Limited studies have indicated that some chemotherapy agents activate the transcription factor nuclear factor-κB (NF-κB), and that this leads to suppression of the apoptotic potential of the chemotherapy. In contrast, it was reported recently that stable inhibition of NF-κB in four different cancer cell lines did not lead to augmentation of the chemotherapy-induced apoptosis. In this study, we have focused on colorectal cancer, which is known to be highly resistant to genotoxic chemotherapy and gamma irradiation. We show that the topoisomerase I inhibitor 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin(CPT-11) activates NF-κB in most colorectal cancer cell lines. We then examine a therapeutic strategy that uses adenovirus-mediated transfer of the super-repressor IκBα to inhibit NF-κB activation as an adjuvant approach to promote chemosensitivity in colorectal tumor cells to treatment with CPT-11. These data demonstrate that the protection from apoptosis induced in response to CPT-11 treatment is effectively inhibited by the transient inhibition of NF-κB in a variety of human colon cancer cell lines and in a tumor xenograft model, resulting in a significantly enhanced tumoricidal response to CPT-11 via increased induction of apoptosis. These findings indicate that the activation of NF-κB by chemotherapy is an important underlying mechanism of inducible chemoresistance.

The transcription factor NF-κB3(1) has been shown to regulate apoptosis in several different settings (reviewed in Ref. 2). Importantly,several groups have shown that NF-κB is activated by exposure of cells to chemotherapy (reviewed in Ref. 3). Recently, we studied the human fibrosarcoma cell line HT1080 and showed that the activation of NF-κB by the chemotherapeutic agent daunorubicin strongly suppressed the apoptotic potential of this chemotherapy(4). Furthermore, tumors derived from HT1080 cells were induced to undergo apoptosis and regression when animals were treated with the topoisomerase I inhibitor CPT-11 in parallel with adenoviral delivery of a modified form of IκBα, the natural inhibitor of NF-κB. These data argued that the activation of NF-κB by chemotherapy provides an important mechanism for inducible chemoresistance. In apparent contrast with these studies, it was reported recently (5) that four different cell lines that stably express the modified form of IκBα did not show enhanced cytotoxicity in response to chemotherapy, although the chemotherapies activated NF-κB. We report here continued studies on the role of NF-κB in controlling inducible chemoresistance, and we report a therapeutic regimen involving the inhibition of NF-κB that leads to dramatic antitumor responses. Additionally, we show that the stable inhibition of NF-κB via IκB expression is not a consistent experimental approach to test the role of NF-κB in chemoresistance.

To extend our original studies, we have focused on colorectal cancers. Like most solid tumors, colorectal cancers are frequently resistant to chemotherapy and irradiation (6). Several of the cellular mechanisms that determine sensitivity of cancer cells to genotoxic therapies have been elucidated recently (7, 8, 9, 10). For example, one mechanism for chemoresistance is the up-regulation of the multidrug resistance gene product (MDR1), which is responsible for pumping chemotherapy agents from the cells (11). Other mechanisms of resistance appear to affect the ability of a cancer cell to undergo apoptosis, the major mechanism by which chemotherapy and radiation induce the killing of tumor cells (12, 13, 14). Thus, mutation in the p53 tumor suppressor gene leads to acquired resistance that impairs p53-dependent responses to apoptotic stimuli and may promote cancer cell survival and proliferation in some cancer cells (15). Another important mechanism for resistance to chemotherapy is inducible chemoresistance,a process whereby exposure of tumor cells to cancer therapy leads to their resistance to apoptosis (11). As described above, we have proposed that a major mechanism for inducible chemoresistance is the up-regulation of the transcription factor NF-κB.

The objectives of this report are to determine whether the principle of reversing inducible chemotherapy resistance, as a means of enhancing the apoptotic response to chemotherapy treatment, can be applied broadly to the treatment of colorectal cancer in preclinical models. Specifically, these experiments explore whether the topoisomerase I inhibitor CPT-11, a chemotherapy showing promise for cancers of different tissue origin (6, 16, 17, 18, 19, 20), induces activation of NF-κB in a variety of human colorectal cancer cells. We also examine whether transient inhibition of NF-κB activation concurrent with CPT-11 exposure enhances the anticancer effect of CPT-11 among different colorectal cancer cell lines, and several of the cell lines used by Bentires-Alj et al.(5). Finally, in an effort to determine the optimal dosing schedule necessary to maximize tumoricidal response in a colon cancer xenograft model, we examine a variety of treatment schedules that combine inhibition of NF-κB activation with CPT-11 administration. It is hoped that the results of these studies will contribute to the design and implementation of a novel therapeutic approach that improves patients’ responses to systemic treatment for metastatic colorectal cancer as well as other forms of cancer.

Adenovirus Vectors.

The recombinant adenovirus vectors used in this study were replication-defective Ad5-based vectors constructed with the transgene expression driven by the CMV early/intermediate promoter/enhancer. All vectors were expanded in 293 cells and purified and titered as described previously (21). The vector Ad.CMV.IκBαexpresses the super-repressor form of IκBα that is mutated at serine residues 32 and 36 and functions as a potent and specific repressor of NF-κB-mediated events (4, 22). The control vector Ad.CMV3, generously provided by J. A. Roth (University of Texas M. D. Anderson Cancer Center, Houston, TX), contains a CMV promoter similar to Ad.CMV.IκBα but lacks a transgene insert. Studies of transduction efficiency of the cell lines used in these experiments indicate that moderate levels of transgene expression may be accomplished using a MOI ranging from 20 to 100 (data not shown).

Chemotherapy Agents.

Camptothecin is a specific inhibitor of mammalian DNA topoisomerase I. The camptothecin analogue CPT-11 and its active metabolite SN38 were generously provided by J. Malczyn (Pharmacia and Upjohn Co., Kalamazoo,MI).

Cell Culture.

The human colon cancer cell line LOVO was obtained from ATCC(Rockville, MD). The LOVO cells were grown in F-12 (Ham) with 20% FBS. The colon cancer cell lines SW1463, SW837, SW620, and SW480 were obtained from ATCC and grown in L-15 with 10% FBS. The colon cancer cell lines KM12-L4 and KM12-SM (generous gifts of J. Fidler, The University of Texas M. D. Anderson Cancer Center, Houston, TX) and HT-29, WiDR, and CL188 (obtained from ATCC) were grown in MEM with 10%FBS. The colon cancer cell line CCD841 (ATCC) was grown in DMEM with 10% FBS, and NCI H508 was grown in RPMI with 10% fetal bovine serum. All media were obtained from Life Technologies, Inc. (Gaithersburg, MD)and supplemented with 100 μg/ml penicillin G and 100 μg/ml streptomycin. MCF-7 and HCT116 were obtained from ATCC and grown in Eagle’s MEM with 10% FBS and McCoy’s 5A medium with 10% FBS,respectively. Cell cultures were maintained at 37°C.

