Bax Induction Activates Apoptotic Cascade via Mitochondrial Cytochrome c Release and Bax Overexpression Enhances Apoptosis Induced by Chemotherapeutic Agents in DLD-1 Colon Cancer Cells

Cancer cells express different levels of apoptosis-promoting Bax protein. The present study evaluated whether induction of Bax initiates apoptosis and whether Bax overexpression enhances apoptosis induced by several chemotherapeutic agents in DLD-1 colon cancer cells, which originally express a high level of endogenous Bax protein and a low level of Bcl-2 protein. To investigate these two points, parental DLD-1 cells were transfected with the Tet-On Bax induction system ( pTet-On and pTRE-Bax plasmids), and stable transduced cells were obtained. Induction of Bax by the Tet-On system initiated cytochrome c release from mitochondria, caspase-3 activation, and apoptosis to some extent in DLD-1 cells. Apoptosis induced by a chemotherapeutic agent, 5-fluorouracil, mitomycin C, paclitaxel, doxorubicin, or cisplatin, was enhanced by Bax overexpression. These findings suggest that Bax -overexpression-based gene therapy combined with chemotherapy would be effective in the treatment of colon cancer.     

Bax-induction alone is sufficient to activate apoptosis cascade in wild-type Bax-bearing K562 cells, and the initiation of apoptosis requires simultaneous caspase activation

It has been reported that expression of Bax by Tet-On system induces apoptosis in Jurkat cells. The parental Jurkat cells have mutation of Bax gene and do not express Bax protein. Wild-type Bax-bearing cells express endogenous Bax protein and it is still unclear whether overexpression of Bax alone can sufficiently induce apoptosis in these cells in the absence of any cytotoxic stimulus. To investigate this, wild-type Bax-bearing K562 cells were transfected with Tet-On Bax-inducible system (pTet-On and pTRE-Bax plasmids), and Bax-inducible stable cell lines were established. Overexpression of Bax in wild-type Bax-bearing K562 cells without any cyctotoxic signal resulted in increase of apoptotic cells, caspase-3 activation, mitochondrial release of cytochrome c, and mitochondrial membrane potential change. Western blotting and confocal microscopy revealed that overexpression of Bax was detected in mitochondria. A pancaspase inhibitor, zVAD-fmk, which has no effect on mitochondrial cytochrome c release and mitochondrial membrane potential change inhibited the apoptotic events in the presence of overexpressed Bax in mitochondria. These findings suggest that Bax protein, when present above a threshold level, is sufficient to trigger an apoptosis cascade, and its initiation requires simultaneous caspase activation probably not mediated by mitochondrial cytochrome c release and mitochondrial membrane potential change in K562 cells.     

Bax overexpression enhances cytochrome c release from mitochondria and sensitizes KATOIII gastric cancer cells to chemotherapeutic agent-induced apoptosis

To evaluate whether overexpression of Bax, an apoptosis-promoting gene, sensitizes KATOIII gastric cancer cells to apoptosis induced by chemotherapeutic agents, three stable cell lines of KATOIII transfected with Bax (KATOIII-Bax), Bcl-2 (KATOIII-Bcl-2), or control pCI-neo expression vector (KATOIII-pCI-neo) were established. The cells were treated with paclitaxel, 5-fluorouracil, or doxorubicin, and the apoptotic response was measured. Our results showed that the sensitivity of the KATOIII-Bax cells to chemotherapeutic agents was enhanced compared with that of the KATOIII-pCI-neo cells, and the KATOIII-Bcl-2 cells were more resistant to these agents. Western blotting revealed that cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells. Significant increase of cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was detected 24 h after exposure to chemotherapeutic agents, when apoptotic cells were less than 10%. The cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells at all time points examined after exposure to chemotherapeutic agents. Marked activation of caspase-3 in the KATOIII-Bax cells was observed 48 h and 72 h after exposure to chemotherapeutic agents compared with that in the KATOIII-pCI-neo cells. Consistently, zVAD-fmk, a pancaspase inhibitor, repressed the paclitaxel-induced apoptosis. In addition, Bcl-2 overexpression strongly blocked KATOIII cell apoptosis by inhibiting the cytochrome c release from mitochondria and caspase-3 activation. These findings suggest that cytochrome c release is a major mechanism of apoptotic response and Bax overexpression sensitizes KATOIII cells to chemotherapeutic agent-induced apoptosis through enhancing the release of cytochrome c from mitochondria.     

