- Email: [email protected]
Relationship between cytotoxicity and dna strand break-age produced by adriamycin and other intercalating agents
Inr. J. Rudicrlion &wo/ogy Biol. Phys.,, Vol. 5, pp. 1221-1224 ’ Pergamon Press Ltd., 1979. Printed m the U.S.A.
0360-3016/79/081221-04$02.00/O
??Adriamycin
RELATIONSHIP BETWEEN CYTOTOXICITY AND DNA STRAND BREAKAGE PRODUCED BY ADRIAMYCIN AND OTHER INTERCALATING AGENTS-t E. Ross, M.D.
W. Department
of Medicine,
University
of Florida,
Gainesville,
FL
and L. A. ZWELLING, Laboratory
of Molecular
M.D. and K. W. KOHN,
Pharmacology,
National Cancer Institute,
M.D. NIH, Bethesda,
MD
Intercalating
agents were compared with respect to cytotoxicity and DNA damage in order to further clarify the mechanism of action of this important group of anti-tumor drugs. Using the DNA alkaline elution technique, we have previously reported that when L1210 cells are exposed to a variety of intercalators, DNA single strand breaks are formed. The single strand breaks are spatially associated with DNA-protein crosslinks and the breaks and cross-links occur with approximately equal frequency. In this work, the cytotoxic potency of these drugs were compared using a soft agar colony forming assay. Ellipticine, which had produced the greatest frequency of single strand breaks, was only weakly toxic while adriamycin and actinomytin D exhibited potent cytotoxicity but produced fewer single strand breaks per unit drug concentration. To test the possibility that DNA repair processes may be involved in the cytotoxicity of intercalating agents, drug-treated cells were allowed to form colonies in the presence of caffeine, an inhibitor of DNA repiication repair. Cytotoxicity of ellipticine, actinomycin D and hycanthone were significantly increased while that of adriamycin was diminished. We conclude that intercalating agents cause the formation of protein-associated DNA breaks but the role of these breaks in drug-induced cytotoxicity is unclear. Response to post-treatment incubation with caffeine suggests that the mechanism of cytotoxicity with adriamycin may differ in important respects from that of other intercalating agents. Extensions of these studies may be useful in elucidating the molecular basis for the biological interaction between radiation and intercalators. Adriamycin,
Caffeine,
DNA repair, Hycanthone,
Ellipticine,
INTRODUCTION (ADR)
D.
studies designed to examine this relationship by comparing a variety of intercalating agents with respect to cytotoxic potency and the formation of strand breaks. In addition, it was of interest to determine if caffeine, an inhibitor of DNA repair in rodent cells, would enhance cell killing by the intercalators.
to a group of drugs which exert their anti-tumor effect by intercalation between DNA base pairs, thus causing distortions in the helix and reduction in template activity. Recently, single strand breaks in DNA have been described following treatment of mammalian cells with a variety of intercalating agents.‘,s,8,s,10,11,12We have studied this phenomenon using the alkaline elution technique and found that these breaks appear unique in that each is spatially associated with a single DNA-protein cross-link.‘O This suggests the possibility that the single strand break may result from the action of an intracellular protein responding to the binding of drug to DNA. The relationship between the drug-induced strand breaks and antitumor activity is unknown. In this paper we report Adriamycin
Actinomycin
belongs
METHODS
AND MATERIALS
Mouse leukemia L1210 cells, grown in suspension, were used in all experiments. Details of tissue culture techniques have been published.7 Adriamycin and hycanthone were dissolved in distilled H,O. Ellipticine was dissolved in lo-*N HCl and actinomytin D in ethanol. Cells were resuspended in fresh warm medium at 9 x lo5 cells/ml just pior to drug exposure. Drug treatment was for one hour, at which