Cell Growth Inhibition.

Human cancer cells (5–8 × 104)were seeded in six-well plates and infected with Ad.CMV.IκBα at a MOI of 20 per target cell when cells reached 20% confluence. The control adenovirus vector Ad.CMV3 was used to infect the control group. Drug treatment with SN38, the active metabolite of CPT-11, was administered 24 h after virus infection at a final concentration of 1000, 500, or 100 ng/ml. Daily cell counts were performed for 4 days. Experiments were performed in triplicate.

NF-κB Activation Assay.

Activation of NF-κB in response to treatment with chemotherapy was determined by the EMSA as described previously (23). For in vitro experiments, cancer cells were cultured in 100-mm dishes until 50–70% confluence was achieved. Cells were infected with Ad.CMV.IκBα (MOI, 100) for 1 h and then washed with PBS and refed medium. Cells were treated with SN38 (1 μg/ml) 24 h after adenovirus infection. Cells were then harvested at times 0, 1, 2, and 6 h after treatment with SN38. Nuclear extracts were prepared by collecting cells and then washing and suspending them in hypotonic buffer. The nuclear pellet was separated by centrifugation, and the cytoplasmic supernatant was discarded. The nuclei were then resuspended in a low-salt buffer to high-salt buffer, and the soluble protein was released by centrifugation, collected, and stored at −70°C. The DNA probe used contains an NF-κB site (underlined) from the H-2κb gene(5′-CAGGGCTGGGGATTCCCATCTCCACAGTTTCACTTC-3′; Ref.3). In brief, 7 μg of nuclear extracts were preincubated with 1 μg of poly(deoxyinosinic-deoxycytidylic acid) in binding buffer (10 mm Tris, 50 mmNaCl, 20% glycerol, 0.5 mm EDTA, and 1 mm DTT) for 10 min at room temperature. Approximately 20,000 cpm of 32P-labeled DNA probe was then added and allowed to bind for 15 min. The complexes were then separated on a 5% polyacrylamide gel and autoradiographed.

The procedure for obtaining nuclear extracts from cancer cells was modified for tumor samples as follows. s.c. tumors were harvested after treatment and snap-frozen in liquid nitrogen. Frozen tumor tissues were morselized over liquid nitrogen and scraped into 10-ml conical tubes containing 5 ml of solution A [0.3 m sucrose, 60 mm KCl, 15 mm NaCl, 15 mm HEPES (pH 7.5), 2 mm EDTA, 0.5 mm EGTA, 14 mm2-ME, and 0.1% NP40]. The mixture was ground into a slurry, and the nuclear solution was layered. On the top of the slurry, 2.5 ml of solution B [0.88 m sucrose, 60 mm KCl, 15 mm NaCl, 15 mm HEPES (pH 7.5), 2 mmEDTA, 0.5 mm EGTA, and 14 mm 2-ME] were added,and the mixture was spun down for 10 min at 3000 rpm. The pellet was brought up to 1 ml of solution D [1 m sucrose, 15 mm KCl, 15 mm NaCl, 15 mm HEPES (pH 7.5), 0.1 mm EDTA, 0.1 mm EGTA, and 14 mm 2-ME] and layered on top of 2.5 ml of solution D and then spun down at 3000 rpm for 10 min. The supernatant was aspirated,and the pellet was resuspended in NE buffer [20 mm Tris(pH 8.0), 120 mm NaCl, 1.5 mm MgCl, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride,and 25% glycerol] with proteinase inhibitor (10 μg/ml leupeptin, 25μg/ml aprotinin, and 5 μg/ml pepstatin). The mixture was incubated on ice for 10 min and then spun down at full speed for 15 min at 4°C. The nuclear protein remaining in solution was then collected.

In Vivo Evaluation of Tumor Growth.

The ability of the super-repressor IκBα to enhance sensitivity to CPT-11 was assessed in a LOVO xenograft model. The tumors were established by injecting 5 × 106LOVO cells s.c. into the flank of nude mice (NCR nu/nuathymic nude mice, 5–6 weeks of age, female, and weight of 18–20 g;Taconic Germantown, NY). Once tumors reached a mean diameter of 8–10 mm, treatment was initiated. Animals were treated on days 0, 7, and 14 with a single-pass intratumoral injection of adenovirus expressing the super-repressor IκBα (Ad.CMV.IκBα at a concentration of 1 × 1010 pfu/200 μl), control adenovirus Ad.CMV3 (1 × 1010pfu/200 μl), or vehicle alone using a technique described previously(21). On the basis of previous published reports of tumoricidal response to CPT-11 in xenograft models (24, 25), a dosage of 33 mg/kg every 4 days was selected and administered i.v. via tail-vein injection during the 20-day treatment period. A total of 5 drug treatments was administered. PBS was administered i.v. as a control vehicle. Tumor diameters along two orthogonal axes were recorded every other day until tumors approached 20 mm of mean diameter, at which point animals were sacrificed according to protocol. Tumor volume was calculated by assuming a spherical shape of the tumor, using the formula: volume = 4/3 πr3, where r is 1/2 (mean diameter of the tumor).

The effect of differing schedules of administration of Ad.CMV.IκBαon tumoricidal response was assessed in a xenograft model. Nude mice bearing s.c. LOVO tumors (mean diameter, 8–10 mm) were treated over a 50-day period with i.v. CPT-11 (33 mg/kg every 4 days) and differing schedules (every 5, 10, 15, and 28 days) of intratumoral injection of the adenovirus expressing the super-repressor IκBα (Ad.CMV.IκBαat a dosage of 1 × 1010 pfu/200μl).

Assays to Detect Apoptosis and Transgene Expression in Vivo.