Relative level of expression of Bax and Bcl-XL determines the cellular fate of apoptosis/necrosis induced by the overexpression of Bax.

The Bax protein plays a critical role in the apoptosis of cancers induced by radiotherapy or chemotherapy, which induce both apoptosis and necrosis. We transduced various glioblastoma cells with the Bax gene via an adenoviral vector and found that A-172 cells led to necrotic cell death, while U251 cells apoptotic cell death, even though a similar level of Bax protein was introduced. A-172 cells displayed a much higher constitutive expression of the Bcl-XL protein compared with that of U251 cells. Upon simultaneous overexpression of the Bcl-XL and Bax proteins in the U251 cells, Bax-induced apoptosis of U251 cells was suppressed and an increase in the number of necrotic cells was seen. Moreover, induction of a higher amount of Bax protein in A-172 cells increased the percentage of apoptotic cells. In conclusion, if a cancerous cell expresses a high enough amount of Bax to undergo death, apoptosis will be induced. If a cancerous cell expresses a level of Bcl-XL which prevents Bax-induced apoptosis, the overexpression of Bax leads to necrotic cell death.     

Bax:Bcl-2 ratio modulation by bryostatin 1 and novel antitubulin agents is important for susceptibility to drug induced apoptosis in the human early pre-B acute lymphoblastic leukemia cell line, Reh

The ratio of Bax to Bcl-2 protein can determine whether cells will die via apoptosis or be protected from it. Reh was found to express a high basal level of Bcl-2 but was lacking of Bax protein expression. Treatment with bryostatin 1 induced a down-regulation in Bcl-2 protein that was not accompanied by an obvious Bax protein induction or apoptosis. These results suggest that a decreased level of Bcl-2 alone in this cell line is not sufficient for apoptosis induction. In an effort to identify the mechanism whereby apoptosis could be induced in this ALL model, we treated Reh cells with three microtubule inhibitors: dolastatin 10, auristatin PE and vincristine, in the presence and absence of bryostatin 1. When used alone, only dolastatin 10 induced apoptosis that was detected morphologically, and by flow cytometry. Western blots revealed that dolastatin 10-induced apoptosis was accompanied by the induction of Bax protein and the reduction in Bcl-2 protein. Auristatin PE and vincristine induced both Bax and Bcl-2 protein, leaving the Bax:Bcl-2 ratio constant. Reh cells pretreated for 24 h with bryostatin 1 followed by dolastatin 10, auristatin PE or vincristine showed significant apoptosis which was accompanied by Bcl-2 protein down regulation and Bax protein up regulation. We conclude that: (1) expression of bax is necessary for apoptosis-induction in this model; (2) a decrease in Bcl-2 level alone is not sufficient and might not be necessary for apoptosis-induction; and (3) the ratio of Bax:Bcl-2 plays a critical role in susceptibility to apoptosis in Reh cells. The results from this study should prove useful in guiding the clinical application of these novel agents in the treatment of acute lymphoblastic leukemia.     

Bax translocation is crucial for the sensitivity of leukaemic cells to etoposide-induced apoptosis

Bax translocation from cytosol to mitochondria is believed to be a crucial step for triggering cytochrome c release from mitochondria. However, it is unclear whether Bax translocation is associated with Bax induction by DNA damaging agents. The induction of Bax in response to DNA damaging agents has been considered to be linked with p53. In this study, we used the p53 negative human chronic myeloid leukaemia K562 cell line. Bax up-regulation occurred at the whole cell level after DNA damage induced by etoposide. However, after incubation with etoposide, Bax failed to translocate to mitochondria and as a result, the apoptotic process was blocked. A Bax stable transfectant, the K/Bax cell line, expressed more Bax protein in the cytosol, mitochondria and nuclei. This Bax overexpression induced cytochrome c release, a reduction of cytochrome c oxidase activity and mitochondrial membrane potential (Delta(Psi)m). However, Bax-induced apoptosis was blocked downstream of mitochondria in K562 cells. The increased levels of mitochondrial Bax sensitized cells to etoposide-induced activation of caspases-2, -3 and -9 and apoptosis. However, after transient transfection with the Apaf-1 gene, K/Bax cells were sensitized to etoposide-induced caspase activation and apoptosis to a larger extent compared with Bax or Apaf-1 transfection alone. We therefore conclude that two mechanisms contribute to the resistance of K562 cells to etoposide-induced apoptosis; firstly failure of Bax targeting to mitochondria and, secondly, deficiency of Apaf-1. Uncoupling of Bax translocation from Bax induction can occur in response to etoposide-induced DNA damage.     

