Leukemia Research Vol. 16, No. 5, pp. 491495, 1992. Printed in Great Britain.
0145-2126/92 $5.00 + .00 © 1992 Pergamon Press Ltd
THE LINKING OF A N T I C A N C E R DRUGS, CELL CYCLE BLOCKS, A N D D I F F E R E N T I A T I O N : IMPLICATIONS IN THE S E A R C H F O R ANTINEOPLASTIC D R U G S RICHARD D. DINNEN and KANEY EBISUZAKI Departments of Microbiology and Immunology and Biochemistry, Health Sciences Centre, The University of Western Ontario, London, Ontario, Canada
(Received 30 September 1991. Revision accepted 6 February 1992) Abstract--The quest for anticancer drugs has been primarily directed at agents that interfere with cell replication, yet the basis for drug-induced cytotoxicity remains unsolved. In our previous studies we noted a relationship between a mitotic block and commitment to terminal differentiation in the murine (Friend) erythroleukemia (FEL) cell. Since anticancer drugs are known to often block cell cycle transit typically in G2/mitosis, we tested a number of anticancer drugs with various modes of action and found that they all committed FEL cells to differentiate. Furthermore, other G 2/mitosis-blocking drugs were also effective in inducing commitment. These results suggest (1) a causal relationship involving anticancer drugs, cell cycle block and differentiation, (2) that the search for new anticancer drugs utilize a differentiation assay and include G2/mitosis-blocking agents.
Key words: Anticancer drugs, cell cycle, differentiation, commitment, erythroleukemia, murine.
CANCER has often been depicted as a disease in which cell proliferation and differentiation become uncoupled. One proposed strategy of cancer chemotherapy derived from this viewpoint involves the induction of differentiation of the malignant cell resulting in a cell with limited capacity for proliferation [1-3]. Such a strategy is attractive because the drug is targeted to a select cell population, but this approach has had limited application [1, 4]. The main thrust of cancer chemotherapy has been directed at cytotoxic drugs that limit the proliferation of cells in general but are particularly effective with rapidly growing tumor cells. This report is concerned specifically with in vitro studies which show that anticancer drugs with various modes of action were all effective in inducing differentiation commitment of F E L cells.
Dulbecco's (Iscoves) medium supplemented with 10% FBS (Bocknek, BDH, Canada), penicillin (50 units/ml), and streptomycin (50 ~tg/ml) in a humidified atmosphere containing 5% CO2. Commitment was assayed as described [5]. Briefly, logarithmically grown FEL cells (K clone) were treated with 1.5% DMSO and 8 mM 3AB (Pfaltz and Bauer, Waterbury, CT) for 64 h, centrifuged, washed, and exposed to various concentrations of drugs for 20 h. The cells were subsequently transferred to fresh medium containing cytochalasin B (1.0 ~tg/ml; Aldrich Chemicals, Milwaukee, WI) for 24 h to allow for the expression of the differentiated phenotype in the absence of further cell division and finally stained for Bz+ (hemoglobin-producing, differentiated) cells [6]. A minimum of 300 cells were counted for each sample. Cell viability was assayed using the trypan blue exclusion assay [7] at the end of the experiment. Mitotic indices were measured as described [8]. Cytofluorometry analysis was performed using nuclei stained with propidium iodide as described [9]. For soft agar colony analysis, the anticancer drugs were removed from culture, the cells were washed, plated on soft agar for 4-5 days [10], stained, and examined for Bz+ colonies.
