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Overexpression of Bcl-xL Promotes Chemotherapy Resistance of Mammary Tumors in a Syngeneic Mouse Model

1999; Elsevier BV; Volume: 155; Issue: 6 Linguagem: Inglês

10.1016/s0002-9440(10)65505-8

1525-2191

Rebecca Liu, Carmen Page, David Beidler, Max S. Wicha, Gabriel Núñez,

Cancer-related Molecular Pathways

Bcl-xL, a prosurvival member of the Bcl-2 family that is expressed in many tumors, represses apoptosis induced by chemotherapeutic drugs in vitro. However, the contribution of apoptosis and prosurvival Bcl-2-related proteins to chemotherapy resistance in vivo is unknown and has been challenged by recent results with clonogenic survival assays. To test the ability of Bcl-xL to provide chemotherapy resistance to tumors, we transfected the mouse bcl-xL gene into the tumorigenic SCK mammary cell line and assessed the response of tumor cells to chemotherapeutic drugs in clonogenic assays and in a syngeneic mouse model. Bcl-xL conferred protection on SCK cells against methotrexate at certain drug concentrations, but not at all against 5-fluorouracil in clonogenic survival assays in vitro. Injection of SCK cells transfected with Bcl-xL or control plasmid in the mammary fat pads of syngeneic recipient mice resulted in tumors of similar size. However, although the volume of control tumors regressed up to 80% after 4 to 5 days of chemotherapy, SCK tumors expressing Bcl-xL did not regress and continued to grow in the presence of methotrexate or 5-fluorouracil. In addition, numbers of apoptotic cells were significantly higher in control tumors as compared to Bcl-xL-expressing tumors in animals treated with methotrexate or 5-fluorouracil. These results provide evidence that inhibition of apoptosis through Bcl-xL overexpression can promote resistance to chemotherapy in tumors in vivo. Bcl-xL, a prosurvival member of the Bcl-2 family that is expressed in many tumors, represses apoptosis induced by chemotherapeutic drugs in vitro. However, the contribution of apoptosis and prosurvival Bcl-2-related proteins to chemotherapy resistance in vivo is unknown and has been challenged by recent results with clonogenic survival assays. To test the ability of Bcl-xL to provide chemotherapy resistance to tumors, we transfected the mouse bcl-xL gene into the tumorigenic SCK mammary cell line and assessed the response of tumor cells to chemotherapeutic drugs in clonogenic assays and in a syngeneic mouse model. Bcl-xL conferred protection on SCK cells against methotrexate at certain drug concentrations, but not at all against 5-fluorouracil in clonogenic survival assays in vitro. Injection of SCK cells transfected with Bcl-xL or control plasmid in the mammary fat pads of syngeneic recipient mice resulted in tumors of similar size. However, although the volume of control tumors regressed up to 80% after 4 to 5 days of chemotherapy, SCK tumors expressing Bcl-xL did not regress and continued to grow in the presence of methotrexate or 5-fluorouracil. In addition, numbers of apoptotic cells were significantly higher in control tumors as compared to Bcl-xL-expressing tumors in animals treated with methotrexate or 5-fluorouracil. These results provide evidence that inhibition of apoptosis through Bcl-xL overexpression can promote resistance to chemotherapy in tumors in vivo. The treatment of cancer cells with chemotherapy and irradiation is limited by the emergence of cancer cells resistant to these therapies. Frequently, many tumors respond to chemotherapy during initial treatment but develop a multidrug resistance phenotype with continued therapy. The molecular events responsible for this resistance remain largely unknown. Overexpression of the P-glycoprotein and other members of the ATP-binding cassette (ABC) superfamily has been shown to induce multidrug resistance.1Endicott JA Ling V The biochemistry of P-glycoprotein-mediated multidrug resistance.Annu Rev Biochem. 1989; 58: 137-171Crossref PubMed Scopus (2165) Google Scholar, 2Ling V Multidrug resistance: molecular mechanisms and clinical relevance.Cancer Chemother Pharmacol. 1997; 40: 3-8Crossref Scopus (477) Google Scholar, 3Grant CE Valdimarsson G Hipfner DR Almquist KC Cole SPC Deeley RG Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs.Cancer Res. 1994; 54: 357-361PubMed Google Scholar However, development of multidrug resistance is often observed in the absence of ABC protein overexpression,4Futscher BW Abbaszadegan MR Domann F Dalton WS Analysis of MRP mRNA in mitoxantrone-selected, multidrug-resistant human tumor cells.Biochem Pharmacol. 