tDrugs were obtained from the Developmental Therapeutics Program, Division of Cancer Treatment,
NCI. Reprint requests to: W. E. Ross, M.D. 1221
1222
Radiation Oncology 0 Biology 0 Physics
August 1979, Volume 5, Number 8
time the cells were washed twice with fresh warm medium and resuspended. All incubations were at 37°C and in the dark. DNA strand break frequency was determined by the alkaline elution technique. Details of this methodology have been published.7,10 Assays were performed immediately upon drug removal. Colonyforming ability following drug treatment was determined by the soft agar technique of Chu and Fischer.3 Colonies were counted 14-17 days after seeding. Cloning efficiency of untreated L1210 cells was 8%9570. The effect of caffeine on colony-forming ability of drug-treated L1210 cells was determined by resuspending the cells in medium containing 2mM caffeine following drug treatment. This concentration of caffeine was maintained in the agar tubes. Non-drug treated cells which were seeded in the presence of caffeine exhibited ~60% cloning efficiency. Survival of cells seeded with or without caffeine was normalized to the appropriate control value. Each point on Fig. 1 and 2 represents the mean of duplicate values obtained in a single experiment. 0.1 -
t
RESULTS
DNA strand break frequency and colony forming ability were determined for various doses of ADR, ellipticine and actinomycin D (Fig. 1). Ellipticine (320pM) produced a high frequency of DNA single strand breaks but only a small reduction in clonogenic potential. In contrast, ADR (0.2-1.4 pg/ml) and actinomycin D (0.2-0.75 pg/ml) caused far fewer strand breaks but were highly lethal. The effect of caffeine on intercalator-induced cytotoxicity is demonstrated in Figs. 2 and 3. When cells are exposed to 2 mM caffeine following removal of ellipticine, hycanthone, or actinomycin D, there is a significant enhancement of cytotoxicity. In the case of hycanthone and actinomycin D, this appears to be primarily due to loss of the shoulder region. An increase in the slope of the linear portion of the survival curve is suggested as well but the data does not allow precise quantitation. There is a clear increase in the slope of the ellipticine survival curve when the cells are exposed to caffeine. The cytotoxicity of ADR is demonstrated in Fig. 3. In contrast to ellipticine, hycanthone, and actinomycin D, incubation of cells in the presence of caffeine following ADR treatment reduces the cytotoxic effect of adriamycin at the higher concentrations .
In order to define the concentration dependence of the caffeine effect, cells were treated for one hour with either ellipticine (80 PM) or ADR (1.25 E.Lg/ml). The effect on ellipticine cytotoxicity increased with
ActinomydnD 0.05
-
0.03-
\
Adriamycin
0.011
0.1
0.2
0.3
0.4
BREAK FREQUENCY PER 106 NUCLEOTIDES
Fig. 1. Relationship between cytotoxicity and induction of DNA single strand breaks. Colony forming ability was determined by soft agar cloning assay. DNA single strand break frequency was determined by alkaline elution. Each point represents the result obtained at a single drug concentration.
caffeine concentration over a 0.1-2.0 mM range while the effect of adriamycin toxicity exhibited a plateau at approximately 0.5 mM caffeine (Fig. 4). In other studies, cells were exposed to 2 mM caffeine for 3 hours prior to drug treatment, washed, treated with ellipticine, and then seeded in agar without caffeine. The caffeine was without effect under these circumstances (data not shown). Similarly, if cells were exposed to caffeine for less than 3 hours following drug treatment, then washed prior to seeding in agar, no enhancement of cytotoxicity was seen (data not shown). Caffeine has potent solubilizing activity.13 Thus, it was important to demonstrate that the caffeine was
Toxicity and DNA breaks by intercalators 0 W. E. Ross rt trl.
0.11
0
,
I
1
I
20
40
m
80
rm ELLlPTlClNE
1223
&“I
ACTINOMYCIN
0.
