(CANCER RESEARCH 49, 3134-3138, June 1, 1989] Modulation of Human Lymphocyte 06-Alkylguanine-DNA AlkyItransferase by Streptozotocin in Vivo1
Stanton L. Gerson2
Hematology-Oncology Division, Department of Medicine, and the R. L. Ireland Cancer Center, University Hospitals of Cleveland and Case Western Reserve University Medical School, Cleveland, Ohio 44106
ABSTRACT transferase activity takes advantage of the mechanism of action of the protein. Specifically, the alkyltransferase covalently The ability to modulate DNA repair has been proposed as an effective transfers the alkyl group from the Opposition of guanine to a method to overcome cytotoxic drug resistance in human tumors. However, cysteine moiety in the active site of the protein (4). Because the no studies have shown that it is possible to achieve modulation of DNA repair in humans in vivo. This study analyzes modulation of O'-alkyl- transfer reaction involves a covalent bond between the alkyl guanine-DNA alkyltransferase, a DNA repair protein that protects cells group and the active site of the protein, the alkyltransferase from cytotoxic DNA adducts formed by nitrosoureas. Streptozotocin has becomes irreversibly inactivated in stoichiometric proportion been shown to inactivate the alkyltransferase in vitro and sensitize tumor to the number of 06-alkylguanine adducts repaired (4, 6). cells to other nitrosoureas. Thus, we determined whether biochemical Two classes of modulators have been described for the alkyl modulation of alkyltransferase activity could be documented in patients transferase. First are methylating agents such as Streptozotocin, receiving therapeutic doses of Streptozotocin and whether the modulation /V-methyl-N-nitrosourea, and Af-methyl-N-nitro-^V-nitrosoguan- was specific to Streptozotocin or occurred in patients undergoing treat idine, which form O6-methylguanine-DNA adducts that are ment with other DNA-damaging agents as well. Normal peripheral blood transferred to the protein during the repair process (7, 8). lymphocytes were used to analyze modulation of the alkyltransferase. Second is the modified base 06-methylguanine, which is a We found that lymphocyte alkyltransferase activity was significantly decreased 20 h after treatment with Streptozotocin (500 mg/m2) or high substrate for the alkyltransferase (9,10). It appears that transfer dose l,3-bis-(2-chloroethyl)-l-nitrosourea (350 mg/m1) but not after of the methyl group from O6-methylguanine inactivates the treatment with the other DNA-damaging agents or lower doses of 1,3- protein directly (9, 10). Although chloroethylnitrosoureas also bis-(2-chloroethyl)-l-nitrosourea. A cumulative decline in lymphocyte inactivate the alkyltransferase by forming DNA adducts, these alkyltransferase activity occurred with daily Streptozotocin treatment, agents are not efficient modulators because fewer 06-alkylgua- reaching 26 ±9%of control after the third day of treatment (P < 0.0005). nine adducts are formed than with methylating agents and the Thus, the alkyltransferase DNA repair protein can be modulated in vivo larger adducts are repaired more slowly than the methyl adduct in humans given systemic drug treatment. While further studies are (11-13). Consequently, chloroethylnitrosoureas are very cyto needed to document that biochemical modulation can be achieved in the toxic at doses required to achieve modulation in vitro (7, 12). target tumor in humans, this study supports the development of clinical Interest in biochemical modulation of the alkyltransferase is trials using Streptozotocin as a biochemical modulator of nitrosourea great because in vitro inactivation of the alkyltransferase will resistance in human malignancies. sensitize human tumor cells to chloroethylnitrosoureas by 2- to 12-fold (2, 9, 10, 12, 14, 15). In addition, many human tumor INTRODUCTION cells possess higher levels of alkyltransferase than their normal counterparts (14), suggesting that biochemical modulation of Biochemical modulation of tumor drug resistance is an excit the alkyltransferase may have clinical application. In these ing area of experimental cancer