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Experimental Therapeutics |
Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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400 mm3 (1.5% of body weight). Little or no overt toxicity of the repeated CPA treatment regimen was observed. The same CPA schedule was much less effective in inducing regression of 9L tumors that were not transduced with P450/P450 reductase. Repeated CPA treatment of mice bearing large, late-stage P450/P450 reductase-transduced tumors (9–16% of body weight) resulted in major (
95%) regression in 15 of 16 tumors, with tumor eradication observed in 2 cases. Although CPA resistance was found to emerge in the population of P450/P450 reductase-transduced tumors, this resistance primarily involved a loss of expression of the transduced P450 and/or P450 reductase gene, rather than development of intrinsic cellular resistance to the activated form of CPA. These findings demonstrate that repeated CPA treatment on a 6 day schedule can be highly effective when combined with P450/P450 reductase gene therapy and suggest that repeated transduction of tumors with prodrug-activation genes may be necessary to achieve tumor eradication and a sustained therapeutic response. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Another approach to improving the therapeutic efficiency of P450-based cancer gene therapy, investigated in the present study, involves the introduction of novel drug scheduling regimens to enhance the antitumor effect in an in vivo setting. The potential importance of drug scheduling for CPA-based cancer gene therapy is suggested by the short plasma elimination half-life values for CPA seen in some cancer patients (e.g., t1/2 of 1.5 h in children and young adults; Ref. 4
), which may limit the availability of CPA for intratumoral drug activation. The bioavailability of CPA and the isomeric ifosfamide may also be influenced by the ability of these drugs to induce their own metabolism in cancer patients (e.g., Refs. 13
, 14
) by induction of liver P450s that contribute to prodrug activation (15)
. Recent preclinical investigations of CPA scheduling reported by Folkman and colleagues demonstrated that the administration of moderate doses of CPA on a schedule of repeated drug treatment every 6 days without interruption is much more effective than conventional high-dose drug schedules, both in the case of drug-sensitive tumors and drug-resistant tumors (16)
. The improved efficacy of this schedule is a result of the sustained activity of CPA against endothelial cells lining the blood vessels feeding the tumor cells. This sustained antiangiogenic effect is not obtained using classic regimens (16)
, which use maximally tolerated doses of CPA, necessitating a relatively long drug-free period for recovery of bone marrow and other sensitive host tissues (17)
.
The present study investigates whether the effectiveness of CPA-based P450 gene therapy can be improved by implementing the 6 day repeated cycle of CPA administration introduced by Browder et al. (16) . Our findings reveal that this drug scheduling approach leads to long-term suppression of tumor growth, resulting in the eradication of midstage and a portion of late-stage tumors transduced with P450 and P450 reductase. Substantial regression of tumors that are very large at the beginning of drug treatment (up to 16% of the animal’s body weight) could also be achieved with remarkably low host toxicity. During the course of these studies, we observed the emergence of a population of P450/P450 reductase-transduced tumor cells that was resistant to CPA but not to activated CPA. We identified, as the mechanism of this resistance, the loss of the transduced P450 or P450 reductase gene, indicating a need for repeated transduction of tumors with P450/P450 reductase for effective gene therapy. Overall, our findings support the conclusion that the every-6 day schedule of CPA treatment (16) can be superior to classical CPA treatment regimens and demonstrate that this therapeutic regimen can be particularly effective when given in the context of P450/P450 reductase-based gene therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retroviral Transduction of 9L Gliosarcoma Cells with Human P450 and P450 Reductase cDNAs.
Cloning of CYP2B6 cDNA (18)
into the retroviral vector pBabe-puro and preparation of P450 2B6-expressing 9L gliosarcoma cells (9L/2B6 cells) by retroviral transduction were described previously (5)
. cDNA encoding hRed cloned into the EcoRI site of pUV1 (19)
was obtained from Dr. F. Gonzalez, National Cancer Institute. This cDNA was subcloned into the EcoRI site of the retroviral vector pWZL-Blast (obtained from Dr. D. White, Millenium Pharmaceuticals, Cambridge, MA). pWZL-Blast is based on the pBabe plasmid (20)
and encodes a blasticidin-resistance gene transcribed from the retroviral 3'-long terminal repeat. The presence of the correct ATG initiation codon in the cloned P450 reductase cDNA was verified by DNA sequencing.
