Synergistic antitumor activity of oncolytic reovirus and chemotherapeutic agents in non-small cell lung cancer cells
© Sei et al; licensee BioMed Central Ltd. 2009
Received: 29 January 2009
Accepted: 14 July 2009
Published: 14 July 2009
Reovirus type 3 Dearing strain (ReoT3D) has an inherent propensity to preferentially infect and destroy cancer cells. The oncolytic activity of ReoT3D as a single agent has been demonstrated in vitro and in vivo against various cancers, including colon, pancreatic, ovarian and breast cancers. Its human safety and potential efficacy are currently being investigated in early clinical trials. In this study, we investigated the in vitro combination effects of ReoT3D and chemotherapeutic agents against human non-small cell lung cancer (NSCLC).
ReoT3D alone exerted significant cytolytic activity in 7 of 9 NSCLC cell lines examined, with the 50% effective dose, defined as the initial virus dose to achieve 50% cell killing after 48 hours of infection, ranging from 1.46 ± 0.12 ~2.68 ± 0.25 (mean ± SD) log10 pfu/cell. Chou-Talalay analysis of the combination of ReoT3D with cisplatin, gemcitabine, or vinblastine demonstrated strong synergistic effects on cell killing, but only in cell lines that were sensitive to these compounds. In contrast, the combination of ReoT3D and paclitaxel was invariably synergistic in all cell lines tested, regardless of their levels of sensitivity to either agent. Treatment of NSCLC cell lines with the ReoT3D-paclitaxel combination resulted in increased poly (ADP-ribose) polymerase cleavage and caspase activity compared to single therapy, indicating enhanced apoptosis induction in dually treated NSCLC cells. NSCLC cells treated with the ReoT3D-paclitaxel combination showed increased proportions of mitotic and apoptotic cells, and a more pronounced level of caspase-3 activation was demonstrated in mitotically arrested cells.
These data suggest that the oncolytic activity of ReoT3D can be potentiated by taxanes and other chemotherapeutic agents, and that the ReoT3D-taxane combination most effectively achieves synergy through accelerated apoptosis triggered by prolonged mitotic arrest.
The use of viral vectors for cancer gene therapy has been vigorously explored for the last two decades. The overall goal of the strategy is to promote cancer cell death through various means, such as tumor suppressor gene replacement, oncogene inactivation, suicide gene delivery, drug sensitization or enhancement of anticancer immunity. The extensive research efforts to develop tumor cell death-inducing viral vectors have reignited the interest in oncolytic viruses in recent years as a promising group of viral therapeutics that can directly induce tumor cell lysis through viral replication. The latest multidisciplinary research in cancer genomics and proteomics further provides an opportunity to discern various molecular pathways specifically upregulated (or dysregulated) in cancers that can be exploited as part of viral replication and destruction machinery. Indeed, many replication-competent oncolytic viruses currently in development are recombinant viruses engineered to become reliant on such cancer-specific molecules and signaling pathways for viral entry and replication, thus rendering cancer cells more selectively susceptible to virus-mediated oncolysis . Unlike chemical entity-based anticancer agents, these viruses can propagate in susceptible tumor cells, re-target, infect, and destroy remaining cancer cells within the primary tumor or in the metastases, repeating the cycle until viral spread is halted by the host antiviral response or by mechanical barriers such as loss of vasculature and necrotic tissues.
Mammalian reoviruses are ubiquitous, non-enveloped dsRNA viruses, normally associated with relatively benign pathology in humans. The Dearing strain of reovirus serotype 3 (ReoT3D) is a non-engineered wild type reoviral strain and belongs to a growing number of the new generation of oncolytic viruses because of its innate ability to preferentially kill transformed cells [2, 3]. The oncolytic potency of ReoT3D has been extensively demonstrated against various cancers in vitro and in vivo, including colon, pancreatic, ovarian and breast cancers, as well as malignant gliomas and lymphoid malignancies [4–9]. The safety, feasibility and potential efficacy of ReoT3D cancer therapy are currently being investigated in phase I/II clinical trials . As with other emerging therapeutics for cancer, the combined regimen of ReoT3D and conventional chemotherapeutic agents is expected to play a significant role in future clinical applications. However, it is currently unknown whether conventional chemotherapeutic agents can augment or interfere with the oncolytic effect of ReoT3D. In this study, we evaluated the oncolytic activity of ReoT3D in non-small cell lung cancer (NSCLC), and explored the therapeutic feasibility of ReoT3D-chemotherapeutic combination regimens against NSCLC.
