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[Cancer Research 65, 4987-4992, June 15, 2005]
© 2005 American Association for Cancer Research

Priority Reports

DNA-Dependent Protein Kinase Is a Molecular Target for the Development of Noncytotoxic Radiation–Sensitizing Drugs

Eric T. Shinohara1, Ling Geng1, Jiahui Tan1, Heidi Chen2, Yu Shir2, Eric Edwards1, James Halbrook5, Edward A. Kesicki5, Adam Kashishian5 and Dennis E. Hallahan1,3,4

Departments of 1 Radiation Oncology, 2 Biostatistics, and 3 Cancer Biology, Vanderbilt University School of Medicine; 4 Vanderbilt-Ingram Cancer Center, Nashville, Tennessee; and 5 ICOS Corp., Bothell, Washington

Requests for reprints: Dennis E. Hallahan, Department of Radiation Oncology, Vanderbilt University,1301 22nd Avenue South, B-902 The Vanderbilt Clinic, Nashville, TN 37232-5671. Phone: 615-343-9244; Fax: 615-343-3075; E-mail: dennis.hallahan{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
DNA-dependent protein kinase (DNA-PK)–defective severe combined immunodeficient (SCID) mice have a greater sensitivity to ionizing radiation compared with wild-type mice due to deficient repair of DNA double-strand break. SCID cells were therefore studied to determine whether radiosensitization by the specific inhibitor of DNA-PK, IC87361, is eliminated in the absence of functional DNA-PK. IC87361 enhanced radiation sensitivity in wild-type C57BL6 endothelial cells but not in SCID cells. The tumor vascular window model was used to assess IC87361-induced radiosensitization of SCID and wild-type tumor microvasculature. Vascular density was 5% in irradiated SCID host compared with 50% in C57BL6 mice (P < 0.05). IC87361 induced radiosensitization of tumor microvasculature in wild-type mice that resembled the radiosensitive phenotype of tumor vessels in SCID mice. Radiosensitization by IC87361 was eliminated in SCID tumor vasculature, which lack functional DNA-PK. Irradiated LLC and B16F0 tumors implanted into SCID mice showed greater tumor growth delay compared with tumors implanted into either wild-type C57BL6 or nude mice. Furthermore, LLC tumors treated with radiation and IC87361 showed tumor growth delay that was significantly greater than tumors treated with radiation alone (P < 0.01 for 3 Gy alone versus 3 Gy + IC87361). DNA-PK inhibitors induced no cytotoxicity and no toxicity in mouse normal tissues. Mouse models deficient in enzyme activity are useful to assess the specificity of novel kinase inhibitors. DNA-PK is an important target for the development of novel radiation-sensitizing drugs that have little intrinsic cytotoxicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Limitations of cytotoxic radiosensitizers, such as cisplatin, Taxol, and mytomycin C, are the inadvertent systemic toxicity. Recent advances in the development of molecular targeted therapy provide novel radiosensitizers that have limited inherent cytotoxicity. One such molecular target is the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which participates in nonhomologous repair of DNA double-strand breaks (DSB; refs. 1–4). DNA-PK is a member of the phosphatidylinostitol 3-kinase (PI3-K) superfamily that includes ATM, ATR, PI3-K, and TRRAP (5, 6). In fact, DNA-PK inhibitors, such as wortmannin and LY297004, inhibit each of these enzymes. The DNA-PK complex is composed of Ku80, Ku70, and the DNA-PKcs (7–10). Ku80 and Ku70 bind to DNA DSB to facilitate the binding of and perhaps the activation of DNA-PKcs. Mutations involving any of these three components of the DNA-PK complex result in a radiation-sensitive phenotype (11). DNA-PK-defective severe combined immunodeficient (SCID) mice have increased radiosensitivity in comparison with normal mice (2, 11–14). Moreover, small interfering RNAs (siRNA), which specifically down-regulate DNA-PK expression, induce radiosensitization of tumor models (15, 16).

