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TNF-α promotes Doxorubicin-induced cell apoptosis and anti-cancer effect through downregulation of p21 in p53-deficient tumor cells
BBRC Biochemical and Biophysical Research Communications 330 (2005) 1034–1040 www.elsevier.com/locate/ybbrc
TNF-a promotes Doxorubicin-induced cell apoptosis and anti-cancer effect through downregulation of p21 in p53-deficient tumor cells Wei Cao a, Wan-Hao Chi a, Jun Wang b, Juan-Juan Tang b, Yan-Jun Lu b,* a b
Laboratory of Medical Molecular Virology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China Cancer Research Center, Celstar Bio-Pharmaceutical Co. Ltd., 1 Juli Road Pudong, Shanghai 201203, China Received 25 February 2005 Available online 22 March 2005
Abstract p53 is a key regulator in cell apoptosis, and cancer cells deficient in p53 expression fail to respond to chemotherapy. Here we show that effective Doxorubicin (DOX)-induced apoptosis is p53-dependent. However, an alternative treatment of DOX/TNF-a/ DOX restored sensitivity of p53-deficient cells to DOX-induced apoptosis. Treatment of cells with TNF-a resulted in a decrease of p21 (waf1/cip1/sdi1) expression following second dose of DOX. In previous work, we demonstrated that p21 suppressed DOX-induced apoptosis via its (cyclin-dependent kinase) CDK-binding and CDK-inhibitory activity. Thus, we propose that TNF-a enhances the anti-cancer effect of DOX through suppressing the anti-apoptotic activity of p21, and that a combined treatment TNF-a/Dox is an effective chemotherapeutic strategy for p53-deficient cancers. Ó 2005 Elsevier Inc. All rights reserved. Keywords: p53; p21; CDK2; TNF-a; NFjB/RelA/p65
TNF-a stimulates the cell response by binding to its receptor I and II. The NFjB family of transcription factors are key mediators of intracellular signaling events initiated by TNF-a [1]. NFjB generally exists as an inactive dimer sequestered in the cytoplasm by an inhibitor protein termed IjB. In response to TNF-a, activation of NFjB involves phosphorylation of IjB on two critical serine residues at codon 32 and 36. Once phosphorylated, IjB is subjected to ubiquitination at two lysine residues and subsequent degradation by the 26S proteasome. Unmasking IjB from NFjB allows it to translocate into the nucleus, where it binds to the target genes, and promotes transcription [2]. TNF-a shows a strong anti-tumor effect on many solid tumors. But its clinical application is limited by its se*
Corresponding author. Fax: +86 21 5080 3128. E-mail address: [email protected] (Y.-J. Lu).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.188
vere systemic side effects, before therapeutic doses could be reached [3,4]. Much effort has been paid on the development of less toxic recombinant TNF-a for a safe and systemic administration [5,6]. Our clinical phase II and phase III trials showed that systemic administration of TNF-a in combination with a chemotherapy agent Doxorubicin (DOX) for lung tumors remarkably enhanced the anti-tumor effect, as compared to the DOX treatment alone. In total, 44% of the 133 patients exhibited complete or partial remission of lung tumors in response to this combination therapy strategy [7–9]. Since it has been approved by the Chinese SDA, this innovative clinical application of TNF-a in combination with DOX may offer great opportunities for cancer patients. However, the molecular basis for the combination treatment has not been elucidated. DOX is the most widely used chemotherapy agent in the treatment of tumor. Recent data suggest that
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DOX-induced apoptosis is interconnected with the machinery of cell cycle control. Progression through G1 or G2 phase and entry into the S or M phase are tightly regulated by cyclin-dependent kinases (CDKs) [10]. P21 (waf1/cip1/sdi1), which acts primarily as a transcription target of p53, is an important cellular checkpoint protein for inhibition of complex cyclin-CDKs activities, which contributes to its suppressing effect on DOX-mediated apoptosis in human tumor cells [11,12]. In this study, we used HCT116 (p53+/+) cells and their matched HCT116 (p53 / ) cells in which the p53 gene has been deleted by homologous recombination. We demonstrated that unlike HCT116 (p53+/+) cells, HCT116 (p53 / ) cells were resistant to DOX-induced apoptosis. However, treatment with DOX followed by TNF-a sensitized HCT116 (p53 / ) cells to DOX-induced cell death by enhancing the CDK2 kinase activity. We present evidence that TNF-a may be involved in augmenting DOX-induced apoptosis.