Oncolytic activity of ReoT3D and progeny virion production in NSCLC cell lines
Oncolytic activity of ReoT3D in 9 NSCLC cell lines included in the NCI-60 cell line panel
NSCLC Cell Line
ED50 (log10 pfu/cell: mean ± SD)
2.68 ± 0.25
2.51 ± 0.27
2.56 ± 0.19
2.47 ± 0.34
2.47 ± 0.15
1.46 ± 0.12
1.96 ± 0.27
In vitro combination effects of ReoT3D and chemotherapeutic agents against NSCLC cells
Combination index values for ReoT3D-chemotherapeutic combination regimens in NSCLC cells
Combination Index Values§
Combination Index Values§
Drugs & Cell Lines
Drug Sensitivity ¶ IC50 (μM)
0.003 ± 0.001
38.50 ± 16.01
0.08 ± 0.04
56.06 ± 15.87
77.79 ± 25.08
5.31 ± 1.21
6.82 ± 3.22
3.51 ± 1.15
29.34 ± 6.62
14.71 ± 6.60
0.01 ± 0.003
0.83 ± 0.61
0.02 ± 0.01
17.69 ± 10.16
0.92 ± 0.35 (nM)
48.80 ± 23.08
1.49 ± 1.09 (nM)
28.46 ± 14.13
These data demonstrated that while the oncolytic activity of ReoT3D could be generally potentiated by the chemotherapeutic agents that alone were cytotoxic to the tested cell lines, paclitaxel appeared to exert unique effects on the cell-death process induced by ReoT3D, even in the cells with reduced sensitivity to the compound.
Progeny virion production from NSCLC cells treated with the combination of ReoT3D and chemotherapeutic agents
PARP cleavage in NSCLC cells treated with ReoT3D-chemotherapeutic combination regimens
Activation of caspases in NSCLC cells treated with ReoT3D alone or in combination with paclitaxel
In order to evaluate whether the extent of caspase activation induced by the ReoT3D-paclitaxel combination was significantly different from ReoT3D single treatment in these cell lines, the caspase activity dose-response curves were fitted by non-linear regression using GraphPad Prism (GraphPad Software Inc., San Diego, CA) (Figure 6b). The best-fit values of a variable, LogEC50, obtained by the Prism analysis from 3 independent experiments were then compared between the two treatment regimens, ReoT3D alone vs. ReoT3D-paclitaxel combination, using a paired t-test. The analysis demonstrated that the activation of caspases was significantly enhanced with the ReoT3D-paclitaxel combination therapy as compared to ReoT3D alone in NCI-H460, NCI-H23, EKVX and NCIH322M (P = 0.004, 0.03, 0.04, and 0.02, respectively). These data suggested that enhanced apoptotic cell death most likely constituted the synergistic cell killing induced by the combination of ReoT3D and paclitaxel in NSCLC cells.
Dynamic effects of ReoT3D and paclitaxel on cell cycle progression and caspase activation
ReoT3D infection has been shown to induce cell cycle arrest at G1/S and G2/M [19, 20]. Antimicrotubule agents, including taxanes and vinca alkaloids, activate the spindle-assembly checkpoint and induce mitotic arrest at the metaphase and anaphase transition, which ultimately leads to cell death by apoptosis . A certain proportion of cells exposed to these antimitotic agents may undergo an aberrant mitotic exit without cytokinesis, forming multinucleated cells in interphase . To gain mechanistic insight into the enhanced apoptotic cell death induced by the ReoT3D-paclitaxel combination, we investigated the effects of ReoT3D and paclitaxel on cell cycle and caspase-3 activation in NSCLC cells using flow cytometry.
