fnr.
./. Rudrurirm
” Pergamon
Ot~co/~~g~
Press Ltd.,
Bid.
P/TM.,,
1979. Printed
036@3016/79/081305-04$02.00/O
Vol. 5, pp. 1305-1308 I” the U.S.A.
??Adriamycin
EFFECT OF ADRIAMYCIN IN VZVO ON SYNCHRONIZED MURINE FIBROSARCOMA CELLS-t
CYTOTOXIC
DAVID J. GRDINA, Ph.D., CURTIS P. SIGDESTAD, Ph.D.* and JEAN A. JOVONOVICH, B.A. Section of Experimental Radiotherapy, The University of Texas System Cancer Center M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Drive, Houston, Texas 77030 The cytotoxic effect of Adriamycin (ADR) on synchronized fibrosarcoma (FSa) cells lodged in the lungs of specific pathogen free C,Hf/Bu mice was determined. FSa cells from primary asynchronous cultures were separated and synchronized on the basis of cell size by centrifugal elutriation. Cell cycle parameters were determined by flow micro-fluorometry (FMF). Viable tumor cells from each elutriator fraction were injected intravenously into recipient mice. Twenty minutes later, ADR (10 mg/kg) was administered intravenously into half of the animals in each group. After fourteen days the animals were killed, and the resulting lung colonies were counted. Under these conditions, the S-phase enriched populations were found to be the most sensitive to ADR. Cell survival ranged from a maximum of 8% to a minimum of 0.3% for the GI and S-phase enriched populations, respectively. Dose response curves were also determined for selected populations treated in vivo. While no great difference in Do was observed between the survival curves representing each of these populations (range, 0.65-0.71 mg/kg), the extrapolation numbers varied from 0.41 (51% S-phase) to 2.1 (9% S-phase). Cytotoxicity,
Adriamycin,
Murine fibrosarcoma
cells.
Tumor System: Female C,Hf/Bu mice, 10 to 12 weeks old, and a methylcholanthrene-induced FSa were used in this study. Single cell suspensions were prepared by mincing and trypsinization, and cell viability, determined by phase contrast microscopy, was found routinely to be greater than 9.5%.” To reduce tumor cell heterogeneity and better prepare cells for centrifugal elutriation, the cell suspension was incubated in vitro for 48 hr.2 Cell Separation: FSa cells (3x 10R in exponential growth) were separated and synchronized at 4°C by
centrifugal elutriation using a Beckman JE-6 elutriator rotor.’ Centrifugation speed was kept constant (- 1200 RPM) and medium flow rates were increased by increments of 2 ml/mm In this manner, 12 fractions were routinely collected. Cells in each fraction were characterized with respect to size (Coulter counter and Channelyzer II) and DNA content (flow microfluorometer).’ Lung Colony Assay: Mice, with hind legs shielded, were whole body irradiated with 1000 rad. Twenty-four hours later these animals were injected with 6x lo3 viable FSa cells from each of the elutriator fractions along with 10” heavily irradiated (10,000 rad) FSa cells. After 14 days the mice were killed, lungs removed, and colonies counted. Adriamycin Testing in viva: Adriamycin was made up with sodium chloride injection to give a final concentration of 1 mgicc. To characterize its cytotoxic effect on synchronized FSa cells in viva, ADR was injected intravenously at a concentration of 10 mgikg body weight 20 min following tumor cell injection. Drug was injected into half of the animals in each experimental group. Under these conditions, no drug toxicity was observed in the recipient animals. Selected fractions of cells were exposed to
tThis investigation was supported in part by grant numbers CA-18628, CA-23270, and CA-06294, awarded by the National Cancer Institute, DHEW.