Transfection of the ecotropic packaging cell line Bosc 23 (21)
with retroviral plasmid DNA encoding hRed, harvesting of the retroviral supernatant, and infection of 9L gliosarcoma cells transduced with CYP2B6 (9L/2B6 cells; Ref. 5
) were carried out as previously described (5)
. Pools of blasticidin-resistant cells were selected using 2.5 µg/ml blasticidin S-hydrochloride for 2 days. The resultant drug-resistant pools of 9L/2B6/hRed cells exhibited a 4- to 5-fold increase in P450 reductase-catalyzed cytochrome C reduction in isolated microsomes (
A550 nm measured at 30°C) compared with control transfectants. Measured CYP2B6 protein levels in the 9L-P450/Reductase cells were 20 pmol P450 protein/mg microsomal protein by Western blot analysis using a human lymphoblast-expressed CYP2B6 cDNA standard (Gentest, Inc., Woburn, MA). 9L cells transduced with pBabe-puro retrovirus without P450 (9L/pBabe control cells; Ref. 5
) were used as a control tumor cell line for direct comparisons to 9L-P450/Reductase cells. 9L/pBabe cells and the corresponding 9L/pBabe tumors used in these experiments are referred to as "9L control" or simply "9L" cells and tumors.
In Vivo Tumor Experiments.
9L gliosarcoma cells used for tumor implantation (9L-P450/Reductase or 9L control cells) were grown in DMEM containing 10% fetal bovine serum on 100-mm tissue culture dishes until nearly confluent. The cells were trypsinized and resuspended in DMEM without fetal bovine serum to a concentration of 8 x 106 cells/ml and were kept on ice until injection. Four-week-old (28–30 g) male ICR/Fox Chase mice, an outbred scid strain (Ref. 22
; Taconic Farms, Germantown, NY), were given injections of 4 x 106 tumor cells in a volume of 0.5 ml of serum-free DMEM using a 0.5-inch 29-gauge needle and a 1-ml insulin syringe (i.e., two tumors/mouse) at each posterior flank. CPA administration every 6 days (see below) or using an episodic schedule (as described in Ref. 5
), was initiated between 22 and 49 days after tumor implantation as specified in each experiment. CPA was solubilized in PBS at 4.8 mg/ml and kept at 37°C until injection to maintain drug solubility. Untreated control mice were used to monitor tumor doubling times in the absence of drug treatment, which were similar for 9L control and 9L-P450/Reductase tumors (see Table 1
). Autoclaved cages containing sterile food and water were changed twice each week in a laminar flow hood. Mice were housed in the Boston University Laboratory of Animal Care Facility in accordance with approved protocols. Mice were fed Prolab RMH 3000 5P00 diet, which provides 14, 26, and 60% calories from fat, protein, and carbohydrates, respectively. Dietary additives and medications (e.g., ondansetron and dexamethasone), used to alleviate gastrointestinal dysfunction and ameliorate weight loss (16)
, were not used in the present study. Tumor growth experiments were generally terminated when tumor volumes exceeded 11,000 mm3.