Recent cancer drug development has lead to small molecular weight compounds that enhance control of tumors treated with cytotoxic agents (17–19). In particular, DNA-PK inhibitors IC87102, IC87361, and IC86621 are morphilin derivatives that have a much lower IC50 for DNA-PK compared with PI3-K (19). The more highly evolved morpholino-flavonoid, IC87361, is 50-fold more selective for DNA-PK than for p110ß (19). The purpose of this study was to determine whether the DNA-PK-specific inhibitor IC87361 enhances tumor control in irradiated tumors in wild-type mice, which would suggest that DNA-PK is a viable molecular target for the development of radiosensitizers. Moreover, SCID cells served as a model to study the specificity of inhibition of IC87361 for DNA-PK. Our findings indicate that specific DNA-PK inhibitors radiosensitized several cancer cell lines and induce a radiosensitive phenotype in wild-type mice that resembles that of tumors in SCID mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor models. The B16F0 cell line was obtained from American Type Culture Collection (Manassas, VA). B16F0 cells were maintained in Hyclone (VWR) MEM/Earle's balanced salt solution with 10% FCS and 0.5% penicillin/streptomycin from Life Technologies, Inc. (Gaithersburg, MD) LLC cells were obtained from American Type Culture Collection and were maintained in high-glucose (4.5 g) DMEM supplemented with 10% FCS and 1% penicillin/streptomycin, and 106 cells were injected s.c. into the hind limbs of mice.

Apoptosis and mitotic assays. B16F0 and LLC cells were maintained in complete medium. Cells were plated onto glass slides until 85% confluent. A 200 nmol/L concentration of IC87361 in complete medium was added to cells 30 minutes before irradiation with 6 Gy. Slides were then fixed with methanol and stained with H&E staining. Experiments were done in triplicate and four low-power fields (200x) were counted for each slide. Number of apoptotic and mitotic cells were counted in each field and the mean and SE were calculated.

Clonogenic assay. CD31+ vascular endothelial cells were harvested from the pulmonary vasculature of SCID mice. SCID and wild-type endothelial cells were plated onto fibronectin-coated plates and treated with IC87361 (200 nmol/L) followed 1 hour later by irradiation (0, 2, 4, and 6 Gy). LLC and B16F0 cells were plated on uncoated culture plates and treated with IC87361 alone or with 6 Gy. Cells were incubated for 8 days, fixed for 15 minutes with 3:1 methanol/acetic acid, and stained for 15 minutes with 0.5% crystal violet (Sigma, St. Louis, MO) in methanol. Colonies were counted when they contained ≥50 cells. Surviving fraction was calculated as (mean colony counts) / (cells plated) x (plating efficiency).

Comet assay. Comet assay of DNA fragmentation was done on SCID and wild-type endothelial cells. Cells were grown to 80% confluence on slides and pretreated with 200 nmol/L of IC87361 2 hours before irradiation with 6 Gy. Six hours later, cells were scraped, washed, and placed into a low-melting agarose gel according to Trevigen protocol (20). Slides were then placed in an electrophoresis chamber and run for 20 minutes. Slides were stained with green florescence dye (Trevigen, Gaithersburg, MD) and imaged by fluorescence microscopy.

Tumor vascular window model. The dorsal skin fold window is a 3-g plastic frame applied to the skin of an animal and remains attached for the duration of the study. The window frame was marked with coordinates, which were used to photograph the same microscopic field each day. Vascular windows were photographed using 4x objective to obtain a 40x total magnification. Color slides were used to catalogue the appearance of blood vessels on days 0 to 7. Color slides were scanned into Photoshop software and analyzed by the Optimas software. Vascular center lines were positioned by the Optimas software and verified by an observer blinded to the treatment groups. Tumor blood vessels were quantified by the use of the Optimas software at 40x magnification, which quantifies the vascular length density (VLD) of blood vessel within the microscopic field. VLD is a summation of the total vascular length in a given window model. The mean and SE of VLD was calculated for each treatment group of five mice.

Tumor volume assessment. C57BL6, SCID, and nude mice received s.c. injections of 106 LLC cells suspended in 0.1 mL of medium. An equal number of large and intermediate size tumors were present in each group of five mice. Mouse tumors were stratified into groups so that the mean tumor volume of each group was comparable. The mean volume of the tumors in mice at the start of treatment (day 0) averaged 200 mm3 in all groups, including control C57BL6, irradiated C57BL6, control nude, irradiated nude, control SCID, and irradiated SCID mice. A total dose of 21 Gy was given in seven fractionated doses of 3 Gy given on days 0 to 4, 7, and 8. A second experiment was done to confirm these findings in B16F0 tumors implanted into 10 SCID and C57BL6. The mean volume of the tumors in mice at the start of treatment (day 0) was ~100 mm3 in all groups, including control C57BL6, irradiated C57BL6, control SCID, and irradiated SCID mice.