Materials and methods Cells and transfection. Human colorectal cancer cell lines HCT116 (p53+/+) and HCT116 (p53 / ) were generous gifts of Dr. B. Vogelstein. The p21 and CDK2 expression plasmids have been described previously [10,11]. The plasmid expressing the IjB mutant/ IjBSR was provided by Dr. Yamagishi (Kyoto University, Kyoto, Japan). IjB super repressor (IjB-SR) containing the mutations at Ser 32, Ser 36 phosphorylation residues which cannot be phosphorylated and degraded is used as a dominant negative inhibitor of NF-jB. HCT116 cells were transiently transfected with expression constructs for p21, CDK2, and IjBSR, using the Effectene Transfection Kit (Qiagen, Chatsworth, CA), as described previously [10]. The efficiency of transfection was higher than 98%, which was detected by the proportion of green cells after transfection of GFP-expressing plasmid (pEGFP-c1) (Invitrogen, US) with the same protocol. All cells were cultured in DulbeccoÕs modified EagleÕs minimum essential medium (Hyclone, logan, UT) supplemented with 10% fetal bovine serum (Hyclone, logan, UT). Western blotting. Western blotting has been described previously [10,11]. A 40 lg aliquot of cell lysate was separated by 12% polyacrylamide–SDS gel electrophoresis and transferred to a Hybond-C Super membrane. p21 and CDK2 were detected with an anti-p21 antibody and an anti-CDK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. CDK2 kinase assay. Cell extracts (100 lg) were incubated with an anti-CDK2 antibody (SC-163), and the complexes were collected with 80 ll protein G-plus–agarose (Santa Cruz Biotechnology, Santa Cruz. CA). The cyclin-dependent kinases activity was measured by CDK assay kit (Upstate Biotechnology, Lake Placid, NY). Briefly, the immune complexes (40 lg) were mixed with [c-32P]ATP (3000 Ci/mmol), histone H1 (2 lg) as a substrate, and the reaction buffer containing inhibitors of other kinases. After incubation for 10 min at 30 °C, phosphorylated histone H1 was resolved by 16% polyacrylamide–SDS gel electrophoresis and detected by autoradiography. Measurement of cell apoptosis. Cells were treated with DOX at doses of 0.2 and 0.8 lg/ml, and with TNF-a (500 U/ml), respectively. Cells were washed with PBS, stained with the DNA-binding dye, Hoechst 33258 (0.5 lg/ml in PBS), and examined under a fluorescence microscope. Cells showing condensed nuclear morphology and nuclear lobulations were regarded as undergoing apoptosis.
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Results TNF-a sensitized HCT116 (p53 / ) cells to DOX-induced apoptosis P53 tumor suppressor is known to play an important role in potentiating apoptosis through a variety of mechanisms [13–15]. To confirm this point, we treated HCT116 (p53+/+) cells and HCT116 (p53 / ) cells with DOX for 72 h. And the nuclear morphology of the cells was examined by staining them with DNAbinding dye Hoechst 33258 and by microscopy, that was indicated as apoptotic cell death. As shown in Fig. 1A, DOX-induced apoptosis was higher in p53+/+ cells than in p53 / cells. This result suggests a requirement of p53 for DOX-induced cell apoptosis. To explore a condition for a killing effect on p53-deficient cells, we treated cells with different combinational use of alternating DOX and TNF-a treatment (i.e., Day1, DOX; Day2, DOX; Day3, DOX; Day1, DOX; Day2, TNF-a; Day3, DOX; Day1, TNF-a; Day2, DOX; Day3, DOX, Day1, DOX; Day2, DOX; Day3, TNF-a; and Day1, TNF-a; Day2, TNF-a; Day3, TNF-a). The results are shown in Fig. 1B. Treatment with DOX, and then incubating the cells with TNF-a treatment, was followed by another treatment of DOX (DOX–TNF–DOX treatment). This experimental protocol was not typical but based on our clinical application of TNF-a in combination with DOX [7–9]. Interestingly, HCT116 (p53 / ) cells were the most sensitive of all to the apoptotic effect of this DOX–TNF–DOX alternative treatment. To determine whether the cell death in HCT116 (p53 / ) cells caused by DOX was due to apoptosis, DNA fragmentations were examined in HCT116 (p53 / ) cells after treatment. The DNA ladders, a typical apoptotic morphology, were indicated as apoptotic cell death in the treatment with DOX–DOX–DOX, DOX–TNF–DOX, and DOX–DOX–TNF (Fig. 1C). Together, our data suggest that loss of p53 impairs DOX-induced cell apoptosis, but treatment of TNF-a enhanced the sensitivity to DOX-induced apoptosis in p53-deficient cells. This observation led us to suggest that alternative administration of TNF-a with DOX may be a more effective protocol for therapeutic treatment of p53-deficient tumors. The cells with functional p53 are more sensitive to DOX-induced apoptosis due to activation of p53-dependent apoptotic pathway. TNF-a enhanced CDK2 kinase activity in HCT116 (p53 / ) cells In previous studies, we showed that DOX is a cell cycle-dependent anti-tumor drug. P21 suppresses DOX-induced apoptosis by arresting cells at G1 or G2 phase [10,16]. The expression level of p21 is increased in
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Fig. 1. Treatment of TNF-a reversed HCT116 (p53 / ) cellsÕ sensitivity to DOX-induced apoptosis. (A) Fraction of apoptotic cells after 72 h of DOX (0.2 and 0.8 lg/ml) treatment in HCT116 (p53+/+) cells (d) and HCT116 (p53 / ) cells (s). (B) fraction of apoptotic cells after Day1, Day2, and Day3 treatment of DOX (0.8 lg/ml), or TNF-a (500 U/ml) in HCT116 (p53 / ) cells. (C) Agarose gel electrophoresis pattern of DNA obtained from HCT116 (p53 / ) cells with treatment of DOX (0.8 lg/ml) or TNF-a (500 U/ml). Cells were stained with Hoechst 33258 after treatments. Apoptotic cells were counted under a microscope. Error bars indicate the standard deviation of three experimental results.