1994; 47: 1601-1606Crossref PubMed Scopus (74) Google Scholar, 5Zaman GJR Versantvoort CHM Smit JJM Eijdems EWHM deHaas M Smith AJ Broxterman HJ Mulder NH de Vries EGE Baas F Borst P Analysis of the expression of MRP, the gene for a new putative transmembrane drug transporter, in human multidrug resistant lung cancer cell lines.Cancer Res. 1993; 53: 1747-1750PubMed Google Scholar suggesting that other mechanisms play a role in drug resistance. Another mechanism responsible for this pleotropic drug resistance may involve expression of antiapoptotic proteins, as multiple chemotherapy drugs have been shown to kill tumor cells by apoptosis.6Barry MA Behnke CA Eastman A Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins, and hyperthermia.Biochem Pharmacol. 1990; 40: 2343-2362Crossref PubMed Scopus (932) Google Scholar, 7Kaufmann SH Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note.Cancer Res. 1989; 49: 5870-5878PubMed Google Scholar Members of the Bcl-2 family of proteins are important regulators of apoptosis induced by a wide array of stimuli, including chemotherapeutic agents.8Miyashita T Reed JC Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line.Blood. 1993; 81: 151Crossref PubMed Google Scholar, 9Minn AJ Rudin CM Boise LH Thompson CB Expression of bcl-xL can confer a multidrug resistance phenotype.Blood. 1995; 86: 1903Crossref PubMed Google Scholar, 10Simonian PL Grillot DAM Nuñez G Bcl-2 and Bcl-xL can differentially block chemotherapy-induced cell death.Blood. 1997; 90: 1208-1216Crossref PubMed Google Scholar Two members of this family, Bcl-2 and Bcl-xL, function as repressors of cell death and are expressed in a wide variety of human tumors derived from epithelial, hematopoietic, and soft tissue lineages.11Yang E Korsmeyer SJ Molecular thanatopsis: a discourse on the Bcl-2 family and cell death.Blood. 1996; 88: 386-401Crossref PubMed Google Scholar Expression levels of Bcl-2-related proteins change as tumors become less differentiated, or after treatment,12Castle VP Heidelberger KP Bromberg J Ou X Dole M Nuñez G Expression of the apoptosis-suppressing protein bcl-2, in neuroblastoma is associated with unfavorable histology and N-myc amplification.Am J Pathol. 1993; 143: 1543-1550PubMed Google Scholar, 13Weller M Malipiero U Aguzzi A Reed JC Fontana A Protooncogene bcl-2 gene transfer abrogates Fas/APO-1 antibody-mediated apoptosis of human malignant glioma cells and confers resistance to chemotherapeutic drugs and therapeutic irradiation.J Clin Invest. 1995; 95: 2633-2643Crossref PubMed Scopus (268) Google Scholar, 14Krajewska M Krajewski S Epstein JI Shabaik A Sauvageot J Song K Kitada S Reed JC Immunohistochemical analysis of bcl-2, bax, bcl-x, and mcl-1 expression in prostate cancers.Am J Pathol. 1996; 148: 1567-1576PubMed Google Scholar, 15Tu Y Renner S Xu F Fleishman A Taylor J Weisz J Vescio R Rettig M Berenson J Krajewski S Reed JC Lichtenstein A Bcl-x expression in multiple myeloma: possible indicator of chemoresistance.Cancer Res. 1998; 58: 256-262PubMed Google Scholar suggesting that expression of Bcl-2 family members may play an important role in tumor progression and/or resistance to therapy. Although the precise mechanism by which Bcl-2 and Bcl-xL inhibit apoptosis is controversial, it is thought that these prosurvival proteins act by interfering with the activation of initiator (upstream. caspases.16Adams JM Cory S The Bcl-2 protein family: arbiters of cell survival.Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4855) Google Scholar Because chemotherapeutic drugs induce caspase activation,17Eischen CM Kottke TJ Martins LM Basi GS Tung JS Earnshaw WC Leibson PJ Kaufmann SH Comparison of apoptosis in wild-type and Fas-resistant cells: chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions.Blood. 