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[Cancer Research 61, 5078-5082, July 1, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Sensitization of Human Tumor Cells to CPT-11 via Adenoviral-mediated Delivery of a Rabbit Liver Carboxylesterase1

Monika Wierdl, Christopher L. Morton, James K. Weeks, Mary K. Danks, Linda C. Harris and Philip M. Potter2

Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) is activated by carboxylesterases (CE) to yield the potent topoisomerase I inhibitor, SN-38. We have demonstrated previously that a rabbit liver CE is approximately 100-1000-fold more efficient at drug activation than a highly homologous human CE. In an attempt to use rabbit CE expression in combination with CPT-11 for gene therapy approaches for the treatment of cancer, we have developed an adenoviral vector expressing this intracellular CE. After transduction, this virus produces very high levels of CE activity in a panel of human tumor cell lines and results in marked sensitization to CPT-11 of all of the transduced cells. Reductions in IC50 values for this drug ranged from 11–127-fold. Additionally, comparison with an adenovirus expressing a secreted form of the rabbit CE indicated that a collateral effect could be achieved with reductions in the IC50 values ranging from 4–19-fold. These data suggest that the described reagents may be suitable for use in vivo in a viral-directed enzyme prodrug therapy approach using CPT-11.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CPT-113 is an anticancer camptothecin prodrug that has demonstrated good antitumor activity both in animal xenograft models of human tumors and in patients undergoing therapy with this drug (1, 2, 3, 4, 5, 6, 7) . This prodrug is metabolized by esterases to yield SN-38, a potent topoisomerase I inhibitor, which is cytotoxic (8, 9, 10, 11) . Activation of CPT-11 by human esterases is poor, resulting in very low plasma SN-38 levels after administration to patients (7, 8, 9, 10, 11) . In contrast, very high levels of SN-38 are observed in mice and rats after treatment with CPT-11, indicating that the enzymes involved in drug catalysis are either much more efficient or much more abundant (8 , 12 , 13) .

We have demonstrated recently (9 , 14) that, similar to rodent enzymes, a rCE is highly proficient at drug activation, being greater than 100-fold more efficient than a homologous human enzyme. Because low level drug metabolism is observed in patients treated with CPT-11, selective activation of the drug at the tumor site may be a viable approach to increase antitumor activity. As a prelude to these types of gene therapy experiments, we describe here the construction and characterization of adenoviral vectors designed to express either an intracellularly localized or a secreted form of the rabbit CE and determine the ability of these vectors to sensitize human tumor cell lines to CPT-11.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
CPT-11.
CPT-11, kindly provided by Dr. J. Patrick McGovren (Pharmacia Upjohn, Kalamazoo, MI), was dissolved in methanol and stored at -20°C. For growth inhibition assays, the drug was diluted in media immediately before use.

Cell Lines and Plasmids.
The rhabdomyosarcoma cell line Rh30 was provided by Dr. Peter Houghton (St. Jude Children’s Research Hospital, Memphis, TN), and the neuroblastoma cell line, NB-1691, was obtained from the Pediatric Oncology Group. The lung carcinoma line A549, the colon adenocarcinoma line HT29, the neuroblastoma line SK-N-AS, the glioblastoma line U373MG, and the embryonal kidney cell line 293 were all purchased from the American Type Culture Collection (Rockville, MD). All of the cell lines were cultured in DMEM containing 10% FCS at 37°C under an atmosphere of 10% CO2/90% air. For studies with adenovirus, FCS was heat inactivated at 56°C for 30 min. U373MG cell lines transfected with either the full-length rCE cDNA (U373MGpIRESRABFL) or the truncated cDNA (U373MGpIRESRABC) were generated by electroporation as described previously (9 , 15 , 16) . The adenoviral vector pAVS6a was obtained from Genetic Therapy Inc. (Gaithersburg, MD), and Addl327 was kindly provided by Dr. Elio Vanin (St. Jude Children’s Research Hospital).