therapeutics (1). While the studies, we determined whether biochemical modulation of basis for modifying drug resistance can often by established in alkyltransferase activity occurs in peripheral blood lymphocytes vitro, it can be difficult to document effective modulation in from patients receiving therapeutic doses of Streptozotocin. humans, making it hard to judge the therapeutic efficacy of the Because Streptozotocin is relatively well tolerated at clinically modulator. This study evaluated biochemical modulation of used doses (16), it has potential use as a biochemical modulator cellular resistance to nitrosoureas in human peripheral blood of the alkyltransferase in cancer therapy (17). To determine the lymphocytes in vivo. The aim was to document whether mod specificity of alkyltransferase modulation following chemother ulation could be achieved in vivo, so that future clinical trials apy exposure, lymphocytes from patients receiving other DNA- of biochemical modulation of tumor nitrosourea resistance damaging agents are also evaluated. We found that Streptozo could be planned. tocin caused a significant dose-dependent decline in lymphocyte In vitro studies have shown that nitrosourea resistance in malignant tumor cells is mediated by efficient repair of drug- alkyltransferase activity, supporting its potential role as a bio induced DNA adducts (2-4). The DNA repair protein involved chemical modulator of nitrosourea resistance in cancer treat is 06-alkylguanine-DNA alkyltransferase (alkyltransferase), ment. which repairs O6-alkylguanine-DNA adducts, one of the cyto toxic DNA adducts formed by chloroethyl-nitrosoureas (5, 6). MATERIALS AND METHODS Tumors which are resistant to nitrosoureas often have high Patient Population. All patients had malignant tumors [Hodgkin's levels of alkyltransferase activity (1, 2). Modulation of alkyl- disease (5), melanoma (6), adenocurcinoma (13), carcinoid (4), islet cell tumor (2), glioma (11), or non-Hodgkin's lymphoma (4)] and were Received 10/31/88; revised 2/16/89; accepted 2/24/89. The costs of publication of this article were defrayed in part by the payment undergoing treatment with chemotherapy following a number of differ of page charges. This article must therefore be hereby marked advertisement in ent protocols. Patients receiving Streptozotocin, CCNU,3 or conven accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tional dose BCNU (40-200 mg/m2) were treated as outpatients. Pa 1Supported in part by Grants CA43688, CA45609, and P30-CA43703 from tients receiving cisplatin, thiotepa, or high dose BCNU (350 mg/m2) the National Cancer Institute and Grant ESCA 00134 from the NIEHS. S. L. G. were enrolled in intensive chemotherapy treatment protocols, given in is the'To recipient whom of requests an Edward for reprintsMallinckrodt should Jr. be Foundation addressed, Scholar at Department Award. of Medicine, University Hospitals of Cleveland, 2074 Abington Rd., Cleveland, OH 3The abbreviations used are: CCNU, l-(2-chloroethyl)-l-nitroso-3-cyclohex- 44106. ylureu; BCNU, l,3-bis(2-chloroethyl)-l-nitrosourea. 3134
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. MODULATION OF DNA REPAIR ¡NVIVO conjunction with bone marrow reconstitution as part of the R. L. receiving a variety of chemotherapeutic agents. Specifically, Ireland Cancer Center Autologous Bone Marrow Transplantation Pro seven groups of patients were studied: those receiving (a) cis- gram ( 18). Patients had not received chemotherapy for at least 3 weeks platin (median age, 41; dose, 40 mg/m2; five patients); (b) high prior to being studied. All patients received antiemetic therapy during dose thiotepa (median age, 40; dose, 40-270 mg/m2; seven chemotherapy administration, which consisted of one or more of the patients); (c) low dose BCNU (median age, 43; dose, 40-100 following: dexamethasone, metachlopromide, prochlorperazine, and mg/m2; nine patients); (d) intermediate dose BCNU (median haloperidol. age, 47; dose, 120-200 mg/m2; six patients); (e) high dose Reagents. Standard chemical reagents were obtained from Sigma. RPMI 1640 culture medium was obtained from