Reductive activation of mitomycins A and C by vitamin C.

The anticancer drug mitomycin C produces cytotoxic effects after being converted to a highly reactive bis-electrophile by a reductive activation, a reaction that a number of 1-electron or 2-electron oxidoreductase enzymes can perform in cells. Several reports in the literature indicate that ascorbic acid can modulate the cytotoxic effects of mitomycin C, either potentiating or inhibiting its effects. As ascorbic acid is a reducing agent that is known to be able to reduce quinones, it could be possible that the observed modulatory effects are a consequence of a direct redox reduction between mitomycin C and ascorbate. To determine if this is the case, the reaction between mitomycin C and ascorbate was studied using UV/Vis spectroscopy and LC/MS. We also studied the reaction of ascorbate with mitomycin A, a highly toxic member of the mitomycin family with a higher redox potential than mitomycin C. We found that ascorbate is capable to reduce mitomycin A efficiently, but it reduces mitomycin C rather inefficiently. The mechanisms of activation have been elucidated based on the kinetics of the reduction and on the analysis of the mitosene derivatives formed after the reaction. We found that the activation occurs by the interplay of three different mechanisms that contribute differently, depending on the pH of the reaction. As the reduction of mitomycin C by ascorbate is rather inefficiently at physiologically relevant pH values we conclude that the modulatory effect of ascorbate on the cytotoxicity of mitomycin C is not the result of a direct redox reaction and therefore this modulation must be the consequence of other biochemical mechanisms.

DNA adducts of decarbamoyl mitomycin C efficiently kill cells without wild-type p53 resulting from proteasome-mediated degradation of checkpoint protein 1.

The mitomycin derivative 10-decarbamoyl mitomycin C (DMC) more rapidly activates a p53-independent cell death pathway than mitomycin C (MC). We recently documented that an increased proportion of mitosene1-beta-adduct formation occurs in human cells treated with DMC in comparison to those treated with MC. Here, we compare the cellular and molecular response of human cancer cells treated with MC and DMC. We find the increase in mitosene 1-beta-adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53. DMC caused more DNA damage than MC in the nuclear and mitochondrial genomes. Checkpoint 1 protein (Chk1) was depleted following DMC, and the depletion of Chk1 by DMC was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Gene silencing of Chk1 by siRNA increased the cytotoxicity of MC. DMC treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity, suggesting that DMC mediated DNA adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis. Thus, the mitosene-1-beta stereoisomeric DNA adducts produced by the DMC signal for a p53-independent mode of cell death correlated with reduced nuclear size, persistent DNA damage, increased ubiquitin proteolysis and reduced Chk1 protein.

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.

Effects of mitomycin on human colon carcinoma cells.

Subpopulations of malignant cells from primary cultures of human colon carcinoma were characterized with respect to their response to mitomycin (MMC). Growth inhibition assays indicated values of 2.06, 0.93, and 0.33 microM for the concentration of drug giving 50% inhibition of growth for sublines HCT 116b, HCT 116, and HCT 116a, respectively. Alkaline elution of filter-bound DNA from cells exposed to MMC in vitro showed a positive correlation between the amount of DNA cross-linking and growth inhibition as a function of drug concentration. Comparable DNA cross-linking was obtained at MMC concentrations of 10 microM for HCT 116b and 5 microM and HCT 116. The cross-linking of DNA from HCT 116a cells at 5 microM MMC was approximately equal to that from HCT 116 cells at doses between 10 and 20 microM MMC. Cross-link removal as a function of time after drug removal of MMC-treated cells was also measured. There was little difference in the rates of alkaline DNA elution after drug removal between HCT 116b and HCT 116a, suggesting that the ability to repair cross-links was not responsible for the differential sensitivities of the cells to MMC. The relative sensitivities of the subpopulations to MMC were reflected in vivo by MMC treatment of nude BALB/c mice bearing xenografts of the cultured sublines.

DNA interstrand cross-link and free radical formation in a human multidrug-resistant cell line from mitomycin C and its analogues.