1997; 90: 935-943Crossref PubMed Google Scholar, 18Sun X-M MacFarlane M Zhuang J Wolf BB Green DR Cohen GM Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis.J Biol Chem. 1999; 274: 5053-5060Crossref PubMed Scopus (782) Google Scholar Bcl-2 and Bcl-xL may inhibit chemotherapy-induced apoptosis, at least in part, by repressing chemotherapy-induced caspase activity. However, it remains to be determined whether inhibition of apoptosis in tumor cells could lead to resistance to chemotherapy in vivo. Because Bcl-2 and Bcl-xL can inhibit chemotherapy-induced apoptosis in vitro, it has been suggested that the expression of these proteins plays a role in chemotherapy resistance. However, the contribution of Bcl-2 and Bcl-xL to tumor chemotherapy resistance remains unclear. Bcl-2 expression has been associated with poor response to chemotherapy in acute myeloid leukemia19Campos L Rouault JP Sabido O Oriol P Roubi N Vasselon C Archimbaud E Magaud JP Guyotat D High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy.Blood. 1993; 81: 3091Crossref PubMed Google Scholar, 20Maung ZT MacLean FR Reid MM Pearson AD Proctor SJ Hamilton PJ Hall AG The relationship between bcl-2 expression and response to chemotherapy in acute leukaemia.Br J Haematol. 1994; 88: 105-109Crossref PubMed Scopus (151) Google Scholar and large-cell lymphoma.21Hill ME MacLennan KA Cunningham DC Vaughan Hudson B Burke M Clarke P Di Stefano F Anderson L Vaughan Hudson G Mason D Selby P Linch DC Prognostic significance of Bcl-2 expression and bcl-2 major breakpoint region rearrangement in diffuse large cell non-Hodgkin's lymphoma: a British National Lymphoma Investigation Study.Blood. 1996; 88: 1046-1051PubMed Google Scholar, 22Hermine O Haioun C Lepage E d'Agay MF Briere J Lavignac C Fillet G Salles G Marolleau JP Diebold J Reyas F Gaulard P Prognostic significance of bcl-2 protein expression in aggressive non-Hodgkin's lymphoma. Groupe d'Etude des Lymphomes de l'Adulte (GELA).Blood. 1996; 87: 265-272Crossref PubMed Google Scholar Yet the significance of Bcl-2 or Bcl-xL expression is complicated by the observation that these survival proteins inhibit chemotherapy- and irradiation-induced apoptosis in short-term assays, but in certain systems do not affect clonogenic survival of tumor cells in vitro.23Lock RB Stribiniskiene L Dual modes of death induced by etoposide in human epithelial tumor cells allow Bcl-2 to inhibit apoptosis without affecting clonogenic survival.Cancer Res. 1996; 56: 4006-4012PubMed Google Scholar, 24Yin DX Schimke RT Bcl-2 expression delays drug-induced apoptosis but does not increase clonogenic survival after drug treatment in HeLa cells.Cancer Res. 1995; 55: 4922-4928PubMed Google Scholar, 25Kyprianou N King ED Bradbury D Rhee JG Bcl-2 overexpression delays radiation-induced apoptosis without affecting the clonogenic survival of human prostate cancer cells.Int J Cancer. 1997; 70: 341-346Crossref PubMed Scopus (154) Google Scholar These observations suggest that the antiapoptotic effect of Bcl-2 may not necessarily translate into increased survival of tumor cells in vivo, as the capacity of Bcl-2 or Bcl-xL to modulate chemotherapy-induced apoptosis in growing tumors remains to be determined. In the present study, we have expressed Bcl-xL in the murine mammary adenocarcinoma cell line SCK and determined the ability of Bcl-xL to regulate the response of tumor cells to chemotherapy in clonogenic assays in vitro and in vivo using a syngeneic mouse model. SCK mouse mammary carcinoma cells,26Lin JC Song SW Influence of vascular thermotolerance on the heat-induced changes in blood flow, pO2, and cell survival in tumors.Cancer Res. 1993; 53: 2076-2080PubMed Google Scholar kindly provided by Dr. C. W. Song of the University of Minnesota, were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, and 100 μg/ml streptomycin. SCK cells (5 × 106) were transfected using lipofectamine (Gibco BRL, Rockville, MD) with 6 μg of the pSFFV-m-Bcl-xL to produce Flag-tagged mouse Bcl-xL27González-García M Ballestero R Ding L Duan L Boise L Duan L Boise LH Thompson CB Nuñez G Bcl-xL is the major Bcl-x in RNA from expressed during murine development, and its product localizes to mitochondria.Development. 