell senescence. However, BU treatment strongly induced extracellular signal-regulated kinase (Erk) activation followed by sequential activation of p38 mitogen-activated protein kinase (MAPK or p38) and c-Jun NH2-terminal kinase (JNK) and up-regulation of p16Ink4a (p16). Inhibition of Erk and/or p38, but not that of JNK, significantly attenuated the BU-induced increase in senescence-associated 
-galactosidase (SA--gal) staining, up-regulation of p16, and development of senescent morphology, even though neither inhibitor immediately released the cells from BU-induced growth inhibition. Upon the removal of Erk and/or p38 inhibitors from the WI38 cells that had been treated with BU, the cells proceeded to cellular proliferation and 5-bromo-2'-deoxyuridine (BrdU) incorporation, indicating that suppression of Erk and/or p38 inhibits BU-induced senescence. To our knowledge, this is the first study demonstrating that BU is a distinctive chemotherapeutic agent that can selectively induce cellular senescence in normal human fibroblasts through the Erk and p38 MAPK pathways.
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-pifithrin (-PFT)], Erk (PD98059), p38 (SB203580), and JNK (SP600125) were purchased from Calbiochem (San Diego, CA). BU, Etop, and BrdU were obtained from Sigma (St. Louis, MO). Antibodies against phosphorylated p53 and Erk and those against total p53, Erk, p38, JNK, and p21Cip1/WAF1 (p21) were obtained from Cell Signaling (Beverly, MA); antiphosphorylated p38 and JNK were from Promega (Madison, WI); anti--actin was from Santa Cruz (Santa Cruz, CA); anti-p16 was from BD PharMingen (San Diego, CA); anti-BrdU was from Sigma; antiphosphorylated histone 2AX (
H2AX) was from Upstate (Lake Placid, NY); and goat anti-rabbit IgG-horseradish peroxidase, rabbit anti-goat IgG-horseradish peroxidase, Texas Red Dye-conjugated goat anti-mouse IgG, and fluorescein isothiocyanate-conjugated anti-mouse IgG were from Jackson ImmunoResearch (West Grove, PA). Cell Culture. WI38 cells (human embryonic lung diploid fibroblasts) originally obtained from American Type Culture Collection (Manassas, VA) were cultured in minimum essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified incubator at 37°C and 5% CO2. Cells at early passages (<25 passages) were used in all experiments to avoid complications of replicative senescence because WI38 cells have a mean lifespan of approximately 45 to 60 population doublings. For the induction of senescence, cells at approximately 70% confluence were exposed to varying concentrations of BU (7.5–120 µM) or Etop (20 or 80 µM) for 24 h. The cells were washed once with PBS to remove drug and recultured in fresh medium for various durations as indicated in each experiment.
Small Interference RNA Treatment. WI38 cells were transfected with 20 µM scrambled sequence (siCONTROL NonTargeting siRNA no. 1) or p53 small interference RNA (siRNA) (siGENOME SMART pool reagent for Human TP53) (Dharmacon, Chicago, IL) using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Two days after the transfection, they were exposed to either BU (120 µM) or Etop (20 µM) as described above.
Analysis of Cell Proliferation. WI38 cells were seeded in wells of a 24-well plate at 60,000 cells/well and cultured overnight. They were treated with BU for 24 h as described before, after which the drug was removed, and the cells were cultured in fresh medium for up to 11 days. Every 3 days the cells were trypsinized, counted, and subcultured again in a fresh medium at 1:5 and 1:2 dilutions for control untreated and BU- or Etop-treated cells, respectively.
Cell Cycle Analysis. Cell cycle analysis was done using propidium iodide staining and a flow cytometer as described previously (Wang et al., 2004).