Fig. 2. Effect of caffeine on cytotoxicity of hycanthone, ellipticine, and actinomycin D. L1210 cells were treated with intercalator for one hour, washed twice, and seeded in soft agar in the presence (solid circles) or absence (open circles) of 2 mM caffeine. Each point is the mean of duplicate values obtained in a single experiment. not simply dislodging intercalators from non-specific cellular binding sites into the extracellular milieu thereby effecting a prolonged drug exposure. To test for this, untreated cells were added to agar tubes containing caffeine and cells which had been treated with lethal concentrations of intercalator. Each drug was tested. The colony-forming efficiency of the untreated cells was similar to that of cells incubated in agar containing caffeine only.
0.25
0.50
0.75
1.0
1.25
pg/ml ADRIAMYCIN Fig. 3. Effect of caffeine on cytotoxicity See legend, Fig. 2.
of Adriamycin.
DISCUSSION The basis of the anti-tumor activity exhibited by intercalating agents has been ascribed to reduction of DNA template activity. Using isolated DNA and the appropriate polymerase it is possible to demonstrate that inhibition of RNA and DNA synthesis is dependent on the extent of drug-DNA binding.4 Recent studies have shown that treatment of mammalian cells with a variety of intercalating agents results in DNA single strand breaks. Further, it has been suggested that DNA strand breakage may be an additional factor in intercalator-induced cytotoxicity.‘,” Since these strand breaks cannot be demonstrated using only isolated DNA, it is likely that some additional feature of the drug-cell interaction is necessary to account for their presence. One means of assessing the significance of these strand breaks would be to compare several intercalating agents of differing cytotoxic potency with respect to the formation of DNA single strand breaks. The data we have reported herein suggest that the lethality of adriamytin, actinomycin D and ellipticine cannot be accounted for simply on the basis of DNA strand
1224
Radiation Oncology 0 Biology 0 Physics 30
I
I
I
breakage and raises the possibility that these breaks may have other biological implications. Incubation of cells with caffeine following treatment with intercalating agents represents another means of defining the relationship between the cytotoxicity of these drugs and their effects on DNA. We have shown that caffeine enhances the cytotoxic effect of the intercalating agents ellipticine, actinomycin D, and hycanthone on L1210 cells. The
I
10
< >,
5.0
h
3.0
August 1979, Volume 5, Number 8
effect is dependent on the caffeine concentration and appears to require prolonged exposure. The simplest interpretation of these data is that caffeine interferes with one or more intracellular events which are required for the cells to survive exposure to the inter-
2 T#
calator . 1.0
0.5
0.3
0.1
0
0.5
1.0
1.5
2.0
mM CAFFEINE Fig. 4. Dose dependence of caffeine effect on Adriamycin and ellipticine cytotoxicity. L1210 cells were treated with either Adriamycin (1.25 pg/ml) (closed circles) or ellipticine (80 PM) (open circles) for one hour, washed twice, and seeded in soft agar containing various concentrations of caffeine.
Caffeine inhibits DNA replication repair in rodents and in exicision repair-deficient human cells following exposure to UV radiation and a number of drugs which bind covalently to DNA.6 The mechanism is unknown. DNA repair following exposure of cells to intercalating agents has not been conclusively demonstrated. However, the presence of protein-associated DNA single strand breaks following intercalator exposure suggests that a repair process may be operative. Further examination of molecular events which accompany the caffeine potentiation of intercalator-induced cytotoxicity will be required to determine if this results from inhibition of DNA repair. If is of great interest to note that, in contrast to the other intercalators, the cytotoxicity of ADR was somewhat diminished by caffeine. It is not obvious what feature of ADR causes it to differ from the other drugs in this respect but further exploration of this phenomenon may reveal important differences in mechanism of action.
