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The influence of cathepsin B and leupeptin on potentially lethal damage repair in mammalian cells
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THE INFLUENCE OF CATHEPSIN B AND LEUPEPTIN ON POTENTIALLY LETHAL DAMAGE REPAIR IN MAMMALIAN CELLS M. OSMAK, PH.D.,’ M. KORBELIK,
PH.D.,’ A. SUHAR, PH.D.,* J. SKRK, PH.D.~ AND V. TURK, PH.D.*
‘Department of Experimental Biology and Medicine, Ruder BoSkovic Institute, 4 1000 Zagreb, *Department of Biochemistry, Josef Stefan Institute, 6 1000 Ljubljana; and ‘Department of Radiobiology, The Institute of Oncology, 61000 Ljubljana, Yugoslavia Cell response to irradiation depends on many micro-environmental and intracellular factors. It is known that proteinases control many physiological functions and are also involved in progression of the cell cycle. They also could be involved in cell response to irradiation. In this work the influence of cathepsin B, which is one of the important lysosomal proteinases, and one of its inhibitors, leupeptin, on the potentially lethal damage repair (PLDR) was studied. Chinese hamster V79 cells were irradiated with gamma rays in the plateau-phase of growth. Immediately after irradiation cathepsin B or leupeptin were added to the growth medium. Four hours later, a determined sufficient period of time for maximal PLDR, the cells were replated to assess survival and mutation induction. Mutation frequency was determined at the hypoxanthine-guanine phosphorihosyltrausferase (HGPRT) locus using resistance to 6-thioguanine (6-TG). Simultaneously, the activity of cysteine, aspartic and serine proteinases were determined at different postirradiation intervals. The results show that when plateau-phase cells were incubated with cathepsin B during the postirradiation interval strong inhibition of PLDR was observed, accompanied with a reduced number of 6-TG resistant mutants. If leupeptin was added, more modest inhibition of PLDR was observed, accompanied with only slight reduction in the mutation frequency. The addition of cathepsin B or leupeptin to irradiated cells modified the activities of intracellular proteinases. As the highest alterations in proteinase activities were observed at the time when maximum repair of DNA lesions occurred, the biological consequences could involve a series of sequential steps in intracellular proteinase activities. Cathepsin B, Leupeptin, PLDR, Cell-culture.
INTRODUCTION Proteinases have many very important functions. They control not only intracellular protein turnover, but participate also in many other physiological functions ineluding: blood coagulation, fibrinolysis, inflammation, control of blood pressure, release of hormones, and pharmacologically active peptides from precursors proteins, transport of secretory proteins across membranes, and other vital processes (21). The intracellular proteinases are also involved in cell growth and multiplication (35), in response of bacteria to DNA damage (46), and in carcinogenesis (25, 33, 44). The ability of mammalian cells to repair potentially lethal damage induced by radiation was first observed with exponentially growing cells exposed to suboptimal growth
conditions (4,30). Later it was discovered that cells in the plateau phase of growth can also repair this kind of damage: an increase in the cell-survival was obtained when a time interval was allowed to elapse between irradiation and subculturing for colony forming assay (14, 15, 17, 20). In all these experiments treatments were involved which delayed or postponed progression of cells through the cell-cycle, like the use of conditioned media. In vivo, many cells are in a nonproliferative state (11). Their response to irradiation will depend on micro-environmental and intracellular factors (11). Some of these could involve the intracellular proteinases because of their broad function in many processes in cells. In previous work (27) the influence of different proteinases and their inhibitors on the repair of potentially lethal damage in irradiated cells was examined. In this paper the influence
The data from this manuscript were presented at the 20th Annual Meeting of the European Society for Radiation Biology, Pisa (Italy), September 15-19, 1986, and at the 18th FEBS Meeting, Ljubljana (Yugoslavia), June 2%July 3, 1987. Reprint requests to: Maja Osmak, Ph.D., Department of Experimental Biology and Medicine, Ruder BoSkoviC Institute, 4 1000 Zagreb, Yugoslavia. Acknowledgements-The authors are grateful to Prof. J. Boiikov
for mathematical analysis of the data. They would like also to thank M. Fiolic, Lj. Krajcar, and S. Turk for their excellent technical assistance. This work was supported by Project No 58 from the Research Council of the Socialistic Republic of Croatia and by Project Oncology, 27 B, from the Research Council of the Socialistic Republic of Slovenia. Accepted for publication 29 September 1988. 101
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of the intracellular proteinase cathepsin B on PLDR was studied in more detail, as well as the influence of leupeptin, one of the cathepsin B inhibitors. It was examined whether the addition of cathepsin B or leupeptin to the cells irradiated in the plateau-phase of growth could influence the PLDR in respect to cell survival and mutation induction. Since proteinases usually act through a cascade of events (44) the activities of cysteine, aspartic and serine proteinases were simultaneously determined. These proteinases were chosen because from the literature is known that they play an important role in cell biology. METHODS AND MATERIALS Cells and culture conditions Chinese hamster V79 cells were used in this study. They were grown as monolayer cultures in modified Eagle’s minimum essential medium, supplemented with 10 percent fetal calf serum and antibiotics. The doubling time of these cells incubated at 37°C in a humid atmosphere and 5% CO* was 11 hours. Survival assay
To obtain plateau-phase cultures, 2 X 1O6exponentially growing cells were seeded per 60 mm Petri-dishes. The following day the cells reached plateau-phase of growth. Confluence was maintained for 2 days with daily refeeding, with the last refeeding 24 hr before irradiation. The cultures were irradiated on ice with gamma rays. Irradiation was carried out at Gammacell 220, Atomic Energy of Canada LTD at the dose rate of 4.33 Gy/min. Some samples were plated immediately after irradiation (zero time), whereas the others were left at 37°C for various times prior to plating. The cells were detached from Petridishes by pipetting the medium onto them repeatedly until single cell suspension was obtained. In this set of experiments, the dose of 10 Gy was used. In the second set of experiments the dose range of 1-12 Gy of gamma rays was used. In these experiments the samples were plated immediately or 4 hr after irradiation. If the influence of cathepsin B or leupeptin was examined, the same procedure was used, only that cathepsin B at activity 0.39 nmol/h/ml or leupeptin at a concentration of 50 &ml were added to the growth medium immediately after irradiation and were present in the medium during the postirradiation period prior to colony plating. After the colony plating, the cells were incubated for 8 days. The resulting colonies were then stained and counted. Cathepsin B was isolated from human brain tissue (39), while leupeptin was obtained from the Peptide Institute Inc, Japan. For each experimental curve the experiments were done at least twice, in triplicate dishes. Mutation assay
The induction of mutation was determined at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus using resistance to 6-thioguanine (6-TG) (45). The