Construction of Recombinant Adenovirus.
Adenoviral vectors were generated by homologous recombination of pAVS6a with adenoviral DNA (Addl327; Ref. 17 ) after transfection into 293 cells. An agarose overlay was applied to the cells, and plaques were allowed to form (typically 10–14 days). Individual plaques were isolated, DNA was prepared, and the presence of the rabbit CE cDNA was determined by PCR. Additionally, the absence of wild-type adenoviral DNA was monitored by PCR analysis of the E1a gene (18) . Plaques that did not contain wild-type sequences were subjected to two more rounds of plaque purification before large-scale amplification and preparation by cesium chloride ultracentrifugation. Adenoviral titers were determined using 293 cells, and aliquots of virus were stored at -80°C. moi is defined as the number of plaques produced in 1 x 106 293 cells in a total volume of 1 ml of media after incubation with virus for 1 h.

CE Assays.
CE activity was determined in sonicated cell extracts or culture media by a spectrophotometric assay using o-nitrophenyl acetate as a substrate (9 , 19) . Briefly, samples were incubated with 3 mM o-nitrophenyl acetate in 50 mM HEPES (pH 7.4), and the change in A420 was determined spectrophotometrically. Data were transferred to an Excel spreadsheet, and the specific activity of the sample was calculated from the extinction coefficient for o-nitrophenol (13.4 x 103 M-1 cm-1). A minimum of four assays were performed for each sample, and values for cell extracts were corrected for protein concentration. Data were expressed as µmol o-nitrophenol produced/min/mg of protein or /ml of culture media.

CPT-11 Conversion Assays.
Conversion of CPT-11 to SN-38 was monitored by incubating either cell extracts or media samples with 5 or 25 µM CPT-11 for 18 h in 50 mM HEPES (pH 7.4) at 37°C. Reactions were terminated by the addition of an equal volume of acidified methanol, and particulate matter was removed by centrifugation at 100,000 x g for 30 min at 4°C. Concentrations of both drugs in the supernatant were then determined by high performance liquid chromatography.

Detection of CPT-11 and SN-38.
CPT-11 and SN-38 concentrations in biological samples were determined by high performance liquid chromatography as described previously (13 , 20) . Detection was achieved using a Jasco 920 fluorescence detector, and the sensitivity of this system was 20 pg/µl and 1.5 pg/µl for CPT-11 and SN-38, respectively.

Western Analysis.
Whole cell extracts were separated in 4–20% precast SDS-PAGE gels (Invitrogen, Carlsbad, CA), proteins were transferred to Immobilon-P membrane by electroblotting, (21) and Western analysis was performed as described previously (22) . CEs were detected using a horseradish peroxidase-conjugated rabbit antiporcine esterase antibody (Research Diagnostics, Inc., Flanders, NJ) and enhanced chemiluminescence (Amersham Life Sciences, Arlington Heights, IL). As a control for loading, an anti-TFIID antibody was used to reprobe the membrane (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Prestained molecular weight protein markers (Invitrogen) were used to visualize the molecular mass of the immunoreactive protein species.

Growth Inhibition Assays.
Growth inhibition assays were performed in triplicate in 6-well multiwell plates. Cells were plated at the following densities: A549, 5 x 104 cells/well; HT29, 6 x 104 cells/well; NB-1691, 1 x 105 cells/well; Rh30, 5 x 104 cells/well; SK-N-AS, 5 x 104 cells/well; and U373MG, 7 x 104 cells/well. Then, the cells were transduced with adenovirus for 24 h. The following day, the medium was removed, and the cells were allowed to grow until the CE activity no longer increased, typically 3–4 days. At this time, the cells were exposed to CPT-11 for 2 h, and after replacing with fresh media, the cells were allowed to grow for a additional three cell doublings (determined from a nondrug-treated well) or a maximum of 6 days. Cell number was measured using a Coulter Z2 (Coulter Electronics, Luton, England), and IC50 values were calculated from data plots using Prism software (Graphpad Software, San Diego, CA).