REFERENCES 1. Bases, R., Mendez, F, Elequin, F, Liebeskind, D., Kozin, A., Neubort, S.: Early steps in mutagenesis by hycanthone. Cancer Res. 38: 781-786, 1978. 2. Byfield, J.E., Lee, Y.C., Tu, L.: Molecular interactions between adriamycin and x-ray damage in mammalian tumor cells. Znt. J. Cancer 19: 186-193, 1977. 3. Chu, M.Y., Fischer, G.A.: Incorporation of cytosine3H arabinoside and its effect on murine leukemia cells 17: 753-767, 1%8. (15178Y). Biochem. Pharmacol. 4. DiMarco, A.: Adriamycin: Mode and mechanism of action. Cancer Chemo. Rep. 6: 91-106, 1975. 5. Furlong, N.B., Suto, J., Brown, T., Chavez, F., Hurlbert, R.B.: Induction of limited DNA damage by the anti-tumor agent Cain’s acridine. Cancer Res. 38: 132P1335,
1978.
6. Kihlman, B.A.: Caffeine and Chromosomes. Amsterdam, Elsevier Scientific Publishing Company, 1977, pp. 26&268. 7. Kohn, K.W., Erickson, L.C., Ewig, R.A.G., Friedman, C.A.: Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry 15: 4629-4637,
1976. 8. Lee, Y.C., Byfield, J.E.: Induction of DNA degradation in vivo by adriamycin. J. Natl. Cancer Inst. 57: 221-224,
1976.
9. Pater, M.M., Mak, S.: Actinomycin D induced breakage of human KB cell DNA. Nature 250: 786788, 1974.
10. Ross, W.E., Glaubiger, D.L., Kohn, K.W.: Quantitative and qualitative aspects of intercalator-induced
DNA damage. Biochem.
Biopys.
Acta
562: 41-50,
1979.
11. Schwartz, H.S.: Alkali-labile regions and strand breaks in DNA from cells treated with daunorubicin. J. Medicine 12. Spataro,
7: 33-46,
1976.
A., Kessel, D.: Studies on camptothecininduced degradation and apparent reaggregation of DNA from L1210 cells. Biochem. Biophys. Res.
Comm.
48: 643-648,
1972.
H.: The solubilization of polycyclic 13. Weil-Malherbe, aromatic hydrocarbons by purines. Biochem. J. 40: 351-363,
1946.
0360-3016/79/081221-04$02.00/O
??Adriamycin
RELATIONSHIP BETWEEN CYTOTOXICITY AND DNA STRAND BREAKAGE PRODUCED BY ADRIAMYCIN AND OTHER INTERCALATING AGENTS-t E. Ross, M.D.
W. Department
of Medicine,
University
of Florida,
Gainesville,
FL
and L. A. ZWELLING, Laboratory
of Molecular
M.D. and K. W. KOHN,
Pharmacology,
National Cancer Institute,
M.D. NIH, Bethesda,
MD
Intercalating
agents were compared with respect to cytotoxicity and DNA damage in order to further clarify the mechanism of action of this important group of anti-tumor drugs. Using the DNA alkaline elution technique, we have previously reported that when L1210 cells are exposed to a variety of intercalators, DNA single strand breaks are formed. The single strand breaks are spatially associated with DNA-protein crosslinks and the breaks and cross-links occur with approximately equal frequency. In this work, the cytotoxic potency of these drugs were compared using a soft agar colony forming assay. Ellipticine, which had produced the greatest frequency of single strand breaks, was only weakly toxic while adriamycin and actinomytin D exhibited potent cytotoxicity but produced fewer single strand breaks per unit drug concentration. To test the possibility that DNA repair processes may be involved in the cytotoxicity of intercalating agents, drug-treated cells were allowed to form colonies in the presence of caffeine, an inhibitor of DNA repiication repair. Cytotoxicity of ellipticine, actinomycin D and hycanthone were significantly increased while that of adriamycin was diminished. We conclude that intercalating agents cause the formation of protein-associated DNA breaks but the role of these breaks in drug-induced cytotoxicity is unclear. Response to post-treatment incubation with caffeine suggests that the mechanism of cytotoxicity with adriamycin may differ in important respects from that of other intercalating agents. Extensions of these studies may be useful in elucidating the molecular basis for the biological interaction between radiation and intercalators. Adriamycin,
Caffeine,
DNA repair, Hycanthone,
Ellipticine,
INTRODUCTION (ADR)
D.