DNA-PK inhibitors were also studied in C57BL6 mice separated into control, irradiated, IC87361 alone and IC87361 given before irradiation, with five mice per group. LLC and B16F0 tumors were implanted into the hind limbs of C57BL6 mice and were grown to a volume of 300 mm3, at which time treatment was initiated (day 0). Mice treated with i.p. injection of 75 µg IC87361. Radiation was given as 3 Gy fractions per day given 1 hour after drug administration on days 0, 1, 2, 3, 4, 7, and 8. Tumor volumes were measured 3 days per week using skin calipers, as previously described (17, 18). Tumor volumes were calculated using the formula (a x b x c / 2) that was derived from the formula for an ellipsoid (d3 / 6). Data were calculated as the fold increase of original (day 0) tumor volume and graphed as fold increase in volume ± SD for each treatment group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IC87361 radiosensitizes wild-type but not severe combined immunodeficient tumor vasculature. Mutations involving DNA-PKcs result in a radiation-sensitive phenotype (10). To determine whether the DNA-PK-defective SCID host is a useful model to study the specificity of inhibitors of DNA-PK, the dorsal skin fold tumor vascular window model was used to measure VLD. Tumor vascular window studies in SCID mice showed markedly greater regression of irradiated tumor VLD compared with that of tumors in C57BL6 mice. At 72 hours after irradiation, tumor VLD was 5% in SCID compared with 50% in C57BL6 mice (P < 0.05; Fig. 1A and B). These findings indicate that the SCID host is a useful model to study the specificity of inhibitors of DNA-PK.



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  		Figure 1. IC87361 radiosensitizes tumor vasculature in wild-type but not SCID host. Dorsal skin fold windows were applied to C57BL6 and SCID mice to study tumor vascular response to radiation. LLC tumor cells were pelleted and implanted into the skin fold within the window. Angiogenesis into the window occurred over the course of 7 days. Tumor vasculature was irradiated with 3 Gy and photographed by microscopy directly after irradiation (0 hour). A, representative photographs at 40x magnification of tumor vascular window at 0 and 48 hours in C57BL6 and SCID mice. VLD was quantified by use of the Optima software analysis of window data. B, columns, mean of percent change in VLD values; bars, SE. C, mice were treated with IC87361, 3 Gy, or IC87361 given 1 hour before 3 Gy. D, columns, mean of percent change in VLD in wild-type C57BL6 mice; bars, SE. *, P < 0.05. E, columns, mean of percent change in VLD in SCID mice; bars, SE.

 
To determine whether the radiosensitizing effect of IC87361 is specific to DNA-PK in the mouse model, we studied the response of SCID blood vessels by use of the dorsal skin fold vascular window model. Tumor vascular windows were placed onto C57BL6 mice and LLC cells were injected into the skin fold. This induced the formation of blood vessels which were subsequently treated with IC87361, 3 Gy alone, or IC87361 before 3 Gy (Fig. 1C). VLD in C57BL6 mice treated with IC87361 before 3 Gy showed significant enhancement of radiation-induced tumor vascular destruction in wild-type host with VLD of 5% at 72 hours (P < 0.05; Fig. 1D). IC87361 alone and 3 Gy alone produced minimal reduction in tumor VLD to 85% and 90%, respectively (Fig. 1D). The radiosensitizing effect of IC87361 was not observed in tumor vasculature in SCID mice (Fig. 1E). VLD was significantly reduced in response to 3 Gy alone compared with untreated SCID mice (P < 0.05). IC87361 given before irradiation produced no further radiosensitizing effect in SCID microvasculature in the tumor vascular window model (Fig. 1E).

