Cell Cycle Phase Specificity of Antitumor Agentsl

[CANCER RESEARCH 32, 398-407, February 1972] Cell Cycle Phase Specificity of Antitumor Agentsl

B. K. Bhuyan, L. G. Scheldt, and T. J. Fraser Research Laboratories, The Upjohn Company, Kalamazoo, Michigan 49001

SUMMARY correlating the biochemical effect of the drug to its lethal effect. For example, many drugs are maximally toxic to cells The sensitivity to drugs of synchronous and asynchronous at the G]-S boundary, and a correlation between the lethal populations of DON cells was studied. Agents that were and biochemical effects of the drug may help to delineate the cytotoxic at a specific phase of the cell cycle gave dose-survival biochemical reaction that triggers the cell across the Gi-S curves that decreased to a constant saturation value. DNA boundary, (b) They may help in designing combinations of synthesis inhibitors such as 1-0-D-arabinofuranosylcytosine, drugs on a rational basis. Thus, 2 drugs affecting the same 5-azacytidine, 5-hydroxy-2-formylpyridinethiosemicarbazone phase of the cell cycle would not give additive effects, if (NSC 107392), sodium camptothecin (NSC 100880), combined. 5-fluorodeoxyuridine, and pseudourea (NSC 56054) were Our studies were intended to determine the phase most cytotoxic to cells in the S phase. However, the DNA specificity of several clinically active agents and of agents with synthesis inhibitor, neocarzinostatin, was most cytotoxic to known biochemical activity. Parts of this study were reported cells in the G! phase. The protein synthesis inhibitors, previously (7). pactamycin and sparsomycin, were also most cytotoxic to cells in the S phase. Cells in the Gi-S border region were most MATERIALS AND METHODS sensitive to the RNA synthesis inhibitors, actinomycin D and nogalamycin, and to the alkylating agents, Cell Culture. DON cells, from a Chinese hamster fibroblast 1 ,3-bis(2-chloroethyl)-l-nitrosourea, and line (ATCC CCL16), were grown at 37Â° in McCoy's 1-(2-chloroethyl)-3-cyclohexyl-1 -nitrosourea. Streptozotocin 5A medium modified by the addition of lactalbumin and tubercidin, which markedly inhibit the synthesis of DNA, hydrolysate (0.8 g/liter) and fetal calf serum (200 RNA, and protein, were cytotoxic to cells in all phases of the ml/liter). The medium was obtained from the Grand cell cycle. 5-Fluorouracil was also not phase specific. Cells in Island Biological Company, Grand Island, N. Y. The mitosis and in the Gt phase were most sensitive to cells were grown in 8-oz prescription bottles planted with chlorambucil, L-phenylalanine mustard, and ellipticine. about 2 X IO6 cells in 25 ml of medium and were maintained in logarithmic growth by subculture every 2 days. With renewal of the medium every 2 days, an 8-oz bottle could INTRODUCTION support logarithmic cell growth, with a generation time of 10 to 12 hr, until there were about IO8 cells/bottle. For Bruce et al. (12) have classified a number of subculturing, the cell monolayer was detached from glass by chemotherapeutic agents into 3 groups. The 1st group, consisting of -y-radiation and nitrogen mustard, killed both treatment with a 0.1% trypsin solution; the cells were then dispersed in medium, and an aliquot was planted in bottles. proliferative and resting cells in all portions of the generation Synchronous DON Cells. A modification of the method of cycle. In contrast, the agents in the 3rd group killed only Stubblefield et al. (42) was used to obtain a population of proliferative cells in all phases of the cell cycle. Agents mitotic cells. Logarithmically growing cells were planted in belonging to both of these groups gave exponential curves, roller bottles (11 x 28.5 cm; 840-sq cm growth surface; BÃ©lico when survival was measured at different drug concentrations. Glass, Inc., Vineland, N. J.) at about IO7 cells in 200 ml of The 2nd group of agents gave dose-survival curves that medium. The bottles were gassed with a 5% C02 :95% air decreased to a constant saturation value at high doses, mixture and were rotated for 2 days on a roller apparatus indicating that they killed cells in 1 portion of the cell cycle; (BÃ©lico)at 0.6 rpm. After 2 days of incubation, Colcemid i.e., these agents were phase specific. (demecolcine; Ciba Pharmaceutical Company, Summit, N. J.) The variation in the sensitivity of cultured mammalian cells was added to the medium to give 0.06 Mg/ml, and the bottles through the division cycle to different agents has been were incubated for 3 more hr. The medium was then poured reported. The recent paper by Mauro and Madoc-Jones (33) off, and we selectively removed the mitotic cells by rolling the summarizes the results obtained with a large number of agents. bottles with 40 ml of trypsin (at 4Â°;0.125 mg/ml) and 1 ml of Such studies are useful for 2 reasons: (a) They may enable us NaHC03 (7.5%). The mitotic cells were centrifuged and better to understand the parameters of the cell cycle by resuspended in medium at 4Â°, and about IO6 cells were planted in 3-oz bottles containing medium at 37Â°.The mitotic 'This study was supported in part by Contract PH43-68-1023 with cells constituted between 85 and 95% of the harvested cells. Chemotherapy, National Cancer Institute, NIH, Bethesda, Md. 20014. Determination of Percentage of Cell Survival after Exposure Received December 21, 1970; accepted November 3, 1971. of DON Cells to Drug. Cells growing in synchrony were