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) were treated with CPA by i.p. injection at 140 mg/kg BW, repeated every 6 days (CPA-6 day schedule). The first CPA injection was given 22 days after tumor cell implantation. This treatment corresponds to the schedule developed by Browder et al. (16)
for C57/BL6/J mice, except that the dose of CPA was decreased from 170 to 140 mg/kg/injection to take into account the greater chemosensitivity of the scid mouse host used in the present study [compare with the maximally tolerated dose of CPA, 265 mg/kg BW, when given as a single i.p. injection in the same scid mouse model (22)
]. CPA treatments were halted after 11 cycles of CPA injection, at which point none of the 9L-P450/Reductase tumors were detectable. CPA-6 day treatment was resumed 32 days later for all of the mice, at which time regrowth was detected in 4 of 8 tumors. CPA was given to some of the tumor-bearing mice in this study using the conventional schedule of 150 mg CPA/kg body weight/injection x 2 injections, spaced by a 24-h interval, as described previously (5)
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2200–2400 mm3 at the time of first drug treatment; 4–5 mice per group) were treated with CPA at 140 mg/kg every 6 days beginning 32–35 days after tumor implantation. Drug treatment was continued for a total of 16 CPA injections. In Study C, the tumors were grown to late-stage (4400–5300 mm3, corresponding to day 45–49 after tumor implantation) prior to beginning the CPA-6 day injection schedule. Mice in this study (n = 3–4/group) received a total of 14 CPA injections. In Study D, three of the late-stage 9L-P450/Reductase tumor-bearing mice that served as drug-free controls for Study A were treated with CPA at a fixed dose (8.4 mg of CPA/injection) every 6 days beginning on day 44 after tumor implantation when the mean tumor size was 7200 mm3 (n = 6) and the BW was 40 g. This fixed CPA dose was continued for a total of seven CPA injections without adjustment for the subsequent decrease in overall BW that accompanied tumor regression.
Analysis of Tumor Growth Rates.
Tumor growth was monitored in measurements taken twice a week using Vernier calipers (Manostat Corp., Switzerland) and tumor areas (length x width) were calculated. Tumor volumes were calculated from the measured tumor areas using the formula: volume = 
/6 (L x W)3/2, and in some cases are expressed as a percentage of initial BW (28 g) assuming a tumor density of 1.0 (23)
. Tumor growth delay data were analyzed as described previously (8)
, except that tumor volumes were used for the calculations in place of tumor areas. Tumor-doubling time was defined as the time in days required for the tumor volume to double from the time of first drug treatment. Tumor growth delay values were then calculated as the difference in tumor-doubling time between the drug-treated and control groups. Specific growth delay values were calculated as (T2 - T1)/T1, where T1 and T2 are times in days required for the control and the drug-treated tumors, respectively, to double in volume. The specific growth delay parameter provides an estimate of the number of doubling times by which tumor growth is delayed by drug treatment and enables comparisons of therapeutic responses between tumors that differ in their growth rates (24)
.
Tumor Excision and in Vitro Analysis of Chemosensitivity to CPA and 4-OOH-CPA.
Tumor-bearing mice were killed at the end of each experiment by cervical dislocation. Animals were sprayed with 70% ethanol around the vicinity of the tumor, and individual tumors were then explanted for characterization of the tumor cell population in tissue culture. One-half of each tumor was immediately frozen in liquid nitrogen, whereas the remaining portion was cut into small pieces in 0.5 ml of sterile-filtered DMEM containing collagenase type IV (4500 units/ml). Samples were incubated in sterile tubes for 5 min at room temperature with vigorous pipetting to help digest the tumor. Samples were allowed to stand without pipetting for an additional 3 min to precipitate the undigested tissue and the supernatants were then plated in 100-mm tissue culture dishes with 15 ml of DMEM containing 10% FBS. A total of 14 tumors were explanted using this protocol, each yielding a stable population of 9L tumor cells that could be propagated in cell culture. The efficiency of tumor excision from the CPA-6 day treated mice was 5- to 10-fold higher for 9L control than for 9L-P450/Reductase tumors, as judged by the yield of 9L cell colonies in cell culture.
To evaluate the chemosensitivity of the explanted 9L-P450/Reductase tumor cells, cells were plated in triplicate at 4000 cells/well of a 48-well plate 18–24 h prior to drug treatment. Cells were then treated with 0–1.5 mM CPA or 0–5 µM 4OOH-CPA, which spontaneously decomposes in aqueous media to yield the cytotoxic metabolite 4-OH-CPA. Cells were incubated for 4 days in a tissue culture incubator maintained under normoxic conditions (19.6% O2, 5% CO2, 75.4% N2). Cells remaining after this time were quantitated using a crystal violet-staining/alcohol-extraction assay (5) . Data are presented as cell number relative to drug-free controls, mean ± SD values for triplicate samples, unless indicated otherwise. Error bars not visible in the individual figures are too small to be seen.