A subline of the human breast tumor cell line (MCF-7), selected for resistance to Adriamycin and having the multidrug resistance phenotype, also developed significant cross-resistance to mitomycin C and its two analogues, BMY 25282 and BMY 25067. Because mitomycin C and the analogues contain both quinone and aziridine moieties, the mechanism of tumor cell kill is thought to involve alkylation and cross-linking of DNA molecules, hence they are not expected to show cross-resistance to cells selected for resistance to a DNA intercalator. Studies to understand this novel observation show that the resistant MCF-7 cells form significantly less hydroxyl radical and DNA cross-linking in the presence of mitomycin C and BMY 25282 than the sensitive cells. Although BMY 25067 formed less free radicals in the resistant cells, similar to the other two drugs, the formation of DNA cross-links was identical in both cell lines, indicating a somewhat different mechanism of tumor cell kill by this analogue. DNA cross-link formation increased slightly with time in the sensitive cells while there was a small decrease in the resistant cells. This difference in the formation of toxic intermediates appeared to result from enhanced detoxification of reactive species (hydrogen peroxide and alkylating intermediates) as a result of significantly higher glutathione peroxidase (14-fold) and glutathione S-transferase (44-fold) activities in the resistant cell line. These events, i.e., free radical formation and DNA alkylation, showed a good correlation with the cytotoxicity in drug-sensitive cells, indicating that both mechanisms contribute to cell killing of human breast tumor cells.

Chinese hamster ovary cell lines resistant to mitomycin C under aerobic but not hypoxic conditions are deficient in DT-diaphorase.

We have previously reported the isolation of CHO cell lines resistant to mitomycin C under aerobic conditions of drug exposure. Here it is reported that these cell lines have the same response to mitomycin C under hypoxic conditions as do controls. The cells are shown to have lower levels of DT-diaphorase activity than controls, but similar levels of activity of NADPH:cytochrome c reductase, another enzyme involved in the metabolism of mitomycin C. Evidence for molecular defects in the DT-diaphorase gene or gene transcript is presented for the deficient cell lines. The consequences of this DT-diaphorase deficiency is further explored by testing the toxicity of menadione, an established enzyme substrate. The isolation of CHO cell lines deficient in DT-diaphorase activity and resistant to mitomycin C under aerobic but not hypoxic conditions suggests that mitomycin C reduction by this enzyme has a significant impact on cytotoxicity under aerobic but not hypoxic conditions. Similarly, DT-diaphorase metabolism of menadione does not appear to have a significant impact on cytotoxicity in CHO cells.

Enhancement by hyperthermia of the in vitro cytotoxicity of mitomycin C toward hypoxic tumor cells.

Mitomycin C and hyperthermia are both toxic to chronically hypoxic EMT6 tumor cells. Combinations of this drug and heat were tested in vitro in normally aerated and chronically hypoxic EMT6 mouse mammary tumor cells to establish whether greater than additive cytotoxicity could be achieved by combined treatment. Cell survival was measured at four concentrations of mitomycin C (0.01, 0.1, 1.0, and 10 microM) at 37 degrees or at elevated temperatures (41, 42, and 43 degrees) for durations of 1, 2, 3, and 6 hr. At 42 degrees, exposure to mitomycin C for 3 and 6 hr produced a 2- to 3-fold increase in hypoxic tumor cell kill at all drug concentrations over that expected for strict additivity. A 15-fold enhancement in the kill of hypoxic tumor cells was obtained at 1.0 and 10 microM mitomycin C at 43 degrees for 6 hr of exposure. Under most conditions, additivity was observed for the antibiotic and heat in oxygenated cells, except at 43 degrees with 0.01 and 0.1 microM mitomycin C following 3 and 6 hr of treatment, conditions under which a 5- to 10-fold potentiation of tumor cell kill was obtained. The rate of formation of reactive metabolites from mitomycin C under anaerobic conditions in EMT6 cell-free preparations was measured. A 30 to 50% increase in alkylating activity was observed at elevated temperatures, suggesting that the enhanced cytotoxicity of mitomycin C with heat toward hypoxic cells may, in part, be due to an increase in activation of the drug.

pH dependence of mitomycin C-induced cross-linking activity in EMT6 tumor cells.