Radiosensitization with DNA-dependent protein kinase–specific inhibitor. The recent development of specific inhibitors of DNA-PK has provided a model to study the role of DNA-PK in vascular endothelial cells (19). Clonogenic assays were conducted on primary culture CD31-positive endothelial cells from SCID mice. SCID endothelial cells showed a statistically significant reduction in clonogenic survival compared with wild-type endothelial cells (Fig. 2). To determine the effectiveness and specificity of DNA-PK inhibitors, clonogenic survival was studied in irradiated SCID and wild-type endothelial cells following treatment with IC87361. DNA-PK-defective SCID endothelial cells treated with IC87361 and radiation showed no reduction in clonogenic survival compared with SCID cells treated with radiation alone (Fig. 2A). Primary culture wild-type endothelial cells treated with IC87361, on the other hand, showed a significant increase in radiosensitivity that was similar to the results in SCID mice (P < 0.05; Fig. 2B).



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  		Figure 2. Clonogenic assay of SCID and wild-type endothelial cells treated with IC87361. CD31+ SCID vascular endothelial cells were harvested from SCID mice. SCID and wild-type endothelial cells were plated onto fibronectin-coated plates in quadruplicate and treated with 200 nmol/L IC87361 followed 1 hour later by irradiation (0, 2, 4, and 6 Gy). Medium was changed after 1 hour. Fraction of surviving (A) SCID endothelial cells and (B) wild-type endothelial cells after treatment with DMSO (solid line) or IC87361 (dotted line). C, comet assays of DNA fragmentation in SCID and wild-type endothelial cells treated with IC87361; SCID and wild-type endothelial cells were grown to 80% confluence on slides and pretreated with 200 nmol/L of IC87361 1 hour before irradiation with 6 Gy. Six hours later, cells were scraped, washed, and placed into a low-melting gel according to Trevigen protocol. Slides were then placed in an electrophoresis chamber and run for 20 minutes. Slides were stained with florescent dye (Trevigen) and imaged by fluorescence microscopy. Columns, percentage of cells that contained "comet tails" indicating fragmented DNA in SCID endothelial cells (C; left) and wild-type endothelial cells after treatment with 6 Gy or IC87361 1 hour before 6 Gy (C; right). *, P < 0.05.

 
DNA DSBs are the primary determinant of cytotoxicity in cells treated with ionizing radiation (20). The comet assay was used to measure fragmented DNA in irradiated cells (21, 22). SCID cells treated with IC87361 with 6 Gy showed no significant increase in DNA fragmentation compared with SCID treated with 6 Gy alone (Fig. 2C) at 34% compared with 31%, respectively (Fig. 2D). Conversely, wild-type endothelial cells showed 7% DNA fragmentation following 6 Gy alone (Fig. 2D) and showed a significant increase in the percentage of cells showing DNA fragmentation (18%) when pretreated with IC87361 before irradiation (P < 0.05; Fig. 2C). IC87361 alone showed no DNA fragmentation in wild-type endothelial cells (Fig. 2C). These findings indicate that DNA-PK inhibition before irradiation leads to a substantial increase in fragmented DNA in vascular endothelium.

IC87361 affects radiation response. To determine whether the radiosensitizing effects of IC87361 are also observed in cancer cells in vitro, we studied cell lines that were later implanted into wild-type host as well as SCID mice. Clonogenic assay of B16F0 melanoma and LLC lung cancer cells were treated with IC87361, 6 Gy, or both IC87361 and 6 Gy. Clonogenic assays showed that IC87361 alone had no significant effect on plating efficiency in either cancer cell line. Radiation had a modest reduction in survival for LLC and B16F0 (Fig. 3A-B). However, combined IC87361 with 6 Gy showed a significant reduction in cell survival for LLC and B16F0 cells (P < 0.05, compared with 6Gy alone). Quantification of apoptotic and mitotic cells using H&E staining showed similar results with little effect on mitosis or apoptosis in either cell line with IC87361 alone; 6 Gy alone induced a >13-fold reduction in mitotic cells and ~31-fold increase in apoptosis in B16F0 cancer cells (Fig. 3C). IC87361 with 6 Gy showed 70-fold increase in apoptosis with no visible mitotic cells (P < 0.05, compared with 6Gy alone). LLC cells treated with radiation showed a 17-fold decrease in mitosis and an 8.6-fold increase in apoptosis (Fig. 3D). LLC treated with IC87361 and 6 Gy showed a 22-fold increase in apoptosis with no observed mitotic cells (P < 0.05, compared with 6Gy alone).