LOVO tumor xenografts (1-cm diameter) were treated with a single intratumoral injection of Ad.CMV.IκBα (1 × 1010 pfu/200 μl). Twenty-four h after virus administration, CPT-11 (33 mg/kg) was given i.v. by tail-vein injection. Tumors were harvested at times 0, 1, 2, and 6 h after drug treatment, embedded in OCT mounting medium (VWR, Sakura Finetec U.S.A., Inc., Torrance, CA), snap-frozen in 2-methylbutane (Aldrich Chemical Co., Milwaukee, WI) over liquid nitrogen, and then stored at−70°C. Four-μm-thick sections were cut and collected on charged and precleaned microscope slides (Fisher Scientific, Pittsburgh, PA). Cryosections were stained using a double immunohistological staining technique to detect the presence of exogenous IκBα in tumor cells and to determine the level of apoptosis, determined by the TUNEL assay in tumors. Exogenous IκBα was detected using a murine antibody(anti-HA monoclonal antibody at the dilution of 1:200) to the HA tag(fluorescent red) present on the exogenous IκBα expressed by the Ad.CMV.IκBα. The reaction mixture was fixed with 10% neutral buffered formalin and blocked with 2% BSA, followed by incubation for 1 h. Binding was detected with a 1:100 dilution of rhodamine-conjugated goat antimouse secondary antibody. Apoptosis was detected using the In Situ Cell Death kit (Boehringer Mannheim Corp., Indianapolis, IN) and a fluorescent green antibody to detect TUNEL-positive cells. Analysis of tumor sections was performed using a two-color fluorescent microscope at ×100.

NF-κB Activation Is Induced by SN38 in Most Colorectal Cancer Cell Lines.

Previously, we have analyzed the ability of daunorubicin and the topoisomerase I inhibitor CPT-11/SN38 to activate NF-κB in fibrosarcoma cells (3, 4). Although others have reported that different chemotherapy agents activate NF-κB in different cell types, a recent report suggested that stable inhibition of NF-κB did not lead to enhanced cytotoxicity in several cancer cell lines,including the colorectal cancer cell line HCT116 and the breast cancer cell line MCF-7 (5). We explored the ability of SN38, the active metabolite of CPT-11, to activate NF-κB in a panel of colorectal cancer cell lines plus MCF-7 cells. All of the cell lines tested were relatively resistant to treatment with SN38 (data not shown). To inhibit NF-κB, we used transient inhibition via the adenoviral expression of the super-repressor IκBα. Our data from EMSAs demonstrated that NF-κB activation, as measured by nuclear translocation of NF-κB, is induced at 1 h after SN38 treatment and persisted up to 6 h in 10 of 11 cancer cell lines tested and in MCF-7 cells (Fig. 1,A). One of the cell lines, HT-29, did not show activation of NF-κB in response to SN38 treatment. The serine-to-alanine mutations at serine residues 32 and 36 of the super-repressor form of IκBαinhibit signal-induced phosphorylation and subsequent proteasome-mediated degradation of IκBα. Cytoplasmic binding of the mutant IκBα protein to the nuclear localization sequences of NF-κB thereby blocks nuclear translocation and subsequent binding of NF-κB to κB binding sites on DNA, effectively blocking NF-κB-mediated transcription (4). We have shown previously in vitro that pretreatment of LOVO cells with Ad.CMV.IκBα prior to SN38 administration markedly decreased nuclear translocation of NF-κB in response to SN38 (26). We show here that adenoviral delivery of the super-repressor IκB, but not control adenoviral infection, suppressed NF-κB activation (Fig. 1 A).

The Activation of NF-κB Inhibits SN38-induced Cytotoxicity.

We determined whether inhibition of NF-κB activation by SN38 would augment the cytotoxic response of this chemotherapy. We analyzed cell numbers for six of the colorectal cancer cell lines and for MCF-7 breast cancer cells after SN38 treatment in the presence or absence of adenovirus encoding the super-repressor form of IκBα. Treatment with the adenovirus expressing the super-repressor IκBα inhibited the growth of some cell lines compared with treatment with the control vector (Fig. 1 B). However, in all cell lines tested, the addition of super-repressor IκBα to SN38 treatment resulted in a significant increase in the level of growth inhibition compared with treatment with chemotherapy alone, super-repressor IκBα alone,control vector alone, and control vector combined with SN38(P < 0.001, analyzed by ANOVA). Furthermore,the reduced cell counts resulting from combined treatment of the super-repressor IκBα combined with SN38 was associated with a markedly increased level of apoptosis relative to controls receiving either chemotherapy alone or Ad.CMV.IκBα alone (data not shown). Previously, Bentires-Alj et al.(5) established several cancer cell lines, including HCT116 and MCF-7, stably expressing super-repressor IκBα and found that cytotoxicity was not enhanced when NF-κB was inhibited. Our data contradict those conclusions, at least in two of these cell lines, because we show that transient inhibition of NF-κB activation by adenovirus expression of the super-repressor IκBα strongly augments the cytotoxic response to SN38 (see “Discussion”). Furthermore, because the response is seen in both p53 mutated (KM12-L4, KM12-SM, and WiDR) and p53 wild-type(CCD841, MCF-7, HCT116, and LOVO) cell lines, our data suggest that the enhancement of cytotoxicity induced by inhibition of NF-κB is independent of p53 function (see “Discussion”).

Sensitivity of Colorectal Tumors to CPT-11 Is Markedly Enhanced by NF-κB Inhibition.

Experiments were performed to determine whether the super-repressor IκBα could promote enhanced sensitivity of LOVO xenograft tumors to CPT-11 (Fig. 2,A). As described previously (3), tumor growth after combined treatment with super-repressor IκBα and CPT-11 was significantly less than treatment with the super-repressor IκBα or CPT-11 alone (P < 0.0001, analyzed by ANOVA). Delivery of IκB once a week for 3 weeks combined with CPT-11 treatment twice a week for 3 weeks led to dramatic inhibition of tumor growth (Fig. 2,A). Interestingly, tumor size increased after 50 days and ultimately achieved a rapid growth rate. However,modification of the treatment schedule to treat tumors every 5 or 10 days in combination with CPT-11 administration over a 50-day period resulted in a persistent tumoricidal response (Fig. 2,B) and ultimately long-term cures after cessation of treatment at day 50. Tumor growth in groups receiving treatment with Ad.CMV.IκBα every 5 or 10 days was significantly less compared with groups receiving viral treatment every 15, 21, or 28 days (P < 0.0001, analyzed by ANOVA). Tumor regrowth in the groups treated with virus every 15, 21, or 28 days suggests that IκBα played an important role in the enhancement of the tumoricidal response to CPT-11. Long-term follow-up of treatment groups after cessation of both viral and CPT-11 treatment on day 50 demonstrated that between 50 and 66% of animals receiving viral treatment every 5 or 10 days remained tumor-free 5 months after discontinuation of all treatments (Fig. 2 C).

Super-Repressor IκBα Inhibits Nuclear Translocation of NF-κB induced by CPT-11 in Vivo.