*Present address: Curtis P. Sigdestad, Ph.D., University of Louisville, Department of Therapeutic Radiology, Radiation Center, Louisville, Kentucky 40202
INTRODUCTION We have previously described a new and rapid procedure for characterizing the phase specificity of chemotherapeutic drugs in vi~o.~ In this communica-
tion, we describe the application of this method to determine the cytotoxic effect of ADR on synchronized murine fibrosarcoma (FSa) cells arrested in the lungs of C,,Hf/Bu mice. The tumor cells were separated and synchronized by centrifugal elutriation and characterized with respect to cell age distribution by flow microfluorometry analysis. MATERIALS
AND
METHODS
1305
1306
Radiation Oncology 0 Biology 0 Physics
different concentrations of drug in ~$0, and dose response curves were constructed. The untreated control animals were used to compare to treated animals and calculate percent surviving cells. RESULTS The recovery of cells following centrifugal elutriation was routinely greater than 95%. The relative synchrony of cells recovered in each fraction is presented in Figure 1. No normal cells, as determined by FMF analysis, were detected in any of the recovered elutriator fractions.z The lung cloning efficiencies in these experiments ranged from 1.4 to 1.9% (83 to 115 colonies, respectively). The effect of ADR on synchronized FSa populations lodged in the lungs of test animals was characterized (see Figure 1). While ADR was effective to varying degrees in reducing the number of lung colonies in all experimental groups, it was most effective against the cell population containing the greatest proportion of S-phase cells (i.e., fraction 9). To further characterize the effectiveness of ADR in viva, unseparated cells (USC) and selected elutriator fractions of FSa cells, designated fractions A, B, and C, were injected into mice and dose response curves were determined (see Figure 2). No significant difference in D,, was observed between any of the populations (range of 0.65 to 0.71 mgikg). The extrapolation numbers, however, varied significantly from 0.41 (51% S-phase cells) to 2.1 (9% S-phase cells). DISCUSSION The cytotoxic effect of ADR on various synchronized murine fibrosarcoma cells lodged in the lungs of C,Hf/Bu mice has been characterized. Cell synchrony was achieved by centrifugal elutriation. This method is rapid and effective for isolating relatively pure populations of G, cells on the basis of size. While populations of S and G, phase cells were also significantly enriched by this procedure, they were not isolated free from G, cell contamination. Whole body irradiated mice and heavily irradiated tumor cells were used in the lung colony procedure. Under these conditions, tumor cell retention and colony forming efficiency is independent of either cell size or position within the division cycle.3 Finally, ADR was injected intravenously because this route of administration has been reported to be the most effective against animals bearing pulmonary tumor colonies.6 Following injection, ADR is rapidly cleared from the plasma, deposited in tissues, such as the lung, and excreted slowly.‘. Cell killing by ADR, as evidenced by a reduction in lung colony number in treated as compared to untreated animals, increased with increasing propor-
August 1979, Volume 5, Number 8
Adriamycin
(10 mglkg I.V.)
10 1
PUSC 1:
80 60 40 20 3
4
5
6
Elutriator
7
8
91011
Fraction Number
FIG. 1. The percent of surviving FSa cells following exposure in V~IYIto adriamycin (ADR; top) and the percent of cells distributed among the various cell.cycle phases (FMF analysis; bottom) are plotted as a function of elutriator fraction number for comparison. USC refers to unseparated control cells. The vertical bars represent 1 standard error of the mean.
tion of S-phase cells present in each of the cell fractions. While cell killing was observed in all cell fractions, the greatest effect was seen for cells collected in fraction 9. This fraction contained the largest percentage of S-phase cells (i.e., 74%). These data compare favorably with data acquired using in vitro cell systems.” The effects of drug induced progression delay on cells and drug retention in lung tissue on eventual lung colony formation is at present unclear. Because ADR is not metabolized in mice, its cytotoxic activity should be expressed immediately following injection.’ At this time, under the conditions described, the access of drug to tumor cells is presumably maximized. Finally, the relative increased sensitivity of S-phase FSa cells to ADR following in vivo exposure is also reflected in the reduced extrapolation numbers determined from the dose response curves presented in Figure 2. However, because of contamina-
Adriamycin
and synchronized
murine
fibrosarcoma
cells
0 GRDINA
c/ rrl.
1307
Adrlamycin In VW0 Exposure
2 4
6
6 10 12
2 4
MglKg
2
4 6
6 10 12
2
MglKg
6
6 10 12
6
6 10 12
I.V.
4
I.V.
