- Email: [email protected]
Selective killing of transformed cells in combination with inhibitors of polyamine biosynthesis and S-phase specific drugs
CellBiologylnternationalReports,
Vol. 5, No. 10, October 7981
991
SELECTIVE KILLING OF TRANSFORMED CELLS IN COMBINATIONWITH INHIBITORS OF POLYAMINRBIOSYNTRESISAND S-PHASE SPECIFIC DRUGS %rasad
S. Sunkara,
lSusan K. Fowler and 2Kenji Nishioka
Departments of lDevelopmenta1 Therapeutics and 'Surgery The University of Texas System Cancer Center M.D. Anderson Hospital and Tumor Institute Houston, Texas 77030
SUMMARY: The objective of this study was to examine the effect of inhibition of polyamine biosynthesis on the cell cycle traverse of normal and transformed cells using a-difluoromethyl ornithine (DFMO), a catalytic Irreversible inhibitor of ornithine decarboxylase. The results of this study indicate that DFMObrings about a significant decrease in the intracellular levels of putrescine and spermidine followed by an inhibition of cell growth in both the normal and transformed cells. The DFMOtreatment results in a differential effect on the cell cycle traverse of nor9~1 and transformed cells. A majority of the normal cells were arrested in Gl phase whereas their transformed counterparts were found to be accumulated in S phase of the cell cycle. Further, treatment of both types of cells with an S phase specific drug like Ara-C after polyamine depletion with DPMOresulted in a preferential synergistic killing of the transformed cells while the normal cells were protected from the cytotoxicity of Ara-C. These observations suggest the use of inhibitors like DPMOmight help in designing better therapeutic regimens in combination with other cytotoxic drugs. INTRODUCTION The naturally occurring polyamlnes, putrescine, spermidine and spermine seem to play an important role in the proliferation of mammalian cells (1). With the availability of specific inhibitors of polyamine biosynthesis, it has been possible to demonstrate that polyamines play important roles in the regulation of *
Present
and correspondence
030%1651/61/100661-06/$02.00/0
address: Merrell Research Center Merrell Dow Pharmaceuticals Inc. 2110 East Galbraith Road Cincinnati, Ohio 45215 @ 1661 Academic
Press Inc. (London)
Ltd.
992
Cell Biology International
Reports, Vol. 5, No. 10, October 7981
DNA synthesis (2, 3, 4) and cytokinesis (5, 6) of mammalian cells. Inhibition of polyamine biosynthesis either by methyl glyoxal bis(guanyl hydrazone) (MGBG) or a-methyl ornithine has been reported to have a differential effect on the cell cycle traverse of normal and transformed cells (4, 7, 8, 9). The normal human, mouse and rat embryo fibroblasts were found to be blocked in early Gl phase (4, 7) whereas the majority of the transformed cells were found to accumulate in late Gl of S phase of the cell cycle (4, 8). Recently a-difluoromethyl ornithine, a catalytic irreversible inhibitor of ornithine decarboxylase was synthesized (10) and shown to bring about a significant depletion of putrescine and spermidine levels in a number of cell systems both in vitro, and in who (11, 12, 13). In earlier study (12) we have showed that DFMJ causes a rapid inhibition of HeLa cell growth as a result of polyamine depletion and also arrests a majority of the cells in S phase. Further a sequential administration of DPMOand Ara-C, an S phase specific drug, results in a synergistic antiproliferative effect on HeLa cells. Similar results were obtained by Rupniak and Paul (14) wherein they have reported a selective killing of transformed cells when hydroxyurea was used in combination with MGBG. In the present study, we have investigated the effect of DPMO on the growth and cell cycle traverse of a number of normal and transformed Cells. The results of this study indicate that DPMO brings about inhibition of cell proliferation as a result of depletion of intracellular levels of putreecine and spermidine and also brings about an arrest of a majority normal cells (WI-38 and (SV-WI3T3) in Gl phase, whereas their transformed counterparts 38 (2RA); SV-3T3 and HeLa) were accumulated in S phase of the cell Further treatment of both normal and transformed cells cycle. with Ara-C after polyamine depletion with DPMOresulted in a preferential synergistic killing of the transformed cells while the normal cells were protected from the cytotoxicity of the Ara-C. MATERIALSANDMRTHODS Cell and cell synchrony. HeLa cells were routinely grown as monolayer cultures a8 described earlier (15). WI-38, 3T3, SV-WI-38 (2RA) and SV-3T3 were kindly supplied by Dr. B. R. Brinkley. All these cells were grown as monolayers in McCoy's medium supplemented with'161 fetal calf serum. To obtain mitotic cells for cell fusion experiments, an exponentially growing culture of HeLa cells was partially synchronized by a single thymidine block that was followed by N20 block for 9 hr , and the separation of mitotic cells by selective detachment (16). Cells thus harvested had a mitotic index of about 98X. Cell kinetics. Cells from different normal and transformed cell lines were plated in 150 mn culture plates at a cell density of The cells were grown either in the 2.5 X 105 cells per plate.
