Stem and progenitor cell-mediated tumor selective gene therapy.

The poor prognosis for patients with aggressive or metastatic tumors and the toxic side effects of currently available treatments necessitate the development of more effective tumor-selective therapies. Stem/progenitor cells display inherent tumor-tropic properties that can be exploited for targeted delivery of anticancer genes to invasive and metastatic tumors. Therapeutic genes that have been inserted into stem cells and delivered to tumors with high selectivity include prodrug-activating enzymes (cytosine deaminase, carboxylesterase, thymidine kinase), interleukins (IL-2, IL-4, IL-12, IL-23), interferon-beta, apoptosis-promoting genes (tumor necrosis factor-related apoptosis-inducing ligand) and metalloproteinases (PEX). We and others have demonstrated that neural and mesenchymal stem cells can deliver therapeutic genes to elicit a significant antitumor response in animal models of intracranial glioma, medulloblastoma, melanoma brain metastasis, disseminated neuroblastoma and breast cancer lung metastasis. Most studies reported reduction in tumor volume (up to 90%) and increased survival of tumor-bearing animals. Complete cures have also been achieved (90% disease-free survival for >1 year of mice bearing disseminated neuroblastoma tumors). As we learn more about the biology of stem cells and the molecular mechanisms that mediate their tumor-tropism and we identify efficacious gene products for specific tumor types, the clinical utility of cell-based delivery strategies becomes increasingly evident.

An improved human carboxylesterase for enzyme/prodrug therapy with CPT-11.

CPT-11 is a potent antitumor agent that is activated by carboxylesterases (CE) and intracellular expression of CEs that can activate the drug results in increased cytotoxicity to the drug. As activation of CPT-11 (irinotecan-7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) by human CEs is relatively inefficient, we have developed enzyme/prodrug therapy approaches based on the CE/CPT-11 combination using a rabbit liver CE (rCE). However, the in vivo application of this technology may be hampered by the development of an immune response to rCE. Therefore, we have developed a mutant human CE (hCE1m6), based on the human liver CE hCE1, that can activate CPT-11 approximately 70-fold more efficiently than the wild-type protein and can be expressed at high levels in mammalian cells. Indeed, adenoviral-mediated delivery of hCE1m6 with human tumor cells resulted in up to a 670-fold reduction in the IC(50) value for CPT-11, as compared to cells transduced with vector control virus. Furthermore, xenograft studies with human tumors expressing hCE1m6 confirm the ability of this enzyme to activate CPT-11 in vivo and induce antitumor activity. We propose that this enzyme should likely be less immunogenic than rCE and would be suitable for the in vivo application of CE/CPT-11 enzyme/prodrug therapy.

Structural constraints affect the metabolism of 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) by carboxylesterases.

7-Ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin [CPT-11 (irinotecan)] is a water-soluble camptothecin-derived prodrug that is activated by esterases to yield the potent topoisomerase I poison SN-38. We identified a rabbit liver carboxylesterase (CE) that was very efficient at CPT-11 metabolism; however, a human homolog that was more than 81% identical to this protein activated the drug poorly. Recently, two other human CEs have been isolated that are efficient in the conversion of CPT-11 to SN-38, yet both demonstrate little homology to the rabbit protein. To understand this phenomenon, we have characterized a series of esterases from human and rabbit, including several chimeric proteins, for their ability to metabolize CPT-11. Computer predictive modeling indicated that the ability of each enzyme to activate CPT-11 was dependent on the size of the entrance to the active site. Kinetic studies with a series of nitrophenyl and naphthyl esters confirmed these predictions, indicating that activation of CPT-11 by a CE is constrained by size-limited access of the drug to the active site catalytic amino acid residues.

Sensitization of human tumor cells to CPT-11 via adenoviral-mediated delivery of a rabbit liver carboxylesterase.

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 IC(50) 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 IC(50) 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.

A virus-directed enzyme prodrug therapy approach to purging neuroblastoma cells from hematopoietic cells using adenovirus encoding rabbit carboxylesterase and CPT-11.

