Relationship between cytotoxicity and DNA damage in mammalian cells treated with anthracenedione derivatives
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Chem.-Biol. Inte~+ions. 46 (1983) 369-379 Elsevier Scientific Publishers Ireland Ltd.
RELATIONSIIIP BETWEEN CYTOTOXICITY AND DNA DAMAGE IN IAN CELLS TREATED WITHI ANTHRACENEDIONE
SARAH E. LOCHE:R and RAYMOND E. MEYN
Deparlment of fiysics. Uniwrsity of Texas. M.D. Anderson Hospital and Tumor Institute at Houston, Houston. TX 77030 U.S.A.
(Received December 15th. 1982) (Revision received .4pril4th. 1983) (Accepted April 7th. 1963)
The effects of two anthracenedione derivatives on in vitro cell survival and DNA of Chines,2 hamster ovary (CHT)) cells were investigated. The two drugs studied were 1,4dihydroxy-5,&brs-(2-((2-hydroxyethyl)amino)ethylamino)- 9.10.anthracenedione (DHAQ, NSC No. 279836) and 1,4bis-(2.((2-
hydroxyethyl)-cunino)ethylamino)-9,1O-anthracenedione (HAQ, NSC No. 287513). DHAQ was lOO=fold more potent in re&;ring cell survival than HAQ. DNA strand breaks were assayed by alkaline elution. DHAQ (10 ng/ml) caused more strand breakage than 1000 ng/ml HAQ. This difference correlates well with their differences in ability to kill cells.
Key words: DNA damage -Single-strand breaks - Anthracenedione deriva- tives - Mammalian cells
Efforts to reduce the dose-limiting cardiac toxicity of t.he anthracycline antitumor agents have stimulated the recent interest in aminoalkylanth- raqumone chemotherapeutic drugs [l]. The cardiac toxicity appears to be related to the aminosugar moiety of the anthracyclines. Thus, the anth-
*Present address: Biophysical Sciences Department. Roswell Park Memorial Institute Buffalo, New York 14263. l To whom correspondence should be sent. Abbreviations: CHO, Chinese hamster ovary; DHAQ. 1,4-dihy~xy-5,sbis-(2-((2. hydroxyethyl)amino)ethylamino)-9,Wanthracenedione (NSC No. 279836): HAQ, l&bis-(2-((2. hydroxyethyl)amina~thylamino)-9.10-anthre diacetate (NSC No. 287513).
0009.2797163/$03.00 ,q 1963 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
NHtCH2)2NHCH2CH2OH II I
HA ii I NHICH&NHCH$H2OH
Fig. 1. Structures of DHAQ and HAQ.
raquinone agens preserve a planar chromophorc similar to the anthracy- clines, which is necessary for intercalation; however, they substitute amino- alkyl side chainIs for an aminosugar side group to stabilize the DNA inter- calation [2,31.
Recent evaluation of a series of anthraquiuone derivatives indicates that two possess significant antitumor effect in anima.1 tumor systems 131: HAQ and DHAQ, (Fig. 1).
These compounds have been evaluated for cell survival and cell cycle progression effects in vitro [US] and for genotoxicity 171. DHAQ is tenfold more @ent in in vivo tumor systems and several hundred-fold more potent for in vitro cell Jsilling than HAQ [2,3]. DHAQ has a higher therapeutic index than I&IQ in vi..0 since it exhibits less toxicity ard greater antitumor effects 131. DHAQ also shows greater genotoxicity as measured by cytogen,etic techniques 1.71.
DN,4 has be,zn implicated as an important intracellular target for the anthracycline a ntitumor agents [8,9]. Thus, the differences in effectiveness of HAQ and DHAQ may be related to di,Rering ability of the agents to induce DNA damage. T.he studies reported here address this question by assaying the amount of DNA damage induced by these dr.rgs in CHO cells treated in vitro, using the alkaline elution technique developed by Kohn and coworkers [lO,li].
Cdl growth and labelling CHO cells were grown in monolayer cultures in McCoys 5A medium sup- plemented with 15% fetal calf serum. %Icubationra were carried out at 37C in
a humidified atmosphere of 95% air/58 COP. Under these conditions, cells have a doubling time of approx. 12 h. Cells in the exknential growth phase were used in all experiments. DNA was labelled by incubation with [2- Wlthymidine (50 Ci/mM, 0.02 pCi/ml) for 18 h. This labelling period was followed by a 6h incubation in medium without label to chase the label into high molecular weight DNA.
Drug treatment HAQ and DHAQ were obtained from the Division of Cancer Treatment,
National Cancer Institute, Bethesda, MD. A solution of HAQ was made by dissolving 10 mg of drug in 10 ml of solution A (0.8 g NaCl, 0.4 g KCl, 1.0 g glucose, 0.35g NaHCO?/l). DHAQ solution was made by dissolving 10 mg of the drug in 0.1 M HCl. Drug solutions were filter sterilized and then ap- propriate dilutions made in solution A. All drug solutions were made up immediately before each experiment.
