Inr. .I. Radiation Oncology Bid Phys., Vol. Prmted in the U.S.A. All rights reserved.
0360-3016192 $5.00 + .OO Copyright Q 1992 Pergamon Press plc
22, pp. 489494
0 Session B: Biochemical Modification of Therapeutic Response
IODODEOXYURIDINE INCORPORATION AND RADIOSENSITIZATION IN THREE HUMAN TUMOR CELL LINES VALERYUHL, M.D. ,* THEODORE L. PHILLIPS, M.D. ,*t GLENDAY. Ross, B.A. ,* WILLIAMJ. BODELL,FkD.*$ ANDJYTTE RASMUSSEN, B.S.+ UCSF, San Francisco,
CA 94143, USA
Iododeoxyuridine is a halogenated pyrimidine and non-hypoxic cell radiosensitizer currently being used in clinical trials. The amount of radlosensitization by IdUrd is related to the amount of incorporation of the drug into a cell’s DNA. These experiments were carried out in three human tumor cell lines (lung, glioma, and melanoma) in monolayer culture exposed to concentrations of IdUrd from 0.1-10 FM for one and three cell cycles before irradiation to determine incorporation and sensitization as a function of drug exposure. Except for the lung cell line, which required greater than 1 PM IdUrd, these cells demonstrate radiosensitlzation when exposed to 0.1 )J,Mor greater of IdUrd. Maximum sensitization occurred at 10 p.M IdUrd for all the cell lines at three cell cycles. The percent thymidine replacement by IdUrd increased with increasing concentrations, but was cell line dependent. Maximum percent replacement occurred at 10 FM at three cell cycles for all the cell lines: lung = 22.4%) glioma = 32.0%, and melanoma = 39.1%. The relationships between percent thymidine replacement and sensitization are not identical across these human tumor cell lines. If IdUrd is going to be a successful radiosensitlzer in clinical trials, sustained plasma levels of 10 FM or greater for at least three cell cycles should be achieved during irradiation. This may be best accomplished’ with repeated short exposures to IdUrd (three cell cycles or approximately 4 days in these cell lines) every l-2 weeks during radiation. Measurements of thymidine replacement in a tumor biopsy should be attempted prior to radiation to develop a predictive assay for radiosensitization. Radiosensitization, Incorporation, Iododeoxyuridine, AS49 cells, US7 mg cells, Ul cells.
Iododeoxyuridine (IdUrd) is a halogenated pyrimidine that competes with thymidine for incorporation into DNA and has limited cytotoxicity as an antineoplastic agent (18). However, it has been shown to be a non-hypoxic cell radiosensitizer (3, 4, 17), inhibiting to some extent the recovery of ionizing radiation-induced damage (6). Phase I/II trials with intravenous IdUrd demonstrate minimal systemic toxicity, high steady-state plasma levels (l-8 p.M), and encouraging clinical results when used in tumor sites where the surrounding normal tissue has a low mitotic rate compared to the tumor (5, 8, 9, 10). The mechanism of sensitization involves direct incorporation of IdUrd into the DNA by replacing thymidine. The degree of radiosensitization depends on the extent of IdUrd incorporation (4, 19). Sensitization appears to be caused by enhanced radiochemical events with greater DNA strand break frequencies (6, 7). This is supported by evidence
that sensitization does not occur with very high LET radiations (13). Pre-treatment of exponentially growing cells in virro with IdUrd demonstrates changes in radiation survival parameters: decreased shoulder of the curve (n) at high concentrations and decreased slope (Do) (4, 6, 7). Extensive work has been accomplished with rodent cell lines and IdUrd incorporation (14), demonstrating that significant thymidine replacement can be achieved with IdUrd concentrations as low as 0.1 FM and that the degree of enhancement is related directly to concentration times cell cycle times (micromolar hours), as well as to percent thymidine replacement. Recently, interest has been revived in the investigation of IdUrd incorporation in human tumor cell lines (11) because of the success of in vivo Phase I testing (10). Our report expands the knowledge of the influence of IdUrd on incorporation into DNA and the resulting radiosensitization in three additional cultured human tumor cell lines. It is hoped that these experiments will aid in improving the design of clinical trials with IdUrd.
Presented at the 7th International Meeting of Chemical fiers of Cancer Treatment, Clearwater, FL, 2-6 February *Dept. of Radiation Oncology. TLaboratory of Radiobiology. *Dept. of Neurological Surgery. Reprint requests to: Valery Uhl, M.D., Department of tion Oncology, UCSF-Long 75, San Francisco, CA 94143,
Acknowledgements-The authors wish to express their appreciation to Ms. Hiroko Kowta for her assistance in preparing the manuscript. Supported by NIH Training Grant #CA 09215, Office of Health and Environmental Research, U.S. Department of Energy, Contract #DE-AC03-76-SF01012 and #CA 13525. Accepted for publication 3 July 1991.
INTRODUCTION
Modi1991.
