Chlorozotocin. Mechanism of reduced bone marrow toxicity in mice.

Chlorozotocin is a chloroethyl nitrosourea with a glucose carrier that has curative activity for the murine L1210 leukemia, but is nonmyelosuppressive in mice. To determine the mechanism for this unique property of reduced bone marrow toxicity, comparative studies were conducted with chlorozotocin and CCNU, a myelotoxic chloroethyl nitrosourea. Suspensions of L1210 leukemia and murine bone marrow cells were incubated for 2 h with 0.1 mM [(14)C]-chloroethyl chlorozotocin or CCNU. Chlorozotocin demonstrated a fourfold increased covalent binding of the chloroethyl group to L1210 nuclei when compared to equimolar CCNU. Chlorozotocin alkylation of L1210 cells resulted in the binding of 57 pmol of [(14)C]ethyl group/mg of DNA, which represented a 2.3-fold increased alkylation when compared to CCNU. In marked contrast, the binding of the chloroethyl group to bone marrow nuclei was equivalent for both drugs. In addition, chlorozotocin alkylation of murine bone marrow DNA, 45 pmol of [(14)C]ethyl group/mg of DNA, was equivalent to that of CCNU. The ratio of L1210:bone marrow DNA alkylation was 1.3 for chlorozotocin compared to 0.6 for CCNU. The intracellular carbamoylation of L1210 and bone marrow protein by CCNU was 400- to 600-fold greater than that produced by chlorozotocin. After a 2-h exposure to 0.1, 0.05, or 0.01 mM drug, both chlorozotocin and CCNU produced a reduction in the cloning efficiency of L1210 cells that was dose dependent. However, chlorozotocin was significantly more cytotoxic than CCNU at all three molar concentrations (P < 0.01). Chlorozotocin, 0.1 mM, reduced L1210 DNA synthesis to 1% of control by 48 h, in contrast to 16% with equimolar CCNU (P < 0.01). In mice bearing 10(5) L1210 cells, chlorozotocin produced its optimal antitumor activity (332% increased life span [ILS]) at doses of 48-64 mumol/kg, with >50% indefinite survivors. In contrast, CCNU at the same molar doses resulted in only a 191% ILS; a CCNU dose of 128 mumol/kg was required for comparable optimal L1210 antitumor activity, 413% ILS. On a molar basis, the dose of chlorozotocin that produced optimal in vivo L1210 antitumor activity was one-third to one-half that of CCNU. Chlorozotocin, unlike CCNU, produced no murine bone marrow toxicity at its optimal therapeutic dose. This unique combination of antitumor activity without myelosuppression can be correlated with the advantageous ratio of L1210:bone marrow in vitro DNA alkylation by chlorozotocin (1.3) as compared to equimolar CCNU (0.6).

Increased mutagenicity of chloroethylnitrosoureas in the presence of a rat liver S9 microsome mixture.

The mechanism for an enhanced effect of Aroclor 1254-induced Sprague-Dawley rat liver 9,000 x g supernatant (S9) microsome preparation on the mutagenicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-N-nitrosourea (CCNU), and 2-[3-(2-chloroethyl)-3-nitrosoureido]-2-deoxy-D-glucopyranose (chlorozotocin) for Salmonella typhimurium strain TA1535 was studied. Although all three compounds were direct-acting mutagens, rat liver S9 increased the mutagenic response to BCNU, CCNU, and chlorozotocin. The enhanced mutagenic effect was independent of NADPH. Heat-denatured S9 enhanced the mutagenicity of BCNU and CCNU, but not that of chlorozotocin. Mutagenic enhancement, however, was less than that observed with untreated S9. The substitution of extractable S9 lipid and bovine serum albumin for S9 in the reaction mixture resulted in an enhanced mutagenicity of CCNU with little or no effect on BCNU or chlorozotocin mutagenicity. These results suggest that the enhanced mutagenicity of CCNU, and possibly that of BCNU, in the presence of S9 was due in part to nonspecific factors that are present in the S9 preparation.

Enhanced cloning efficiency of murine rhabdomyosarcoma cells after chlorozotocin treatment: relationship with enhanced lung metastasis.

