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Laboratory of Apoptosis Research [M. A. S.] and Laboratory of Tumor Biology [S. U., B. V.], Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-fodrin, and cytokeratins-18 and -19, thereby facilitating the preparatory dismantling of dying cells for efficient phagocytosis by neighboring cells or antigen-presenting cells in a process that circumvents physiologically unnecessary inflammation. Both caspase-dependent and -independent pathways of cell death may be relevant for cancer research (reviewed in Ref. 1 ). Cell death induced by ionizing radiation is believed to occur through classical reproductive death (mitotic catastrophe) and/or apoptosis, depending on cell type. Reproductive cell death results from a failure of DNA-damaged cells to faithfully repair DNA lesions, eventually resulting in imbalances in biochemical reactions, and can occur as late as four to five cell divisions after treatment with ionizing radiation (reviewed in Refs. 2 , 3 ). Interestingly, reproductive cell death frequently culminates with secondary intrinsic apoptosis in many cell types (reviewed in Ref. 4 ).
Apoptosis can be induced through two main pathways: (a) release of apoptogenic molecules from mitochondria on recognition of intracellular stress (the intrinsic pathway), and (b) ligation of death receptors on the cell surface (the extrinsic pathway; reviewed in Ref. 5
). In the intrinsic pathway, release of cytochrome c into the cytosol activates caspase-9 in a complex with the APAF-1 protein, subsequently inducing activation of downstream effector caspases. In one well-studied example of the extrinsic pathway, aggregation of the CD95 death receptor induces recruitment of the protein FADD and subsequent activation of caspase-8 in a death-inducing signaling complex (reviewed in Ref. 6
). Active caspase-8 truncates and thereby activates the protein Bid, which then translocates to mitochondria and induces release of cytochrome c. In some cell types, caspase-8 can also directly activate a sufficient amount of caspase-3 in a more direct route to apoptosis induction. In addition, CD95 may mediate caspase-independent cell death through recruitment of the protein RIP (7)
. Many tumor cell types are relatively resistant to signaling through CD95, although they express CD95 on their cell surface (8)
. Treatment with IFN-
(9
, 10)
, IL-123
(11
, 12)
, IL-18 (13)
, or agents that inhibit protein synthesis, such as cycloheximide and actinomycin D, can sensitize most resistant cell types, partially through CD95 up-regulation (in response to IFN-, IL-12, and IL-18) but also through a poorly understood mechanism that is independent of CD95 expression levels. Through production of cytokines, CTLs can induce CD95 expression on target cells that can subsequently facilitate exocytosis-independent induction of apoptosis (14)
. Increased surface expression of CD95 endows an enhanced "sentinel" function, allowing target cells to more readily receive apoptotic signals delivered during encounter with CTLs and natural killer cells expressing CD178, the natural ligand for CD95 (5)
.
In a subset of normal tissues, cell death can be induced by activation of the p53 tumor suppressor protein (4) . Activation of p53 protein induces its function as a target-specific regulator of gene transcription, consequently modulating the expression of downstream proteins, many of which can promote apoptosis, cell cycle arrest, or DNA repair, depending on cell type and context. The p53 protein is strongly activated by DNA double-strand breaks. Double-strand breaks occur after treatment with ionizing radiation or genotoxic drugs as well as during DNA replication. Because neither cell cycle arrest nor apoptosis favors tumorigenesis, the p53 gene is mutated or lost in more than half of human tumors, and p53 protein activity may be inhibited in some of the tumors that retain exclusively wild-type alleles of the p53 gene (15 , 16) . In most tumor types, however, a lethal dose of ionizing radiation induces cell death regardless of p53 functional status (4 , 17) . Furthermore, ionizing radiation appears to induce a primary wave of apoptosis only in tumor types that originate from tissues that are susceptible to p53-dependent apoptosis, i.e., cells of the hematopoietic system, epithelium of the small intestine, hair follicles, and spermatogonia (4) . In addition to directly inducing cell cycle arrest and DNA repair, p53 may exert additional effects in cells that survive its activation, but these effects are not well characterized.