RadiaUSA. 489
490
I. J. Radiation Oncology 0 Biology 0 Physics
METHODS
AND MATERIALS
Cell culture Human lung adenocarcinoma (A549) and malignant melanoma (Ul) cells were obtained from Dr. James B. Mitchell at the National Cancer Institute (NCI). Human malignant glioma cells (U87mg) were obtained from Dr. Dennis Deen at the Brain Tumor Research Center (BTRC) of the Department of Neurosurgery at the University of California, San Francisco. A549 was grown in RPM1 1640 medium supplemented with 10% fetal bovine serum, L-giutamine, penicillin, and streptomycin. U87mg and Ul were grown in the same fashion, except RPM1 1640 medium was supplemented with 5% fetal bovine serum and 5% bovine calf serum supplemented with iron. Exponentially growing stock cultures were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. Under these conditions, the doubling times and plating efficiencies of the human tumor cell lines were 22 hr and 80-95% for A549, 33 hr and 20% for U87mg, and 33 hr and 50% for Ul , respectively. Cells were plated in 60 mm plastic culture dishes the day prior to experimentation in order for the cells to settle and attach to the plate before exposure to IdUrd and/or irradiation. The number of cells plated was adjusted to yield lo6 cells per dish at time of irradiation. Drug treatment IdUrd was supplied by the Drug Development Branch, NCI. A 10 mM stock solution in phosphate buffered solution (PBS) was made initially and fresh solutions of IdUrd in complete medium were made just prior to each experiment. After the cells had attached to the plates, the supernatant was removed from the dishes and approximately 5 ml of complete medium containing varying concentrations of IdUrd (0, 0.1, 1.0 and 10 pM) were added to the dishes for one and three cell cycles before irradiation. At these concentrations and times, IdUrd was not toxic and did not cause significant changes in growth rate for A549. The U87mg cell line exhibited no change in plating efficiency (PE); however, the growth rate progressively slowed after two cell cycles at 10 FM. The Ul melanoma cell line was the most sensitive to IdUrd, demonstrating a decrease in PE at 1 pM even at one cell cycle. Growth rate was unaffected by 5 10 pM IdUrd over these cell cycle times. Irradiation conditions Cell samples were irradiated using a Sheppard 137-cesium irradiator that produces gamma rays at 2.45 Gray per min. Dosimetry was carried out by TLD measurements using lithium fluoride crystals calibrated against standard sources and ion chambers. Analysis of survival curves After irradiation, the supernatant was removed and the cells were washed with PBS, trypsinized with STV, resus*Beckman
170 isotope detector.
Volume 22, Number 3, 1992
pended in complete medium and counted on a Model Zf Coulter Counter. A known quantity was re-plated in 60 or 100 mm dishes with 5 or 15 ml of normal medium at densities to yield 50-100 colonies. After incubation for 12 days, the plates were fixed in ethanol, stained with methylene blue, and scored for colony formation. Any colony consisting of 50 or greater cells was counted. Each survival point was plated in triplicate and repeated multiple times. Plating efficiency was calculated as the ratio of the number of colonies counted to the number of cells plated. Programs for the analysis of cellular survival data developed by N. Albright were used (1). The program weighted the data points, calculated the best fit to the linear quadrant (LQ) formula, and calculated the sensitizer enhancement ratio (SER) values with standard errors. The percent thymidine replacement versus SER and concentration of IdUrd times cycle time curves were fit by the TELLAGRAF linear regression program. Incorporation of IdUrd After IdUrd treatment, the cells were washed with PBS, trypsinized, centrifuged, and quick frozen. The amount of IdUrd incorporation into cellular DNA was analyzed using the 32P post-labelling procedure (2). Cellular DNA was isolated and enzymatically digested to 3’-monophosphate nucleotides that were then phosphorylated to 32P-5’, 3-bis phosphorylated nucleotides with gamma-32P-ATP, and polynucleotide kinase. The 3’-phosphate was removed by P-l nuclease to form 32P-5’-phosphorylated nucleotides. Mononucleotides were separated from 32P-ATP on an ion exchange column, and 32P-IdUrd-5’-monophosphate (IdUrdMP) was separated from the other 5’-monophosphates by high performance liquid chromatography. Peaks were identified with an in-line radiation detector.* The radioactivity in each monophosphate peak was summed and the percentage of each peak was calculated by dividing by the total radioactivity and multiplying by 100. Percent IdUrd substitution for thymidine monophosphate (TMP) was calculated as: % IdUrdMP % IdUrdMP
+ % TMP
x 100.
RESULTS Radiation survival curves based on clonogenic survival of the three human tumor lines exposed to varying concentrations (0, 0.1, 1 and 10 p.M) of IdUrd are shown in Figures l-6 for exposure times of one and three cell cycles. These have been fitted by the LQ formula. Individual data points have been omitted for clarity. Survival decreases as a function of radiation dose and as the concentration of IdUrd increases above 0.1 p,M for all the cell lines except
Iododeoxyuridine incorporation 0 V. AS49
ld6-' 0
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IdUrd
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1oun
--_-
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___
O.lun
._.._......
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s
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UHL et al. USitrq+ Id&d
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--_
1Vn
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.~
O.lun
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“‘I
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s "'I
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,J
10-4
20
16
0
Radiation Dose (Gyl
2
4
6
8
10
12
Radiation Dose (Gy) Fig. 1. Human lung adenocarcinoma A549 cell survival after irradiation with 137-Cs and 22 hr (one cell cycle) exposure to IdUrd.
A549,
in which
a concentration
greater
than
1 FM
is
needed at one cell cycle to see a decrease in survival. The SER values at 10% survival and percent thymidine replacement (%TR) for the three cell lines are shown in Table 1. Human lung adenocarcinoma A549 cells demonstrate radiosensitization when exposed to greater than 1 PM
Fig. 3. Human brain glioma U87 mg cell survival after irradiation with 137-Cs and 33 hr (one cell cycle) exposure to IdUrd.
IdUrd. Human glioma U87mg and melanoma Ul demonstrate radiosensitization when exposed to p.M. Maximal sensitization occurs with 10 p,M three cell cycles for all three cell lines: 1.61 t
U87m~ + ldllrd
AS49
+
Id&d
10&m
----
l.Oun o.,un
----
for
3 Cell
Cycles
0
’ ”
a
’ 6
”
~~..