The effect of chlorozotocin [(CZT) CAS: 54749-90-5; 2-(3-(2-chloroethyl)-3-nitrosoureido)-D-gluco-pyranose] was studied on a series of tumor cells, cultured or extracted fresh primary or transplanted tumors, by means of clonogenic assay. The ability of most rat rhabdomyosarcoma cells to form colonies in soft agar was enhanced when exposed to the water-soluble nitrosourea chloride CZT. The tumor cells tested were derived from a) several primary tumors induced in WAG rats by colloidal nickel, then cultured and exposed to CZT early during in vitro passage; b) the 9-4 tumor, also Ni-induced but maintained in long-term culture; and c) the Ni-induced 9-4/0 tumor, maintained by transplantation in syngeneic rats. No inhibition of colony formation was observed in any of the cell lines even at high concentrations of CZT. Adriamycin, chosen as a control treatment, strongly inhibited the cloning efficiency (CE) of the tumor cells. In vivo, the weekly injection of 10 mg CZT/kg body weight into syngeneic rats bearing transplanted tumors led to an enhancement of lung metastasis formation. The CZT enhancement of CE of tumor cells and its relationship to increased in vivo tumor metastasis is discussed.

Mutagenic activity of nitrosourea antitumor agents.

The relative mutagenic activities of chloroethyl-nitrosourea and methylnitrosourea antitumor agents in active clinical use were determined with the use of the Ames Salmonella typhimurium assay. The results indicated that the drugs induced base substitutions. 2-Deoxy-2-[[(methylnitrosoamino)carbonyl]amino]-D-glucopyranose (also called streptozotocin), a glucose-containing methylnitrosourea, was the most mutagenic of all compounds tested and showed at least a 250-fold increase in activity when compared to that of its chloroethyl analog, 2-[[[(2-chloroethyl) nitrosoamino]carbonyl]-amino]-2-deoxy-D-glucose (also called chlorozotocin). All nitrosoureas, with the exception of N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2-chloroethyl)-N-nitrosourea monohydrochloride, a pyrimidine chloroethyl analog, demonstrated an increase in mutagenicity after incubation with induced Sprague-Dawley rat liver microsomes. No correlation between in vitro chemical alkylating activity and mutagenic potential was observed. Mutagenic activity was not observed to be of predictive value for antitumor activity in the L1210 leukemia model system.

Enhancement of pulmonary metastases induced by decreased lung natural killer cell activity.

Administration of repeated high doses of 7.5 mg chlorozotocin [(CZT) CAS: 54749-90-5]/kg to syngeneic WAG rats bearing the rhabdomyosarcoma 9-4/0 enhanced the incidence of spontaneous metastasis compared to its incidence in untreated rats. This enhancement was observed concomitantly with an increase in the survival of 9-4/0 rhabdomyosarcoma and P 77 fibrohistiocytoma tumor cells, labeled with [125I]5-iodo-2'-deoxyuridine or 51Cr and injected iv. Within the first 24 hours after P 77 cell injection, the lungs retained 10% of the cells while the lungs of controls or of rats given one CZT injection only retained 0.06%. The natural killer (NK) cell cytotoxicity of cells flushed out from lung capillaries [lung intracapillary cells (LIC)] was studied concomitantly in a 4-hour 51Cr release assa against YAC-1 and P 77 target cells. A large reduction was again observed in NK cytotoxicity but only after repeated injections of 7.5 mg CZT/kg. Lung defenses were gradually restored after treatment stopped. Administration of polyinosinic-polycytidylic acid with CZT restored the NK cell cytotoxicity of LIC and inhibited lung metastases amplification. The close relationship between metastasis and NK activity indicates the need for caution as regards the effects of chemotherapy on NK activity.

Immunomodulation by various nitrosoureas and its effect on the survival of the murine host bearing a syngeneic tumor.