K.C Biologicals (Ke- BCNU (median age, 51; dose, 350 mg/m2; seven patients); (/) nesa, KS) and was supplemented to a final concentration of 25 HIM4- CCNU (median age, 51; dose, 100 mg/m2; five patients); and (2-hydroxyethyl)-l-piperazineethanesulfonicacid, 1 HIMglutamine, and (g) streptozotocin (median age, 53; dose, 500 mg/m2; six 15% fetal calf serum (Hyclone Laboratories, Logan, UT). A43H]Meth- patients). There was no significant difference between groups ylnitrosourea was obtained from Amersham (Arlington Heights, IL) in the age distribution or the proportion of patients having had (specific activity, 4.7 Ci/mmol). prior chemotherapy or prior radiation therapy. Diagnoses var Preparation of Peripheral Blood Lymphocytes from Patients. All patient donors gave informed consent prior to having venipuncture. ied between groups, as expected due to protocol eligibility, but consisted of solid tumors and lymphomas. Patients receiving Samples (20 ml) of blood were drawn at specified time points before high dose BCNU (350 mg/m2) also received cisplatin, 40 mg/ and after chemotherapy administration. Normal peripheral blood lym phocytes were isolated as previously described (19). Briefly, blood cells m2, concurrently. Three of six patients receiving streptozotocin were separated by Ficoll-Hypaque isopycnic centrifugation. The mono- also received 5-fluorouracil, 400 mg/m2. nuclear cells at the interface were collected, diluted to 1 x IO6cells/ml Table 1 shows the change in human lymphocyte alkyltrans in culture medium, and incubated in tissue culture flasks for 2 h at 37°Cto remove monocytes by adherence. The nonadherent lympho ferase activity 20 h after the first drug exposure in vivo. The cytes were then washed twice in phosphate-buffered saline with 1 HIM mean pretreatment alkyltransferase level in patient lympho EDTA and were resuspended at 4 x IO7cells/ml in cell extract buffer, cytes was similar to the mean in normal donor lymphocytes which consisted of 70 mM 4-(2-hydroxyethyl)-l-piperazineethanesul- that we have previously reported (8, 27), 8.4 ±4.9 (mean ± fonic acid (pH 7.8), 0.1 mM EDTA, 5% glycerol, and 1 mM dithiothre- SD) units/Mg of DNA in patients compared to 7.28 ±2.8 units/ itol (19). Cells were then frozen at -80°Cuntil used. Cell suspensions fig of DNA in normal donors, despite the fact that most of the were sonicated 3 times for 5 s at 4"C to complete cell disruption, using patients had received prior chemotherapy and were older (me a Branson sonifier, followed by centrifugation at 10,000 x g for 2 min dian age, 45 years; range, 16-72 years) than the normal donors to remove cellular debris. Samples of extracts were taken for determi (median age, 30 years; range 21-38 years). A large range in nation of protein and DNA content, using previously described proce enzyme activity among patients was observed, similar to that dures (19, 20), and were assayed directly for alkyltransferase activity. O'-Alkylguanine-DNA Alkyltransferase Assay. The alkyltransferase noted previously in normal donors (19). activity in cell extracts was measured as removal of the methyl-3li No statistically significant change in lymphocyte alkyltrans adduct from O6-[3H]methylguanine in [me
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. MODULATION OF DNA REPAIR IN VIVO findings are consistent with in vitro studies which indicate that As shown in Fig. 2, all patients treated with streptozotocin the only chemotherapeutic agents which deplete cellular alkyl- experienced a time-related, cumulative dose-dependent decline transferase activity are the nitrosoureas (12, 22). in lymphocyte alkyltransferase activity. Of note, four of six In vitro studies further suggested that nitrosourea-induced patients had a large decline after the first dose of streptozotocin, changes in alkyltransferase activity are dependent on dose and while two others required two or three doses to achieve the time after treatment (11, 12, 19). For this reason, we examined same effect. The rate of decline in activity was the same in the lymphocyte alkyltransferase activity in patients receiving mul three patients