Cell Biology International
Reports, Vol. 5, No. 10, October 1981
993
presence or absence of DPMO(2.5 mM) for 96 hr at 37OC in a CO2 At the end of the,incubation period cells were trypsiincubator. nized, counted and portions of the cells (2 x 106 cells) were used for determining the cell cycle kinetics and polyaminelevels of both control and DPMOtreated populations. To find where the DPMOtreated cells were blocked, we applied the technique of premature chromosome condensation (17). This method, which involves the Sendai Virus-mediated fusion between mitotic and interphase cells makes it possible to determine the position of an interphase cell in the cell cycle on the basis of the morphology of it's prematurely condensed chromosomes (PCC). This method has been used for cell cycle analysis (4, 18). After 96 hr of incubation the DPMOtreated and untreated cells were fused with synchronized population of mitotic HeLa cells to induce PCC, chromosome preparations were made and scored for the frequency of the various typee of PCC, i.e., Gl, S and G2. The percentage of cells in S phase was further confirmed by pulse labelling the interphase cells for 20 min just before fusion and then determining the labelling indices after autoradiography. The cell pellet was resuspended Measurement of polyamine levels. in 0.2 ml of 4% sulfosalisylic acid and sonicated using a Branson The homogenates was then centrifuged at 10,000 x g in a Sonifier. Sorvall RC 2B refrigerated centrifuge at 4'C. The supernatant so obtained was used to estimate the polyamide levels in a Durrum amino acid analyzer according to Marton and Lee (19). The results presented represent the average of at least two estimates. Effect of combination of DPMOand Ara-C on WI-38 and SV-WI-38 (2RA) cell growth. For these studies, the WI-38 and SV-WI-38 cells were grown with or without a 2.5 mM concentration of DPMOfor 4 days. At the end of the incubation period one half of both the control and DF'MOtreated cells were subjected to a 16 hr Ara-C (50 pg/ml) treatment followed by incubation in regular medium. The other half grown in regular medium served as control. The increase in cell number was determined over a period of 5 days by trypsinizing the cells and counting them by the use of coulter particle counter. The viability of the cells were determined by trypan blue dye exclusion method. The data presented are an average of three experiments. RESULTSAND DISCUSSION Effect of DEMOon the growth and polyamine levels of normal and The inhibition of polyamine biosynthesis by transformed cells. DPMOresulted in a complete disappearance of intracellular putreseine and a significant reduction in both the spermidine and spermine levels in most of the cell lines investigated (Table 1). This inhibition of polyamine synthesis was followed by a significant reduction in the cell growth ranging from 36% to 70% of the This effect control cultures depending on the cell line studied. on the cell growth by DPMOcould easily be reversed by replacing
994
Cell BiologylnternationalReports,
Vol. 5, No. 10, October 7981
TABLE 1. Differential
Cell Line
1
Effects of DFMO Treatment on the Cell Traverse of Normal and Transformed Cells*
Creatment
Cell No. x 106
Frequency PCC in Gl
S
Cycle
(%>
Polyamine Levels (n.mol./106 Cells)
G2
Put.
Spd.
Spn.