Human colon cancer cells lacking Bax resist curcumin-induced apoptosis and Bax requirement is dispensable with ectopic expression of Smac or downregulation of Bcl-XL

Multiple apoptotic stimuli induce conformational changes in Bax, a proapoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemoresistance in colon adenocarcinomas. Curcumin, a dietary compound from turmeric, is known to induce apoptosis in a variety of cancer cells. To understand the role of Bax in curcumin-induced apoptosis we used HCT116 human colon cancer cells with one allele of Bax gene (Bax+/-) and Bax knockout HCT116 (Bax-/-) cells in which Bax gene is inactivated by homologous recombination. Cell viability decreased in a concentration-dependent manner in Bax+/- cells treated with curcumin (0-50 microM) whereas only minimal changes in viability were observed in Bax-/- cells upon curcumin treatment. In Bax-/- cells curcumin-induced activation of caspases 9 and 3 was blocked and that of caspase 8 remained unaltered. Curcumin-induced release of cytochrome c, Second mitochondria derived activator of caspase (Smac) and apoptosis inducing factor (AIF) was also blocked in Bax-/- cells and reintroduction of Bax, downregulation of the antiapoptotic protein Bcl-XL by antisense DNA as well as the overexpression of Smac, highly sensitized the Bax-/- cells toward curcumin-induced apoptosis. There was no considerable difference in the percentage of apoptotic cells in Bak RNAi transfected Bax+/- or Bax-/- cells treated with curcumin when compared with their corresponding vector transfected cells treated with curcumin. The present study demonstrates the role of Bax but not Bak as a critical regulator of curcumin-induced apoptosis and implies the potential of targeting antiapoptotic proteins like Bcl-XL or overexpression of proapoptotic proteins like Smac as interventional approaches to deal with Bax-deficient chemo-resistant cancers for curcumin-based therapy.     

Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1's inhibitory effect on Bak

The intrinsic mitochondrial apoptotic pathway acts through two core pro-apoptotic proteins Bax (Bcl2-associated X protein) and Bak (Bcl2-antagonist/killer 1). Although Bax and Bak seem to have redundant roles in apoptosis, accumulating evidence also suggests that they might not be interchangeable under certain conditions, at least in some human cell lines. Here we report the generation of Bak knockout as well as BaxBak double knockout HCT116 human colon carcinoma cells. We show that Bak is dispensable for apoptosis induced by a variety of stimuli including ABT-737 but not for fluorouracil-induced apoptosis. In addition, Bax deficiency only provides partial protection against camptothecin and cisplatin-induced apoptosis and no protection against killing by Puma or ABT-737 plus Noxa overexpression. Moreover, Bak is activated normally in response to many chemotherapeutic drugs in the presence of Bax, but remains kept in check by Mcl-1 in the absence of Bax. Our data suggest that Bax and Bak are functionally redundant, but they are counteracted by distinct anti-apoptotic Bcl-2 family proteins in different species.     

Pinocembrin triggers Bax-dependent mitochondrial apoptosis in colon cancer cells

Bioflavanoids are the major pigments in plants with multitude of biological activities including inhibition of proliferation or induction of apoptosis in tumor cells. Even though the safety records of most flavanoids are exceptional, its therapeutic use is still in its infancy. We have isolated pinocembrin (5,7-dihydroxyflavanone) from Alpinia galanga that showed cytotoxicity against a variety of cancer cells including normal lung fibroblasts with relative nontoxicity to human umbilical cord endothelial cells. The compound induced loss of mitochondrial membrane potential with subsequent release of cytochrome c and processing of caspase-9 and -3 in colon cancer cell line HCT 116. Processing of caspase-8 was minimal. The initial trigger for mitochondrial apoptosis appears to be by the translocation of cytosolic Bax protein to mitochondria. Overexpression of proapoptotic Bax protein sensitized the colon cancer cells to pinocembrin-induced apoptosis and Bax knockout cells were resistant to pinocembrin-induced apoptosis. Antiapoptotic protein Bcl-X(L) only partially prevented apoptosis induced by this compound. The Bax-dependent cell death involving classical cytochrome c release and processing of caspase-9 and -3 suggests that pinocembrin is a classical mitochondrial apoptosis inducer. But the failure of Bcl-X(L) overexpression to completely prevent apoptosis induced by this compound suggests that pinocembrin is capable of triggering mitochondrial-independent cell death that needs to be clarified. The existence of cell death upon Bcl-X(L) overexpression is a promising feature of this compound that can be exploited against drug resistant forms of cancer cells either alone or in combination with other drugs.     