1994; 120: 3033-3042PubMed Google Scholar or control pSFFV plasmid (Invitrogen, Carlsbad, CA). pcDNA-3-m-Bcl-xL was constructed by ligation of Flag-tagged mouse Bcl-xL cDNA into the plasmid pcDNA-3 (Invitrogen). Authenticity of the constructs was confirmed by dideoxy sequencing. Individual cell clones were selected for growth in the presence of G418 (0.5 mg/ml) by limiting dilution. Expression of Flag-Bcl-xL in single cell clones was analyzed by flow cytometry using anti-Flag and protein expression was confirmed by Western blot analysis as described below. Cells were seeded in rows of 750, 250, and 100 cells/well in 6-well plates (Costar, Corning, NY) and further incubated for 18 hours. Concentrated stock solutions of methotrexate and 5-fluorouracil were serially diluted and added to each of the rows. After a 6-hour exposure, the medium was aspirated and the wells were washed in drug-free medium for 1 hour, followed by a 10-day incubation in drug-free medium to allow colony formation. At the end of this incubation, medium was aspirated and the cells were fixed and stained by the addition of 0.5% methylene blue in 50% ethanol for 45 minutes at room temperature. The plates were gently washed with water and allowed to air-dry. Visible colonies were counted to determine the percent colony formation of plated cells for each drug treatment. Colony formation percentages for each drug treatment were compared to colony-formation values of untreated controls. Values were expressed as the mean ± SE from triplicate experiments. The expression of Flag-murine Bcl-xL was determined by Western blot analysis as described previously28Merino R Grillot DAM Simonian PL Muthukkumar S Fanslow WC Bondada S Nuñez G Modulation of anti-IgM-induced B cell apoptosis by Bcl-xL and CD40: dissociation from cell cycle arrest and dependence on the avidity of the antibody-IgM receptor interaction.J Immunol. 1995; 155: 3830-3838PubMed Google Scholar using anti-Flag mAb (5 μg/ml). After incubation with rabbit anti-mouse IgG secondary antibody, the reaction was developed by enhanced chemiluminescense using the ECL kit (Amersham, Arlington Heights, IL) and exposed to film (Eastman Kodak). For in vitro apoptosis assays, cells were seeded at 1 × 105 cells in triplicate wells in media containing methotrexate 1 μg/ml (Immunex, Seattle, WA) or 5-fluorouracil 1 μg/ml (Hoffman-LaRoche, Nutley, NJ). The percentage of apoptotic cells was determined at different time points in triplicate cultures by nuclear propidium iodide staining followed by flow cytometric analysis as described previously.28Merino R Grillot DAM Simonian PL Muthukkumar S Fanslow WC Bondada S Nuñez G Modulation of anti-IgM-induced B cell apoptosis by Bcl-xL and CD40: dissociation from cell cycle arrest and dependence on the avidity of the antibody-IgM receptor interaction.J Immunol. 1995; 155: 3830-3838PubMed Google Scholar Results were based on the analysis of at least 5 × 104 events from each triplicate culture. Values were expressed as the mean ± SE from triplicate cultures. Apoptosis in tumors was evaluated by analysis of tumor sections stained with hematoxylin and eosin using histological criteria as described.29Nishimura R Nagao K Miyayama H Matsuda M Baba K Matsuoka Y Yamashita H Fukuda M Higu Chi A Apoptosis in breast cancer and its relationship to clinic pathological characteristics and prognosis.J Surg Oncol. 1999; 71: 226-234Crossref PubMed Scopus (23) Google Scholar Apoptosis was quantitated in 10 consecutive high power fields (HPF; 40× lens objective) in each tumor sample. Number of apoptotic cells was evaluated in viable tumor at the outer cortex of the tumor mass in a blinded fashion. Each HPF consisted of solid sheets of tumor cells, with the same approximate number of cells noted in both control and Bcl-xL expressing tumors. Number of mitotic figures per HPF were quantitated in a similar fashion. SCK tumors were established in syngeneic female A/J mice as described above. The incision was closed with wound clips and tumors were allowed to grow for 9 to 10 days. Methotrexate (0.9 mg/kg/day) or 5-fluorouracil (23 mg/kg/day) diluted in sterile normal saline was administered intraperitoneally (i.p.) q.d. × 3 to 4 days. Control animals received sterile normal saline alone, and tumor measurements were taken in parallel. Tumor volume was measured every day with linear calipers and calculated in cubic millimeters as (a × b2/2), where a is the larger diameter and b the smaller diameter of the tumor. For in vitro chemotherapy-induced death assays, statistical significance was calculated by two-way analysis of variance using SYSTAT software (Chicago, IL). For in vivo chemotherapy response assays, statistical significance was calculated using a general linear model with two-way analysis of variance. Post hoc comparisons of mean values from different groups using Bonferroni adjustment were performed for both in vitro and in vivo assays. We selected the SCK mammary