Mutation Research, 263 (1991) 69-75

69

© 1991 ElsevierSciencePublishers B.V. 0165-7992/91/$03.50 ADONIS 0165799291001M7N MUTLET 0492

Biochemical evidence for two different mechanisms for bleomycin-induced cell killing Amato J. Giaccia, Joseph Shieh, Annette Cholon and J. Martin Brown Department of Radiation Oncology, Division of Radiation Biology. Stanford University School of Medicine, Stanford. CA 94305 (U.S.A.)

(Received28 November 1990) (Revisionreceived9 January 1991) (Accepted 11 January 1991)

Keywords: Bleomycinhypersensitivecell; AFIGE; DNA double-strand breaks

Summary Using pulsed-field gel electrophoresis, we have measured the ability o f two bleomycin-sensitive mutants, XR-1 and BL-10, to repair DNA double-strands breaks (DSB). XR-1 was originally isolated by its hypersensitivity to killing with ionizing radiation, but we have also shown that it is sensitive to killing with bleomycin. In contrast, BL-10 was isolated by its extreme sensitivity to killing with bleomycin, and it is not crosssensitive to other DNA breaking agents. A 1-h treatment o f bleomycin induces a similar number of DNA double-strand breaks in XR-1, BL-10 and C H O cells. However, XR-1 is unable to repair bleomycin-induced D N A double-strand breaks, whereas BL-10 possesses the same kinetics o f repair as parental CHO. These data lead us to conclude that at least two mechanisms of killing exist for bleomycin; one of them is D N A DSB-dependent, and the other seems to be DNA DSB-independent.

Past work has suggested that the critical target for killing cells by ionizing radiation and radiomimetic drugs is DNA (Painter, 1980; Sikic, 1986). Supporting evidence for DNA as the target is that the mutants which are most sensitive to killing by radiation are also defective in the repair o f DNA double-strand breaks (DSBs) (Jeggo, 1990). Radio-

Correspondence: Dr. Amato J. Giaccia, Stanford Medical Center, CBRL, GK 115, Divisionof Radiation Biology,Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305-5468 (U.S.A.).

mimetic drugs, such as bleomycin, adriamycin and neocarzinostatin, also produce DNA strand breaks, but vary in the proportion of single-strand breaks to double-strand breaks (Ward, 1988). Several mutants tested to date, which are sensitive to killing by ionizing radiation and defective in rejoining DNA DSBs, exhibit cross-sensitivity to killing by other DNA-breaking agents. Presumably and as yet untested, these cell lines are also unable to repair bleomycin-induced DNA double-strand breaks. Similarly, mutants originally isolated by their sensitivity to bleomycin killing, which are defective in DNA double-strand break repair, are

70 also cross-sensitive to ionizing radiation (Hickson et al., 1988). Analysis of 19 bleomycin-sensitive mutants that we have previously isolated demonstrated that it was possible to uncouple bleomycin sensitivity from ionizing radiation sensitivity (Stamato et al., 1987). This is somewhat of an unexpected finding in that bleomycin supposedly exerts its cytotoxic effects by producing free-radicals through a reversible oxidation-reduction reaction of bleomycin chelated with iron (Fe2 ÷ is oxidized to Fe 3 ÷ ) intercalated into DNA, thereby producing single and double strand breaks (Sausville et al., 1976). Also, 6 of these mutants did not possess any dramatic cross-sensitivity to killing by cross-linking agents (MMC), intercalating/frameshift mutagens (ICR-191) or alkylating agents (EMS). Although the spectrum of DNA damage caused by bleomycin is similar to that caused by ionizing radiation, bleomycin may have other cellular targets. Membrane-interacting chemicals, such as ethanol, polyhydric alcohols and heat, can potentiate bleomycin cell killing, suggesting that membrane damage is also important for bleomycin cell killing (Magin et al., 1979; Suzuki et al., 1990; Urano et al., 1988). Using pulsed-field gel electrophoresis (Denko et al., 1989; Stamato et al., 1990), we have analyzed both the induction and repair of DNA doublestrand breaks in wild-type CHO-K1 cells, in XR-1 cells (sensitive to killing by ionizing radiation and bleomycin), and in BL-10 cells (sensitive to killing by bleomycin, but not X-rays). Induction of bleomycin-induced breaks is similar in all three cell lines, eliminating the possibility that cellular cytotoxicity results from unequal initial DSB formation. We found that BL-10 was able to rejoin bleomycin-induced DNA DSBs with the same kinetics as wild-type CHO cells. In contrast, XR-1 cells were only able to rejoin 20070 of their DNA breaks after 24 h of repair incubation. These data suggest that the sensitivity of BL-10 cells to bleomycin cannot be attributed to decreased DNA DSB repair, and imply that there may be targets other than DNA which can contribute to cell killing by bleomycin.