who also received 5-flourouracil as in those who tiple doses of chemotherapy. The chemotherapy regimens ex received streptozotocin alone. After three or four doses (1500- amined were: (a) thiotepa, 40-270 mg/m2 i.v. daily, days 1-3 2000 mg/m2) of streptozotocin, the lymphocyte alkyltransferase (six patients); (¿>)BCNU, 80 mg/m2 i.v. daily, days 1-3 (five activity in all six patients had declined to less than 40% of patients); (c) BCNU, 350 mg/m2 i.v. daily, days 1-3 (six pa control (range, 18-36%). The initial level of alkyltransferase tients); and (d) streptozotocin, 500 mg/m2 i.v. daily, days 1-4 activity did not predict the time course or extent of change in (six patients). The peripheral blood counts of these patients activity after streptozotocin treatment. remained relatively stable during the treatment period, although patients in the high dose chemotherapy groups uniformly ex perienced a decline in their lymphocyte counts 4-8 days after DISCUSSION lymphocyte sampling was complete (18). Fig. 1 shows the These data indicate that therapeutic administration of strep percentage change in lymphocyte alkyltransferase activity 20 h tozotocin or high dose BCNU inactivates the DNA repair after each daily drug treatment. No cumulative change in al protein O6-alkylguanine-DNA alkyltransferase in normal hu kyltransferase activity was noted in patients receiving thiotepa man peripheral blood lymphocytes. Other agents, including or 80 mg/m2 BCNU. However, a significant decline was ob CCNU, thiotepa, and cisplatin, did not cause significant served in lymphocyte alkyltransferase activity in patients re changes in lymphocyte alkyltransferase activity at the doses ceiving either 350 mg/m2 BCNU (P < 0.05) or 500 mg/m2 used. Because CCNU was used at much lower doses than streptozotocin (P < 0.0005). While the decline in lymphocyte BCNU, it is possible that higher doses of CCNU might also alkyltransferase activity in patients receiving high dose BCNU have produced changes in alkyltransferase activity. The great was significant, it varied widely between patients and did not est, most consistent inactivation of the alkyltransferase was appear to be cumulative. In contrast, a clear dose- and time- observed with streptozotocin, where a cumulative effect oc dependent decline in lymphocyte alkyltransferase was observed curred that was both time and dose dependent. Given the in the streptozotocin group, reaching 26 ±9% of control on constraints of this study, we were unable to separate out the day 3, compared to a mean of 58 ±31% on day 3 in the high effects of a single treatment on alkyltransferase activity over dose BCNU group. The full effect of the streptozotocin treat time and we had too few patients to determine whether disease, ment appeared to require at least 20 h since, on each day of age, or other confounding variables such as concurrent non- treatment, lesser changes in lymphocyte alkyltransferase activ malignant disease (24) influenced the change in alkyltransferase ity were observed 2 and 6 h compared to 20 h after treatment activity. Thus, we have not determined the optimal dose or (data not shown). schedule to achieve inactivation of the alkyltransferase in pa tients. Nonetheless, these data suggest that streptozotocin may 160 be a specific and effective modulator of the alkyltransferase in vivo and may have clinical application as a biochemical modu o OC lator of nitrosourea resistance in human tumors. Many human tumors are resistant to nitrosoureas and con- O U s? Ili 3 a oc a» to co
LU CO < DC LU U. V)
DAY OFTREATMENT 1- Fig. 1. Lymphocyte alkyltransferase activity after chemotherapy exposure in vivo. Lymphocytes were collected from patients daily, 20 h after chemotherapy treatment. Day 0 indicates pretreatment values. Alkyltransferase activity was measured as described in "Materials and Methods" and was converted to the percentage of pretreatment activity (% control) for each patient. Values shown are mean ±SD for each treatment groups. •.Thiotepa, 40-270 mg/m2 daily, 0- 2 days (seven patients); O, BCNU, 120 mg/m2 daily, 0-2 days (rive patients); A, DAY OF TREATMENT BCNU, 3SO mg/m2 daily, 0-2 days (seven patients); and •,streptozotocin, 500 Fig. 2. Change in lymphocyte alkyltransferase activity during daily strepto mg/m2 daily, 0-4 days (six patients). For the BCNU at 350 mg/m2 group, P < zotocin treatment. Six patients receiving 500 mg/m2 streptozotocin daily for 4 0.05 on days 1 and 3; for the streptozotocin group, P < 0.05 on day 1, P < 0.005 days had lymphocyte alkyltransferase activity measured before treatment (day 0) on day 2, and P < 0.0005 on day 3. and 20 h after each dose of streptozotocin infusion (days 1-4). 3136