Chemotherapeutic efficacies of the nitrosoureas 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), chlorozotocin (CLZ), and streptozotocin (STZ) were investigated against the LSA tumor which is syngeneic to C57BL/6 mice. It was observed that a single injection of 20 mg/kg body weight of BCNU or CLZ, even at an advanced stage of tumor growth, completely cured greater than 90% of the tumor-bearing mice. Furthermore, BCNU-cured or CLZ-cured mice could specifically reject secondary rechallenge with the LSA tumor. In contrast, a single dose treatment with STZ at 20-200 mg/kg body weight failed to cure the tumor-bearing mice (0% survival). The failure of STZ to cure tumor-bearing mice was next addressed considering three possible mechanisms: (a) STZ was less tumoricidal; (b) STZ suppressed the immunity of the host; and (c) STZ failed to eliminate tumor-specific suppressor T-cells. The failure of STZ to cure tumor-bearing mice was not totally related to its tumoricidal properties since STZ at higher doses did possess significant tumoricidal activity in vitro and in vivo, comparable to that of BCNU or CLZ. When spleen cells from normal mice treated with BCNU, CLZ, or STZ were assayed for their responsiveness to the T-cell mitogens concanavalin A or phytohemagglutinin, it was observed that STZ was in fact less immunosuppressive than BCNU or CLZ. The fact that STZ did not suppress the immunity of the host was also suggested by the findings that BCNU-cured mice treated with STZ or CLZ could still reject secondary rechallenge with the specific tumor LSA. Following treatment of tumor-bearing mice with BCNU or CLZ, tumor-specific delayed type hypersensitivity responses were demonstrable in these mice but not in STZ-treated mice. The inability of STZ-treated tumor-bearing mice to elicit a delayed type hypersensitivity response was not due to selective depletion of delayed type hypersensitivity-inducing CD4+ T-cells but was probably due to failure of STZ to eliminate tumor-specific suppressor cells. Together these findings suggested that the failure of STZ to cure LSA tumor-bearing mice was not due to lack of tumoricidal activity or related to suppression of tumor-specific effector T-cell function but may be due to the failure of STZ to eliminate tumor-specific T suppressor cells. The present study suggests that the outcome of chemotherapy with nitrosoureas depends, in addition to the tumoricidal activity of the drug, on the immunomodulating action on the immune mechanisms of the host.

DNA damage and repair in the bone marrow of rats treated with four chloroethylnitrosoureas.

DNA is considered to be an important target for the antitumor and toxic properties of the chloroethylnitrosoureas. Since the main target for their dose-limiting toxicity and the antileukemic efficacy is believed to be the bone marrow, we have compared the formation and subsequent removal of DNA-DNA interstrand cross-links in the bone marrow of rats which had received a single i.p. injection (100 mumol/kg) of four chloroethylnitrosoureas. The kinetics of cross-link removal was identical for chlorozotocin, which is known to have low chemical carbamoylating activity, to that of 1,3-bis(2-chloroethyl)-1-nitrosourea, a drug with a relatively high carbamoylating capacity. The differential bone marrow toxicity exhibited by these two agents could not, therefore, be explained by a carbamoylation-mediated difference in the rate and extent of DNA-DNA interstrand cross-link removal. The peak level and overall magnitude of cross-links were, however, found to vary considerably with the chemical structure of the analogues. Both 1-(2-chloroethyl)-1-nitroso-3-(methylene-carboxamido)urea and 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea, were much more effective in inducing interstrand cross-links than 1,3-bis(2-chloroethyl)-1-nitrosourea or chlorozotocin. This differential cross-linking did not, however, parallel the single-dose acute toxicity of these agents but reflected to a greater extent differences in their antileukemic activity. Considering the widely differing biological properties of this class of compounds, the measurement of DNA-DNA interstrand cross-linking in vivo might prove relevant in the evaluation of novel nitrosoureas.

Uptake and decomposition of chlorozotocin in L5178Y lymphoblasts in vitro.

Uptake and metabolism of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin) by L5178Y lymphoblasts in vitro was investigated, using both glucose- and chloroethyl-labeled chlorozotocin. A time course of uptake of total radioactivity revealed a greater cell/medium distribution ratio of activity in cells treated with chloroethyl-labeled chlorozotocin compared to cells treated with the glucose-labeled compound. Thin-layer chromatographic analysis showed that uptake of intact chlorozotocin was identical in cells treated with either glucose- or chloroethyl-labeled drug and that the cell/medium distribution ratio never exceeded unity. Accumulation of 14C-chlorozotocin was not inhibited by an excess of unlabeled chlorozotocin, the structural analogs glucose and glucosamine, or several metabolic inhibitors or by sodium ion depletion. These observations, together with the relatively low temperature quotient for the uptake process, suggested that chlorozotocin uptake occurs by passive diffusion. In cells treated with glucose-labeled chlorozotocin, a bicyclic urethan derivative and polar metabolites soluble in trichloroacetic acid were formed. In cells exposed to chloroethyl-labeled drug, nonpolar as well as polar metabolites were noted. Formation of metabolites from the glucose moiety was impeded by the presence of an excess of unlabeled chlorozotocin, the structural analogs glucose and glucosamine, the glucose transport inhibitors phlorizin and phloretin, the metabolic inhibitor m-chlorophenyl carbonyl cyanide hydrazone and by sodium depletion. Appearance of metabolites arising from the chloroethyl moiety was also blocked by the presence of m-chlorophenyl carbonyl cyanide hydrazone and by sodium ion depletion. These results suggested that metabolism of chlorozotocin in L51789Y lymphoblasts appears to be enzyme mediated.