FIG. 2. The response of selected FSa tumor populations, treated while retained in the lungs of C,Hf/Bu mice, to different doses of adriamycin is described. Populations are designated USC for unseparated control cells, and A, B, and C for selected elutriator separated cells. D,,‘s and extrapolation numbers (n), along with associated standard errors of the mean, determined by computer analysis, are: USC: D,,, 0.71 (0.74, 0.68); n, 0.78 (1.12, 0.54). A: D,,, 0.65 (0.72, 0.59); n, 2.1 (4.17, 0.97). B: D,,, 0.68 (0.73, 0.63); n, 0.65 (1.19, 0.36). C: D,,, 0.66 (0.73, 0.59): n, 0.41 (0.93, 0.18). Vertical error bars represent 1 standard error of the mean.
tion by relatively drug resistant G, cells in each of the elutriator fractions (see Figure l), the Do’s of the survival curves did not vary significantly from one another. This is expected since at the higher doses, the major contribution to the survivors is provided by the most resistant fraction of cells. In conclusion, we have described the usefulness
of a new and rapid procedure for characterizing the phase specificity of chemotherapeutic drugs in view. In this manner it has been demonstrated that while ADR is effective in vivo against cells in all phases of the cell cycle, it is most effective against S-phase cells.
1308
Radiation Oncology 0 Biology 0 Physics
August 1979, Volume 5, Number 8
REFERENCES I. DiFronzo, G., Gambetta, R.A., Lenaz, L.: Distribution and metabolism of adriamycin in mice. Comparison with daunomycin. Rev. Europ. Etudes Clin. et Biol. 16: 572-576, 197 1. 2. Grdina, D.J., Peters, L.J., Jones, S., Chan, E.: Separation of cells from a murine fibrosarcoma on the basis of size. I. Relationship between cell size and age as modified by growth in \Av or in vitro. .I. Natl. Cancer Inst. 61: 209-214, 1978. 3. Grdina, D.J., Peters, L.J., Jones, S., Chart, E.: Separation of cells from a murine fibrosarcoma on the basis of size. II. Differential effects of cell size and age on lung
retention and colony formation in normal and preconditioned mice. .I. Nat/. Cancer Inst. 61: 215-220, 1978. 4. Grdina, D.J., Sigdestad, C.P., Peters, L.J.: Phasespecific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma cells synchronized by centrifugal elutriation. Br. J. Cuncer 39: 152-158, 1979. 5. Kim, S.H., Kim, J.H.: Lethal effect of adriamycin on the division cycle of HeLa cells. Cancer Research 32: 323-325, 1972. 6. Shaeffer, J., Attanasio, M.A., El-Mahdi, A.M.: Route dependence of adriamycin effectiveness in murine osteosarcoma. Cancer Letters 3: 237-241, 1977.
./. Rudrurirm
” Pergamon
Ot~co/~~g~
Press Ltd.,
Bid.
P/TM.,,
1979. Printed
036@3016/79/081305-04$02.00/O
Vol. 5, pp. 1305-1308 I” the U.S.A.
??Adriamycin
EFFECT OF ADRIAMYCIN IN VZVO ON SYNCHRONIZED MURINE FIBROSARCOMA CELLS-t
CYTOTOXIC
DAVID J. GRDINA, Ph.D., CURTIS P. SIGDESTAD, Ph.D.* and JEAN A. JOVONOVICH, B.A. Section of Experimental Radiotherapy, The University of Texas System Cancer Center M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Drive, Houston, Texas 77030 The cytotoxic effect of Adriamycin (ADR) on synchronized fibrosarcoma (FSa) cells lodged in the lungs of specific pathogen free C,Hf/Bu mice was determined. FSa cells from primary asynchronous cultures were separated and synchronized on the basis of cell size by centrifugal elutriation. Cell cycle parameters were determined by flow micro-fluorometry (FMF). Viable tumor cells from each elutriator fraction were injected intravenously into recipient mice. Twenty minutes later, ADR (10 mg/kg) was administered intravenously into half of the animals in each group. After fourteen days the animals were killed, and the resulting lung colonies were counted. Under these conditions, the S-phase enriched populations were found to be the most sensitive to ADR. Cell survival ranged from a maximum of 8% to a minimum of 0.3% for the GI and S-phase enriched populations, respectively. Dose response curves were also determined for selected populations treated in vivo. While no great difference in Do was observed between the survival curves representing each of these populations (range, 0.65-0.71 mg/kg), the extrapolation numbers varied from 0.41 (51% S-phase) to 2.1 (9% S-phase). Cytotoxicity,
Adriamycin,
Murine fibrosarcoma
cells.