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. MODULATION OF DNA REPAIR IN VIVO tain high levels of alkyltransferase activity (1-3, 14). We and showed that chronic exposure to a variety of chemotherapeutic others have shown that inactivation of the alkyltransferase with agents including the nitrosoureas results in a modest decline in 06-methylguanine, streptozotocin, or other methylating agents alkyltransferase activity. Whether repeated chemotherapeutic sensitizes tumor cells to chloroethylnitrosoureas, resulting in a exposure inactivates the alkyltransferase specificallyor whether 2- to 12-fold reduction in the 50% lethal dose of these com chronic drug exposure affects the alkyltransferase nonspecifi- pounds (2, 9, 10, 12, 15). When measured, the decrease in cally by, for instance, an alteration in the subtype of circulating alkyltransferase activity associated with sensitization has been lymphocytes, a change in their proliferative status, or a de 60-90% (3, 9, 10, 12). Thus it would appear that, if streptozo creased ability to synthesize the alkyltransferase protein, which tocin can modulate the alkyltransferase activity in human tu can influence the level of alkyltransferase in lymphocytes (19, mors in vivoto at least the extent we observed in lymphocytes, 34), remain to be determined. then the combination of streptozotocin and another nitrosourea In summary, streptozotocin administered at clinically used may be expected to be effective against tumors which are doses significantly decreases the activity of an important DNA otherwise resistant to nitrosoureas. repair protein that provides resistance to nitrosoureas. Given There are three issues that should be addressed in anticipation that streptozotocin can enhance the cytotoxic effect of nitro of clinical trials with this combination: (a) whether the changes soureas in vitro through depletion of the alkyltransferase and in lymphocyte alkyltransferase activity will be predictive of that many human tumors have high levels of alkyltransferase changes in the target tumor; (/>)whether the combination of activity, our data suggest that this modulation may be an streptozotocin and another nitrosourea will lead to unaccepta important mechanism to overcome tumor cell resistance to ble toxicity; (c) whether inactivating the alkyltransferase will nitrosoureas in vivo. improve the therapeutic index of the nitrosoureas. Human tissues and tumors differ greatly in alkyltransferase activity (21, ACKNOWLEDGMENTS 25, 26) and there are only a few studies to suggest that the level of activity in one tissue is predictive of that in another tissue The expert technical assistance of K. Miller, S. Kessen, and C. (20). Furthermore, the level of DNA alkylation by streptozo Donavan is greatly appreciated. The author thanks Drs. H. Lazarus tocin varies between tissues (27), suggesting that, ultimately, and J. Trey and other members of the R. L. Ireland Cancer Center for documentation of biochemical modulation in the target tumor referral of patient material, Drs. H. Lazarus and N. A. Berger for will be necessary for optimal use of the combination of strep thoughtful review of the manuscript, and L. Lucas for preparation of the manuscript. tozotocin followed by BCNU. 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3138 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. Modulation of Human Lymphocyte O6-Alkylguanine-DNA Alkyltransferase by Streptozotocin in Vivo
Stanton L. Gerson
Cancer Res 1989;49:3134-3138.
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