All treatments were performed on cells in suspension. CHO cells were trypsinized, peleted and resuspended in 5ml fresh medium at a density of approx. 1.25 x lo6 cells/ml. Drugs were added in the smE!llest possible volume and thoroughly mixed. Drug treatments were carried 3ut. in a 37C water bath for 1 h. After incubation, cell suspensions were placed on ice and spun down in the cold. Cells were resuspended in fresh ice-cold medium and spun down again. Then the cells were resuspended in ice-cold medium and held on ice until used for alkaline elution or plated in an in vitro survival assay.
Alkaline elution analysis The alkaline elution analysis procedures used in this irAvestigation were
essentially identical to those developed by Kohn and coworkers [lO,ll]. Briefly, 1 x 10 a 14C-lat~elled cells were diluted into 20 ml cold phosphate- buffered saline (0.15 m NaCl, 0.014 M KH2POI, 0.088 M KzHPOJ and gently impinged ontc p blycarbonate filters (25 mm diameter, 2 pm pore size). The cells were then rinsed with 10 ml cold phosphate-buffered saline and lysed by addition of 5 ml of lysir; solution with proteinase K (2% SDS, ~3.025 M EDTA, 0.25 mg/ml proteinase K, pH 9.7). The lysis solution was intubated on the filters for 30min at room temperature.
After cell lysis and incubation, the filters were rinsed ooce with 5ml 0.02M EDTA @H 10.3). In this step, as in the lysis procedur,?, the solution was allowed to pass through the filter by gravity. The DNA on the filters was then eluted with 0.1 M tetrapropylammonium hydroxide, 0.02 M EDTA (acid form) and 0.1% SDS (pH 12.1) at a constant flow rate of O.C14ml/min and collected into ten 40ml fractions. The radioactivity in these fracGons and that remaining on the filter was assayed by the procedure of Kohn et al. [lo].
The alkaline elution technique described above was developed by Kohn et al. [ll] to minimize the influence of protein adhesion to the filter or DNA in order to yield the most accurate estimate of DNA strand brerilkage. This particular method was chosen for the studies reported here when we obser- ved that DHAQ produced DNA-protein cross-links in the DNA of mammalian
cells. This w,m demonstrated using an alkaline elution procedure slightly different ,from th.e one detailed previously. In this case, the filters were made of polyvinyl chloride (25mm diameter, 2 pm pore size, Millipore Corp., Bedford, MA) and the lysis solution contained Sarcosyl instead of SDS (0.2% Sarcosyl, 0.04 M EDTA, 2 M NaCl, pH 10.0) and did not contain proteinase K. The SDS was also removed from the tetrapropylammonium hydroxide solut\on.
In order to observe the cross-links, it was necessary to introduce single- strand breaks into the DNA using ionizing radiation. Cross-links were then observed as a reduction in the elution of the DNA from the cells that had been trefated with the drug plus radiation compared to that from control C&J that received radiation alone [ll]. For this purpose, the cells were placed on ice and irradiated with 400 rad of X-rays (250 KVP) at 383 rad/min just prior to analysis by alkaline elution. After irradiation, the cells were kept on ice until lysis in order to prevent repair of the single-strand breaks. In some experiments, the cell lysates were subjected to proteolytic digestion prior to elution. Proteinase K was added to the Sarcosyl lysis solution and was allowed to remain in contact with the cells or the filter for 30 min at room temperature.
Lktenination &of cell survival To assay survival of cells following I&+ug treatment, we plated appropriate
numbers of cell:3 in triplicate into 60=m:nl culture dishes containing 5 ml of fresh culture medium. The dishes were incubated for 7 days for colony formation.
Survival curves for CHO cells treabed in suspension with HAQ or DHAQ for 1 h at 37C are presented in Fig. 2. A log-log scale was used to accommodate both survival curves on the same graph since there was a very large differenc(e in the ability of the two drugs to kill cells. Treatment with 20 ng/ml DHAiQ resulted in 50% survival, whereas a dose of 2000 nglml HAQ was necessary to produce the same effect (0.047 mM DHAQ vs. 4.86 mM HAQ). Thus, DHAQ was about lOGfold more potent for killing in vitro. These survival data for DHAQ are very similar to previously published data for several mammalian cell lines [4,51.