Mitomycin C (MC), a quinone-containing bioreductive alkylating agent, is cytotoxic to aerobic EMT6 tumor cells despite the fact that little bioactivation of MC occurs in EMT6 cell homogenates in the presence of O2. Because spontaneous activation of MC at acidic pH has been reported in chemical systems, aerobic EMT6 tumor cells were incubated in serum-free 2-(N-morpholino)ethanesulfonic acid or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH 5.7, 6.4, and 7.5 and exposed to MC for 2 h. As the extracellular pH was lowered, MC-induced DNA-DNA cross-linking, as measured by alkaline elution techniques, was enhanced. This effect was dose dependent at the three pH values tested. Measurement of intracellular pH by flow cytometric analysis indicated that the decrease in extracellular pH was paralleled by a fall in intracellular pH. The alteration of the extracellular pH had no effect on the colony-forming ability of control cells. The survival of cells treated with MC, however, was decreased as the pH was lowered. These data suggest that the intracellular and/or the extracellular pH is an important determinant of MC activity in aerobic EMT6 tumor cells.

Secretion of proteinase inhibitors by tumorigenic and nontumorigenic guinea pig and Syrian hamster fibroblasts: evidence for autocrine regulation of local proteolysis.

A cytokine that inhibits fibrinolysis has been detected in the serum-free culture medium of guinea pig and hamster fibroblasts. This proteinase inhibitor was also present in Triton X-100 extracts of guinea pig cells. It was stable at pH 3.0 for 2 hr and was produced by cells rather than assimilated from serum in the culture medium as evidenced by: (a) an apparent molecular size (less than 45 kilodaltons) less than that of the principal serum-derived proteinase inhibitors; (b) its continued secretion after several passages of the cells in serum-free medium; and (c) the lack of inhibitory activity in the medium of mitomycin C-treated cells. The cytokine inhibited the proteinase activity of human urokinase, soluble TPA-stimulated guinea pig plasminogen activator, and the cell-associated plasminogen activator of tumorigenic guinea pig cells. Soluble plasminogen activator appeared to be inhibited to a greater degree than the cell-associated enzyme. The fluorogenic substrate (7-(N-carbobenzoxyglycylglycylargininamido)-4-methylcoumarin was used in a direct assay of proteinase activity and demonstrated that the cytokine inhibited both plasminogen activator and plasmin, the two proteinases of the fibrinolytic cascade. Tumorigenic guinea pig and hamster fibroblasts as well as nontransformed guinea pig fibroblasts were found to produce the inhibitory cytokine, and the amount of inhibitor secreted was independent of the tumorigenic potential of the cells. Production of the inhibitor by normal cells may be related to contact inhibition of growth, and this cytokine may contribute to the fine regulation of local proteolysis within tissues.

Inhibition of cyclophosphamide and mitomycin C-induced sister chromatid exchanges in mice by vitamin C.

Ascorbic acid (vitamin C) is known to act as an antimutagen and anticarcinogen in several test systems. However, there is no report of its effect on carcinogen-induced chromosomal damage in vivo in animals. The present study was performed to determine whether or not ascorbic acid affects sister chromatid exchanges (SCEs) induced by cyclophosphamide (CPA) and mitomycin C (MMC) in bone marrow and spleen cells in mice. The results indicate that ascorbic acid per se did not cause a significant increase in SCEs in mice. However, increasing concentrations of ascorbic acid caused decreasing levels of CPA- and MMC-induced SCEs in both cell types in vivo. At the highest concentration of ascorbic acid, 6.68 g/kg, approximately 75 and 40% SCE inhibition in both cell types was noted for CPA and MMC, respectively. Likewise, under in vivo/in vitro conditions (exposure of animals to experimental chemicals followed by culturing of cells), ascorbic acid caused a dose-related decrease in CPA- and MMC-induced SCEs, up to a dose of 3.34 g/kg At this concentration, approximately 50% CPA- and MMC-induced SCE inhibition was observed in both cell types studied. Thus, ascorbic acid acts as an anti-SCE agent in both in vivo and in vivo/in vitro conditions in mice.