398 CANCER RESEARCH VOL. 32

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1972 American Association for Cancer Research. Cell Cycle Phase Specificity exposed to drug for 1 hr at different times after the planting cycloheximide) were developed by The Upjohn Company, of mitotic cells to expose cells in different parts of the cell Kalamazoo, Mich. The solubilities and structures of the drugs cycle. Asynchronous cells were also exposed to the drug for 1 are as follows. FUdR, neocarzinostatin (NSC 69856; Ref. 32), hr. thio-TEPA (NSC 6396), cycloheximide, ara-C, 5-azaCR, After exposure to drug, the supernatant medium was sodium camptothecin (alkaloid; NSC 100880; Ref. 20), and poured off, and the cells were detached with trypsin and streptozotocin (22) were soluble in H2O, Tu, and sparsomycin resuspended in medium at 37Â°.When it was suspected that the (45) were soluble in hot H2O. FU was soluble at 20 mg/ml in drug might also detach cells, the supernatant medium was also 7.5% NaHC03. Nogalamycin (46) and actinomycin D were centrifuged, and these cells were added to the total cell pool. dissolved in acetone at 1 mg/ml. The cells were diluted in medium at 37Â°,and 2 ml of cells 5-Hydroxy-2-formylpy ridine thiosemicarbazone (NSC were planted in plastic Petri plates (Linbro Chemical Co., New 107392), pactamycin (44), BCNU (NSC 409962), CCNU (NSC Haven, Conn.) to give 10 to 100 colonies per plate. Twelve 79037), and chlorambucil [4 {p- [bis(2-chlore- plates were planted for each sample. After incubation for 7 to thyl)amino] phenyl} butyric acid; NSC 3088] were dissolved 8 days in an atmosphere of 8% C02:92% air at 37Â°, the in ethanol at 5 to 10 mg/ml. Pseudourea (NSC 56054; Ref. 13) medium was poured off, and the colonies were stained with was dissolved at 10 mg/ml dimethyl sulfoxide. Ellipticine 0.2% mÃ©thylÃ¨neblue in 70% ethanol. The colonies were (NSC 71795; Ref. 20) and L-phenylalanine mustard counted with a Quebec colony counter (Spencer Lens Co., [3-(p-[bis(2-chloroethyl)amino]phenyl-L-alanine; NSC 8806] Buffalo, N. Y.). The plating efficiencies were about 50% for were dissolved at 10 mg/ml in 0.1 N HC1. synchronous cells and 70% for asynchronous cells. For When we used solvents for solutions of drugs, we attempted calculation of the percentage of survivals, the control (no drug to prepare a 10 mg/ml solution. Thus, it was possible to dilute treatment) samples were normalized to 100% survival. In these out the solvent in the process of diluting the drug to the experiments, the coefficient of variation in determining cell desired concentration. The drug concentrations used to survival was about 15%, the coefficient of variation being the determine sensitivity of different phases were chosen to show standard deviation expressed as a percentage of the mean. a well-defined age response. In many cases, 2 or 3 different Thus, if the percentages of survival of 2 different samples were levels of the drug were used (see Charts 2, 4, etc.), but the 50 and 30%, respectively, then they would be statistically sensitivity of different phases to the drug pertains only to the significantly different at the 95% confidence level. Almost all concentrations used and is not necessarily true at much higher experiments were repeated, and the results were found to be drug concentrations. reproducible; in duplicate determinations, the individual values were within 10% of the mean value. Determination of Macromolecule Synthesis. Asynchronous DON cells were planted at IO6 cells/3-oz bottle in 10 ml of medium. After overnight incubation, the cells were refed with fresh medium, and drug and labeled precursors were added for g 2 1 hr. To stop uptake of radioactivity, cells were detached with l-H- 0.1% trypsin containing unlabeled precursors (100 jug/ml), and the cells were suspended in 0.9% NaCl solution. One aliquot "â€¢1 was counted in the Coulter counter to give the cell number, while another (1-ml) aliquot of cells was filtered through WO 0.45-M Millipore filters. The filter was washed 4 times with cold 10% trichloroacetic acid and once with ethanol. The filter was then incubated with 0.5 ml of 0.5 N perchloric acid at 70Â° 80 for 20 min. Diotol (15 ml) was added, and the filter was u> counted in a scintillation counter. o 60 Autoradiography and Mitotic Index Determination. The slides were prepared for autoradiography and mitotic index determinations, as previously described (28). g 40 Drug Samples. The drugs with the NSC numbers were obtained from Chemotherpy, National Cancer Institute, Bethesda, Md. The other drugs (ara-C,2 5-azaCR, Tu, 20 streptozotocin, nogalamycin, pactamycin, sparsomycin, and

0 10 12 2The abbreviations used are: aia-C, 1-0-D-arabinofuranosylcytosine HOURS (Cytosar); 5-azaCR, 5-azacytidine; Tu, tubercidin; FUdR, 5-flurodeoxyuiidine; thio-TEPA, tris(l-aziridinyl)phosphine sulfide; Chart 1. Cell cycle of synchronized DON cells. Mitotic cells, planted in 3-oz bottles, were pulsed with TdR-3H (l /iCi/ml, 2 Ci/mmole) for FU, 5-fluorouracil; NSC 107392, S-hydroxy^-formylpyridinethiosemi- caibazone; BCNTJ, l,3-bis(2-chloroethyl)-l-nitrosourea; CCNU, 10 min at different times after planting. The cells were then harvested, l-(2-chloroethyl)-3-cyclohexyl-l-nitrosouiea; pseudourea, and cell counts, mitotic index, and percentage of3 H-labeled cells were 2,2' -(9,10-anthrylenedimethylene)bis(2-thiopseudourea)di- determined. The curves show the mean Â±average deviation from the hydrochloride dihydrate; TdR, thymidine. mean of 4 separate experiments.