Tumor cells that contaminate hematopoietic cell preparations contribute to the relapse of neuroblastoma patients who receive autologous stem cell rescue as a component of therapy. Therefore, effective purging methods are needed. This study details in vitro experiments to develop a viral-directed enzyme prodrug purging method that specifically targets neuroblastoma cells. The approach uses an adenovirus to deliver the cDNA encoding a rabbit liver carboxylesterase that efficiently activates the prodrug irinotecan,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11). The data show that an adenoviral multiplicity of infection of 50 transduces 100% of cultured neuroblastoma cells and primary tumor cells, irrespective of the level of tumor cell line contamination. Exposure of neuroblastoma cell lines or of mixtures of these cell lines with CD34(+) cells at a ratio of 10:90 to replication-deficient AdRSVrCE for 24 h and subsequent exposure of cells to 1-5 microM CPT-11 for 4 h increased the toxicity of CPT-11 to three neuroblastoma cell lines (SJNB-1, NB-1691, and SK-N-SH) from approximately 20-50-fold and eradicated their clonogenic potential. Also, after "purging," RNA for neuroblastoma cell markers (tyrosine hydroxylase, synaptophysin, and N-MYC) was undetectable by reverse transcription-PCR. In contrast, the purging protocol did not affect the number or type of colonies formed by CD34(+) cells in an in vitro progenitor cell assay. No bystander effect on CD34(+) cells was observed. The method described is being investigated for its potential clinical utility, particularly its efficacy for use with patients having relatively high tumor burdens, because no published methods have been shown to be efficacious when the tumor burden exceeds 1%.

Development, characterization and therapy of a disseminated model of childhood neuroblastoma in SCID mice.

PURPOSE: To develop a highly reproducible model of disseminated childhood neuroblastoma in mice to allow secondary evaluation of therapeutics against microscopic disseminated disease. METHODS: CB17/Icr SCID were injected i.v. with 10(3) to 5 x 10(6) human NB-1691 neuroblastoma cells. NB-1691 cells were detected by PCR for synaptophysin and tyrosine hydroxylase in peripheral blood, and bone marrow. Therapeutic studies evaluated topotecan and vincristine as single agents or in combination. Topotecan was administered i.v. daily for 5 days on two consecutive weeks. Courses were repeated every 21 days for three cycles. Vincristine (1 mg/kg) was administered i.v. every 7 days for nine consecutive weeks. Treatment started 11-21 days after tumor cell inoculation. RESULTS: Following injection of > or = 1 x 10(5) cells 100% of mice developed disease. Mice inoculated with 10(7) cells survived a median of 42 days. Survival time was a linear function of the cell inoculum. At autopsy, gross tumor was routinely detected in many organs in particular liver, ovaries, kidneys and adrenals. NB-1691 cells were detected by PCR in peripheral blood, and bone marrow. Immunohistochemical staining showed that lesions were strongly positive for synaptophysin, chromogranin A and negative for leukocyte common antigen. Topotecan (0.6 mg/kg) alone extended median survival from 44 days (controls) to 95 days. When treatment was started 21 days after inoculation of NB-1691 cells, topotecan extended median survival from 39 days (controls) to 91 and 99 days at dose levels of 0.3 and 0.6 mg/kg, respectively. Vincristine (1 mg/kg) extended survival by a median of 9.5 days. In combination with vincristine (1 mg/kg), median survival was increased to 141 days (topotecan 0.6 mg/kg) and 159 days (topotecan 1.0 mg/kg). CONCLUSION: This model of disseminated neuroblastoma is highly reproducible. As this model may more closely simulate childhood disease it may be a valuable adjunct in developing new approaches to advanced stage, poor prognosis neuroblastoma.

Use of a modified ornithine decarboxylase promoter to achieve efficient c-MYC- or N-MYC-regulated protein expression.

One of the advantages of viral-directed enzyme prodrug therapy (VDEPT) is its potential for tumor-specific cytotoxicity. However, the viruses used to deliver cDNAs encoding prodrug-activating enzymes transduce normal cells as well as tumor cells, and several approaches to achieve tumor-specific expression of the delivered cDNAs are being investigated. One such approach is to regulate transcription of the prodrug-activating enzyme with a promoter that is preferentially activated by tumor cells. Published data suggest that the most promising transcription factor/promoter/enhancer combinations are those activated by a tumor-specific transcription factor to retain tumor cell specificity but that are equal in strength to nonspecific viral promoters in their ability to up-regulate target cDNAs. This report identifies MYC-responsive, modified ornithine decarboxylase (ODC) promoter/enhancer sequences that up-regulate target protein expression in tumor cells overexpressing either N-MYC or c-MYC protein. The most efficient of the four constructs assessed contained six additional CACGTG MYC binding sites 5' to the endogenous ODC promoter (R6ODC). Reporter assays with this chimeric promoter/enhancer regulating expression of chloramphenicol acetyltransferase demonstrated 50-250-fold more activity in MYC-expressing cells compared with similar assays with promoterless plasmids. The R6ODC regulatory sequence was approximately equivalent to the CMV promoter in inducing expression of the neomycin resistance gene in c-MYC-expressing SW480 and HT-29 colon carcinoma cells and in N-MYC-expressing NB-1691 neuroblastoma cells. The modified ODC promoter may, therefore, be useful in achieving tissue-specific expression of target proteins in tumor cells that overexpress c- or N-MYC.