p53-dependent and p53-independent pathways after DNA damage [11]. The combinational use of treatment with DOX and TNF-a resulted in significant different apoptosis induction in HCT116 (p53 / ) cells (Fig. 1). It led us to suggest that the sequence of DOX– TNF-a or TNF-a–DOX treatment plays an important role in this study. Then HCT116 (p53 / ) cells were treated with DOX alone or it was followed by a treatment with TNF-a and with TNF-a alone or followed by a treatment with DOX. The p21 protein amounts were assessed by Western blotting analysis. As shown in Fig. 2, the p21 expression level was elevated in HCT116 (p53 / ) cells after DOX treatment alone. Moreover, the amount of p21 expression was low in the treatment with DOX followed by TNF-a compared to the treatment with DOX alone. We previously reported that p21 prevented cell apoptosis, in correlation with its ability to bind CDK2 and to inhibit CDK2 kinase activity [11]. To examine the
CDK2 kinase activity, HCT116 (p53 / ) cell extracts were immunoprecipitated with an antibody against CDK2 and then CDK2 kinase activity was measured by using histone H1 as a substrate. A high level of CDK2 activity was detected in HCT116 (p53 / ) cells, following a treatment with DOX and then TNF-a, compared to other treatments. However, the amount of CDK2 expression was of the same extent after the treatment (Fig. 2). These results suggest that after exposure to DOX, surviving HCT116 (p53 / ) cells that showed a low degree of CDK2 activity are resistant to continuous DOX-induced apoptosis. However, when followed by stimulation of TNF-a increased the CDK2 activity. NFjB/RelA activity may be required for DOX-induced apoptosis in HCT116 (p53 / ) cells NFjB is a key mediator of signaling events to TNF-a and it plays an anti-apoptotic role in TNF-a-induced
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Fig. 2. TNF-a potentiates the CDK2 kinase activity. HCT116 (p53 / ) cells were treated with DOX (0.8 lg/ml) in Day1 alone or followed by a treatment with TNF-a (500 U/ml) in Day2, and with TNF-a (500 U/ml) in Day1 alone or followed by a treatment with DOX (0.8 lg/ml) in Day2. P21 and CDK2 expression was determined by Western blotting analysis. CDK2 kinase activity was measured by phosphorylation of histone H1 in anti-CDK2 immunoprecipitates in HCT116 (p53 / ) cells.
Fig. 3. Modulation of cell apoptosis by mutated IjB. HCT116 (p53 / ) cells were transiently transfected with vector (h) and plasmid containing mutated IjB (IjBSR) (j) in the presence of DOX (0.8 lg/ml) on Day1, TNF-a (500 U/ml) treatment on Day2, and treated with DOX on Day3. Cells were stained with Hoechst 33258. Apoptotic cells were counted under a microscope. Error bars indicate the standard deviation of three experimental results.
apoptosis [17,18]. As expected, transient expression of IjBSR efficiently enhanced TNF-a-induced apoptosis (data not shown). However, when HCT116 (p53 / ) cells were forced to transiently express IjBSR, the percentage of apoptotic cells following the treatment of DOX–TNF-a–DOX was remarkably reduced (26.0%) as compared to 41.3% in control cells without over-expression of IjBSR (Fig. 3). This suggests that NFjB/ RelA activation may be required for DOX-induced apoptosis. DOX plus TNF-a-induced apoptosis is prevented by over-expression of p21 To detect whether p21 protein played an important role in this pathway, here we transiently transfected cells with a p21 expression vector and examined the direct effect of p21 over-expression on the treatment of HCT116
(p53 / ) cells with DOX–TNF-a (Fig. 4A). Upon overexpression of p21, cell apoptosis rate (25.7%) was remarkably decreased in comparison to that in cells transfected with a control vector (43.1%). To examine whether high level of p21 restored a survival advantage by inhibiting CDK2 kinase activity, HCT116 (p53 / ) cell extracts were immunoprecipitated with an antibody against CDK2 and then CDK2 kinase activity was measured by using histone H1 as a substrate. A low level of CDK2 activity was detected in HCT116 (p53 / ) cells, after over-expression of exogenous p21. These results also indicate that CDK2 is a key player in cell apoptosis, and indeed transient over-expression of CDK2 and then enhanced CDK2 activity made cells hypersensitive to DOX-induced apoptosis (Fig. 4B).
Discussion In this study, we report that p53+/+ tumor cells were more sensitive to DOX-induced apoptosis than p53-deficient tumor cells, in agreement with previous studies. We also document here that treatment of p53-deficient cells with TNF-a between two DOX administrations remarkably increases the cytotoxic effect of chemotherapy agent DOX on tumor cells. Treatment of TNF-a resulted in decreased expression of p21 (waf1/cip1/sdi1) and an enhanced CDK2 activity. We present evidence that TNF-a may be involved in augmenting DOX-induced apoptosis. Tumor cells that have wild-type p53 were more sensitive to DOX-induced cell apoptosis, probably due to a plasma membrane protein whose induction by DOX correlated with activation of the p53-dependent apoptotic pathway [19]. The mechanism through which p53
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Fig. 4. P21 and CDK2 effects on cell apoptosis. (A) HCT116 (p53 / ) cells were transiently transfected with vector (h) and plasmid containing p21 (j) in the presence of DOX (0.8 lg/ml) on Day1, TNF-a (500 U/ml) treatment on Day2, and DOX treatment on Day3. (B) HCT116 (p53 / ) cells were treated with DOX (0.8 lg/ml) for 72 h following transfection with vector (h) and plasmid containing CDK2 (j). Cells were stained with Hoechst 33258. Apoptotic cells were counted under a microscope. Error bars indicate the standard deviation of three experimental results. P21 and CDK2 expression was determined by Western blotting analysis, while CDK2 kinase activity was measured by phosphorylation of histone H1 in antiCDK2 immunoprecipitates in HCT116 (p53 / ) cell extracts.