FEBRUARY 1972 399

Table l subdivided into classes on the basis of their inhibition of Inhibition of macromolecule synthesis in DON cells macromolecule synthesis and known sites of action. This by various agents division does not, however, imply that a given agent has only 1 (%)DNA9296918560778437753740446074079808052.681RNA102487145364860688464-20-30877446913535.190Protein24179510881979041-9-310119899982540.4biochemical mode of action. The subdivision merely indicates that the agent in question markedly inhibits the Agentara-Cbara-C5-AzaCRcNSC synthesis of a particular macromolecule. Thus both ara-C and Tu inhibit DNA synthesis (Table 1) and are classified as DNA synthesis inhibitors. However, only ara-C specifically inhibits DNA synthesis, while Tu inhibits almost equally the synthesis 107392bSodium of DNA, RNA, and protein. The sites of action of many of camptothecinbSodium these agents have recently been compiled by Livingston and camptothecinTudStreptozotocin6StreptozotocinFUCFUdRcNeocarzinostatincNeocarzinostatinActinomycinbNogalamycinbPactamycinbPactamycinSparsomycin0Pseudourea6PseudoureaDose(Mg/ml)100100010021.859.22100050001001005501211001001.110Inhibition0Carter (29). Other pertinent publications on their sites of action are listed below after each compound. The inhibition of macromolecule synthesis by several of the agents is shown in Table 1. ara-C (14, 19), 5-azaCR (28), NSC 107392 (11), sodium camptothecin (23), Tu (l), Streptozotocin (5), FU (21, 31), FUdR (21), and neocarzinostatin (27, 34) have been classified in the following section as DNA synthesis inhibitors. Among them only ara-C, NSC 107392, and neocarzinostatin markedly inhibited DNA synthesis without simultaneous marked inhibiton of RNA and protein synthesis. The other agents in this group markedly inhibited DNA, RNA, and/or protein synthesis. Actinomycin D (38) and nogalamycin (8) have been 0 In all cases, the DON cells were incubated with radioactive precursors for 1 hr. TdR-3H, uridine-5-3H, and DL-valine-' 4C were classified as RNA synthesis inhibitors. Actinomycin D was a added to give 5 /uCi/2 Mg/ml, 5 MCi/5 Mg/ml, and 0.1 Â¿tCi/23Mg/ml, much more specific inhibitor of RNA synthesis than was respectively. The specific activities of control (no drug) samples of nagalamycin. DNA, RNA, and protein were (per IO6 cells): 13.5 X 10s, 20 X IO3, Pactamycin (Ref. 18, pp. 169-173) and sparsomycin (Ref. and 16.1 X IO2 cpm, respectively. All samples were in duplicate. b DON cells were exposed to agent and radioactive precursor for 1 hr. 2i 2b c Cells were exposed to agent for 5 hr prior to addition of 100 radioactive precursor for 1 hr. d Cells were exposed to Tu for 2 hr prior to addition of labeled precursor for 1 hr. e Cells were exposed to agent for 1 hr prior to addition of labeled metabolities. The agents in Group b rapidly inhibited macromolecule so synthesis, while the other agents needed a longer period of contact with cells. Neocarzinostatin stimulated the incorporation of precursors into RNA and protein. (100 vig/ml) (1000 ug/ml)

v> RESULTS if. \5-AZA-CR ,'(100 ug/ml) Cell Cycle of Synchronous DON Cells. The results obtained when mitotic DON cells were planted as a monolayer are shown in Chart 1. The mitotic index in different experiments ranged from 0.85 to 0.95. Mitosis was completed within 1 hr, as shown by decrease in mitotic index from about 0.9 to ARA-C < 0.05. Cell division was not complete until 2 hr after mitotic S-AZA-CR cells were planted, and the relative cell number increased from 20 40 60 80 100 4 8 12 1 to 1.7 during this period. Within 3 hr after planting, about 80% of the cells had entered S phase (labeled by TdR-3H lig/ml HOURS pulse), and they remained in S for 7 hr. After 7 hr, cells Chart 2. Sensitivity of DON cells to 5-azaCR and ara-C. In a, started entering G2 with some loss of synchrony. Cell division asynchronous cells were exposed to different levels of drugs for 2 hr. commenced about 9 hr from the time of planting of mitotic The drug was removed, and the cells were diluted and planted. Colonies were counted after 7 to 8 days of incubation. In b, mitotic cells were cells. Therefore, under these conditions, the approximate time exposed to the drugs for 1-hr periods after planting. The drugs were taken for the different phases (after planting of mitotic cells) removed after 1 hr, and the cells were diluted and planted in 12 plates is from 0 to 2 hr to complete mitosis and GÃŒ,from 2 to 8 hr for each sample. Colonies were counted after 7 to 8 days of incubation. for S, and from 8 to 11 hr for G2 and mitosis. The coefficient of variation in percentage of survival was about 15%. Inhibition of Macromolecule Synthesis by Several of the The times shown are halfway points in the period of exposure, i.e., cells Agents. For convenience in presentation, the agents have been exposed at 1 hr after planting and harvested at 2 hr are shown at 1.5 hr.

400 CANCER RESEARCH VOL. 32

100E " 3k decreased to a constant saturation value at high doses, indicating that ara-C is cytotoxic to a specific phase or phases l '

\ \ 107392 (10 n/Ml) / of the cell cycle. Results obtained with synchronous cells (Chart 2b) showed that, at low levels, both ara-C and 5-azaCR were specifically cytotoxic to S-phase cells. High levels of 5-azaCR (100 Mg/ml) were lethal to cells in GÃŒ,S, G2, and M,

NSC 107392 although cells in S phase were still most sensitive. However, ara-C at high level (1 mg/ml) was still lethal only to cells in S. The dose-survival curve for NSC 107392 (Chart 3a) decreased to a constant saturation value, indicating that this

IN Sb

FU (SO wg/ml)

XX 1008SO (2 ug/nl) NSC 100880 (ug/ml) FUdR 2 4 I 2Â» W 20 30 70 4 1 12 NSC 107392 {m/ml) HOURS FUdR (20 iig/ml) Chart 3. Sensitivity of asynchronous (a) and synchronous (b) DON V cells to NSC 107392 and sodium camptothecin (NSC 100880). Protocol same as Chart 2, except drug exposure was for 1 hi.

4b STREPTOZOTOCIN (0.2 mg/ml) M 20 40 60 80 100 4 8 FUdR (ug/ml) HOURS

Chart 5. Sensitivity of asynchronous (a) and synchronous (b) DON cells to FU and FUdR. Protocol same as in Chart 3.

NEOCARZINOSTATIN

1NEOCARZINOSTATIN (1 ug/ml)

0.4 0.8 1.2 Tu (ug/ml)

Chart 4. Sensitivity of asynchronous (a) and synchronous (b) DON cells to streptozotocin and Tu. Protocol same as in Chart 3.