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cells were irradiated in the plateau-phase of growth and replated either immediately after irradiation, or incubated for 4 hr with or without cathepsin B or leupeptin present. The samples containing 2 X 1O6surviving cells (calculated from the survival curves) were plated in a-MEM growth medium. The cells were allowed to grow 6 days with one replating in between. Following this expression period, the cells (1 X lo5 cells/Petri dish; 20 plates per experimental point) were plated in medium containing 5 pg/ ml of 6-TG for determination of 6-TG resistant mutants. Simultaneously, the cells were plated in normal growth medium to determine the plating efficiency for each experimental point. After 7-10 days the colonies were stained and counted. The calculation of mutation frequency was based on the number of mutants scored and the number of cells which survived. For each experimental curve the experiments were done twice. Proteinase activity
The activities of cysteine, aspartic and serine proteinases were determined by the following procedure: the cells were harvested, concentrated by centrifugation, lysed in distilled water, sonicated and homogenized. The proteinase activities in homogenate were determined using specific substrates: N-cr-benzoyl-DL-arginine- 1-naphtylamine (BANA) for cysteine proteinases (40), 2% bovine hemoglobine at pH 3.5 for aspartic proteinases, and 1% calf thymus histones at pH 7.5 for determination of serine proteinase activity (38). It was examined whether the addition of cathepsin B or leupeptin changed the activities of proteinases during the postirradiation time intervals in cells irradiated with gamma rays in the plateau-phase of growth. Simultaneously, the changes in proteinase activities were determined in non-irradiated controls incubated with the same concentrations of cathepsin B or leupeptin, for given time intervals. RESULTS Survival data
Chinese hamster V79 cells irradiated in the plateauphase of growth are capable of repairing PLD (Fig. 1). The recovery capacity is expressed as the ratio of survival of the cells plated after postirradiation incubation at 37°C and survival of cells plated immediately after irradiation. It can be seen that the recovery factor increased with the increase of time interval between irradiation and plating, reaching the plateau at about 4 hr of postirradiation incubation at 37’C. If cathepsin B was presented during the whole postirradiation incubation, the recovery factor was significantly reduced. The addition of leupeptin also modified PLDR, although the recovery factor was reduced to a lesser degree than with cathepsin B. The response of plateau-phase cells which were plated immediately after irradiation with gamma rays or were left undisturbed for 4 hr prior to plating is shown in Figure 2. If cathepsin B was added to growth medium immedi-
Cathespin B and leupeptin in PLDR 0 M. OSMAK
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Fig. 1. Kinetics of potentially lethal damage repair in Chinese hamster V79 cells in the plateau-phase of growth irradiated with 10 Gy gamma rays (0). Cathepsin B (A) or leupeptin (Cl) were present in growth medium during the whole postirradiation incubation.
ately after irradiation and was incubated with cells during the 4 hr postirradiation period, the increase in survival due to PLDR was significantly reduced. Similar, but more modest changes were observed with the addition of leupeptin. The regression analysis of the data indicate (I), that the slopes of the survival curves for the cells which were allowed to repair PLDR for 4 hr, with or without cathepsin B or leupeptin added, were not significantly different: Do had the values of 2.58 f 0.15 Gy (control cells), 2.48 + 0.12 Gy (with cathepsin B) and 2.54 f 0.09 Gy (with leupeptin). However, the changes in the shoulder region had shown to be significant (p < 0.05). The Dq value for the cells which were left undisturbed for 4 hr prior to plating was 4.15 + 0.32 Gy. This value decreased
z 0
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to 2.84 + 0.13 Gy for the cells to which cathepsin B was
added immediately after irradiation and was incubated with cells during the 4 hr postirradiation period, while addition of leupeptin decreased this value to 3.45 zk 0.21 Gy.
Mutation data The induction of mutation at the HGPRTase locus was determined using selection in medium containing 6-TG for the cells irradiated in the plateau-phase of growth. Mutation frequencies were corrected for background mutants. Neither addition of cathepsin B nor leupeptin increased the number of 6-TG resistant mutants in unirradiated cells: The mutation frequencies averaged 7.72 -+ 0.48 X 10-6/survivor for control cultures, 6.89 + 0.5 1 X 10-6/survivor for cathepsin B treated cultures, and 7.08 k 0.41 X 10-6/survivor for leupeptin treated cultures. If the cells were plated immediately after irradiation, the upward bending curve was obtained (Fig. 3A). If the cells were allowed to repair the PLD for 4 hr, the number of mutants induced was lower. When during the 4 hr postirradiation incubation at 37°C cathepsin B was presented in the growth medium, the frequency of 6-TG’ mutants was lower than with cells which were not incubated with cathepsin B after irradiation. The frequency of 6-TG’ mutants was also slightly lower if leupeptin was added, but due to standard deviations, this decrease was not significant (Fig. 3B).
Proteinase activities
Fig. 2. Dose survival curves of V79 cells irradiated in the plateauphase of growth. The cells were plated either immediately after irradiation (0) or 4 hr after irradiation (0); cathepsin B (A) or leupeptin (Cl) were present in the culture medium during the 4 hr postirradiation incubation prior to colony plating. 0: Do =2.13+0.11Gy,Dq= 1.95+0.15Gy;O:Do=2.58-t0.15 Gy, Dq = 4.15 f 0.32 Gy; A: Do = 2.48 f 0.12 Gy, Dq = 2.84 k 0.13 Gy; Cl: Do = 2.54 + 0.09 Gy, Dq = 3.45 zk 0.21 Gy.
The activities of three types of proteinases (cysteine, aspartic, and serine) were determined in cells irradiated with 10 Gy of gamma rays in the plateau-phase of growth, with or without addition of cathepsin B or leupeptin. Simultaneously, the activities of proteinases were determined in unirradiated control plateau-phase cells after addition of cathepsin B or leupeptin. Figure 4 shows the effect of cathepsin B on the activities of cysteine proteinases. The addition of cathepsin B to nonirradiated plateauphase cells induced an increase in the cysteine proteinase
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8
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2
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4
2
6
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Fig. 3. Induction of 6-TG resistant mutants in the plateau-phase cells as a function of gamma-ray dose: A) the cells were plated immediately after irradiation (0) or 4 hr later (0). B) During 4 hr of postirradiation incubation prior to plating cathepsin B (A) or leupeptin (Cl) were present in the growth medium. The dashed line represents the mutation induction curve of cells plated 4 hr after irradiation.
value (about 11 fold activity (CPA) with the maximum higher than the control value) at 30 min after the addition
of cathepsin B. Later, CPA returned to initial values and 4 hr after the addition of cathepsin B they were not significantly different from the control value. The CPA increased slightly during the first 30 minutes after irradiation, but later on did not differ from the control. If immediately after irradiation cathepsin B was added, again only during the first 30 postirradiation minutes was a small increase in CPA observed.