Control
__
3 Call
Cyclar
~~
‘\
' L""
16
20
Radiation Dose (Gy) Fig. 2. Human lung adenocarcinoma A549 cell survival after irradiation with 137-Cs and 66 hr (three cell cycles) exposure to
IdUrd .
o.,m
for ~_
. .
a n "",
10
----~
r
\ 1o-5
1oml ,m
cells can only 0.1 IdUrd at 0.17 for
1o-5L--
0
2
4
6
8
10
12
Radiation Dose (Gy) Fig. 4. Human brain glioma U87 mg cell curvival after irradiation with 137-Cs and 99 hr (three cell cycles) exposure to IdUrd.
492
I.J. Radiation Oncology 0 Biology 0 Physics
Volume 22, Number 3, 1992
Table 1. SER (10% survival + standard error) and % thymidine replacement (%TR) at one and three cell cycles for human lung cancer (A549), glioma (487 mg), and melanoma (Ul) cells treated with varying concentrations of IdUrd before irradiation A549 IdUrd Concentration One cell cycle 10 uM 1 uM 0.1 uM Three cell cycles 10 uM 1 uM 0.1 uM
U87 mg
Ul
SER c SE
%TR
SER 2 SE
%TR
SER + SE
%TR
1.26 k .14 1.12 IL .13 0.87 -r- .06
13.3 4.5 0.9
1.61 2 .17 1.22 k .16 1.39 ? .39
21.0 7.6 0.7
1.35 ? .lO 1.17 2 .07 1.08 k .lO
11.4 2.6 0.6
1.61 2 .17 0.99 k .09 0.94 f .09
22.4 7.6 2.3
1.63 c .09 1.07 k .lO 1.19 ? .lO
32.0 12.8 2.0
2.07 k .I0 1.34 k .06 1.18 ? .06
39.1 3.1 1.8
A549, 1.63 + 0.09 for U87mg, and 2.07 + 0.10 for Ul. The melanoma Ul line demonstrates the greatest sensitization and the highest %TR at three cell cycles. The degree of radiosensitization depends on the extent of IdUrd incorporation into the DNA. As the %TR increases, the SER values increase for all cell lines (Fig. 7), though not at the same rate. Ul appears to be more sensitive to a given %TR demonstrating SER’s above 1 for all levels of %TR. Just as the SER’s are maximum at three cell cycles for 10 PM IdUrd, the percent IdUrd replacement is also maximum at three cell cycles but across all concentrations for all three human tumor lines. The %TR increases to a maximum of 22.4% for A549, 32.0% for U87 mg, and 39.1% for Ul at 10 p,M. The incorporation of IdUrd is related to the concentration of the drug in the medium and the num-
Ul
+
1olm
IdUrd
for
1 Cell
ber of cell cycles (CXT). Overall, A549 demonstrated less incorporation than the other cell lines for the same CXT (Fig. 8). DISCUSSION Phillips et al. found that significant thymidine replacement can be achieved with IdUrd concentrations as low as 0.1 p,M for Chinese hamster V-79 cells. They found that the degree of enhancement is related to concentration times length of exposure of the cells to IdUrd as well as to %TR. Peak incorporation and SER required at least three cell cycles. The maximum thymidine relacement was 49% after 48 hr (six cell cycles) exposure to 30 FM yielding an SER of 2.7.
Cycle
Ul + (Om
----.-._ .- .-..___._
0
6
10
16
20
Radiation Dose (Gy) Fig. 5. Human melanoma Ul cell survival after irradiation with 137-Cs and 33 hr (one cell cycle) exposure to IdUrd.
6
Id&d for ----_
lun
---
O.lIm
-----~
10
3 Cell
Cycles
--~
16
Radiation Dose (Gy) Fig. 6. Human melanoma Ul cell survival after irradiation with 137~Cs and 99 hr (three cell cycles) exposure to IdUrd.
Iododeoxyuridineincorporation 0 V.
UHL
493
ef al.
CXT VS %TR
SER VS %TR
I
0
V U87mg
..
CONCENTRATION
Fig. 7. Sensitizer Enhancement Ratio (SER) at 10% survival as a function of percent Thymidine replacement (%TR) by IdUrd.
X CELL CYCLE TIME
Fig. 8. Concentration of IdUrd times cycle time (CXT in micromolar cycle time) as a function of percent Thymidine replacement (%TR) by IdUrd.
Lawrence et al. investigated the influence of the duration of exposure to 10 p,M IdUrd on incorporation into DNA and the resulting radiosensitization in cultured human colon cancer HT29 cells. In their system, incorporation of IdUrd plateaued at 30% after 4 days of exposure. Prolonging the time of exposure to 7 days increased cytotoxicity without affecting either incorporation or radiosensitization. They suggested that short, repeated exposures to IdUrd might produce sustained radiosensitization with less thrombocytopenia and myelosuppression than longer treatment periods. This approach was used by UCSF/NCCC in a recently completed trial of BrdUrd in malignant gliomas with some success (12, 15). In our study, the effect of pre-treatment with clinically achievable levels of IdUrd (O-10 PM) on three human tumor cell lines is compared by in vitro clonogenic survival. Significant thymidine replacement can be achieved in these cells and, as found by others (11, 14), peak incorporation and SER occurred at three cell cycles. There is variation between the cell lines in maximal %TR at a given concentration, but there are insufficient data to determine whether SER varies among the lines at similar %TR. Overall, A549 demonstrated less enhancement than the other two cell lines at all concentrations and cell cycles. The apparent resistance of these cells to sensitization, as well as their lower incorporation rates, is interesting. They are known to contain high concentrations of glutathione, which may make
them resistant, but this may not be the major cause (16). It may be possible that at low levels of IdUrd there is enhanced repair of potentially lethal damage that results in minimal sensitization or that at the lower concentrations of IdUrd, the A549 cells are depleting the medium of drug with time. The thymidine pathways in these cell lines have not been analyzed. However, differences in the enzyme activities (Km for thymidine kinase) or nucleotide pools may account for the lower %TR in A549 despite its shorter cell cycle time compared to Ul . Clinical trials should strive to obtain sustained plasma levels of 10 pM IdUrd for optimal radiosensitization. As suggested by Lawrence et al., multiple short exposures would present repeated opportunities for tumor cells to come into S phase and incorporate IdUrd with the potential to administer a higher concentration of IdUrd and thus provide a higher level of sensitization. This would be particularly important in tumors that do not demonstrate sensitization at low doses of IdUrd like the lung tumor line investigated in this paper. Due to the apparent variability of sensitization to IdUrd and to predict the usefulness of IdUrd in humans, it is going to be necessary to study the percent IdUrd incorporation and radiation response with and without IdUrd of a wide range of human tumor cells prepared directly from fresh tumor specimens after in vivo drug infusion.