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  		Figure 3. IC87361 effects cancer cell survival. Clonogenic assay of B16F0 melanoma and LLC lung cancer cells were treated with IC87361, 6 Gy, or both IC87361 and 6 Gy. Clonogenic assay for (A) LLC and (B) B16F0 cells. Quantification of apoptotic and mitotic cells using H&E staining of cancer cell lines treated with IC87361 alone, 6 Gy, or both IC87361 and 6 Gy in (C) B16F0 and (D) LLC cancer cells. *, P < 0.05, compared with radiation alone.

 
IC87361 enhances radiation-induced tumor growth delay. To determine whether the enhanced radiation response in tumor blood vessels of SCID host translates into tumor responsiveness, we studied tumor growth delay following fractionated radiotherapy. Tumors were implanted into C57BL6 and SCID mice, whereas nude mice were studied to control for the immunosuppressed phenotype (Fig. 4). Tumors were treated with daily fractionated radiotherapy using 3-Gy doses. B16F0 tumors in C57BL6 mice showed growth of 381 ± 23 mm3/d (mean ± SE) from days 0 to 11 after irradiation (days 14 and 25 post-implantation). Radiation induced a significant increase in tumor growth delay in SCID mice, which grew nearly 10 times slower at 38 ± 10 mm3/d (P < 0.001; Fig. 4A). There was also a 4-fold greater radiation-induced growth delay in SCID mice implanted with B16F0 compared with tumors in C57BL6 mice (13 days compared with 3 days to reach a 10-fold increase in size; Fig. 4A). Likewise, LLC tumors in SCID mice irradiated with 3 Gy/d to a total dose of 21 Gy slowed tumor growth to 61 ± 10 mm3/d (P < 0.01) from treatment days 0 to 11 (Fig. 4D). LLC tumors in Nude and C57BL6 hosts showed a far smaller response to radiation. Untreated tumors in nude mice had growth of 276 ± 28 mm3 compared with irradiated LLC in Nude mice that had a growth of 210 ± 11 mm3 (P < 0.002; Fig. 4C). Growth in untreated LLC in C57BL6 was 353 ± 11 mm3 compared with irradiated tumors in C57BL6 which showed growth of 267 ± 24 mm3 (P < 0.01) from treatment days 0 to 11 (Fig. 4B). There was a 2-fold increase in growth delay resulting tumors that were five times the original volume in SCID (requiring 7 days) compared with C57BL6 mice (14 days) or nude mice (14 days). Athymic nude mice were used to control for the immunocompromised status of SCID mice.



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  		Figure 4. IC87361 recapitulates tumor radiosensitivity observed in SCID mice. B16F0 cells were implanted into the hind limb of SCID and C57BL6 mice. Tumors were grown to an average of 100 mm3. LLC cells implanted into the hind limb of SCID, C57BL6, and nude mice were grown to an average volume of 200 mm3. Tumors were measured with calipers at the indicated time points for each of the groups. Control mice received no therapy ({bullet}). Radiation ({circ}) was given at 3-Gy fractions per day on days 0 to 4 and days 7 and 8 (21 Gy total dose). Points, mean of the tumor volumes from five animals per treatment group for C57BL6 and SCID mice implanted with B16F0 (A); bars, SE. Tumor growth curves compare irradiated and unirradiated LLC tumors in (B) C57BL6, (C) nude, and (D) SCID mice. *, P < 0.001, comparing SCID with C57BL6. E and F, tumor growth curves compare control, irradiated, and IC87361 given before irradiation. LLC or B16F0 cells were pelleted and implanted into the hind limb of C57BL6 mice. Tumors were grown to a volume of 300 mm3 volume, at which time treatment was initiated (day 0). Mice treated with IC87361 received 75 µg i.p. Radiation was given 1 hour after drug administration in those mice treated with IC87361. Tumors were measured at the indicated time points for each of the groups. Control mice received no therapy. Radiation was administered as 3-Gy fractions per day on days 0 to 4 and days 7 and 8. Points, mean of eight animals in each treatment group for B16F0 (E) and LLC (F); bars, SE. *, P < 0.01 for radiation alone versus IC87361 and radiation.