Collateral Effect Assays.
To distinguish the activation of CPT-11 by an extracellular enzyme as opposed to intracellular activation and transfer of SN-38 to neighboring cells, i.e., a bystander effect, we have termed the former mechanism a collateral effect. To assess a collateral effect from secreted CEs, ~1 x 107 cells were transduced with adenovirus at a moi of 5, and CE expression in the media was monitored. After 4 days, the medium was harvested and applied to untransduced cells in 6-well multiwell plates. CPT-11 was added for 2 h, and after replacing with fresh media, the cells were allowed to grow for three cell doublings of untreated control cells or a maximum of 6 days. Cell number and IC50 values were then determined as described above.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Construction of an Adenoviral Vector Expressing rCE.
To assess the ability of viral vectors to deliver the rabbit CE cDNA to human tumor cells, we constructed an adenovirus designed to express this enzyme. To generate this vector, we ligated the 1.7-kb EcoRI fragment containing the full length rabbit liver cDNA (9) into pSP65Cla (23) , allowing the removal of the cDNA with ClaI overhangs to facilitate ligation into pAVS6a (Fig. 1)Citation . This plasmid was cotransfected into 293 cells with ClaI-restricted Addl327 DNA (17) . After analysis of plaque DNA for the presence of the cDNA and the absence of the E1a gene (18) , large-scale preparations of AdRSVrCE were performed. Titers of 3 x 1010 plaque-forming units/ml were achieved for AdRSVrCE. A control virus containing no cDNA (AdVC) was also generated and demonstrated a similar titer (2.2 x 1010) to AdRSVrCE.



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  		Fig. 1. Construction of adenovirus-expressing rCE. The rabbit CE present as a 1.7-kb EcoRI fragment (9) was ligated into pSP65Cla (23) and excised with ClaI to facilitate ligation into pAVS6a. This plasmid was then cotransfected with Addl327 DNA into 293 cells, and the virus was prepared as described in "Materials and Methods." ITR, inverted terminal repeat; HRF, homologous recombination fragment.

 
Adenoviral-mediated Expression of the rCE in Rh30 Cells.
To confirm that expression of the rCE could be achieved from adenoviral vectors, Rh30 cells were transduced with AdRSVrCE at an moi of 5, and cells were harvested at 24-h intervals. Extracts were prepared for CE assays and CPT-11 conversion analyses. As can be seen in Table 1Citation , high levels of CE activity (up to ~1100 µmol/min/mg) were present in cell extracts after viral transduction that peaked at 72 h. Similarly, the yields of SN-38 produced from these extracts increased and peaked at the same time point. After 72 h, the levels of CE activity dropped and the conversion of CPT-11 decreased in parallel. This reduction may be attributable to viral-mediated or CE-mediated cytotoxicity. SDS-PAGE analysis of these cell extracts confirmed the above data, with the presence of the expected Mr 65,000 immunoreactive band in all of the samples exhibiting CE activity (Fig. 2)Citation . Approximate equal loading of the Western analysis was confirmed by analysis with an anti-TFIID antibody.


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  		Table 1 CE activities and CPT-11 conversion by Rh30 cell extracts after transduction with AdRSVrCE at an moi of 5

 


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  		Fig. 2. Western analysis of Rh30 cells after transduction with AdRSVrCE. Cell extracts were prepared 24–144 h after transduction and subject to Western analysis with an anti-CE (top) or anti-TFIID antibody (bottom). Arrow, the expected position of the rabbit CE.