The role of drug transport in resistance to nitrogen mustard and other alkylating agents in L518Y lymphoblsts.

An investigation was undertaken of the mechanism of resistance to nitrogen mustard (HN2) and other alkylating agents, with particular emphasis on the interaction between cross-resistance and drug transport mechanisms in L5178Y lymphoblasts. Dose-survival curves demonstrated that the D0 for HN2-sensitive cells (L5178Y) treated with HN2 in vitro was 9.79 ng/ml and the D0 for HN2-resistant cells (L5178Y/HN2) was 181.11 ng/ml; thus, sensitive cells were 18.5-fold more responsive than were resistant cells and the difference was highly significant (p less than 0.001). A similar evaluation of 5 additional alkylating agents, including chlorambucil, melphalan, 1,3-bis(2-chloroethyl)-l-nitrosourea, Mitomycin C, and 2,3,5-tris(ethyleneimino)-1,4-benzoquinone, revealed that L5178Y/HN2 cells were also cross-resistant, in part, to each of these compounds. Furthermore, the degree of cross-resistance was remarkably similar; for each drug, dose-survival studies showed that HN2-resistant cells were approximately 2- to 3-fold more resistant to therapy than were sensitive cells. L5178Y/HN2 cells were also cross-resistant to cyclophosphamide in vivo; after treatment with cyclophosphamide, DBA/2 female mice that were given inoculations of L5178Y cells, but not those given transplants of L5178Y/HN2 cells, showed a significant prolongation of survival time (p less than 0.01). Transport of HN2, hydrolyzed derivative of HN2 and choline by L5178Y lymphoblasts in vitro was not competitively inhibited by any of the other alkylating agents, suggesting that transport of these compounds was by an independent mechanism. These findings suggest that the mechanism whereby L5178Y/HN2 cells are cross-resistant to other alkylating agents may involve nontransport factors and that these other drugs may bypass a major portion of HN2 resistance by using independent transport systems.

Enhancement of mitomycin C cytotoxicity to hypoxic tumor cells by dicoumarol in vivo and in vitro.

Previous work by our laboratories demonstrated that dicoumarol can increase the enzymatic activation of mitomycin C (MC) to alkylating species by tumor cell sonicates under hypoxic conditions. To determine whether this increased generation of reactive metabolites would result in increased cytotoxicity, we examined the effect of this combination on the viability of EMT6 cells treated in vitro under hypoxic and oxygenated conditions. Dicoumarol increased the cytotoxicity of MC to these neoplastic cells under hypoxic conditions and decreased the toxicity of the antibiotic to aerobic cells. These findings suggested that dicoumarol might enhance the toxicity of MC to the hypoxic cells of solid tumors, without increasing the toxic side effects of the antibiotic to the host. Treatment of EMT6 tumor-bearing animals with both dicoumarol and MC significantly decreased the survival of the radioresistant hypoxic tumor cells from that obtained with MC alone. In contrast, the leukopenia produced by the antibiotic was not exacerbated by the addition of dicoumarol. These results suggest that a treatment regimen combining dicoumarol and MC might be a useful adjunct to radiation therapy for the eradication of the radioresistant hypoxic cells in solid tumors.

Activity of mitomycin C for aerobic and hypoxic cells in vitro and in vivo.