studies designed to examine this relationship by comparing a variety of intercalating agents with respect to cytotoxic potency and the formation of strand breaks. In addition, it was of interest to determine if caffeine, an inhibitor of DNA repair in rodent cells, would enhance cell killing by the intercalators.
to a group of drugs which exert their anti-tumor effect by intercalation between DNA base pairs, thus causing distortions in the helix and reduction in template activity. Recently, single strand breaks in DNA have been described following treatment of mammalian cells with a variety of intercalating agents.‘,s,8,s,10,11,12We have studied this phenomenon using the alkaline elution technique and found that these breaks appear unique in that each is spatially associated with a single DNA-protein cross-link.‘O This suggests the possibility that the single strand break may result from the action of an intracellular protein responding to the binding of drug to DNA. The relationship between the drug-induced strand breaks and antitumor activity is unknown. In this paper we report Adriamycin
Actinomycin
belongs
METHODS
AND MATERIALS
Mouse leukemia L1210 cells, grown in suspension, were used in all experiments. Details of tissue culture techniques have been published.7 Adriamycin and hycanthone were dissolved in distilled H,O. Ellipticine was dissolved in lo-*N HCl and actinomytin D in ethanol. Cells were resuspended in fresh warm medium at 9 x lo5 cells/ml just pior to drug exposure. Drug treatment was for one hour, at which
tDrugs were obtained from the Developmental Therapeutics Program, Division of Cancer Treatment,
NCI. Reprint requests to: W. E. Ross, M.D. 1221
1222
Radiation Oncology 0 Biology 0 Physics
August 1979, Volume 5, Number 8
time the cells were washed twice with fresh warm medium and resuspended. All incubations were at 37°C and in the dark. DNA strand break frequency was determined by the alkaline elution technique. Details of this methodology have been published.7,10 Assays were performed immediately upon drug removal. Colonyforming ability following drug treatment was determined by the soft agar technique of Chu and Fischer.3 Colonies were counted 14-17 days after seeding. Cloning efficiency of untreated L1210 cells was 8%9570. The effect of caffeine on colony-forming ability of drug-treated L1210 cells was determined by resuspending the cells in medium containing 2mM caffeine following drug treatment. This concentration of caffeine was maintained in the agar tubes. Non-drug treated cells which were seeded in the presence of caffeine exhibited ~60% cloning efficiency. Survival of cells seeded with or without caffeine was normalized to the appropriate control value. Each point on Fig. 1 and 2 represents the mean of duplicate values obtained in a single experiment. 0.1 -
t
RESULTS
DNA strand break frequency and colony forming ability were determined for various doses of ADR, ellipticine and actinomycin D (Fig. 1). Ellipticine (320pM) produced a high frequency of DNA single strand breaks but only a small reduction in clonogenic potential. In contrast, ADR (0.2-1.4 pg/ml) and actinomycin D (0.2-0.75 pg/ml) caused far fewer strand breaks but were highly lethal. The effect of caffeine on intercalator-induced cytotoxicity is demonstrated in Figs. 2 and 3. When cells are exposed to 2 mM caffeine following removal of ellipticine, hycanthone, or actinomycin D, there is a significant enhancement of cytotoxicity. In the case of hycanthone and actinomycin D, this appears to be primarily due to loss of the shoulder region. An increase in the slope of the linear portion of the survival curve is suggested as well but the data does not allow precise quantitation. There is a clear increase in the slope of the ellipticine survival curve when the cells are exposed to caffeine. The cytotoxicity of ADR is demonstrated in Fig. 3. In contrast to ellipticine, hycanthone, and actinomycin D, incubation of cells in the presence of caffeine following ADR treatment reduces the cytotoxic effect of adriamycin at the higher concentrations .