promotes apoptosis is dependent on the Apaf-1/caspase9 pathway and involves cytochrome c release from mitochondria [20]. Several potential downstream mediators such as Bax [21], CD95 (Fas/Apo-1) [22], Killer/DR5 [23], PERP [19], and p53AIP1 [24] appear to be involved in p53-dependent apoptosis. p53 plays an important role in the tumor therapy. However, it is reported that more than 60% human tumors have lost their wild-type p53 function. Increasing evidence suggests that deficiency of p53 in tumors constitutes resistance to chemotherapy and anti-angiogenic treatment [25–27]. Hence, efforts have been made to improve the efficacy of anti-tumor therapy on the tumors deficient in p53 expression, such as a combination of chemotherapy with transduction of E2F1, a cell cycle progression signal [28,29]. However, the molecular basis for the p53 / tumor resistance to chemotherapy has to be well defined. In this study, we found that treatment of DOX–TNF-a–DOX reversed the sensitivity to DOXinduced apoptosis in HCT116 (p53 / ) cells. This might represent a new strategy to improve the efficacy of therapeutic response on p53-deficient tumors. DOX, a widely used anti-tumor agent, targets the DNA topoisomerase II enzyme activity, which involves sequential DNA binding, cleavage of DNA phosphodiester backbone, and subsequently causes DNA breaks. It induces cell apoptosis due to aberrant progression of cell cycle [30,31]. Our previous data suggested that after DOX treatment, damaged cells that arrested their cell cycle did not undergo apoptosis. Whereas abnormal progression of cell cycle is responsible for DOX-induced apoptosis [10], CDK2 phosphorylation of pRB and
triggering cell cycle proceeded. CDK2 activity is required for DOX-induced apoptosis [10,11]. When HCT116 (p53 / ) cells were exposed to DOX, abnormal progression of cell cycle led to apoptosis due to accumulation of damaged DNA. As shown in Fig. 2, low CDK2 kinase activity was detected in surviving cells after 24 h DOX treatment. The present findings indicate that resting cells from the cycling are at least in part responsible for resistance to DOX treatment in HCT116 (p53 / ) cells. This idea is reinforced by promoting DOX-induced apoptosis by transient expression of wild-type CDK2 (Fig. 4), but not by that of dominant negative mutant CDK2 (data not shown). Unlike DOX, TNF-a-induced apoptosis through death receptor signaling but was independent of cell cycle. Activation of NFjB functions to protect cells in response to TNF-a stimulation. In other situations, however, NFjB contributes to apoptosis [18,32,33]. DNA damaging agents mediate the activation of NFjB; induction of topoisomerase II is correlated with NFjB activation and Fas ligand expression in response to some anti-tumor drugs is dependent on a functional NFjB activation [34]. Moreover, PerkinsÕ group found that expression of RelA/p65 cDNA increased the histone H1 kinase activity by association with the p300 coactivator [35]. In combination with our previous data it suggested that after 24 h treatment of DOX, the surviving HCT116 (p53 / ) cells resting at G1 or G2 phase resisted further DOX-induced apoptosis. A clear elevation of CDK2 activity regulation by TNF-a reversed the HCT116 (p53 / ) cellsÕ sensitivity to DOX-induced apoptosis. Thus, treatment of TNF-a
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increased CDK2 kinase activity (Fig. 2). The functional importance of NFjB activity in DOX-induced apoptosis was confirmed with transient over-expression of IjBSR resulting in a blocking of the effect on promoting DOXinduced apoptosis (Fig. 3). Together, our data suggested that activation of NFjB by TNF-a modulated the cell cycle regulatory protein CDK2 kinase activity in HCT116 (p53 / ) cells, in augmenting an effective therapeutic response to DOX. p21 is an inhibitor of all CDKs, which provides checkpoints that control entry into cell cycle. In the previous study, we demonstrated a decrease of histone H1 kinase activity by wild-type p21 in association with CDK2 but not by mutant p21s [11]. In this study, we observed that the p21 expression level was a key player in this pathway. As shown in Fig. 2 the amount of p21 expression was lower in the treatment with DOX followed by TNF-a than in the treatment with DOX alone. Lowing expression of p21 in HCT116 (p53 / ) cells may provide a good rationale for TNF-a treatment of tumors in combination with chemotherapeutic agent, such as DOX treatment. It is the same model as that recently proposed by Dr. Bendjennat [36]. It showed that low doses of UV led to p21 degradation and resulted in PCNA free from the sequestration of p21 and permitting PCNA to participate in DNA repair. We propose that the stimulation by TNF-a may induce p21 protein degradation. However, the biochemical basis of this hypothesis remains to be clarified in the further study. In this study, we found that p21Õs negative regulation on CDK2 was partly blocked by DOX–TNF-a treatment. In combination with the previously reported that the CDK2 activity was regulated by NFjB/RelA through interaction with the coactivator p300 and p21 disturbed the NFjB/RelA-associated cyclin-CDKs activity [19], we propose that TNF-a may play an important role in histone H1 kinase activity by decreasing the ability of p21 with CDK2. It is widely accepted that activation of NFjB functions to protect cells from apoptosis in response to TNF-a treatment. In this report, we found after exposing to DOX, treatment of TNF-a enhanced CDK2 activity. Elevation of CDK2 activity resulted in cellsÕ sensitization to DOX-mediate cell apoptosis. We propose that TNF-a/NFjB/RelA may play an important role in histone H1 kinase activity by blocking the interaction of p21 with CDK2. However, further studies should be extended to detect the interaction between TNF-a/NFjB/RelA and p21.