Proficient metabolism of irinotecan by a human intestinal carboxylesterase.

Irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11)] is metabolized by esterases to yield the potent topoisomerase I poison 7-ethyl-10-hydroxycamptothecin. One of the major side effects observed with CPT-11 is gastrointestinal toxicity, and we supposed that this might be due to local activation of CPT-11 within the gut. Carboxylesterase (CE) activity was detected in human gut biopsies, and extracts of these tissues converted CPT-11 to 7-ethyl-10-hydroxycamptothecin in vitro. Expression of a human intestinal CE cDNA in COS-7 cells produced extracts that demonstrated proficient CPT-11 activation and conferred sensitivity of cells to CPT-11. These results suggest that gut toxicity from CPT-11 may be due in part to direct drug conversion by CEs present within the small intestine.

Use of the ornithine decarboxylase promoter to achieve N-MYC-mediated overexpression of a rabbit carboxylesterase to sensitize neuroblastoma cells to CPT-11.

Overexpression of specific transcription factors by tumor cells can be exploited to regulate expression of proteins that induce apoptosis or activate prodrugs, thereby producing tumor-selective toxicity. A majority of advanced-stage neuroblastomas overexpress the transcription factor N-MYC, and this overexpression is associated with poor prognosis. This study describes regulation of expression by N-MYC, via the ornithine decarboxylase (ODC) promoter, of a rabbit liver carboxylesterase (CE) that activates the prodrug CPT-11. Chloramphenicol acetyltransferase reporter assays and CE activity assays in transiently transfected neuroblastoma cell lines (SJNB-1, SJNB-4, NB-1691) and rhabdomyosarcoma cell lines (JR1neo20, JR1Nmyc6, JR1Nmyc9) support this approach as a potential method for sensitizing tumor cells to CPT-11. Clonogenic assays with IMR32 human neuroblastoma cells which express N-MYC and that had been stably transfected with a plasmid containing an ODC promoter/CE cassette corroborated results of enzyme activity assays. Specifically, IMR32.ODC.CE cells expressed approximately eightfold more CE activity than IMR32.CMV.neo cells; and 5 microM CPT-11 reduced the clonogenic potential of IMR32.ODC.CE cells to zero, while 50 microM CPT-11 was required to produce the same effect with IMR32.CMV.neo cells. Current experiments focus on adenoviral delivery of an ODC promoter/CE cDNA cassette for potential virus-directed enzyme prodrug therapy applications.

Activation of CPT-11 in mice: identification and analysis of a highly effective plasma esterase.

The camptothecin prodrug CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is converted by esterases to yield the potent topoisomerase I poison SN-38 (7-ethyl-10-hydroxycamptothecin). Recently, a mouse strain (Es1(e)) has been identified that demonstrates reduced plasma esterase activity, and we have monitored the ability of plasma from these mice to metabolize CPT-11. Total plasma esterase activity was reduced 3-fold in Esl(e)mice in comparison to control mice, and this resulted in a 200-fold reduction in SN-38 production after incubation with CPT-11 in vitro. In addition, pharmacokinetic studies of CPT-11 and SN-38 in these animals demonstrated approximately 5-fold less conversion to SN-38. However, extracts derived from tissues from Es1(e) animals revealed total esterase activities similar to those of control mice, and these extracts metabolized CPT-11 with equal efficiency. Northern analysis of RNA isolated from organs indicated that the liver was the primary source of Es-1 gene expression and that very low levels of Es-1 RNA were present in Es1(e) mice. These results suggest that the reduced levels of Es-1 esterase present in Es1(e) mice are due to down-regulation of gene transcription, and that this plasma esterase is responsible for the majority of CPT-11 metabolism in mice.

P53 mutation and MDM2 amplification frequency in pediatric rhabdomyosarcoma tumors and cell lines.