18, pp. 410â€”415) almost completely inhibited protein synthesis, although DNA and RNA synthesis were also markedly inhibited. BCNU (Ref. 29, pp. 360-365), CCNU, chlorambucil (Ref. 12 29, pp. 81-98), thio-TEPA, and phenylalanine mustard (Ref. HOURS 29, pp. 99-111), have been classified as alkylating agents. Chart 6. Sensitivity of asynchronous (a) and synchronous (b) DON Response to DNA Synthesis Inhibitors. The dose-survival cells to neocarzinostatin. Protocol same as in Chart 3. The results of 2 curves of asynchronous cells exposed to ara-C (Chart 2a) separate experiments are shown.

FEBRUARY 1972 401

drug is cytotoxic to a specific phase or phases of the cell cycle. to cells in the S phase. The percentage ol cell kill obtained in The dose-survival curve for sodium camptothecin (Chart 3a) different phases with sodium camptothecin, 10 Mg/ml, was suggests the presence of cell populations with varying approximately the same as the percentage of cell kill at 2 sensitivities to the drug. The results with synchronous cells jug/ml. Although both DNA and RNA synthesis were markedly (Chart 3Z>)show that both of these agents are most cytotoxic inhibited by sodium camptothecin, 10 Mg/ml (Table 1), the drug was still maximally lethal to cells in S. However, unlike NSC 107392, cells in M, d, and G2 were killed by sodium camptothecin. The results with asynchronous cells exposed to streptozotocin are shown in Chart 4a. The results with synchronous cultures (Chart 4b) showed that cells in all phases of the cell cycle were equally sensitive to the drug. This becomes particularly evident at the higher concentration (0.5 mg/ml) of streptozotocin.

Â»l\\ivi\ 8b; Ã›â€”A ACTINOMYCIN (0.2 |ig/ml) |ig/ml)\PACTAMYCIN (100 STREPTOVITACINn\V â€¢¿ 11x1 / \\ Â¿r"Ã¯l \\ fi1 \PACTAMYCIN\ \/>'1 \X \ .:i ~~SPARSOMYCINSTREPTOVITACIN \/â€¢'i \-1 'â€¢/;! Â»,x/SPARSOMYCIN

A (iig/ml) (100 pg ml) 5 1.0 15A. SO 100 150 4 8 12 241 12 PACTAMYCIN.SPARSOMYCIN(iig/ml) HOURS NOGALAMYCIN (gg/ml) HOURS Chart 8. Sensitivity of asynchronous (a) and synchronous (b) DON Chart 7. Sensitivity of asynchronous (a) and synchronous (b) DON cells to streptovitacin, pactamycin, and sparsomycin. Protocol same as cells to actinomycin D and nogalamycin. Protocol same as in Chart 3. in Chart 3.

Table 2 Correlation of cell kill with percentage of inhibition of protein synthesis before and after washing off of the drug Cell kill" Inhibition of protein synthesis (%) hrb (before hrÂ°(after h^ (after DrugPactamycin Ihr17.2 washing)99.9 washing)1954.5washing)52.5

Pactamycin 1.0 24 64.9 99.97 62 Pactamycin 10100 27.5 80.5 99.9 91.9 91.2 Puromycin 23.2 64.8 99.6 0 38.5 CycloheximideDose(Mg/ml)0.1100AT 21.2At4hr45.457.51 99.91 204 78 0 After 1- or 4-hr exposure to the drug, the cells were washed, diluted, and planted to determine the percentage of surviving cells. 6 The cells were exposed to the drugs for 1 hr. Immediately after drug exposure, the cells were given a 20-min pulse with DL-valine-l-14C (1 MCi/23 Mg/ml medium) to determine the percentage of inhibition of protein synthesis before washing. A similar percentage of inhibition of protein synthesis (before washing) was obtained when cells were exposed to the drug for 4

c For determination of the percentage of inhibition of protein synthesis after washing, the cells were washed after 1 hr of drug exposure, were allowed a 1-hr recovery period, and then were given a 20-min pulse of DL-valine-l-'4C. Control cells incorporated 10" cpm/2 X 10 cells into the acid-insoluble fraction. " Same as above, except cells were exposed to drug for 4 hr.

402 CANCER RESEARCH VOL. 32

â€¢¿vu101_J>IcosÃ•0.10.010001Ik In synchronous cultures, Tu at low levels (0.2 jug/ml) was 10aA\\\ approximately equally cytotoxic to cells in all phases of the cell cycle (Chart 4Z>).However, at high levels (0.4/^g/ml), cells |ig/ml)As^s/ (10 at the G!-S boundary region seemed to be more sensitive to k\THIO-TEPA\ Tu than were the cells in GÂ¡or S. k^\* FU was equally cytotoxic to cells in all phases of the cell Â»/ & |oc cycle (Chart 5b). With asynchronous cells, the dose-survival A~Ã»'Ã» o \" 2u^1 curve for FUdR had a shoulder preceding the exponential \ A^^//Ã»CHLORAMBUCIL \ \\ component of the curve, since there was no cell kill with \8806\ FUdR at 5 Â¿ig/mlor less. The exponential component of the pg/ml).,AA.8806 (26 \\ Itajf0.2100 dose-survival curve had 2 different slopes, indicating cell X\\\CHLORAMBUCIIAACHLORAMBUCILTHIO-TEPA populations with different sensitivities to the drug (Chart 5a). This was reflected in the greater sensitivity of cells in the S phase to FUdR.