DNA-protein cross-linking and DNA interstrand cross-linking by haloethylnitrosoureas in L1210 cells.

Bifunctional alkylating agents are known to produce cross-links between DNA and protein and between paired DNA strands. The possibility of discriminating these two classes of cross-links in L1210 cells treated with haloethylnitrosoureas or nitrogen mustard was explored with the alkaline elution technique. Two classes of cross-links were demonstrated, based on sensitivity to proteinase K; the proteinase-sensitive cross-links appear to be DNA-protein cross-links, and the proteinase-resistant class may include interstrand cross-links. Proteinase-sensitive cross-links form more rapidly than do proteinase-resistant cross-links in cells treated with chloroethylnitrosoureas, perhaps because these agents can chloroethylate protein sulfhydryl or amino groups followed by rapid reaction of these chloroethylated groups with DNA. Although both types of cross-links produced by nitrogen mustard disappeared or were repaired after 24 hr, the removal of cross-links produced by chloroethylnitrosoureas either did not occur or was incomplete in 24 hr. In addition to cross-links, cells treated with haloethylnitrosoureas exhibited DNA strand breaks; a method is suggested for estimating the apparent frequencies of strand breaks and cross-links in the DNA.

A comparison of the biological and biochemical properties of 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea and 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose.

1-4-Amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) is a water-soluble nitrosourea that has produced delayed hematological toxicity in man during Phase 1 clinical trials. ACNU has in vitro alkylating activy 40% less than that of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin) but shares the property of negligible carbamoylating activity with the latter compounds. ACNU is highly active against murine L1210 leukemia. However, the maximum therapeutic dose, 30 mg/kg (a dose lethal to 10% of the animals), produced a 64% reduction in peripheral WBC and an 85% decrease in circulating neutrophils in normal mice. This was correlated with a 76% inhibition of normal mouse bone marrow DNA synthesis within 24 hr after treatment, followed by full recovery within 48 hr after treatment, followed by full recovery within 48 hr. In contrast, DNA synthesis in L1210 cells was suppressed to less than 10% of control for a minimum of 72 hr. While ACNU, a pyrimidine analog, possesses many of the chemical properties of chlorozotocin, it does not share with the latter compound its reduced myelotoxicity at therapeutic doses. The glucose carrier of the chlorozotocin molecule appears to impart the selective sparing of normal bone marrow.

DNA damage in cultured L1210 cells by 2-haloethyl esters of (methylsulfonyl)methanesulfonic acid.

The effects of the 2-chloroethyl, 2-bromoethyl, and 2-fluoroethyl esters of (methylsulfonyl)methanesulfonic acid upon the DNA of cultured L1210 cells have been measured and compared with each other and with the effects of chlorozotocin. Results obtained by the alkaline elution method indicated that, at equimolar and equitoxic concentrations, the esters caused more strand scission than chlorozotocin, but at compound concentrations that caused a 50% reduction in colony formation by cells following an exposure period of 2 h, they caused no detectable cross-linking, whereas chlorozotocin did cause cross-linking. Two in vitro experimental methods that are based upon the complexing of ethidium to calf thymus DNA also yielded data showing that, at equimolar concentrations, chlorozotocin caused cross-linking of calf thymus DNA, but the 2-chloroethyl ester did not. These results indicate that these esters might not kill cells by producing DNA-DNA cross-links. The three esters caused qualitatively similar effects, but the fluoro esters caused less strand scission than the chloro and bromo esters, which caused about the same extent of strand scission.

Application of in vivo and in vitro pharmacokinetics for physiologically relevant drug exposure in a human tumor clonogenic cell assay.