BACKGROUND: The p53 tumor suppressor gene is the most commonly mutated gene in human cancer, and mutations arise in a wide variety of tumor types. Wild-type p53 functions as a regulator of apoptosis, so mutations in the p53 gene are generally associated with aggressive tumors and a poor prognosis. PROCEDURE: We have investigated the p53 mutation and MDM2 amplification frequencies in biopsies from pediatric rhabdomyosarcoma (RMS) tumors and cell lines by SSCP and Southern analyses. RESULTS: A mutation was detected in only 1 of 20 tumor specimens (5%), whereas the frequency in established RMS cell lines was significantly higher (6/10, 60%). p53 Mutations were more common in cell lines derived from tumors previously exposed to chemotherapy compared to those derived from tumors at di-agnosis, and it is likely that these mutations enhanced the probability of successful long-term culture. The frequency of MDM2 gene amplification in patient biopsies was also low (2/20, 10%). Interestingly, complete responses to treatment were obtained in the two patients with tumors that demonstrated amplification of MDM2. The response to treatment of patients with tumors wild-type for p53 and without MDM2 amplification was quite varied, indicating that expression of a wild-type p53 gene at diagnosis cannot always facilitate a favorable outcome. CONCLUSIONS: p53 mutation and MDM2 gene amplification frequencies are extremely low in RMS tumors, but a wild-type p53 genotype is not always associated with a favorable prognosis.

Isolation and characterization of a cDNA encoding a horse liver butyrylcholinesterase: evidence for CPT-11 drug activation.

Butyrylcholinesterases (BuChEs; acylcholine acylhydrolase; EC 3.1.1.8) have been demonstrated to convert the anticancer agent CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) into its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin). In addition, significant differences in the extent of drug metabolism have been observed with BuChEs derived from different species. In an attempt to understand these differences, we have isolated the cDNA encoding a horse BuChE. Based upon the NH2-terminal amino acid sequence of a purified horse BuChE, we designed degenerate primers to amplify the coding sequence from horse liver cDNA. Following polymerase chain reaction and rapid amplification of the cDNA ends, we generated an 1850-bp DNA fragment, containing an 1806-bp open reading frame. The cDNA encodes a protein of 602 amino acid residues, including a 28-amino-acid NH2-terminal signal peptide. Furthermore, the DNA sequence and the deduced amino acid sequence revealed extensive homology to butyrylcholinesterase genes from several other species. In vitro transcription-translation of the cDNA produced a 66-kDa protein, identical to the size of native horse serum BuChE following removal of carbohydrate residues with endoglycosidase F. Additionally, transient expression of the cDNA in Cos-7 cells yielded extracts that exhibited cholinesterase activity and demonstrated a Km value for butyrylthiocholine of 106+/-9 nM. This extract converted the anticancer drug CPT-11 into SN-38, demonstrating that this drug can be activated by enzymes other than carboxylesterases.

Topoisomerase enzymes as drug targets.

DNA topoisomerases catalyze changes in the topology of DNA. Recently, other functions have also been reported for these enzymes. For example, topoisomerase I participates in transcription by RNA polymerases I, II, and III, and also has a kinase activity. Topoisomerase I binds directly to at least two helicases, nucleolin and SV40 T antigen, and mechanistic studies show that T antigen alters the function of topoisomerase I. Additional protein and nucleotide interactions for both topoisomerases I and II suggest that each protein is multifunctional. It may be that the multifunctional nature of these enzymes is the basis for the antitumor activity seen with inhibitors of these enzymes. Clinical trials with combinations of CPT-11 and 5-fluorouracil for the treatment of colon cancer, and preclinical studies with CPT-11 and vincristine are particularly encouraging. Protracted schedules of administration of topoisomerase inhibitors will likely have greater antitumor effect than more concentrated, higher dose exposures, but a systematic determination of optimal schedules of administration is needed.

The anticancer prodrug CPT-11 is a potent inhibitor of acetylcholinesterase but is rapidly catalyzed to SN-38 by butyrylcholinesterase.

Patients treated with high doses of CPT-11 rapidly develop a cholinergic syndrome that can be alleviated by atropine. Although CPT-11 was not a substrate for acetylcholinesterase (AcChE), in vitro assays confirmed that CPT-11 inhibited both human and electric eel AcChE with apparent K(i)s of 415 and 194 nM, respectively. In contrast, human or equine butyryl-cholinesterase (BuChE) converted CPT-11 to SN-38 with K(m)s of 42.4 and 44.2 microM for the human and horse BuChE, respectively. Modeling of CPT-11 within the predicted active site of AcChE and BuChE corroborated experimental results indicating that, although the drug was oriented correctly for activation, the constraints dictated by the active site gorge were such that CPT-11 would be unlikely to be activated by AcChE.