 
Adenoviral-mediated Sensitization of Human Tumor Cell Lines to CPT-11.
Having determined that adenoviral-mediated delivery of the rCE generated high levels of enzyme activity in Rh30 cell extracts, we assessed the ability of these reagents to induce cytotoxicity in cells in combination with CPT-11. Cells were transduced at a moi of 5, and sensitivity to CPT-11 was assessed after 4 days. An example of a typical growth inhibition curve is demonstrated in Fig. 3Citation , in which Rh30 cells expressing the rCE were sensitized >60-fold to CPT-11. Table 2Citation demonstrates the change in IC50 for a panel of human tumor cell lines after treatment with AdRSVrCE and CPT-11. A minimum of an 11-fold reduction in the IC50 was observed, consistent with the hypothesis that adenoviral-mediated delivery and expression of the rCE can sensitize cells to CPT-11. Indeed, in some cell lines such as Rh30 and U373MG, very high levels of CE activity were detected in cell extracts, and this resulted in 64–127-fold changes in the IC50 values, respectively (Table 2)Citation .



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  		Fig. 3. Growth inhibition curve of Rh30 cells treated with CPT-11 after transduction with AdRSVrCE ({blacktriangleup} ) or AdVC (----{blacksquare}-----).

 

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  		Table 2 CE activity and IC50 values of cells after treatment with AdRSVrCE and CPT-11

 
rCES Activates CPT-11.
Having demonstrated that sensitization to CPT-11 for a panel of human tumor cells lines could be achieved by adenoviral-mediated delivery of the rabbit CE cDNA, we attempted to achieve a collateral effect by engineering the protein to be secreted from the cell. To determine whether the rCES (15) could activate CPT-11, we generated stable U373MG cell lines expressing this protein by transfection with a plasmid encoding the secreted CE. After 7 days, the medium was harvested, and both CE activity and CPT-11 conversion assays were performed. As can be seen (Table 3)Citation , approximately 2-fold more CE activity was detected in media derived from cells transfected with the rabbit liver cDNA. The activity present in media from the control cells (U373MG) is attributable to esterases present in FCS. An increased amount of SN-38 (~21-fold) was also present in media containing the rabbit CE, confirming that the secreted protein can activate CPT-11.


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  		Table 3 CE activity and SN-38 production by media containing the rCES

 
To determine whether this level of CE was sufficient to activate CPT-11 and kill untransfected cells, i.e., produce a collateral effect, we mixed different ratios of media derived from U373MG and U373MGRABFL cell lines and applied them to untransfected U373MG cells. Cells were drug treated for 2 h, and cell numbers were determined after untreated cells had undergone three cell doublings. Table 4Citation demonstrates that as the percentage of CE-containing media was increased, the enzyme activity increased, and the IC50 values for CPT-11 decreased concomitantly. Additionally, there is a correlation between the levels of SN-38 produced and the amount of CE activity with the latter inversely proportional to the IC50 value (Table 4)Citation . These data confirm that the rabbit CE is capable of converting CPT-11 to SN-38 extracellularly and that this level of SN-38 is sufficient to induce growth inhibition in untransfected U373MG cells.


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  		Table 4 Collateral effect in U373MG cells from media containing rCE

 
Collateral Effect with Adenovirus Expressing the rCES.
Because we observed a collateral effect from media containing rCES, we constructed an adenovirus expressing this protein to determine its ability to sensitize cells to CPT-11. Because we have described previously (15) the construction of the 1.7-kb cDNA encoding the COOH-terminally deleted rabbit CE, adenoviral production was achieved in an identical fashion to that for AdRSVrCE, to generate AdRSVrCES. After large-scale amplification, adenovirus with a titer of 5 x 1010 plaque-forming units/ml was produced that lacked wild-type virus as determined by PCR-mediated analysis for the E1a gene (18) .

