Kinetic Response of Cultured Human Lymphoid Cells to Rubidazone 1, 2 Barthel Barlogie, 3 Benjamin Drewinko, 4 Robert S. Benjamin, 3, 5 and Ti Li Loo 3, 6
ABSTRACT-Analysis of rubldazone, the benzoylhydrazone de· rivatlve of daunorublcln, for Its eHects on cell cycle progression of a human lymphoid cell line showed a kinetic response pattern similar to that of adrlamycln. Thus rubldazone Induced a G2 block, the magnitude and duration of which were dependent on concentration and Incubation time. However, In contrast to adrlamycln, a marked phase-dependent sensitivity for the Induction of G2 -accumulatlon was observed; cells treated In early and midS-phase were most sensitive. This age-dependent kinetic response may account for the smaller G2 -accumulatlon In asynchronous cultures and the closer correlation of the magnitude of this kinetic eHect with concentration and duration of rubldazone treatment. Prolonged exposure to high concentrations of rubldazone also delayed the traverse through G, and/or the G,-S transition, whereas the S-phase transit was not Impaired. Interference with cell cycle progression through G, Into S-phase caused a stepwise accumulation of cells In G2 -phase.-J Natl Cancer Inst 60: 279-282, 1978.
The anthracycline antibiotics daunorubicin and ADR have considerably advanced treatment of solid tumors and leukemias (1-3), but dosage is limited by the cardiotoxicity of these agents (4). Conjugation of both daunorubicin and ADR to DNA may lessen the cardiac side effects and permit administration of higher doses (5, 6). However, a differential effect of lysosomotropic compounds such as these on tumor versus normal cells has not yet been substantiated in clinical trials (7). Recently, the effectiveness of the benzoylhydrazone derivative of daunorubicin, RU, has been investigated in vitro, in animal tumor systems, and in human leukemia and solid tumors (8-11). Studies by Jacquillat et al. (10) suggest that RU is at least as effective as daunorubicin for treatment of acute leukemia, but less toxic for normal hematopoietic cells. Recent reports by Benjamin et al. (12) and Keating et al. (13) demonstrate that RU is highly active in the treatment of patients with acute leukemia who are over 50 years of age. In animal toxicology studies at the National Cancer Institute (14), RU appeared to be less cardiotoxic than daunorubicin and adriamycin both on a mg/m 2 basis and in terms of time to cardiotoxicity. Previously, we reported the effects of ADR and the ADR-DNA complex on the cell cycle traverse of a human lymphoma cell line (15, 16). We have used the same concentration of ADR in the complex and demonstrated that, in contrast to free ADR, the lysosomotropic complex displays a marked phase sensitivity for the induction of G 2-block (16). In this paper we show that, like ADR and the ADR-DNA complex, RU induces an effective G 2-block, the magnitude and duration of which are dependent on concentration and exposure time. The induction of G 2-block is a function VOL. 60, NO.2, FEBRUARY 1978
of cell age; cells in early and mid-S-phase are most sensitive to this kinetic effect of RU. In addition, long incubation (~12 hr) with high concentrations (~0.5 ILg/ ml) of R U reversibly blocks cell cycle progression in G t and/or the Gt-S transition.
MATERIALS AND METHODS Our studies were done on cultured human lymphoid cells (Trcells) (17) grown as monolayer cultures in Ham's FlO medium supplemented with 20% fetal calf serum, vitamins, and antibiotics. With the percent-labeled mitoses technique, the cell cycle time was determined to be 31 hours, with t Gt =15 hours, ts=lO hours, and t G2 =6 hours (18). Drug-induced perturbation effects of the mitotic cycle were analyzed through sequential DNA histograms produced by a pulse cytophotometer ICP 11 (Phywe Co., Gottingen, Germany) (19). After ethanol fixation, cell suspensions were stained with 50 ILg mithramycin/ml for 5 minutes (20, 21). For each DNA histogram, 50,000-100,000 cells were measured; the histogram evaluation was done with a modification of Fried's model (22, 23). The standard deviation between duplicate samples for compartment distribution in Gt~, So, and G+ M phases did not exceed 2.5%. The effects of drug concentration and incubation time on cell cycle perturbation by RU were investigated in asynchronous populations in exponential phase of growth. Monolayer cells were harvested at intervals of 6 to 24 hours both during drug incubation and after drug release (two washings with Hanks' balanced salt solution and renewal of medium) for a total of 6-7 days from drug addition. The influence of cell age on the kinetic response pattern was examined in TdR-synchronized cultures (single 3-mM TdR block for 24 hours) (21, 24); at regular intervals after release of the TdR block, Tt-cells were incubated for 1 hour with 1 ILg RU/ ABBREVIATIONS USED: RU = rubidazone; ADR = adriamycin; TdR = thymidine; Do = mean lethal dose necessary to reduce survivors to 37%; PCP = pulse cytophotometry. , Received June 13, 1977; accepted August 12, 1977. Supported by Public Health Service (PHS) contract NOI-CM53773 from the Division of Cancer Treatment, National Cancer Institute (NCI), and by PHS grant CA14528 from the NCI. 3 Department of Developmental Therapeutics, University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Avenue, Houston, Tex. 77030. 4 Department of Laboratory Medicine, University of Texas System Cancer Center. • Junior Faculty Fellow of the American Cancer Society. 6 We thank Susan Sumners for technical help and Pearl Brown for secretarial assistance. 2
279
J NATL CANCER INST
BARLOGIE, DREWINKO, BENJAMIN, AND LOO
280
ml, and DNA histogram analyses were done over a period of 7 days after TdR release. RU, supplied by the Societe des Usines Chimiques Rhone-Poulenc, was dissolved in 0.9% NaCI immediately before each experiment. Appropriate dilutions were made with growth medium.