The results obtained in 2 different experiments with g |ig/ml)p.*' (2.5 neocarzinostatin are shown in Chart 6. The difference between eou10zSLUio the 2 experiments with neocarzinostatin could be due to the pg/ml)^jr*^4Â£,THIO-TEPA (10 variation in the neocarzinostatin preparation. With synchronous cells, both experiments (in spite of the /r**' difference in percentage of survival values) show that the drug *y ""Â«.v' iXNÂ¿goc3 \/ * was maximally cytotoxic to cells in M and GÃŒphases. The \-*-8806! sensitivity decreased as the cells progressed through S and uÃ /mi)/f-*\. ' (5 increased again as the cells entered G2 and M. TV\Â« RNA Synthesis Inhibitors. The dose-survival curves with (|ig/ml) /THIO-TEPA^â€”i.4 10502 20 30 40 CO'" asynchronous cells (Chart la) showed that actinomycin D, 0.1 10NSC4 6 8 812HOURSIUU010 Â¿ig/ml,and nogalamycin, 1 i/g/ml, did not kill any cells, 8806 (iig/ml)lObCHLORAMBUCIL indicating the presence of a shoulder region preceding the Chart 10. Sensitivity of asynchronous (a) and synchronous (b) DON exponential component. The results with synchronous cells cells to chlorambucil, thio-TEPA, and L-phenylalanine mustard. (Chart 7Â¿>)indicated that cells in the G^S boundary region Protocol same as in Chart 3.

rao were most sensitive to the agent. These results confirm those 9b of Elkind et al. (15) and Mauro and Madoc-Jones (33). BCNU (2 iig/ml) Protein Synthesis Inhibitors. The dose-survival curves of asynchronous cells exposed to pactamycin, sparsomycin, and streptovitacin A are shown in Chart 8a. Only sparsomycin 10 showed a shoulder region prior to the exponential component of the curve. Streptovitacin A and sparsomycin curves reached saturation values for percentage of survival at high doses, indicating that these agents are phase specific. The results

CCNU 18 |ig/ml) obtained with synchronous cells are given in Chart Sb. Cells in S phase were most sensitive to pactamycin and sparsomycin. Cells in S phase have been shown to be sensitive to streptovitacin A by Mauro and Madoc-Jones (33). The dose (in AmÃ³les/mi) for 50% cell kill after 1 hr of BCNlT, exposure to several protein synthesis inhibitors was: 0.1 streptovitacin A, 0.0018; pactamycin, 0.025; sparsomycin, 0.095; puromycin, >0.2; and cycloheximide, >0.7. These results indicate that, except for streptovitacin A, the protein synthesis inhibitors tested (pactamycin, puromycin, toi cycloheximide, and sparsomycin) were relatively ineffective in killing cells after 1 hr of exposure. The results in Table 2 may indicate a possible explanation of this effect. Cells exposed to protein synthesis inhibitors for 1 hr have greater than 99% of their protein synthesis inhibited, and yet only 25% of the cells 0.001 were killed. This was due to the fact that when the agents were 20 30 8 12 (ig/ml HOURS removed by washing after 1 hr of exposure, the cells recovered their protein synthesizing ability. This was not true when high Chart 9. Sensitivity of asynchronous (a) and synchronous (b) DON levels of pactamycin were used, possibly indicating that all of cells to BCNU and CCNU. Protocol same as in Chart 3. the pactamycin was not removed by washing. However, after 4

FEBRUARY 1972 403

DISCUSSION PSEUDOUREA ,., (0.33 (ig/ml) / The selective detachment method of Terasima and Tolmach (43) for obtaining mitotic cells does not subject the cells to any perturbations and is therefore the ideal method for synchronizing cells. However, the low yield of mitotic cells forced us to use the present method of selectively removing mitotic cells from a culture treated with Colcemid. Stubblefield et al. (42) reported that the cells suffered no lasting effects from a 2-hr Colcemid (0.06 ;ug/ml) treatment. However, Kato and Yosida (24) have shown that chromosomal nondisjunction occurred frequently after Colcemid treatment and that the plating efficiency of synchronized cells was lower than that of untreated control cells. We found that synchronized cells had about 70% of the plating efficiency of asynchronous cells. Also, when an asynchronous population of DON cells was exposed to Colcemid (0.06 jug/ml) for 4 hr, only about 80% of the cells survived. These results indicate that our synchronized population may contain a certain percentage of dead or dying cells. However, the measurement of reproductive cell survival (or colony-forming ability) after treatment with cytotoxic agents is not affected by the presence of dead cells in the culture at the time of drug treatment. Although 20% of the cells had lost their reproductive potential, they still were able to synthesize DNA, 20 40 60 80 100 since almost 90% of the cells were labeled with TdR-3H pulse ELLIPTICINE (Mg/ml) HOURS (Chart 1). Chart 11. Sensitivity of asynchronous (a) and synchronous (b) DON The index of synchrony (F) was determined by the method cells to ellipticine and pseudourea. Protocol same as in Chart 3. of Blumenthal and Zahler (9) to be 0.59. This compared favorably with the F values of 0.6 of Kim and Stambuck (26) hr of exposure to the drug, the protein synthesis machinery and the calculated F value of 0.65 for the synchronized had been severely damaged so that the cell did not completely cultures of Pfeifer and Tolmach (35). recover its synthetic capacity. Therefore, the percentage of During the 1st cell division, the relative cell number inhibition of protein synthesis after the drug had been washed increased from 1 to 1.7 (instead of the expected 2) during a off compared favorably with the percentage of cell kill. 2-hr period. During the 1st hr, mitotic division was completed, Alkylating Agents. BCNU and CCNU (Chart 9Z>)were most and the mitotic index decreased from 0.9 to <0.05. This cytotoxic to cells in GÃŒ-Sborder or early S. With both BCNU result might indicate that some of the cells completed mitotic and CCNU, the sensitivity of cells decreased as the cells division but either did not complete cell division or did not progressed into S. However, with CCNU and to a lesser extent separate after cytokinesis and were counted as 1 cell in the with BCNU, the cell sensitivity increased again as the cells Coulter counter. Pfeiffer and Tolmach (35) found that their entered late S or G2. relative cell number increased from 1 to 1.8. Thio-TEPA, phenylalanine mustard, and chlorambucil were The cells stayed in S till about 7 hr, and then they began most cytotoxic to cells in the M and GÃŒphase(Chart 10Z>).At entering G2 with some loss of synchrony. The loss of the higher concentration of phenylalanine mustard and with synchrony increased as the cells progressed through the cell thio-TEPA, cells in G2 were as sensitive as the G, cells. cycle from GÃŒto S to G2. This is indicated by the steeper Miscellaneous Agents. The results with pseudourea and slope of the percentage-labeled cell curve as the cells go from ellipticine, 2 agents with unknown sites of action, are shown in GÃŒtoS, compared with the slope of the curve as the cells go Chart 11. Pseuodourea was most cytotoxic to cells in S, while from S to G2. Such loss of synchrony is due to the variation in ellipticine was most cytotoxic to cells in M and d. As the the intermitotic times of individual cells, as shown by Sisken cells progressed through S, their sensitivity to ellipticine (40). decreased, but ellipticine toxicity increased again as the cells In our experiments, the cells were exposed to drug usually passed through late S or early G2. The greater sensitivity to for 1 hr. Such a short period of exposure was chosen for 2 ellipticine of cells in M and G! as compared with cells in S or reasons: (a) the GÃŒand G2 phases of DON cells are of less G2 was seen at drug doses ranging from 0.36 p.g/m\ (not shown than 2-hr duration. Therefore, in order to expose cells in these in chart) to 10/ug/ml. phases of the cell cycle, it was necessary to use a short The inhibition of macromolecule synthesis by pseudourea is exposure period. (Â¿>)Manydrugs have a short plasma half-life, shown in Table 1. Pseudourea inhibited DNA synthesis more such that usually the cells in vivo are exposed to drug for a than RNA or protein synthesis at the lower concentrations, short time. For example, ara-C (10), pactamycin (6), 5-azaCR while at 10 Mg/ml of the drug, DNA and RNA syntheses were (37), actinomycin D (36), and streptozotocin (39) have plasma equally inhibited. half-lives of about 20 min, < 5 min, < 5 min, < 5 min, and 5