Formation of nuclear Bax/p53 complexes is associated with chemotherapy induced apoptosis

Mechanisms by which chemotherapeutic agents induce apoptosis are not completely understood. Current knowledge of the actual pharmacologic effects of chemotherapy and their biochemical mechanisms are better understood than the downstream events, which initiate the apoptotic cascade. The chemotherapeutic agent cisplatin causes DNA damage and can induce apoptosis in several types of human cancers. We found the formation of previously unreported nuclear complexes between the tumor suppressor protein p53 and the pro-apoptotic protein Bax, in human melanoma cell lines induced into apoptosis following cisplatin exposure. These detergent resistant complexes were detected: after wild type (wt) p53 and Bax increased in the nucleus; at the same time when active cytoplasmic apoptosis related protease, caspase 3/CPP32 appeared; and prior to the detection of apoptotic DNA fragmentation. Three channel fluorescence laser scanning confocal image microscopy revealed that the nuclear Bax/p53 complexes remained in the nucleus and localized proximal to DNA fragmentation sites as assayed by TUNEL after cisplatin exposure. Two human melanoma cell lines, expressing wt p53, were induced into apoptosis after cisplatin exposure, however they differed in the timing of this induction. In both cell lines the formation of nuclear Bax/p53 co-immunoprecipitable complexes correlated with the timing of the induction of apoptosis. The degree of apoptosis induced by different concentrations of cisplatin correlated with the amount of nuclear Bax/p53 complexes. The co-immunoprecipitation of Bax and p53 was found regardless of the antibodies tested and was specific since Bcl-xL/p53 complexes were not detected. Additionally, the human prostate cancer cell line, LNCaP, also formed nuclear Bax/p53 complexes only after apoptosis was induced by paclitaxel.     

hTERT: a novel endogenous inhibitor of the mitochondrial cell death pathway

hTERT is the catalytic subunit of the telomerase and is hence required for telomerase maintenance activity and cancer cell immortalization. Here, we show that acute hTERT depletion has no adverse effects on the viability or proliferation of cervical and colon carcinoma cell lines, as evaluated within 72 h after transfection with hTERT-specific small interfering RNAs (siRNAs). Within the same time frame, hTERT depletion facilitated the induction of apoptotic cell death by cisplatin, etoposide, mitomycin C and reactive oxygen species, yet failed to sensitize cells to death induction via the CD95 death receptor. Experiments performed with p53 knockout cells or chemical p53 inhibitors revealed that p53 was not involved in the chemosensitizing effect of hTERT knockdown. However, the proapoptotic Bcl-2 family protein Bax was involved in cell death induction by hTERT siRNAs. Depletion of hTERT facilitated the conformational activation of Bax induced by genotoxic agents. Moreover, Bax knockout abolished the chemosensitizing effect of hTERT siRNAs. Inhibition of mitochondrial membrane permeabilization by overexpression of Bcl-2 or expression of the cytomegalovirus-encoded protein vMIA (viral mitochondrial inhibitor of apoptosis), which acts as a specific Bax inhibitor, prevented the induction of cell death by the combination of hTERT depletion and chemotherapeutic agents. Altogether, our data indicate that hTERT inhibition may constitute a promising strategy for facilitating the induction of the mitochondrial pathway of apoptosis.     

