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Tumor Uptake and Elimination of 2',2'-Difluoro-2'-deoxycytidine (Gemcitabine) after Deoxycytidine Kinase Gene Transfer

Correlation with in Vivo Tumor Response¹

Departments of Radiation Oncology [A. W . B., S. G. S., S. M. H.] and Division of Medical Oncology [A. W. B., L. D. C.], Wake Forest University School of Medicine, Winston-Salem, North Carolina 27156; and Departments of Surgery [H. L.], Radiation Oncology [J. E. T.], Radiology [S. K. M.], and Medicine [B. S. M.], University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS HPLC-determined Effects of dCK... F-19 MRS-determined Effects of... Effects of dFdCyd on... DISCUSSION REFERENCES

 
Purpose: We hypothesized that tumor uptake and elimination of 2',2'-difluoro-2'-deoxycytidine/2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (dFdCyd/dFdCTP) would be altered after dCK gene transfer and that this change would result in an enhanced cytotoxic effect. To test this hypothesis, we examined dFdCyd/dFdCTP uptake and clearance in HT-29 human colon carcinoma xenografts in nude mice by high-performance liquid chromatography (HPLC) and fluorine-19 magnetic resonance spectroscopy (F-19 MRS).

Experimental Design: HT-29 tumors were grown from cells infected with either the retroviral vector alone (LNPO-LacZ) or vector containing the dCK gene (LNPO-dCK). HPLC and F-19 MRS analyses were performed after a single 160 mg/kg i.p. injection of dFdCyd. Tumor response was determined in animals receiving a similar dosing schedule of dFdCyd.

Results: HPLC experiments revealed an increased tumor accumulation of dFdCTP in xenografts overexpressing dCK compared with wild-type controls (P 0.05). dFdCTP in the dCK-infected tumors was easily identified at 24 h postinjection. Conversely, no dFdCTP could be detected in the control xenografts 14 h postinjection. Subsequent F-19 MRS experiments confirmed an altered uptake, revealing a 2.5-fold greater accumulation of dFdCyd/dFdCTP in the dCK xenografts. Whereas a modest tumor growth delay was observed in the wild-type tumors receiving dFdCyd, dCK xenografts demonstrated a marked tumor growth delay following treatment (P 0.05).

Conclusions: These data support the hypothesis that increased expression of dCK cDNA in HT-29 xenografts results in an enhanced dFdCTP accumulation and prolonged elimination kinetics, and ultimately a potentiated in vivo tumor response to dFdCyd. Related to these effects, changes in the overall tumor metabolism of dFdCyd/dFdCTP was detectable by noninvasive F-19 MRS. These data are relevant to future preclinical and clinical studies evaluating dCK gene transfer and dFdCyd therapy.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS HPLC-determined Effects of dCK... F-19 MRS-determined Effects of... Effects of dFdCyd on... DISCUSSION REFERENCES

 
Gemcitabine (dFdCyd)³ is a cytotoxic dCyd analogue that has demonstrated significant antitumor activity in a number of human tumor models (1, 2, 3, 4) . Several Phase I and II clinical trials have reported encouraging results using a variety of infusion schedules and dosing schemes (5, 6, 7, 8) . dFdCyd requires conversion to an active metabolite by phosphorylation to a triphosphate derivative, dFdCTP, which is then incorporated into DNA. dCK is the rate-limiting enzyme in this phosphorylation. In vitro studies involving the retroviral transfer of the dCK gene, reported by Hapke et al. (9) , demonstrated the significance of dCK activity on HT-29 tumor cell sensitivity to dFdCyd and other nucleoside analogues. As reported, HT-29 cells stably infected with LNPO-Lac Z had dCK activity equivalent to that of uninfected cells. In contrast, the mean increase in dCK activity for cells infected with LNPO-dCK was 2.3-fold. The dCK levels in these cells correlated with both message and protein levels. A central limitation in most in vivo gene therapy studies is the difficulty in monitoring the efficacy of gene delivery, as well as gene function. Although in vitro studies using Northern and Western blot analysis can demonstrate significant increases in dCK mRNA and protein levels, respectively, after the retroviral transfer of dCK, direct evidence that the gene has altered the metabolic fate of dFdCyd and whether this translates into a greater antitumor effect in vivo needs to be explored.