It was important to show that CPT-11 actually induced NF-κB in tumors and that the adenoviral delivery of IκBα into tumors successfully inhibited NF-κB activation. EMSA assay for nuclear protein obtained from tumor tissue extracts demonstrated that NF-κB activation was induced by CPT-11 at 1 h after systemic drug administration with a peak activation of NF-κB observed at 2 h (Fig. 3). NF-κB activation in tumors was blocked by pretreatment with the adenovirus expressing the super-repressor IκBα. The results demonstrate that in vivo activation of NF-κB after treatment with CPT-11 was inhibited by the expression of the super-repressor IκBα but not the control vector.

Apoptosis Is Rapidly Induced by CPT-11 after Pretreatment with the NF-κB Super-Repressor IκBα.

We measured viral infectivity and IκBα expression along with the induction of apoptosis in LOVO tumors after systemic CPT-11 treatment. Two-color immunohistochemical staining of tumors injected with the adenovirus expressing the super-repressor IκBα and treated with CPT-11 demonstrated HA-positive cells diffusely throughout the tumor at all time points sampled, indicating successful adenovirus-mediated transfer of the super-repressor IκBα gene to tumor cells(Fig. 4). Staining for TUNEL-positive cells demonstrated that only a few tumor cells were undergoing apoptosis prior to CPT-11 treatment and at 1 h after treatment. In comparison, increasing levels of apoptosis were observed at both 2 and 6 h after drug treatment. Importantly,treatment with the control virus Ad.CMV3 and CPT-11 or with Ad.CMV.IκBα and PBS induced apoptosis in <1% of tumor cells at all time points sampled (data not shown). These data demonstrate that the activation of NF-κB in colorectal tumors suppresses the apoptotic potential of the chemotherapeutic response.

NF-κB Activation Induced by Cancer Therapies Blocks the Induction of Apoptosis.

Most cancer therapeutics function by killing cells through the induction of the apoptotic pathway. In fact, resistance to the induction of the apoptotic response is a principle mechanism by which cancer cells protect against cell killing (12). We have reported previously that the activation of NF-κB by TNF-α, ionizing radiation, and the cancer chemotherapeutic compound daunorubicin leads to an inhibition of the apoptotic response induced by these stimuli in fibrosarcoma cells (4). Similar results were obtained by others (27, 28, 29) relative to TNF-α. Thus, we and others have proposed that potential apoptotic stimuli initiate two distinct signaling pathways, one that leads to activation of apoptosis and one that leads to NF-κB activation, which induces a cell survival response through the inhibition of apoptosis (2, 4, 30, 31, 32, 33, 34). Contrasting findings such as those reported by Kasibhatla et al.(35), in which stress-induced expression of Fas ligand (leading to Fas-mediated apoptosis) in human leukemic Jurkat cells required NF-κB activation, have led some authors to conclude that NF-κB serves multiple functional roles under different conditions (36). The mechanism by which chemotherapy activates NF-κB is presently unknown and is the focus of ongoing investigation in our laboratory. However, the mechanism whereby NF-κB suppresses apoptosis is better understood and involves the induction of expression of genes that block the caspase cascade(2). We have shown previously that inhibition of inducible NF-κB activation by transient expression of the super-repressor IκBα leads to a dramatic improvement in the killing response of tumor cells when exposed to apoptotic stimuli (3). On the basis of these studies, we conclude that the apoptotic response to conventional chemotherapy and irradiation may be augmented by the inhibition of NF-κB activation in resistant cancer cells. The objective of this report was to evaluate the role of transient inhibition of NF-κB as an adjunct to the topoisomerase I inhibitor CPT-11 for the treatment of colorectal cancer cells.

Use of CPT-11 in the Treatment of Colorectal Cancer.

Colorectal cancer is the second most common cause of mortality from malignancy in the United States, accounting for ∼57,000 deaths in the United States in 1997 (37). Approximately 50% of these patients will eventually die of metastatic disease (38). The relatively high mortality rate of patients who are not cured by surgical treatment results from the resistance that most cancers have to conventional chemo- and radiation therapies (39, 40, 41). Recently, efforts to overcome the 70–80% rate of resistance of colorectal cancers to conventional therapies have directed the use of new compounds with reported higher levels of sensitivity in clinical trials. CPT-11 is one such promising agent used to treat a variety of solid tumors including colorectal cancer and lung cancer (6, 17, 18, 19, 20, 42). In vitro studies have demonstrated CPT-11 to have sustained activity against chemotherapy-resistant colon cancer cell lines, including those having the multi-drug resistance(MDR) phenotype (6). In addition, results from clinical trials indicate CPT-11 to be a promising anticancer agent, used as a single agent or in combination with other agents, with a duration of response from several months to 1 year (17, 18, 19, 20, 43). Because of its lack of cross-resistance with 5-FU,promising clinical responses suggest that CPT-11 may be an effective second-line agent in the treatment of patients who have failed first-line treatment with 5-FU-based regimens (6). A recently completed multicenter randomized clinical trial found that CPT-11 increased the 1-year overall survival rate 2.6 times greater than supportive care in patients who had failed conventional treatment with 5-FU (16).

SN38 Induces Activation of NF-κB in a Variety of Human Colorectal Cancer Cell Lines.

In our previous studies, we have demonstrated that NF-κB activation may result from exposure of cancer cells to a variety of apoptotic stimuli including TNF-α, chemotherapy, and irradiation(4). In this report, we have evaluated a wide variety of human colorectal cancer cells to determine whether this inducible response is widely observed or incidental. To evaluate this response,we have selected a variety of resistant colorectal cancer cell lines including those that are mutated for the p53 gene (WiDR,KM12-L4, KM12-SM, SW480, and SW620), contain the K-rasoncogene (LOVO, HCT116, SW480, and SW620), as well as those that overexpress Bcl-2 (KM12-L4 and SW480). The enhanced cytotoxicity responses attained through inhibition of NF-κB were found to be independent of the status of p53, K-ras, or Bcl-2 expression. These results do not imply that expression of these proteins is not relevant to cancer therapy, but that the enhancement of cytotoxicity can be attained in their absence of expression. In preliminary studies, anticancer agents that traditionally have been used to treat patients with metastatic colorectal cancer, including 5-FU and mitomycin C, were found to only weakly induce NF-κB activation in a variety of colorectal cancer cell lines tested (data not shown). In some cell lines, no activation of NF-κB was observed after treatment with these agents. In contrast,SN38, the active metabolite of CPT-11, was found to activate NF-κB in 10 of 11 colorectal cancer cell lines tested. Importantly, in all cases in which NF-κB activation was induced by SN38, inhibition of activation was facilitated by pretreatment with super-repressor IκBα but not the control vector. These findings are consistent with our observations in a variety of different cancer subtypes including pancreatic cancer, sarcoma, and breast cancer in which SN38 was found to be a very potent and consistent inducer of NF-κB activation (data not shown). In addition, relative to a variety of different anticancer agents we have tested, the level of inducible NF-κB activation after treatment with SN38 is surpassed only by TNF-α (data not shown).