p53 (18)
, as well as nuclear factor-
B (19
, 20)
, is a transcriptional regulator of the cd95 gene. It has recently become clear that loss of p53 activity prevents DNA-damaged tumor cells from increasing their expression of CD95 (21, 22, 23)
. To date, however, the functional relevance of this loss of regulation of CD95 expression has been examined almost exclusively through correlative data. Genotoxin-induced, p53-dependent up-regulation of CD95 in vitro has previously been reported to correlate with increased apoptosis in response to treatment with agonistic anti-CD95 antibody (24
, 25) . Additionally, we have recently reported that infection of cells with simian virus-40 results in loss of p53-dependent up-regulation of CD95 expression that correlates with loss of enhancement of anti-CD95 antibody-induced apoptosis after DNA damaging treatment (26)
. However, correlative data are not definitive in these systems because at least one arm of the CD95 signaling pathway can be sensitized by p53-activating agents in a manner that may be independent of p53 (27
, 28)
; i.e., DNA damage-induced enhancement of responsiveness to signals through CD95 might be partially unrelated to p53 activity. Thus, although DNA-damaging drugs (22
, 27)
and ionizing radiation (28, 29, 30)
enhance the response of sensitive cells or presensitized cells to CD95-mediated cell death, the relative contribution of p53 activity is unclear, and direct evidence is needed to firmly establish a causative relationship between p53-dependent regulation of CD95 expression and increased function. To address these issues, we used two models of p53 inactivation to examine the role of wild-type p53 activity in regulating the constitutive level and the DNA damage-induced enhancement of CD95 expression and function.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40% confluency, and fresh medium was added every 3 days and 24 h before harvesting. In experiments examining dose-fractionated radiation-induced enhancement of CD95-mediated apoptosis, cells were plated at 80% confluency, and fresh medium was added every 3 days and 8 h after the final irradiation. Cells were subjected to fractionated doses of X-radiation Tuesday through Friday of the first week and Monday through Thursday of the second week. Irradiation was administered with a Clinac 600C linear accelerator (Varian, Palo Alto, CA) at a dose rate of 2.4 Gy/min
Reagents.
IFN-, cycloheximide, and anti-actin mAb clone AC-40 were obtained from Sigma (St. Louis, MO). mAb clone M30 (Roche/Boehringer Mannheim, Mannheim, Germany) was used to recognize a neo-epitope expressed on a cytokeratin-18 fragment generated specifically by caspase activity. Agonistic anti-CD95 IgM mAb (clone CH11), FITC-conjugated anti-CD95 mAb (clone UB2), and FITC-conjugated isotype-matched (IgG1) irrelevant antibody were purchased from Immunotech (Westbrook, ME). mAb DO-1 recognizes the epitope 20SDLWKL25 in the NH2-terminal region of human p53 (31
, 32)
. mAb Bp53-10 is specific for the COOH-terminal region of p53 (33
, 34)
. mAb 118 is specific for p21waf1/cip1 (35)
. mAb PC-10 specifically binds PCNA (36)
. Anti-caspase-8 mAb clone 12F5, linked/FLAG-tagged recombinant CD178 (SUPERFas-Ligand), and Z-VAD-fmk were from Alexis (San Diego, CA).
Quantitation of Cell Death.
Cells were harvested with 0.1% trypsin plus 0.25% EGTA in PBS (pH 7.4; 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4 in distilled water). Analyses of sub-G1 DNA content were performed as described previously (28)
after pooling of adherent and floating cells. Cell viability was calculated as the frequency of cells excluding the vital dye trypan blue as determined by counting of a minimum of 200 cells under a light microscope for each sample. Cell survival was determined according to clonogenic survival in standard colony-forming assays with crystal violet as the stain. Colonies with >50 cells were counted.
Cells containing fragments of intracellular cytokeratin-18 were detected as described previously (26) . The M30 antibody was diluted 1:250 in staining solution for MCF-7 cells and derived clones and 1:50 for HCT116 cells. Cells were quantitatively analyzed on an EPICS XL flow cytometer (Coulter, Hialeah, FL) using the manufacturer’s analysis software. Subcellular debris was eliminated from analysis by appropriate gating of the forward- and side-scatter parameters.
Stable Transfection.