Analysis of Activated Caspase 3. WI38 cells were cultured on chamber slides to subconfluence and then exposed to BU or Etop for 24 h. Activation of caspase 3 was determined using a kit containing the SR-DEVD-FMK-FLICA reagent (Invitrogen) per the manufacturer's instructions. The cells were viewed using an Olympus BX61 fluorescent microscope (Olympus, Tokyo, Japan). Images were captured with a Hamamatsu C4742-95 digital camera (Hamamatsu Corporation, Bridgewater, NJ). Captured images were processed using Side Book 4.0 Digital Microscopy software (Intelligent Imaging Innovations, Denver, CA) and displayed using Adobe Photoshop version 6.0 (Adobe Systems, San Jose, CA).
SA--gal Staining. SA--gal activity was determined using a SA--gal staining kit from Cell Signaling according to the manufacturer's instructions. Senescent cells were identified as blue-stained cells by standard light microscopy, and a minimum of 1000 cells was counted in 10 random fields to determine the percentage of SA--gal-positive cells.
BrdU Incorporation Assay. DNA synthesis in control untreated and BU- or Etop-treated cells was determined by measuring BrdU incorporation into DNA as described previously (Wang et al., 2004). Images of BrdU and Hoechst 33342 nuclear staining were captured and processed similarly as described above for the analysis of activated caspase 3. To calculate the percentage of BrdU-positive cells, a minimum of 1000 cells was counted in eight random fields on a slide, and the number of BrdU-positive cells was divided by the total number of cells counted.
Analysis of DNA Double-Strand Breaks. WI38 cells were cultured on chamber slides to subconfluence and then exposed to BU or Etop for up to 24 h. For determination of DNA double-strand breaks (DSBs), cells were fixed with ice-cold 95% ethanol/5% acetic acid for 5 min, blocked with 3% bovine serum albumin in PBS for 30 min, incubated with 2 µg/ml H2AX (at Ser 139) antibody (Upstate) for 1 h, and washed three times with PBS. Fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody was then added, and the cells were incubated in the dark for 1.5 h, rinsed with PBS, and counterstained with Hoechst 33342 for nuclear detection as described previously. The total number of H2AX foci and number of H2AX-positive cells were quantified for each treatment by counting a minimum of 1000 cells from eight randomly chosen fields and expressed as a percentage of H2AX-positive cells and average H2AX foci per cell. The number of H2AX foci per cell closely correlates to the number of DNA DSBs induced in a cell (Rogakou et al., 1998, 1999).
Western Blot Analysis. Lysates of WI38 cells were prepared and analyzed as described previously (Wang et al., 2004).
Statistical Analysis. The data were analyzed by analysis of variance. In the event that analysis of variance justified post hoc comparisons between group means, these were conducted using the Student-Newman-Keuls test for multiple comparisons. For experiments in which only single experimental and control groups were used, group differences were examined by unpaired Student's t test. Differences were considered significant at p < 0.05. All of these analyses were done using GraphPad Prism (GraphPad Software, Inc., San Diego, CA).
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-gal staining (Fig. 1, C and D). In contrast, WI38 cells underwent apoptosis after exposure to a high dose of Etop (80 µM) (Fig. 1B; data not shown) and senescence to a low dose of Etop (20 µM), respectively (Fig. 1, B–D). These findings indicate that the lack of apoptotic response to BU is not due to an inherited deficiency of the apoptotic machinery in WI38 cells but suggest that that BU is a distinctive chemotherapeutic agent that can selectively induce cellular senescence but not apoptosis in WI38 cells.