To determine whether a collateral effect could be achieved with AdRSVrCES, medium was harvested from cells transduced with this virus at an moi of 5 and applied to untransduced cells in combination with CPT-11. Because only basal levels of intracellular CE were present, the toxicity of CPT-11 was attributable to the extracellular conversion to SN-38 by the secreted rabbit liver enzyme. This approach was adopted as opposed to mixing of transduced cells because adenoviral gene expression might affect growth rates and, hence, apparent cytotoxicity to CPT-11. Table 5Citation indicates the IC50 values for a panel of cell lines after incubation with media derived from AdRSVrCES-transduced cells and CPT-11. Significant differences in the levels of CE activity in the media paralleled a corresponding reduction in the IC50 values for CPT-11, and sensitization to the drug was observed in A549, HT29, Rh30, and U373MG cells. In contrast, although AdRSVrCE allowed expression of intracellular CE and sensitized the two neuroblastoma lines NB-1691 and SK-N-AS to CPT-11, no CE activity was detected in the media after transduction with AdRSVrCES. The possibility that the protein secretory pathway is disrupted in neuroblastoma cells is currently being investigated.


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  		Table 5 IC50 values after treatment with media derived from AdRSVrCES transduced cells and CPT-11

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates the sensitization of a panel of cell lines to CPT-11 after adenoviral-mediated delivery of a rCE cDNA. We have reported previously (9 , 14 , 15 , 24) that cells transfected with plasmids expressing the rCE are sensitized cells to the drug. In this manuscript and the accompanying study, we have expanded these studies by using adenovirus to deliver the prodrug-activating CE to potentiate the toxicity of CPT-11 for proposed gene therapy experiments. We have demonstrated in six of six cell lines that very high intracellular levels of CE activity can be achieved after adenoviral transduction at modest mois (<=5) and result in significant sensitization to CPT-11. Our results are in contrast to data presented by Kojima et al. (25) who used adenovirus to transfer and express a human CE in tumor cell lines. Of 11 cell lines tested, only three demonstrated more than a 5-fold decrease in the IC50 values for CPT-11. The results of Kojima et al. (25) were achieved using a moi of 50.

In our study, experiments performed at mois of 50 with AdRSVrCE resulted in exceedingly high levels of CE activity (>30,000 µmol/min/mg) and led to growth arrest of the cells (data not shown). In the two cell lines common to both studies, A594 and HT29, Kojima et al. (25) achieved changes in the IC50 values of ~17-fold and zero, respectively. In our studies with the rabbit CE, we observed reductions in the IC50 of 46- and 11-fold using a moi of 5. These differences may be attributable to the ability of the rabbit CE to preferentially activate CPT-11 in comparison with the human CE. We have reported previously (14) that the rabbit enzyme is at least 100-fold more efficient at drug activation as compared with a highly homologous human CE.

Data in Tables 2Citation and 5Citation indicate that although all of the cell lines expressing or exposed to medium containing the rabbit CE were sensitized to CPT-11, there was no direct comparison in the levels of CE activity and the fold change in the IC50 value. This is probably attributable to inherent differences in the sensitivity of the cells to SN-38, because we have observed a 200-fold range in the IC50 values with this drug with several cell lines. However, as indicated in Table 4Citation , there is a direct correlation between the level of CE activity and the amount of SN-38 produced. Hence, we cannot a priori predict the IC50 values of cells to CPT-11 before or after transduction with AdRSVrCE or AdRSVrCES, but it is probable that exposure to these adenoviral vectors will significantly increase the toxicity to cells by this drug.

This manuscript describes the construction of two different adenoviruses for use in VDEPT with CPT-11. AdRSVrCE, which encodes an intracellular CE, would be suitable for the elimination of tumor cells from bone marrow (26) . Cytotoxicity would be limited to cells transduced with the virus. However, for the treatment of solid tumors, AdRSVrCES would be the reagent of choice. This virus encodes the secreted form of the rabbit CE and should induce a collateral effect by producing SN-38 in the extracellular fluid. Hence, untransduced cells would be subjected to the cytotoxic effects of the drug. To what extent this occurs would depend upon many factors including the level of CE expression, the elimination of CE from the extracellular fluid, binding of SN-38 to extracellular proteins, and exogenous metabolism of CPT-11.