MATERIALS AND METHODS
RESULTS AND DISCUSSION
INTRODUCTION
The F E L cell is a virally transformed murine cell line that has been a useful model for studying the differentiation process. It differentiates following exposure to a variety of agents, including some antitumor drugs [11-13]. In a previous study, we demonstrated that treatment of F E L cells with the inducer, DMSO and the poly(ADP-ribose) polymerase inhibitor, 3AB for 56 h blocked the differentiation pathway
FEL cells were cultured at 37°C in Iscoves modified
Abbreviations: FEL, Friend erythroleukemia; FBS, fetal bovine serum; DMSO, dimethylsulfoxide; 3AB, 3-aminobenzamide; Bz, benzidine. Correspondence to: Departments of Microbiology and Immunology and Biochemistry, Health Sciences Centre, The University of Western Ontario, London, Ontario, N6A 5C1, Canada. 491
492
R.D. D1NNENand K. EBISUZAKI
just prior to commitment [10]. When these cells were washed to remove DMSO and 3AB and incubated in medium containing DMSO, commitment was rapid, indicating that many of the preliminary steps in the differentiation pathway had already occurred during the initial incubation with DMSO and 3AB. On the other hand, if these cells were returned to medium containing no additions, the cells resumed normal growth, and few differentiated cells were evident [5]. Surprisingly, when the DMSO/3AB-treated cells were incubated with the mitotic inhibitors nocodazole or colcemid, the cells became committed to terminal differentiation [14]. However, further incubation in the absence of the mitotic inhibitors was required for the expression of the differentiated phenotype. Since the number of mitotic cells (in medium containing cytochalasin B, see [5]), were approximately equivalent to the number of cells that differentiated, we suggested that mitosis may be a requirement for commitment. Extension of these studies to the antimitotic, anticancer drugs vincristine and vinblastine, indicated that they were also effective in commitment (Table 1). The antineoplastic drug adriamycin, known to damage DNA and depress mitotic activity [15] was also effective. A further survey of clinically used anticancer drugs, indicated that they all induced commitment (Table 1). Since antineoplastic drugs are known to cause a cell cycle delay in Gz [16] or mitosis, [17] we tested Gz/mitosis blocking drugs [18-23] and found that they were also active in commitment (Table 2). The results shown in Tables 1 and 2 summarize data obtained from a larger experiment involving a range of drug concentrations. The data shown represents a single drug concentration, in which the effect on differentiation is clearly evident and where viability is reasonably maintained. The drug concentrations were selected on the basis of previously reported studies, and where the data was available, included concentrations which were attained during cancer chemotherapy ]24-26]. Examples of these more detailed studies are shown in Fig. 1, in which the relationship between increasing drug concentrations (adriamycin, vincristine, or bleomycin) and the appearance of differentiated (Bz+) cells is apparent. The in vivo concentrations of adriamycin (approx. 1-27 nM), vincristine (approx. 1015 nM) and bleomycin (0.13-10 ~t/ml) reported to be attained during chemotherapy [24-26] also differentiate FEL cells under in vitro conditions. We also noted that at higher drug concentrations there is decreased cell viability. One feature that distinguishes these anticancer drugs from other inducers is that the concentrations of drug required for commitment is low, and in many cases similar to levels attained in chemotherapy [24-26]. Although many
agents induce FEL cell differentiation, [11-13]* an important consideration for a potential anticancer drug is that differentiation occurs at physiologically tolerable concentrations. The following agents were found to be ineffective in the commitment assay: butyryl cAMP (0.05-2.5 mM), EGTA (0.146.75mM), acetic acid (0.1-2mM), ethanol (0.52.5%), penicillin/streptomycin (500u/ml; 500 ~tg/ ml), antimycin A (0.02-0.9 ~M), rifampin (0.011.5 ~tM), N-ethylmaleimide (4-20~M) and AZT (zidovudine) (1-200 ~tM). The concentrations selected were in the range used, and usually in excess of other studies [12, 27, 28]. A few of the anticancer drugs were studied in more detail. Cytofluorometric analysis of FEL cells treated with vincristine, adriamycin, or bleomycin under the conditions described in Tables 1 and 2 indicated accumulation of G2/mitosis-delayed cells (Fig. 2). These drugs were previously observed to cause G2/ mitosis delay in other cell lines [15, 17, 29]. After vincristine treatment, metaphase cells accumulated, as indicated by staining for mitotic