In our experiments, we attempted to use HPLC and F-19 MRS to monitor the tumor anabolism of dFdCyd. If the tumor conversion of dFdCyd to its active metabolite, dFdCTP, is altered in tumors overexpressing the dCK gene, as we and others have postulated, F-19 MRS should provide a noninvasive method to identify these changes in situ. Although F-19 MRS has provided unique insights into the metabolic fate of 5-fluorouracil in vitro and in vivo, there are only limited reports using this tool in dFdCyd studies, and none of these were in the setting of gene-directed therapies (10 , 11) . In addition to providing pharmacological information, the F-19 MRS findings, at least in this setting, could also provide indirect information regarding the relative efficacy of the dCK gene delivery and function.

This report describes experiments performed in the HT-29 xenograft model to determine (a) whether increased dCK activity, mediated by dCK gene transfer, results in enhanced tumor catabolism of dFdCyd to dFdCTP, as measured by standard HPLC techniques; (b) whether the observed changes in dFdCyd metabolism can be detected by F-19 MRS; and (c) whether the anticipated enhanced conversion of dFdCyd to the active metabolite dFdCTP results in an improved dFdCyd antitumor effect.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS HPLC-determined Effects of dCK... F-19 MRS-determined Effects of... Effects of dFdCyd on... DISCUSSION REFERENCES

 
Cells and Cell Culture.
dFdCyd and dFdCTP were obtained from Eli Lilly and Co. (Indianapolis, IN). The HT-29 colon carcinoma cells were cultured as described previously (12) . HT-29-LNPO-LacZ and LNPO-dCK cell lines after retrovirus transfer of the dCK cDNA were obtained from the laboratory of Dr. Beverly Mitchell (University of North Carolina at Chapel Hill, Chapel Hill, NC) and maintained as described previously (9) .

Animal Model.
Experiments were performed in 6–10-week-old female athymic (nude) mice (Harlan Sprague Dawley, Inc., Madison, WI) maintained in the Division of Laboratory Animal Medicine at University of North Carolina at Chapel Hill. Each animal (average body weight, 25 g) recieved an s.c. injection, in the flank, containing 1 x 10⁷ wild-type HT-29 cells, HT-29 cells infected with the retroviral vector (LNPO-Lac-Z) only, or HT-29 cells infected with retrovirus containing dCK (LNPO-dCK) for the experiments outlined (9) .

HPLC Analysis of dNTPs.
Tumor dFdCTP and dNTPs were extracted and analyzed by a previously described method that had been modified for use with solid tumors (13) . Briefly, frozen tumors were weighed and then homogenized (15–20 strokes) in a 10-ml Potter-Elvehjem tissue homogenizer containing 1.0 ml of ice-cold 0.2 N HClO₄ and 50 µl of a 5.0 x 10^-5 M stock of dITP as an internal standard. The aqueous phase was decanted into a microcentrifuge tube and then centrifuged at 4°C for 10 min at 15,000 rpm. The supernatant was decanted into a fresh microcentrifuge tube containing 15 µl of 10 N NaOH and centrifuged again for 10 min at 15,000 rpm. The supernatant (0.9 ml) was transferred to a fresh microcentrifuge tube containing 50 µl of 1 M NH₄HCO₃ (pH 8.95) and 50 µl of 0.3 M MgCl₂. To remove ribonucleotide 5'-triphosphates, the aqueous extract was loaded on a column [Bio-Gel 601 boronate affinity gel (Bio-Rad Laboratories); 8 x 17 mm in a 3-ml plastic syringe] and eluted with 2.0 ml of 0.05 M NH₄HCO₃-0.015 M MgCl₂. An aliquot (1.85 ml) of the eluate was transferred to a test tube and acidified with 150 µl of 1 N HCl. The dFdCTP and dNTP in the 2.0 ml of acidified eluate were quantitated by HPLC methods on a Beckman 126 dual pump gradient system with a Beckman 166 UV detector. The dFdCTP, dNTPs, and residual ribonucleotide 5'-triphosphates were separated on a Whatman Partisphere SAX anion-exchange column (4.6 x 250 mm; 5-µm particle size, 120 Å pore size) with a linear gradient of NH₄H₂PO₄ (pH 3.35; 0.15–0.60 M) over 75 min followed by isocratic elution with 0.60 M NH₄H₂PO₄ for 10 min. The dFdCTP and dNTPs were detected at 272 nm and quantitated against the dITP internal standard with a detection limit of 5 pmol for dFdCTP. The amounts of dFdCTP and dNTPs in the tumors were normalized to the weights of the extracted tumors.