Materials and methods

Cell lines and bleomycin treatment The bleomycin-sensitive cell line, BL-10, was derived from CHO-K1 cells following a single mutagen treatment by ethyl methanesulfonate (Stamato et al., 1987). XR-1 was initially isolated on its hypersensitivity to killing by ionizing radiation and has also been found to be sensitive to killing by bleomycin (Giaccia et al., 1990). All cell lines were routinely maintained at 37°C in 5°70 CO2 with Alpha MEM (Gibco) media supplemented with 10070 fetal calf serum, 200 units/ml of penicillin and 100/~g/ml of streptomycin. BL-10's sensitivity to killing by bleomycin was determined by plating known quantities of cells into 6 well costar plates, allowing them to attach for 4-6 h and then exposing them to varying concentrations of drug for 1 h. The cells were then washed 3 × and refed with fresh F12 media and allowed to grow into colonies for 6-8 days. After this time interval, the colonies were fixed and stained, and colonies with greater than 50 cells were counted. PFGE assay for induction and repair of DNA double-strand breaks The assay for induction of DNA DSBs is essentially that described by Stamato and Denko (1990) for ionizing radiation with slight modifications. Briefly, exponentially growing cells were plated at a density of 2 × 106 cells/100-mm dish and allowed to grow for 2-3 population doublings in media containing 0.02 /~Ci/ml [14C]thymidine (40 mCi/ mmole, Amersham) to label both DNA strands. Before bleomycin treatment, the cells were washed twice with radioactive-free media and allowed to incubate 4 h to remove unincorporated thymidine. The cells were then treated in media with varying concentrations of bleomycin for 1 h. After washing the treated cells 3 times with drug-free media, the cells were either allowed to incubate at 37°C to allow DNA break rejoining or immediately trypsinized and neutralized with serum, centrifuged and washed once with phosphate-buffered saline without Ca 2 + and Mg 2+ . The cells were centrifuged once again and resuspended in phosphate

71 buffered saline containing 1070 agarose (Insert agarose-FMC, Rockland, ME) which had been equilibrated at 37°C. The agarose solution with the cells was cast into 3 mm in diameter glass tubes, cut into 5 mm lengths and then lysed for 16 h in 5 volumes of lysis buffer (0.5 M E D T A pH 7.9, 1% sarkosyl, 1 mg proteinase K/ml, Boehringer Mannheim, Indianapolis) at 50°C. After lysis, the plugs are dialyzed against 25 vol. o f TE (10 mM Tris pH 7.4, 1 mM EDTA) twice and then incubated for 1 h in T E containing 0.1 mg ribonuclease A/ml. The agarose plugs which contain purified DNA are loaded into 2 mm wells that were cast in a 0.8% agarose gel and subjected to asymmetric field inverted gel electrophoresis (AFIGE) (Denko et al., 1989) for 16-24 h. The conditions for AFIGE were 900 sec at + 1.25 V / c m and 75 sec at - 5.0 V/cm. Under these conditions, DNA fragments o f < 6 Mb will enter the gel while all larger DNA fragments will remain in the well. For quantitation, the lanes were separated from the wells with a scalpel and placed into a scintillation vial. 50 #1 of 10 N HCI was added, and the samples were melted on a hot plate. Upon the addition of scintillation fluid, the liquefied samples were counted in a Beckman LS 1800 (approximately 5000-10 000 cpm/lane). The induction o f DNA breaks is proportional to the fraction o f DNA eluted from the gel (Stamato and Denko, 1990) which was determined by the equation: Percentage of DNA releated from gel = I number of cpm in lane ~ × 100 total cpm in lane + well ) The rejoining of DNA strand breaks was determined by the equation: For repair: Percentage of DNA repaired = [1 - [(% DNA released after treatment at time t) - (*70DNA released after no treatment at time t)]/ [(aT0DNA released after treatment at 0 h) - (~/0DNA released after no treatment at 0 h)]l x 100 For untreated bleomycin controls, only 2 - 3 % o f the DNA was released from the well. Results are