We suspected that DHAQ might interact with DNA in an analogous manner to Adriamycin, based on structural similarity. Thus, as Adriamycin had been previously shown to produce DNA-protein cross-links [121, we investigated whether DHAQ induced such lesions using the alkaline elution technique. The results of these experiments are shown in Fig. 3. Polyvinyl chloride filters and Sarcosyl lysis :aolution were used, as the,se facilitate observation of DNA-protein cross-links [ll]. As can be seen, DNA from cells treated with 20 nglml of DHAQ eluted in a manner similar to the untreated
I I111111 1 IW 5 10 50 100 500 lcKJ0 5(r(xI
DRUG CONCENTRATION hg/ml)
Fig. 2. Survival curves for CHO cells exposed in suspension for 1 h at 37C to DHAQ and H-46. Data points are the average of three experiments. Standard deviations are indicated when larger than the representative symbol.
control. However. if the cells were given 400 rad of X-rays, producing single-strand DNA breaks, DNA from DHAQ-treated cells eluted conr;ider- ably slower than DNA from cells that only received X-rays. Such finqiings suggest that cross-links in the DNA retard the expression of the X,.-ray- induced breaks [ll]. The effect was even more dramatic when a higher dose of DHAQ (100 ng/ml) was used.
Proteinase K was introduced in some experiments to test whethe - the DHAQ-induced cross-links were of the DNA-protein type. The results (Fig. 3) demonstrated that most of the cross-links made in DNA by DHAQ were susceptible to proteinase digestion. Furthermore, after proteinase treat,ment the early rate of DNA elution of DHAQ-treated cells was faster than the X-ray control, suggesting that some DHAQ-induced DNA strand bleaks originally masked by DNA-protein cross-links were now expressed. 011 the other hand, that the rate of elution of DNA from DHAQ-treated cells was slower than that of the X-ray control at longer elution times suggests that either the proteinase did not remove all of the DNA-protein cross-1inl.s or DHAQ produced some DNA interstrand cross-links in addition. Results with HAQ were qualitatively similar (data not shown); however, much higher doses of drug were necessary to produce such effects.
A different alkaline elution procedure was used in all subsequent experi- ments so that the numbers of DNA strand breaks produced in cells trtbated
s of D
with DHAQ or HAQ could be compared. This method, described in Materi- als and Methods, minimizes the influence of DNA-protein cross-links on the kinetics of alkaline elution of DNA containing strand breaks Ill]. The DNA alkaline elution profiles resulting from this approach for CHO cells treated with HAQ or DHAQ are presented in Fig. 4. DNA strand breaks were observed as an increased rate of elution from the filter. There was a dose-dependent increase in strand scission caused by either agent. DHAQ induced far more strand breaks than HAQ:20ng/ml HDAQ produced ap- proximately the same level of strand scission as a dose of 5000 ng/ml HAQ. Thus, DHAQ was about 250-fold more effective for inducing strand scission in DNA than was HAQ at the same concentration.
Possible differences in the repairability of these strand breaks were asses- sed by allowing the cells to undergo repair incubation at 37C for varying periods after drug treatment. These data are presented in Fig. B as the percentage of strand breaks remaining after repair incubation versus in- cubation time, where 100% was the level of strand scission seen immediately after drug treatment. Equitoxic drug doses that reduced survival to 15% were chosen for these studies. The fraction of unrepaired DNA strand breaks wa.5
I I I I I I I J
2 4 6 8
Fig. 4. Alkaline elution profiles for DNA from CHO cells treated with various doses of DHAQ or HAQ. Conditions for alkaline elution in these experiments were chosen to msPinize visualiza- tion of single-strand breaks and minimize the influence of DNA-protein cross-1ia;ks.
I I I I I 0.5 1 2 3
REPAIR INCUBATION TIME (hr)
Kinetics of rejoining of single-strand breaks following treatment of CHO cells with 50 nglml DHAQ or 5000 ng/ml HAQ. The percent DNA strand breaks remaining as a function of time after drug treatment was calculated from alkaline elution profiles as described in Results.
calculated as follows:
unrepaired breaks = ln(fdt/fc) ln(fdolfc)
where fdo and fdt are the fractions of DNA retained on the filter from drug treated cells at fraction 5 after zero time or t repair time, respectively, and fc is the fraction of control cell DNA retained on the filter at fraction 5.
A rapid repair component appeared in the first 30 min of HAQ repair kinetics. It was absent in the DHAQ recovery process. This rapid repair was followed by a plateau in which minimum further repair occurred during up to 3h incubation. DHAQ recovery kinetics showed a slower but more constant rate of repair of strand breaks. Residual strand scission was present after 24-h repair for both agents (data not shown).