Comparison of activation of CPT-11 by rabbit and human carboxylesterases for use in enzyme/prodrug therapy.

Several recent studies have examined the possibility of producing tumor-specific cytotoxicity with various enzyme/ prodrug combinations. The enzymes are targeted to tumor cells either with antibodies (ADEPT, antibody directed enzyme prodrug therapy) or with viruses (VDEPT). The goal of the present study was to identify an appropriate enzyme for use in activating the prodrug 7-ethyl-10-[4-(1-piper-idino)-1-piperidino]carbonyloxycamptothe cin (CPT-11). In this study, we compared the efficiency of CPT-11 metabolism by rabbit and human carboxylesterases in in vitro and in situ assays. Although the rabbit and human enzymes are very similar (81% identical; 86% homologous) and the active site amino acids are 100% identical, the rabbit enzyme was 100-1000-fold more efficient at converting CPT-11 to SN-38 in vitro and was 12-55-fold more efficient in sensitizing transfected cells to CPT-11. In vivo, Rh30 rhabdomyosarcoma cells expressing the rabbit carboxylesterase and grown as xenografts in immune-deprived mice were also more sensitive to CPT-11 than were control xenografts or xenografts expressing the human enzyme. Each of the three types of xenografts regressed when the mice were treated with CPT-11 given i.v. at 2.5 mg of CPT-11/kg/daily for 5 days/week for 2 weeks [(dx5)2] (one cycle of therapy), repeated every 21 days for a total of three cycles. However, following cessation of treatment, recurrent tumors were detected in seven of seven mice bearing control Rh30 xenografts and in two of seven mice bearing Rh30 xenografts that expressed the human enzyme. No tumors recurred in mice bearing xenografts that expressed the rabbit carboxylesterase. We conclude that rabbit carboxylesterase/CPT-11 may be a useful enzyme/prodrug combination.

Relationship between tumor extracellular fluid exposure to topotecan and tumor response in human neuroblastoma xenograft and cell lines.

PURPOSE: We have reported a 6-fold difference in the topotecan (TPT) lactone systemic exposure achieving a complete response in the human neuroblastoma xenografts NB-1691 and NB-1643. However, the relationship between tumor extracellular fluid (ECF) exposure to TPT and the antitumor activity in xenograft and in vitro models has not been established. METHODS: TPT was given i.v. to mice bearing NB-1691 and NB-1643 tumors. Prior to dosing, microdialysis probes were placed in tumors of mice bearing NB-1691 and NB-1643 tumors. Plasma and tumor ECF concentrations of TPT lactone were assayed by high performance liquid chromatography. The inhibitory concentration (IC50) was determined for NB-1691 and NB-1643 cell lines in vitro. RESULTS: The TPT AUC(ECF) values determined for NB-1691 (n = 10) and NB-1643 (n = 11) were 7.3 +/- 0.84 and 25.6 +/- 0.76 ng h ml(-1), respectively (P < 0.05). TPT tumor ECF penetration in NB-1691 and NB-1643 was 0.04 +/- 0.04 and 0.15 +/- 0.11 (P < 0.05), respectively. The IC50 values recorded after 6 h of TPT exposure daily for 5 consecutive days for NB-1691 and NB-1643 were 2.7 +/- 1.1 and 0.53 +/- 0.19 ng/ml, respectively (P < 0.05). CONCLUSIONS: NB-1643 was more sensitive in vitro than NB-1691, and at similar plasma TPT exposures, NB-1643 had a greater degree of TPT tumor ECF exposure and penetration as compared with NB-1691. Potential factors affecting tumor TPT ECF disposition include tumor vascularity, capillary permeability, and interstitial pressure. The clinical importance of this study is underscored by the need to select anticancer agents with a high capacity for tumor penetration and to optimize drug administration to increase tumor penetration.