WI-38
Control DFMO
5.4 2.3
42.0 70.9
51.0 29.1
7.0 0
0.704 0
2.896 0.016
3.36 1.80
3T3
Control DFMO
1.04 0.46
46.0 60.0
44.0 26.7
10.0 13.3
0.56 0
3.61 0.816
0.665 0.384
HeLa
Control DFMO
5.16 1.50
52.0 24.0
40.0 74.0
8.0 2.0
0.224 0
2.36 0.136
1.272 1.104
SV3T3
Control DFMO
3.72 2.10
38.0 6.0
56.0 84.0
6.0 10.0
0.852 0
3.224 0.20
0.892 1.008
2FA
Control DFMO
6.10 2.2
45.0 20.0
50.0 76.0
5.0 4.0
0.608 0
3.936 0.640
.1.65 9.79
c
*
Exponentially growing cells (0.25 x 106> were treated with DFMO (2.5 mM) for 96 hr. At the end of the treatment, the cells were collected, counted, and fused with mitotic HeLa cells and the frequency of different types of PCC were scored. Put: Putrescine; Spd: Spermidine; Spn: Spermine.
the drug with medium containing putrescine (50 pM) (data not presented) as shown earlier (11, 12). These results further confirm the requirements of polyamines for the optimal cell proliferation (2, 4, 20). Effect of DPMOon the cell cycle traverse of normal and trepsTo determine the cell cycle kinetics of the DPMO formed cells. treated cultures we have employed the technique of premature that inhibichromosome condensation (PCC). The results indicate tion of polyamine biosynthesis by DPMGresulted in an accumulation of normal cells (WI-38 and 3T3) in Gl phase of the cell cycle with a concomitant decrease in S and G2 populations in the case of WI38 and S phase population with 3T3 cells. A majority of the Gl PCC were of early Gl type suggesting that DPMGbrings about an accumulation of normal cells in early Gl phase of the cell cycle. On the contrary, a significant percentage of the DPHI treated transformed cells (Hela, SV-3T3 and 2RA) exhibited an S phase
CellBiologylnternationalReports,
Vol. 5, No. 10, October 1981
995
4.0, "I! 25 3.0 E s z 2.0
.‘
i
0
2
i
3
4
DAYS AFTER DFMO TREATMENT
Fig.
1:
Synergistic cytotoxicity of Ara-C to SV transformed WI-38 (2RA) cells following DFMO treatment. 0, control; 0, control cultures with Ara-C (50 ug/ml) for 16 hr and replaced with regular medium,A, DFMO-treated cultures replaced with regular medium;A, DFMO-treated cultures treated with Ara-C (50 ug/ml) for 16 hr and replaced with regular medium.
8.0 7.0 m;; ;; -8
6.0
g1
4.0
3 E
30
5.0
2.0 1.0
I 0
1 I
I
I
I
I
2
3
4
5
DAYS AFTER DFMO TREATMENT
Fig.
2:
Cytotoxicity of Ara-C and DFMO to normal human diploid fibroblasts (WI-38). The abbreviations and symbols are the same as in Fig. 1.
996
Cell Biolog y International
Repotis,
Vol. 5, No. 70, October 198 1
morphology of the PCC suggesting an accumulation of these cells in S phase of the cell cycle. Similar observations on the differential cell cycle traverse of normal and transformed cells as a result of polyamine limitation with other inhibitors like MGBGand a-MO were reported (4, 7, 8). These results confirm our earlier suggestion that normal cells recognize the deprivation polyamines and stop before the "restriction point" in Gl (4), whereas the transformed cells do not recognize and slowly go through the cell cycle with a low rate of DNA synthesis (2, 3, 4) resulting in an accumulating S phase cells. Effect of sequential administration of DFMOand Ara-C on normal and transformed cells. Since we have observed a Gl accumulation in normal cells and enrichment of transformed cells in S phase of the cell cycle after DPMOtreatment, we have tested whether we can preferentially increase the toxicity of Ara-C to transformed cells. The results presented in Figure 1 clearly indicate a potentiation of cytotoxicity of Ara-C to SV-transformed human fibroblasts (2 RA cells) following the DPMOtreatments. On the other hand the pretreatment with DFMOresulted in a protection from the cytotoxicity of Ara-C in case of normal human diploid fibroblasts (WI-38) (Figure 2). Although there was an initial lag after the Ara-C the