In order to define the concentration dependence of the caffeine effect, cells were treated for one hour with either ellipticine (80 PM) or ADR (1.25 E.Lg/ml). The effect on ellipticine cytotoxicity increased with
ActinomydnD 0.05
-
0.03-
\
Adriamycin
0.011
0.1
0.2
0.3
0.4
BREAK FREQUENCY PER 106 NUCLEOTIDES
Fig. 1. Relationship between cytotoxicity and induction of DNA single strand breaks. Colony forming ability was determined by soft agar cloning assay. DNA single strand break frequency was determined by alkaline elution. Each point represents the result obtained at a single drug concentration.
caffeine concentration over a 0.1-2.0 mM range while the effect of adriamycin toxicity exhibited a plateau at approximately 0.5 mM caffeine (Fig. 4). In other studies, cells were exposed to 2 mM caffeine for 3 hours prior to drug treatment, washed, treated with ellipticine, and then seeded in agar without caffeine. The caffeine was without effect under these circumstances (data not shown). Similarly, if cells were exposed to caffeine for less than 3 hours following drug treatment, then washed prior to seeding in agar, no enhancement of cytotoxicity was seen (data not shown). Caffeine has potent solubilizing activity.13 Thus, it was important to demonstrate that the caffeine was
Toxicity and DNA breaks by intercalators 0 W. E. Ross rt trl.
0.11
0
,
I
1
I
20
40
m
80
rm ELLlPTlClNE
1223
&“I
ACTINOMYCIN
0.
Fig. 2. Effect of caffeine on cytotoxicity of hycanthone, ellipticine, and actinomycin D. L1210 cells were treated with intercalator for one hour, washed twice, and seeded in soft agar in the presence (solid circles) or absence (open circles) of 2 mM caffeine. Each point is the mean of duplicate values obtained in a single experiment. not simply dislodging intercalators from non-specific cellular binding sites into the extracellular milieu thereby effecting a prolonged drug exposure. To test for this, untreated cells were added to agar tubes containing caffeine and cells which had been treated with lethal concentrations of intercalator. Each drug was tested. The colony-forming efficiency of the untreated cells was similar to that of cells incubated in agar containing caffeine only.
0.25
0.50
0.75
1.0
1.25
pg/ml ADRIAMYCIN Fig. 3. Effect of caffeine on cytotoxicity See legend, Fig. 2.
of Adriamycin.
DISCUSSION The basis of the anti-tumor activity exhibited by intercalating agents has been ascribed to reduction of DNA template activity. Using isolated DNA and the appropriate polymerase it is possible to demonstrate that inhibition of RNA and DNA synthesis is dependent on the extent of drug-DNA binding.4 Recent studies have shown that treatment of mammalian cells with a variety of intercalating agents results in DNA single strand breaks. Further, it has been suggested that DNA strand breakage may be an additional factor in intercalator-induced cytotoxicity.‘,” Since these strand breaks cannot be demonstrated using only isolated DNA, it is likely that some additional feature of the drug-cell interaction is necessary to account for their presence. One means of assessing the significance of these strand breaks would be to compare several intercalating agents of differing cytotoxic potency with respect to the formation of DNA single strand breaks. The data we have reported herein suggest that the lethality of adriamytin, actinomycin D and ellipticine cannot be accounted for simply on the basis of DNA strand
1224
Radiation Oncology 0 Biology 0 Physics 30
I
I
I
breakage and raises the possibility that these breaks may have other biological implications. Incubation of cells with caffeine following treatment with intercalating agents represents another means of defining the relationship between the cytotoxicity of these drugs and their effects on DNA. We have shown that caffeine enhances the cytotoxic effect of the intercalating agents ellipticine, actinomycin D, and hycanthone on L1210 cells. The
I
10
< >,
5.0
h
3.0
August 1979, Volume 5, Number 8
effect is dependent on the caffeine concentration and appears to require prolonged exposure. The simplest interpretation of these data is that caffeine interferes with one or more intracellular events which are required for the cells to survive exposure to the inter-
2 T#
calator . 1.0
0.5
0.3
0.1
0
0.5
1.0
1.5
2.0
mM CAFFEINE Fig. 4. Dose dependence of caffeine effect on Adriamycin and ellipticine cytotoxicity. L1210 cells were treated with either Adriamycin (1.25 pg/ml) (closed circles) or ellipticine (80 PM) (open circles) for one hour, washed twice, and seeded in soft agar containing various concentrations of caffeine.