indices (Table 3). Furthermore, when FEL cells treated with vincristine were subsequently plated in soft agar [10], 55/133 were one-celled Bz+ colonies and the remainder composed of colonies with 2-10Bz+ cells (summation of two experiments). This indicated that terminal differentiation had occurred. There were 23 mixed colonies composed of a few Bz+ cells and a large number of B z - cells. The purely undifferentiated colonies were also composed of large numbers of cells. Similar results were obtained in experiments involving bleomycin, amphotericin-B and hydroxyurea (data not shown). The possibility that terminal differentiation might be the primary basis for drug-induced cell killing is not obviously supported by the observation that most anticancer drugs including vincristine are weak inducers of FEL cell differentiation [11-13]*. However, hemoglobin-production is an indirect assay for differentiation, and the total number of differentiated cells might be quite different by a more direct assay. We have observed that vincristine treatment of FEL cells (745A line) yielded approximately 5% Bz+ cells. However, randomly selected clones derived from 745A gave wide variations in the number of Bz+ cells (
0145-2126/92 $5.00 + .00 © 1992 Pergamon Press Ltd
THE LINKING OF A N T I C A N C E R DRUGS, CELL CYCLE BLOCKS, A N D D I F F E R E N T I A T I O N : IMPLICATIONS IN THE S E A R C H F O R ANTINEOPLASTIC D R U G S RICHARD D. DINNEN and KANEY EBISUZAKI Departments of Microbiology and Immunology and Biochemistry, Health Sciences Centre, The University of Western Ontario, London, Ontario, Canada
(Received 30 September 1991. Revision accepted 6 February 1992) Abstract--The quest for anticancer drugs has been primarily directed at agents that interfere with cell replication, yet the basis for drug-induced cytotoxicity remains unsolved. In our previous studies we noted a relationship between a mitotic block and commitment to terminal differentiation in the murine (Friend) erythroleukemia (FEL) cell. Since anticancer drugs are known to often block cell cycle transit typically in G2/mitosis, we tested a number of anticancer drugs with various modes of action and found that they all committed FEL cells to differentiate. Furthermore, other G 2/mitosis-blocking drugs were also effective in inducing commitment. These results suggest (1) a causal relationship involving anticancer drugs, cell cycle block and differentiation, (2) that the search for new anticancer drugs utilize a differentiation assay and include G2/mitosis-blocking agents.
Key words: Anticancer drugs, cell cycle, differentiation, commitment, erythroleukemia, murine.
CANCER has often been depicted as a disease in which cell proliferation and differentiation become uncoupled. One proposed strategy of cancer chemotherapy derived from this viewpoint involves the induction of differentiation of the malignant cell resulting in a cell with limited capacity for proliferation [1-3]. Such a strategy is attractive because the drug is targeted to a select cell population, but this approach has had limited application [1, 4]. The main thrust of cancer chemotherapy has been directed at cytotoxic drugs that limit the proliferation of cells in general but are particularly effective with rapidly growing tumor cells. This report is concerned specifically with in vitro studies which show that anticancer drugs with various modes of action were all effective in inducing differentiation commitment of F E L cells.
Dulbecco's (Iscoves) medium supplemented with 10% FBS (Bocknek, BDH, Canada), penicillin (50 units/ml), and streptomycin (50 ~tg/ml) in a humidified atmosphere containing 5% CO2. Commitment was assayed as described [5]. Briefly, logarithmically grown FEL cells (K clone) were treated with 1.5% DMSO and 8 mM 3AB (Pfaltz and Bauer, Waterbury, CT) for 64 h, centrifuged, washed, and exposed to various concentrations of drugs for 20 h. The cells were subsequently transferred to fresh medium containing cytochalasin B (1.0 ~tg/ml; Aldrich Chemicals, Milwaukee, WI) for 24 h to allow for the expression of the differentiated phenotype in the absence of further cell division and finally stained for Bz+ (hemoglobin-producing, differentiated) cells [6]. A minimum of 300 cells were counted for each sample. Cell viability was assayed using the trypan blue exclusion assay [7] at the end of the experiment. Mitotic indices were measured as described [8]. Cytofluorometry analysis was performed using nuclei stained with propidium iodide as described [9]. For soft agar colony analysis, the anticancer drugs were removed from culture, the cells were washed, plated on soft agar for 4-5 days [10], stained, and examined for Bz+ colonies.