Inhibition of SN38-induced NF-κB Activation Enhances Sensitivity to SN38 in a Variety of Resistant Colorectal Cancer Cell Lines.

To evaluate the role of SN38-induced NF-κB activation on the chemosensitivity of a variety of resistant colon cancer cell lines, we pretreated these cells with the super-repressor IκBα. In all cell lines tested, sensitivity to SN38 was markedly enhanced by transient inhibition of NF-κB activation. Furthermore, enhanced chemosensitivity was observed at all concentrations of SN38 tested. This has important clinical relevance because of the inability to achieve the requisite therapeutic dosages of chemotherapy in patients and dose-limiting toxicity. At the lower tolerated levels of chemotherapy attained in patients, we would predict, based on these findings, that enhanced chemosensitivity may be achieved by effective NF-κB inhibition in those cells. Although the ability to predict sensitivity of a patient’s tumor to a specific chemotherapy agent is limited, the observed ability to augment the sensitivity of the variety of cell lines tested suggests that application of this combination therapy approach may have a broad impact on colorectal cancer patients receiving CPT-11.

Enhanced sensitivity to SN38 after inhibition of NF-κB activation was observed in cell lines that ranged in SN38 sensitivity from high (WiDR)to low (HCT116). The response of the various cell lines to combination therapy using the super-repressor IκBα and SN38 may reflect differences in sensitivity to chemotherapy, small differences in adenovirus infectivity, as well as variations in the level of activation of NF-κB after chemotherapy exposure. The mechanism underlying chemotherapy resistance is likely multifactorial(44), as demonstrated in the wide range of genetic errors represented in the cell lines tested. Previous analysis of CPT resistance in colorectal cancer cell lines from the NCI Anticancer Screen reported by Goldwasser et al.(45)suggested that CPT uptake and expression of DNA topoisomerase I did not predict cytotoxicity in response to CPT, although the formation of cleavable complexes did reasonably predict CPT sensitivity. Our results suggest that inducible chemotherapy resistance, mediated by the transcription factor NF-κB, may also be a major determinant of sensitivity to CPT-11 and appears to be a shared survival mechanism among a wide variety of colon cancer cell lines. As described above, we observed enhanced chemosensitivity in both p53 mutated and wild-type cell lines, cells with and without oncogenic K-ras, and independent of high or low levels of Bcl-2 expression.

Our findings using transient inhibition of NF-κB via adenovirus-mediated delivery of the super-repressor IκBα are in contrast to those reported recently by Bentires-Alj et al.(5), in which clones of HCT116 and the highly CPT-resistant breast cancer cell line MCF-7 were selected for stable expression of mutated (super-repressor) IκBα. Our data shown here and published previously suggest that the resistance to chemotherapy-induced apoptosis is mediated by the activation of NF-κB. However, in apparent contrast to our findings, in which inhibition of NF-κB activation in the parental cells led to dramatically enhanced sensitization to SN38, the selected clones that were stably transfected with mutated IκBα in the Bentires-Alj study were not more sensitive to the various chemotherapy agents and TNF-α,despite activation of NF-κB by these stimuli. This suggests that the process of selecting clones that contain stable expression of mutated IκBα leads to the acquisition of alternative survival mechanisms,necessary to overcome the NF-κB inhibition that occurs in the presence of constitutively expressed, mutated IκBα. In contrast to the conclusions of the Bentires-Alj report, our data suggest that, in fact, NF-κB does play a central role in inducible chemoresistance. Furthermore, our findings indicate that transient inhibition of NF-κB activation is a potent adjuvant to the treatment of colon cancer with CPT-11. Additional studies are indicated to further evaluate the mechanisms of chemotherapy-induced NF-κB activation and the resulting antiapoptotic response.

Dose Intensification Using Serial Administration of Combined CPT-11 and NF-κB Inhibition Leads to Complete Tumor Eradication in a Colorectal Cancer Xenograft Model.

Application of combined NF-κB inhibition with CPT-11 for the treatment of human colorectal cancer appears to be most promising for chemotherapy-resistant tumors. Cell lines such as WiDR, which were most sensitive to SN38, were found to have the minimum degree of enhanced response to NF-κB inhibition. Similarly, a minimal amount of enhanced sensitivity was observed in vivo in WiDR tumors that received combined treatment (data not shown). In contrast, the moderate resistance of LOVO to SN38 and CPT-11 in vitro and in vivo, respectively, was dramatically overcome by inhibition of NF-κB activation. By inhibiting the inducible survival response that occurs with each individual drug treatment, through the activation of NF-κB, the maximum apoptotic response to CPT-11 was obtained. Although inhibition of constitutive NF-κB activation as a single therapeutic intervention may have potential in the treatment of some types of malignancy, our findings suggest that inhibition of inducible NF-κB activation when combined with chemotherapy may improve the response to conventional chemotherapies. Furthermore, the results from these preclinical studies provide a rational basis for how to most effectively apply this combination therapeutic strategy to optimize the apoptotic response to CPT-11 in patients receiving treatment for metastatic colorectal cancer. Currently, studies are under way to evaluate the potential role of small molecule inhibitors of NF-κB to enhance the apoptotic response to chemotherapy.

Fig. 1.

A, the EMSA was used to evaluate NF-κB activation induced by 1 μg/ml SN38 in human colorectal and breast(MCF-7) cancer cell lines. Chemotherapy-induced activation of NF-κB was observed in 11 of 12 cancer cell lines tested, suggesting that NF-κB activation is induced by SN38 in most colorectal cancer cell lines. Positive control (+) was KM12L4 cells treated for 2 h with 10 ng/ml TNF-α (a potent activator of NF-κB). The figure shown is representative of the findings from two experiments. B,effect of NF-κB inhibition on human cancer cells treated with different concentrations of SN38. Cells were treated with adenovirus control vector alone (CMV) or with SN38 (CMV/SN38),adenovirus vector expressing the super-repressor IκBα alone(IκBα) or with SN38 (IκBα/SN38), SN38 alone(SN38) or vehicle alone (Cont.). All virus infections were performed 24 h prior to drug treatment. Cell counts obtained at 96 h after drug treatment are reported as the mean of triplicate cultures; bars, SD.