Similar effects of ReoT3D-paclitaxel combination on cell cycle and apoptosis induction were also demonstrated in 3 other NSCLC cell lines, NCIH460, EKVX and NCI-H322M (Figure 7b). Paclitaxel treatment invariably increased the proportion of post-G1 cells in these cell lines, although the percentages of cells positive for activated caspase-3 were significantly smaller than in NCI-H23. The ReoT3D-paclitaxel combination consistently led to substantial increases in cells expressing active caspase-3, including in paclitaxel-resistant EKVX and NCI-H322M cells, and these increases appeared more prominent in post-G1 phase (Figure 7b). Indeed, statistical analysis demonstrated that the proportions of active caspase-3-positive cells were significantly different among the treatment groups in the post-G1 cell population (P < 0.005, one-way ANOVA), but not in the population with DNA content of 2N or less (sub-G1 and G1) (P = 0.05). In this subpopulation of post-G1 cells, the proportions of active caspase-3-expressing cells were significantly increased with the ReoT3D-paclitaxel combination treatment as compared to single therapy (P = 0.02 for both between-group comparisons, ReoT3D alone vs. ReoT3D+paclitaxel 0.1 μM, and paclitaxel 0.1 μM alone vs. ReoT3D+paclitaxel 0.1 μM; P = 0.02 and < 0.04 for between ReoT3D alone vs. ReoT3D+paclitaxel 1 μM, and paclitaxel 1 μM alone vs. ReoT3D+paclitaxel 1 μM, respectively). These data suggested that the combination of ReoT3D and paclitaxel enhanced apoptotic cell death, which was linked to cell cycle perturbation in NSCLC cells.
Ultrastructural analysis of NSCLC cells treated with ReoT3D and paclitaxel
Summary of EM findings from survey of 100 adherent NCI-H23 cells
Cells with viral particles
Paclitaxel 1 μM
ReoT3D (MOI = 20)
ReoT3D (MOI = 20) + Paclitaxel 1 μM
These data from flow cytometric and electron microscopic analyses strongly suggested that treatment with the combination of ReoT3D and paclitaxel caused prolonged mitotic arrest, which triggered accelerated apoptosis, resulting in synergistic cell killing in dually treated NSCLC cells.
Lung cancer is the leading cause of cancer mortality in both men and women in the United States  and all cancer deaths worldwide . The most common form of lung cancer is NSCLC that includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Despite the tremendous efforts and progress in lung cancer research, treatment outcomes for non-localized NSCLC remain poor . New treatment strategies are urgently needed to improve survival for advanced NSCLC patients. In the current study, we uncovered a potent oncolytic activity of ReoT3D against a panel of human NSCLC cell lines, in particular, NSCLC cell lines of adenocarcinoma or large cell carcinoma origin. The susceptibility of cancer cells to ReoT3D-mediated cytolysis has been attributed to increased Ras activity [11, 12]. However, we did not observe any significant association between the ReoT3D-permissibility and the presence of Ras-activating gene mutations or activated Ras in human NSCLC cells. The lack of association has also been reported by others in human colon cancers . It is possible that in addition to the activation status of Ras-associated pathways [11, 12], there are other molecular determinants of ReoT3D-sensitivity, such as the cell surface density of putative ReoT3D receptors/coreceptors [27–29] and intracellular virion uncoating processes [30, 31], all of which can affect ReoT3D infection efficiency.
The combination effects of herpesvirus or adenovirus-based oncolytic viral vectors and chemotherapeutic agents have previously been evaluated against different human cancers [32–37]. Synergistic activity was reported in the majority of these studies. However, combination regimens selected for the previous studies were mostly limited in scope in terms of dose range and the number of chemotherapeutic agents investigated. In the current study, we demonstrated that the oncolytic activity of ReoT3D against NSCLC cells could be significantly potentiated by a number of chemotherapeutic agents used in the treatment of NSCLC, including paclitaxel, cisplatin, gemcitabine and vinblastine. Combination analysis based on the Chou-Talalay's method [16, 17] clearly showed significant levels of synergy between ReoT3D and each chemotherapeutic agent tested. Interestingly, we found that the drug sensitivity of each NSCLC cell line was an important determinant for the in vitro synergistic effect of ReoT3D-chemotherapeutic combination regimens, with the exception of ReoT3D-paclitaxel combination. It is conceivable that certain molecular changes conferring drug resistance can antagonize the process of ReoT3D-mediated cell killing. Our data, therefore, caution against the use of chemotherapeutic agents combined with ReoT3D for the treatment of NSCLC that have developed resistance to the agents. In contrast, the level of ReoT3D sensitivity did not appear to compromise the combination effects in NSCLC cells. Rather, the addition of chemotherapeutic agents may help accelerate ReoT3D-induced cell death process, which is otherwise slowed in NSCLC cells with low susceptibility to ReoT3D infection.