We have observed the selective toxicity of mitomycin C toward hypoxic as compared to aerobic cells in vitro for three established cell lines (Chinese hamster ovary, Chinese hamster V-79, and human HeLa) and for cells from the transplantable KHT murine tumor. The magnitude of the selective toxicity was cell line dependent. We have studied the in vivo effects of mitomycin C against aerobic and hypoxic cells of two transplantable murine tumors: the KHT fibrosarcoma and the 16/C mammary carcinoma. Either mitomycin C was given with radiation to kill most of the aerobic cells, or it was given alone. Endpoints of response were cell survival assessed by lung colony assay for the KHT tumor, and growth delay for the 16/C tumor. In some experiments, mitomycin C appeared more effective when used with radiation than when used alone, but the results of combined treatment fell just within the range of additivity as defined by isobologram analysis. The effects of combined treatment were independent of the order in which drug and radiation were given. Mitomycin C was also used in combination with Adriamycin to treat the 16/C tumor, since we have found previously that Adriamycin spares hypoxic cells in this tumor. In three of four experiments, combined drug effects were slightly greater than predicted by an additive relationship. We conclude that mitomycin C is active against hypoxic cells in two murine tumors, but that it has at most minor specificity for hypoxic as compared to aerobic cells in vivo.

Myeloprotective effect of diethyldithiocarbamate treatment following 1,3-bis(2-chloroethyl)-1-nitrosourea, adriamycin, or mitomycin C in mice.

The effect of diethyldithiocarbamate (DDTC) on myelotoxicity induced by 1,3-bis(2-chloroethyl)-1-nitrosourea, Adriamycin, or mitomycin C in C57BL/6J x DBA/2J mice is reported here. All drugs were administered i.v. Myelotoxicity was assessed, 24 h after administration of the myelotoxic drug, using bone marrow stem cell (spleen colony-forming unit) and granulocyte/macrophage progenitor cell (granulocyte/macrophage colony-forming unit in culture) clonogenic assays. Administration of DDTC alone had no effect on spleen colony-forming units or granulocyte/macrophage colony-forming units in culture. 1,3-Bis(2-chloroethyl)-1-nitrosourea showed a dose-dependent toxicity for both cell types, and subsequent treatment with DDTC (300 mg/kg i.v. 3 h after 1,3-bis(2-chloroethyl)-1-nitrosourea) ameliorated this toxicity. The same dosing regimen of DDTC ameliorated Adriamycin-induced toxicity to bone marrow stem cells at the two higher doses tested. However, the myelosuppressive effects of mitomycin C were not altered by DDTC administration (300 mg/kg i.v. 3 h after or 30 min before mitomycin C). These results demonstrate that DDTC ameliorates myelotoxicity induced by several, but not all, chemotherapeutic agents and suggest a broad role for DDTC in cancer chemotherapy.

Antitumor activity and toxicity in animals of RR-150 (7-cysteaminomitosane), a new mitomycin derivative.

The experimental antitumor activity of a new mitomycin derivative, 7-cysteaminomitosane (RR-150), was evaluated in mice. When administered i.p. to mice bearing i.p.-implanted tumors, RR-150 was superior to mitomycin C (MMC) in increasing the life span of animals bearing P388 leukemia, B16 melanoma, and a line of L1210 leukemia partially resistant to MMC. RR-150 appeared comparable to MMC in increasing life span of mice bearing Madison 109 lung carcinoma, Colon 26 carcinoma, or parental (nonresistant) L1210 leukemia. Mice immunosuppressed with 550 rads whole-body irradiation prior to i.p. implantation of B16 still benefited (e.g., 40% cure rate) following optimal RR-150 therapy when compared to nonirradiated, B16-implanted mice given RR-150 (e.g., 70% cure rate). RR-150 had inconsistent activity in the treatment of s.c.-implanted tumors. In toxicity evaluations, RR-150 was comparable to MMC in suppression of total while blood cell counts but appeared to be less neutropenic. RR-150 also caused less cumulative leukopenia than did MMC in a weekly chronic dose experiment. Based on serum chemistries, RR-150 did not have significant nephrotoxicity, but there was evidence of possible liver toxicity at doses near the 50% lethal dose. Because of the balance of favorable antitumor and toxicity properties of RR-150, work is under way to prepare a more bioavailable form for advanced evaluation.