Tumor System: Female C,Hf/Bu mice, 10 to 12 weeks old, and a methylcholanthrene-induced FSa were used in this study. Single cell suspensions were prepared by mincing and trypsinization, and cell viability, determined by phase contrast microscopy, was found routinely to be greater than 9.5%.” To reduce tumor cell heterogeneity and better prepare cells for centrifugal elutriation, the cell suspension was incubated in vitro for 48 hr.2 Cell Separation: FSa cells (3x 10R in exponential growth) were separated and synchronized at 4°C by
centrifugal elutriation using a Beckman JE-6 elutriator rotor.’ Centrifugation speed was kept constant (- 1200 RPM) and medium flow rates were increased by increments of 2 ml/mm In this manner, 12 fractions were routinely collected. Cells in each fraction were characterized with respect to size (Coulter counter and Channelyzer II) and DNA content (flow microfluorometer).’ Lung Colony Assay: Mice, with hind legs shielded, were whole body irradiated with 1000 rad. Twenty-four hours later these animals were injected with 6x lo3 viable FSa cells from each of the elutriator fractions along with 10” heavily irradiated (10,000 rad) FSa cells. After 14 days the mice were killed, lungs removed, and colonies counted. Adriamycin Testing in viva: Adriamycin was made up with sodium chloride injection to give a final concentration of 1 mgicc. To characterize its cytotoxic effect on synchronized FSa cells in viva, ADR was injected intravenously at a concentration of 10 mgikg body weight 20 min following tumor cell injection. Drug was injected into half of the animals in each experimental group. Under these conditions, no drug toxicity was observed in the recipient animals. Selected fractions of cells were exposed to
tThis investigation was supported in part by grant numbers CA-18628, CA-23270, and CA-06294, awarded by the National Cancer Institute, DHEW.
*Present address: Curtis P. Sigdestad, Ph.D., University of Louisville, Department of Therapeutic Radiology, Radiation Center, Louisville, Kentucky 40202
INTRODUCTION We have previously described a new and rapid procedure for characterizing the phase specificity of chemotherapeutic drugs in vi~o.~ In this communica-
tion, we describe the application of this method to determine the cytotoxic effect of ADR on synchronized murine fibrosarcoma (FSa) cells arrested in the lungs of C,,Hf/Bu mice. The tumor cells were separated and synchronized by centrifugal elutriation and characterized with respect to cell age distribution by flow microfluorometry analysis. MATERIALS
AND
METHODS
1305
1306
Radiation Oncology 0 Biology 0 Physics
different concentrations of drug in ~$0, and dose response curves were constructed. The untreated control animals were used to compare to treated animals and calculate percent surviving cells. RESULTS The recovery of cells following centrifugal elutriation was routinely greater than 95%. The relative synchrony of cells recovered in each fraction is presented in Figure 1. No normal cells, as determined by FMF analysis, were detected in any of the recovered elutriator fractions.z The lung cloning efficiencies in these experiments ranged from 1.4 to 1.9% (83 to 115 colonies, respectively). The effect of ADR on synchronized FSa populations lodged in the lungs of test animals was characterized (see Figure 1). While ADR was effective to varying degrees in reducing the number of lung colonies in all experimental groups, it was most effective against the cell population containing the greatest proportion of S-phase cells (i.e., fraction 9). To further characterize the effectiveness of ADR in viva, unseparated cells (USC) and selected elutriator fractions of FSa cells, designated fractions A, B, and C, were injected into mice and dose response curves were determined (see Figure 2). No significant difference in D,, was observed between any of the populations (range of 0.65 to 0.71 mgikg). The extrapolation numbers, however, varied significantly from 0.41 (51% S-phase cells) to 2.1 (9% S-phase cells). DISCUSSION The cytotoxic effect of ADR on various synchronized murine fibrosarcoma cells lodged in the lungs of C,Hf/Bu mice has been characterized. Cell synchrony was achieved by centrifugal elutriation. This method is rapid and effective for isolating relatively pure populations of G, cells on the basis of size. While populations of S and G, phase cells were also significantly enriched by this procedure, they were not isolated free from G, cell contamination. Whole body irradiated mice and heavily irradiated tumor cells were used in the lung colony procedure. Under these conditions, tumor cell retention and colony forming efficiency is independent of either cell size or position within the division cycle.3 Finally, ADR was injected intravenously because this route of administration has been reported to be the most effective against animals bearing pulmonary tumor colonies.6 Following injection, ADR is rapidly cleared from the plasma, deposited in tissues, such as the lung, and excreted slowly.‘. Cell killing by ADR, as evidenced by a reduction in lung colony number in treated as compared to untreated animals, increased with increasing propor-
August 1979, Volume 5, Number 8
Adriamycin
(10 mglkg I.V.)