DPMOtreated normal cell cultures recovered fast treatment, and started logarithmic growth whereas the control cells showed a much longer lag period. The results of this study indicate a differential cell cycle regulation of normal and transformed cells to polyamine limitation induced by DFMO, an irreversible catalytic inhibitor of ODC. The DFMOtreatment resulted in an arrest of normal cells (3T3, WI-38) in Gl phase whereas a majority of the transformed cells were found to be accumulated in late Gl and S phase of the cell cycle. This differential cell cycle regulation could be exploited to preferentially kill the tumor cells with S phase specific drugs like Ara-C or hydroxyurea (12, 14) and at the same time protect the normal cells from the cytotoxicity of these drugs. Since Ara-C brings about gastrointestinal and bone marrow toxicity, it will be interesting to see whether pretreatment with DFMOmight arrest these normal cell populations in Gl phase and make the host insensitive to Ara-C toxicity while preferentially killing the tumor cells. Currently we are testing whether this hypothesis holds true in experimental animals. Recently Prakash et al (13) have shown that a combination of DFMOwith cyclophosphsmide brought about a greater inhibition of proliferation of mammary sarcoma(RMT6) in mice. Similar observations were made by Marton (21) wherein he found that pretreatment of 9L rat brain fibrosarcoma with DFMOenhances the cytotoxicity of BCNUboth in titro and in trim. These observations along with ours suggest the use of inhibitors of polyamine biosynthesis like DFMOmight help in designing better therapeutic regimens in combination with other cytotoxic drugs.
Cellhology
InternationalReports,
Vol. 5, No. 70, October 1981
997
ACKNOWLRDGHMgNTS The authors thank Dr. Potu N. Rao for his helpful discussions. This study was supported by grant CA-23878 from National Cancer Institute, DHHW. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Janne, J., Poso, H. and Raina, A. (1978). Biochem. Biophys. Acta m:241-293. Fillingame, R.H., Jorstad, C.M. and Morris, D.R. (1975). Proc. Natl. Acad. Sci. 72:4042;4045. Krokan, H. and Eriksen,A. Eur. J. Biochem. -72:501(1977). 508. Sunkara, P.S., Pargac, M.B., Nishiolca, K. and Rao, P.N. (1979) J. Cell Physiol. 98:451-458. Sunkara, P.S., RaK P.N., Nishioka, K. and Brinkley, B.R. Exp. Cell Res. 119:63-68. (1979). Oriol-Audit, C. (1978). Eur. J. Biochem. 87:371-376. Rupniak, HT. and Paul, D. (1978). J. CelrPhysiol. %:161170. Rupniak, H.T. and Paul, D. (1978). In Advances in Polyamine Res. (Campbell, et al., eds). 1:117-126, Raven Press, New York. Sunkara, P.S. and Rae, P.N. (1981). In Advances in Polyamine Res. (Caldarera, C.M., Zappia, V. and Bachrach, U., eds). 2:347-356, Raven Press, New York. Metcalf, B.W., Bey, P., Danzin, C., Jung, M.J., Casara, P. and Vevert, J.P. J. Am. Chem. Sot. 100:2551-2553. (1978). Mamont, P.S., Duchesne, M.C., Grove, J. arBey, P. (1978). Biochem. Biophys. Res. Conunun. 81:58-66. Sunkara, P.S., Fowler, S.K., Nixioka, K., and Rao, P.N. (1980). Biochem. Biophys. Res. Commun. 95:423-430. Prakash, N.J., Schechter, P.J., Mamont, KS., Grove, J., KochWeser, J. and Sjoerdsma, A. (1980). Life Sci. 26:181-194. Rupniak, H.T. and Paul, D. (1980). Cancer Res.40:293-297. Rao, P.N. and Engelberg, J. (1965). Science 148x092-1094. Rao, P.N. (1968). Science 160:774-776. Johnson, R.T. and Rae, P.N. (1970). Nature %:717-722. Rao, P.N., Wilson, B., Puck, T.T. J. Cell. Physiol. (1977). 91:131-141. &ton, L.J. and Lee, P.L.Y. Clin. Chem. -21:1721(1975). 1724. Mamont, P.S., Bohlen, P., McCann, P.P., Bey, P., Schuber, F. Proc. Natl. Acad. Sci. 73:1626-1630. and Tardif, C. (1976). In Advances in Polyamine %s. Marton, L.J. (1981). 2:425(Caldarera, CM., Zappia, V. and Bachrach, U., eds.). 430, Raven Press, New York.
