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Experimental Therapeutics, Preclinical Pharmacology |
Department of Molecular Pharmacology [M. K. D., C. L. M., E. J. K., P. J. C., L. B. R., C. A. P., P. J. H., P. M. P.] and Center for Biotechnology [C. W. N.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first consideration in designing potential treatment schema based on the above approaches is the choice of an appropriate enzyme. With each enzyme/prodrug combination, an enzyme efficient in converting the prodrug to the active compound in situ must be identified. Enzymes most commonly used in previous studies have been Escherichia coli cytosine deaminase in combination with 5-fluorocytosine and HSVtk to activate ganciclovir (9, 10, 11, 12) . We have recently isolated and sequenced a cDNA encoding a rabbit liver carboxylesterase that converts the topoisomerase I inhibitor CPT-11 to its active metabolite SN-38 (13 , 14) .
The therapeutic advantage to overexpressing cytosine deaminase or thymidine kinase derives from unique characteristics of mammalian cells or enzymes compared with the bacterial or viral enzymes. Mammalian cells do not express cytosine deaminase (12 , 15) ; the overexpression of the E. coli enzyme is thought, therefore, to confer selective cytotoxicity of 5-fluorocytosine by converting it to 5-fluorouridine in tumor cells induced to express the exogenous deaminase (16) . In contrast, mammalian cells do express thymidine kinase. However, the affinity of the human enzyme for ganciclovir is 4 to 15 times lower than that of the HSVtk (17) . Ganciclovir is thereby preferentially phosphorylated by HSVtk, inhibiting DNA synthesis and producing selective toxicity in cells that overexpress the viral enzyme (18 , 19) . Similarly, both human and rabbit carboxylesterases are known to convert CPT-11 to SN-38 (13 , 14 , 20 , 21) , but unlike the thymidine kinase enzymes, the relative efficiency of catalysis by each enzyme is not known.
In patients, SN-38 is detectable in the plasma within minutes after CPT-11 is administered (22) . The amount of CPT-11 that is converted to SN-38 is <5%, as determined by concentrations of each compound in the plasma (22) . In vitro, overexpression of a human liver enzyme sensitized the A549 human lung tumor cell line to CPT-11 17-fold (23) , and overexpression of the rabbit liver enzyme sensitized human rhabdomyosarcoma and glioma cell lines to CPT-11 by 9- and 56-fold, respectively (13 , 14) . A side-by-side comparison of the two enzymes to demonstrate the relative efficiencies of each enzyme in activating CPT-11 has not been reported. To facilitate the choice of an enzyme for use in ADEPT and VDEPT, we compared the amino acid sequences of rabbit and human carboxylesterases, the activation of CPT-11 by each enzyme in vitro and in situ, the relative ability of each enzyme to sensitize Rh30 rhabdomyosarcoma cells and U-373 MG glioma cells to CPT-11, and the ability of each enzyme to sensitize human tumor xenografts to CPT-11 in a preclinical in vivo model.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
U-373 MG glioblastoma cells were purchased from American Type Culture Collection. U-373 MG cells and Rh30 rhabdomyosarcoma cells were grown in DMEM supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 1 mM sodium pyruvate, 1 mM nonessential amino acids, and 2 mML-glutamine and kept at 37°C in a humidified atmosphere of 5% CO2, as reported previously (13
, 14)
. Each of the above cell lines was transfected with the mammalian expression vector pIRESneo (Rh30pIRES and U373pIRES; Clontech, Palo Alto, CA) or pIRESneo containing the cDNA encoding a rabbit liver carboxylesterase (U373pIRESrabbit and Rh30pIRESrabbit) as reported previously (14)
. Briefly, 107 cells were electroporated with 20 µg of plasmid DNA in a volume of 200 µl of 0.9% NaCl using a Bio-Rad (Hercules, CA) electroporator and a capacitance extender. U-373 MG and Rh30 cells were also transfected with pIRESneo containing the coding sequence for a human carboxylesterase (U373pIREShuman and Rh30pIREShuman) by the same method as above, also as reported previously (14)
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Isolation and Characterization of the Human Carboxylesterase cDNA.
This cDNA was obtained by designing PCR primers based on the published sequence for human alveolar macrophage carboxylesterase (GenBank Accession No. M73499) and performing PCR reactions with cDNA prepared from human liver poly(A)+ RNA mRNA (Clontech) using random primers and a cDNA cycle kit (Invitrogen, Carlsbad, CA). Pfu polymerase (Stratagene, La Jolla, CA) was used to minimize PCR-induced mutations. Preparation of plasmid DNA, ligations, and transformations have all been described previously in detail (14)
. cDNA sequences of selected clones were characterized by automated sequencing in the St. Jude Center for Biotechnology.