Acknowledgments We thank Dr. B. Vogelstein (Johns Hopkins University School of Medicine) for providing cell lines and
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Dr. Yamagishi (Kyoto University, Japan) for providing plasmids for this study.
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TNF-a promotes Doxorubicin-induced cell apoptosis and anti-cancer effect through downregulation of p21 in p53-deficient tumor cells Wei Cao a, Wan-Hao Chi a, Jun Wang b, Juan-Juan Tang b, Yan-Jun Lu b,* a b
Laboratory of Medical Molecular Virology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China Cancer Research Center, Celstar Bio-Pharmaceutical Co. Ltd., 1 Juli Road Pudong, Shanghai 201203, China Received 25 February 2005 Available online 22 March 2005
Abstract p53 is a key regulator in cell apoptosis, and cancer cells deficient in p53 expression fail to respond to chemotherapy. Here we show that effective Doxorubicin (DOX)-induced apoptosis is p53-dependent. However, an alternative treatment of DOX/TNF-a/ DOX restored sensitivity of p53-deficient cells to DOX-induced apoptosis. Treatment of cells with TNF-a resulted in a decrease of p21 (waf1/cip1/sdi1) expression following second dose of DOX. In previous work, we demonstrated that p21 suppressed DOX-induced apoptosis via its (cyclin-dependent kinase) CDK-binding and CDK-inhibitory activity. Thus, we propose that TNF-a enhances the anti-cancer effect of DOX through suppressing the anti-apoptotic activity of p21, and that a combined treatment TNF-a/Dox is an effective chemotherapeutic strategy for p53-deficient cancers. Ó 2005 Elsevier Inc. All rights reserved. Keywords: p53; p21; CDK2; TNF-a; NFjB/RelA/p65
TNF-a stimulates the cell response by binding to its receptor I and II. The NFjB family of transcription factors are key mediators of intracellular signaling events initiated by TNF-a [1]. NFjB generally exists as an inactive dimer sequestered in the cytoplasm by an inhibitor protein termed IjB. In response to TNF-a, activation of NFjB involves phosphorylation of IjB on two critical serine residues at codon 32 and 36. Once phosphorylated, IjB is subjected to ubiquitination at two lysine residues and subsequent degradation by the 26S proteasome. Unmasking IjB from NFjB allows it to translocate into the nucleus, where it binds to the target genes, and promotes transcription [2]. TNF-a shows a strong anti-tumor effect on many solid tumors. But its clinical application is limited by its se*
Corresponding author. Fax: +86 21 5080 3128. E-mail address: [email protected] (Y.-J. Lu).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.188
vere systemic side effects, before therapeutic doses could be reached [3,4]. Much effort has been paid on the development of less toxic recombinant TNF-a for a safe and systemic administration [5,6]. Our clinical phase II and phase III trials showed that systemic administration of TNF-a in combination with a chemotherapy agent Doxorubicin (DOX) for lung tumors remarkably enhanced the anti-tumor effect, as compared to the DOX treatment alone. In total, 44% of the 133 patients exhibited complete or partial remission of lung tumors in response to this combination therapy strategy [7–9]. Since it has been approved by the Chinese SDA, this innovative clinical application of TNF-a in combination with DOX may offer great opportunities for cancer patients. However, the molecular basis for the combination treatment has not been elucidated. DOX is the most widely used chemotherapy agent in the treatment of tumor. Recent data suggest that
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DOX-induced apoptosis is interconnected with the machinery of cell cycle control. Progression through G1 or G2 phase and entry into the S or M phase are tightly regulated by cyclin-dependent kinases (CDKs) [10]. P21 (waf1/cip1/sdi1), which acts primarily as a transcription target of p53, is an important cellular checkpoint protein for inhibition of complex cyclin-CDKs activities, which contributes to its suppressing effect on DOX-mediated apoptosis in human tumor cells [11,12]. In this study, we used HCT116 (p53+/+) cells and their matched HCT116 (p53 / ) cells in which the p53 gene has been deleted by homologous recombination. We demonstrated that unlike HCT116 (p53+/+) cells, HCT116 (p53 / ) cells were resistant to DOX-induced apoptosis. However, treatment with DOX followed by TNF-a sensitized HCT116 (p53 / ) cells to DOX-induced cell death by enhancing the CDK2 kinase activity. We present evidence that TNF-a may be involved in augmenting DOX-induced apoptosis.