Received:
27th May 1981
Accepted:
29th June 1981
Vol. 5, No. 10, October 7981
991
SELECTIVE KILLING OF TRANSFORMED CELLS IN COMBINATIONWITH INHIBITORS OF POLYAMINRBIOSYNTRESISAND S-PHASE SPECIFIC DRUGS %rasad
S. Sunkara,
lSusan K. Fowler and 2Kenji Nishioka
Departments of lDevelopmenta1 Therapeutics and 'Surgery The University of Texas System Cancer Center M.D. Anderson Hospital and Tumor Institute Houston, Texas 77030
SUMMARY: The objective of this study was to examine the effect of inhibition of polyamine biosynthesis on the cell cycle traverse of normal and transformed cells using a-difluoromethyl ornithine (DFMO), a catalytic Irreversible inhibitor of ornithine decarboxylase. The results of this study indicate that DFMObrings about a significant decrease in the intracellular levels of putrescine and spermidine followed by an inhibition of cell growth in both the normal and transformed cells. The DFMOtreatment results in a differential effect on the cell cycle traverse of nor9~1 and transformed cells. A majority of the normal cells were arrested in Gl phase whereas their transformed counterparts were found to be accumulated in S phase of the cell cycle. Further, treatment of both types of cells with an S phase specific drug like Ara-C after polyamine depletion with DPMOresulted in a preferential synergistic killing of the transformed cells while the normal cells were protected from the cytotoxicity of Ara-C. These observations suggest the use of inhibitors like DPMOmight help in designing better therapeutic regimens in combination with other cytotoxic drugs. INTRODUCTION The naturally occurring polyamlnes, putrescine, spermidine and spermine seem to play an important role in the proliferation of mammalian cells (1). With the availability of specific inhibitors of polyamine biosynthesis, it has been possible to demonstrate that polyamines play important roles in the regulation of *
Present
and correspondence
030%1651/61/100661-06/$02.00/0
address: Merrell Research Center Merrell Dow Pharmaceuticals Inc. 2110 East Galbraith Road Cincinnati, Ohio 45215 @ 1661 Academic
Press Inc. (London)
Ltd.
992
Cell Biology International
Reports, Vol. 5, No. 10, October 7981
DNA synthesis (2, 3, 4) and cytokinesis (5, 6) of mammalian cells. Inhibition of polyamine biosynthesis either by methyl glyoxal bis(guanyl hydrazone) (MGBG) or a-methyl ornithine has been reported to have a differential effect on the cell cycle traverse of normal and transformed cells (4, 7, 8, 9). The normal human, mouse and rat embryo fibroblasts were found to be blocked in early Gl phase (4, 7) whereas the majority of the transformed cells were found to accumulate in late Gl of S phase of the cell cycle (4, 8). Recently a-difluoromethyl ornithine, a catalytic irreversible inhibitor of ornithine decarboxylase was synthesized (10) and shown to bring about a significant depletion of putrescine and spermidine levels in a number of cell systems both in vitro, and in who (11, 12, 13). In earlier study (12) we have showed that DFMJ causes a rapid inhibition of HeLa cell growth as a result of polyamine depletion and also arrests a majority of the cells in S phase. Further a sequential administration of DPMOand Ara-C, an S phase specific drug, results in a synergistic antiproliferative effect on HeLa cells. Similar results were obtained by Rupniak and Paul (14) wherein they have reported a selective killing of transformed cells when hydroxyurea was used in combination with MGBG. In the present study, we have investigated the effect of DPMO on the growth and cell cycle traverse of a number of normal and transformed Cells. The results of this study indicate that DPMO brings about inhibition of cell proliferation as a result of depletion of intracellular levels of putreecine and spermidine and also brings about an arrest of a majority normal cells (WI-38 and (SV-WI3T3) in Gl phase, whereas their transformed counterparts 38 (2RA); SV-3T3 and HeLa) were accumulated in S phase of the cell Further treatment of both normal and transformed cells cycle. with Ara-C after polyamine depletion with DPMOresulted in a preferential synergistic killing of the transformed cells while the normal cells were protected from the cytotoxicity of the Ara-C. MATERIALSANDMRTHODS Cell and cell synchrony. HeLa cells were routinely grown as monolayer cultures a8 described earlier (15). WI-38, 3T3, SV-WI-38 (2RA) and SV-3T3 were kindly supplied by Dr. B. R. Brinkley. All these cells were grown as monolayers in McCoy's medium supplemented with'161 fetal calf serum. To obtain mitotic cells for cell fusion experiments, an exponentially growing culture of HeLa cells was partially synchronized by a single thymidine block that was followed by N20 block for 9 hr , and the separation of mitotic cells by selective detachment (16). Cells thus harvested had a mitotic index of about 98X. Cell kinetics. Cells from different normal and transformed cell lines were plated in 150 mn culture plates at a cell density of The cells were grown either in the 2.5 X 105 cells per plate.