HPLC Analysis for CPT-11 and SN-38.
This procedure has been reported previously (22)
. Briefly, a Nova-Pak C18 column was equilibrated with 75 mM ammonium acetate, 25% acetonitrile, pH 4.0, and acidified methanolic extracts of reaction mixtures or cell pellets were eluted at a flow rate of 1 ml/min. Under these conditions, CPT-11 and SN-38 eluted at 5.2 and 7.5 min, respectively. Each compound was detected with a Jasco 821-FP fluorescence detector at an excitation wavelength of 375 nm and an emission wavelength of 550 nm. Products were detected with a Jasco 821-FP fluorescence detector. Data were analyzed using System Gold software. The limit of detection for CPT-11 and SN-38 was 20 and 2 pg/µl, respectively (24)
. Quantitations of CPT-11 and SN-38 produced by this method are reported as total number of fluorescence units/peak based on HPLC internal standards or as pg or molar concentrations based on standard curves of fluorescence peak areas generated with solutions of known concentrations of each drug. The variability of the assay between duplicate samples and among replicate experiments was <1%.
Carboxylesterase Activity.
Carboxylesterase activity was quantitated as described previously using o-NPA as a substrate (14
, 25)
. The reaction monitored was the conversion of o-NPA to nitrophenol. Production of nitrophenol was detected spectrophotometrically at 420 nm. Activity is expressed as µmol of nitrophenol produced/mg protein/min. One unit of enzyme activity is defined as mg of protein required to produce 1 µmol of nitrophenol/min.
Enzymatic Conversion of CPT-11 to SN-38.
These analyses were carried out as described previously (24)
. In brief, cell sonicates were incubated with 25 µM CPT-11 in a final volume of 200 µl of 50 mM HEPES (pH 7.4) at 37°C for 22 h. The reaction was terminated by the addition of one volume of acidified methanol, vortexed, and centrifuged at 14,000 x g for 2 min. SN-38 in the supernatant was quantitated by HPLC (22)
.
Growth Inhibition Assays.
These assays were performed as reported previously (13
, 14)
. Cells were plated at a density of 2 x 104 cells/ml, 2 ml/dish. The cells were allowed to attach overnight, and drug was added in a volume of 20 µl/2-ml tissue culture medium. After control cultures had doubled two to three times, the number of cells remaining in each dish was determined using a Coulter Counter. Data were analyzed using the program for a sigmoidal dose response with a variable or fixed slope of GraphPad Prism software. The concentration of CPT-11 required to inhibit growth of cells by 50% compared with vehicle-treated controls is defined as the IC50.
SDS-PAGE and Immunoblot Analyses.
SDS-PAGE and immunoblot analyses of whole-cell sonciates were performed as described previously (26, 27, 28)
. Gels were either stained with Coomassie Blue R-250, or proteins were transferred by electroblotting to Immobilon-P (Millipore, Bedford, MA) for Western analysis. Filters were incubated with primary antibody, and specific immunoreactivity was detected by chemiluminescence (ECL; Amersham, Arlington Heights, IL).
Xenografts Studies.
Methods for immune deprivation of CBA/CaJ mice, establishment of human tumor cell lines as xenografts, administration of CPT-11, and statistical analysis of xenograft studies were performed as published previously (29)
. Parental Rh30 cells or Rh30 cells transfected with the pIRES plasmid encoding either the rabbit or the human carboxylesterase were established in vitro and subsequently as xenografts by injection 107 cells s.c. in the mice. Drug treatment was begun when the tumors had reached an average volume of 
0.5–1.0 cm3. Any tumor <0.2 cm3 in size at the time drug treatment was initiated was excluded from the analysis. Tumor size at the beginning of the experiment was equal among all six control and drug-treated groups shown in Fig. 5
; tumor volumes of each group are specified in the legend to this figure.
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/6) x d3], where d is the mean diameter. |
RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These four amino acids occur in areas of the protein that are not in the active site of the enzyme. The cDNA sequences of the three human enzymes are >99% identical (data not shown). The sequence of the rabbit liver carboxylesterase, which has been published recently (14)
, is 86% homologous to each of the three human enzymes (see below).