We envisage that each of the described vectors may be best applied for specific applications. The use of an intracellular enzyme might be most appropriate for ex vivo purging of tumor cells before autologous stem cell rescue (26) . In contrast, for the treatment of solid tumors in situ, a collateral effect may be beneficial. Because of the characteristics of currently available adenoviral vectors, it is unlikely that 100% of cells in a tumor will be transduced after local application of virus. Therefore, approaches that increase the effectiveness of extracellular CPT-11 activation should result in increased antitumor activity. Hence, in addition to the adenovirus encoding the intracellular CE, we generated a second adenovirus expressing a secreted form of the rCE (15) . Studies with this virus demonstrated that in four of six cell lines, sufficient CE was secreted into the media to sensitize untransduced cells to CPT-11. Because cultured cells are incubated in considerably more media than would be present in the extracellular fluid of a solid tumor, these in vitro studies may underestimate the effectiveness of the described approach to induce a collateral effect in vivo. We are currently performing human tumor xenograft experiments to assess the efficacy of both AdRSVrCE and AdRSVrCES in inducing tumor regressions in combination with CPT-11.

A second consideration for the use of VDEPT in cancer therapy concerns the biological characteristics of the solid tumor. As mentioned previously, the neuroblastoma cell lines transduced with AdRSVrCES did not secrete the CE, although high levels of enzyme were present after transduction with AdRSVrCE, and these cells were sensitized to CPT-11. Additional experiments have indicated that less protein is synthesized in NB1691 cells after transduction with AdRSVrCES, although this is partially dependent upon the density at which the cells were plated and transduced (data not shown). We are currently characterizing other neuroblastoma cell lines to determine whether this result is unique to this cell type. If so, the production of a collateral effect using the secretion of a drug-activating enzyme may be difficult to achieve in neuroblastoma tumors.

In summary, there are several potential uses of replication-deficient viral vectors in VDEPT cancer therapy. These include the direct injection of solid tumors with virus, the elimination of residual tumor cells after surgical resection or chemotherapy, and the ex vivo purging of tumor cells from bone marrow preparation before autologous stem cell rescue. It is our opinion that direct administration of virus to large tumors either to induce apoptosis or to sensitize cells to drugs is probably not the most effective use of these reagents for cancer treatment. Although such studies may be applicable in very defined clinical situations such as nonmetastatic brain tumors where surgery is not possible, it is unlikely that this approach would be applicable to solid malignancies in general. Hence, we are pursuing alternative strategies to maximize the efficacy of these gene therapy approaches.

We believe that the greatest clinical utility for replication-deficient vectors will be for the treatment of residual disease at the primary site after resection of solid tumors and for the purging of tumor cells from bone marrow used for autologous transplant (26) . This latter procedure uses replication-deficient adenovirus expressing the intracellular form of the rabbit CE (AdRSVrCE, described in this study) to sensitize tumor cells to CPT-11 ex vivo, and preclinical studies describing such an approach are found in Meck et al. (26) . In the case of purging, selectivity of gene expression is afforded by the ability of the viral vectors to transduce tumor cells preferentially to CD34+ cells. In the case of residual disease, however, it will be necessary to design tumor-specific expression vectors to effect tumor-selective cytotoxicity.

In conclusion, we have constructed and monitored the efficacy of adenoviral vectors designed to express either an intracellular localized or a secreted rCE and demonstrated that both can sensitize cells to CPT-11. Additionally, a collateral effect has been observed with some type of tumor cells with the latter virus indicating that this reagent may be suitable for gene therapy approaches for the treatment of solid tumors in combination with CPT-11.


   ACKNOWLEDGMENTS
 
We thank Drs. J. P. McGovren, Elio Vanin, and Peter Houghton for the provision of reagents for these studies and the latter for critical review of this manuscript.