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RESULTS |
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compares the antitumor effect of CPA administered at a moderate dose, repeated every 6 days, to standard high-dose CPA treatment for both 9L control (Fig. 1A)
and 9L-P450/Reductase tumors (Fig. 1B)
. The conventional high-dose CPA regimen [two injections totaling 300 mg/kg of BW over 24 h (5)
] induced a tumor growth delay of 20 days in the case of 9L-P450/Reductase tumors as compared with only 3 days in the case of 9L control tumors (Table 1)
. Repeated injection of CPA 3–4 weeks later using the same high dose prolongs the antitumor effect against 9L-P450/Reductase tumors, but does not lead to substantial tumor regression (5)
]. In contrast, treatment of the 9L-P450/Reductase tumors with CPA repeatedly each 6 days (140 mg CPA/kg of BW/injection) resulted in an almost immediate response to drug treatment, as evidenced by the cessation of tumor growth, generally within 3–7 days. This was followed by major tumor regression and an apparent elimination of all eight tumors after 50–55 days (9–10 CPA injections). Overall, the CPA-6 day treatment schedule effected a specific growth delay of >23 doubling times (>134 days) in the case of the 9L/P450/Reductase tumors (Table 1)
. By contrast, P450-deficient 9L control tumors treated with the same CPA-6 day schedule continued to grow without regression until after the third CPA injection (day 15–17; Table 2
, column 2). During this initial period of CPA-6 day treatment, 9L control tumors increased in volume by an additional 320% and did not exhibit any growth delay compared with drug-free controls (6.4 versus 5.9 days; Table 1
). A similar delay in the onset of CPA responsiveness of 9L control compared with 9L-P450/Reductase tumors was seen in two other experiments, where the tumors were up to 10-fold larger in volume at the time of initial drug treatment (Table 2
; Study B and Study C; also see Fig. 3A
and Fig. 4A
, below).
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. CPA-6 day-treated mice ate normally, in contrast to mice given the conventional high-dose CPA treatment, where a loss of appetite and weight loss frequently occurs for a period of several days following CPA administration (data not shown).
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20–25 days. Subsequently, 4 of the 8 9L-P450/Reductase tumors began to regrow, at which time a second series of CPA/6 day injections was initiated (day 92). This CPA retreatment led to either cessation of growth or regression of all 4 tumors, although 1 tumor resumed growth at day 128 (tumor 20L, Fig. 1B
, inset). Overall, 6 of the 8 tumors in this group regressed to the point where they were undetectable (Table 2
, last column). By contrast, a rapid resumption of 9L control tumor growth was observed shortly after CPA treatment was halted (day 68). This resulted in tumors 4700 ± 952 mm3 in volume (mean ± SD, n = 8) by the time the CPA-6 day injection regimen was resumed on day 92 (c.f., 93 ± 41 mm3 (n = 8) mean volume of 9L-P450/Reductase tumors at that same time point). Moreover, in contrast to mice bearing 9L-P450/Reductase tumors, which tolerated the second series of CPA-6 day injections fairly well, all 4 remaining mice in the 9L control group died after either the second (n = 1) or the third CPA injection in the series (n = 3).
Effect of CPA-6 day Schedule on Growth of Large Tumors.