presented as the mean o f at least 3 independent experiments. Results

Survival o f BL-IO cells following a 1-h bleomycin treatment Fig. 1 depicts the survival of BL-10 cells and C H O cells following a 1-h treatment o f bleomycin. Since previous BL-10 sensitivity testing was performed on 16-h chronic bleomycin exposures, we thought it necessary to verify that BL-10 was sensitive to a 1-h bleomycin exposure; especially since BL-10 is able to repair bleomycin-induced DNA double-strand breaks. Indeed, BL-10 is approximately 5-fold more sensitive than C H O to a 1-h exposure of bleomycin by comparison o f their D37s (dose o f bleomycin required to reduce survival to 37°7o o f the untreated population). Induction o f bleomycin-induced D N A doublestrand breaks The AFIGE technique has several advantages over neutral elution in that 80 samples can be run on one gel. Also, ethidium bromide staining of the D N A permits visualization of the induction and repair of DNA strand breaks, and a molecular weight size of the DNA that has been released from the well can be estimated. This technique has been useful in measuring induction and repair o f DNA double-strand breaks induced by ionizing radiation (Giaccia et al., 1990) and by restriction enzymes (Giaccia et al., 1990). Fig. 2 is a photograph o f an ethidium bromide stained agarose gel illustrating the induction o f bleomycin-induced DNA doublestrand breaks. Fig. 3 is a graphical representation o f the data from Fig. 2 and shows the percent o f DNA released from the well as a function o f dose. For a 1-h bleomycin treatment, a linear relationship exists between the DNA released from the well and dose o f bleomycin for all 3 cell lines. Since the induction o f DNA double-strand breaks is the same in all 3 cell lines, we can exclude the possibility that the different sensitivities o f the cells to bleomycin are the result of different levels o f initial DNA double-strand breaks.

72

stable. After 24 h, only 20°70 of XR-I's DNA DSBs are repaired. In contrast, BL-10 ceils exhibit similar biphasic kinetics of repair to CHO ceils, although they are as sensitive to killing by bleomycin as XR-1 cells. Both cell lines possess a 7"]/2 of 30 min for the fast component and continue repairing their breaks for 24 h, reaching a maximum of 90070 rejoined breaks. This finding eliminates the possibility that DNA double-strand breaks uniquely produced by bleomycin (Mirabelli et al., 1982) are responsible for BL-10's hypersensitivity to killing by bleomycin.

1001

1 HR

,,,,,J


_. ¢1: 03

BL10-1 HR

BLEOMYCIN CONC ( ~g/ml )

MARKERS

0

S.C. S.P. ;~

50 100 250

A CHO I 20

I 40

L 60

810

I

I 100

I

20

CONCENTRATION OF BLEOMYCIN (ug/ml) Fig. I. Survival of CHO and BL-I0 for varying concentrations of bleomycin for 1 h treatment.

Repair of bleomycin-induced DNA double-strand breaks We have previously shown by both neutral elution (Giaccia et al.. 1985) and AFIGE (Giaccia et al., 1990) that XR-1 cells are unable to repair ionizing radiation-induced DNA double-strand breaks. Here, we also find that the hypersensitivity of XR-1 to killing by bleomycin (Giaccia et al., 1990) appears to be a result of the inability to repair bleomycin-induced DNA double-strand breaks. Although all 3 cell lines show an immediate initial repair of DNA breaks, XR-I cells fail to continue to repair their DNA and are devoid of the slow component of repair. In fact, our data suggest that as XR-1 cells are allowed time to repair their breaks, either some DNA degradation occurs or a certain subset of DNA breaks rejoined are un-

B BL10

C XR-1

Fig. 2. Photograph of an agarose gel after asymmetric field inversion gel electrophoresis (AFIGE) depicting the release of DNA from the well into the gel as a function of bleomycin concentration for CHO, BL-10 and XR-1 cells.

73

,=1 .d W

6O

Y

50 =E

0

4O

14.

ua ill

..a

ill g=

3020-

Z a

,~

0

0

!

!

100

200

300

DOSE OF BLEOMYClN (lxg/ml) Fig. 3. Induction o f bleomycin-induced DNA DSBs graphed as percentage of DNA released as a function of bleomycin dose. • CHO, • BL-10, and • XR-1 cells.