REFERENCES Albright, N. Computer programs for the analysis of cellular survival data. Radiat. Res.l12:331-340; 1987. Bodell, W. J.; Rasmussen, J. A 32P postlabeling determining the incorporation of bromodeoxyuridine lular DNA. Anal. B&hem. 142:525-528; 1984. Djordjevic,
B.; Szybalski,
assay for into cel-
W. Genetics of human cell lines.
III. Incorporation of 5-bromo- and 5-iododeoxyuridine into the deoxyribonucleic acid of human cells and its effect on radiation sensitivity. J. Exp. Med. 112:509-531; 1960. 4. Erickson, R. L.; Szybalski, W. Molecular radiobiology of human cell lines. V. Comparative radiosensitizing properties of 5-halodeoxycytidines and 5-halodeoxyuridines. Radiat.
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Res. 20:252-262; 1963. 5. Jackson, D.; Kinsella, T.; Rowland, J.; Wright, D.; Katz, D.; Main, D.; Collins, J.; Komblith, P.; Glatstein, E. Halogenated pyrimidines as radiosensitizers in the treatment of glioblastoma multiforme. Am. J. Clin. Oncol. 10:437443; 1987. 6. Kelland, L. R.; Steel, G. G. Inhibition of recovery from damage induced by ionizing radiation in mammalian cells. Radiother. Oncol. 13:285-299; 1988. 7. Kinsella, T. J.; Dobson, P. P.; Mitchell, J. B.; Fomace, A. J. Enhancement of x ray induced DNA damage by pre-treatment with halogenated pyrimidine analogs. Int. J. Radiat. Oncol. Biol. Phys. 13:733-739; 1987. 8. Kinsella, T. J.; Glatstein, E. Clinical experience with intravenous radiosensitizers in unresectable sarcomas. Cancer 59: 908-915; 1987. 9. Kinsella, T. J.; Mitchell, J. B.; Russo, A.; Morstyn, G.; Glatstein, E. The use of halogenated thymidine analogs as clinical radiosensitizers: rationale, current status, and future prospects: non-hypoxic cell sensitizers. Int. J. Radiat. Oncol. Biol. Phys. 10:1399-1406; 1984. 10. Kinsella, T. J.; Russo, A.; Mitchell, J. B.; Collins, J. M.; Rowland, J.; Wright, D.; Glatstein, E. A Phase I study of intravenous iododeoxyuridine as a clinical radiosensitizer. Int. J. Radiat. Oncol. Biol. Phys. 11:1941-1946; 1985. 11. Lawrence, T. S.; Davis, M. A.; Maybaum, J.; Stetson, P. L.; Ensminger, W. D. The dependence of halogenated pyrimidine incorporation and radiosensitization on the duration of drug exposure. Int. J. Radiat. Oncol. Biol. Phys. 18:1393-1398; 1990. 12. Levin, V. A.; Wara, W. M.; Gutin, P. H.; Wilson, C. B.; Phillips, T. L.; Prados, M.; Flam, M. S.; Ahn, D. K. Initial
Volume 22, Number 3, 1992
13.
14.
15.
16.
17.
18.
19.
analysis of NCOG 6682-I: Bromodeoxyuridine (BUdR) during irradiation followed by CCNU, procarbazine, and vincristine (PCV) chemotherapy for malignant gliomas. Proc. Am. Sot. Clin. Oncol. 9:91; 1990. Linstadt, D.; Blakely, E.; Phillips, T. L.; Castro, J. R. Radiosensitization produced by iododeoxyuridine with high linear energy transfer heavy ion beams. Int. J. Radiat. Oncol. Phys. 15:703-710; 1988. Phillips, T. L.; Bodell, W. J.; Uhl, V.; Ross, G. Y.; Rasmussen, J.; Mitchell, J. B. Correlation of exposure time, concentration and incorporation of IdUrd in V-79 cells with radiation response. Int. J. Radiat. Oncol. Biol. Phys. 16: 1251-1255; 1989. Phillips, T. L.; Levin, V. A.; Ahn, D. K.; Gutin, P. H.; Davis, R. L.; Wilson, C. B.; Prados, M. D.; Wara, W. M.; Flam, M. S. Evaluation of bromodeoxyuridine in glioblastoma multiforme: A Northern California Cancer Center Phase II study. Int. J. Radiat. Oncol. Biol. Phys. 21:709-714; 1991. Phillips, T. L.; Mitchell, J. B.; de Graff, W.; Russo, A.; Glatstein, E. Variation in sensitizing efficiency for SR 2508 in human cells dependent on glutathione content. Int. J. Radiat. Oncol. Biol. Phys. 12:1627-1635; 1986. Piro, A. J.; Taylor, C. C.; Belli, J. A. Interaction between radiation and drug damage in mammalian cells. II. The effect of actinomycin-D on the repair of sublethal radiation damage in plateau phase cells. Cancer 37:2697-2702; 1976. Prusoff, W. F. Synthesis and biological activities of iododeoxyuridine, an analog of thymidine. Biochem. Biophys. Acta. 32:295-296; 1959. Szybalski, W. X-ray sensitization by halopyrimidines. Cancer Chemother. Rep. 58:539-557; 1974.