Wild-type p53 can induce p21 and apoptosis in neuroblastoma cells but the DNA damage-induced G1 checkpoint function is attenuated.

p53 is a tumor suppressor protein important in the regulation of apoptosis. Because p53 functions as a transcription factor, cellular responses depend upon activity of p53 localized in the nucleus. Cytoplasmic sequestration of p53 has been proposed as a mechanism by which the function of this protein can be suppressed, particularly in tumor types such as neuroblastoma in which the frequency of mutations of p53 is low. Data presented here demonstrate that nuclear p53 protein is expressed in a panel of neuroblastoma cell lines, and after exposure to DNA damage, transcriptionally active p53 expression can be induced. After exposure to both equitoxic IC80 and 10-Gy doses of ionizing radiation, both p53 and p21 were induced, but G1 cell cycle arrest was attenuated. To investigate whether the DNA damage signaling pathway was incapable of inducing sufficient p53 in these cells, we expressed additional wild-type p53 after adenoviral vector transduction. This exogenous p53 expression also resulted in p21 induction but was unable to enhance the G1 arrest, suggesting that the pathway downstream from p53 is nonfunctional. Although p53-mediated G1 arrest is attenuated in neuroblastoma cells, the ability of p53 to induce apoptosis appears functional, consistent with its chemosensitive phenotype. This work demonstrates that p53 is expressed in the nucleus of neuroblastoma cells and can mediate induction of p21. However, this cell type appears to have an attenuated ability to mediate a DNA damage-induced G1 cell cycle arrest.

Conversion of the CPT-11 metabolite APC to SN-38 by rabbit liver carboxylesterase.

The anticancer drug CPT-11 (7-ethyl-[4(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is a water-soluble derivative of camptothecin. We report here the conversion of APC (7-ethyl-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin), an inactive metabolite of CPT-11, to SN-38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT-11, by a rabbit liver carboxylesterase. This reaction is not catalyzed by any known human enzyme. The formation of SN-38 from APC was characterized by an apparent Km of 37.9 +/- 7.1 microM and a Vmax of 16.9 +/- 0.9 pmol/units/min. SN-38 was confirmed as a reaction product by high-performance liquid chromatography and mass spectrometry. A 24-h incubation of 10 microM APC with 500 units/ml of rabbit carboxylesterase produced 4 microM SN-38. The product of this reaction inhibited the growth of U373 MG human glioblastoma cells in vitro. The IC50 for a 24-h exposure of U373 MG cells to APC in the presence of 50 units/ml of rabbit carboxylesterase was 0.27 +/- 0.08 microM, whereas APC alone demonstrated no inhibition of growth at concentrations up to 1 microM. The IC50 of U373 MG cells transfected with the cDNA encoding the rabbit carboxylesterase (U373pIRESrabbit) and exposed to APC for 24 h was 0.8 +/- 0.1 microM APC, whereas the growth of cells transfected with vector control (U373pIRES) was unaffected by up to 1 microM APC. Because APC is nontoxic to human cells, we are investigating the possibility of using APC/rabbit carboxylesterase in a prodrug/enzyme therapeutic approach.

Studies on the mechanism of resistance to rapamycin in human cancer cells.

Rapamycin is a potent cytostatic agent that arrests cells in the G1 phase of the cell cycle. The relationships between cellular sensitivity to rapamycin, drug accumulation, expression of mammalian target of rapamycin (mTOR), and inhibition of growth factor activation of ribosomal p70S6 kinase (p70(S6k)) and dephosphorylation of pH acid stable protein I (eukaryotic initiation factor 4E binding protein) were examined. We show that some cell lines derived from childhood tumors are highly sensitive to growth inhibition by rapamycin, whereas others have high intrinsic resistance (>1000-fold). Accumulation and retention of [14C]rapamycin were similar in sensitive and resistant cells, with all cells examined demonstrating a stable tight binding component. Western analysis showed levels of mTOR were similar in each cell line (<2-fold variation). The activity of p70(S6k), activated downstream of mTOR, was similar in four cell lines (range, 11.75-41. 8 pmol/2 x 10(6) cells/30 min), but activity was equally inhibited in cells that were highly resistant to rapamycin-induced growth arrest. Rapamycin equally inhibited serum-induced phosphorylation of pH acid stable protein I in Rh1 (intrinsically resistant) and sensitive Rh30 cells. In serum-fasted Rh30 and Rh1 cells, the addition of serum rapidly induced c-MYC (protein) levels. Rapamycin blocked induction in Rh30 cells but not in Rh1 cells. Serum-fasted Rh30/rapa10K cells, selected for high level acquired resistance to rapamycin, showed >/=10-fold increased c-MYC compared with Rh30. These results suggest that the ability of rapamycin to inhibit c-MYC induction correlates with intrinsic sensitivity, whereas failure of rapamycin to inhibit induction or overexpression of c-MYC correlates with intrinsic and acquired resistance, respectively.