Materials and methods Cells and transfection. Human colorectal cancer cell lines HCT116 (p53+/+) and HCT116 (p53 / ) were generous gifts of Dr. B. Vogelstein. The p21 and CDK2 expression plasmids have been described previously [10,11]. The plasmid expressing the IjB mutant/ IjBSR was provided by Dr. Yamagishi (Kyoto University, Kyoto, Japan). IjB super repressor (IjB-SR) containing the mutations at Ser 32, Ser 36 phosphorylation residues which cannot be phosphorylated and degraded is used as a dominant negative inhibitor of NF-jB. HCT116 cells were transiently transfected with expression constructs for p21, CDK2, and IjBSR, using the Effectene Transfection Kit (Qiagen, Chatsworth, CA), as described previously [10]. The efficiency of transfection was higher than 98%, which was detected by the proportion of green cells after transfection of GFP-expressing plasmid (pEGFP-c1) (Invitrogen, US) with the same protocol. All cells were cultured in DulbeccoÕs modified EagleÕs minimum essential medium (Hyclone, logan, UT) supplemented with 10% fetal bovine serum (Hyclone, logan, UT). Western blotting. Western blotting has been described previously [10,11]. A 40 lg aliquot of cell lysate was separated by 12% polyacrylamide–SDS gel electrophoresis and transferred to a Hybond-C Super membrane. p21 and CDK2 were detected with an anti-p21 antibody and an anti-CDK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. CDK2 kinase assay. Cell extracts (100 lg) were incubated with an anti-CDK2 antibody (SC-163), and the complexes were collected with 80 ll protein G-plus–agarose (Santa Cruz Biotechnology, Santa Cruz. CA). The cyclin-dependent kinases activity was measured by CDK assay kit (Upstate Biotechnology, Lake Placid, NY). Briefly, the immune complexes (40 lg) were mixed with [c-32P]ATP (3000 Ci/mmol), histone H1 (2 lg) as a substrate, and the reaction buffer containing inhibitors of other kinases. After incubation for 10 min at 30 °C, phosphorylated histone H1 was resolved by 16% polyacrylamide–SDS gel electrophoresis and detected by autoradiography. Measurement of cell apoptosis. Cells were treated with DOX at doses of 0.2 and 0.8 lg/ml, and with TNF-a (500 U/ml), respectively. Cells were washed with PBS, stained with the DNA-binding dye, Hoechst 33258 (0.5 lg/ml in PBS), and examined under a fluorescence microscope. Cells showing condensed nuclear morphology and nuclear lobulations were regarded as undergoing apoptosis.
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Results TNF-a sensitized HCT116 (p53 / ) cells to DOX-induced apoptosis P53 tumor suppressor is known to play an important role in potentiating apoptosis through a variety of mechanisms [13–15]. To confirm this point, we treated HCT116 (p53+/+) cells and HCT116 (p53 / ) cells with DOX for 72 h. And the nuclear morphology of the cells was examined by staining them with DNAbinding dye Hoechst 33258 and by microscopy, that was indicated as apoptotic cell death. As shown in Fig. 1A, DOX-induced apoptosis was higher in p53+/+ cells than in p53 / cells. This result suggests a requirement of p53 for DOX-induced cell apoptosis. To explore a condition for a killing effect on p53-deficient cells, we treated cells with different combinational use of alternating DOX and TNF-a treatment (i.e., Day1, DOX; Day2, DOX; Day3, DOX; Day1, DOX; Day2, TNF-a; Day3, DOX; Day1, TNF-a; Day2, DOX; Day3, DOX, Day1, DOX; Day2, DOX; Day3, TNF-a; and Day1, TNF-a; Day2, TNF-a; Day3, TNF-a). The results are shown in Fig. 1B. Treatment with DOX, and then incubating the cells with TNF-a treatment, was followed by another treatment of DOX (DOX–TNF–DOX treatment). This experimental protocol was not typical but based on our clinical application of TNF-a in combination with DOX [7–9]. Interestingly, HCT116 (p53 / ) cells were the most sensitive of all to the apoptotic effect of this DOX–TNF–DOX alternative treatment. To determine whether the cell death in HCT116 (p53 / ) cells caused by DOX was due to apoptosis, DNA fragmentations were examined in HCT116 (p53 / ) cells after treatment. The DNA ladders, a typical apoptotic morphology, were indicated as apoptotic cell death in the treatment with DOX–DOX–DOX, DOX–TNF–DOX, and DOX–DOX–TNF (Fig. 1C). Together, our data suggest that loss of p53 impairs DOX-induced cell apoptosis, but treatment of TNF-a enhanced the sensitivity to DOX-induced apoptosis in p53-deficient cells. This observation led us to suggest that alternative administration of TNF-a with DOX may be a more effective protocol for therapeutic treatment of p53-deficient tumors. The cells with functional p53 are more sensitive to DOX-induced apoptosis due to activation of p53-dependent apoptotic pathway. TNF-a enhanced CDK2 kinase activity in HCT116 (p53 / ) cells In previous studies, we showed that DOX is a cell cycle-dependent anti-tumor drug. P21 suppresses DOX-induced apoptosis by arresting cells at G1 or G2 phase [10,16]. The expression level of p21 is increased in
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Fig. 1. Treatment of TNF-a reversed HCT116 (p53 / ) cellsÕ sensitivity to DOX-induced apoptosis. (A) Fraction of apoptotic cells after 72 h of DOX (0.2 and 0.8 lg/ml) treatment in HCT116 (p53+/+) cells (d) and HCT116 (p53 / ) cells (s). (B) fraction of apoptotic cells after Day1, Day2, and Day3 treatment of DOX (0.8 lg/ml), or TNF-a (500 U/ml) in HCT116 (p53 / ) cells. (C) Agarose gel electrophoresis pattern of DNA obtained from HCT116 (p53 / ) cells with treatment of DOX (0.8 lg/ml) or TNF-a (500 U/ml). Cells were stained with Hoechst 33258 after treatments. Apoptotic cells were counted under a microscope. Error bars indicate the standard deviation of three experimental results.