0360-3016192 $5.00 + .OO Copyright Q 1992 Pergamon Press plc
22, pp. 489494
0 Session B: Biochemical Modification of Therapeutic Response
IODODEOXYURIDINE INCORPORATION AND RADIOSENSITIZATION IN THREE HUMAN TUMOR CELL LINES VALERYUHL, M.D. ,* THEODORE L. PHILLIPS, M.D. ,*t GLENDAY. Ross, B.A. ,* WILLIAMJ. BODELL,FkD.*$ ANDJYTTE RASMUSSEN, B.S.+ UCSF, San Francisco,
CA 94143, USA
Iododeoxyuridine is a halogenated pyrimidine and non-hypoxic cell radiosensitizer currently being used in clinical trials. The amount of radlosensitization by IdUrd is related to the amount of incorporation of the drug into a cell’s DNA. These experiments were carried out in three human tumor cell lines (lung, glioma, and melanoma) in monolayer culture exposed to concentrations of IdUrd from 0.1-10 FM for one and three cell cycles before irradiation to determine incorporation and sensitization as a function of drug exposure. Except for the lung cell line, which required greater than 1 PM IdUrd, these cells demonstrate radiosensitlzation when exposed to 0.1 )J,Mor greater of IdUrd. Maximum sensitization occurred at 10 p.M IdUrd for all the cell lines at three cell cycles. The percent thymidine replacement by IdUrd increased with increasing concentrations, but was cell line dependent. Maximum percent replacement occurred at 10 FM at three cell cycles for all the cell lines: lung = 22.4%) glioma = 32.0%, and melanoma = 39.1%. The relationships between percent thymidine replacement and sensitization are not identical across these human tumor cell lines. If IdUrd is going to be a successful radiosensitlzer in clinical trials, sustained plasma levels of 10 FM or greater for at least three cell cycles should be achieved during irradiation. This may be best accomplished’ with repeated short exposures to IdUrd (three cell cycles or approximately 4 days in these cell lines) every l-2 weeks during radiation. Measurements of thymidine replacement in a tumor biopsy should be attempted prior to radiation to develop a predictive assay for radiosensitization. Radiosensitization, Incorporation, Iododeoxyuridine, AS49 cells, US7 mg cells, Ul cells.
Iododeoxyuridine (IdUrd) is a halogenated pyrimidine that competes with thymidine for incorporation into DNA and has limited cytotoxicity as an antineoplastic agent (18). However, it has been shown to be a non-hypoxic cell radiosensitizer (3, 4, 17), inhibiting to some extent the recovery of ionizing radiation-induced damage (6). Phase I/II trials with intravenous IdUrd demonstrate minimal systemic toxicity, high steady-state plasma levels (l-8 p.M), and encouraging clinical results when used in tumor sites where the surrounding normal tissue has a low mitotic rate compared to the tumor (5, 8, 9, 10). The mechanism of sensitization involves direct incorporation of IdUrd into the DNA by replacing thymidine. The degree of radiosensitization depends on the extent of IdUrd incorporation (4, 19). Sensitization appears to be caused by enhanced radiochemical events with greater DNA strand break frequencies (6, 7). This is supported by evidence
that sensitization does not occur with very high LET radiations (13). Pre-treatment of exponentially growing cells in virro with IdUrd demonstrates changes in radiation survival parameters: decreased shoulder of the curve (n) at high concentrations and decreased slope (Do) (4, 6, 7). Extensive work has been accomplished with rodent cell lines and IdUrd incorporation (14), demonstrating that significant thymidine replacement can be achieved with IdUrd concentrations as low as 0.1 FM and that the degree of enhancement is related directly to concentration times cell cycle times (micromolar hours), as well as to percent thymidine replacement. Recently, interest has been revived in the investigation of IdUrd incorporation in human tumor cell lines (11) because of the success of in vivo Phase I testing (10). Our report expands the knowledge of the influence of IdUrd on incorporation into DNA and the resulting radiosensitization in three additional cultured human tumor cell lines. It is hoped that these experiments will aid in improving the design of clinical trials with IdUrd.
Presented at the 7th International Meeting of Chemical fiers of Cancer Treatment, Clearwater, FL, 2-6 February *Dept. of Radiation Oncology. TLaboratory of Radiobiology. *Dept. of Neurological Surgery. Reprint requests to: Valery Uhl, M.D., Department of tion Oncology, UCSF-Long 75, San Francisco, CA 94143,
Acknowledgements-The authors wish to express their appreciation to Ms. Hiroko Kowta for her assistance in preparing the manuscript. Supported by NIH Training Grant #CA 09215, Office of Health and Environmental Research, U.S. Department of Energy, Contract #DE-AC03-76-SF01012 and #CA 13525. Accepted for publication 3 July 1991.
INTRODUCTION
Modi1991.