F-19 MRS.
The spectroscopy experiments were performed 3 weeks after inoculation of the tumor cells. Prior to all spectroscopy experiments, mice were anesthetized with an i.p. injection of ketamine at 100 mg/kg of body weight to allow for immobilization and proper placement within the magnet (12) . Tumor-bearing mice (5, 6, 7) with comparably sized tumors (5–7 mm in diameter) were pooled in each group for MRS analysis. Each animal received a single i.p. bolus injection of dFdCyd at a dose of 160 mg/kg of body weight. This dFdCyd dose was selected based on data from Boven et al. (1) and has been shown to be a well-tolerated, nonlethal dose in similar mouse model systems. This dose also maximizes the signal-to-noise ratio for the MRS experiments. No fluorine signal was detected when the coil was placed over the opposite (nontumor-bearing) flank at the time of the dFdCyd injection.

All animals were placed in a 20-cm horizontal bore spectroscopy system (2.0 T; Otsuka Imaging, Fort Collins, CO) with the magnet tuned to fluorine (81.3 MHz) and a one-turn home-built surface coil placed over the tumor. Immediately after the i.p. administration of dFdCyd, F-19 spectra were acquired using a repetition time of 1.5 s and a sweep width of 20 KHz; a total of 1200 transients were collected per animal study. F-19 MRS acquisitions were routinely collected at 30-min intervals and carried out for up to 48 h. The spectra were processed using a left-shift on the free induction decay curve to eliminate the first two points because of a direct current frequency transient that introduced a rolling baseline in the Fourier transform of the free induction decay. A 40-Hz exponential multiplier was then applied, and a Fourier transform was performed to obtain the F-19 spectra. Zero-order phase and baseline corrections were applied to obtain the final F-19 spectrum. Peak areas were determined from the phased and baseline-corrected spectra by use of a peak-area analysis program that assumes a Gaussian shape for the resonance peak (NMR1; New Methods, Inc.; Syracuse, NY). The relative fluorine content was determined by use of a 5-fluorouracil standard placed on the coil and measurement of the area of the dFdCyd peak in the F-19 spectra for comparison. The 5-fluorouracil standard is the concentration unit applied for quantifying the relative concentrations of intratumor fluorine in equally sized tumors. Experiments at mM concentrations reported by Edzes et al. and others (10 , 11) have indicated that the fluorine signals for the parent compound dFdCyd and the two major metabolites dFdCTP and dFdUrd are overlapping. Hence, only the relative contributions from these three compounds could be distinguished in these experiments.

In Vivo Tumor Response.
Tumor size was determined twice weekly by caliper measurements, and tumor volumes were calculated as follows: volume = (a x b²)/2, where a and b are orthogonal tumor diameters. Data are expressed as the ratio of the tumor volume at varying times after treatment compared with the day treatment was started (day 10). All tumors measured 2–3 mm in diameter at the start of all experiments. The dFdCyd dosing was identical to that described for the MRS experiments, except that the animals received a second i.p. injection of dFdCyd given 3 days (day 4) after the initial dose. This dFdCyd dose and dosing schedule had demonstrated predictable antitumor effects in prior studies performed in our laboratory (14) .