mouse tumor model to assess the ability of Bcl-xL to promote chemotherapy resistance in vivo, as SCK carcinoma cells were derived spontaneously and established as tumors in the absence of any exposure to chemotherapeutic drugs.26Lin JC Song SW Influence of vascular thermotolerance on the heat-induced changes in blood flow, pO2, and cell survival in tumors.Cancer Res. 1993; 53: 2076-2080PubMed Google Scholar Furthermore, SCK form tumors in female syngeneic A/J hosts, which mimics the clinical setting more closely than xenograft models. Before determining the ability of Bcl-xL to regulate chemotherapy resistance of tumors in the animal, we determined if Bcl-xL provides protection to SCK carcinoma cells against chemotherapy-induced apoptosis in vitro. In these experiments, we used one of two different Bcl-xL expression plasmids, pSFFV- Bcl-xL or pcDNA3-Bcl-xL, to overexpress Flag-tagged mouse Bcl-xL. As a control, we transfected SCK cells with the corresponding empty vectors. After selection in G418, three independently derived SCK clones that stably overexpress Bcl-xL were identified (Figure 1A). All three Bcl-xL clones exhibited reduced apoptosis after incubation 5-fluorouracil (Figure 1B) and methotrexate (Figure 1C), two drugs commonly used to treat patients with breast cancer, when compared with SCK clones transfected with control plasmids. These results are in agreement with a large body of evidence indicating that Bcl-xL inhibits or delays chemotherapy-induced apoptosis of tumor cells in short-term assays in vitro.8Miyashita T Reed JC Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line.Blood. 1993; 81: 151Crossref PubMed Google Scholar, 9Minn AJ Rudin CM Boise LH Thompson CB Expression of bcl-xL can confer a multidrug resistance phenotype.Blood. 1995; 86: 1903Crossref PubMed Google Scholar, 10Simonian PL Grillot DAM Nuñez G Bcl-2 and Bcl-xL can differentially block chemotherapy-induced cell death.Blood. 1997; 90: 1208-1216Crossref PubMed Google Scholar The clonogenic ability of tumor cells is thought to be a more reliable assay than short-term survival in predicting the response of tumor cells to chemotherapy in vivo. We determined next whether Bcl-xL would provide increased clonogenic survival in vitro after transient exposure to chemotherapeutic drugs. SCK cells expressing Bcl-xL, or control cells stably transfected with control plasmid, were treated with increasing concentrations of methotrexate, 5-fluorouracil, or vehicle control for 6 hours, then washed and cultured in drug-free medium to permit colony formation. Colonies that formed in triplicate cultures after 10 days were stained with methylene blue and counted. The ability of both SCK-Bcl-xL and control SCK-pSFFV cells to form colonies declined after treatment with methotrexate or 5-fluorouracil in a dose-dependent manner when compared to untreated cells (Figure 2, A and B). In three separate experiments, Bcl-xL-expressing cells demonstrated significantly increased colony formation after exposure to certain concentrations of methotrexate when compared to control cells (Figure 2A), but Bcl-xL did not have any significant effect on colony formation at any 5-fluorouracil concentration (Figure 2B). To determine whether expression of Bcl-xL would confer resistance to chemotherapy in vivo, we used SCK murine mammary adenocarcinoma cells grown in female syngeneic A/J recipient mice as an in vivo tumor model. Dose-response experiments revealed that i.p. injections of methotrexate at 0.9 mg/kg/day or 5-fluorouracil at 23 mg/kg/day were optimal to assess the response of SCK tumors to chemotherapy (data not shown). We injected 1 × 106 SCK cells stably transfected with Bcl-xL or control plasmid in the right and left mammary fat pads of the same mice to adequately control for the amount of chemotherapy given to both groups of tumors. Tumors averaging 60 mm3 were established by day 8 to 9 after injection of 1 × 106 SCK cells transfected with Bcl-xL or control plasmid into the mammary fat pads. No significant difference in volume was observed between tumors established from SCK-Bcl-xL and SCK-control plasmid (59.5 ± 28 versus 61.9 ± 27 mm3, n = 27). Mice bearing SCK tumors expressing Bcl-xL (one mammary pad) and control plasmid (contralateral mammary pad) were treated with daily i.p. injections of 5-fluorouracil or methotrexate, two chemotherapeutic drugs commonly used to