Antitumor activity and toxicity in animals of BMY-25282, a new mitomycin derivative.

BMY-25282, 7-N-(dimethylamino methylene)mitomycin C, is one of a novel series of amidino mitomycin derivatives. Some of these were discovered as intermediates in a synthetic program being conducted to find improved procedures for modifying the structure of mitomycin C (MMC). Markedly superior in vivo antitumor effects have been observed with BMY-25282 compared to MMC in initial tests against i.p.-implanted P388 leukemia and B16 melanoma. When administered i.v. to mice bearing s.c. B16 melanoma, BMY-25282 was also superior to MMC. The derivative was fully active against a line of L1210 leukemia which was partially resistant to MMC treatment but had little or no activity against a line of L1210 fully resistant to MMC. It is also 2 to 4 times more potent than MMC based on a comparison of doses required to achieve optimum antitumor effects. The superior antitumor efficacy of BMY-25282 over MMC against both i.p. and s.c. B16 melanoma was maintained when the drug was given in pluronic acid formulation. Against P-388 leukemia, however, the efficacy of the drugs was equivalent when BMY-25282 was administered in the pluronic vehicle. In an in vitro clonogenic assay involving freshly explanted human tumors, BMY-25282 was consistently more potent in cytotoxic effects than MMC. With human colorectal carcinoma samples, BMY-25282 was 13.8 times more potent than MMC. The i.v. 50% lethal dose values of BMY-25282 and MMC in C57BL/6 X DBA/2 F1 mice were 2.1 mg/kg and 8.6 mg/kg, respectively. Leukopenic effects of the drugs in mice were comparable at doses up to their respective 50% lethal dose values. Hematology studies in ferrets revealed a similar pattern of depression and recovery of lymphocytes, neutrophils, and platelets for BMY-25282 and MMC; however, with BMY-25282 there was earlier recovery of platelet counts. BMY-25282 is being further developed toward possible clinical trial.

Role of NADPH:cytochrome c reductase and DT-diaphorase in the biotransformation of mitomycin C1.

Hypoxic cells of solid tumors are difficult to eradicate by X-irradiation or chemotherapy; as an approach to this problem, our laboratories are investigating the effects of the bioreductive alkylating agent mitomycin C (MC) on hypoxic cells. This antibiotic was preferentially toxic to EMT6 mouse mammary tumor cells and V79 Chinese hamster lung fibroblasts under hypoxic conditions, but it was equitoxic to Chinese hamster ovary cells in the presence and absence of oxygen. All cell lines catalyzed the formation of reactive metabolites under hypoxic conditions and contained NADPH:cytochrome c reductase and DT-diaphorase, two enzymes which may be responsible for the cellular activation of MC. Although a correlation existed between enzymatic activities and the formation of reactive metabolites from MC, there was no correspondence between these parameters and the degree of cytotoxicity expressed by MC under hypoxic conditions. Purified NADPH:cytochrome c reductase reduced MC in the absence of oxygen, with addition of cytochrome P-450 enhancing, but not participating directly in, the reduction reaction. Addition of NADP+ to cell sonicates substantially reduced NADPH:cytochrome c reductase activity, while the formation of reactive metabolites was affected only slightly; converse results were observed using mersalyl. Exposure of cell sonicates to dicumarol inhibited DT-diaphorase activity, while the rate of formation of reactive metabolites of MC was enhanced. The findings suggest that NADPH:cytochrome c reductase and some as yet to be identified enzyme(s) are important for the reductive activation of MC. DT-diaphorase and cytochrome P-450 are not directly involved in the activation of MC, but they appear to modulate the degree of activation to reactive species, which are presumably responsible for the observed cytotoxicity.

Mitomycin C resistance in a human colon carcinoma cell line associated with cell surface protein alterations.