TIME IttOURS
The addition of leupeptin induced similar changes in CPA (Fig. 5). Leupeptin added to Unix-radiated plateauphase cells induced a very high increase in CPA, reaching maximum value ( 14-fold increase) at 15 minutes after the addition of leupeptin. Two hr after the addition of leupeptin the CPA returned to the control value. If leupeptin was added to irradiated cells, a significant increase in CPA was again observed, reaching maximum 30 minutes after the addition of this drug, and it returned slowly to the control value at 4 hr postirradiation.
1
Fig. 4. Kinetics of cysteine proteinase activities in the plateau-phase cells after the addition of cathepsin B (A), irradiation with 10 Gy (0) or irradiation and immediate addition of cathepsin B (A). The standard errors of control samples are indicated (=). The points without error bars are those in which the errors were smaller than the symbols.
Cathespin B and leupeptin
in
PLDR 0 M. OSMAK
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et al.
I6
0
I
2
5
3
TIME (HOURS
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1
Fig. 5. Kinetics of cysteine proteinase activities in the plateau-phase cells after the addition of leupeptin (Cl), irradiation with 10 Gy (0) or irradiation and immediate addition of leupeptin (H). The standard errors of control samples are indicated (=). The points without error bars are those in which the errors were smaller than the symbols.
The effects of cathepsin B and leupeptin on activities of aspartic proteinases were also determined. Figure 6 shows that following the addition of cathepsin B to nonirradiated plateau-phase cells the increase in aspartic proteinase activities (APA) occurred, reaching maximum value 45 minutes after the cathepsin B addition. Similar changes in APA could be observed in irradiated cells. If cathepsin B was added to irradiated cells, an increase in APA value was again observed 45 minutes after irradiation, but it was lower than in irradiated cells not treated with cathepsin B.
Figure 6 shows also the APA changes in irradiated and nonirradiated cells following the addition of leupeptin. If leupeptin was added to nonirradiated cells, the maximum increase (5.5 hold) was observed 15 minutes after the drug addition. The APA remained high also 2 hr following the addition of leupeptin. If however, leupeptin was added to irradiated cells, again an increase in APA was observed (lower than in leupeptin treated nonirradiated cells but higher than in irradiated cells with no drug added). In this case the maximum value was reached 30 minutes after postirradiation.
#-
‘IS-
TIME(HOURS I
Fig. 6. Kinetics of aspartic proteinase activities in the plateau-phase cells after the addition of cathepsin B (A), leupeptin (Cl), irradiation with 10 Gy (O), or after irradiation and immediate addition of cathepsin B (A) or leupeptin (m). Standard errors of control samples are indicated (=). The points without error bars are those in which the errors were smaller than the symbols.
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The changes in activities of serine proteinases in nonirradiated and irradiated cells after the addition of cathepsin B or leupeptin were also examined (Fig. 7). Preliminary data show that addition of cathepsin B to plateauphase cells significantly decreased the serine proteinase activity (SPA) the 2nd hr after irradiation, but later on reached the control value. Irradiation of cells, reduced their SPA value which remained much lower than controls up to 6 hr after exposure. Cathepsin B added to the cells immediately after irradiation did not significantly influence the SPA of irradiated cells. Leupeptin added to plateau-phase cells lowered their SPA for the examined time intervals. If leupeptin was added to irradiated cells, their SPA also decreased and stayed lower even 6 hr after irradiation.
added to irradiated cells neither induces DNA damage, nor interferes with DNA repair or replication. If the same concentrations of cathepsin B or leupeptin were added to irradiated exponentially growing cells, no change in cell survival was observed (data not shown). This indicates, that the physiological status of cells at the moment of irradiation may determine the survival of cathepsin B or leupeptin treated cells. Potentially lethal lesions are supposed to be doublestrand breaks (5,9,29). The repair of PLD probably takes place in the interval between irradiation and the next S phase or mitosis and it is therefore minimal for cells irradiated at the G,/S border or in mitosis (23). Therefore, allowing more time for repair of DNA lesions before the cells enter the S phase of growth would result in higher cell survival. Indeed, as shown in Figure 2, the cells, incubated for 4 hr after irradiation in the plateau-phase of growth, showed an increase in cell survival. It is known that proteinases usually act through a cascade of events. Some of them are involved in cell multiplication (35). One possible mechanism to explain the observed effects could be that addition of cathepsin B indirectly promoted cells entering into the S phase of their cell cycle, thus shortening the time period available for repair of lesions before the damaged DNA is replicated. If so, the increase in activity of neutral serine proteinases would be expected (7, 16, 18, 19) because of the positive correlation between cellular growth rate and neutral serine proteinase activities. However, as shown in Figure 7 neither addition of cathepsin B nor leupeptin included an increase in serine proteinase activities. Evermore, both of them reduced the activities of serine proteinases. The role of proteinases in the mutagenesis of animal
DISCUSSION The importance of cysteine lysosomal proteinases (cathepsin B, H, L) in the intracellular protein degradation was recognized many years ago (24). Recently the interest has been focused on these proteinases again while some seem to be involved in the process of neoplastic transformation and tumor metastasis (2, 13, 32, 34, 36, 37). In this work we studied the influence of cathepsin B on the potentially lethal damage repair. The addition of cathepsin B to plateau-phase irradiated cells leads to reduction in potentially lethal damage repair. The concentration added to the growth medium had no effect on the survival of unirradiated cells. The reduction in PLDR was also observed in leupeptin treated cells. According to Borek and Cleaver (6) the concentration of leupeptin
Oh-
I
I
2
L
6
TIME IHOURS) Fig. 7. Kinetics of serine proteinase activities in the plateau-phase cells after the addition of cathepsin B (A), leupeptin (Cl), irradiation with 10 Gy (0) or a&x irradiation and immediate addition of cathepsin B (A) or leupeptin (m). Standard errors of control samples are indicated (=). The points without error bars are those in which the errors were smaller than the symbols.