RadiaUSA. 489
490
I. J. Radiation Oncology 0 Biology 0 Physics
METHODS
AND MATERIALS
Cell culture Human lung adenocarcinoma (A549) and malignant melanoma (Ul) cells were obtained from Dr. James B. Mitchell at the National Cancer Institute (NCI). Human malignant glioma cells (U87mg) were obtained from Dr. Dennis Deen at the Brain Tumor Research Center (BTRC) of the Department of Neurosurgery at the University of California, San Francisco. A549 was grown in RPM1 1640 medium supplemented with 10% fetal bovine serum, L-giutamine, penicillin, and streptomycin. U87mg and Ul were grown in the same fashion, except RPM1 1640 medium was supplemented with 5% fetal bovine serum and 5% bovine calf serum supplemented with iron. Exponentially growing stock cultures were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. Under these conditions, the doubling times and plating efficiencies of the human tumor cell lines were 22 hr and 80-95% for A549, 33 hr and 20% for U87mg, and 33 hr and 50% for Ul , respectively. Cells were plated in 60 mm plastic culture dishes the day prior to experimentation in order for the cells to settle and attach to the plate before exposure to IdUrd and/or irradiation. The number of cells plated was adjusted to yield lo6 cells per dish at time of irradiation. Drug treatment IdUrd was supplied by the Drug Development Branch, NCI. A 10 mM stock solution in phosphate buffered solution (PBS) was made initially and fresh solutions of IdUrd in complete medium were made just prior to each experiment. After the cells had attached to the plates, the supernatant was removed from the dishes and approximately 5 ml of complete medium containing varying concentrations of IdUrd (0, 0.1, 1.0 and 10 pM) were added to the dishes for one and three cell cycles before irradiation. At these concentrations and times, IdUrd was not toxic and did not cause significant changes in growth rate for A549. The U87mg cell line exhibited no change in plating efficiency (PE); however, the growth rate progressively slowed after two cell cycles at 10 FM. The Ul melanoma cell line was the most sensitive to IdUrd, demonstrating a decrease in PE at 1 pM even at one cell cycle. Growth rate was unaffected by 5 10 pM IdUrd over these cell cycle times. Irradiation conditions Cell samples were irradiated using a Sheppard 137-cesium irradiator that produces gamma rays at 2.45 Gray per min. Dosimetry was carried out by TLD measurements using lithium fluoride crystals calibrated against standard sources and ion chambers. Analysis of survival curves After irradiation, the supernatant was removed and the cells were washed with PBS, trypsinized with STV, resus*Beckman
170 isotope detector.
Volume 22, Number 3, 1992
pended in complete medium and counted on a Model Zf Coulter Counter. A known quantity was re-plated in 60 or 100 mm dishes with 5 or 15 ml of normal medium at densities to yield 50-100 colonies. After incubation for 12 days, the plates were fixed in ethanol, stained with methylene blue, and scored for colony formation. Any colony consisting of 50 or greater cells was counted. Each survival point was plated in triplicate and repeated multiple times. Plating efficiency was calculated as the ratio of the number of colonies counted to the number of cells plated. Programs for the analysis of cellular survival data developed by N. Albright were used (1). The program weighted the data points, calculated the best fit to the linear quadrant (LQ) formula, and calculated the sensitizer enhancement ratio (SER) values with standard errors. The percent thymidine replacement versus SER and concentration of IdUrd times cycle time curves were fit by the TELLAGRAF linear regression program. Incorporation of IdUrd After IdUrd treatment, the cells were washed with PBS, trypsinized, centrifuged, and quick frozen. The amount of IdUrd incorporation into cellular DNA was analyzed using the 32P post-labelling procedure (2). Cellular DNA was isolated and enzymatically digested to 3’-monophosphate nucleotides that were then phosphorylated to 32P-5’, 3-bis phosphorylated nucleotides with gamma-32P-ATP, and polynucleotide kinase. The 3’-phosphate was removed by P-l nuclease to form 32P-5’-phosphorylated nucleotides. Mononucleotides were separated from 32P-ATP on an ion exchange column, and 32P-IdUrd-5’-monophosphate (IdUrdMP) was separated from the other 5’-monophosphates by high performance liquid chromatography. Peaks were identified with an in-line radiation detector.* The radioactivity in each monophosphate peak was summed and the percentage of each peak was calculated by dividing by the total radioactivity and multiplying by 100. Percent IdUrd substitution for thymidine monophosphate (TMP) was calculated as: % IdUrdMP % IdUrdMP
+ % TMP
x 100.
RESULTS Radiation survival curves based on clonogenic survival of the three human tumor lines exposed to varying concentrations (0, 0.1, 1 and 10 p.M) of IdUrd are shown in Figures l-6 for exposure times of one and three cell cycles. These have been fitted by the LQ formula. Individual data points have been omitted for clarity. Survival decreases as a function of radiation dose and as the concentration of IdUrd increases above 0.1 p,M for all the cell lines except
Iododeoxyuridine incorporation 0 V. AS49
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s
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491
UHL et al. USitrq+ Id&d
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--_
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.~
O.lun
\
“‘I
10
s "'I
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,J
10-4
20
16
0
Radiation Dose (Gyl
2
4
6
8
10
12
Radiation Dose (Gy) Fig. 1. Human lung adenocarcinoma A549 cell survival after irradiation with 137-Cs and 22 hr (one cell cycle) exposure to IdUrd.
A549,
in which
a concentration
greater
than
1 FM
is
needed at one cell cycle to see a decrease in survival. The SER values at 10% survival and percent thymidine replacement (%TR) for the three cell lines are shown in Table 1. Human lung adenocarcinoma A549 cells demonstrate radiosensitization when exposed to greater than 1 PM
Fig. 3. Human brain glioma U87 mg cell survival after irradiation with 137-Cs and 33 hr (one cell cycle) exposure to IdUrd.
IdUrd. Human glioma U87mg and melanoma Ul demonstrate radiosensitization when exposed to p.M. Maximal sensitization occurs with 10 p,M three cell cycles for all three cell lines: 1.61 t
U87m~ + ldllrd
AS49
+
Id&d
10&m
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----
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Cycles
0
’ ”
a
’ 6
”
~~..
Control
__
3 Call
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~~
‘\
' L""
16
20
Radiation Dose (Gy) Fig. 2. Human lung adenocarcinoma A549 cell survival after irradiation with 137-Cs and 66 hr (three cell cycles) exposure to
IdUrd .
o.,m
for ~_
. .
a n "",
10
----~
r
\ 1o-5
1oml ,m
cells can only 0.1 IdUrd at 0.17 for
1o-5L--
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8
10
12
Radiation Dose (Gy) Fig. 4. Human brain glioma U87 mg cell curvival after irradiation with 137-Cs and 99 hr (three cell cycles) exposure to IdUrd.