The biological half-lives and decay rate constants under the conditions of a human brain tumor clonogenic cell assay were determined for six clinically used anticancer agents. The agents studied were: 1,3-bis(2-chloroethyl)-1-nitrosourea; 3-(2-chloroethyl-3-nitrosoureido-2-deoxy-D-glucopyranose; cis-diaminedichloroplatinum(II); 2,5-diaziridinyl-3,6-bis-(carboethoxyamino)-1,4-benzoquinone; 4-demethylepipodophylotoxin-D-thylidene glucoside; and 9-hydroxy-2-N-methylellipticine. In vitro decay of all six drugs was found to be according to first order kinetics. The half-lives of two drugs, namely, 1,3-bis(2-chloroethyl-1-nitrosourea and 3-(2-chloroethyl-3-nitrosoureido-2-deoxy-D-glucopyranose under the human tumor clonogenic cell assay (HTCA) conditions were found to be similar to their terminal in vivo half-lives in humans. For the other drugs, however, there was a very large difference between their in vitro and in vivo pharmacokinetics. In the case of 2,5-diaziridinyl-3,6-bis(carboethoxyamine)-1,4-benzoquinone, we observed about an 80-fold difference between its in vitro half-life of 40.76 h and its in vivo terminal half-life of 0.52 h. We describe the principles upon which these data can be used to design clinically more relevant in vitro drug exposure protocols in HTCAs. Since, generally, tumor cells are exposed to drugs in the HTCA either continuously or for a specified duration, e.g., 1 or 2 h, we computed the initial in vitro drug concentrations to which tumor cells should be exposed such that the resulting in vitro (c X t) after a 2-h or a continuous exposure will be within clinically achievable levels. The application of these in vivo and in vitro pharmacokinetic principles will provide for more physiological testing of patient tumor cell sensitivity to anticancer drugs in the HTCA, and is likely to result in lower rates of false positive responses in clinical trials using clonogenic cell assays.

Repair of DNA alkylation induced in L1210 leukemia and murine bone marrow by three chloroethylnitrosoureas.

The removal of DNA adducts is an essential step of DNA repair following exposure to chloroethylnitrosoureas. Adduct removal was evaluated in both L1210 and murine bone marrow DNA for lesions induced by three chloroethylnitrosoureas. 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea, a marrow-toxic agent with high carbamoylating activity, was not removed in either system for at least 6 to 12 hr. These results were compared with those obtained with two glucose-linked chloroethylnitrosoureas, chlorozotocin and 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea. Both of these agents have low marrow toxicity at therapeutic doses. Chlorozotocin, which has very low chemical carbamoylating activity, was found to permit approximately 40% removal of drug-derived DNA adducts in both systems within the first 6 hr and approximately 50% by 18 hr. The second glucose-linked analog, 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, has relatively high carbamoylating activity and was found to inhibit early removal of DNA adducts as effectively as does 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. It would thus appear that the selective marrow-sparing property of the sugar-linked chloroethylnitrosoureas is not dependent upon carbamoylation-mediated differences in the rate and extent of DNA adduct removal. In view of the comparable therapeutic activity of the three drugs for L1210 leukemia, therapeutic efficacy does not appear to be impaired by the increased rate of adduct removal observed with chlorozotocin in this system.

Effects of N,N'-bis(2-chloroethyl)-N-nitrosourea and 2-[3-(2-chloroethyl)-3-nitrosoureido]-2-deoxy-D-glucopyranose upon the proliferation, DNA synthesis, and viability of cultured sensitive and resistant L1210 cells.

The effects of N,N'-bis(2-chloroethyl)-N-nitrosourea and chlorozotocin upon the proliferation, DNA synthesis, and viability of cultured cells of a sensitive line of L1210 leukemia, a line partially resistant to N,N'-bis(2-chloroethyl)-N-nitrosourea, and a line resistant to cyclophosphamide were determined. The results indicate that neither the effect upon proliferation nor the effect upon DNA synthesis is a good predictor of the extent of cell kill. The similarity of the effects of N,N'-bis(2-chloroethyl)-N-nitrosourea upon these two parameters for the three cell lines indicates that the sensitive and resistant cells are affected to approximately the same extent, but more of the resistant cells survive. Additional studies are required to seek the reasons for this differential survival.

Biological and biochemical properties of 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea (NSC D 254157), a nitrosourea with reduced bone marrow toxicity.