Caffeine inhibits DNA replication repair in rodents and in exicision repair-deficient human cells following exposure to UV radiation and a number of drugs which bind covalently to DNA.6 The mechanism is unknown. DNA repair following exposure of cells to intercalating agents has not been conclusively demonstrated. However, the presence of protein-associated DNA single strand breaks following intercalator exposure suggests that a repair process may be operative. Further examination of molecular events which accompany the caffeine potentiation of intercalator-induced cytotoxicity will be required to determine if this results from inhibition of DNA repair. If is of great interest to note that, in contrast to the other intercalators, the cytotoxicity of ADR was somewhat diminished by caffeine. It is not obvious what feature of ADR causes it to differ from the other drugs in this respect but further exploration of this phenomenon may reveal important differences in mechanism of action.
REFERENCES 1. Bases, R., Mendez, F, Elequin, F, Liebeskind, D., Kozin, A., Neubort, S.: Early steps in mutagenesis by hycanthone. Cancer Res. 38: 781-786, 1978. 2. Byfield, J.E., Lee, Y.C., Tu, L.: Molecular interactions between adriamycin and x-ray damage in mammalian tumor cells. Znt. J. Cancer 19: 186-193, 1977. 3. Chu, M.Y., Fischer, G.A.: Incorporation of cytosine3H arabinoside and its effect on murine leukemia cells 17: 753-767, 1%8. (15178Y). Biochem. Pharmacol. 4. DiMarco, A.: Adriamycin: Mode and mechanism of action. Cancer Chemo. Rep. 6: 91-106, 1975. 5. Furlong, N.B., Suto, J., Brown, T., Chavez, F., Hurlbert, R.B.: Induction of limited DNA damage by the anti-tumor agent Cain’s acridine. Cancer Res. 38: 132P1335,
1978.
6. Kihlman, B.A.: Caffeine and Chromosomes. Amsterdam, Elsevier Scientific Publishing Company, 1977, pp. 26&268. 7. Kohn, K.W., Erickson, L.C., Ewig, R.A.G., Friedman, C.A.: Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry 15: 4629-4637,
1976. 8. Lee, Y.C., Byfield, J.E.: Induction of DNA degradation in vivo by adriamycin. J. Natl. Cancer Inst. 57: 221-224,
1976.
9. Pater, M.M., Mak, S.: Actinomycin D induced breakage of human KB cell DNA. Nature 250: 786788, 1974.
10. Ross, W.E., Glaubiger, D.L., Kohn, K.W.: Quantitative and qualitative aspects of intercalator-induced
DNA damage. Biochem.
Biopys.
Acta
562: 41-50,
1979.
11. Schwartz, H.S.: Alkali-labile regions and strand breaks in DNA from cells treated with daunorubicin. J. Medicine 12. Spataro,
7: 33-46,
1976.
A., Kessel, D.: Studies on camptothecininduced degradation and apparent reaggregation of DNA from L1210 cells. Biochem. Biophys. Res.
Comm.
48: 643-648,
1972.
H.: The solubilization of polycyclic 13. Weil-Malherbe, aromatic hydrocarbons by purines. Biochem. J. 40: 351-363,
1946.