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; Ref. 30
). This human carboxylesterase is 81% identical and 86% similar to the rabbit enzyme at the amino acid level. The regions of greatest disparity between the rabbit and human enzymes occur between amino acids 250 and 272 and 303 and 344 (Fig. 1)
. The three reported active site residues (serine 221, glutamic acid 353, and histidine 467) are identical in all previously reported carboxylesterases, as well as in the carboxylesterases used in this study.
Catalysis of o-NPA and CPT-11 by Human and Rabbit Carboxylesterases.
Rh30 rhabdomyosarcoma cells expressing human or rabbit carboxylesterases were harvested, resuspended in 50 mM HEPES buffer, and sonicated to prepare lysates. Each lysate was then incubated with 3 mM o-NPA, and the conversion of o-NPA to nitrophenol was monitored spectrophotometrically. Equal amounts of enzymatic activity (20, 200, or 2000 units) from sonicates of Rh30pIREShuman or Rh30pIRESrabbit cells were then incubated with 25 µM CPT-11 for 22 h. Methanolic extracts of each reaction mixture were analyzed by HPLC (Fig. 2)
to quantitate the enzymatic conversion of CPT-11 to SN-38. Quantitation of data in HPLC profiles (Fig. 3)
shows a concentration-dependent increase from 70 nM to 17 µM SN-38 in reaction mixtures containing CPT-11 and increasing amounts of rabbit carboxylesterase activity. In each case, the concentration of CPT-11 decreased commensurately with an increase in SN-38 production. In contrast, with sonicates from cells expressing the human carboxylesterase, only 15 nM SN-38 was detected in reaction mixtures containing 200 or 2000 units of carboxylesterase activity. Similar results were obtained with enzymes expressed in baculovirus and with cell extracts from other carboxylesterase transfected cell lines and xenografts (data not shown). The kinetics of conversion of CPT-11 to SN-38 by the rabbit enzyme were as reported previously by Guichard et al. (24)
. It was not possible to calculate kinetic parameters for activation of CPT-11 by the human enzyme due to the very low level of SN-38 produced.
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, PAGE), and immunoblots with anti-HA antibody (Fig. 4
, HA) and anti-tubulin (data not shown) were done. Specific HA-reactive and TUB-reactive bands were quantitated, and the amount of immunodetectable tagged carboxylesterase was normalized to amount of tubulin. The levels of tagged carboxylesterase expressed by Rh30pIRESrabbitHA and Rh30pIREShuman HA cells (bands indicated by arrow) differed by <2-fold. One nonspecific immunoreactive band was also detected in sonicates from all three cell lines. Because it is already known that Rh30pIRESrabbitHA and Rh30pIREShuman HA cells have equal levels of enzymatic activity (Ref. 27
and Fig. 4
legend), the results indicate that the ability of the two enzymes to metabolize o-NPA was very similar. Taken together, the above results show that the rabbit carboxylesterase activates CPT-11 more efficiently than the human carboxylesterase when compared either on the basis of units of enzymatic activity using o-NPA as a substrate or on the basis of amount carboxylesterase protein.
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compare the amount of SN-38 produced by U373pIRESrabbit and U373pIREShuman cells incubated with 250 µM CPT-11. The data indicate that in intact cells, the rabbit carboxylesterase is 7-fold more efficient than the human carboxylesterase in metabolizing CPT-11.
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Sensitization of Rh30 Xenografts to CPT-11 by Rabbit or Human Carboxylesterase in a Preclinical Mouse Model.