Fig. 1.

A, the EMSA was used to evaluate NF-κB activation induced by 1 μg/ml SN38 in human colorectal and breast(MCF-7) cancer cell lines. Chemotherapy-induced activation of NF-κB was observed in 11 of 12 cancer cell lines tested, suggesting that NF-κB activation is induced by SN38 in most colorectal cancer cell lines. Positive control (+) was KM12L4 cells treated for 2 h with 10 ng/ml TNF-α (a potent activator of NF-κB). The figure shown is representative of the findings from two experiments. B,effect of NF-κB inhibition on human cancer cells treated with different concentrations of SN38. Cells were treated with adenovirus control vector alone (CMV) or with SN38 (CMV/SN38),adenovirus vector expressing the super-repressor IκBα alone(IκBα) or with SN38 (IκBα/SN38), SN38 alone(SN38) or vehicle alone (Cont.). All virus infections were performed 24 h prior to drug treatment. Cell counts obtained at 96 h after drug treatment are reported as the mean of triplicate cultures; bars, SD.

Close modal
Fig. 2.

The ability of the super-repressor IκBα to enhance sensitivity to CPT-11 was assessed in a LOVO xenograft model. A, tumoricidal response of LOVO tumors to CPT-11 administered in combination with the adenovirus vector expressing the super-repressor IκBα (Ad.CMV.IκBα) compared with control adenovirus (Ad.CMV3), or vehicle alone. Adenovirus was administered as a weekly intratumoral injection of 1 × 1010pfu/200 μl of virus for 3 weeks. CPT-11 (33 mg/kg) was administered i.v. every 4 days during the 20-day treatment period. PBS was administered i.v. as a control for treatment with CPT-11. Tumor diameters along two orthogonal axes were recorded every other day. Volume was calculated by assuming a spherical shape of the tumor, using the formula: volume = 4/3 πr3, where r is 1/2 (mean diameter of the tumor), and recorded as the mean for each treatment group(n = 15–19); bars, SE. B, the effect of differing schedules of Ad.CMV.IκBαadministration on tumoricidal response was assessed in vivo. Nude mice bearing s.c. LOVO tumors (mean diameter, 8–10 mm) were treated over a 50-day period with i.v. CPT-11 (33 mg/kg every 4 days) and differing schedules (every 5, 10, 15, and 28 days) of intratumoral injection of the adenovirus expressing the super-repressor IκBα (Ad.CMV.IκBα at a dosage of 1 × 1010 pfu/200 μl). Tumor volume was calculated by assuming a spherical shape of the tumor, using the formula: volume = 4/3 πr3, where r is 1/2(mean diameter of the tumor measured along two orthogonal axes). Tumor volume (Y axis) was recorded as the mean volume for each treatment group (n = 10); bars, SE. C, representative animals from two treatment groups after 50 days of treatment as described in B. Top row, mice that have received intratumoral treatments with Ad.CMV.IκBα administered every 15 days compared with adenovirus treatment every 5 days (bottom row).

Fig. 2.

The ability of the super-repressor IκBα to enhance sensitivity to CPT-11 was assessed in a LOVO xenograft model. A, tumoricidal response of LOVO tumors to CPT-11 administered in combination with the adenovirus vector expressing the super-repressor IκBα (Ad.CMV.IκBα) compared with control adenovirus (Ad.CMV3), or vehicle alone. Adenovirus was administered as a weekly intratumoral injection of 1 × 1010pfu/200 μl of virus for 3 weeks. CPT-11 (33 mg/kg) was administered i.v. every 4 days during the 20-day treatment period. PBS was administered i.v. as a control for treatment with CPT-11. Tumor diameters along two orthogonal axes were recorded every other day. Volume was calculated by assuming a spherical shape of the tumor, using the formula: volume = 4/3 πr3, where r is 1/2 (mean diameter of the tumor), and recorded as the mean for each treatment group(n = 15–19); bars, SE. B, the effect of differing schedules of Ad.CMV.IκBαadministration on tumoricidal response was assessed in vivo. Nude mice bearing s.c. LOVO tumors (mean diameter, 8–10 mm) were treated over a 50-day period with i.v. CPT-11 (33 mg/kg every 4 days) and differing schedules (every 5, 10, 15, and 28 days) of intratumoral injection of the adenovirus expressing the super-repressor IκBα (Ad.CMV.IκBα at a dosage of 1 × 1010 pfu/200 μl). Tumor volume was calculated by assuming a spherical shape of the tumor, using the formula: volume = 4/3 πr3, where r is 1/2(mean diameter of the tumor measured along two orthogonal axes). Tumor volume (Y axis) was recorded as the mean volume for each treatment group (n = 10); bars, SE. C, representative animals from two treatment groups after 50 days of treatment as described in B. Top row, mice that have received intratumoral treatments with Ad.CMV.IκBα administered every 15 days compared with adenovirus treatment every 5 days (bottom row).

Close modal
Fig. 3.

A, EMSA of nuclear protein extracts from LOVO tumors after treatment with the super-repressor IκBα and CPT-11 was used to evaluate the ability of Ad.CMV.IκBα to inhibit NF-κB activation in vivo. Tumors were treated with a single intratumoral injection of Ad.CMV.IκBα (labeled IκBαabove) or control vector Ad.CMV3 (labeled CMV above) as described in“Materials and Methods.” Tumors were treated with CPT-11 24 h after adenovirus injection and harvested at time 0 (24 h after virus treatment), 1, 2, and 6 h after drug treatment. The positive control for this experiment was obtained from the colorectal cell line WiDr treated with SN38 at 2 h.

Fig. 3.

A, EMSA of nuclear protein extracts from LOVO tumors after treatment with the super-repressor IκBα and CPT-11 was used to evaluate the ability of Ad.CMV.IκBα to inhibit NF-κB activation in vivo. Tumors were treated with a single intratumoral injection of Ad.CMV.IκBα (labeled IκBαabove) or control vector Ad.CMV3 (labeled CMV above) as described in“Materials and Methods.” Tumors were treated with CPT-11 24 h after adenovirus injection and harvested at time 0 (24 h after virus treatment), 1, 2, and 6 h after drug treatment. The positive control for this experiment was obtained from the colorectal cell line WiDr treated with SN38 at 2 h.

Close modal
Fig. 4.

Two-color immunohistochemical staining of tumor sections was performed to evaluate adenovirus-mediated transfer of the super-repressor IκBα gene into tumor cells and the level of apoptosis induced by combined treatment of tumors with the super-repressor IκBα and CPT-11. Tumor cells expressing the HA-tagged super-repressor IκBα (fluorescent red) and cells staining TUNEL positive (fluorescent green, see arrows) were detected using a two-color fluorescent microscope (×100) at times 0 (T0h), 1(T1h), 2 (T2h), and 6(T6h) h after CPT-11 treatment.