Cathespin B and leupeptin in PLDR 0 M. OSMAK et al.
and human cells is still unclear. Involvement
of proteinases has been documented for mutagenesis in bacteria: proteinase inhibitors were shown to inhibit SOS responses in the bacteria Escherichia coli: mutagenesis, filamentation as well as induction of prophage lambda by DNA damaging agents through proteolysis of repressor protein (46). The involvement of similar error-prone DNA repair (SOS) function in mutagenesis of animal cells has not yet been resolved. The effects of proteinase inhibitors: antipain, chymostatin, elastatinal, leupeptin, and pepstatin on induction of mutations, as determined by Qzaserine (AZA) resistance and ouabain (OUA) resistance in Chinese hamster cells exposed to ultraviolet light (UV) or treated with N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and 3-methylcholantrene (MCA) was examined (28). Little or no effect on mutation frequency was observed at a comparative dose. Only pepstatin reduced the frequency of AZA resistant mutants obtained, following treatment with MNNG. Similarly, the addition of leupeptin had no influence on the number of 6-TG resistant mutants in MNNG treated cells (26). The mutation data indicate (Fig. 3) that conditions allowing PLD repair reduced the number of mutants induced in plateau-phase irradiated cells. These data are in agreement with those in the literature (22,3 1). If cathepsin B was added immediately after irradiation and was present in growth medium for 4 hr prior to plating, the number of mutants induced was reduced, while the addition of leupeptin did not significantly change the number of mutants induced. Noting that the addition of both, cathepsin B or leupeptin caused the reduction in cell-survival, one can suppose that potentially lethal lesions responsible for cell killing may not be identical to those leading to mutations; this was also shown by Iliakis (22). Alternatively, cathepsin B and leupeptin may not affect the repair of lesions responsible for cell killing and mutation induction in the same way. Our finding that cathepsin B reduces the frequency of 6-TG resistant mutants while leupeptin does not, could be because leupeptin inhibits the activities of not only cathepsin B, but also cathepsin A, D, H, L, calcium activated neutral proteinases, trypsin, plasmin, kallikrein, and trombokinase (3). It is interesting to note that both cathepsin B and its
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inhibitor leupeptin exhibited similar effects on PLDR. These results are further supported by the finding that both these agents modified the activities of the three types of proteinases examined in a similar way (also, the modification of proteinase activities induced by leupeptin were more expressed) (Figs. 4-6). A similar paradoxical effect of leupeptin was also observed in vivo. Leupeptin was tested as a potential antidystrofic agent supposing to exert its curing activity through inhibition of cathepsin B activity. It was found, however, that leupeptin, if given to mice, stimulate the cathepsin B activity (4 1). The mechanism involving this unexpected finding is not clear. One possibility which arises is, that if natural inhibitor of cathepsin B is present in the initial lysosomal site, but not in the autolysosome, increased proteinase activity might be noted because it was shown that in rat, lysosomal proteinases move from their original organelles into autolysosomes following leupeptin administration. The complexity of proteinase activities is further sup ported by finding, that the addition of one proteinase inhibitor could modify the activity of an unrelated proteinase: the addition of the cysteine proteinase inhibitor (leupeptin) causes the induction of acid proteinase (hemoglobin hydrolase) in primary cultures of hepatocytes (42, 43). It was a true induction, since the addition of cyclohexamide, an inhibitor of protein synthesis, prevents a leupeptin-induced increase in hemoglobin hydrolase activity. This and similar data support the concept of a cascade chain of proteinase activation and the interdependence of the whole proteinase system. The changes in proteinase activities observed in irradiated plateau phase cells following the addition of cathepsin B or leupeptin are most evident in the period of maximal DNA repair, that is during the first 2 hr following irradiation (8, 10, 12, 47). The changes in proteinase activities at the time when repair of the DNA lesion takes place could therefore modify cellular response to irradiation. We can conclude, that cathepsin B, an intrinsic intracellular proteolytic enzyme, whose primary role in mammalian cells is degradation of physiologically important proteins, could be also involved in the cellular response to irradiation, probably through a cascade of intracellular proteinase activation.
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mechanism for variations in sensitivity in vivo (Abstr.). Int. J. Radiat. Biol. 49:701; 1986. Kuroki, T.; Drevon, C. Inhibition of chemical transformation in C3H 10 T l/2 cells by protease inhibitors. Cancer Res. 39:2755-2761; 1979. Leenhouts, H. P.; Chadwick, K. H. The crucial role of DNA double-strand breaks in cellular radiobiological effects. In: Lett, J. T.; Adler, T. T., eds. Advances in radiation biology. New York: Academic Press; 1979:56-101. Phillips, R. A.; Tolmach, L. J. Repair of potentially lethal damage in x-irradiated HeLa cells. Radiat. Res. 29:413432; 1966. Rao, B. R. S.; Hopwood, L. E. Modification of mutation in plateau-phase Chinese hamster ovary cells exposed to gamma irradiation during recovery from potentially lethal damage. Int. J. Radiat. Biol. 42:501-508; 1982. Recklies, A. D.; Mart, J. S.; Poole, A. R. Secretion of a thiol proteinase from mouse mammary carcinomas and its characterisation. Cancer Res. 42: 1026- 1032; 1982. Rossman, T. G.; Troll, W. Protease inhibitors in carcinogenesis: Possible sites of action. In: Slaga, T. S., ed. Carcinogenesis, Vol. 5. Modifiers of chemical carcinogenesis. New York: Raven Press; 1980: 127-143. Ryan, R. E.; Crissman, J. D.; Honn, K. V.; Sloane, B. F. Cathepsin-B like activity in viable tumor cells isolated from rodent tumors. Cancer Res. 45:3636-3641; 1985. Scott, G. K. Mini-review: Proteinases and eucaryotic cell growth. Comp. Biochem. Physiol. 87B:l-10; 1987. Sloane, B. F.; Dunn, J. R.; Honn, K. V. Lysosomal cathepsin B: Correlation with neoplastic potential. Science 2 12: 115 l1153; 1981. Sloane, B. F.; Honn, K. V. Cysteine proteinascs and metastasis Reviews 3:249-263; 1984. Suhar, A., Kopitar, M.; Turk, V. The isolation of liver serine proteinase by isolation of liver serine proteinase by affinity chromatography on 4-phenyl-buthylamine-sepharose. Acta Biol. Med. Germ. 41:61-68; 1982. Suhar, A.; Marks, N. Purification and properties of brain cathepsin B. Evidence for cleavage of pituitary lipoproteins. Europ. J. Biochem. 101:23-30; 1979. Suhar, A.; Marks, N.; Turk, V.; Benuck, M. On the metabolism of opiate peptides by brain proteolytic enzymes. In: Turk, V.; Vitale, Lj., eds. Proteinases and their inhibitors. Ljubljana, Oxford: Mladinska knjiga, Pergamon Press; 198 1: 33-43. Sutherland, J. H. R.; Greenbaum, L. M. Paradoxical effect of leupeptin in vivo on cathepsin B activity. B&hem. Biophys. Res. Corn. 110:332-338; 1983. Tanaka, K.; Ikegaki, N.; Ichihara, A. Induction of hemoglobin-hydrolase activity by the thiol-proteasc inhibitors leupeptin and antipain in adult rat liver cells in primary culture. Biochem. Biophys. Res. Corn. 91: 102-107; 1979. Tanaka, K.; Ikegaki, N.; Ichihara, A. Effects of leupeptin and pepstatin on protein turnover in adult rat hepatocytes in primary culture. Arch. Biochem. Biophys. 208:296-304; 1983. Troll, W.; Frenkel, K.; Wiesner, R. Protease inhibitors as anticarcinogens. J N C I 73:1245-1250; 1984. Van Zeeland, A. A,; Simons, J. W. I. M. Linear dose-response relationships after prolonged expression times in V79 Chinese hamster cells. Mutat. Res. 35: 129-138; 1976. Walker, G. C. Inducible DNA repair systems. Ann. Rev. Biochem. 54~425-457; 1985. Weibezahn, K. F.; Sexauer, C.; Coquerelle, T. Negative pion irradiation of mammalian cells. III. A comparative analysis of DNA strand breakage, repair and cell survival after exposure to ?r-mesons and X rays. Int. J. Radiat. Biol. 38:365371; 1980.