Because the long-range goal of these experiments is the application of an appropriate enzyme for use in ADEPT or VDEPT, we next determined whether expression of carboxylesterases could sensitize Rh30 rhabdomyosarcoma human tumor cells grown as xenografts in immune-deprived mice to CPT-11. In this preclinical model, expression of the transfected cDNAs produced carboxylesterase activities of 212 µmol/min/mg for the human enzyme and 132 µmol/min/mg protein for the rabbit enzyme when xenografts were assessed at weeks 7–8, indicating that roughly equivalent amounts of both enzyme activities were present throughout the treatment cycle. Carboxylesterase activity is also readily detectable in xenografts that have been propagated s.c. for as long as a year, with the level of enzyme activity in xenografts expressing the human carboxylesterase cDNA being consistently slightly higher (2–3-fold) than the xenografts expressing the rabbit cDNA (data not shown). Importantly, tumors were advanced (an average of 0.5–1.0 cm3 in volume) before CPT-11 treatment was begun. Mice bearing xenografts (seven animals per group; two tumors/mouse) were treated with 2.5 mg of CPT-11/kg/day for 5 days each week for 2 weeks (one cycle of therapy), repeated every 21 days for a total of three cycles (over 8 weeks). Week 8 was the final week of treatment. Mice were observed for an additional 6 weeks after cessation of treatment to assess tumor recurrence. Using this regimen, all tumors regressed. However, xenografts not expressing either the rabbit or the human carboxylesterase (Rh30) recurred in seven of seven mice immediately after cessation of CPT-11 administration (Fig. 5
, arrow). Xenografts expressing the human carboxylesterase regressed, but tumors recurred in two of seven mice. In contrast, xenografts expressing the rabbit carboxylesterase regressed completely and did not regrow during the 14 weeks of the study. We conclude that although the human carboxylesterase does activate CPT-11, the rabbit carboxylesterase is more efficient in vitro, in situ, and in vivo.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to the enzymes used in this study, several other carboxylesterases have been partially purified or their cDNAs isolated. The purified or partially purified enzymes that catalyze CPT-11 include those from human liver microsomes (21)
and rat serum (20
, 31)
; but the specific enzyme(s) or isozyme(s) that activate CPT-11 in vivo has not been determined. Neither have cDNAs encoding these proteins been isolated. The coding sequences for 13 carboxylesterases are, however, available in the GenBank database. As part of our long-range goal to identify enzymes useful in ADEPT or VDEPT, we also examined the expression in human tumor cells of the cDNAs encoding a rat serum carboxylesterase and a mouse carboxylesterase. The methods used were identical to those detailed in this report, and the sequences of those carboxylesterases were, as with the sequences above, remarkably homologous to the sequences shown in Fig. 1
. However, for reasons unknown, repeated efforts to establish cells lines that stably or transiently expressed either the rat or the mouse protein in human tumor cells lines were unsuccessful. Because it was not possible to express these proteins, we could not assess the ability of either the mouse or the rat protein to metabolize CPT-11.
Additionally, a human liver carboxylesterase has been reported recently to sensitize A549, H157, and SK-MES1 cells 6–17-fold to CPT-11 (23)
but to have a minimal effect in eight other human tumor cell lines (
IC50 1.2–2.5). It is not known whether the cDNA used in that study corresponded to the liver (1) or liver (2) enzyme as designated in Table 1
. It remains to be determined, because of the minimal differences among the sequences reported for the three human enzymes, whether these different sequences represent products of three separate genes, polymorphisms, or mutations or were introduced by in vitro manipulations. The >99% identity among these proteins indicates that the possibility that they represent products of unique genes is somewhat unlikely.
Present studies focus on delivery of the rabbit liver carboxylesterase cDNA with adenoviral vectors and induction of expression of this enzyme by tumor-specific transcription factors to achieve tumor-specific toxicity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NIH Grants CA-23099, CA-63512, CA-76202, CA-66124, the Cancer Center Core Grant P30-CA-21765, and by the American Lebanese and Syrian Associated Charities. 
2 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Phone: (901) 495-3440; Fax: (901) 521-1668; E-mail: phil.potter{at}stjude.org
3 The abbreviations used are: ADEPT, antibody directed enzyme prodrug therapy; VDEPT, virus directed enzyme prodrug therapy; HSVtk, Herpes simplex virus thymidine kinase; APC, 7-ethyl-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin; CPT-11, irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin; HPLC, high performance liquid chromatography; HA, hemagglutinin; IC50, concentration of drug that inhibits the growth of cells in tissue culture by 50%; o-NPA, ortho-nitrophenyl acetate; SN-38, 7-ethyl-10-hydroxycamptothecin.
Received 12/ 4/98; revised 1/21/99; accepted 1/22/99.
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REFERENCES |
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, Rh30; •, Rh30pIRESrabbit; 
, Rh30pIREShuman. Untreated xenografts: 
, Rh30; 
, Rh30pIRESrabbit; 
, Rh30pIREShuman. The mean volume in cm3 of the tumors at the beginning of treatment (time 0) in the control groups were: Rh30, 0.85 ± 0.23; Rh30pIRESrabbit, 0.59 ± 0.41; Rh30pIREShuman, 0.57 ± 0.23; bars, SD. The volumes of tumors at time 0 in the drug-treated groups were: Rh30, 0.67 ± 0.31; Rh30pIRESrabbit, 0.90 ± 0.42; Rh30pIREShuman, 0.54 ± 0.23; bars, SD.