Fig. 4.

Two-color immunohistochemical staining of tumor sections was performed to evaluate adenovirus-mediated transfer of the super-repressor IκBα gene into tumor cells and the level of apoptosis induced by combined treatment of tumors with the super-repressor IκBα and CPT-11. Tumor cells expressing the HA-tagged super-repressor IκBα (fluorescent red) and cells staining TUNEL positive (fluorescent green, see arrows) were detected using a two-color fluorescent microscope (×100) at times 0 (T0h), 1(T1h), 2 (T2h), and 6(T6h) h after CPT-11 treatment.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by grants from the University of North Carolina Lineberger Comprehensive Cancer Center (to J. C. C.); NCI Grant CA75528 (to J. C. C.) and Grants CA72771, CA75080, and CA73756(to A. S. B.); and the American Cancer Society Clinical Oncology Career Development Award 96-21 (to J. C. C.). Additional support for this project was provided by the University of North Carolina Specialized Program of Research Excellence program in breast cancer NCI Grant CA58223.

3

The abbreviations used are: NF-κB, nuclear factor-κB; CPT-11,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin;CMV, cytomegalovirus; MOI, multiplicity of infection; SN38,7-ethyl-10-hydroxycamptothecin; ATCC, American Type Culture Collection;FBS, fetal bovine serum; EMSA, electrophoretic mobility shift assay;2-ME, 2-mercaptoethanol; pfu, plaque-forming unit(s); TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; HA,hemagglutinin; 5-FU, 5-fluorouracil; CPT, camptothecin; TNF, tumor necrosis factor; NCI, National Cancer Institute.

We thank Yuhua Lin for assistance with statistical analysis and Gail Somodi for assistance in the preparation of the manuscript.