Statistical Analysis.
To analyze dFdCTP accumulation and tumor growth curves, we used a repeated-measures ANOVA model with a serial correlation structure. The transformation log (dFdCTP + 0.5) was used to stabilize the variances and increase adherence to the assumptions of the ANOVA model. For the in vivo tumor-response experiments, we compared the relative tumor sizes on the last day of observation (day 21) using the Wilcoxon rank-sum test. P 0.05 was considered statistically significant.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS HPLC-determined Effects of dCK... F-19 MRS-determined Effects of... Effects of dFdCyd on... DISCUSSION REFERENCES

 
The in vitro histological and growth characteristics (population doubling time) for the HT-29 wild-type, HT-29-LNPO-LacZ (vector only), and HT-29-LNPO-dCK cell lines were indistinguishable. With the exception of the HPLC experiments, all other experiments were performed in all three cell lines, and in each case, no difference in drug accumulation or tumor growth kinetics could be detected among the HT-29 wild-type, HT-29-LNPO-LacZ, or the HT-29-LNPO-LacZ systems. The results reported, therefore, will focus on observed differences between the HT-29 wild-type and the HT-29-LNPO-dCK cell lines.

	HPLC-determined Effects of dCK Expression on dFdCTP Levels.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS HPLC-determined Effects of dCK... F-19 MRS-determined Effects of... Effects of dFdCyd on... DISCUSSION REFERENCES

 
dFdCTP levels in the HT-29 wild-type tumors gradually increased during the first 4 h post i.p. injection of dFdCyd and gradually decreased until undetectable after 14 h (Fig. 1) . The dFdCTP levels also increased following the i.p. injection in the HT-29-LNPO-dCK tumors, but at a greatly increased rate: the maximum dFdCTP levels detected at 2 h were 4-fold higher than those observed in the HT-29 wild-type tumors. This observed difference in accumulation and retention of dFdCTP was statistically significant (P = 0.0023). In contrast to the HT-29 wild-type tumors, dFdCTP levels were clearly detectable 24 h after the injection in all HT-29-LNPO-dCK tumors.

View larger version (14K): [in this window] [in a new window]   Fig. 1. dFdCTP levels detected in HT-29 wild-type (•) and HT-29-LNPO-dCK xenografts (). The dFdCTP levels (nmoles/g of tumor) are significantly greater in the xenografts overexpressing dCK compared with the controls (P 0.05). The dFdCTP levels also persisted beyond 24 h in the HT-29-LNPO-dCK xenografts. Each data point reflects the mean dFdCTP level detected in three to six tumors. Bars, SE.

	F-19 MRS-determined Effects of dCK Expression on dFdCyd Pharmacokinetics.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS HPLC-determined Effects of dCK... F-19 MRS-determined Effects of... Effects of dFdCyd on... DISCUSSION REFERENCES

View larger version (22K): [in this window] [in a new window]   Fig. 2. dFdCTP levels detected in HT-29 wild-type () and HT-29-LNPO-dCK (•) xenografts. The fluorine signal is significantly greater in the xenografts overexpressing dCK compared with the controls. The dFdCyd fluorine signal persisted beyond 36 h in the HT-29-LNPO-dCK xenografts, but was undetectable in the HT-29 wild-type xenografts after 14 h. Each data point reflects the mean fluorine signal detected in three to six tumors. Bars, SE.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Representative F-19 spectra demonstrating the increased retention of fluorine signal observed in the HT-29-LNPO-dCK tumors overexpressing dCK (middle) versus the HT-29 wild-type control tumor (left) and HT-29-LNPO-Lac Z tumors (right). Each series of spectra reflects the fluorine signal collected over time (hours). The shaded peak at 0.0 p.p.m. is the fluorine signal from the fluorouracil control. The second peak upfield from the control peak represents fluorine signal from intratumor dFdCyd.