The most intriguing finding from our study was the synergistic effect of ReoT3D-paclitaxel combination consistently observed in all the NSCLC cell lines examined, regardless of the level of sensitivity to the compound. Because previous studies of oncolytic virus-chemotherapeutic combinations, in particular with paclitaxel, did not address the impact of drug resistance on the combination effects, we cannot ascertain whether our finding is unique to reovirus-containing combination therapy. Mammalian reoviruses are known to exploit microtubules for the formation of viral replication complexes (inclusion bodies) . Based on our initial findings that the addition of paclitaxel to ReoT3D significantly increased the level of progeny virion production from all the NSCLC cell lines tested, we speculated that microtubule-stabilizing paclitaxel might have enhanced reoviral replication, resulting in a more efficient and synergistic oncolytic effect. However, the increased progeny virion production was not necessarily a unique outcome of ReoT3D-paclitaxel combination, but was also observed with ReoT3D-vinblastine combination in vinblastine-resistant NCI-H322M cells, where the combination of ReoT3D and vinblastine was found to be strongly antagonistic. Moreover, the addition of gemcitabine to ReoT3D treatment was not associated with an increased progeny virion production, regardless of the combination effects (synergy or antagonism) attained. These data suggested that the synergistic effect of ReoT3D and chemotherapeutic agents was not the direct result of enhanced lytic cell death, but more likely the manifestation of accelerated programmed cell death, which was triggered before virion assembly and release.
Reovirus has been shown to induce apoptotic cell death in a variety of cell types, including cancer cells [18, 39]. Indeed, increased caspase activity and apoptotic cleavage of PARP were readily detectable in ReoT3D-treated NSCLC cells within 24 hours in the current study, as has been shown in ReoT3A-exposed cancer cells . The combination treatment with ReoT3D and paclitaxel led to more robust caspase activation than ReoT3D alone with a significant leftward shift of the dose response curve in both paclitaxel-sensitive and -resistant NSCLC cell lines, suggesting that the enhanced apoptosis most likely constituted the synergistic cell killing by the combination, regardless of the level of paclitaxel sensitivity. To gain more insight into the mechanistic basis of accelerated apoptosis associated with the ReoT3D-paclitaxel combination, we examined the effects of ReoT3D and paclitaxel on caspase-3 activation in relation to cell cycle progression. While taxanes and other antimicrotubule agents are known to activate the spindle checkpoint and induce mitotic arrest , ReoT3D infection has been shown to effect cell cycle arrest at G1/S and G2/M [19, 20]. Because arrests in cell cycle progression induced by anticancer agents are commonly followed by apoptosis , we hypothesized that these two agents with differing effects on cell cycle progression may have synergistically activated apoptotic pathways in dually treated NSCLC cells.
We found that the proportion of cells expressing activated caspase-3 was significantly increased by the ReoT3D-paclitaxel combination as compared to either ReoT3D or paclitaxel single treatment in each NSCLC cell line tested. Interestingly, the activation of caspase-3 was found more prominent in post-G1 cell population with ≥ 4N DNA content. Escape from mitotic arrest induced by spindle poisons such as taxanes and other antimicrotubule agents is commonly observed in cancer cells with impaired (weakened) mitotic checkpoint [21, 41]. These cells that prematurely exit mitosis without proper cell division form large multinucleated cells with DNA content of 4N or greater, as demonstrated by EM in our study. After such mitotic slippage, some cells may undergo p53-dependent apoptosis, while others survive through senescence or continuing cell cycle (endoreduplication) , depending on the functions of p53, MAP kinase pathways, and p21-activated kinase [43, 44]. In the current study, we found that NSCLC cells could efficiently escape from mitotic arrest induced by paclitaxel at 0.1 ~1 μM and survive at least for the first 24 hours of exposure, with the exception of NCI-H23 cells that were more prone to apoptosis upon paclitaxel exposure than 3 other NSCLC cell lines examined. While paclitaxel-treated NCIH460 cells ultimately underwent dramatic cell death after 48 hours, EKVX and NCI-H322M demonstrated considerable levels of resistance to paclitaxel-induced CPE. Nonetheless, the combination of ReoT3D and paclitaxel consistently accelerated the apoptotic process in post-G1 cells, including in paclitaxelresistant EKVX and NCI-H322M cells. This enhanced apoptosis appeared to have resulted from prolonged mitotic arrest, as corroborated by the EM data demonstrating that treatment with the ReoT3D-paclitaxel combination resulted in increased numbers of both mitotically arrested and apoptotic cells while decreasing the number of multinucleated cells as compared to paclitaxel alone.