Clones of MCF-7 cells were derived by transfection with a plasmid coding for a truncated, dominant-negative mouse p53 protein including amino acid residues 1–14 and 302–390 under the control of the cytomegalovirus promoter (pCMV-p53DD); expression of this miniprotein specifically inhibits p53 activity (37
, 38) . This miniprotein should not interact with p53 family members p63 and p73 because the COOH-terminal oligomerization domain of wild-type p53 does not bind these proteins (39)
. As controls, Neo clones were derived by transfection of a bacterial gene for neomycin phosphotransferase under the control of the cytomegalovirus promoter (pCMV-Neo). Stable transfectants were selected by use of 2 mg/ml G418 sulfate (Life Technologies, Inc., Paisley, Scotland). Independently isolated clones expressing high levels of p53DD miniprotein were examined to control for unexpected effects of DNA insertion sites.
Protein Detection.
Immunoblotting and immunofluorescent staining were performed as described previously (26)
. Immunocytochemistry was performed as described (23)
.
Cell Cycle Analysis.
DNA content was quantitated as described previously (23
, 28)
. The percentages of cells in each phase of the cell cycle were determined with use of MultiCycle-AV software (Phoenix Flow Systems, San Diego, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Similar results were obtained for MCF-7 cells (Fig. 1A)
. A single large dose of 16 Gy induced delayed occurrence of sub-G1 events, which appeared by 4 days after irradiation in both cell lines (data not shown). Interestingly, continuous incubation with Z-VAD reduced the percentage of fractionated radiation-induced sub-G1 events by >50% (Fig. 1A)
. The fraction of irradiated cells that exhibited sub-G1 DNA content in the presence of Z-VAD may have died a caspase-independent death because DNA fragmentation is known to occur during late necrosis and so-called paraptosis and is therefore not completely specific to apoptosis (1)
.
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. Furthermore, although Z-VAD inhibited fragmentation of cytokeratin-18 in irradiated cells, it did not inhibit the emergence of a large subset of cells exhibiting reduced side scattering of laser light (Fig. 1C)
. Finally, in clonogenic assays, Z-VAD did not rescue cells from radiation-induced reduction in long-term survival (Fig. 1D)
. Thus, inhibition of caspase activity altered the characteristics of radiation-induced death, but not the frequency (1
, 17)
. These results imply that although apoptosis was dispensable for radiation-induced death, fractionated radiation-induced death of these carcinoma cells preferentially ended with apoptosis, consistent with the generally held belief that radiation-induced reproductive cell death ends with secondary apoptosis in many cell types (4)
.
p53 Regulates Constitutive and DNA Damage-Inducible CD95 Expression in HCT116 Cells.
Because a small subset of cells survived radiation treatment (Fig. 1D)
, we examined a potentially complementary route for killing surviving cells. We evaluated whether p53 enhanced the response of surviving cells to CD95-mediated apoptosis. It has previously been reported that the response to CD95-mediated signals can be regulated at the level of surface expression (40)
. However, after DNA damage, the relative contribution of p53-dependent up-regulation of CD95 surface expression to enhancement of CD95-mediated cell death is poorly defined because cells with mutant p53 might also become more responsive to CD95-mediated signals (27
, 28)
. To address this question, we used as a model p53-deficient (-/-) HCT116 cells (41)
. The status and function of p53 in these cells were confirmed by examining the expression and function of human p53 and p21waf1/cip1 proteins. p53+/+ HCT116 cells constitutively expressed a detectable level of human p53 that was elevated 2 h after X-irradiation (8 Gy), and this elevation was partially reduced after 8 h (Fig. 2A)
. As expected, p53-/- cells expressed no p53 protein, and no induction was observed after irradiation. Induction of wild-type p53 expression 2 h after irradiation of p53+/+ cells corresponded to a weak induction of the protein product of the p53-regulated gene p21waf1/cip1 and was followed by strong up-regulation 8 h after irradiation (Fig. 2A)
. On the other hand, p21 protein in p53-/- cells was virtually undetectable, and no induction was observed (Fig. 2A)
. Elevated expression of p21 in p53+/+ cells correlated with a modest arrest of the cell cycle in G1 phase relative to p53-/- cells (Fig. 2B)
, as shown previously (41)
.
|
. Interestingly, constitutive surface expression of CD95 was substantially reduced in p53-/- cells (Fig. 3A)
. Furthermore, X-irradiation of p53-/- cells did not induce a detectable increase in surface CD95 expression (Fig. 3A)
. This latter result is consistent with the reported requirement for p53 in DNA damage-induced up-regulation of CD95 expression (21
, 22
, 30)
.