	Effects of dFdCyd on Tumor Growth in HT-29 Tumors.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS HPLC-determined Effects of dCK... F-19 MRS-determined Effects of... Effects of dFdCyd on... DISCUSSION REFERENCES

View larger version (21K): [in this window] [in a new window]   Fig. 4. Effect of dCK gene expression on the response of HT-29 human tumor xenografts to dFdCyd. Tumor growth curves for untreated mice bearing HT-29 wild-type control () and HT-29-LNPO-dCK () demonstrate no difference in the growth kinetics for the two xenografts. Growth curves for HT-29 wild-type (•) and HT-29-LNPO-dCK xenografts receiving 160 mg/kg of dFdCyd () i.p. on days 1 and 4 are also shown. The difference in the tumor volumes for the treated HT-29 wild-type versus the HT-29-LNPO-dCK xenografts on day 21 was statistical (P 0.03). Each data point reflects the mean of five to seven xenografts per treatment group. Bars, SE.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS HPLC-determined Effects of dCK... F-19 MRS-determined Effects of... Effects of dFdCyd on... DISCUSSION REFERENCES

As shown in Fig. 2 , the maximum accumulation of dFdCTP, as determined in our HPLC experiments, occurred at 2–4 h following the i.p. injection of dFdCyd. This was true in both the control xenografts and tumors overexpressing dCK. A 4-fold increase in the initial dFdCTP level was detected in the HT-29-LNPO-dCK-infected tumors compared with the control tumors. Our F-19 MRS results were consistent with our HPLC observations; the fluorine signal reached a maximum between 1 and 3 h postinjection, and tumors overexpressing dCK displayed an 4-fold increase in fluorine signal compared with the control tumors. Because the fluorine signals for dFdCyd, its three phosphorylated metabolites, and dFdUrd overlap at a field strength of 2.0 tesla, the fluorine signal detected in our F-19 MRS experiments is not specific for dFdCTP, which prevents a detailed correlation of the data. A similar observation of a rapid accumulation of dFdCyd has been reported by Krisjansen et al. (11) , who used F-19 MRS in an in vivo model of two human lung cancer cell lines. The authors observed peak intratumor signals within 50–70 min after the i.p. injection of dFdCyd, and the elimination of fluorine followed first-order kinetics. Our HPLC results, when reviewed in concert with the F-19 MRS data, support the postulate that the initial accumulation of luorine signal is, to some extent, related to the enhanced anabolism of dFdCyd to dFdCTP in the HT-29-LNPO-dCK xenografts. This concept is supported by data reported by Heinemann et al. (22) for in vitro experiments in CEM cells incubated with increasing concentrations of dFdCyd. HPLC analysis of the intracellular metabolites revealed that the predominant metabolite at 0–2 h. posttreatment was dFdCTP; dFdCTP comprised >75% of the total metabolites detected during the interval of accumulation. In vivo data in support of the in vitro results of Heinemann et al. (22) come from Ruiz van Haperen et al. (4) : peak dFdCTP tumor concentrations were observed between 0 and 240 min in WiDR xenografts receiving an i.p. injection of 120 mg/kg dFdCyd. Our HPLC and F-19 MRS analyses revealed similar results. These data taken together support our conclusion that the origin of the increased fluorine signal observed during our F-19 MRS experiments resulted, in part, from the enhanced catabolism of dFdCyd to dFdCTP.