Overexpression of histone deacetylase 1 confers resistance to sodium butyrate-mediated apoptosis in melanoma cells through a p53-mediated pathway

Melanoma cells typically express wild-type p53, yet they are notoriously resistant to DNA-damaging agents. Here, we show that sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, induced apoptosis in human melanoma cells in a dose- and time-dependent manner. Apoptosis was associated with HDAC1-dependent induction of Bax and acetylation of p53. Down-regulation of HDAC1 by an antisense vector sensitized the cells to NaB-induced apoptosis, whereas its overexpression conferred resistance to this agent. Increased HDAC1 levels and activity impaired NaB-mediated activation of Bax promoter and Bax protein levels. Finally, using p53-null melanoma cell line and RNA interference in cells expressing wild-type p53 protein, we show that Bax induction and NaB-mediated apoptosis is p53 dependent.     

The in vitro and in vivo effects of 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), a specific inhibitor of phosphatidylinositol 3'-kinase, in human colon cancer cells.

PURPOSE: Phosphatidylinositol 3'-kinase (PI3K) and Akt/protein kinase B(PKB) allow for escape from apoptosis in various human cancer cells. We postulated that 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), a PI3K inhibitor, should inactivate Akt/PKB, consequently inhibiting cell proliferation and inducing apoptosis in vitro and in vivo. EXPERIMENTAL DESIGN: Human colon cancer cell lines (DLD-1, LoVo, HCT15, and Colo205) and their mouse xenografts (DLD-1 and LoVo) were used in this study. The expression of phosphorylated Akt (Ser(473)) and apoptosis in cancer cells were determined by immunoblotting and immunohistochemistry. To evaluate the activity of caspase-3 in culturing cells, the caspase colorimetric assay was also performed. RESULTS: LY294002 demonstrated a remarkable growth-inhibitory and apoptosis-inducing effect in these colon cancer cell lines, with decreased expression of phosphorylated Akt (Ser(473)). However, there was a great discrepancy between the sensitivity for LY294002 and the level of expression of phosphorylated Akt. Although the LoVo and Colo205 cells exhibited high sensitivity to LY294002 with increased apoptosis, the DLD-1 and HCT15 cells did not show rapid induction of apoptosis. The caspase-3 activity was significantly high in the LoVo cells but not in the DLD-1 cells. In the experiments using mouse xenografts, we found that LY294002 administration in vivo also resulted in suppression of tumor growth and induction of apoptosis, especially in the LoVo tumors, and therefore showed remarkable effectiveness in the mouse peritonitis carcinomatosa model. CONCLUSIONS: PI3K-Akt/PKB plays an important role in colon cancer development and progression by helping to promote cell growth and allowing cells to escape apoptosis. These results propose the usefulness of LY294002 as an antitumoral agent for patients with colorectal cancer.     

Multidomain Bcl-2 homolog Bax but not Bak mediates synergistic induction of apoptosis by TRAIL and 5-FU through the mitochondrial apoptosis pathway

The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.     

Dimerization of mitochondrial Bax is associated with increased drug response in Bax-transfected A253 cells.

Human head and neck squamous cell carcinoma A253 cells, which do not express p53 and p21 proteins, were engineered to stably express about 50-fold higher level of Bax protein (A253/Bax) than the mock-transfected (A253/vec) or parental cells. Using these cell lines, studies were carried out to evaluate the role of Bax in response to anticancer drugs and to study the associated mechanisms. A253/Bax cells exhibited a significant increase in in vitro sensitivity to various anticancer drugs, including tomudex (9.5-fold), SN-38 (13.8-fold), doxorubicin (7.9-fold), taxol (3.1-fold), 5-FU (2.7-fold), and 5-FU/LV (4.5-fold). Increased level of drug-induced apoptosis was observed in A253/Bax cells in a drug concentration-dependent manner. In untreated A253/Bax cells, Bax was expressed in a monomeric state. Treatment with tomudex induced the formation of Bax dimer in a drug concentration-dependent manner. Dimerization of Bax occurred only in mitochondria, while the cytosolic Bax was retained in the monomeric state. Low level of Bax dimerization was also detected in parental A253 cells following tomudex exposure. In addition, Bax dimer formation was associated with mitochondrial cytochrome c release and activation of caspases in A253/Bax cells. The data suggest that Bax overexpression increases drug response by enhancing drug-induced apoptosis. Furthermore, dimerization of mitochondrial Bax and downstream mechanisms are associated with drug-induced apoptotic cell death and increased drug sensitivity.     