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). Unexpectedly, however, loss of radiation-induced surface up-regulation (Fig. 3A)
had little or no functional consequence; after X-irradiation (8 Gy), there was a relatively small increase in anti-CD95 antibody-induced apoptosis in both p53+/+ and -/- cells (Fig. 3B)
. These findings indicate that radiation pretreatment modestly increased the amount of apoptosis induced by anti-CD95 antibody but do not support a major role for p53 in mediating this effect in HCT116 cells.
To further characterize the role of p53 in regulating the constitutive level and the radiation-induced enhancement of CD95-mediated apoptosis, we examined activation of downstream caspase-8. After treatment with anti-CD95 antibody, the decreases in the 54–55-kDa full-length inactive forms of caspase-8 were more pronounced in unirradiated p53+/+ cells than in -/- cells (Fig. 3C)
. Radiation enhanced this effect and induced a small but detectable increase in the active 41–43-kDa forms of caspase-8 in both groups of cells (Fig. 3C)
.
p53 Regulates Constitutive and Inducible CD95 Expression in MCF-7 Cells, with a Major Effect on Function.
We next used a model of stable transfection of MCF-7 cells with a truncated version of the mouse wild-type p53 gene. The inactive miniprotein product of this gene (p53DD) has been shown to specifically bind and stabilize wild-type human p53, prolonging its usually transient expression and inhibiting its function (37
, 38)
. Two clones of MCF-7 cells expressing the miniprotein were established and named DDp53-9 and DDp53-12. Expression of the p53DD miniprotein was confirmed by detection of an 19-kDa band by use of a COOH-terminal-specific anti-p53 antibody (Fig. 4A)
. We confirmed inactivation of wild-type p53 by the miniprotein by examining human p53 and p21waf1/cip1 protein expression and function after irradiation. Parental MCF-7 and vector-transfected control cells (clones Neo-3 and Neo-7) constitutively expressed a low level of human p53 protein that was transiently elevated 2 h after X-irradiation (8 Gy), and the elevation was decreased by 8 h (Fig. 4A)
, as reported previously (42)
.
|
. Elevated expression of p21 protein in irradiated MCF-7 cells and Neo-transfected clones correlated with G1 arrest of the cell cycle, whereas miniprotein-expressing cells exhibited no detectable arrest of their cell cycle (Fig. 4B)
. Immunocytochemistry indicated that the miniprotein had stabilized human p53 expression in 100% of cells (Fig. 4C)
, indicative of the clonal origin of these cultures.
MCF-7 cells and Neo clones expressed a low constitutive level of CD95 on their cell surfaces and exhibited significant up-regulation of CD95 after a single dose of X-radiation (8 Gy; Fig. 5A
). Constitutive expression of surface CD95 was substantially reduced in both miniprotein-expressing clones (Fig. 5A)
. X-irradiation of miniprotein-expressing clones did not induce a detectable increase in surface CD95 expression (Fig. 5A)
.
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25% of unirradiated MCF-7 cells and Neo clones specifically underwent apoptosis relative to untreated controls after aggregation of CD95 with agonistic antibody, whereas <10% of unirradiated p53DD-expressing clones underwent anti-CD95 antibody-induced apoptosis (P 
0.05, Student’s t test; Fig. 5B
). Thus, as in HCT116 cells, regulation of constitutive expression of CD95 by p53 had a measurable functional consequence in MCF-7 cells. Unlike HCT116 cells, however, loss of radiation-induced surface up-regulation had a substantial functional effect in MCF-7 cells. After X-irradiation (8 Gy), >50% of MCF-7 cells and Neo clones specifically underwent anti-CD95 antibody-induced cell death, an increase of 25–30% above the rate for unirradiated cells treated with anti-CD95 antibody. On the other hand, the percentage of specific CD95-mediated death in equivalently treated p53-expressing clones was >20%, representing an increase of only 5–10% above that of unirradiated cells treated with anti-CD95 antibody (Fig. 5B)
. These findings indicate that more than half of the single-dose radiation-induced enhancement of anti-CD95 antibody-induced apoptosis in MCF-7 cells is dependent on wild-type p53 activity.