p53-dependent and p53-independent pathways after DNA damage [11]. The combinational use of treatment with DOX and TNF-a resulted in significant different apoptosis induction in HCT116 (p53 / ) cells (Fig. 1). It led us to suggest that the sequence of DOX– TNF-a or TNF-a–DOX treatment plays an important role in this study. Then HCT116 (p53 / ) cells were treated with DOX alone or it was followed by a treatment with TNF-a and with TNF-a alone or followed by a treatment with DOX. The p21 protein amounts were assessed by Western blotting analysis. As shown in Fig. 2, the p21 expression level was elevated in HCT116 (p53 / ) cells after DOX treatment alone. Moreover, the amount of p21 expression was low in the treatment with DOX followed by TNF-a compared to the treatment with DOX alone. We previously reported that p21 prevented cell apoptosis, in correlation with its ability to bind CDK2 and to inhibit CDK2 kinase activity [11]. To examine the
CDK2 kinase activity, HCT116 (p53 / ) cell extracts were immunoprecipitated with an antibody against CDK2 and then CDK2 kinase activity was measured by using histone H1 as a substrate. A high level of CDK2 activity was detected in HCT116 (p53 / ) cells, following a treatment with DOX and then TNF-a, compared to other treatments. However, the amount of CDK2 expression was of the same extent after the treatment (Fig. 2). These results suggest that after exposure to DOX, surviving HCT116 (p53 / ) cells that showed a low degree of CDK2 activity are resistant to continuous DOX-induced apoptosis. However, when followed by stimulation of TNF-a increased the CDK2 activity. NFjB/RelA activity may be required for DOX-induced apoptosis in HCT116 (p53 / ) cells NFjB is a key mediator of signaling events to TNF-a and it plays an anti-apoptotic role in TNF-a-induced
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Fig. 2. TNF-a potentiates the CDK2 kinase activity. HCT116 (p53 / ) cells were treated with DOX (0.8 lg/ml) in Day1 alone or followed by a treatment with TNF-a (500 U/ml) in Day2, and with TNF-a (500 U/ml) in Day1 alone or followed by a treatment with DOX (0.8 lg/ml) in Day2. P21 and CDK2 expression was determined by Western blotting analysis. CDK2 kinase activity was measured by phosphorylation of histone H1 in anti-CDK2 immunoprecipitates in HCT116 (p53 / ) cells.
Fig. 3. Modulation of cell apoptosis by mutated IjB. HCT116 (p53 / ) cells were transiently transfected with vector (h) and plasmid containing mutated IjB (IjBSR) (j) in the presence of DOX (0.8 lg/ml) on Day1, TNF-a (500 U/ml) treatment on Day2, and treated with DOX on Day3. Cells were stained with Hoechst 33258. Apoptotic cells were counted under a microscope. Error bars indicate the standard deviation of three experimental results.
apoptosis [17,18]. As expected, transient expression of IjBSR efficiently enhanced TNF-a-induced apoptosis (data not shown). However, when HCT116 (p53 / ) cells were forced to transiently express IjBSR, the percentage of apoptotic cells following the treatment of DOX–TNF-a–DOX was remarkably reduced (26.0%) as compared to 41.3% in control cells without over-expression of IjBSR (Fig. 3). This suggests that NFjB/ RelA activation may be required for DOX-induced apoptosis. DOX plus TNF-a-induced apoptosis is prevented by over-expression of p21 To detect whether p21 protein played an important role in this pathway, here we transiently transfected cells with a p21 expression vector and examined the direct effect of p21 over-expression on the treatment of HCT116
(p53 / ) cells with DOX–TNF-a (Fig. 4A). Upon overexpression of p21, cell apoptosis rate (25.7%) was remarkably decreased in comparison to that in cells transfected with a control vector (43.1%). To examine whether high level of p21 restored a survival advantage by inhibiting CDK2 kinase activity, HCT116 (p53 / ) cell extracts were immunoprecipitated with an antibody against CDK2 and then CDK2 kinase activity was measured by using histone H1 as a substrate. A low level of CDK2 activity was detected in HCT116 (p53 / ) cells, after over-expression of exogenous p21. These results also indicate that CDK2 is a key player in cell apoptosis, and indeed transient over-expression of CDK2 and then enhanced CDK2 activity made cells hypersensitive to DOX-induced apoptosis (Fig. 4B).
Discussion In this study, we report that p53+/+ tumor cells were more sensitive to DOX-induced apoptosis than p53-deficient tumor cells, in agreement with previous studies. We also document here that treatment of p53-deficient cells with TNF-a between two DOX administrations remarkably increases the cytotoxic effect of chemotherapy agent DOX on tumor cells. Treatment of TNF-a resulted in decreased expression of p21 (waf1/cip1/sdi1) and an enhanced CDK2 activity. We present evidence that TNF-a may be involved in augmenting DOX-induced apoptosis. Tumor cells that have wild-type p53 were more sensitive to DOX-induced cell apoptosis, probably due to a plasma membrane protein whose induction by DOX correlated with activation of the p53-dependent apoptotic pathway [19]. The mechanism through which p53
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Fig. 4. P21 and CDK2 effects on cell apoptosis. (A) HCT116 (p53 / ) cells were transiently transfected with vector (h) and plasmid containing p21 (j) in the presence of DOX (0.8 lg/ml) on Day1, TNF-a (500 U/ml) treatment on Day2, and DOX treatment on Day3. (B) HCT116 (p53 / ) cells were treated with DOX (0.8 lg/ml) for 72 h following transfection with vector (h) and plasmid containing CDK2 (j). Cells were stained with Hoechst 33258. Apoptotic cells were counted under a microscope. Error bars indicate the standard deviation of three experimental results. P21 and CDK2 expression was determined by Western blotting analysis, while CDK2 kinase activity was measured by phosphorylation of histone H1 in antiCDK2 immunoprecipitates in HCT116 (p53 / ) cell extracts.