404 CANCER RESEARCH VOL. 32

min, respectively. Therefore, cytotoxicity of a drug chromosomes, S-phase cells also synthesize a new complement determined after 1 hr of exposure might correlate better with of histones. The process of DNA and histone synthesis is the in vivo effects of the drug than might the usual method of tightly coupled so that interruption of histone synthesis results cytotoxicity determination, wherein the cells are exposed to in the inhibition of DNA synthesis. This would account for the the drug for 2 to 3 days. sensitivity of cells in S to protein synthesis inhibitors. Mauro and Madoc-Jones (33) suggested that most of the (f) Our results, together with those of Mauro and drugs studied gave dose-survival curves in which a shoulder Madoc-Jones (33), show that alkylating agents are region preceded the exponential portion of the curve. The characterized by toxicity to cells in M, GÃŒ,or Gi-S transition. presence of a shoulder region suggests that the cells Whether this common site for the alkylating agents indicates accumulate damage which ultimately leads to a lethal effect vulnerability of the mitotic chromosomes or of DNA before and that survivors have sublethal damage which is repaired replication is not known. (16). Of all the agents reported here, only FuDR, In contrast to our results with FUdR, Lozzio (30) reported nogalamycin, actinomycin D, and sparsomycin gave that FUdR was equally cytotoxic to cells in all phases of the dose-survival curves in which a shoulder region was obviously cell cycle except the mitotic stage. However, he did find that IO"5 M FUdR killed 97% of the cells in early S, compared to present. At the present state of our knowledge, it may not be only 70% of the cells killed in GÃŒorlate S. However, at higher possible to predict the most susceptible phase from a levels of the drug, cells in all phases were equally sensitive. knowledge of the biochemical site of action. However, Our results with BCNU differ from those of Barranco and attempts have been made to explain the sensitivity of different Humphrey (3), who found that Chinese hamster ovary cells phases on the basis of the site of action of the drug; this is were most sensitive in mid-S phase. Their method differed discussed below. from ours in 2 aspects: (a) For a valid correlation, exposure to a given drug should (a) Our DON cells were about 14 times more sensitive than be completely terminated by washing the cells to remove the were the ovary cells. Thus, 90% cell kill of asynchronous cells drug. However, nucleosides such as Tu, 5-azaCR, FU, etc., are after 1-hr exposure required BCNU, in amounts of 85 and 6 converted to their respective nucleotides inside the cell and jug/ml for the ovary cells and DON cells, respectively. may be held in the intracellular pool. For example, the lethal Therefore, in our phase specificity experiments, DON cells action of FU can be expressed even 80 hr after the exposure of were exposed to BCNU, 2 Â¿tg/ml,compared with 100 Â¿tg/ml cells to FU (31). Therefore even if FU killed only S-phase for the ovary cells (3). cells, GÃŒandG2 cells exposed to the drug will accumulate the (b) Their method for synchronizing cells consisted of a nucleotide and be killed when the cells enter S. double treatment with excess TdR and so differed from ours. (b) It would be logical to suppose that agents that Both of us found that cells in the Gi-S border were highly specifically inhibit DNA synthesis will be lethal to cells in S sensitive to BCNU. The cells decreased in sensitivity as they phase only. ara-C, NSC 107392, and neocarzinostatin are 3 progressed through S, with another zone of great sensitivity in such agents. The first 2 compounds are specifically lethal to mid-S (3). Here, our results differ from theirs. Whether the cells in S, while neocarzinostatin is more cytotoxic to cells in difference in the method of synchrony and the levels of BCNU M and GÃŒthan in S. The strange behavior of neocarzinostatin used would account for the difference in our results needs might be explained by the observation (27) that cells in GÃŒ,investigation. exposed to the drug, were unable to synthesize DNA on Bruce et al. (12) differentiated between certain reaching S, which probably resulted in cell death. Drug added (phase-nonspecific) agents, such as nitrogen mustard and to cells in S did not inhibit DNA synthesis. â€¢¿y-rays,thatkilled cells in all phases of the cell cycle, and (c) Sodium camptothecin inhibits equally both DNA and phase-specific agents such as ara-C, that killed cells in a specific RNA synthesis and kills cells in all phases of the cycle, phase of the cell cycle. The studies reported here and those of although the cells in S were always more sensitive to the drug. Mauro and Madoc-Jones (33) and others (15, 16) have shown In contrast, streptozotocin, Tu, FU, and FUdR at high that there is a marked difference in the sensitivity of cells in concentration (30) all inhibited DNA and RNA and/or protein different phases of the cell cycle toward "phase-nonspecific" synthesis but were toxic to cells in all phases. (12) agents. Thus, cells at Gi-S transition are 50 times more (d) Actinomycin D (38) and nogalamycin (8) bind to the sensitive to BCNU than the cells in G2 (Chart 9b). The cells in DNA template and inhibit RNA synthesis more than DNA M and GÃŒwere250 times more sensitive to ellipticine (Chart synthesis (Table 1). Cells in Gi-S or early S are most sensitive 116) than cells in G2. By proper manipulation, this differential to both these drugs (this paper and Refs. 15 and 33). Fujiwara sensitivity may be utilized in cancer chemotherapy. (17) found that, when actinomycin D was added in GÃŒ,the We suggest that knowlege of the phase specificity of agents synthesis of early replicating DNA was inhibited. However, may enable us to devise rational combinations of drugs. Thus, actinomycin D added in the middle of the S phase had little 2 drugs inhibiting in the same phase, if given together, will not effect on the late replicating DNA. The results indicate that result in improved cell kill, although they may delay the inhibition of RNA synthesis in GÃŒled to the inhibition of development of resistance to either drug. However, it is early replicating DNA, which probably led to cell death. essential that we also know the effect of the drug on (e) Cells in the S phase were most sensitive to protein progression of the cells through the cell cycle. Thus, it has synthesis inhibitors, such as pactamycin and sparsomycin. been suggested that ara-C and HU block cells at the Gi-S Spalding et al. (41) showed that, during replication of interface (2,19, 25). Our studies (B. K. Bhuyan, W. N. Vorhof,