Cell Biology International
Reports, Vol. 5, No. 10, October 1981
993
presence or absence of DPMO(2.5 mM) for 96 hr at 37OC in a CO2 At the end of the,incubation period cells were trypsiincubator. nized, counted and portions of the cells (2 x 106 cells) were used for determining the cell cycle kinetics and polyaminelevels of both control and DPMOtreated populations. To find where the DPMOtreated cells were blocked, we applied the technique of premature chromosome condensation (17). This method, which involves the Sendai Virus-mediated fusion between mitotic and interphase cells makes it possible to determine the position of an interphase cell in the cell cycle on the basis of the morphology of it's prematurely condensed chromosomes (PCC). This method has been used for cell cycle analysis (4, 18). After 96 hr of incubation the DPMOtreated and untreated cells were fused with synchronized population of mitotic HeLa cells to induce PCC, chromosome preparations were made and scored for the frequency of the various typee of PCC, i.e., Gl, S and G2. The percentage of cells in S phase was further confirmed by pulse labelling the interphase cells for 20 min just before fusion and then determining the labelling indices after autoradiography. The cell pellet was resuspended Measurement of polyamine levels. in 0.2 ml of 4% sulfosalisylic acid and sonicated using a Branson The homogenates was then centrifuged at 10,000 x g in a Sonifier. Sorvall RC 2B refrigerated centrifuge at 4'C. The supernatant so obtained was used to estimate the polyamide levels in a Durrum amino acid analyzer according to Marton and Lee (19). The results presented represent the average of at least two estimates. Effect of combination of DPMOand Ara-C on WI-38 and SV-WI-38 (2RA) cell growth. For these studies, the WI-38 and SV-WI-38 cells were grown with or without a 2.5 mM concentration of DPMOfor 4 days. At the end of the incubation period one half of both the control and DF'MOtreated cells were subjected to a 16 hr Ara-C (50 pg/ml) treatment followed by incubation in regular medium. The other half grown in regular medium served as control. The increase in cell number was determined over a period of 5 days by trypsinizing the cells and counting them by the use of coulter particle counter. The viability of the cells were determined by trypan blue dye exclusion method. The data presented are an average of three experiments. RESULTSAND DISCUSSION Effect of DEMOon the growth and polyamine levels of normal and The inhibition of polyamine biosynthesis by transformed cells. DPMOresulted in a complete disappearance of intracellular putreseine and a significant reduction in both the spermidine and spermine levels in most of the cell lines investigated (Table 1). This inhibition of polyamine synthesis was followed by a significant reduction in the cell growth ranging from 36% to 70% of the This effect control cultures depending on the cell line studied. on the cell growth by DPMOcould easily be reversed by replacing
994
Cell BiologylnternationalReports,
Vol. 5, No. 10, October 7981
TABLE 1. Differential
Cell Line
1
Effects of DFMO Treatment on the Cell Traverse of Normal and Transformed Cells*
Creatment
Cell No. x 106
Frequency PCC in Gl
S
Cycle
(%>
Polyamine Levels (n.mol./106 Cells)
G2
Put.
Spd.
Spn.