The differences in the level of DNA lesions and cell survival resulting from treatment with these two drugs are striking. Neither the data presented here nor any other report rule out the possibility that the analogs differ in their ability to penetrate to the nucleus. In fact, the observed differences could be
most easily explained if more DHAQ reached the DNA for a given external cellular concentration. However, it seems unlikely that the substitution of two hydroxyl groups for hydrogens on the chrr.I.nophore would alter penetrability sufficiently to account for the lOO.fold &tierence in survival or 250~fold enhancement of strand scission. On the other hand, the nature of these substituents does increase the electron density of the chromophore and hence the avidity of binding to DNA. Binding avidity is thought to be critical in the mechanism of DNA strand scission by anthracyclines . Since anthracenediones are, in general, believed to act by a similar mechanism [I], the alteration could possibly play a role in the increased strand scission by DHAQ. On the other hand, Johnson et al.  have reported that, among various aminoanthraquinones, the ability of the different compounds LO bind to DNA does not correlate with their relative antitumor activity in vivo. Thus, other factors must be considered in the attempt to relate the molecular effects of these agents to their biological effects.
Bachur et al.  have shown that anticancer drugs containing quinone groups can react with DNA in solution via a free radical mechanism follow- ing chemical reduction. This interaction can lead to single strand breaks in the DNA under these conditions ; however, it is not known if the same reactions cause the DNA strand breaks observed in living cells treated with such drugs. Ross et al. 1121 have alternatively suggested that the breaks in cells may represent a cellular response to the intercalation perhaps involving DNA breaking-rejoining enzymes like topoisomerases. This model would explain why the cellular breaks are found associated with DNA-protein crosslinks provided that the topoisomerase remains bound to the DNA for some period of time following the incision [12,15]. In addition to adriamycin and ellipticine 1121, such breaks have been observed in cells treated with m-AMSA , an acridine intercalator and 5iminodaunorubicine 1171. an anthracycline modified in the quinone ring which possesses a lower potential for free radical formation than adriamycin.
The fact that DNA strand breaks could only be observed in cells treated with DHAQ under conditions where DNA-protein cross-links were eliminated (Fig. 3) suggests that these lesions are similar to the protein-associated DNA breaks in cells treated with the anthracyclines and other intercalators mentioned above. These results are confirmatory of a previous study with DHAQ 1181.
It is not completely clear, however, that these protein-concealed DNA strand breaks represent the cytotoxic lesions. That the differences in cyto- toxicity (lOO.fold) and induction of DNA strand scission (250-fold) between DHAQ and HAQ do not correlate exactly suggests that at the very least the relationship between cytotoxicity and DNA damage is slightly digerent for the two drugs. This may be due to subtle differences in the types of DNA lesions pro&& but not distinguished by alkaline elution. The different repair kinetics for DNA strand breaks produced by the two agents @ig. 5) suggest that the type of breaks may be different. In a study of three intercalating agents, Ross et a]. 1151 also noted that cytotoxicity did not
correlate with st,rad scission levels. This finding bas been extended to other intercalators by Zwelling and coworkers 116,171 who also reported that the persistence of these lesions in cellular DNA varies considerably among the difierent drugs studied.
This overall lack of correlation supports the idea that different inter- calaters may form different types of strand break lesions, and thus, lesion repairability may be an additional critical factor for cell survival. The repair kinetics shown ip Fig. 5 for DHAQ and HAQ indicate that repair of intercalator-indud DNA strand breaks does occur and that even highly similar &ugs cm iipduce DNA ]esions that are distinguished by the cellular repair system. It iaalso possible that the drugs are selective for different sites in DNA, which may differ in frequency or accessibility to repair enzymes and th us cytotexic potential.
Gn the other hand, the induction of DNA lesions may not be the sole mechanism responsible for the cell-killing effects of these drugs. Many other intercalating drugs, including anthracyclines, associate with phospholipids and affect membrane stability . ?n fact, adriamycin has been shown by Myers et al. 1201 to produce lipid per-oxidation in vivo via a free radical mechanism. Furthermore, at least in animal systems, these drugs are actively metabolized but to differing degrees [Sl]. HAQ has also been shown to inhibit NADPH-cytochrome P-450 reductase activity and microsomal oxidative drug metabohsm .
In summary, our data indicate that, in addition to their structural similarities to anthracyclines, the anthracenedione derivatives HAQ and DHAQ induce a particular type of lesion in DNA, protein-associated DNA breaks, that was first demonstrated in cells treated with intercakating agents such as adriamycin 1121. These findings coupled with the observed higher antitumor and lower cardiotoxicity characteristics of the anthracenedione derivatives generally support the hypothesis that the antitumor effects of such agents is due to their intercalation and subsequent action on DNA, while the toxicity of drugs such as adriamycin may be due to membrane effects 1191. MO&ever, the differences in cytotoxicity and DNA damage production between HAQ and DHAQ reported here demonstrate the large potential importance of otherwise simple structural modifications to the potency and mechanism of action of such drugs. Investigations by alkaline elution of the type+and relative amounts of DNA lesions in mammalian cells caused by new anticencer agents may aid in drug development.
This work was supported by MH Grant No. CA-23270.
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