10 1
PUSC 1:
80 60 40 20 3
4
5
6
Elutriator
7
8
91011
Fraction Number
FIG. 1. The percent of surviving FSa cells following exposure in V~IYIto adriamycin (ADR; top) and the percent of cells distributed among the various cell.cycle phases (FMF analysis; bottom) are plotted as a function of elutriator fraction number for comparison. USC refers to unseparated control cells. The vertical bars represent 1 standard error of the mean.
tion of S-phase cells present in each of the cell fractions. While cell killing was observed in all cell fractions, the greatest effect was seen for cells collected in fraction 9. This fraction contained the largest percentage of S-phase cells (i.e., 74%). These data compare favorably with data acquired using in vitro cell systems.” The effects of drug induced progression delay on cells and drug retention in lung tissue on eventual lung colony formation is at present unclear. Because ADR is not metabolized in mice, its cytotoxic activity should be expressed immediately following injection.’ At this time, under the conditions described, the access of drug to tumor cells is presumably maximized. Finally, the relative increased sensitivity of S-phase FSa cells to ADR following in vivo exposure is also reflected in the reduced extrapolation numbers determined from the dose response curves presented in Figure 2. However, because of contamina-
Adriamycin
and synchronized
murine
fibrosarcoma
cells
0 GRDINA
c/ rrl.
1307
Adrlamycin In VW0 Exposure
2 4
6
6 10 12
2 4
MglKg
2
4 6
6 10 12
2
MglKg
6
6 10 12
6
6 10 12
I.V.
4
I.V.
FIG. 2. The response of selected FSa tumor populations, treated while retained in the lungs of C,Hf/Bu mice, to different doses of adriamycin is described. Populations are designated USC for unseparated control cells, and A, B, and C for selected elutriator separated cells. D,,‘s and extrapolation numbers (n), along with associated standard errors of the mean, determined by computer analysis, are: USC: D,,, 0.71 (0.74, 0.68); n, 0.78 (1.12, 0.54). A: D,,, 0.65 (0.72, 0.59); n, 2.1 (4.17, 0.97). B: D,,, 0.68 (0.73, 0.63); n, 0.65 (1.19, 0.36). C: D,,, 0.66 (0.73, 0.59): n, 0.41 (0.93, 0.18). Vertical error bars represent 1 standard error of the mean.
tion by relatively drug resistant G, cells in each of the elutriator fractions (see Figure l), the Do’s of the survival curves did not vary significantly from one another. This is expected since at the higher doses, the major contribution to the survivors is provided by the most resistant fraction of cells. In conclusion, we have described the usefulness
of a new and rapid procedure for characterizing the phase specificity of chemotherapeutic drugs in view. In this manner it has been demonstrated that while ADR is effective in vivo against cells in all phases of the cell cycle, it is most effective against S-phase cells.
1308
Radiation Oncology 0 Biology 0 Physics
August 1979, Volume 5, Number 8
REFERENCES I. DiFronzo, G., Gambetta, R.A., Lenaz, L.: Distribution and metabolism of adriamycin in mice. Comparison with daunomycin. Rev. Europ. Etudes Clin. et Biol. 16: 572-576, 197 1. 2. Grdina, D.J., Peters, L.J., Jones, S., Chan, E.: Separation of cells from a murine fibrosarcoma on the basis of size. I. Relationship between cell size and age as modified by growth in \Av or in vitro. .I. Natl. Cancer Inst. 61: 209-214, 1978. 3. Grdina, D.J., Peters, L.J., Jones, S., Chart, E.: Separation of cells from a murine fibrosarcoma on the basis of size. II. Differential effects of cell size and age on lung
retention and colony formation in normal and preconditioned mice. .I. Nat/. Cancer Inst. 61: 215-220, 1978. 4. Grdina, D.J., Sigdestad, C.P., Peters, L.J.: Phasespecific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma cells synchronized by centrifugal elutriation. Br. J. Cuncer 39: 152-158, 1979. 5. Kim, S.H., Kim, J.H.: Lethal effect of adriamycin on the division cycle of HeLa cells. Cancer Research 32: 323-325, 1972. 6. Shaeffer, J., Attanasio, M.A., El-Mahdi, A.M.: Route dependence of adriamycin effectiveness in murine osteosarcoma. Cancer Letters 3: 237-241, 1977.