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TJ-cells in exponential phase of growth were exposed to increasing concentrations of RU for 1,3,12,24, and 48 hours. Treatment with 0.05 Itg/ml for 2=24 hours produced a transient accumulation of 38% of the cells in the G 2 + M compartment 32 hours after drug addition, mainly at the expense of the GJ~ fraction. The mitotic index never exceeded control values in this and all subsequent experiments, so that the G 2 + M fraction represented a virtually pure G 2 -population. Treatment of TJ-cells with 0.1 Itg RU/ml for 1 and 3 hours did not produce significant compartment shifts. Exposure for 2= 12 hours increased G 2 -accumulations as a function of increasing incubation time (text-fig. 1).
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Hours (O=time of drug addition) i.-PCP distribution in G, and G 2 of asynchronous T,lymphoma cells treated with 0.1 ILg RU/ml for different exposure times. Solid lines indicate continuous presence of the drug; broken lines connect values obtained after drug release. RU induces accumulation of cells in G 2 at the expense of the G1-
ABSTRACT-Analysis of rubldazone, the benzoylhydrazone de· rivatlve of daunorublcln, for Its eHects on cell cycle progression of a human lymphoid cell line showed a kinetic response pattern similar to that of adrlamycln. Thus rubldazone Induced a G2 block, the magnitude and duration of which were dependent on concentration and Incubation time. However, In contrast to adrlamycln, a marked phase-dependent sensitivity for the Induction of G2 -accumulatlon was observed; cells treated In early and midS-phase were most sensitive. This age-dependent kinetic response may account for the smaller G2 -accumulatlon In asynchronous cultures and the closer correlation of the magnitude of this kinetic eHect with concentration and duration of rubldazone treatment. Prolonged exposure to high concentrations of rubldazone also delayed the traverse through G, and/or the G,-S transition, whereas the S-phase transit was not Impaired. Interference with cell cycle progression through G, Into S-phase caused a stepwise accumulation of cells In G2 -phase.-J Natl Cancer Inst 60: 279-282, 1978.
The anthracycline antibiotics daunorubicin and ADR have considerably advanced treatment of solid tumors and leukemias (1-3), but dosage is limited by the cardiotoxicity of these agents (4). Conjugation of both daunorubicin and ADR to DNA may lessen the cardiac side effects and permit administration of higher doses (5, 6). However, a differential effect of lysosomotropic compounds such as these on tumor versus normal cells has not yet been substantiated in clinical trials (7). Recently, the effectiveness of the benzoylhydrazone derivative of daunorubicin, RU, has been investigated in vitro, in animal tumor systems, and in human leukemia and solid tumors (8-11). Studies by Jacquillat et al. (10) suggest that RU is at least as effective as daunorubicin for treatment of acute leukemia, but less toxic for normal hematopoietic cells. Recent reports by Benjamin et al. (12) and Keating et al. (13) demonstrate that RU is highly active in the treatment of patients with acute leukemia who are over 50 years of age. In animal toxicology studies at the National Cancer Institute (14), RU appeared to be less cardiotoxic than daunorubicin and adriamycin both on a mg/m 2 basis and in terms of time to cardiotoxicity. Previously, we reported the effects of ADR and the ADR-DNA complex on the cell cycle traverse of a human lymphoma cell line (15, 16). We have used the same concentration of ADR in the complex and demonstrated that, in contrast to free ADR, the lysosomotropic complex displays a marked phase sensitivity for the induction of G 2-block (16). In this paper we show that, like ADR and the ADR-DNA complex, RU induces an effective G 2-block, the magnitude and duration of which are dependent on concentration and exposure time. The induction of G 2-block is a function VOL. 60, NO.2, FEBRUARY 1978
of cell age; cells in early and mid-S-phase are most sensitive to this kinetic effect of RU. In addition, long incubation (~12 hr) with high concentrations (~0.5 ILg/ ml) of R U reversibly blocks cell cycle progression in G t and/or the Gt-S transition.