WI-38
Control DFMO
5.4 2.3
42.0 70.9
51.0 29.1
7.0 0
0.704 0
2.896 0.016
3.36 1.80
3T3
Control DFMO
1.04 0.46
46.0 60.0
44.0 26.7
10.0 13.3
0.56 0
3.61 0.816
0.665 0.384
HeLa
Control DFMO
5.16 1.50
52.0 24.0
40.0 74.0
8.0 2.0
0.224 0
2.36 0.136
1.272 1.104
SV3T3
Control DFMO
3.72 2.10
38.0 6.0
56.0 84.0
6.0 10.0
0.852 0
3.224 0.20
0.892 1.008
2FA
Control DFMO
6.10 2.2
45.0 20.0
50.0 76.0
5.0 4.0
0.608 0
3.936 0.640
.1.65 9.79
c
*
Exponentially growing cells (0.25 x 106> were treated with DFMO (2.5 mM) for 96 hr. At the end of the treatment, the cells were collected, counted, and fused with mitotic HeLa cells and the frequency of different types of PCC were scored. Put: Putrescine; Spd: Spermidine; Spn: Spermine.
the drug with medium containing putrescine (50 pM) (data not presented) as shown earlier (11, 12). These results further confirm the requirements of polyamines for the optimal cell proliferation (2, 4, 20). Effect of DPMOon the cell cycle traverse of normal and trepsTo determine the cell cycle kinetics of the DPMO formed cells. treated cultures we have employed the technique of premature that inhibichromosome condensation (PCC). The results indicate tion of polyamine biosynthesis by DPMGresulted in an accumulation of normal cells (WI-38 and 3T3) in Gl phase of the cell cycle with a concomitant decrease in S and G2 populations in the case of WI38 and S phase population with 3T3 cells. A majority of the Gl PCC were of early Gl type suggesting that DPMGbrings about an accumulation of normal cells in early Gl phase of the cell cycle. On the contrary, a significant percentage of the DPHI treated transformed cells (Hela, SV-3T3 and 2RA) exhibited an S phase
CellBiologylnternationalReports,
Vol. 5, No. 10, October 1981
995
4.0, "I! 25 3.0 E s z 2.0
.‘
i
0
2
i
3
4
DAYS AFTER DFMO TREATMENT
Fig.
1:
Synergistic cytotoxicity of Ara-C to SV transformed WI-38 (2RA) cells following DFMO treatment. 0, control; 0, control cultures with Ara-C (50 ug/ml) for 16 hr and replaced with regular medium,A, DFMO-treated cultures replaced with regular medium;A, DFMO-treated cultures treated with Ara-C (50 ug/ml) for 16 hr and replaced with regular medium.
8.0 7.0 m;; ;; -8
6.0
g1
4.0
3 E
30
5.0
2.0 1.0
I 0
1 I
I
I
I
I
2
3
4
5
DAYS AFTER DFMO TREATMENT
Fig.
2:
Cytotoxicity of Ara-C and DFMO to normal human diploid fibroblasts (WI-38). The abbreviations and symbols are the same as in Fig. 1.
996
Cell Biolog y International
Repotis,
Vol. 5, No. 70, October 198 1
morphology of the PCC suggesting an accumulation of these cells in S phase of the cell cycle. Similar observations on the differential cell cycle traverse of normal and transformed cells as a result of polyamine limitation with other inhibitors like MGBGand a-MO were reported (4, 7, 8). These results confirm our earlier suggestion that normal cells recognize the deprivation polyamines and stop before the "restriction point" in Gl (4), whereas the transformed cells do not recognize and slowly go through the cell cycle with a low rate of DNA synthesis (2, 3, 4) resulting in an accumulating S phase cells. Effect of sequential administration of DFMOand Ara-C on normal and transformed cells. Since we have observed a Gl accumulation in normal cells and enrichment of transformed cells in S phase of the cell cycle after DPMOtreatment, we have tested whether we can preferentially increase the toxicity of Ara-C to transformed cells. The results presented in Figure 1 clearly indicate a potentiation of cytotoxicity of Ara-C to SV-transformed human fibroblasts (2 RA cells) following the DPMOtreatments. On the other hand the pretreatment with DFMOresulted in a protection from the cytotoxicity of Ara-C in case of normal human diploid fibroblasts (WI-38) (Figure 2). Although there was an initial lag after the Ara-C the DPMOtreated normal cell cultures recovered fast treatment, and started logarithmic growth whereas the control cells showed a much longer lag period. The results of this study indicate a differential cell cycle regulation of normal and transformed cells to polyamine limitation induced by DFMO, an irreversible catalytic inhibitor of ODC. The DFMOtreatment resulted in an arrest of normal cells (3T3, WI-38) in Gl phase whereas a majority of the transformed cells were found to be accumulated in late Gl and S phase of the cell cycle. This differential cell cycle regulation could be exploited to preferentially kill the tumor cells with S phase specific drugs like Ara-C or hydroxyurea (12, 14) and at the same time protect the normal cells from the cytotoxicity of these drugs. Since Ara-C brings about gastrointestinal and bone marrow toxicity, it will be interesting to see whether pretreatment with DFMOmight arrest these normal cell populations in Gl phase and make the host insensitive to Ara-C toxicity while preferentially killing the tumor cells. Currently we are testing whether this hypothesis holds true in experimental animals. Recently Prakash et al (13) have shown that a combination of DFMOwith cyclophosphsmide brought about a greater inhibition of proliferation of mammary sarcoma(RMT6) in mice. Similar observations were made by Marton (21) wherein he found that pretreatment of 9L rat brain fibrosarcoma with DFMOenhances the cytotoxicity of BCNUboth in titro and in trim. These observations along with ours suggest the use of inhibitors of polyamine biosynthesis like DFMOmight help in designing better therapeutic regimens in combination with other cytotoxic drugs.