FEBRUARY 1972 405

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1972 American Association for Cancer Research. B. K. Bhuyan, L. G. Scheldt, and T. J. FrÃ¤ser and T. J. FrÃ¤ser,paper in preparation) also indicate that NSC 17. Fujiwara, Y. Role of RNA Synthesis in DNA Replication of 107392 blocks at the Gj-S interface. Thus, it is possible that Synchronous Population of Cultured Mammalian Cells. J. Cellular these drugs (ara-C, HU, and NSC 107392) could be Comp. Physiol., 70: 291-3,00, 1967. 18. Gottlieb, D., and Shaw, P. D. Antibiotics. I. Mechanism of Action. beneficially combined with agents that acted either in M and Berlin: Springer-Verlag, 1967. GÃŒ(e.g., neocarzinostatin) or at the G]-S interface (e.g., 19. Graham, F. L., and Whitmore, G. F. The Effect of actinomycin D). Such studies are in progress. 1-0-D-Arabinofuranosylcytosine on Growth, Viability, and DNA Synthesis of Mouse L-cells. Cancer Res., JO: 2627-2635, 1970. 20. Hartwell, J., and Abbott, B. Antineoplastic Principles in Plants. ACKNOWLEDGMENTS Advan. Pharmacol. Chemotherapy, 7: 117-209, 1969. 21. Heidelberger, C. Fluorinated Pyrimidines. Progr. Nucleic Acid Res. The help of Dr. T. F. Brodasky in setting up the computer program Mol. Biol., 4: 2-50, 1965. for the calculation of percentage of survival is gratefully acknowledged. 22. Herr, R. R. Jahnke, H. K., and Argoudelis, A. D. The Structure of The help of Dr. E. Stubblefield in setting up synchronous cultures is Streptozotocin. J. Am. Chem. Soc., Â£9.4808-4809, 1967. acknowledged. 23. Horowitz, S. B., Chang, C., and Grollman, A. P. Mechanism of Action of Camptothecin. Pharmacologist, 12: 282, 1970. REFERENCES 24. Kato, H., and Yosida, T. H. Nondisjunction of Chromosomes in a Synchronous Cell Population Initiated by Reversal of Colcemid 1. Acs, G., Reich, E., and Mori, M. Biological and Biochemical Inhibition. Exptl. Cell Res., 60: 459-464, 1970. Properties of the Analogue Antibiotic Tubercidin. Proc. Nati. 25. Kim, J. H., and Eidinoff, M. L. Action of 1-0-D-Arabinofuranosyl- Acad. Sei. U. S., 52: 493-501, 1964. cytosine on Nucleic Acid Metabolism and Viability of HeLa Cells. 2. Bacchetti, S., and Whitmore, G. F. The Action of Hydroxyurea on Cancer Res., 25: 698-702, 1965. Mouse L-Cells. Cell Tissue Kinet., 2: 193-211, 1969. 26. Kim, J. H., and Stambuck, B. K. Synchronization of HeLa Cells by 3. Barranco, S. C., and Humphrey, R. M. The Effects of Vinblastine Sulfate. Exptl. Cell Res., 44: 631-633, 1966. l,3-Bis(2-chloroethyl)-l-nitrosourea on Survival and Cell 27. Kumagai, K., Koide, T., Kikuchi, M., and Ishida, N. Specific Progression in Chinese Hamster Cells. Cancer Res., 31: 191-195, Inhibition of the Initiation of DNA Synthesis in HeLa Cells by 1971. Neocarzinostatin. Abstracts of the Tenth International Cancer 4. Bertalanffy, F. D., and Gibson, M. H. L. The in Vivo Effects of Congress, Houston, p. 401. Houston: Univ of Texas Press, 1970. Arabinosylcytosine on the Cell Proliferation of Murine B16 28. Li, L. H., Olin, E. J., Fraser, T. J., and Bhuyan, B. K. Phase Melanoma and Ehrlich Ascites Tumor. Cancer Res., 31: 66-77, Specificity of 5-Azacytidine against Mammalian Cells in Tissue 1971. Culture. Cancer Res., 30: 2770-2775, 1970. 5. Bhuyan, B. K. Action of Streptozotocin on Mammalian Cells. 29. Livingston, R. B., and Carter, S. K. Single Agents in Cancer Cancer Res., 30: 2017-2023, 1970. Chemotherapy. New York: IFI Plenum Publishing Corp., 1970. 6. Bhuyan, B. K., and Johnston, R. L. Metabolic Studies on 30. Lozzio, C. B. Lethal Effects of Fluorodeoxyuridine on Cultured Pactamycin. Biochem. Pharmacol., 12: 1001-1010, 1963. Mammalian Cells at Various Stages of the Cell Cycle. J. Cell 7. Bhuyan, B. K., Scheidt, L. G., and Fraser, T. J. Cell Cycle Physiol., 74: 57-62, 1969. Specificity of Several Antitumor Agents. Proc. Am. Assoc. Cancer 31. Madoc-Jones, H., and Bruce, W. R. On the Mechanism of the Res., 11: 8, 1970. Lethal Action of 5-FU on Mouse L-Cells. Cancer Res., 28: 8. Bhuyan, B. K., and Smith, C. G. Differential Interaction of 1976-1981, 1968. Nogalamycin with DNA of Varying Base Composition. Proc. Nati. 32. Maeda, H., and Ishida, N. Conformational Study of Antitumor Acad. Sei. U. S., 54: 566-572, 1965. Proteins, Neocarzinostatin, and a Deaminated Derivative. Biochim. 