1-(2-Chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea (GANU), a water-soluble nitrosourea, differs from 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin) by the placement of the cytotoxic group on C-1 of glucose. Its biological and biochemical properties are compared with those of chlorozotocin. At a 10% lethal dose (10 mg/kg i.p.), GANU demonstrates minimal myelosuppression. This dose failed to depress normal bone marrow DNA synthesis, in contrast to a 96% inhibition in L1210 DNA synthesis. In L1210 cell suspension, equimolar doses of GANU and chlorozotocin produced equivalent degrees of inhibition in DNA synthesis. GANU has significant L1210 activity in BALB/c X DBA/2 F1 mice treated on Day 2 of tumor growth. A 117% increased life-span and 15% 45-day survivors are atained with 15 mg/kg i.p., a 50% lethal dose. However, in concurrent studies using randomly selected littermate groups of mice, GANU proved less active than chlorozotocin which produced a 306% increased life-span (15 mg/kg i.p.). GANU and chlorozotocin have similar in vitro alkylating activity but the in vitro carbamoylating activity of GANU is sevenfold that of chlorozotocin. On a molar basis, the lethal toxicity of GANU is twice that of chlorozotocin. The significant carbamoylating activity of GANU may contribute to its greater toxicity and therefore limit the mumoles of alkylating agent that can be administered to the tumor. These structure-activity studies further confirm that the addition of a glucose carrier to a cytotoxic nitrosourea moiety can selectively reduce bone marrow toxicity while retaining antitumor activity.

Response of cultured human cell lines from rhabdomyosarcoma xenografts to treatment with chloroethylnitrosoureas.

DNA interstrand cross-links are thought to be the cytotoxic lesion resulting from treatment of cells with the chlorethylnitrosoureas (CENUs). We showed in an earlier study that the resistance of xenografts of pediatric rhabdomyosarcoma to therapy with CENUs correlates with levels of O6-alkylguanine-DNA alkyltransferase. We now demonstrate a relationship between levels of the alkyltransferase and CENU-induced cytotoxicity and DNA-interstrand cross-link formation in two cell lines recently established from such rhabdomyosarcoma xenografts. Rh18 cells were derived from the HxRh18 xenograft line, which contains the alkyl-transferase and is relatively resistant to CENUs, and Rh28 cells were derived from the HxRh28 xenograft line, which lacks detectable alkyltransferase activity and is sensitive to treatment with the CENUs. In vitro, Rh28 cells were 5- to 6-fold more sensitive to growth inhibition by 1,3-bis(2-chloroethyl)nitrosourea than Rh18 cells. Extracts of Rh18 cells contained 3.8 units of the alkyltransferase per mg of protein, whereas such activity was undetectable in Rh28 cells, a unit of the alkyltransferase being defined as 1 pmol of [3H] methyl transferred from [3H]methyl-labeled DNA to protein. DNA interstrand cross-links, measured by alkaline elution 6 h after a 1-h pulse treatment with CENU, could not be detected in Rh18 cells but were found in the Rh28 line. The phenotypes of the parental xenograft lines defined by their alkyltransferase levels and by responses to CENU therapy of the mice have clearly been retained in the cultured cell lines, and as predicted, cross-link formation was inhibited in the alkyltransferase-containing Rh18 cells. These two new cell lines thus provide a useful model for studying the role of DNA repair in rhabdomyosarcoma resistance to these alkylating agents.

Measurements of DNA damage in Chinese hamster cells treated with equitoxic and equimutagenic doses of nitrosoureas.

The DNA of V-79 Chinese hamster cells was examined by alkaline elution following treatment of cultures with eight different nitrosoureas. Drug incubations were performed under consistent biological conditions of equal toxicity and equal mutation induction at the hypoxanthineguanine phosphoribosyltransferase locus. The goals of this study were to determine whether DNA damage could be detected in cells treated with biologically relevant doses of nitrosoureas and to determine whether the type and number of observed DNA lesions could be correlated with the cytotoxic and mutagenic effects of the drugs. All of the compounds tested produced, to some degree, lesions that were observed as DNA strand breaks upon exposure of the DNA to alkali. The levels of DNA strand breaks and/or alkali-labile lesions were comparable for all of the drugs at the equimutagenic doses. DNA cross-linking was observed at both the equitoxic and the equimutagenic concentrations of the haloethylnitrosoureas, but cross-linking was not observed with methylnitrosourea or streptozotocin. Methylnitrosourea and streptozotocin required approximately 40 times the drug concentration to produce toxicity equal to the haloethylnitrosoureas. These data suggest that the ability to cross-link DNA confers increased cytotoxicity to the haloethylnitrosoureas.