Agonistic anti-CD95 antibody induced significant cleavage of the inactive 54–55-kDa full-length forms of caspase-8 to its 41–43-kDa forms in unirradiated MCF-7 cells and Neo clones, but not in DDp53 clones (Fig. 5C)
. Furthermore, CD95-mediated cleavage of caspase-8 was considerably more prominent in irradiated MCF-7 and Neo cultures than in unirradiated cultures. In contrast, cleavage was virtually undetectable in irradiated DDp53 clones.
To exclude the possibility that these findings might occur only under conditions that do not mimic the clinical situation, we used a dose-fractionated regimen because this is the type of treatment provided to human patients (4)
. To further approximate in vivo conditions, IFN- was used as a sensitizing agent instead of cycloheximide. Because up-regulation of CD95 expression is one of the mechanisms activated by IFN- to sensitize cells to CD95-mediated apoptosis (9
, 10)
, we began by examining CD95 surface expression 24 h after treatment with IFN-, fractionated X-radiation, or both. In Neo cells, surface CD95 expression was up-regulated by either IFN- or fractionated irradiation, and an additive effect was consistently observed when these agents were combined (Fig. 6A)
. A similar additive effect was seen when IFN- was combined with etoposide or 5-fluorouracil (data not shown). In p53DD-expressing cells, IFN-, but not fractionated radiation, substantially increased CD95 surface expression, and no additive effect was observed (Fig. 6A)
.
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-sensitized cells, regardless of whether the CD95-aggregating modality was anti-CD95 antibody or recombinant CD178. In the experiment shown in Fig. 6B
, treatment of Neo cultures with IFN- plus anti-CD95 antibody specifically induced 17% cell death in unirradiated cultures (26% - 9%) and 35% in fractionally irradiated cultures (68% - 33%) relative to cultures receiving only IFN-. In p53DD-expressing cells, IFN- plus anti-CD95 mAb specifically induced 12% cell death in unirradiated cultures and only 15% in fractionally irradiated cultures. IFN- plus recombinant CD178 induced 20% specific cell death in unirradiated Neo cells and 43% in irradiated cells. In p53DD-expressing cultures, IFN- plus recombinant CD178 induced 15% specific cell death in unirradiated cells and 19% in irradiated cells. These findings in DDp53 cells suggest that IFN- can partially restore CD95 expression and function to tumor cells that have reductions in these characteristics caused by obstruction of normal p53 activity. In addition, these results indicate that most of the radiation-induced enhancement of the response to CD95-mediated signals in MCF-7 cells is dependent on wild-type p53 activity, in contrast to the situation in HCT116 cells.
A measurable difference might have been expected between the effects of anti-CD95 antibody and recombinant CD178 in the absence of sensitizing pretreatment, particularly in CD95-resistant cells such as MCF-7, because it is known that the amount of cell death induced by anti-CD95 agonistic antibodies does not necessarily correlate well with results obtained with CD178 protein (reviewed in Ref. 43
). Indeed, in the absence of presensitization with IFN- in Neo cells, anti-CD95 antibody had little detectable effect on viability, whereas recombinant CD178 induced 12% specific cell death in unirradiated cells (18% - 6%) and 27% in irradiated cells (54% - 27%; Fig. 6B
). In contrast, in p53DD-expressing cells in the absence of IFN- presensitization, neither recombinant CD178 nor anti-CD95 antibody had a significant effect on viability in either unirradiated or fractionally irradiated cultures, presumably because of the nearly negative level of constitutive CD95 surface expression in these clones (Fig. 6A)
. These results with recombinant CD178 support the conclusion that regulation of surface expression of CD95 by p53 can have significant functional consequences in resistant cells even in the absence of previous sensitization. Furthermore, these findings indicate that X-radiation can behave as a partial sensitizing agent (without IFN- presensitization) for apoptosis induced by recombinant CD178 protein (but not by anti-CD95 agonistic antibody), at least for cell types exhibiting p53-dependent CD95 up-regulation.