Induction of apoptosis and down-regulation of Bcl-XL in cancer cells by a novel small molecule, 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol

In a search for new anticancer agents, we identified that 2[[3-(2,3-dichlorophenoxy) propyl]amino]ethanol (2,3-DCPE) induced apoptosis more effectively in various cancer cells than in normal human fibroblasts. We further evaluated the cell-killing effects of this compound in vitro in several human cancer cell lines and normal human fibroblasts. A cell viability assay showed that IC(50)s for human colon cancer cell lines LoVo and DLD-1, for human lung cancer cell lines H1299 and A549, and for normal human fibroblasts were 0.89, 1.95, 2.24, 2.69, and 12.6 micro M, respectively. Subsequent studies revealed that 2,3-DCPE could cause cleavage of caspase-8, caspase-3, caspase-9, and poly(ADP-ribose) polymerase and release of cytochrome c in cancer cells but not in normal human fibroblasts. Our data also showed that 2,3-DCPE attenuated the protein level of Bcl-XL and that apoptosis induction by 2,3-DCPE could be blocked by enforced overexpression of Bcl-XL. Our results suggest that 2,3-DCPE might be a potential new anticancer agent.     

Enhanced sensitivity to cis-diamminedichloroplatinum(II) of a human carcinoma cell line with mutated p53 gene by cyclin-dependent kinase inhibitor p21(WAF1) expression

p53-mediated induction of p21(WAF1), a cyclin-dependent protein kinase inhibitor, is known to protect cancer cells from the cytotoxic effects of anti-cancer drugs or gamma-irradiation. Since the p53 gene is frequently inactivated in cancer cells, we examined whether p21(WAF1) expression may alter the sensitivity of cancer cells with mutated p53 gene to anti-cancer drugs. Cells of a colon cancer cell line DLD-1 were transfected with p21(WAF1) expression vector controlled by a tetracycline-repressable promoter and transfectants were cloned (Dp21-1). p21(WAF1) expression induced by removal of tetracycline from culture media repressed cell proliferation and resulted in altered cell shape, suggesting induction of differentiation. Dp21-1 cells with p21(WAF1) expression were more sensitive to cis-diamminedichloroplatinum(II) (CDDP) (IC(50) value, 10 microM) than those without p21(WAF1) expression (IC(50), 22 microM). Sensitivity to doxorubicin was not different between Dp21-1 cells with and without p21(WAF1) expression. DNA ladder formation was observed in Dp21-1 cells treated with CDDP, indicating that the enhanced sensitivity to CDDP involves apoptosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosolic protein revealed that subunit protein bands with M(r) 55 kDa and 44 kDa were markedly increased in cells with p21(WAF1) expression. By immunoblotting, these proteins were identified as c-Jun N-terminal kinase (JNK) 2 and p38 mitogen-activated protein kinase (MAPK) delta, respectively, both of which are believed to be involved in apoptosis induction by CDDP. These results suggest that p21(WAF1) may enhance the sensitivity of colon cancer cells with mutated p53 gene to CDDP, possibly through the JNK and p38 MAPK pathways.     

Induction of p53-independent Apoptosis Associated with G2M Arrest Following DNA Damage in Human Colon Cancer Cell Lines

The tumor suppressor p53 protein induces apoptosis in response to various kinds of DNA damage in normal cells, but it is still unclear whether or not apoptosis induced by DNA damage correlates with the p53 status in tumor cells. We determined the status of p53 by functional analysis of separated alleles in yeast in five human colon cancer cell lines, SW-480, SW-620, DLD-1, COLO320 and LS174T and investigated whether p53 is necessary for apoptosis and cell cycle arrest after treatment of the cells with a DNA-damaging agent, etoposide (VP-16), or γ-irradiation. Of these cell lines, only LS174T expresses a functional p53. Apoptosis was detected in SW-480 and COLO320 cell lines, but not in the other cell lines, including LS174T cell line with a normal p53 function. Furthermore, cell cycle analysis revealed accumulation in the G2M phase preceding induction of apoptosis in SW-480 and COLO320 cells, but not in the other cells. These results suggest that apoptotic induction by DNA damage is not necessarily related to p53 status and that induction of p53-independent apoptosis following DNA damage may correlate with G2M arrest in the cell cycle, at least in the colon cancer cell lines used in this study.