MATERIALS AND METHODS Our studies were done on cultured human lymphoid cells (Trcells) (17) grown as monolayer cultures in Ham's FlO medium supplemented with 20% fetal calf serum, vitamins, and antibiotics. With the percent-labeled mitoses technique, the cell cycle time was determined to be 31 hours, with t Gt =15 hours, ts=lO hours, and t G2 =6 hours (18). Drug-induced perturbation effects of the mitotic cycle were analyzed through sequential DNA histograms produced by a pulse cytophotometer ICP 11 (Phywe Co., Gottingen, Germany) (19). After ethanol fixation, cell suspensions were stained with 50 ILg mithramycin/ml for 5 minutes (20, 21). For each DNA histogram, 50,000-100,000 cells were measured; the histogram evaluation was done with a modification of Fried's model (22, 23). The standard deviation between duplicate samples for compartment distribution in Gt~, So, and G+ M phases did not exceed 2.5%. The effects of drug concentration and incubation time on cell cycle perturbation by RU were investigated in asynchronous populations in exponential phase of growth. Monolayer cells were harvested at intervals of 6 to 24 hours both during drug incubation and after drug release (two washings with Hanks' balanced salt solution and renewal of medium) for a total of 6-7 days from drug addition. The influence of cell age on the kinetic response pattern was examined in TdR-synchronized cultures (single 3-mM TdR block for 24 hours) (21, 24); at regular intervals after release of the TdR block, Tt-cells were incubated for 1 hour with 1 ILg RU/ ABBREVIATIONS USED: RU = rubidazone; ADR = adriamycin; TdR = thymidine; Do = mean lethal dose necessary to reduce survivors to 37%; PCP = pulse cytophotometry. , Received June 13, 1977; accepted August 12, 1977. Supported by Public Health Service (PHS) contract NOI-CM53773 from the Division of Cancer Treatment, National Cancer Institute (NCI), and by PHS grant CA14528 from the NCI. 3 Department of Developmental Therapeutics, University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Avenue, Houston, Tex. 77030. 4 Department of Laboratory Medicine, University of Texas System Cancer Center. • Junior Faculty Fellow of the American Cancer Society. 6 We thank Susan Sumners for technical help and Pearl Brown for secretarial assistance. 2
279
J NATL CANCER INST
BARLOGIE, DREWINKO, BENJAMIN, AND LOO
280
ml, and DNA histogram analyses were done over a period of 7 days after TdR release. RU, supplied by the Societe des Usines Chimiques Rhone-Poulenc, was dissolved in 0.9% NaCI immediately before each experiment. Appropriate dilutions were made with growth medium.
G,
100 ~
12 } Hours of
*
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~~:
80
Incubation
"E
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...
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RESULTS
Q)
a..
Asynchronous Cells
TJ-cells in exponential phase of growth were exposed to increasing concentrations of RU for 1,3,12,24, and 48 hours. Treatment with 0.05 Itg/ml for 2=24 hours produced a transient accumulation of 38% of the cells in the G 2 + M compartment 32 hours after drug addition, mainly at the expense of the GJ~ fraction. The mitotic index never exceeded control values in this and all subsequent experiments, so that the G 2 + M fraction represented a virtually pure G 2 -population. Treatment of TJ-cells with 0.1 Itg RU/ml for 1 and 3 hours did not produce significant compartment shifts. Exposure for 2= 12 hours increased G 2 -accumulations as a function of increasing incubation time (text-fig. 1).
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Hours (O=time of drug addition) i.-PCP distribution in G, and G 2 of asynchronous T,lymphoma cells treated with 0.1 ILg RU/ml for different exposure times. Solid lines indicate continuous presence of the drug; broken lines connect values obtained after drug release. RU induces accumulation of cells in G 2 at the expense of the G1-