promotes apoptosis is dependent on the Apaf-1/caspase9 pathway and involves cytochrome c release from mitochondria [20]. Several potential downstream mediators such as Bax [21], CD95 (Fas/Apo-1) [22], Killer/DR5 [23], PERP [19], and p53AIP1 [24] appear to be involved in p53-dependent apoptosis. p53 plays an important role in the tumor therapy. However, it is reported that more than 60% human tumors have lost their wild-type p53 function. Increasing evidence suggests that deficiency of p53 in tumors constitutes resistance to chemotherapy and anti-angiogenic treatment [25–27]. Hence, efforts have been made to improve the efficacy of anti-tumor therapy on the tumors deficient in p53 expression, such as a combination of chemotherapy with transduction of E2F1, a cell cycle progression signal [28,29]. However, the molecular basis for the p53 / tumor resistance to chemotherapy has to be well defined. In this study, we found that treatment of DOX–TNF-a–DOX reversed the sensitivity to DOXinduced apoptosis in HCT116 (p53 / ) cells. This might represent a new strategy to improve the efficacy of therapeutic response on p53-deficient tumors. DOX, a widely used anti-tumor agent, targets the DNA topoisomerase II enzyme activity, which involves sequential DNA binding, cleavage of DNA phosphodiester backbone, and subsequently causes DNA breaks. It induces cell apoptosis due to aberrant progression of cell cycle [30,31]. Our previous data suggested that after DOX treatment, damaged cells that arrested their cell cycle did not undergo apoptosis. Whereas abnormal progression of cell cycle is responsible for DOX-induced apoptosis [10], CDK2 phosphorylation of pRB and
triggering cell cycle proceeded. CDK2 activity is required for DOX-induced apoptosis [10,11]. When HCT116 (p53 / ) cells were exposed to DOX, abnormal progression of cell cycle led to apoptosis due to accumulation of damaged DNA. As shown in Fig. 2, low CDK2 kinase activity was detected in surviving cells after 24 h DOX treatment. The present findings indicate that resting cells from the cycling are at least in part responsible for resistance to DOX treatment in HCT116 (p53 / ) cells. This idea is reinforced by promoting DOX-induced apoptosis by transient expression of wild-type CDK2 (Fig. 4), but not by that of dominant negative mutant CDK2 (data not shown). Unlike DOX, TNF-a-induced apoptosis through death receptor signaling but was independent of cell cycle. Activation of NFjB functions to protect cells in response to TNF-a stimulation. In other situations, however, NFjB contributes to apoptosis [18,32,33]. DNA damaging agents mediate the activation of NFjB; induction of topoisomerase II is correlated with NFjB activation and Fas ligand expression in response to some anti-tumor drugs is dependent on a functional NFjB activation [34]. Moreover, PerkinsÕ group found that expression of RelA/p65 cDNA increased the histone H1 kinase activity by association with the p300 coactivator [35]. In combination with our previous data it suggested that after 24 h treatment of DOX, the surviving HCT116 (p53 / ) cells resting at G1 or G2 phase resisted further DOX-induced apoptosis. A clear elevation of CDK2 activity regulation by TNF-a reversed the HCT116 (p53 / ) cellsÕ sensitivity to DOX-induced apoptosis. Thus, treatment of TNF-a
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increased CDK2 kinase activity (Fig. 2). The functional importance of NFjB activity in DOX-induced apoptosis was confirmed with transient over-expression of IjBSR resulting in a blocking of the effect on promoting DOXinduced apoptosis (Fig. 3). Together, our data suggested that activation of NFjB by TNF-a modulated the cell cycle regulatory protein CDK2 kinase activity in HCT116 (p53 / ) cells, in augmenting an effective therapeutic response to DOX. p21 is an inhibitor of all CDKs, which provides checkpoints that control entry into cell cycle. In the previous study, we demonstrated a decrease of histone H1 kinase activity by wild-type p21 in association with CDK2 but not by mutant p21s [11]. In this study, we observed that the p21 expression level was a key player in this pathway. As shown in Fig. 2 the amount of p21 expression was lower in the treatment with DOX followed by TNF-a than in the treatment with DOX alone. Lowing expression of p21 in HCT116 (p53 / ) cells may provide a good rationale for TNF-a treatment of tumors in combination with chemotherapeutic agent, such as DOX treatment. It is the same model as that recently proposed by Dr. Bendjennat [36]. It showed that low doses of UV led to p21 degradation and resulted in PCNA free from the sequestration of p21 and permitting PCNA to participate in DNA repair. We propose that the stimulation by TNF-a may induce p21 protein degradation. However, the biochemical basis of this hypothesis remains to be clarified in the further study. In this study, we found that p21Õs negative regulation on CDK2 was partly blocked by DOX–TNF-a treatment. In combination with the previously reported that the CDK2 activity was regulated by NFjB/RelA through interaction with the coactivator p300 and p21 disturbed the NFjB/RelA-associated cyclin-CDKs activity [19], we propose that TNF-a may play an important role in histone H1 kinase activity by decreasing the ability of p21 with CDK2. It is widely accepted that activation of NFjB functions to protect cells from apoptosis in response to TNF-a treatment. In this report, we found after exposing to DOX, treatment of TNF-a enhanced CDK2 activity. Elevation of CDK2 activity resulted in cellsÕ sensitization to DOX-mediate cell apoptosis. We propose that TNF-a/NFjB/RelA may play an important role in histone H1 kinase activity by blocking the interaction of p21 with CDK2. However, further studies should be extended to detect the interaction between TNF-a/NFjB/RelA and p21.
Acknowledgments We thank Dr. B. Vogelstein (Johns Hopkins University School of Medicine) for providing cell lines and
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Dr. Yamagishi (Kyoto University, Japan) for providing plasmids for this study.
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