The complete regression of midstage 9L-P450/Reductase tumors treated with the CPA-6 day schedule (Fig. 1B)
prompted us to investigate the effectiveness of this treatment regimen when applied to late-stage tumors. Because the doses of CPA are adjusted to the animal’s total weight (BW + tumor weight), we also investigated whether greater host toxicity would be apparent in animals with a larger tumor burden. 9L control and 9L-P450/Reductase tumors were grown to an average size of 2200–2400 mm3 (8–9% of initial body weight; Fig. 3
) or 4400–5300 mm3 (late-stage tumors; 16–19% of initial body weight; Fig. 4
), at which time CPA-6 day treatment was initiated. In both cases the 9L-P450/Reductase tumors responded to the CPA-6 day schedule more rapidly (within 4–7 days) and more completely than 9L control tumors. Of note, tumor regression was delayed (for up to 18 days) and was less extensive with the 9L control tumors, despite the fact that these mice received more total CPA than mice bearing 9L-P450/Reductase tumors, because of their greater tumor burden and hence larger BW (Table 2)
. A specific growth delay of >17 tumor doubling times was achieved with the 9L-P450/Reductase tumors but not the 9L control tumors (Table 3)
. Moreover, the 9L control tumors never disappeared completely (74–85% regression of control tumors versus 96–99% regression of 9L-P450/Reductase tumors; Study B and Study C, Table 2
). The dramatic reductions in tumor size seen with the late-stage tumors on the CPA-6 day schedule are reflected in the profiles of body weights, which had increased from 30 g to as high as 55 g in parallel to the increase in tumor burden (Fig. 4B)
. The substantial reduction in body weight in association with CPA-induced tumor regression evidences the dramatic antitumor effect of the CPA-6 day treatment regimen.
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; Table 2
(Study D)], 9L-P450/Reductase tumors were grown to a size of 7200 mm3 (25% of initial BW), at which time the mice were given a fixed dose of CPA, 8.4 mg CPA/mouse (corresponding to an initial CPA dose of 210 mg/kg) repeated every 6 days for a total of 7 injections. This drug treatment regimen was highly effective, with major tumor regression (93% decrease in tumor volume) seen in 6 of 6 tumors. However, the total CPA dose (59 mg/mouse over the course of 36 days) resulted in cumulative toxicity and lethality to all of the treated mice.
CPA-6 day Schedule Does Not Promote Intrinsic Cellular Resistance to Activated CPA.
Although initially highly responsive to CPA-6 day treatment, 9L-P450/Reductase tumors from the late-stage tumor experiments eventually became resistant to repeated CPA injection (Figs. 3
and 4
). Similarly, an apparent resistance to CPA was seen in 1 of 8 midstage tumors (Fig. 1B
, inset; tumor 20L). The cause of this apparent resistance to CPA was investigated using tumor cells explanted from 9L-P450/Reductase tumor 20L. Fig. 5
shows that the explanted tumor cell population was rather insensitive to CPA in culture, in contrast to the original 9L-P450/Reductase tumor cell line (panel A). Treatment of the cells with chemically activated CPA (i.e., 4OOH-CPA) induced comparable killing of the tumor 20L explanted cells and the original 9L-P450/Reductase cells (Fig. 5B)
. Thus, the insensitivity of the explanted tumor cells to CPA is not attributable to an intrinsic resistance to activated CPA, but results from the inability of these tumor cells to activate CPA efficiently. Further investigation revealed that the explanted tumor cells no longer express the transduced P450 reductase gene, as indicated by the decrease in cytochrome C reduction rates back to the level of 9L controls (Fig. 5C)
and by the loss of resistance to blasticidin, a selection marker for the transduced P450 reductase gene (panel D). A partial (60%) loss of resistance to puromycin, selection marker for the transduced P450 2B6 gene, was also seen (Fig. 5D)
, and correspondingly, there was a detectable decrease in expression of P450 2B6 protein (Fig. 6A
; Lane 10 versus Lane 2). These findings are consistent with the reduced CPA sensitivity of the explanted tumor 20L cells shown in Fig. 5A
.