   FOOTNOTES
 
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 in part by NIH Grants CA76202, CA79763, the Cancer Center Core Grant CA21765 and by the American Lebanese Syrian Associated Charities. Back

2 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Phone: (901) 495-2825; Fax: (901) 521-1668; E-mail: phil.potter{at}stjude.org Back

3 The abbreviations used are: CPT-11, irinotecan 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin; CE, carboxylesterase; moi, multiplicity of infection; rCE, rabbit liver CE; rCES, secreted form of rCE; RSV, Rous sarcoma virus promoter; SN-38, 7-ethyl-10-hydroxycamptothecin; VDEPT, virus-directed enzyme prodrug therapy; TFIID, transcription factor IID. Back

Received 1/11/01. Accepted 5/ 2/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. 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: 2823-2839, 1993.[Abstract/Free Full Text]
  2. Houghton P. J., Cheshire P. J., Hallman J. D., II, Lutz L., Friedman H. S., Danks M. K., Houghton J. A. Efficacy of topoisomerase I inhibitors, topotecan and irinotecan, administered at low dose levels in protracted schedules to mice bearing xenografts of human tumors. Cancer Chemother. Pharmacol., 36: 393-403, 1995.[Medline]
  3. Thompson J., Zamboni W. C., Cheshire P. J., Richmond L., Luo X., Houghton J. A., Stewart C. F., Houghton P. J. Efficacy of oral irinotecan against neuroblastoma xenografts. Anticancer Drugs, 8: 313-322, 1997.[Medline]
  4. Thompson J., Zamboni W. C., Cheshire P. J., Lutz L., Luo X., Li Y., Houghton J. A., Stewart C. F., Houghton P. J. Efficacy of systemic administration of irinotecan against neuroblastoma xenografts. Clin. Cancer Res., 3: 423-431, 1997.[Abstract]
  5. Chabot G. G. Clinical pharmacokinetics of irinotecan. Clin. Pharmacokinet., 33: 245-259, 1997.[Medline]
  6. Furman W. L., Stewart C. F., Poquette C. A., Pratt C. B., Santana V. M., Zamboni W. C., Bowman L. C., Ma M. K., Hoffer F. A., Meyer W. H., Pappo A. S., Walter A. W., Houghton P. J. Direct translation of a protracted irinotecan schedule from a xenograft model to a Phase I trial in children. J. Clin. Oncol., 17: 1815-1824, 1999.[Abstract/Free Full Text]
  7. Rivory L. P., Haaz M-C., Canal P., Lokiec F., Armand J-P., Robert J. Pharmacokinetic interrelationships of irinotecan (CPT-11) and its three major plasma metabolites in patients enrolled in Phase I/II trials. Clin. Cancer Res., 3: 1261-1266, 1997.[Abstract]
  8. Satoh T., Hosokawa M., Atsumi R., Suzuki W., Hakusui H., Nagai E. Metabolic activation of CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin, a novel antitumor agent, by carboxylesterase. Biol. Pharm. Bull., 17: 662-664, 1994.[Medline]
  9. Potter P. M., Pawlik C. A., Morton C. L., Naeve C. W., Danks M. K. Isolation and partial characterization of a cDNA encoding a rabbit liver carboxylesterase that activates the prodrug irinotecan (CPT-11). Cancer Res., 52: 2646-2651, 1998.
  10. Morton C. L., Wadkins R. M., Danks M. K., Potter P. M. CPT-11 is a potent inhibitor of acetylcholinesterase but is rapidly catalyzed to SN-38 by butyrylcholinesterase. Cancer Res., 59: 1458-1463, 1999.[Abstract/Free Full Text]
  11. Tanizawa A., Fujimori A., Fujimori Y., Pommier Y. Comparison of topoisomerase I inhibition, DNA damage, and cytotoxicity of camptothecin derivatives presently in clinical trials. J. Natl. Cancer Inst. (Bethesda), 86: 836-842, 1994.[Abstract/Free Full Text]