MATERIALS AND METHODS
RESULTS AND DISCUSSION
INTRODUCTION
The F E L cell is a virally transformed murine cell line that has been a useful model for studying the differentiation process. It differentiates following exposure to a variety of agents, including some antitumor drugs [11-13]. In a previous study, we demonstrated that treatment of F E L cells with the inducer, DMSO and the poly(ADP-ribose) polymerase inhibitor, 3AB for 56 h blocked the differentiation pathway
FEL cells were cultured at 37°C in Iscoves modified
Abbreviations: FEL, Friend erythroleukemia; FBS, fetal bovine serum; DMSO, dimethylsulfoxide; 3AB, 3-aminobenzamide; Bz, benzidine. Correspondence to: Departments of Microbiology and Immunology and Biochemistry, Health Sciences Centre, The University of Western Ontario, London, Ontario, N6A 5C1, Canada. 491
492
R.D. D1NNENand K. EBISUZAKI
just prior to commitment [10]. When these cells were washed to remove DMSO and 3AB and incubated in medium containing DMSO, commitment was rapid, indicating that many of the preliminary steps in the differentiation pathway had already occurred during the initial incubation with DMSO and 3AB. On the other hand, if these cells were returned to medium containing no additions, the cells resumed normal growth, and few differentiated cells were evident [5]. Surprisingly, when the DMSO/3AB-treated cells were incubated with the mitotic inhibitors nocodazole or colcemid, the cells became committed to terminal differentiation [14]. However, further incubation in the absence of the mitotic inhibitors was required for the expression of the differentiated phenotype. Since the number of mitotic cells (in medium containing cytochalasin B, see [5]), were approximately equivalent to the number of cells that differentiated, we suggested that mitosis may be a requirement for commitment. Extension of these studies to the antimitotic, anticancer drugs vincristine and vinblastine, indicated that they were also effective in commitment (Table 1). The antineoplastic drug adriamycin, known to damage DNA and depress mitotic activity [15] was also effective. A further survey of clinically used anticancer drugs, indicated that they all induced commitment (Table 1). Since antineoplastic drugs are known to cause a cell cycle delay in Gz [16] or mitosis, [17] we tested Gz/mitosis blocking drugs [18-23] and found that they were also active in commitment (Table 2). The results shown in Tables 1 and 2 summarize data obtained from a larger experiment involving a range of drug concentrations. The data shown represents a single drug concentration, in which the effect on differentiation is clearly evident and where viability is reasonably maintained. The drug concentrations were selected on the basis of previously reported studies, and where the data was available, included concentrations which were attained during cancer chemotherapy ]24-26]. Examples of these more detailed studies are shown in Fig. 1, in which the relationship between increasing drug concentrations (adriamycin, vincristine, or bleomycin) and the appearance of differentiated (Bz+) cells is apparent. The in vivo concentrations of adriamycin (approx. 1-27 nM), vincristine (approx. 1015 nM) and bleomycin (0.13-10 ~t/ml) reported to be attained during chemotherapy [24-26] also differentiate FEL cells under in vitro conditions. We also noted that at higher drug concentrations there is decreased cell viability. One feature that distinguishes these anticancer drugs from other inducers is that the concentrations of drug required for commitment is low, and in many cases similar to levels attained in chemotherapy [24-26]. Although many
agents induce FEL cell differentiation, [11-13]* an important consideration for a potential anticancer drug is that differentiation occurs at physiologically tolerable concentrations. The following agents were found to be ineffective in the commitment assay: butyryl cAMP (0.05-2.5 mM), EGTA (0.146.75mM), acetic acid (0.1-2mM), ethanol (0.52.5%), penicillin/streptomycin (500u/ml; 500 ~tg/ ml), antimycin A (0.02-0.9 ~M), rifampin (0.011.5 ~tM), N-ethylmaleimide (4-20~M) and AZT (zidovudine) (1-200 ~tM). The concentrations selected were in the range used, and usually in excess of other studies [12, 27, 28]. A few of the anticancer drugs were studied in more detail. Cytofluorometric analysis of FEL cells treated with vincristine, adriamycin, or bleomycin under the conditions described in Tables 1 and 2 indicated accumulation of G2/mitosis-delayed cells (Fig. 2). These drugs were previously observed to cause G2/ mitosis delay in other cell lines [15, 17, 29]. After vincristine treatment, metaphase cells accumulated, as indicated by staining for mitotic indices (Table 3). Furthermore, when FEL cells treated with vincristine were subsequently plated in soft agar [10], 55/133 were one-celled Bz+ colonies and the remainder composed of colonies with 2-10Bz+ cells (summation of two experiments). This indicated that terminal differentiation had occurred. There were 23 mixed colonies composed of a few Bz+ cells and a large number of B z - cells. The purely undifferentiated colonies were also composed of large numbers of cells. Similar results were obtained in experiments involving bleomycin, amphotericin-B and hydroxyurea (data not shown). The possibility that terminal differentiation might be the primary basis for drug-induced cell killing is not obviously supported by the observation that most anticancer drugs including vincristine are weak inducers of FEL cell differentiation [11-13]*. However, hemoglobin-production is an indirect assay for differentiation, and the total number of differentiated cells might be quite different by a more direct assay. We have observed that vincristine treatment of FEL cells (745A line) yielded approximately 5% Bz+ cells. However, randomly selected clones derived from 745A gave wide variations in the number of Bz+ cells (