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I.J. Radiation Oncology 0 Biology 0 Physics
Volume 22, Number 3, 1992
Table 1. SER (10% survival + standard error) and % thymidine replacement (%TR) at one and three cell cycles for human lung cancer (A549), glioma (487 mg), and melanoma (Ul) cells treated with varying concentrations of IdUrd before irradiation A549 IdUrd Concentration One cell cycle 10 uM 1 uM 0.1 uM Three cell cycles 10 uM 1 uM 0.1 uM
U87 mg
Ul
SER c SE
%TR
SER 2 SE
%TR
SER + SE
%TR
1.26 k .14 1.12 IL .13 0.87 -r- .06
13.3 4.5 0.9
1.61 2 .17 1.22 k .16 1.39 ? .39
21.0 7.6 0.7
1.35 ? .lO 1.17 2 .07 1.08 k .lO
11.4 2.6 0.6
1.61 2 .17 0.99 k .09 0.94 f .09
22.4 7.6 2.3
1.63 c .09 1.07 k .lO 1.19 ? .lO
32.0 12.8 2.0
2.07 k .I0 1.34 k .06 1.18 ? .06
39.1 3.1 1.8
A549, 1.63 + 0.09 for U87mg, and 2.07 + 0.10 for Ul. The melanoma Ul line demonstrates the greatest sensitization and the highest %TR at three cell cycles. The degree of radiosensitization depends on the extent of IdUrd incorporation into the DNA. As the %TR increases, the SER values increase for all cell lines (Fig. 7), though not at the same rate. Ul appears to be more sensitive to a given %TR demonstrating SER’s above 1 for all levels of %TR. Just as the SER’s are maximum at three cell cycles for 10 PM IdUrd, the percent IdUrd replacement is also maximum at three cell cycles but across all concentrations for all three human tumor lines. The %TR increases to a maximum of 22.4% for A549, 32.0% for U87 mg, and 39.1% for Ul at 10 p,M. The incorporation of IdUrd is related to the concentration of the drug in the medium and the num-
Ul
+
1olm
IdUrd
for
1 Cell
ber of cell cycles (CXT). Overall, A549 demonstrated less incorporation than the other cell lines for the same CXT (Fig. 8). DISCUSSION Phillips et al. found that significant thymidine replacement can be achieved with IdUrd concentrations as low as 0.1 p,M for Chinese hamster V-79 cells. They found that the degree of enhancement is related to concentration times length of exposure of the cells to IdUrd as well as to %TR. Peak incorporation and SER required at least three cell cycles. The maximum thymidine relacement was 49% after 48 hr (six cell cycles) exposure to 30 FM yielding an SER of 2.7.
Cycle
Ul + (Om
----.-._ .- .-..___._
0
6
10
16
20
Radiation Dose (Gy) Fig. 5. Human melanoma Ul cell survival after irradiation with 137-Cs and 33 hr (one cell cycle) exposure to IdUrd.
6
Id&d for ----_
lun
---
O.lIm
-----~
10
3 Cell
Cycles
--~
16
Radiation Dose (Gy) Fig. 6. Human melanoma Ul cell survival after irradiation with 137~Cs and 99 hr (three cell cycles) exposure to IdUrd.
Iododeoxyuridineincorporation 0 V.
UHL
493
ef al.
CXT VS %TR
SER VS %TR
I
0
V U87mg
..
CONCENTRATION
Fig. 7. Sensitizer Enhancement Ratio (SER) at 10% survival as a function of percent Thymidine replacement (%TR) by IdUrd.
X CELL CYCLE TIME
Fig. 8. Concentration of IdUrd times cycle time (CXT in micromolar cycle time) as a function of percent Thymidine replacement (%TR) by IdUrd.
Lawrence et al. investigated the influence of the duration of exposure to 10 p,M IdUrd on incorporation into DNA and the resulting radiosensitization in cultured human colon cancer HT29 cells. In their system, incorporation of IdUrd plateaued at 30% after 4 days of exposure. Prolonging the time of exposure to 7 days increased cytotoxicity without affecting either incorporation or radiosensitization. They suggested that short, repeated exposures to IdUrd might produce sustained radiosensitization with less thrombocytopenia and myelosuppression than longer treatment periods. This approach was used by UCSF/NCCC in a recently completed trial of BrdUrd in malignant gliomas with some success (12, 15). In our study, the effect of pre-treatment with clinically achievable levels of IdUrd (O-10 PM) on three human tumor cell lines is compared by in vitro clonogenic survival. Significant thymidine replacement can be achieved in these cells and, as found by others (11, 14), peak incorporation and SER occurred at three cell cycles. There is variation between the cell lines in maximal %TR at a given concentration, but there are insufficient data to determine whether SER varies among the lines at similar %TR. Overall, A549 demonstrated less enhancement than the other two cell lines at all concentrations and cell cycles. The apparent resistance of these cells to sensitization, as well as their lower incorporation rates, is interesting. They are known to contain high concentrations of glutathione, which may make
them resistant, but this may not be the major cause (16). It may be possible that at low levels of IdUrd there is enhanced repair of potentially lethal damage that results in minimal sensitization or that at the lower concentrations of IdUrd, the A549 cells are depleting the medium of drug with time. The thymidine pathways in these cell lines have not been analyzed. However, differences in the enzyme activities (Km for thymidine kinase) or nucleotide pools may account for the lower %TR in A549 despite its shorter cell cycle time compared to Ul . Clinical trials should strive to obtain sustained plasma levels of 10 pM IdUrd for optimal radiosensitization. As suggested by Lawrence et al., multiple short exposures would present repeated opportunities for tumor cells to come into S phase and incorporate IdUrd with the potential to administer a higher concentration of IdUrd and thus provide a higher level of sensitization. This would be particularly important in tumors that do not demonstrate sensitization at low doses of IdUrd like the lung tumor line investigated in this paper. Due to the apparent variability of sensitization to IdUrd and to predict the usefulness of IdUrd in humans, it is going to be necessary to study the percent IdUrd incorporation and radiation response with and without IdUrd of a wide range of human tumor cells prepared directly from fresh tumor specimens after in vivo drug infusion.