9. Blumenthal, L. K., and Zahler, S. A. Index for Measurement of Biophys. Acta, 747: 597-599, 1967. Synchronization of Cell Populations. Science, 135: 724, 1962. 33. Mauro, F., and Madoc-Jones, H. Age Responses of Cultured 10. Borsa, J., Whitmore, G. F., Valeriote, F. A., Collins, D., and Bruce, Mammalian Cells to Cytotoxic Drugs. Cancer Res., 30: W. R. Studies on the Persistence of Methotrexate, Arabinosyl 1397-1408, 1970. Cytosine, and Leucovorin in Serum of Mice. J. Nati. Cancer Inst., 34. Ono, Y. Watanabe, M., and Ishida, N. Mode of Action of 42: 235-242, 1969. Neocarzinostatin: Inhibition of DNA Synthesis and Degradation of 11. Brockman, R. W., Sidwell, R. W., Arnett, G., and Shaddix, S. DNA in Sorcina lutea. Biochim. Biophys. Acta, 119: 46-58, 1966. Heterocyclic Thiosemicarbazones. Correlation between Structure, 35. Pfeiffer, S. E., and Tolmach, L. J. Selecting Synchronous Inhibition of Ribonucleotide ReducÃaseand Inhibition of DNA Populations of Mammalian Cells. Nature, 213: 139-142, 1967. Viruses. Proc. Soc. Exptl. Biol. Med., 133: 609-614, 1970. 36. Phillips, F. S., Schwartz, H. S., Sodergren, S. S., and Tau, C. T. C. 12. Bruce, W. R., Meeker, B. E., and Valeriote, F. A. Comparison of The Toxicity of Actinomycin D. Ann. N. Y. Acad. Sci., 89: the Sensitivity of Normal Hematopoietic and Transplanted 348-360, 1960. Lymphoma Colony-forming Cells to Chemotherapeutic Agents 37. Pittilo, R. F., and Wooley, C. 5-Azacytidine: Microbiological Assay Administered in Vivo. J. Nati. Cancer Inst., 37: 233-245, 1966. in Mouse Blood. Appi. Microbio!., 18: 284-286, 1969. 13. Carter, S. K. 2-2'-(9,10-Anthrylenedimethylene)bis(2-thiopseu- 38. Reich, E., and Goldberg, I. H. Actinomycin D and Nucleic Acid dourea)dihydrochloride, dihydrate. Cancer Chemotherapy Rept., Function. Progr. Nucleic Acid Res. Mol. Biol., 3: 183-234, 1964. 5.2 (Suppl.): 153-163, 1968. 14. Chu, M. Y., and Fischer, G. A. A Proposed Mechanism of Action of 39. Schein, P. S., and Loftus, S. Streptozotocin: Depression of Mouse 1-0-D-Arabinofuranosy Icytosine as an inhibitor of the Growth of Liver Pyridine Nucleotides. Cancer Res., 28: 1501-1506, 1968. Leukemic Cells. Biochem. Pharmacol., //. 423-430, 1962. 40. Sisken, J. E. Analysis of Variations in Intermitotic Times. In: G. G. 15. Elkind, M. M., Kano, E., and Sutton-GUbert, H. Cell-Killing by Rose, (ed.), Cinemicrography in Cell Biology, pp. 143-168. New Actinomycin D in Relation to the Growth Cycle of Chinese York: Academic Press, Inc., 1963. Hamster Cells. J. Cell Biol., 42: 366-377, 1969. 41. Spalding, J., Kajiwara, K., and Mueller, G. C. The Metabolism of 16. Elkind, M. M., and Whitmore, G. F. The Radiobiology of Cultured Basic Proteins in Hela Cell Nuclei. Proc. Nati. Acad. Sei. U. S., 55: Mammalian Cells, Chap. 6. New York: Gordon & Breach, 1967. 1535-1542, 1966.

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42. Stubblefield, E., Klevecz, R., and Deaven, L. Synchronized Argoudelis, A. D. The Structure of Pactamycin. J. Org. Chem.,35: Mammalian Cell Culture: I. Cell Replication Cycle and 1420-1425, 1970. Macromolecular Synthesis following Brief Colcemid Arrest of 45. Wiley, P. F., and MacKellar, F. A. The Structure of Sparsomycin. J. Mitosis. J. CeUPhysiol., 69: 345-354, 1967. Am. Chem. Soc., 92: 417-418, 1970. 43. Terasima, T., and Tolmach, L. J. Growth and Nucleic Acid 46. Wiley, P. F., MacKellar, F. A., CarÃ³n, E. L., and Kelly, R. B. Synthesis in Synchronously Dividing Populations of HeLa Cells. Isolation, Characterization, and Degradation of Nogalamycin. Exptl. Cell Res., 30: 344-362, 1963. Tetrahedron Letters, 6: 663-668, 1968. 44. Wiley, P. F., Jahnke, H. K., MacKellar, F., Kelly, R. B., and

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B. K. Bhuyan, L. G. Scheidt and T. J. Fraser

Cancer Res 1972;32:398-407.

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