To further explore the sensitizing potential of fractionated radiation for CD178-induced cell death, we performed clonogenic assays on fractionally irradiated cells incubated with recombinant CD178 in the absence of IFN- pretreatment. In Neo cells, recombinant CD178 reproducibly exerted only a minor effect on colony formation in unirradiated cells, but importantly, it reduced the number of colonies surviving fractionated irradiation by at least 4-fold (Fig. 6C
, left panels). In p53DD-expressing cells, recombinant CD178 had no detectable effect on colony formation in either unirradiated or fractionally irradiated cultures (Fig. 6C
, right panels). Altogether, these findings provide direct evidence that p53-dependent regulation of CD95 surface expression plays a major role in X-radiation-induced enhancement of CD95-mediated death in MCF-7 cells, regardless of whether signaling through CD95 is achieved by (a) agonistic antibody in the presence of sensitizing pretreatment with either cycloheximide (Fig. 5B)
or IFN- (Fig. 6B)
, or (b) recombinant CD178 in the absence or presence of sensitizing pretreatment (Fig. 6, B and C)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The consequences of p53 activation might be more pronounced in untreated cancer cells than in healthy tissues. In cancer cells, it is possible that cancer-associated alterations, such as oncogene activation, directly enhance the expression or activities of factors that are required for p53 activation (reviewed in Ref. 16
). This suggests the possibility that downstream events in the p53 pathway (such as CD95 up-regulation) might be more profound in tumor cells than in healthy cells, even in tumor cells that survive p53 activation. The results presented here demonstrate that p53 regulates the constitutive expression of CD95 in HCT116 and MCF-7 carcinoma cells derived from colon and breast tissues, whose cells are not susceptible to p53-dependent apoptosis. Similarly, it was recently reported that p53-dependent regulation of constitutive CD95 surface expression occurs in oncogene-transformed mouse embryonic fibroblasts (30)
. In these transformed fibroblasts (30)
as well as in HCT116 and MCF-7 carcinoma cells, constitutive CD95 expression was decreased by the loss of p53 activity to a level that significantly inhibited transmission of death-inducing signals. This indicates that substantial reductions in constitutive CD95 expression and function caused by inhibition of p53 activity during tumorigenesis might be mechanisms that allow tumor cells to partially evade the CD178-dependent arm of CTL- and natural killer cell-mediated killing. Encouragingly however, carcinoma cells that have lost most of their constitutive surface expression of CD95 after loss of p53 activity can nonetheless be partially sensitized to CD178-induced apoptosis by IFN- (Fig. 6B)
.
In addition to the emerging role of p53 in regulating constitutive CD95 expression and function described above, it is becoming clear that p53 regulates inducible CD95 expression and function after DNA damage, at least in reported tumor cell models. Bleomycin was reported to induce p53-dependent enhancement of CD95 expression and function in an artificial p53-expression system in Hep3B cells (22)
. Furthermore, -irradiation was shown to induce transformed mouse embryonic fibroblasts expressing a low level of CD95 to be more responsive to CD95-mediated signals, in a p53-dependent manner (30)
. However, we found that despite exhibiting a similar amount of radiation-induced up-regulation of CD95 expression, the cell lines used in our study differed qualitatively in the level of enhanced responsiveness to CD95 ligation. In HCT116 cells expressing a high constitutive level of CD95, radiation induced up-regulation of CD95 expression but had little or no effect on responsiveness to anti-CD95 antibody, whereas in MCF-7 cells expressing a low constitutive level of CD95, radiation induced both up-regulation of surface expression and significant enhancement of responsiveness. These results suggest that the level of constitutive surface expression may have affected, to some degree, the extent of the functional effect of surface up-regulation of CD95. Another, nonmutually exclusive possibility is that the effect of radiation-induced up-regulation of surface CD95 expression in HCT116 cells may have been limited by a prosurvival balance in the availability of downstream effector and inhibitor molecules. These findings in HCT116 and MCF-7 cells indicate that up-regulation of CD95 expression after p53 activation may substantially increase susceptibility to CD95-mediated signals in only a subset of tumor cell types.