  12. Satoh T., Hosokawa M. Molecular aspects of carboxylesterase isoforms in comparison with other esterases. Toxicol. Lett. (Amst.), 82–83: 439-445, 1995.
  13. Morton C. L., Wierdl M., Oliver L., Ma M., Danks M. K., Stewart C. F., Eiseman J. L., Potter P. M. Activation of CPT-11 in mice: identification and analysis of a highly effective plasma esterase. Cancer Res., 60: 4206-4210, 2000.[Abstract/Free Full Text]
  14. Danks M. K., Morton C. L., Krull E. J., Cheshire P. J., Richmond L. B., Naeve C. W., Pawlik C. A., Houghton P. J., Potter P. M. Comparison of activation of CPT-11 by rabbit and human carboxylesterases for use in enzyme/prodrug therapy. Clin. Cancer Res., 5: 917-924, 1999.[Abstract/Free Full Text]
  15. Potter P. M., Wolverton J. S., Morton C. L., Wierdl M., Danks M. K. Cellular localization domains of a rabbit and a human carboxylesterase: influence on irinotecan (CPT-11) metabolism by the rabbit enzyme. Cancer Res., 58: 3627-3632, 1998.[Abstract/Free Full Text]
  16. Morton C. L., Potter P. M. Comparison of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Spodoptera frugiperda and COS7 cells for recombinant gene expression: application to a rabbit liver carboxylesterase. Mol. Biotechnol., 16: 193-202, 2000.[Medline]
  17. Graham F. L., Prevec L. Methods for construction of adenovirus vectors. Mol. Biotechnol., 3: 207-220, 1995.[Medline]
  18. Zhang W. W., Koch P. E., Roth J. A. Detection of wild-type contamination in a recombinant adenoviral preparation by PCR. Biotechniques, 18: 444-447, 1995.[Medline]
  19. Beaufay H., Amar-Costesec A., Feytmans E., Thines-Sempoux D., Wibo M., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods. J. Cell Biol., 61: 188-200, 1974.[Abstract/Free Full Text]
  20. Guichard S., Morton C. L., Krull E. J., Stewart C. F., Danks M. K., Potter P. M. Conversion of the CPT-11 metabolite APC to SN-38 by rabbit liver carboxylesterase. Clin. Cancer Res., 4: 3089-3094, 1998.[Abstract]
  21. Matsuidaira P. Limited N-terminal sequence analysis. Methods Enzymol., 182: 602-613, 1990.[Medline]
  22. Morton C. L., Potter P. M. Rhabdomyosarcoma-specific expression of the Herpes simplex virus thymidine kinase gene confers sensitivity to ganciclovir. J. Pharmacol. Exp. Ther., 286: 1066-1073, 1998.[Abstract/Free Full Text]
  23. Potter P. M., McKenzie P. P., Hussain N., Morton C. L., Harris L. C. Construction of adenovirus for high level expression of small RNAs in mammalian cells: application to a Bcl-2 ribozyme. Mol. Biotechnol., 15: 105-114, 2000.[Medline]
  24. Danks M. K., Morton C. L., Pawlik C. A., Potter P. M. Overexpression of a rabbit liver carboxylesterase sensitizes human tumor cells to CPT-11. Cancer Res., 58: 20-22, 1998.[Abstract/Free Full Text]
  25. Kojima A., Hackett N. R., Ohwada A., Crystal R. G. In vivo human carboxylesterase cDNA gene transfer to activate the prodrug CPT-11 for local treatment of solid tumors. J. Clin. Investig., 101: 1789-1796, 1998.[Medline]
  26. Meck M. M., Wierdl M., Wagner L. M., Burger R. A., Guichard S. M., Krull E. J., Harris L. C., Potter P. M., Danks M. K. A VDEPT approach to purging neuroblastoma cells from hematopoietic cells using adenovirus-encoding rabbit carboxylesterase and CPT-11. Cancer Res., 61: 5083-5089, 2001.[Abstract/Free Full Text]



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