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. This loss of CPA sensitivity was associated with the loss of the transduced P450 reductase, as judged by the decrease in cytochrome C reductase activity back to the level of the 9L cell controls (Fig. 6C)
. Whereas a loss of P450 2B6 protein was apparent in cells derived from two tumors (43R and 64R; Fig. 6A
), all of the other excised tumor cells retained a level of P450 2B6 protein comparable with that found in the original 9L-P450/Reductase cells. One exception was tumor 66L, in which P450 2B6 protein levels were inexplicably elevated several-fold. When challenged with chemically activated CPA, all of the tumor cells except those derived from tumors 42R and 66L showed strong intrinsic drug sensitivity (Fig. 6B)
, excluding intrinsic cellular resistance to the activated CPA metabolite as the mechanism of CPA resistance in vivo. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When using conventional CPA scheduling, which comprises an initial treatment with high doses of CPA, a tumor growth delay of 25 days is observed in mice using the present P450/P450 reductase gene therapy model (5
, 7)
. Repeated CPA injection after a 3–4 week recovery period prolongs the period of tumor growth delay but does not eliminate the tumors (5)
. Some improvement can be obtained by cotreatment with the antithyroid drug methimazole, which selectively inhibits hepatic P450-catalyzed prodrug activation (8)
, or by combination of CPA with the bioreductive drug tirapazamine (7)
; however, large-scale tumor regression leading to tumor eradication has not been achieved. By contrast, combination of the CPA-6 day scheduling approach of Browder et al. (16)
with P450/P450 reductase gene therapy induced major tumor regression, even in the case of tumors that were very large (up to 16% of BW) at the time of initial CPA treatment (Table 2)
. The antitumor effects of this CPA-6 day schedule were much less extensive in the case of 9L tumors that were not transduced with P450 and P450 reductase. Moreover, whereas P450/P450 reductase-transduced tumors generally responded within a few days to the CPA-6 day treatment regimen, as indicated by the onset of tumor regression, control 9L cells required up to 18 days to respond to the drug, during which time a doubling or tripling of tumor volumes occurred. Moreover, the subsequent regression of the P450-deficient tumors that ultimately did result was not nearly as dramatic and complete as that of the 9L-P450/Reductase tumors (Table 2)
. Although some of the P450-expressing tumors did begin to regrow when CPA treatment was halted, the majority of these tumors were still intrinsically responsive to a second series of CPA-6 day injections. The potential clinical utility of the CPA-6 day schedule is supported by the apparent absence of drug toxicity during the overall period of CPA treatment (up to 130 days), as judged by monitoring food consumption and by the absence of substantial weight loss during the course of the study.
Continued CPA-6 day treatment was associated with an apparent development of resistance to CPA and significant regrowth of a portion of the P450/P450 reductase-containing tumors examined in this study. This resistance resulted from a loss of the transduced P450 and/or P450 reductase gene and, hence, the capacity to activate CPA, rather than de novo acquisition of intrinsic resistance to activated CPA. Presumably, this reflects the selection and regrowth of a population of tumor cells that has spontaneously lost the drug-sensitivity P450/P450 reductase genes under selective pressure of CPA treatment. Loss of either P450 or P450 reductase gene expression may occur with greater frequency in large tumors and would be expected to lead to a decrease in CPA sensitivity, in the latter instance because the endogenous level of P450 reductase, which is found in 9L and many other tumor cells, is insufficient to fully support efficient metabolism by the transduced P450 gene (6) . In the present study, the P450 reductase-expressing 9L tumor cells were derived from a pool of retrovirally transduced cells; and, accordingly, P450 reductase-negative tumor cells were apparently selected under CPA selection pressure. Accordingly, repeated transduction of tumors with a gene therapy vector may be required to achieve tumor eradication using this and probably other prodrug activation-based cancer gene therapies.
The mechanism for the apparent development of CPA resistance seen in the case of 9L control tumors is uncertain. In vitro culture of 9L cells excised from the CPA-6 day-treated mice indicated that the majority of these tumor cells retained a sensitivity to activated CPA similar to that of the initial 9L tumor cell line (data not shown). Conceivably, these cells could exhibit in vivo CPA resistance by a mechanism analogous to the acquired multicellular resistance/tumor compaction mechanism described elsewhere, which is manifest only in an in vivo setting, in which the tumor adapts and becomes less responsive to activated CPA produced in the liver (25, 26, 27)
. Alternatively, although not seen in other studies (28)
, endothelial cells lining the tumor vasculature could potentially develop intrinsic resistance to CPA, e.g., by overexpression of aldehyde dehydrogenase, which inactivates the active CPA metabolite, 4-OH-CPA (4)
. Our inability to eradicate nontransduced 9L gliosarcoma tumors in scid mice contrasts with the findings of Browder et al. (16)
, who found that CPA-6 day scheduling led to the eradication of Lewis lung carcinomas grown as solid tumors in C57Bl6/J mice (16)
. This difference may be explained by contributions of the immune system to tumor surveillance and its active participation in the elimination of apoptotic Lewis lung carcinoma cells in the earlier studies, performed using immunocompetent C57/Bl/6J mice (compare Ref. 29
). This immune surveillance mechanism would not be operative in our study, in which immunodeficient scid mice were used. Other possibilities include the use of a lower CPA dose in our experiments (140 mg/kg of BW/injection in the present study versus 170 mg/kg of BW/injection by Browder et al.) and the fact that the Lewis lung tumors in the prior study were very-early-stage tumors (0.1 cm3) and presumably more sensitive to antiangiogenic CPA scheduling at the time of the first CPA treatment (compare initial 9L control tumor sizes ranging from 0.9 cm3 to 5 cm3 in the present study). Intrinsic differences in the CPA sensitivity of 9L gliosarcoma as compared with Lewis lung carcinoma, which is known to secrete angiostatin (30)
, could also play a role.