treat mammary cancers. Tumors derived from SCK cells transfected with Bcl-xL (clone 46) were significantly larger than control tumors after treatment with daily injections of 5-fluorouracil (Figure 3A). Quantitative analysis revealed that tumors derived from SCK transfected with control plasmid decreased to 20 to 30% of the original tumor volume by day 2, and to less than 20% by day 4 after daily i.p. administration of 5-fluorouracil (Figure 3B). In contrast, tumors derived from SCK cells expressing Bcl-xL cells were resistant to 5-fluorouracil in that they continued to increase in volume over 200% in the presence of the drug (Figure 3B). Mice harboring SCK tumors expressing Bcl-xL had to be sacrificed by day 6 or 7 after administration of chemotherapy due to large tumor burden. To determine whether the ability of Bcl-xL to confer resistance of tumors to 5-fluorouracil could be extended to other chemotherapeutic drugs, we analyzed the susceptibility of Bcl-xL-expressing and control SCK tumors to methotrexate. Tumors from SCK cells transfected with Bcl-xL or control plasmid grew at the same rate in vivo in the absence of methotrexate (Figure 4A). As we observed with 5-fluorouracil, tumors derived from SCK cells transfected with Bcl-xL (clone 46) continued to increase in volume, whereas control SCK tumors decreased by day 2 and 3 after drug administration (Figure 4, A and B). To further verify these observations, we assessed the susceptibility to methotrexate of tumors derived from two additional SCK-Bcl-xL clones (clones 3 and 15) and two SCK control clones (clones 3.3 and 3.15). Tumors established from SCK-Bcl-xL clones were resistant to chemotherapy when compared with SCK clones transfected with empty vector (Figure 4B). These results indicate that these observations with Bcl-xL are not due to clonal variation in that they could be reproduced in three independently derived clones. To determine whether expression of Bcl-xL would inhibit chemotherapy-induced apoptosis in vivo or if the increase in volume seen in Bcl-xL-expressing tumors was due to increased mitotic activity, we assessed the number of apoptotic cancer cells in histological sections of SCK tumors. 1 × 106 SCK cells transfected with Bcl-xL or control plasmid in the right and left mammary fat pads of the same syngeneic A/J mice were established, and the mice were treated with daily injections of i.p. methotrexate or 5-fluorouracil. Tumors were excised at day 0 and day 5 or 6 of treatment. Numbers of apoptotic cells were significantly higher in control tumors by day 5 or 6 of treatment as compared to Bcl-xL-expressing tumors in animals treated with methotrexate or 5-fluorouracil (Figure 5, A and B). Numbers of mitotic figures in control tumors were not significantly different from those in Bcl-xL-expressing tumors at day 0 and day 5 or 6 (data not shown). The results presented here demonstrate that overexpression of Bcl-xL promotes resistance of mammary cancer cells to chemotherapy in a syngeneic mouse model, suggesting that this protein may confer a similar function on primary tumors overexpressing Bcl-xL in humans. A large variety of human tumors derived from different tissues including the breast express Bcl-xL.30Olopade OI Adeyanju MO Safa AR Hagos F Mick R Thompson CB Recant WM Overexpression of Bcl-x protein in primary breast cancer is associated with high tumor grade and nodial metastases.Cancer J Sci Am. 1997; 3: 230-237PubMed Google Scholar Significantly, expression of Bcl-xL in primary breast tumors has been associated with poor prognosis,30Olopade OI Adeyanju MO Safa AR Hagos F Mick R Thompson CB Recant WM Overexpression of Bcl-x protein in primary breast cancer is associated with high tumor grade and nodial metastases.Cancer J Sci Am. 1997; 3: 230-237PubMed Google Scholar suggesting that Bcl-xL plays a role in tumor progression or response to therapy. To our knowledge, this is the first demonstration that a prosurvival Bcl-2 family member can promote resistance of tumor cells to chemotherapy in vivo. The results obtained here with Bcl-xL may also apply to Bcl-2, as both of these structurally related proteins are thought to share a common mechanism that inhibits apoptosis.16Adams JM Cory S The Bcl-2 protein family: arbiters of cell survival.Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4855) Google Scholar Significantly, expression of Bcl-2 and Bcl-xL in untreated primary tumors is often focal, with only a subset of the tumor cells expressing high levels of these proteins.12Castle VP Heidelberger KP Bromberg J Ou X Dole M Nuñez G Expression of the apoptosis-suppressing protein bcl-2, in neuroblastoma is associated with unfavorable histology and N-myc amplification.Am J Pathol. 1993; 143: 1543-1550PubMed Google Scholar, 13Weller M Malipiero U Aguzzi A Reed JC Fontana A Protooncogene bcl-2 gene transfer abrogates Fas/APO-1 antibody-mediated apoptosis of human malignant glioma cells and confers resistance to chemotherapeutic drugs and therapeutic irradiation.J Clin Invest. 1995; 95: 2633-2643Crossref PubMed Scopus (268) Google Scholar, 14Krajewska M Krajewski S Epstein JI Shabaik A Sauvageot J Song K Kitada S Reed JC Immunohistochemical analysis of bcl-2, bax, bcl-x, and mcl-1 expression in prostate cancers.Am J Pathol. 1996; 148: 1567-1576PubMed Google Scholar, 15Tu Y Renner S Xu F Fleishman A Taylor J Weisz J Vescio R Rettig M Berenson J Krajewski S Reed JC Lichtenstein A Bcl-x expression in multiple myeloma: possible indicator of chemoresistance.Cancer Res. 1998; 58: 256-262PubMed Google Scholar, 30Olopade OI Adeyanju MO Safa AR Hagos F Mick R Thompson CB Recant WM Overexpression of Bcl-x protein in primary breast cancer is associated with high tumor grade and nodial metastases.Cancer J Sci Am. 1997; 3: 230-237PubMed Google Scholar A prediction from our results is that chemotherapy would select for tumor clones that overexpress Bcl-2 or Bcl-xL. Consistent with this hypothesis, the percentage of tumor cells that express Bcl-2 and/or the intensity of Bcl-2 staining increases after chemotherapy in primary tumors,12Castle VP Heidelberger KP Bromberg J Ou X Dole M Nuñez G Expression of the apoptosis-suppressing protein bcl-2, in neuroblastoma is associated with unfavorable histology and N-myc amplification.Am J Pathol. 1993; 143: 1543-1550PubMed Google Scholar, 13Weller M Malipiero U Aguzzi A Reed JC Fontana A Protooncogene bcl-2 gene transfer abrogates Fas/APO-1 antibody-mediated apoptosis of human malignant glioma cells and confers resistance to chemotherapeutic drugs and therapeutic irradiation.J Clin Invest. 1995; 95: 2633-2643Crossref PubMed Scopus (268) Google Scholar, 19Campos L Rouault JP Sabido O Oriol P Roubi N Vasselon C Archimbaud E Magaud JP Guyotat D High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy.Blood. 1993; 81: 3091Crossref PubMed Google Scholar, 20Maung ZT MacLean FR Reid MM Pearson AD Proctor SJ Hamilton PJ Hall AG The relationship between bcl-2 expression and response to chemotherapy in acute leukaemia.Br J Haematol. 1994; 88: 105-109Crossref PubMed Scopus (151) Google Scholar and selection of SCC 25 squamous carcinoma cells for resistance to chemotherapy in vitro is associated with expression of Bcl-xL.31Datta R Manome Y Taneja N Boise LH Weichselbaum R Thompson CB Slapak CA Kufe D Overexpression of Bcl-xL by cytotoxic drug exposure confers resistance to ionizing radiation-induced internucleosomal DNA fragmentation.Cell Growth Differ. 1995; 6: 363-370PubMed Google Scholar An important observation derived from these studies is the lack of correlation between the ability of Bcl-xL to affect clonogenic survival in vitro and tumor growth in vivo after chemotherapy. This was particularly observed with the response of SCK tumor cells to 5-fluorouracil. In vivo, tumor cells receive signals from locally produced growth and survival factors, as well as from stromal and cell-to-cell interactions. Because proteins like Bcl-xL are thought to regulate an intracellular survival threshold critical for the induction of apoptosis, these tissue signals are likely to play an important role in determining the apoptotic response in vivo. In addition, clonogenic ability in vitro and tumor growth in vivo require signals that promote both survival and cell cycle progression. Methotrexate and 5-fluorouracil induce both cell cycle arrest and apoptosis in tumor cells.32el Alaoui S Lawry J Griffin M The cell cycle and induction of apoptosis in a hamster fibrosarcoma cell line treated with anti-cancer drugs: its importance to solid tumour chemotherapy.J Neurooncol. 1997; 31: 195-207Crossref PubMed Scopus (25) Google Scholar Bcl-xL is thought to function primarily to block apoptotic signals.16Adams JM Cory S The Bcl-2 protein family: arbiters of cell survival.Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4855) Google Scholar Indeed, we have found that Bcl-xL-expressing tumors exhibit diminished apoptosis as compared to control tumors in animals treated with methotrexate and 5-fluorouracil. By inhibiting apoptosis, Bcl-xL may allow tumor cells to receive signals required to overcome cell cycle arrest induced by chemotherapy drugs. Consistent with this thesis, CD40 ligation and interleukin-4 have been shown to act with Bcl-2 to increase clonogenicity of lymphoma cells.33Walker A Taylor ST Hickman JA Dive C Germinal center-derived signals act with Bcl-2 to decrease apoptosis and increase clonogenicity of drug-treated human B lymphoma cells.Cancer Res. 1997; 57: 1939-1945PubMed Google Scholar Thus, Bcl-xL may cooperate with signals provided by the tumor microenvironment to promote tumor growth in the presence of chemotherapy. Alternatively, to function, Bcl-xL may require signals that are provided in vivo, but not in vitro. These signals may include phosphorylation or other posttranscriptional modifications of Bcl-xL itself or that of Bcl-xL regulatory proteins.16Adams JM Cory S The Bcl-2 protein family: arbiters of cell survival.Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4855) Google Scholar For example, phosphorylation of BAD, an inhibitor of Bcl-xL, is regulated by growth factors and this regulation might be lacking or diminished in vitro.34del Peso L Gonzalez-Garcia M Page C Herrera R Nuñez G Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt.Science. 1997; 278: 687-689Crossref PubMed Scopus (2000) Google Scholar, 35Datta SR Dudek H Tao X Masters S Fu H Gotoh Y Greenberg ME Akt phosphorylation of Bad couples survival signals to the cell-intrinsic death machinery.Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (5003) Google Scholar Chemotherapy resistance in tumor cells is complex and involves many mechanisms that depend in part on the specific drug being used. The contribution of the apoptotic pathway to chemotherapy resistance in tumors is unclear. Our results with Bcl-xL suggest that inhibition of the apoptotic process is an important cause of chemotherapy resistance in vivo. The mechanism by which Bcl-xL and Bcl-2 promote chemotherapy resistance is different from classical drug-target interactions in that these survival proteins provide a multidrug resistance phenotype. The broad-range drug resistance effect of Bcl-2 and Bcl-xL can be explained by their ability to act at a common step in the apoptotic pathway.16Adams JM Cory S The Bcl-2 protein family: arbiters of cell survival.Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4855) Google Scholar In addition to prosurvival Bcl-2 family members, several proteins have been identified that inhibit apoptosis and are expressed in primary tumor cells. They include cellular FLIP36Irmler M Thome M Hahne M Schneider P Hofmann K Steiner V Bodmer JL Schroter M Burns K Mattmann C Rimoldi D French LE Tschopp J Inhibition of death receptor signals by cellular FLIP.Nature. 1997; 388: 190-195Crossref PubMed Scopus (2246) Google Scholar and survivin,37Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar, 38Tamm I Wang Y Sausville E Scudiero DA Vigna N Oltersdorf T Reed JC IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs.Cancer Res. 1998; 58: 5315-5320PubMed Google Scholar proteins that may inhibit apoptosis by selectively targeting caspases.36Irmler M Thome M Hahne M Schneider P Hofmann K Steiner V Bodmer JL Schroter M Burns K Mattmann C Rimoldi D French LE Tschopp J Inhibition of death receptor signals by cellular FLIP.Nature. 1997; 388: 190-195Crossref PubMed Scopus (2246) Google Scholar, 37Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar, 38Tamm I Wang Y Sausville E Scudiero DA Vigna N Oltersdorf T Reed JC IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs.Cancer Res. 1998; 58: 5315-5320PubMed Google Scholar Thus, in addition to Bcl-2-related proteins such as Bcl-xL, it is likely that several antiapoptotic proteins contribute to chemotherapy resistance in primary tumors. Further work should provide insight into the contribution of these apoptosis inhibitors to clinical drug resistance, which may lead to the development of novel approaches to counter such resistance. We thank M. Benedict, M. Clarke, S. Ethier, and L. del Peso for help and suggestions during these studies and C. W. Song for the gift of SCK cells.