Cellhology
InternationalReports,
Vol. 5, No. 70, October 1981
997
ACKNOWLRDGHMgNTS The authors thank Dr. Potu N. Rao for his helpful discussions. This study was supported by grant CA-23878 from National Cancer Institute, DHHW. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Janne, J., Poso, H. and Raina, A. (1978). Biochem. Biophys. Acta m:241-293. Fillingame, R.H., Jorstad, C.M. and Morris, D.R. (1975). Proc. Natl. Acad. Sci. 72:4042;4045. Krokan, H. and Eriksen,A. Eur. J. Biochem. -72:501(1977). 508. Sunkara, P.S., Pargac, M.B., Nishiolca, K. and Rao, P.N. (1979) J. Cell Physiol. 98:451-458. Sunkara, P.S., RaK P.N., Nishioka, K. and Brinkley, B.R. Exp. Cell Res. 119:63-68. (1979). Oriol-Audit, C. (1978). Eur. J. Biochem. 87:371-376. Rupniak, HT. and Paul, D. (1978). J. CelrPhysiol. %:161170. Rupniak, H.T. and Paul, D. (1978). In Advances in Polyamine Res. (Campbell, et al., eds). 1:117-126, Raven Press, New York. Sunkara, P.S. and Rae, P.N. (1981). In Advances in Polyamine Res. (Caldarera, C.M., Zappia, V. and Bachrach, U., eds). 2:347-356, Raven Press, New York. Metcalf, B.W., Bey, P., Danzin, C., Jung, M.J., Casara, P. and Vevert, J.P. J. Am. Chem. Sot. 100:2551-2553. (1978). Mamont, P.S., Duchesne, M.C., Grove, J. arBey, P. (1978). Biochem. Biophys. Res. Conunun. 81:58-66. Sunkara, P.S., Fowler, S.K., Nixioka, K., and Rao, P.N. (1980). Biochem. Biophys. Res. Commun. 95:423-430. Prakash, N.J., Schechter, P.J., Mamont, KS., Grove, J., KochWeser, J. and Sjoerdsma, A. (1980). Life Sci. 26:181-194. Rupniak, H.T. and Paul, D. (1980). Cancer Res.40:293-297. Rao, P.N. and Engelberg, J. (1965). Science 148x092-1094. Rao, P.N. (1968). Science 160:774-776. Johnson, R.T. and Rae, P.N. (1970). Nature %:717-722. Rao, P.N., Wilson, B., Puck, T.T. J. Cell. Physiol. (1977). 91:131-141. &ton, L.J. and Lee, P.L.Y. Clin. Chem. -21:1721(1975). 1724. Mamont, P.S., Bohlen, P., McCann, P.P., Bey, P., Schuber, F. Proc. Natl. Acad. Sci. 73:1626-1630. and Tardif, C. (1976). In Advances in Polyamine %s. Marton, L.J. (1981). 2:425(Caldarera, CM., Zappia, V. and Bachrach, U., eds.). 430, Raven Press, New York.
Received:
27th May 1981
Accepted:
29th June 1981