The molecular mechanism of prolonged mitotic arrest induced by the ReoT3D-paclitaxel combination has yet to be elucidated. It is possible that ReoT3D infection may enhance the mitotic checkpoint activity in cancer cells with weakened mitotic checkpoint, for example, by upregulating the expression of mitotic checkpoint proteins (such as Mad1, Mad2, BubR1/Mad3, Bub1 and Bub3) , Cdk1 and/or cyclin B , or suppressing the anaphase-promoting complex/cyclosome activity . Such ReoT3D-induced alterations in the mitotic regulatory network may reinforce the mitosis-arresting signal of taxanes, leading to prolonged mitotic arrest and apoptosis. Better understanding of the molecular consequences of ReoT3D infection on cell cycle checkpoint function and apoptotic signaling pathways in cancer cells will greatly enhance our ability to design rational combination therapies with proapoptotic oncolytic agent, ReoT3D, and various classes of anticancer agents. Concurrently, it is also of high importance to investigate potential consequences of ReoT3D-chemotherapeutic combinations on normal tissues in order to identify undesirable combination regimens that are associated not only with synergistic oncolytic activity, but also with enhanced toxicity in humans.
The current study showed that the oncolytic activity of ReoT3D could be most effectively potentiated by taxanes through accelerated apoptosis, regardless of the level of taxane-sensitivity. Recently, preliminary findings from ongoing phase I clinical trials evaluating the safety of ReoT3D-taxane combination in patients with chemotherapy-refractory advanced tumors have demonstrated objective anticancer response in some patients without serious side effects [46, 47], corroborating that ReoT3D-taxane combination regimens can achieve a durable oncolytic effect in clinical settings and thus should be further explored as a novel treatment modality for NSCLC.
Cell lines, virus and chemotherapeutic agents
Human NSCLC cell lines included in the NCI-60 cell line panel : NCIH460 (large cell carcinoma), A549/ATCC (adenocarcinoma), HOP-62 (adenocarcinoma), NCI-H322M (bronchoalveolar carcinoma), NCI-H226 (squamous cell carcinoma), EKVX (adenocarcinoma), NCI-H23 (adenocarcinoma), NCI-H522 (adenocarcinoma), and HOP-92 (large cell carcinoma) were obtained from the In Vitro Cell Line Screening Program, DTP, NCI-Frederick. Of the 9 cell lines, NCI-H460, A549/ATCC, HOP-62, and NCIH23 have been found to have K-ras gene mutations . L929 cells (mouse fibroblast L cells NCTC clone 929) were obtained from American Type Culture Collection (Manassas, VA). ReoT3D (REOLYSIN®) was provided by Oncolytics Biotech Inc. (Calgary, AB Canada). Cisplatin, paclitaxel, gemcitabine and vinblastine were obtained through the Drug Synthesis and Chemistry Branch, DTP, NCI.
Determination of virus- and drug-induced cell death
ReoT3D- or drug-induced CPE was assessed by the XTT assay as described previously . ED50 for ReoT3D was defined as the initial virus dose (MOI expressed in pfu/cell) that resulted in 50% cell viability at 48 hours post-inoculation as compared to untreated controls. Sensitivity of NSCLC cell lines to chemotherapeutic agents was expressed as the drug concentration that inhibited cell growth by 50% compared to untreated controls (IC50). Both ED50 and IC50 were calculated by using the software GraphPad Prism (GraphPad Software, Inc.). Cell viability was also assessed by intracellular ATP content (ATPLite™, PerkinElmer, Waltham, MA), when the level of caspases activity was evaluated in selected NSCLC cell lines treated with ReoT3D, paclitaxel or both agents in combination (see below).