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). BU is an alkylating agent that can cause DNA-DNA and DNA-protein cross-links (Iwamoto et al., 2004; Mertins et al., 2004). However, whether the cross-links can lead to DNA strand breaks and the subsequent activation of p53 has not been examined. Histone 2AX (H2AX) phosphorylation is a well recognized marker for DNA DSBs (Rogakou et al., 1998, 1999). By examining H2AX using immunohistochemistry, we investigated the induction of DNA DSBs in WI38 cells that were exposed to 120 µM BU or 20 µM Etop. As shown in Fig. 2, treatment of WI38 cells with either BU or Etop resulted in a time-dependent induction of H2AX phosphorylation, which peaked around 8 h and completely resolved at 24 h after the treatment. However, 8 h after the drug treatment, the percentage of WI38 cells stained positive for H2AX (BU, 35.3% versus Etop, 74.1%) and average number of H2AX foci detected in each cell (BU, 5.1 versus Etop, 14.7) were significantly lower in BU-treated cells than in Etop-treated cells (p < 0.01), indicating that BU is a less potent inducer of DNA DSBs compared with Etop. Consistent with this finding, exposure of WI38 cells to Etop induced a more rapid and robust activation of p53 than BU (Fig. 3A). Specifically, only moderate levels of p53 phosphorylation were detected in BU-treated cells 24 h after BU treatment, whereas high levels of p53 phosphorylation were found in Etop-treated cells within 2 h of Etop treatment. However, both BU and Etop treatment induced the expression of p21 and p16, and the induction of these cyclin-dependent kinase inhibitors has been implicated in the initiation and maintenance of cellular senescence (Sharpless and DePinho, 1999; Campisi, 2005).
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Next, we investigated whether BU induces WI38 fibroblast senescence via the p53 pathway. WI38 cells were treated with either BU (120 µM) or Etop (20 µM) in the presence or absence of 20 µM -PFT (a selective p53 inhibitor) (Komarov et al., 1999) for 24 h, upon which the drug was removed, and the cells were cultured continuously with -PFT for up to 11 days. WI38 cells treated with BU or Etop alone underwent senescence because the cells became permanently growth arrested and exhibited a significant reduction in BrdU incorporation and increase in SA--gal staining at 7 and 11 days after BU or Etop treatment (Fig. 3, B–D). The presence of -PFT in the medium was able to almost abrogate Etop-induced inhibition of cell proliferation and BrdU incorporation and increase in SA--gal staining in WI38 cells, but it had minimal effect on these induced by BU. This finding suggests that Etop induces WI38 cell senescence in part via the p53 pathway, whereas BU induces senescence by a p53-independent mechanism. This suggestion was further supported by the study using p53 siRNA that has the ability to specifically down-regulate the expression of p53. As shown in Fig. 4, transfection of WI38 cells with p53 siRNA, but not that with a scrambled sequence siRNA, reduced the expression of p53 as well as its phosphorylation by BU and Etop (Fig. 4A). In addition, p53 siRNA transfection significantly attenuated Etop-induced increase in SA--gal staining and reduction in BrdU incorporation in WI38 cells but had no significant effect on these induced by BU compared with scrambled siRNA-transfected cells.
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; Wang et al., 2002; Iwasa et al., 2003; Deng et al., 2004). The lack of p53 involvement in BU-induced senescence prompted us to examine whether BU induces WI38 cell senescence via the MAPK pathways. To test this hypothesis, we first examined whether BU activates various MAPKs by analysis of Erk, p38, and JNK phosphorylation using Western blotting. As shown in Fig. 5, BU treatment elicited an immediate activation of Erk, whereas the increases in p38 and JNK phosphorylation occurred 3 days after BU treatment. The activation of all three MAPKs was sustained for up to 11 days after BU treatment.
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). The data presented in Fig. 6A showed that activation of each of these MAPKs by BU was specifically inhibited by their respective inhibitor. In addition, inhibition of Erk by PD98059 also suppressed BU-induced phosphorylation of p38 and JNK, SB203580 attenuated the activation of both p38 and JNK, and SP600125 only inhibited JNK, indicating that BU may cause activation of Erk, p38, and JNK in a cascading fashion as shown in other studies (Serrano et al., 1997; Zhu et al., 1998; Deng et al., 2004). Thereafter, we examined whether inhibition of individual MAPKs attenuates BU-induced senescence. For this study, WI38 cells were incubated with BU for 24 h in the presence or absence of PD98059, SB203580, and PD98059 plus SB203580 or SP600125, after which BU was removed, and SA--gal staining was performed after the cells were cultured for 7 and 11 days with the inhibitor(s) that was added to the initial cultures with BU. The results showed that inhibition of Erk and/or p38 with PD98059 and/or SB203580 significantly inhibited the BU-induced increase in SA--gal staining (Fig. 6B) and prevented the development of senescent morphology in WI38 cells (data not shown), compared with the cells treated with BU alone. However, inhibition of JNK had no such effect. Correspondingly, inhibition of Erk and/or p38 but not that of JNK attenuated the induction of p16 by BU, but none of these inhibitors had any significant effect on BU-induced p21 expression (Fig. 6A). These findings suggest that activation of Erk and p38 mediates BU-induced WI38 cell senescence, probably via up-regulation of p16. Although JNK is also activated by BU, it is not critical to the induction of senescence (Fig. 6B).