REFERENCES Albright, N. Computer programs for the analysis of cellular survival data. Radiat. Res.l12:331-340; 1987. Bodell, W. J.; Rasmussen, J. A 32P postlabeling determining the incorporation of bromodeoxyuridine lular DNA. Anal. B&hem. 142:525-528; 1984. Djordjevic,
B.; Szybalski,
assay for into cel-
W. Genetics of human cell lines.
III. Incorporation of 5-bromo- and 5-iododeoxyuridine into the deoxyribonucleic acid of human cells and its effect on radiation sensitivity. J. Exp. Med. 112:509-531; 1960. 4. Erickson, R. L.; Szybalski, W. Molecular radiobiology of human cell lines. V. Comparative radiosensitizing properties of 5-halodeoxycytidines and 5-halodeoxyuridines. Radiat.
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Res. 20:252-262; 1963. 5. Jackson, D.; Kinsella, T.; Rowland, J.; Wright, D.; Katz, D.; Main, D.; Collins, J.; Komblith, P.; Glatstein, E. Halogenated pyrimidines as radiosensitizers in the treatment of glioblastoma multiforme. Am. J. Clin. Oncol. 10:437443; 1987. 6. Kelland, L. R.; Steel, G. G. Inhibition of recovery from damage induced by ionizing radiation in mammalian cells. Radiother. Oncol. 13:285-299; 1988. 7. Kinsella, T. J.; Dobson, P. P.; Mitchell, J. B.; Fomace, A. J. Enhancement of x ray induced DNA damage by pre-treatment with halogenated pyrimidine analogs. Int. J. Radiat. Oncol. Biol. Phys. 13:733-739; 1987. 8. Kinsella, T. J.; Glatstein, E. Clinical experience with intravenous radiosensitizers in unresectable sarcomas. Cancer 59: 908-915; 1987. 9. Kinsella, T. J.; Mitchell, J. B.; Russo, A.; Morstyn, G.; Glatstein, E. The use of halogenated thymidine analogs as clinical radiosensitizers: rationale, current status, and future prospects: non-hypoxic cell sensitizers. Int. J. Radiat. Oncol. Biol. Phys. 10:1399-1406; 1984. 10. Kinsella, T. J.; Russo, A.; Mitchell, J. B.; Collins, J. M.; Rowland, J.; Wright, D.; Glatstein, E. A Phase I study of intravenous iododeoxyuridine as a clinical radiosensitizer. Int. J. Radiat. Oncol. Biol. Phys. 11:1941-1946; 1985. 11. Lawrence, T. S.; Davis, M. A.; Maybaum, J.; Stetson, P. L.; Ensminger, W. D. The dependence of halogenated pyrimidine incorporation and radiosensitization on the duration of drug exposure. Int. J. Radiat. Oncol. Biol. Phys. 18:1393-1398; 1990. 12. Levin, V. A.; Wara, W. M.; Gutin, P. H.; Wilson, C. B.; Phillips, T. L.; Prados, M.; Flam, M. S.; Ahn, D. K. Initial
Volume 22, Number 3, 1992
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14.
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analysis of NCOG 6682-I: Bromodeoxyuridine (BUdR) during irradiation followed by CCNU, procarbazine, and vincristine (PCV) chemotherapy for malignant gliomas. Proc. Am. Sot. Clin. Oncol. 9:91; 1990. Linstadt, D.; Blakely, E.; Phillips, T. L.; Castro, J. R. Radiosensitization produced by iododeoxyuridine with high linear energy transfer heavy ion beams. Int. J. Radiat. Oncol. Phys. 15:703-710; 1988. Phillips, T. L.; Bodell, W. J.; Uhl, V.; Ross, G. Y.; Rasmussen, J.; Mitchell, J. B. Correlation of exposure time, concentration and incorporation of IdUrd in V-79 cells with radiation response. Int. J. Radiat. Oncol. Biol. Phys. 16: 1251-1255; 1989. Phillips, T. L.; Levin, V. A.; Ahn, D. K.; Gutin, P. H.; Davis, R. L.; Wilson, C. B.; Prados, M. D.; Wara, W. M.; Flam, M. S. Evaluation of bromodeoxyuridine in glioblastoma multiforme: A Northern California Cancer Center Phase II study. Int. J. Radiat. Oncol. Biol. Phys. 21:709-714; 1991. Phillips, T. L.; Mitchell, J. B.; de Graff, W.; Russo, A.; Glatstein, E. Variation in sensitizing efficiency for SR 2508 in human cells dependent on glutathione content. Int. J. Radiat. Oncol. Biol. Phys. 12:1627-1635; 1986. Piro, A. J.; Taylor, C. C.; Belli, J. A. Interaction between radiation and drug damage in mammalian cells. II. The effect of actinomycin-D on the repair of sublethal radiation damage in plateau phase cells. Cancer 37:2697-2702; 1976. Prusoff, W. F. Synthesis and biological activities of iododeoxyuridine, an analog of thymidine. Biochem. Biophys. Acta. 32:295-296; 1959. Szybalski, W. X-ray sensitization by halopyrimidines. Cancer Chemother. Rep. 58:539-557; 1974.