Evaluation of in vitro combination effects by the Chou-Talalay method
The combined effects of ReoT3D and each chemotherapeutic agent on cell survival were analyzed using the software CalcuSyn (Biosoft, Ferguson, MO), which applies the median-effect equation of Chou  and the CI equation of Chou and Talalay . NCI-H460, HOP-92, NCI-H23, EKVX, NCI-H226 and NCI-H322M plated in 96-well microplates as above were exposed in triplicate to a serial dilution of each agent or both in combination using the constant ratio combination design  for 48 hours, followed by the XTT assay for cell viability determination. Calculated CIs were used to ascertain the presence of strong synergism (CI < 0.3), moderate synergism (0.3 < CI < 0.9), additive effect (CI = 1), antagonism (CI > 1) and strong antagonism (CI > 3.3)  between ReoT3D and chemotherapeutic agents.
Reovirus plaque assay
Levels of infectious progeny virions in culture supernatants produced from ReoT3D-infected NSCLC cells were evaluated by plaque assay as previously described . Infectious reovirus titers were expressed as pfu/mL of the original sample.
Determination of activated Ras levels in NSCLC cells
Basal levels of activated Ras in 9 NSCLC cell lines were determined by Ras activation assay Biochem Kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer's instructions. Briefly, cell lysates were prepared from NSCLC cells using the kit lysis buffer, and aliquots of 2 mg protein were incubated with 20 μL of Raf1-RBD beads at 4°C for 1 hr, followed by centrifugation to pellet the Raf1-RBD beads. The pelleted beads were washed with wash buffer twice, and resuspended in 10 μL Laemmli sample buffer. The samples were separated by 12% Tris-glycine SDS-PAGE, and subjected to Western blot analysis with anti-Ras antibody (Cell Signaling Technology, Inc., Danvers, MA).
Western blot analysis of PARP cleavage
NSCLC cells were cultured overnight in 6-well plates at a cell density of 2 × 106 cells/well, followed by incubation with ReoT3D, chemotherapeutic agents (gemcitabine, paclitaxel or vinblastine) or ReoT3D-chemotherapeutics in combination at indicated concentrations. Twenty-four to 30 hours after treatment, both adherent and non-adherent cells were collected and lysed in 150 μL cell lysis buffer (10 mM Tris/pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 μg/mL aprotinin, 1 μg/mL leupeptin and 5 μM pepstatin). Cell lysates containing 20 μg protein were subjected to Western blot analysis with anti-PARP (Cell Signaling Technology, Inc.) and anti-β-actin (Cell Signaling Technology, Inc.) antibodies.
Determination of caspase activity and ATP content by microplate-based assays
The levels of caspase activity and ATP content in NSCLC cells treated with ReoT3D alone or in combination with paclitaxel for 24 hours were evaluated by microplate-based assays, using a fluorometric pan-caspase assay (Homogeneous Caspases Assay; Roche Applied Science, Indianapolis, IN) and luminescence ATP detection assay (ATPLite™; PerkinElmer), respectively.
Flow cytometric analysis of DNA content and caspase-3 activation
Adherent and non-adherent NSCLC cells harvested as above (see Western blot analysis) were fixed and permeabilized with BD Cytofix/Cytoperm™ solution (BD Biosciences, San Jose, CA) and stained with FITC-conjugated anti-active caspase-3 antibody (BD Biosciences), followed by incubation with PI/RNase staining buffer (BD Biosciences) for DNA content determination. The proportion of cells expressing active caspase-3 and DNA content were analyzed using a FACScan™ flow cytometer (BD Biosciences) as previously described .
Transmission electron microscopy
NSCLC cells cultured in 6-well plates were processed in situ for electron microscopic analysis as previously described . Thin sections (60 nm) were examined with a Hitachi H7000 transmission electron microscope.
Results are reported as mean ± SD unless otherwise indicated. Statistical significance of differences was determined by one-way ANOVA or Student's t-test as appropriate. Differences were considered statistically significant when P < 0.05 (two-tailed).
We would like to thank Drs. Jerry Collins, James Zwiebel, Yoshitatsu Sei, and Kevin Harrington for advice and helpful discussions, and Mr. M. Jason De la Cruz for technical assistance. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This research was supported in part by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.
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