Discussion

AFIGE analysis of two CHO mutants which are hypersensitive to killing by bleomycin suggests that at least two separate mechanisms exist for bleomycin cell killing. XR-I's sensitivity to killing by

bleomycin correlates with its inability to repair bleomycin DSBs. This result supports the most commonly held concept that the mechanism of cell killing by bleomycin is through DNA strand breaks. However, BL-10 cells are as sensitive to killing by bleomycin as XR-1 cells, but are as proficient in the repair of their DNA breaks as wild-type CHO cells. It is worthy to note that XR-I's sensitivity to killing by ionizing radiation is cell-cycle dependent (Stamato et al., 1983). XR-I's cell-cycle sensitivity to radiation correlates with its inability to repair DNA DSBs (Giaccia et al., 1985). In the Gl-phase of the cell-cycle, XR-1 cells have a Do of 0.33 Gy and repair only 35°7o of their DNA DSBs, whereas in S-phase, they have a Do of 2.3 Gy and repairs 67070of their breaks. For AFIGE analysis of bleomycin-induced breaks in XR-1, we used exponentially growing cells in which 60-70070 of cells were in Gl-phase. Thus, there is good agreement between the repair of ionizing radiation DNA DSBs and bleomycin DNA DSBs. Our previous work has also demonstrated that

BLEOMYCIN CONCENTRATION ( t~g/ml ) 0 TIME(hr) 0

0.5

1

250 3

24

0

0.5

1

MARKERS 3

24

S.C.S.P. ~.

A CHO

B BL10

C XR-1

Fig. 4. Photograph of an agarose gel after asymmetrical field inverted gel electrophoresis (AFIGE) depicting repair o f bleomycininduced DNA DSBs. Less DNA is eluted from the well into the gel if DSB repair occurs.

74

iit

::°01

40

20

o.f" 0

XR-I~ I

t

10

20

,30

TIME IN HR

Fig. 5. Repair of bleomycin-induced DNA double strand breaks calculated as described in Materials and methods for • CHO, • BL-10, and - XR-I cells.

XR-I ceils are sensitive to killing by restriction enzymes (Giaccia et al., 1990) in addition to ionizing radiatiGn and bleomycin. Since restriction enzyme cleavage of DNA produces ends that possess only 3'-hydroxyl and 5'-phosphate groups, the defect in XR-1 ceils is not confined only to the repair of ionizing radiation-induced DNA DSBs. Although we have not yet identified at the molecular level the gene responsible for the defect in XR-1, it is evident that this gene will participate in the repair of DNA DSBs produced by many different agents. BL-10 cells are not sensitive to killing by ionizing radiation or neocarzinostatin (both agents which kill cells by breakage of DNA). On the other hand, they are sensitive to killing by heat (Anderson et al., in preparation). The mechanism of cell killing by heat has not yet been fully determined, but the initial events in the cellular response to heat occur at the level of the cell membrane (Hahn, 1990). Furthermore, ionizing radiation and other DNAdamaging agents do not induce a ~heat stock response (Anderson et al., 1988), suggesting that these two agents kill by different mechanisms. In contrast, thermotolerance affords some cellular protection against exposure to bleomycin (Hahn et al., 1990), demonstrating a link between the two agents. The genetic defect in BL-10 cells responsible for its bleomycin sensitivity seems to result from a lack of alpha glutathione transferase activi-

ty (Giaccia et al., in preparation). This particular isozyme is responsible for removal of cellular peroxide as well as drug detoxification. The above data therefore lead us to conclude that BL-10's sensitivity to killing by bleomycin is by a different pathway than XR-1. In summary, our data show that two cellular mechanisms exist for bleomycin cell killing; one of them seems to be independent of DNA DSB repair. Further characterization of other bleomycinsensitive cell lines which we and others have isolated will allow us to further delineate the pathway for bleomycin cell killing that is represented by BL-10. Clinically, this may be important in recognizing that bleomycin cell killing may be potentiated by drugs which interfere with other cellular metabolic pathway besides DNA repair.

Acknowledgements This work was supported by U.S.P.H.S. Grant CA 15201 from the National Cancer Institute, D.H.H.S. We thank Dr. Thomas D. Stamato for supplying us with the XR-1 cell line and AFIGE apparatus. We would also like to thank Ms. Chiyoye Adachi for typing the manuscript.