1
Ghosh S., May M. J., Kopp E. B. NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses.
Annu. Rev. Immunol.
,
16
:
225
-260,  
1998
.
2
Wang C. Y., Mayo M. W., Korneluk R. G., Goeddel D. V., Baldwin A. S., Jr. NF-κB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c- IAP2 to suppress caspase-8 activation.
Science (Washington DC)
,
281
:
1680
-1683,  
1998
.
3
Wang C. Y., Cusack J. C., Jr., Liu R., Baldwin A. S., Jr. Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-κB.
Nat. Med.
,
5
:
412
-417,  
1999
.
4
Wang C-Y., Mayo M., Baldwin A. S., Jr. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-κB.
Science (Washington DC)
,
274
:
784
-787,  
1996
.
5
Bentires-Alj M., Hellin A. C., Ameyar M., Chouaib S., Merville M. P., Bours V. Stable inhibition of nuclear factor κB in cancer cells does not increase sensitivity to cytotoxic drugs.
Cancer Res.
,
59
:
811
-815,  
1999
.
6
Cunningham D. Current status of colorectal cancer: CPT-11 (irinotecan), a therapeutic innovation.
Eur. J. Cancer
,
32A
:
S1
-S8,  
1996
.
7
Schmitt C. A., Lowe S. W. Apoptosis and therapy.
J. Pathol.
,
187
:
127
-137,  
1999
.
8
Wu G. S., El-Deiry W. S. Apoptotic death of tumor cells correlates with chemosensitivity, independent of p53 or bcl-2.
Clin. Cancer Res.
,
2
:
623
-633,  
1996
.
9
Kastan M. B. Molecular determinants of sensitivity to antitumor agents.
Biochim. Biophys. Acta
,
1424
:
R37
-R42,  
1999
.
10
el-Deiry W. S. Role of oncogenes in resistance and killing by cancer therapeutic agents.
Curr. Opin. Oncol.
,
9
:
79
-87,  
1997
.
11
Baldini N. Multidrug resistance–a multiplex phenomenon.
Nat. Med.
,
3
:
378
-380,  
1997
.
12
Fisher D. E. Apoptosis in cancer therapy: crossing the threshold.
Cell
,
78
:
539
-542,  
1994
.
13
Friesen C., Herr I., Krammer P. H., Debatin K. M. Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug- induced apoptosis in leukemia cells.
Nat. Med.
,
2
:
574
-577,  
1996
.
14
Fulda S., Susin S. A., Kroemer G., Debatin K. M. Molecular ordering of apoptosis induced by anticancer drugs in neuroblastoma cells.
Cancer Res.
,
58
:
4453
-4460,  
1998
.
15
Kirsch D. G., Kastan M. B. Tumor-suppressor p53: implications for tumor development and prognosis.
J. Clin. Oncol.
,
16
:
3158
-3168,  
1998
.
16
Cunningham D., Pyrhonen S., James R. D., Punt C. J., Hickish T. F., Heikkila R., Johannesen T. B., Starkhammar H., Topham C. A., Awad L., Jacques C., Herait P. Randomised trial of irinotecan plus supportive care versus supportive care alone after fluorouracil failure for patients with metastatic colorectal cancer.
Lancet
,
352
:
1413
-1418,  
1998
.
17
Kudoh S., Fujiwara Y., Takada Y., Yamamoto H., Kinoshita A., Ariyoshi Y., Furuse K., Fukuoka M. Phase II study of irinotecan combined with cisplatin in patients with previously untreated small-cell lung cancer. West Japan Lung Cancer Group.
J. Clin. Oncol.
,
16
:
1068
-1074,  
1998
.
18
Masuda N., Fukuoka M., Takada M., Kusunoki Y., Negoro S., Matsui K., Kudoh S., Takifuji N., Nakagawa K., Kishimoto S. CPT-11 in combination with cisplatin for advanced non-small-cell lung cancer.
J. Clin. Oncol.
,
10
:
1775
-1780,  
1992
.
19
de Forni M., Bugat R., Chabot G. G., Culine S., Extra J. M., Gouyette A., Madelaine I., Marty M. E., Mathieu-Boue A. Phase I and pharmacokinetic study of the camptothecin derivative irinotecan, administered on a weekly schedule in cancer patients.
Cancer Res.
,
54
:
4347
-4354,  
1994
.
20
Oshita F., Noda K., Nishiwaki Y., Fujita A., Kurita Y., Nakabayashi T., Tobise K., Abe S., Suzuki S., Hayashi I., Kawakami Y., Matsuda T., Tsuchiya S., Takahashi S., Tamura T., Saijo N. Phase II study of irinotecan and etoposide in patients with metastatic non-small-cell lung cancer.
J. Clin. Oncol.
,
15
:
304
-309,  
1997
.
21
Cusack J. C., Spitz F. R., Nguyen D., Zhang W. W., Cristiano R. J., Roth J. A. High levels of gene transduction in human lung tumors following intralesional injection of recombinant adenovirus.
Cancer Gene Ther.
,
3
:
245
-249,  
1996
.
22
Jobin C., Panja A., Hellerbrand C., Iimuro Y., Didonato J., Brenner D. A., Sartor R. B. Inhibition of proinflammatory molecule production by adenovirus-mediated expression of a nuclear factor κB super-repressor in human intestinal epithelial cells.
J. Immunol.
,
160
:
410
-418,  
1998
.
23
Haskill S., Beg A. A., Tompkins S. M., Morris J. S., Yurochko A. D., Sampson-Johannes A., Mondal K., Ralph P., Baldwin A. S., Jr. Characterization of an immediate-early gene induced in adherent monocytes that encodes I κB-like activity.
Cell
,
65
:
1281
-1289,  
1991
.
24
Houghton P. J., Cheshire P. J., Hallman J. C., Bissery M. C., Mathieu-Boue A., Houghton J. A. Therapeutic efficacy of the topoisomerase I inhibitor 7-ethyl-10-(4-[1- piperidino]-1-piperidino)-carbonyloxy-camptothecin against human tumor xenografts: lack of cross-resistance in vivo in tumors with acquired resistance to the topoisomerase I inhibitor 9-dimethylaminomethyl-10- hydroxycamptothecin.
Cancer Res.
,
53
:
2823
-2829,  
1993
.
25
Kawato Y., Furuta T., Aonuma M., Yasuoka M., Yokokura T., Matsumoto K. Antitumor activity of a camptothecin derivative, CPT-11, against human tumor xenografts in nude mice.
Cancer Chemother. Pharmacol.
,
28
:
192
-198,  
1991
.
26
Cusack J. C., Wang C.-Y., Liu R., Baldwin A. S. Chemosensitization of colorectal cancer cells by expression of the NF-κB super-repressor IκBa.
Surg. Forum
,
48
:
815
-817,  
1997
.
27
Liu Z. G., Hsu H., Goeddel D. V., Karin M. Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-κB activation prevents cell death.
Cell
,
87
:
565
-576,  
1996
.
28
Van Antwerp D. J., Martin S. J., Kafri T., Green D. R., Verma I. M. Suppression of TNF-α-induced apoptosis by NF-κB.
Science (Washington DC)
,
274
:
787
-789,  
1996
.
29
Beg A. A., Baltimore D. An essential role for NF-κB in preventing TNF-α-induced cell death.
Science (Washington DC)
,
274
:
782
-784,  
1996
.
30
Tartaglia L. A., Goeddel D. V. Two TNF receptors.
Immunol. Today
,
13
:
151
-153,  
1992
.
31
Santana P., Pena L. A., Haimovitz-Friedman A., Martin S., Green D., McLoughlin M., Cordon-Cardo C., Schuchman E. H., Fuks Z., Kolesnick R. Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis.
Cell
,
86
:
189
-199,  
1996
.
32
Hsu H., Huang J., Shu H. B., Baichwal V., Goeddel D. V. TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex.
Immunity
,
4
:
387
-396,  
1996
.
33
Hsu H., Xiong J., Goeddel D. V. The TNF receptor 1-associated protein TRADD signals cell death and NF-κB activation.
Cell
,
81
:
495
-504,  
1995
.
34
Bose R., Verheij M., Haimovitz-Friedman A., Scotto K., Fuks Z., Kolesnick R. Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals.
Cell
,
82
:
405
-414,  
1995
.
35
Kasibhatla S., Brunner T., Genestier L., Echeverri F., Mahboubi A., Green D. R. DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-κB and AP-1.
Mol. Cell
,
1
:
543
-551,  
1998
.
36
Kasibhatla S., Genestier L., Green D. R. Regulation of Fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor κB.
J. Biol. Chem.
,
274
:
987
-992,  
1999
.
37
Parker, S. L., Tong, T., Bolden, S., and Wingo, P. A. Cancer statistics, 1997 [published erratum appears in CA Cancer J. Clin., 47: 68, 1997]. CA Cancer J. Clin., 47: 5–27, 1997.
38
Eisenberg B., Decosse J. J., Harford F., Michalek J. Carcinoma of the colon and rectum: the natural history reviewed in 1704 patients.
Cancer (Phila.)
,
49
:
1131
-1134,  
1982
.
39
Hickman J. A. Apoptosis induced by anticancer drugs.
Cancer Metastasis Rev.
,
11
:
121
-139,  
1992
.
40
Kerr, J. F., Winterford, C. M., and Harmon, B. V. Apoptosis. Its significance in cancer and cancer therapy [published erratum appears in Cancer, 73: 3108, 1994]. Cancer (Phila.), 73: 2013–2026, 1994.
41
Steller H. Mechanisms and genes of cellular suicide.
Science (Washington DC)
,
267
:
1445
-1449,  
1995
.
42
Cunningham D., Pyrhonen S., James R. D., Punt C. J. A., Hickish T. S., Heikkila R., Johannesen T., Starkhammar H., Topham C. A., Ong E., Herait P., Jaques C. A Phase III multicenter randomized study of CPT-11 versus supportive care (SC) alone in patients (Pts) with 5-FU-resistant metastatic colorectal cancer (MCRC).
Proc. Am. Assoc. Clin. Oncol.
,
17
:
1
1998
.
43
Abigerges D., Chabot G. G., Armand J. P., Herait P., Gouyette A., Gandia D. Phase I and pharmacologic studies of the camptothecin analog irinotecan administered every 3 weeks in cancer patients.
J. Clin. Oncol.
,
13
:
210
-221,  
1995
.
44
Nieves-Neira W., Pommier Y. Apoptotic response to camptothecin and 7-hydroxystaurosporine (UCN-01) in the 8 human breast cancer cell lines of the NCI Anticancer Drug Screen: multifactorial relationships with topoisomerase I, protein kinase C, Bcl-2, p53, MDM-2 and caspase pathways.
Int. J. Cancer
,
82
:
396
-404,  
1999
.
45
Goldwasser F., Bae I., Valenti M., Torres K., Pommier Y. Topoisomerase I-related parameters and camptothecin activity in the colon carcinoma cell lines from the National Cancer Institute anticancer screen.
Cancer Res.
,
55
:
2116
-2121,  
1995
.