It has been reported that wild-type p53 activity may hinder chemotherapy in some tumor types by inducing cell cycle arrest, thereby allowing time for tumor cells to recover from genotoxic treatment (46)
. Although p53 does not contribute directly to tumor cell death in many cell types (4)
, p53-dependent enhancement of CD95 expression and function might be detrimental to tumor cells in the presence of an activated antitumor immune response that involves effector cells producing IFN- and expressing CD178.
Regulation of the level of CD95 expression and function by wild-type p53 in wild-type p53-expressing HCT116 and MCF-7 cells (Figs. 3
and 5)
, as well as fibroblasts (30)
, suggests that the act of restoring wild-type p53 activity to cells lacking this activity might be sufficient—in many cell types—to enhance their susceptibility to death signaling through the CD95 receptor. On the basis of this rationale, future therapies aimed at restoring p53 activity might complement treatments that reactivate the stalled antitumor immune response.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic (Grants NC6402-3/2000, NC7133-3/2002, and NC7131-3/2002), and by the Grant Agency of the Czech Republic (301/03/0545). 
2 To whom requests for reprints should be addressed, at Division of Hematology-Oncology, Children’s Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90027. Phone: (323) 669-5633; Fax: (323) 664-9455; E-mail: msheard{at}chla.usc.edu
3 The abbreviations used are: IL, interleukin; mAb, monoclonal antibody; PCNA, proliferating-cell nuclear antigen.
Received 9/27/02. Revised 6/19/03. Accepted 9/ 5/03.
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and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy. J. Clin. Investig., 108: 51-62, 2001.[Medline]
B p50–p65 recruitment. Mol. Cell. Biol., 19: 2098-2108, 1999.B in regulation of multiple immune-response genes and in Fas-induced cell death. J. Exp. Med., 189: 999-1004, 1999.-irradiation renders tumour cells more responsive to apoptotic signals through CD95. Br. J. Cancer, 80: 1689-1696, 1999.[Medline]
and Fas activate complementary Fas-associated death domain-dependent pathways that enhance apoptosis induced by -irradiation. J. Biol. Chem., 275: 8610-8617, 2000.-dependent delay of in vitro tumor progression by Fas overexpression on murine renal cancer cells. J. Immunol., 164: 231-239, 2000.This article has been cited by other articles:
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  A. Gelbard, C. T. Garnett, S. I. Abrams, V. Patel, J. S. Gutkind, C. Palena, K.-Y. Tsang, J. Schlom, and J. W. Hodge Combination Chemotherapy and Radiation of Human Squamous Cell Carcinoma of the Head and Neck Augments CTL-Mediated Lysis. Clin. Cancer Res., March 15, 2006; 12(6): 1897 - 1905. [Abstract] [Full Text] [PDF]
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 C. T. Garnett, C. Palena, M. Chakarborty, K.-Y. Tsang, J. Schlom, and J. W. Hodge Sublethal Irradiation of Human Tumor Cells Modulates Phenotype Resulting in Enhanced Killing by Cytotoxic T Lymphocytes Cancer Res., November 1, 2004; 64(21): 7985 - 7994. [Abstract] [Full Text] [PDF]
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, Z-VAD-fmk; 
, no Z-VAD-fmk added. Data are presented as means of three independent experiments (bars, upper limits of SDs). C, cellular expression of fragments of cytokeratin-18 (CK18). Fixed and permeabilized cells were stained with a mAb that specifically recognized the epitope generated by caspase-dependent cleavage of cytokeratin-18. D, cell survival as determined in colony-forming assays. Continuous incubation with Z-VAD began 24 h after plating of irradiated cells. Colonies of HCT116 cells were counted 22 (±1) days after the final dose of X-radiation; colonies of MCF-7 cells were counted 27 (±2) days after the final dose. Colonies were visualized with crystal violet. Results are representative of four independent experiments. 
, no Z-VAD; 