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Copyright
0360.3016/89 $3.00 + .oO @ 1989 Pergamon Press plc
??Original Contribution
THE INFLUENCE OF CATHEPSIN B AND LEUPEPTIN ON POTENTIALLY LETHAL DAMAGE REPAIR IN MAMMALIAN CELLS M. OSMAK, PH.D.,’ M. KORBELIK,
PH.D.,’ A. SUHAR, PH.D.,* J. SKRK, PH.D.~ AND V. TURK, PH.D.*
‘Department of Experimental Biology and Medicine, Ruder BoSkovic Institute, 4 1000 Zagreb, *Department of Biochemistry, Josef Stefan Institute, 6 1000 Ljubljana; and ‘Department of Radiobiology, The Institute of Oncology, 61000 Ljubljana, Yugoslavia Cell response to irradiation depends on many micro-environmental and intracellular factors. It is known that proteinases control many physiological functions and are also involved in progression of the cell cycle. They also could be involved in cell response to irradiation. In this work the influence of cathepsin B, which is one of the important lysosomal proteinases, and one of its inhibitors, leupeptin, on the potentially lethal damage repair (PLDR) was studied. Chinese hamster V79 cells were irradiated with gamma rays in the plateau-phase of growth. Immediately after irradiation cathepsin B or leupeptin were added to the growth medium. Four hours later, a determined sufficient period of time for maximal PLDR, the cells were replated to assess survival and mutation induction. Mutation frequency was determined at the hypoxanthine-guanine phosphorihosyltrausferase (HGPRT) locus using resistance to 6-thioguanine (6-TG). Simultaneously, the activity of cysteine, aspartic and serine proteinases were determined at different postirradiation intervals. The results show that when plateau-phase cells were incubated with cathepsin B during the postirradiation interval strong inhibition of PLDR was observed, accompanied with a reduced number of 6-TG resistant mutants. If leupeptin was added, more modest inhibition of PLDR was observed, accompanied with only slight reduction in the mutation frequency. The addition of cathepsin B or leupeptin to irradiated cells modified the activities of intracellular proteinases. As the highest alterations in proteinase activities were observed at the time when maximum repair of DNA lesions occurred, the biological consequences could involve a series of sequential steps in intracellular proteinase activities. Cathepsin B, Leupeptin, PLDR, Cell-culture.
INTRODUCTION Proteinases have many very important functions. They control not only intracellular protein turnover, but participate also in many other physiological functions ineluding: blood coagulation, fibrinolysis, inflammation, control of blood pressure, release of hormones, and pharmacologically active peptides from precursors proteins, transport of secretory proteins across membranes, and other vital processes (21). The intracellular proteinases are also involved in cell growth and multiplication (35), in response of bacteria to DNA damage (46), and in carcinogenesis (25, 33, 44). The ability of mammalian cells to repair potentially lethal damage induced by radiation was first observed with exponentially growing cells exposed to suboptimal growth
conditions (4,30). Later it was discovered that cells in the plateau phase of growth can also repair this kind of damage: an increase in the cell-survival was obtained when a time interval was allowed to elapse between irradiation and subculturing for colony forming assay (14, 15, 17, 20). In all these experiments treatments were involved which delayed or postponed progression of cells through the cell-cycle, like the use of conditioned media. In vivo, many cells are in a nonproliferative state (11). Their response to irradiation will depend on micro-environmental and intracellular factors (11). Some of these could involve the intracellular proteinases because of their broad function in many processes in cells. In previous work (27) the influence of different proteinases and their inhibitors on the repair of potentially lethal damage in irradiated cells was examined. In this paper the influence
The data from this manuscript were presented at the 20th Annual Meeting of the European Society for Radiation Biology, Pisa (Italy), September 15-19, 1986, and at the 18th FEBS Meeting, Ljubljana (Yugoslavia), June 2%July 3, 1987. Reprint requests to: Maja Osmak, Ph.D., Department of Experimental Biology and Medicine, Ruder BoSkoviC Institute, 4 1000 Zagreb, Yugoslavia. Acknowledgements-The authors are grateful to Prof. J. Boiikov
for mathematical analysis of the data. They would like also to thank M. Fiolic, Lj. Krajcar, and S. Turk for their excellent technical assistance. This work was supported by Project No 58 from the Research Council of the Socialistic Republic of Croatia and by Project Oncology, 27 B, from the Research Council of the Socialistic Republic of Slovenia. Accepted for publication 29 September 1988. 101
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of the intracellular proteinase cathepsin B on PLDR was studied in more detail, as well as the influence of leupeptin, one of the cathepsin B inhibitors. It was examined whether the addition of cathepsin B or leupeptin to the cells irradiated in the plateau-phase of growth could influence the PLDR in respect to cell survival and mutation induction. Since proteinases usually act through a cascade of events (44) the activities of cysteine, aspartic and serine proteinases were simultaneously determined. These proteinases were chosen because from the literature is known that they play an important role in cell biology. METHODS AND MATERIALS Cells and culture conditions Chinese hamster V79 cells were used in this study. They were grown as monolayer cultures in modified Eagle’s minimum essential medium, supplemented with 10 percent fetal calf serum and antibiotics. The doubling time of these cells incubated at 37°C in a humid atmosphere and 5% CO* was 11 hours. Survival assay