A human colon carcinoma cell line resistant to mitomycin C (MMC) was obtained by repeated exposure of a previously described sensitive parental line, HCT 116, to MMC in vitro. Xenografts grown from the MMC-resistant phenotype were not inhibited in MMC-treated animals, while MMC treatment produced growth inhibition in parental cell xenografts. The MMC-resistant phenotype exhibited a greater amount of a Mr 148,000 cell surface protein than did the parental line. The increase in this Mr 148,000 cell surface protein correlated positively with the degree of MMC resistance. Alkaline elution of filter-bound DNA from resistant cells exposed to MMC in vitro showed a decrease in DNA cross-link formation such that a 10-fold higher MMC concentration was required to produce similar cross-link formation in the resistant cell as compared to the parental cell. The development of MMC resistance was not associated with in vitro cross-resistance to other natural product cytotoxic drugs. This model for resistance to MMC will be useful in future studies to define the mechanisms for MMC action and resistance in human colon carcinoma cells.

Comparison of drug sensitivity among tumor cells within a tumor, between primary tumor and metastases, and between different metastases in the human tumor colony-forming assay.

The human tumor colony-forming assay was used to compare chemosensitivity among tumor cells within a primary tumor, between primary tumor and metastases, and between different metastases. No significant differences in cloning efficiency were found in any of the three comparison studies. However, considerable differences in chemosensitivities were observed between different parts of the same tumor and between the primary tumor and metastases. Two different parts of the same tumor were comparably assayed for nine primary tumors. In nine paired samples which allowed in vitro drug sensitivity testing, there was no satisfactory correlation of sensitivity to cytostatic drugs. Cell suspensions were prepared from 28 primary tumors and from metastases taken from the same patient. In 14 paired samples which formed sufficient colonies for determination of drug effect, the data showed no satisfactory correlation of chemosensitivity between a primary tumor and its metastases. Both tumor samples from different metastatic sites of the same patient formed sufficient colonies in seven of eight instances. In the seven paired samples, there was strong association of chemosensitivity (p less than 0.005). The results indicate that the reported discrepancies of in vitro and in vivo results in clinical trials using the tumor colony-forming assay for predicting resistance or sensitivity to cytostatic drugs may be due to therapeutic heterogeneity among tumor colony-forming units within a primary tumor and between a primary tumor and its metastases.

Mitomycin C skin toxicity studies in mice: reduced ulceration and altered pharmacokinetics with topical dimethyl sulfoxide.

A series of toxicologic and pharmacokinetic studies were performed in BALB/c mice administered intradermal (ID) mitomycin C (MMC) at doses of .015 to 0.25 mg. Dose-dependent skin ulcers were produced at clinically relevant MMC dose levels of .05 and .075 mg (3.6 to 10.7 mg/m2). These doses produced peak ulcers of 0.15 to 0.22 cm2, respectively, one to five days after injection. The integrated ulcer area X time values (area under the curve [AUC] ulceration) were 0.89 and 3.11 cm2 X d. A large number of local pharmacologic adjuvants were found to be ineffective at reducing MMC ulceration after proximal ID injection. These included diphenhydramine, catalase, heparin, hyaluronidase, hydrocortisone, cysteine, N-acetylcysteine, lidocaine, vitamin E, and superoxide dismutase. Also, neither topical heating nor cooling of skin reduced MMC ulcerations. In contrast, a single topical application of a 100% dimethyl sulfoxide (DMSO) solution completely prevented 0.025 mg MMC-induced skin ulceration and significantly reduced .075 mg MMC ulceration (P less than .05 by multiple range tests). Topical DMSO also altered the disposition of ID MMC in mouse skin but not in plasma. Unexpectedly, the DMSO applications slowed MMC elimination from the skin. DMSO significantly increased the AUC for MMC in skin from 0.89 to 2.25 ng/h/mL of tissue (P less than .05). DMSO did not alter the degree of protein binding in skin tissue nor the in vitro chemical stability of MMC in skin tissue homogenates. These results show that experimental MMC-induced skin ulcers in mice can be ameliorated with an immediate application of topical DMSO. This effect is not due to enhanced systemic drug uptake, but may be due to reduced reactivity of MMC with target cellular nucleophiles.