 
To determine whether inhibition of DNA-PK with IC87361 reproduces the radiosensitive phenotype observed in tumors in SCID mice, C57BL6 mice were implanted with B16F0 and LLC tumors and treated with either 3 Gy daily fractions to 21 Gy, IC87361 alone, or IC87361 before each 3 Gy fraction. Tumor growth curves showed a reduction in growth in mice implanted with B16F0 treated with 3 Gy. Untreated tumor growth was 613 ± 29 mm3 compared with tumors treated with radiation alone which showed tumor growth delay of 327 ± 12 mm3 (P < 0.05). IC87361 alone had a growth of 687 ± 28 mm3 compared with 3 Gy + IC87361 growth of 193 ± 12 mm3 (P < 0.001 for 3 Gy alone versus IC87361 + 3 Gy; Fig. 4E). Similarly, untreated LLC tumors showed growth of 533 ± 37 mm3 compared with tumors treated with 3 Gy/d which showed growth of 146 ± 20 mm3 (P < 0.0005). In comparison, LLC treated with 3 Gy and IC87361 showed growth of 49 ± 14 mm3 for (P < 0.01 for 3 Gy alone versus 3 Gy + IC87361; Fig. 4F).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies with siRNA specific for DNA-PK have shown that DNA-PK is a viable target for radiosensitization and chemosensitization (15, 16). However, nonspecific kinase inhibitors such as wortmannin enhance radiosensitivity by inhibition of PI3-K in addition to DNA-PK (23). Here we have shown that small molecular inhibitors can reproduce the effects of a DNA-PK-deficient host. Moreover, DNA-PK inhibition not only radiosensitized cancer cells but also radiosensitized the vascular endothelium. SCID cells represent a model to test the specificity of the radiosensitizing effects of DNA-PK inhibitors. SCID cells contain a mutant kinase domain of the catalytic subunit of DNA-PK and thereby have minimal DNA-PK enzymatic activity (11–14). We found that these DNA-PK functionally depleted cells showed no additional radiosensitization by IC87361. We surmise that the radiation sensitizing effects of IC87361 are abrogated in cells lacking DNA-PK function.

Radiation sensitizing agents, such as cisplatin, mitomycin C, and Taxol, show intrinsic cytotoxic effects unto themselves. In contrast, IC87361 produced no cytotoxicity at doses that were sufficient to induce radiosensitization. Moreover, we found no toxicity in mice with no weight loss and no hypomotility during the 4 weeks of study. Interestingly, radiosensitization by IC87361 requires only brief pharmacokinetics during DNA strand break repair (21). Therefore, sustained drug levels are not essential to achieve the desired radiation enhancement. This is most important when considering that knockout of DNA-PK results in immunosuppression and the development of malignant lymphomas in mice. We found no increase in tumor growth or metastases in mice treated with several daily doses of IC86371 as seen on necropsy. Selective enhancement of the local effects of radiation with limited systemic toxicity is a promising approach in the treatment of solid tumors.

Normal tissues within SCID mice are thrice more sensitive to radiation compared with the radiation sensitivity in normal mouse strains (11, 13, 14). This implies that DNA-PK inhibitors could radiosensitize normal tissues that are within the irradiated field. Presently used radiation sensitizing drugs include cisplatin and Taxol, which enhance radiation sensitivity in normal tissues and also produce systemic toxicities. IC87361 is not cytotoxic and achieves radiation sensitization without systemic toxicity in mouse models. This is not unexpected because DNA-PK knockout mice are viable. Although we did not observe normal tissue toxicity in the hind limb tumor model, clinical trials of DNA-PK inhibitors should consider brachytherapy (e.g., high dose rate) or fractionated stereotactic irradiation.


   Acknowledgments
 
Grant support: NIH grants R01-CA58508, R01-CA70937, R01-CA88076, R21-CA89674, R01-CA89888, R01-CA112385; Vanderbilt Lung Cancer Specialized Programs of Research Excellence grant P50-CA90949; Vanderbilt-Ingram Cancer Center grant CCSG P30-CA68485; NIH training grant T-32CA93240 (E.T. Shinohara).

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.

We thank Allie Fu for technical support.

Received 11/29/04. Revised 3/17/05. Accepted 4/15/05.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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