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However, to our surprise, the inhibition of Erk and/or p38 did not immediately release BU-treated cells from growth inhibition (data not shown), even though the cells were not senescent judged by the lack of SA--gal staining (Fig. 6B), no increase in p16 expression (Fig. 6A), and completely normal cell morphology (data not shown). We postulate that this paradoxical phenomenon is due to the intimate involvement of MAPK signaling in normal cell proliferation (Kyriakis and Avruch, 2001). A prolonged inhibition of Erk and/or p38 along with a brief exposure to BU may cause a temporary cell cycle arrest, possibly mediated by p21 because it was up-regulated in these cells (Fig. 6A). As such, the release of the cells from Erk and/or p38 inhibition should allow the cells to resume proliferation if the cells are not senescent. To test this hypothesis, WI38 cells were cultured with BU in the presence or absence of PD98059, SB203580, and PD98059 plus SB203580 or SP600125 for 24 h. After the removal of BU, the cells were cultured with these inhibitors for 11 days. At this time, none of the cells incorporated BrdU, even though only the cells treated with BU alone or BU plus SP600125 showed senescent morphology and SA--gal staining. Upon releasing the cells from the inhibition by culturing the cells in an inhibitor-free medium, the cells treated with BU and PD98059, SB203580, or PD98059 plus SB203580 started to grow again (Fig. 6C). Three days after the release, these cells showed normal levels of DNA synthesis measured by BrdU incorporation assay (Fig. 6D). However, the cells treated with BU alone or BU plus SP600125 remained growth inhibited and were unable to incorporate BrdU after SP600125 had been removed from the culture. These observations reaffirm that activation of Erk and p38 but not that of JNK plays an important role in mediating BU-induced senescence in WI38 fibroblasts.
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; Robles et al., 1999; Roninson et al., 2001; Han et al., 2002; Roninson, 2002; Rebbaa et al., 2003; Zheng et al., 2004). Low-dose treatment usually induces premature senescence, whereas high-dose treatment causes apoptosis as shown in WI38 cells treated with Etop. However, BU is unique because it can selectively induce WI38 cell senescence but not apoptosis in a dose-independent fashion. This observation is in agreement with our previous finding demonstrating that exposure of mouse HSCs to ionizing radiation (4 Gy), Etop (2 µM), and camptothecin (2 µM) induced apoptosis, whereas exposure of the cells to BU (up to 100 µM) failed to induce HSC apoptosis but effectively inhibited their hematopoietic function (Meng et al., 2003a). Further studies demonstrate that BU inhibits HSCs primarily by induction of senescence. These findings suggest that induction of senescence may be a unique response of normal cells to BU since induction of apoptosis was observed in various leukemia cells after BU treatment (Schwarz et al., 1999; Hassan et al., 2001).