References Anderson, R.L., E. Shiu, G.A. Fisher and G.M. Hahn (1988) DNA damage does not appear to be a trigger for thermotolerance in mammalian cells, Int. J. Radiat. Biol., 54, 285-298. Denko, N., B. Peters, A. Giaccia and T. Stamato (1989) An asymmetric field inversion gel electrophoresis system for the separation of large DNAs, Anal. Biochem., 178, 172-176. Giaccia, A., R. Weinstein and T.D. Stamato (1985) Cell cycledependent repair of double strand breaks in a gamma-ray sensitive Chinese hamster ovary cell, Somat. Cell Mol. Genet., 11,485-491. Giaccia, A.J., N. Denko, R. MacLaren, D. Mirman, C. Waldren, I. Hart and T.D. Stamato (1990a) Human chromosome 5 complements the DNA double-strand breakrepair deficiency and gamma-ray sensitivity of the XR-1 hamster variant, Am. J. Hum. Genet., 47, 459-~469. Giaccia, A.J., R.A. MacLaren, N. Denko, D. Nicolaou and T.D. Stamato (1990b) Increased sensitivity to killing by

75 restriction enzymes in the XR-I DNA double-strand break repair-deficient mutant, Mutation Res., 236, 67-76. Hahn, G.M. (1990) The heat shock response: Events before, during, and after gene activation, in: M. Gautherie (Ed.), Biological Basis of Oncologic Thermotherapy, Springer, Berlin, pp. 135-166. Hahn, G.M., and G.C. Li (1990) Thermotolerance, thermoresistance and thermosensitization, in: R.I. Morimoto, A. Tissieres and C. Georgopoulos (Eds.), Stress Proteins in Biology and Medicine, Cold Spring Harbor Laboratory Press, pp. 79-100. Hickson, I.D., and A.L. Harris (1988) Mammalian DNA repair-use of mutants hypersensitive to cytotoxic agents, Trends Genet., 4, 101-106. Jeggo, P.A. (1990) Studies on mammalian mutants defective in rejoining double-strand breaks in DNA, Mutation Res., 239, 1-16. Magin, R.L., B.I. Sikic and R.L. Cysyk (1979) Enhancement of bleomycin activity against Lewis lung tumors in mice by local hyperthermia, Cancer Res., 39, 3792-3795. Mirabelli, C.K., A. Ting, C.H. Huang, S.K. Mong and S.T. Crooke (1982) Bleomycin and taiisomycin sequence-specific strand scission of DNA: A mechanism of double strand cleavage, Cancer Res., 42, 2779-2785. Painter, R.B. (1980) The role of DNA damage and repair in cell killing induced by ionizing radiation, in: R.E. Meyn and H.R. Withers (Eds.), Radiation Biology in Cancer Research, Raven, New York, pp. 59-68. Sausville, E.A., J. Peisach and S.B. Horwitz (1976) A role for

ferrous ion and oxygen in the degradation of DNA by bleomycin, Biochem. Biophys. Res. Commun., 91,871-877. Sikic, B.I. (1986) Antineoplastic Agents, in: C.R. Craig and R.E. Stitzell (Eds.), Modern Pharmacology, Little, Brown and Company, Boston, pp. 797-832. Stamato, T.D., and N. Denko (1990) Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double strand breaks in mammalian cells, Radiat. Res., 121, 196-205. Stamato, T.D., R. Weinstein, A. Giaccia and L. Mackenzie (1983) Isolation of a cell cycle-dependent gamma-ray sensitive Chinese hamster ovary cell, Somat. Cell Moi. Genet., 9, 165-173. Stamato, T.D., B. Peters, P. Patil, N. Denko, R. Weinstein and A. Giaccia (1987) Isolation and characterization of bleomycin-sensitive Chinese hamster ovary cells, Cancer Res., 47, 1588-1592. Suzuki, T., K. Kohda and Y. Kawazoe (1990) Potentiation of bleomycin cytotoxicity by polyhydric alcohols, Anticancer Res., 10, 97-104. Urano, M., J. Kahn and L.A. Kenton (1988) Effect of bleomycin on murine tumor cells at elevated temperatures and two different pH values, Cancer Res., 48, 615-619. Ward, J.F. (1988) DNA damage produced by ionizing radiation in mammalian cells: Identities, mechanisms of formation, and reparability, in: Progress in Nucleic Acid Research and Molecular Biology, Academic Press, New York, pp. 95-125. Communicated by R.B. Painter