To obtain plateau-phase cultures, 2 X 1O6exponentially growing cells were seeded per 60 mm Petri-dishes. The following day the cells reached plateau-phase of growth. Confluence was maintained for 2 days with daily refeeding, with the last refeeding 24 hr before irradiation. The cultures were irradiated on ice with gamma rays. Irradiation was carried out at Gammacell 220, Atomic Energy of Canada LTD at the dose rate of 4.33 Gy/min. Some samples were plated immediately after irradiation (zero time), whereas the others were left at 37°C for various times prior to plating. The cells were detached from Petridishes by pipetting the medium onto them repeatedly until single cell suspension was obtained. In this set of experiments, the dose of 10 Gy was used. In the second set of experiments the dose range of 1-12 Gy of gamma rays was used. In these experiments the samples were plated immediately or 4 hr after irradiation. If the influence of cathepsin B or leupeptin was examined, the same procedure was used, only that cathepsin B at activity 0.39 nmol/h/ml or leupeptin at a concentration of 50 &ml were added to the growth medium immediately after irradiation and were present in the medium during the postirradiation period prior to colony plating. After the colony plating, the cells were incubated for 8 days. The resulting colonies were then stained and counted. Cathepsin B was isolated from human brain tissue (39), while leupeptin was obtained from the Peptide Institute Inc, Japan. For each experimental curve the experiments were done at least twice, in triplicate dishes. Mutation assay
The induction of mutation was determined at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus using resistance to 6-thioguanine (6-TG) (45). The
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cells were irradiated in the plateau-phase of growth and replated either immediately after irradiation, or incubated for 4 hr with or without cathepsin B or leupeptin present. The samples containing 2 X 1O6surviving cells (calculated from the survival curves) were plated in a-MEM growth medium. The cells were allowed to grow 6 days with one replating in between. Following this expression period, the cells (1 X lo5 cells/Petri dish; 20 plates per experimental point) were plated in medium containing 5 pg/ ml of 6-TG for determination of 6-TG resistant mutants. Simultaneously, the cells were plated in normal growth medium to determine the plating efficiency for each experimental point. After 7-10 days the colonies were stained and counted. The calculation of mutation frequency was based on the number of mutants scored and the number of cells which survived. For each experimental curve the experiments were done twice. Proteinase activity
The activities of cysteine, aspartic and serine proteinases were determined by the following procedure: the cells were harvested, concentrated by centrifugation, lysed in distilled water, sonicated and homogenized. The proteinase activities in homogenate were determined using specific substrates: N-cr-benzoyl-DL-arginine- 1-naphtylamine (BANA) for cysteine proteinases (40), 2% bovine hemoglobine at pH 3.5 for aspartic proteinases, and 1% calf thymus histones at pH 7.5 for determination of serine proteinase activity (38). It was examined whether the addition of cathepsin B or leupeptin changed the activities of proteinases during the postirradiation time intervals in cells irradiated with gamma rays in the plateau-phase of growth. Simultaneously, the changes in proteinase activities were determined in non-irradiated controls incubated with the same concentrations of cathepsin B or leupeptin, for given time intervals. RESULTS Survival data
Chinese hamster V79 cells irradiated in the plateauphase of growth are capable of repairing PLD (Fig. 1). The recovery capacity is expressed as the ratio of survival of the cells plated after postirradiation incubation at 37°C and survival of cells plated immediately after irradiation. It can be seen that the recovery factor increased with the increase of time interval between irradiation and plating, reaching the plateau at about 4 hr of postirradiation incubation at 37’C. If cathepsin B was presented during the whole postirradiation incubation, the recovery factor was significantly reduced. The addition of leupeptin also modified PLDR, although the recovery factor was reduced to a lesser degree than with cathepsin B. The response of plateau-phase cells which were plated immediately after irradiation with gamma rays or were left undisturbed for 4 hr prior to plating is shown in Figure 2. If cathepsin B was added to growth medium immedi-
Cathespin B and leupeptin in PLDR 0 M. OSMAK
i,, BETWEEN
8
i
0
TIME
t-r al.
IRRAMATION
AN0
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Fig. 1. Kinetics of potentially lethal damage repair in Chinese hamster V79 cells in the plateau-phase of growth irradiated with 10 Gy gamma rays (0). Cathepsin B (A) or leupeptin (Cl) were present in growth medium during the whole postirradiation incubation.
ately after irradiation and was incubated with cells during the 4 hr postirradiation period, the increase in survival due to PLDR was significantly reduced. Similar, but more modest changes were observed with the addition of leupeptin. The regression analysis of the data indicate (I), that the slopes of the survival curves for the cells which were allowed to repair PLDR for 4 hr, with or without cathepsin B or leupeptin added, were not significantly different: Do had the values of 2.58 f 0.15 Gy (control cells), 2.48 + 0.12 Gy (with cathepsin B) and 2.54 f 0.09 Gy (with leupeptin). However, the changes in the shoulder region had shown to be significant (p < 0.05). The Dq value for the cells which were left undisturbed for 4 hr prior to plating was 4.15 + 0.32 Gy. This value decreased
z 0
a1
c
v
a CL IL
to 2.84 + 0.13 Gy for the cells to which cathepsin B was
added immediately after irradiation and was incubated with cells during the 4 hr postirradiation period, while addition of leupeptin decreased this value to 3.45 zk 0.21 Gy.
Mutation data The induction of mutation at the HGPRTase locus was determined using selection in medium containing 6-TG for the cells irradiated in the plateau-phase of growth. Mutation frequencies were corrected for background mutants. Neither addition of cathepsin B nor leupeptin increased the number of 6-TG resistant mutants in unirradiated cells: The mutation frequencies averaged 7.72 -+ 0.48 X 10-6/survivor for control cultures, 6.89 + 0.5 1 X 10-6/survivor for cathepsin B treated cultures, and 7.08 k 0.41 X 10-6/survivor for leupeptin treated cultures. If the cells were plated immediately after irradiation, the upward bending curve was obtained (Fig. 3A). If the cells were allowed to repair the PLD for 4 hr, the number of mutants induced was lower. When during the 4 hr postirradiation incubation at 37°C cathepsin B was presented in the growth medium, the frequency of 6-TG’ mutants was lower than with cells which were not incubated with cathepsin B after irradiation. The frequency of 6-TG’ mutants was also slightly lower if leupeptin was added, but due to standard deviations, this decrease was not significant (Fig. 3B).