Two major pathways have been implicated in the induction of senescence (Sharpless and DePinho, 1999; Shay and Wright, 2000; Serrano and Blasco, 2001; Marcotte and Wang, 2002; Campisi, 2005): the p53-p21 pathway triggered by DNA damage or telomere shortening and the p16-retinoblastoma pathway activated by the Ras-Raf-mitogen-activated protein kinase kinase-Erk-p38 cascade (Sharpless and DePinho, 1999; Shay and Wright, 2000; Serrano and Blasco, 2001; Marcotte and Wang, 2002; Campisi, 2005). As an alkylating agent, BU can cause DNA damage by cross-linking DNAs and DNA and proteins (Buggia et al., 1994; Iwamoto et al., 2004; Mertins et al., 2004). The cross-links may be converted into DNA strand breaks that can subsequently activate the p53 pathway to induce senescence. To our knowledge, this is the first report to demonstrate that BU treatment induces DNA DSBs, evidenced by the increase in H2AX staining (Rogakou et al., 1998, 1999). However, the percentage of the cells stained positive for H2AX and number of H2AX foci detected in each cell were significantly lower in BU-treated cells than in Etop-treated cells, indicating that BU induces fewer DNA DSBs than Etop. Moreover, the induction of DNA DSBs by BU failed to elicit an immediate activation of p53, whereas a rapid and robust activation of p53 was observed in Etop-treated cells. A moderate level of p53 phosphorylation was found in WI38 cells at 3 days after BU treatment. However, this activation may not be attributable to DNA damage since at that time H2AX was no longer detectable in the cells. Instead, the activation of p53 may be triggered by p38, which was also activated in the cells by that time and has the ability to phosphorylate p53 (Bulavin et al., 1999; She et al., 2000). Even this moderately delayed activation of p53 seems to play an insignificant role in BU-induced cellular senescence since inhibition of p53 activity by a p53-specific inhibitor (-PFT) or down-regulation of p53 expression using siRNA had no significant effect on BU-induced senescence in WI38 cells. In contrast, inhibition of p53 activity by the same means was able to greatly reduce the induction of senescence by Etop. This finding suggests that BU may induce cellular senescence via a p53-independent mechanism. This suggestion is in agreement with our previous observation that the induction of HSC senescence by BU was not associated with an elevation in p53 activity and p21 expression (Meng et al., 2003b).
Alternatively, BU may induce WI38 cell senescence by activation of the MAPK pathways. Three major MAPK pathways have been implicated in the regulation of cellular senescence in vertebrates (Haq et al., 2002; Wang et al., 2002; Iwasa et al., 2003; Deng et al., 2004). All of them were sequentially activated in WI38 cells by BU: an immediate activation of Erk followed by the activation of p38 and JNK 3 days after BU treatment. The activation of p38 and JNK was downstream of Erk because inhibition of Erk prevented p38 and JNK phosphorylation. However, the delayed activation of p38 and JNK by BU suggests that p38 and JNK are not direct targets of Erk but rather activated indirectly by Erk through a yet to be defined mechanism (Haq et al., 2002; Iwasa et al., 2003; Deng et al., 2004). A similar sequential activation of Erk and p38 was also observed in the cells ectopically transfected with Ras and Raf oncogenes that induce premature senescence in fibroblasts (Serrano et al., 1997; Zhu et al., 1998; Deng et al., 2004). Therefore, we further examined whether specific inhibition of Erk, p38, and/or JNK can attenuate BU-induced senescence in WI38 cells. The results showed that inhibition of Erk and/or p38, but not that of JNK, significantly suppressed BU-induced WI38 cell senescence. These findings are in agreement with previous observations that Ras-induced fibroblast senescence is mediated by the cascading activation of the Erk and p38 pathways and that constitutive activation of p38 induces fibroblast senescence (Serrano et al., 1997; Zhu et al., 1998; Deng et al., 2004). However, inhibition of Erk and/or p38 did not immediately restore cell division after BU treatment. The cell proliferation resumed only after the removal of Erk and/or p38 inhibition, suggesting that continuous inhibition of Erk and/or p38 may prevent BU-treated cells from reentering the cell cycle even though Erk and/or p38 inhibition effectively suppresses BU-induced senescence in the cells. In addition, this phenomenon suggests that the induction of temporary cell cycle arrest and senescence by BU in WI38 cells may be mediated by different mechanisms. The induction of temporary cell cycle arrest is probably mediated by up-regulation of p21, probably in an Erk-p38-independent manner; whereas the induction of senescence may be attributable to the increased expression of p16 resulting from Erk and/or p38 activation. This suggestion is supported by the finding that BU-treated WI38 cells had normal cell morphology and lack of SA--gal staining and p16 up-regulation under continuous Erk and/or p38 inhibition but showed a slight increase in p21 expression. Upon release from Erk and p38 inhibition, the cells proceeded to synthesize DNA and undergo cell division. A similar finding was also observed in ataxia telangiectasia fibroblasts (Naka et al., 2004). It was shown that in ataxia telangiectasia fibroblasts, the temporary cell growth arrest induced by ionizing radiation or H2O2 was mediated by the activation of p53 and induction of p21, whereas activation of the Erk-p38 pathways and subsequent up-regulation of p16 led to the induction of senescence in the cells (Naka et al., 2004).