The precise mechanism whereby P450-expressing tumors dramatically regress in response to the CPA-6 day schedule is not known. In the case of Lewis lung tumors (which were not transduced with a P450 gene), CPA-6 day treatment induces apoptosis of endothelial cells lining the tumor vasculature as a primary response, followed by tumor cell apoptosis after an 3.5-day delay (16)
. Although we did not measure apoptosis directly in the present study, a substantial delay in the apoptosis of P450-deficient 9L tumor cells is strongly indicated by the continued growth of 9L control tumors for up to 18 days after the initiation of CPA-6 day treatment (Table 2)
. This delay suggests that the antiangiogenic mechanism described by Browder et al. (16)
may also be operative in our P450-deficient 9L control tumors. This supposition is further supported by the rapid regrowth of the 9L control tumors after the cessation of CPA-6 day treatment (Fig. 1)
, which is consistent with the residual tumor cells being intrinsically viable but restricted from rapid growth by the antiangiogenic effects of the CPA-6 day schedule. By contrast, 9L-P450/Reductase tumors typically began to regress prior to the second CPA injection on day 6, strongly suggesting a direct cytotoxic response to CPA that was activated locally within the tumor. This direct cytotoxic effect of the CPA-6 day treatment could conceivably be magnified by an endothelial cell-directed, antiangiogenic mechanism analogous to that documented by Browder et al. in their studies of Lewis lung tumors using the same drug schedule (16)
. Although antiangiogenic CPA therapy could potentially compromise a prodrug activation-based gene therapy strategy by inhibiting the efficient entry of prodrug into the solid-tumor mass, the repeated generation of even low levels of activated drug within the tumor by P450 metabolism might be sufficient to kill tumor cells weakened as a consequence of an antiangiogenic drug regimen. We have not, however, directly examined the occurrence of such an antiangiogenic response in the present study, in which CPA-induced apoptosis of the P450-expressing tumor cells is likely to obscure any enhanced apoptosis that may occur in the endothelial cells lining the tumor vasculature as a consequence of localized CPA activation by adjacent P450-containing tumor cells. Indeed, Browder et al. (16)
were only able to visualize and characterize CPA-6 day-induced endothelial cell apoptosis by studying CPA-resistant tumors, for which the background level of drug-induced tumor cell apoptosis is low. Further study will be required to clarify this point, as well as to determine whether enhanced antiangiogenic and antitumor responses can be achieved by targeting CPA-activating P450 genes directly to tumor endothelial cells. Other strategies to be investigated include combination of CPA with antiangiogenesis inhibitors (16
, 31
, 32)
, development of antiangiogenic schedules for other anticancer prodrugs or prodrug combinations (c.f., Ref. 33
), and the design of antiangiogenic schedules for use with CPA in the clinic, where CPA has a substantially longer plasma half-life (up to 12 h in adults) than in the mouse (t1/2, 30 min; Ref. 4
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grant CA49248 (to D. J. W.). Y. J. received fellowship support from the Ernst Schering Research Foundation. 