Proteinase activities
Fig. 2. Dose survival curves of V79 cells irradiated in the plateauphase of growth. The cells were plated either immediately after irradiation (0) or 4 hr after irradiation (0); cathepsin B (A) or leupeptin (Cl) were present in the culture medium during the 4 hr postirradiation incubation prior to colony plating. 0: Do =2.13+0.11Gy,Dq= 1.95+0.15Gy;O:Do=2.58-t0.15 Gy, Dq = 4.15 f 0.32 Gy; A: Do = 2.48 f 0.12 Gy, Dq = 2.84 k 0.13 Gy; Cl: Do = 2.54 + 0.09 Gy, Dq = 3.45 zk 0.21 Gy.
The activities of three types of proteinases (cysteine, aspartic, and serine) were determined in cells irradiated with 10 Gy of gamma rays in the plateau-phase of growth, with or without addition of cathepsin B or leupeptin. Simultaneously, the activities of proteinases were determined in unirradiated control plateau-phase cells after addition of cathepsin B or leupeptin. Figure 4 shows the effect of cathepsin B on the activities of cysteine proteinases. The addition of cathepsin B to nonirradiated plateauphase cells induced an increase in the cysteine proteinase
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L
8
8
M
2
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4
2
6
no% IOy,
DOSEloyb
L
*
m
1)
owe rey1
Fig. 3. Induction of 6-TG resistant mutants in the plateau-phase cells as a function of gamma-ray dose: A) the cells were plated immediately after irradiation (0) or 4 hr later (0). B) During 4 hr of postirradiation incubation prior to plating cathepsin B (A) or leupeptin (Cl) were present in the growth medium. The dashed line represents the mutation induction curve of cells plated 4 hr after irradiation.
value (about 11 fold activity (CPA) with the maximum higher than the control value) at 30 min after the addition
of cathepsin B. Later, CPA returned to initial values and 4 hr after the addition of cathepsin B they were not significantly different from the control value. The CPA increased slightly during the first 30 minutes after irradiation, but later on did not differ from the control. If immediately after irradiation cathepsin B was added, again only during the first 30 postirradiation minutes was a small increase in CPA observed.
TIME IttOURS
The addition of leupeptin induced similar changes in CPA (Fig. 5). Leupeptin added to Unix-radiated plateauphase cells induced a very high increase in CPA, reaching maximum value ( 14-fold increase) at 15 minutes after the addition of leupeptin. Two hr after the addition of leupeptin the CPA returned to the control value. If leupeptin was added to irradiated cells, a significant increase in CPA was again observed, reaching maximum 30 minutes after the addition of this drug, and it returned slowly to the control value at 4 hr postirradiation.
1
Fig. 4. Kinetics of cysteine proteinase activities in the plateau-phase cells after the addition of cathepsin B (A), irradiation with 10 Gy (0) or irradiation and immediate addition of cathepsin B (A). The standard errors of control samples are indicated (=). The points without error bars are those in which the errors were smaller than the symbols.
Cathespin B and leupeptin
in
PLDR 0 M. OSMAK
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et al.
I6
0
I
2
5
3
TIME (HOURS
6
1
Fig. 5. Kinetics of cysteine proteinase activities in the plateau-phase cells after the addition of leupeptin (Cl), irradiation with 10 Gy (0) or irradiation and immediate addition of leupeptin (H). The standard errors of control samples are indicated (=). The points without error bars are those in which the errors were smaller than the symbols.
The effects of cathepsin B and leupeptin on activities of aspartic proteinases were also determined. Figure 6 shows that following the addition of cathepsin B to nonirradiated plateau-phase cells the increase in aspartic proteinase activities (APA) occurred, reaching maximum value 45 minutes after the cathepsin B addition. Similar changes in APA could be observed in irradiated cells. If cathepsin B was added to irradiated cells, an increase in APA value was again observed 45 minutes after irradiation, but it was lower than in irradiated cells not treated with cathepsin B.
Figure 6 shows also the APA changes in irradiated and nonirradiated cells following the addition of leupeptin. If leupeptin was added to nonirradiated cells, the maximum increase (5.5 hold) was observed 15 minutes after the drug addition. The APA remained high also 2 hr following the addition of leupeptin. If however, leupeptin was added to irradiated cells, again an increase in APA was observed (lower than in leupeptin treated nonirradiated cells but higher than in irradiated cells with no drug added). In this case the maximum value was reached 30 minutes after postirradiation.
#-
‘IS-
TIME(HOURS I
Fig. 6. Kinetics of aspartic proteinase activities in the plateau-phase cells after the addition of cathepsin B (A), leupeptin (Cl), irradiation with 10 Gy (O), or after irradiation and immediate addition of cathepsin B (A) or leupeptin (m). Standard errors of control samples are indicated (=). The points without error bars are those in which the errors were smaller than the symbols.
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The changes in activities of serine proteinases in nonirradiated and irradiated cells after the addition of cathepsin B or leupeptin were also examined (Fig. 7). Preliminary data show that addition of cathepsin B to plateauphase cells significantly decreased the serine proteinase activity (SPA) the 2nd hr after irradiation, but later on reached the control value. Irradiation of cells, reduced their SPA value which remained much lower than controls up to 6 hr after exposure. Cathepsin B added to the cells immediately after irradiation did not significantly influence the SPA of irradiated cells. Leupeptin added to plateau-phase cells lowered their SPA for the examined time intervals. If leupeptin was added to irradiated cells, their SPA also decreased and stayed lower even 6 hr after irradiation.
added to irradiated cells neither induces DNA damage, nor interferes with DNA repair or replication. If the same concentrations of cathepsin B or leupeptin were added to irradiated exponentially growing cells, no change in cell survival was observed (data not shown). This indicates, that the physiological status of cells at the moment of irradiation may determine the survival of cathepsin B or leupeptin treated cells. Potentially lethal lesions are supposed to be doublestrand breaks (5,9,29). The repair of PLD probably takes place in the interval between irradiation and the next S phase or mitosis and it is therefore minimal for cells irradiated at the G,/S border or in mitosis (23). Therefore, allowing more time for repair of DNA lesions before the cells enter the S phase of growth would result in higher cell survival. Indeed, as shown in Figure 2, the cells, incubated for 4 hr after irradiation in the plateau-phase of growth, showed an increase in cell survival. It is known that proteinases usually act through a cascade of events. Some of them are involved in cell multiplication (35). One possible mechanism to explain the observed effects could be that addition of cathepsin B indirectly promoted cells entering into the S phase of their cell cycle, thus shortening the time period available for repair of lesions before the damaged DNA is replicated. If so, the increase in activity of neutral serine proteinases would be expected (7, 16, 18, 19) because of the positive correlation between cellular growth rate and neutral serine proteinase activities. However, as shown in Figure 7 neither addition of cathepsin B nor leupeptin included an increase in serine proteinase activities. Evermore, both of them reduced the activities of serine proteinases. The role of proteinases in the mutagenesis of animal
DISCUSSION The importance of cysteine lysosomal proteinases (cathepsin B, H, L) in the intracellular protein degradation was recognized many years ago (24). Recently the interest has been focused on these proteinases again while some seem to be involved in the process of neoplastic transformation and tumor metastasis (2, 13, 32, 34, 36, 37). In this work we studied the influence of cathepsin B on the potentially lethal damage repair. The addition of cathepsin B to plateau-phase irradiated cells leads to reduction in potentially lethal damage repair. The concentration added to the growth medium had no effect on the survival of unirradiated cells. The reduction in PLDR was also observed in leupeptin treated cells. According to Borek and Cleaver (6) the concentration of leupeptin
Oh-
I
I
2
L
6
TIME IHOURS) Fig. 7. Kinetics of serine proteinase activities in the plateau-phase cells after the addition of cathepsin B (A), leupeptin (Cl), irradiation with 10 Gy (0) or a&x irradiation and immediate addition of cathepsin B (A) or leupeptin (m). Standard errors of control samples are indicated (=). The points without error bars are those in which the errors were smaller than the symbols.