The mechanisms by which BU activates the Erk-p38 pathways have yet to be elucidated. The activation of Erk and p38 may result from cross-linking of plasma membrane proteins due to protein alkylation by BU (Buggia et al., 1994; Iwamoto et al., 2004). Alternatively, BU could activate p38 by increasing oxidative stress due to depletion of intracellular glutathione (DeLeve and Wang, 2000; Hassan et al., 2002). When p38 is activated by BU treatment, it can up-regulate the expression of p16 to induce cell cycle arrest and cellular senescence (Haq et al., 2002; Wang et al., 2002; Iwasa et al., 2003; Deng et al., 2004). In addition, activation of p38 can activate p53 or inactivate cdc25 phosphatase (Bulavin et al., 1999, 2001; She et al., 2000; Wang et al., 2000). Activation of p53 in turn induces p21. Induction of p21 and inactivation of cdc25 phosphatase can cause G1 and G2/M cell cycle arrest, respectively (Wang et al., 2000; Bulavin et al., 2001). It has yet to be determined whether induction of p21 and inactivation of cdc25 phosphatase play any role in BU-induced cellular senescence in WI38 cells.
In summary, the data presented in this study demonstrate that BU is a distinctive chemotherapeutic agent. It can selectively induce cellular senescence in normal cells but apoptosis in leukemia cells as shown in previous studies (Schwarz et al., 1999; Hassan et al., 2001). The induction of normal cell senescence is independent of BU-induced DNA damage and p53 activation but relies on the Erk and p38 MAPK pathways. Induction of senescence may contribute to BU-induced normal tissue damage in several different ways. First, when a cell, especially if it is a stem cell such as a HSC, becomes senescent, it cannot proliferate to generate progeny that are normally required for the maintenance of normal tissue homeostasis and for the repair of damaged tissues by a cytotoxic chemotherapeutic agent. Moreover, senescent cells secrete increased levels of proinflammatory cytokines, matrix metalloproteinases, and epithelial growth factors that are known to participate in the pathogenesis of pulmonary fibrosis and other normal tissue injuries (Campisi, 2005). Therefore, a better understanding of the mechanism by which BU and other alkylating agents induce normal cell injury would be beneficial to the effort of developing new therapeutics that could be more effective in depleting leukemia cells while limiting its toxicity to normal tissues.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: BU, busulfan; HSC, hematopoietic stem cell; Etop, etoposide; ERK/Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; SA--gal, senescence associated -galactosidase; BrdU, 5-bromo-2'-deoxyuridine; -PFT, -pifithrin; PD98059, 2'-amino-3'-methoxyflavone; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SP600125, 1,9-pyrazoloanthrone; H2AX, phosphorylated histone 2AX; siRNA, small interference RNA; DSB, double-strand break; TBS, Tris-buffered saline; H2AX, histone 2AX; ANOVA, analysis of variance.
Address correspondence to: Dr. Daohong Zhou, Department of Pathology, Medical University of South Carolina, 165 Ashley Avenue, Suite 309, P.O. Box 250908, Charleston, SC 29425. E-mail: zhoud{at}musc.edu
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