2 To whom requests for reprints should be addressed, at Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215. Fax: (617) 353-7404; E-mail: djw{at}bu.edu
3 The abbreviations used are: P450 or CYP, cytochrome P-450; CPA, cyclophosphamide; 4-OH-CPA, 4-hydroxy-CPA; 4OOH-CPA, 4-hydroperoxy-CPA; CPA-6 day, CPA administered repeatedly every 6 days; hRed, human P450 reductase; 9L-P450/Reductase or 9L/2B6/hRed, rat 9L gliosarcoma cells transduced with retrovirus encoding P450 2B6 and with retrovirus encoding hRed; 9L control, 9L cells transduced with the control retroviral vector pBabe-puro; BW, body weight; scid, severe combined immunodeficient.
Received 10/11/00. Accepted 3/28/01.
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 C.-S. Chen, Y. Jounaidi, and D. J. Waxman ENANTIOSELECTIVE METABOLISM AND CYTOTOXICITY OF R-IFOSFAMIDE AND S-IFOSFAMIDE BY TUMOR CELL-EXPRESSED CYTOCHROMES P450 Drug Metab. Dispos., September 1, 2005; 33(9): 1261 - 1267. [Abstract] [Full Text] [PDF]
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D. S. Riddick, C. Lee, S. Ramji, E. C. Chinje, R. L. Cowen, K. J. Williams, A. V. Patterson, I. J. Stratford, C. S. Morrow, A. J. Townsend, et al. CANCER CHEMOTHERAPY AND DRUG METABOLISM Drug Metab. Dispos., August 1, 2005; 33(8): 1083 - 1096. [Abstract] [Full Text] [PDF]
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 M. A. Rieger, R. Ebner, D. R. Bell, A. Kiessling, J. Rohayem, M. Schmitz, A. Temme, E. P. Rieber, and B. Weigle Identification of a Novel Mammary-Restricted Cytochrome P450, CYP4Z1, with Overexpression in Breast Carcinoma Cancer Res., April 1, 2004; 64(7): 2357 - 2364. [Abstract] [Full Text] [PDF]
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Y. Jounaidi and D. J. Waxman Use of Replication-Conditional Adenovirus as a Helper System to Enhance Delivery of P450 Prodrug-Activation Genes for Cancer Therapy Cancer Res., January 1, 2004; 64(1): 292 - 303. [Abstract] [Full Text] [PDF]
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D. J. Waxman and P. S. Schwartz Harnessing Apoptosis for Improved Anticancer Gene Therapy Cancer Res., December 15, 2003; 63(24): 8563 - 8572. [Abstract] [Full Text] [PDF]
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P. S. Schwartz, C.-S. Chen, and D. J. Waxman Enhanced Bystander Cytotoxicity of P450 Gene-directed Enzyme Prodrug Therapy by Expression of the Antiapoptotic Factor p35 Cancer Res., December 1, 2002; 62(23): 6928 - 6937. [Abstract] [Full Text] [PDF]
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S. Man, G. Bocci, G. Francia, S. K. Green, S. Jothy, D. Hanahan, P. Bohlen, D. J. Hicklin, G. Bergers, and R. S. Kerbel Antitumor Effects in Mice of Low-dose (Metronomic) Cyclophosphamide Administered Continuously through the Drinking Water Cancer Res., May 1, 2002; 62(10): 2731 - 2735. [Abstract] [Full Text] [PDF]
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 R. S. Kerbel, G. Klement, K. I. Pritchard, and B. Kamen Continuous low-dose anti-angiogenic/metronomic chemotherapy: from the research laboratory into the oncology clinic Ann. Onc., January 19, 2002; 13(1): 12 - 15. [Full Text] [PDF]
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G. Klement, P. Huang, B. Mayer, S. K. Green, S. Man, P. Bohlen, D. Hicklin, and R. S. Kerbel Differences in Therapeutic Indexes of Combination Metronomic Chemotherapy and an Anti-VEGFR-2 Antibody in Multidrug-resistant Human Breast Cancer Xenografts Clin. Cancer Res., January 1, 2002; 8(1): 221 - 232. [Abstract] [Full Text] [PDF]
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