Cathespin B and leupeptin in PLDR 0 M. OSMAK et al.
and human cells is still unclear. Involvement
of proteinases has been documented for mutagenesis in bacteria: proteinase inhibitors were shown to inhibit SOS responses in the bacteria Escherichia coli: mutagenesis, filamentation as well as induction of prophage lambda by DNA damaging agents through proteolysis of repressor protein (46). The involvement of similar error-prone DNA repair (SOS) function in mutagenesis of animal cells has not yet been resolved. The effects of proteinase inhibitors: antipain, chymostatin, elastatinal, leupeptin, and pepstatin on induction of mutations, as determined by Qzaserine (AZA) resistance and ouabain (OUA) resistance in Chinese hamster cells exposed to ultraviolet light (UV) or treated with N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and 3-methylcholantrene (MCA) was examined (28). Little or no effect on mutation frequency was observed at a comparative dose. Only pepstatin reduced the frequency of AZA resistant mutants obtained, following treatment with MNNG. Similarly, the addition of leupeptin had no influence on the number of 6-TG resistant mutants in MNNG treated cells (26). The mutation data indicate (Fig. 3) that conditions allowing PLD repair reduced the number of mutants induced in plateau-phase irradiated cells. These data are in agreement with those in the literature (22,3 1). If cathepsin B was added immediately after irradiation and was present in growth medium for 4 hr prior to plating, the number of mutants induced was reduced, while the addition of leupeptin did not significantly change the number of mutants induced. Noting that the addition of both, cathepsin B or leupeptin caused the reduction in cell-survival, one can suppose that potentially lethal lesions responsible for cell killing may not be identical to those leading to mutations; this was also shown by Iliakis (22). Alternatively, cathepsin B and leupeptin may not affect the repair of lesions responsible for cell killing and mutation induction in the same way. Our finding that cathepsin B reduces the frequency of 6-TG resistant mutants while leupeptin does not, could be because leupeptin inhibits the activities of not only cathepsin B, but also cathepsin A, D, H, L, calcium activated neutral proteinases, trypsin, plasmin, kallikrein, and trombokinase (3). It is interesting to note that both cathepsin B and its
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inhibitor leupeptin exhibited similar effects on PLDR. These results are further supported by the finding that both these agents modified the activities of the three types of proteinases examined in a similar way (also, the modification of proteinase activities induced by leupeptin were more expressed) (Figs. 4-6). A similar paradoxical effect of leupeptin was also observed in vivo. Leupeptin was tested as a potential antidystrofic agent supposing to exert its curing activity through inhibition of cathepsin B activity. It was found, however, that leupeptin, if given to mice, stimulate the cathepsin B activity (4 1). The mechanism involving this unexpected finding is not clear. One possibility which arises is, that if natural inhibitor of cathepsin B is present in the initial lysosomal site, but not in the autolysosome, increased proteinase activity might be noted because it was shown that in rat, lysosomal proteinases move from their original organelles into autolysosomes following leupeptin administration. The complexity of proteinase activities is further sup ported by finding, that the addition of one proteinase inhibitor could modify the activity of an unrelated proteinase: the addition of the cysteine proteinase inhibitor (leupeptin) causes the induction of acid proteinase (hemoglobin hydrolase) in primary cultures of hepatocytes (42, 43). It was a true induction, since the addition of cyclohexamide, an inhibitor of protein synthesis, prevents a leupeptin-induced increase in hemoglobin hydrolase activity. This and similar data support the concept of a cascade chain of proteinase activation and the interdependence of the whole proteinase system. The changes in proteinase activities observed in irradiated plateau phase cells following the addition of cathepsin B or leupeptin are most evident in the period of maximal DNA repair, that is during the first 2 hr following irradiation (8, 10, 12, 47). The changes in proteinase activities at the time when repair of the DNA lesion takes place could therefore modify cellular response to irradiation. We can conclude, that cathepsin B, an intrinsic intracellular proteolytic enzyme, whose primary role in mammalian cells is degradation of physiologically important proteins, could be also involved in the cellular response to irradiation, probably through a cascade of intracellular proteinase activation.
REFERENCES P. Statistical methods in medical research. Oxford, Blackwell Scientific Publication: 197 1. 2. Baici, A.; Kntipfel, M. Cysteine proteinases produced by cultured rabbit V2 carcinoma cells and rabbit skin fibroblasts. Int. J. Cancer 38:753-761; 1986. 3. Baugh, R. J.; Schnebli, H. P. Role and potential therapeutic value of proteinase inhibitors in tissue destruction. In: Sttiuli, P., Barrett, A. J., Baici, A., eds. Proteinases and tumor invasion. New York: Raven Press; 1980:157-180. 4. Belli, J. A.; Shelton, M. Potentially lethal radiation damage: Repair by mammalian cells in culture. Science 165:490492; 1969. 1. Amitage,
5. Bliicher, D.; Pohlit, W. DNA double strand breaks in Erhlich ascites tumor cells at low doses of X-ray. II Can cell death be attributed to double strand breaks? Int. J. Radiat. Biol. 42:329-338; 1982. 6. Borek, C.; Cleaver, J. E. Protease inhibitors neither damage DNA nor interfere with DNA repair or replication in human cells. Mutat. Res. 82:373-380; 1981. 7. Bosmann, H. B.; Gutheil, R. L.; Case, K. R. Loss of a critical neutral protease in ageing WI-38 cells. Nature 26 1:499-50 1; 1976. 8. Bradley, M. 0.; Kohn, K. W. X-ray-induced double-strand break production and repair in mammalian cells as mea-
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