It is unlikely that the changes in the observed fluorine signal are related to intracellular dFdCyd levels per se. Heinemann et al. (23) demonstrated in an in vitro system that the accumulation and elimination of dFdCyd are not concentration dependent, whereas the terminal t_1/2 of dFdCTP increased with increasing concentrations: 5, 16, and 43 h for 0.3, 1.0, and 10 µM dFdCyd, respectively. The prolonged retention of dFdCyd/dFdCTP observed in our F-19 MRS and HPLC experiments may also reflect the self-potentiation effect reported with increased intracellular dFdCTP levels. Inactivation and elimination of dFdCTP occur primarily through its dephosphorylation to difluoro-dCMP followed by deamination to difluoro-dUMP by deoxycytidine deaminase and then phosphorylation to dFdUrd; high dFdCTP levels inhibit deoxycytidine deaminase, resulting in dFdCyd as the major metabolite (22) . It has been observed in in vitro systems that higher intracellular concentrations of dFdCTP inhibit this deamination process, further reducing the catabolism of dFdCTP and "self-potentiating" the activity of dFdCyd (22) . Additional data from Heinemann et al. (24) , who investigated the kinetics of metabolite formation in the medium after a 2-h exposure of CEM cells to dFdCyd, suggest that the extracellular catabolites do not contribute significantly to the observed signal. The predominant dFdCyd metabolites observed were dFdUrd and dFdCyd; dFdCTP is dephosphorylated at high concentrations to dFdCyd. Interestingly, the maximum accumulation and plateau values for both catabolites were not reached until 4 h after exposure and remained in plateau for an additional 8 h. Although it is not clear how relevant these in vitro results are to our tumor xenograft observations, these data would at least suggest that the fluorine signal detected in our F-19 MRS experiments probably did not originate from these extracellular metabolites.

Recent work by Hapke et al. (9) has demonstrated the importance of dCK in the activation of dFdCyd. Our in vivo HPLC studies showed that the overexpression of dCK results in enhanced tumor conversion and accumulation of dFdCTP and, ultimately, to a greater tumor response to dFdCyd. These data support our postulate that the antitumor activity of dFdCyd is related to dFdCTP accumulation and retention (4) . F-19 MRS studies evaluating dFdCyd metabolism in two lung cancer cell xenografts observed patterns of fluorine accumulation and retention consistent with those observed in our control tumors (11) . More importantly, and consistent with our results, the authors were able to positively correlate tumor response with tumor accumulation of fluorine signal believed to represent, at least in part, the dFdCTP metabolite.

This is the first demonstration of the in vivo antitumor effect of dFdCyd evaluated in conjunction with dCK cDNA transfer in the HT-29 xenograft model. These findings have potential clinical relevance, suggesting that dCK gene-directed therapies may be useful in enhancing the cytotoxic effects of dFdCyd chemotherapy by effecting the metabolism of the drug. Although gene delivery is a major issue, putting the dCK gene under the control of a promoter with some level of tumor specificity (such as carcinoembryonic antigen in gastrointestinal cancers), would be a logical approach. Another possibility is injecting the gene directly into the tumor and combining dFdCyd with a regionally delivered therapy such as radiation, where dFdCyd is known to be a radiation sensitizer (14 , 25) . Our preliminary data suggest that F-19 MRS techniques, as described here, may be a useful noninvasive tool to determine the efficacy of gene delivery as well as gene function in future dCK gene-directed studies.

	ACKNOWLEDGMENTS

 
We acknowledge Carrie E. Blackstock, Ph.D. (Department of English, University of North Carolina at Chapel Hill) for critical review of the 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 by NIH Grants R0–1-CA34085 and M0–1-RR00046-36.

² To whom requests for reprints should be addressed, at Department of Radiation Oncology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Phone: (336) 716-4981; Fax: (336) 716-5972; E-mail: ablackst{at}wfubmc.edu

³ The abbreviations used are: dFdCyd, 2',2'-difluoro-2'-deoxycytidine; dCyd, deoxycytidine; dFdCTP, 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate; dCK, deoxycytidine kinase; HPLC, high-performance liquid chromatography; MRS, magnetic resonance spectroscopy; dNTP, 2'-deoxynucleotide 5'-triphosphate; dFdUrd, difluorodeoxyuridine.

Received 3/16/01; revised 6/21/01; accepted 6/21/01.

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Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS HPLC-determined Effects of dCK... F-19 MRS-determined Effects of... Effects of dFdCyd on... DISCUSSION REFERENCES

 

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