Biological and biochemical properties of the 2-hydroxyl metabolites of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

The lethal and bone marrow toxicity and antitumor activity of the cis- and trans-2-hydroxylated metabolites of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) have been correlated with their relative in vitro alkylating and carbamoylating activities. Both the isomers have considerably greater alkylating activity and shorter chemical half-lives than the parent compound and on a molar basis have greater antitumor activity against i.p. L1210 leukemia. However, in terms of molar doses resulting in the death of 10% of normal mice, the cis- and trans-2 isomers were 2- and 3-fold more toxic than was CCNU in normal mice. In comparing the antitumor activity produced by a maximum nonlethal dose for each compound, we found that the trans isomer had activity identical to that of CCNU (413 and 410% increased life span compared to control), and the cis isomer had considerably less (152%). Like chlorozotocin, both isomers possess low carbamoylating activity and increased water solubility, two features that have been considered possible contributors to the bone marrow-sparing character of chlorozotocin. However, in the murine model the human bone marrow colony formation (CFU-C) assay neither hydroxylated metabolite of CCNU was associated with reduced myelotoxicity.

Comparison of biochemical and biological effects of four nitrosoureas with differing carbamoylating activities.

Four chloroethylnitrosoureas with differing degrees of carbamoylating activity were compared for their effects on incorporation of radioactive precursors into macromolecules. The comparisons were made with concentrations that, for each drug, produced a defined biological effect, either an 0.5-log or a 2-log reduction in cloning efficiency from a 1-hr drug exposure. Dose-dependent inhibition of incorporation of labeled precursors into nucleic acids and proteins was observed during the 1-hr drug exposure with either of the two strongly carbamoylating nitrosoureas, 1-3-bis(2-chloroethyl)-u-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; the most extensive inhibition involved incorporation into DNA. No inhibitions were observed during exposure to a weakly carbamoylating nitrosourea (chlorozotocin) or during exposure to 1-(2-chloroethyl)-1-nitrosourea, a compound the carbamoylating activity of which is not agreed upon. By 4 hr after removal of 1-3-bis(2-chloroethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea from the extracellular medium, the cells had partially recovered from the earlier inhibition of radioactive thymidine incorporation. This recovery was, however, dependent upon an undefined factor present in serum. The inhibitions that were observed during the 1-hr drug exposure are clearly not essential to the cytotoxic effect of chloroethylnitrosoureas since no inhibitions occurred with two of the four compounds studied.

Effect of O6-benzylguanine analogues on sensitivity of human tumor cells to the cytotoxic effects of alkylating agents.

The effect of O6-benzylguanine, O6-(p-chlorobenzyl)guanine, and O6-(p-methylbenzyl)guanine on the sensitivity of various human tumor cell lines to alkylating agents is evaluated. The sensitivity of human colon tumor cells, HT29, to the chloroethylating agents, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 2-chloroethyl(methylsulfonyl) methanesulfonate (clomesone), and chlorozotocin was increased by pretreatment for 2 h with 25 microM of each analogue. O6-Benzylguanine was slightly more effective as a sensitizer in HT29 cells than the p-chlorobenzyl and p-methylbenzyl analogues. However, all analogues sensitized SF767 glioma cells to the cytotoxic effects of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, and clomesone to the same degree. Both cell lines were sensitized to the methylating agents streptozotocin and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide, the active intermediate of 5-(3,3-dimethyl-1-triazenyl)imidazole-4-carboxamide, by pretreatment with 10 microM O6-benzylguanine for 2 h. The number of Raji cells surviving 50 microM clomesone decreased 3-fold upon pretreatment for 2 h with 1 microM O6-benzylguanine. The degree of enhancement was dependent on the amount of alkyltransferase protein present in cell lines. For example, HT29 cells (alkyltransferase activity, 381 fmol/mg protein) exhibited a greater degree of enhancement when treated with O6-benzylguanine than SF767 (77 fmol/mg protein) and M19-MEL melanoma (36 fmol/mg protein) cells. There was no enhancement observed in mer- cell lines, U251 (less than 2 fmol/mg protein), and BE (3 fmol/mg protein), or with alkylating agents which did not produce a cytotoxic lesion at the O6 position of guanine in DNA such as cisplatin or 4-hydroperoxycyclophosphamide. Our studies suggest that O6-benzylguanine analogues may have utility in mer+ tumors as an adjuvant to a variety of alkylating agents which produce a toxic lesion at the O6 position of guanine.