Hepatic organelle interaction. IV. Mechanism of succinate enhancement of formaldehyde accumulation from endoplasmic reticulum N-dealkylations.

Further evidence for organelle interaction during drug metabolism by the liver is presented. The apparent stimulation by succinate of formaldehyde accumulation in the medium, which was reported to occur with liver slices and homogenates as well as with mitochondria plus microsomes, has been shown to be the result of succinate inhibition of mitochondrial aldehyde dehydrogenase. The mechanism of succinate inhibition is shown to be by reverse electron transport, and an increase in the NADH to NAD+ ratio in the mitochondria; the aldehyde dehydrogenase requires the oxidized form of the pyridine nucleotide as its cofactor. Studies on in vitro N-demethylation by liver microsomes and endoplasmic reticulum segments which cosediment with the mitochondria indicate that formaldehyde produced by the mixed function oxidase is handled differently from formaldehyde added to the medium. The latter is mainly retained in the medium containing 5 mM semicarbazide, while the generated formaldehyde is more than 50% consumed by the mitochondria. Electron microscopy has indicated that the microsomes and the endoplasmic reticulum fragments have a tendency to align themselves close to the mitochondria when present in the same medium. Consequently, it is possible that formaldehyde released to the medium adjacent to the mitochondria, as by N-demethylation, would be exposed to semicarbazide for shorter periods than that added directly to the medium. In agreement with this suggestion, complexing of formaldehyde with semicarbazide was observed spectroscopically not to be an extremely rapid reaction even at 37 degrees C. This is believed to be the reason for the greater extent of consumption of formaldehyde generated by the endoplasmic reticulum.

Distribution of porfiromycin in EMT6 solid tumors and normal tissues of BALB/c mice.

The distribution of porfiromycin was studied in BALB/c mice bearing EMT6 mammary tumors. The levels of 3H in blood and most tissues peaked approximately 15 minutes after intraperitoneal injection of [3H]porfiromycin. The levels of radioactivity present in most of the tissues and in the tumors were similar at 4 hours and 24 hours after administration. Most of the normal tissues showed uniform, low grain densities when analyzed by autoradiography; the liver and the small intestine had the highest labeling densities. Only kidney, bladder, and tumor showed differential distributions of grains from [3H]porfiromycin. In the kidney, higher grain counts were found in cortex than in medullary regions; grains were uniformly distributed within each region. In the bladder, the highest labeling densities were found in regions near the lumen. Tumor regions that had some necrotic features or regions of necrosis that included some viable cells showed higher labeling intensities than healthy-looking tumor regions, probably because the abnormal microenvironments in these regions led to increased rates of activation of porfiromycin to electrophilic species. These findings show that porfiromycin can reach and be activated in tumor regions containing cells resistant to many chemotherapeutic agents and to x rays. The results also support the concept that agents such as porfiromycin can target cells in specific microenvironmental subpopulations of solid tumors.

Correlation between drug uptake and selective toxicity of porfiromycin to hypoxic EMT6 cells.

Mitomycin C and its methylated analogue porfiromycin (Por) have significant potential as adjuncts to regimens presently used for treating solid tumors because of their preferential toxicity to cells existing in an hypoxic environment. An understanding of the factors producing the differential activity of these drugs under aerobic and hypoxic conditions would facilitate the development of new agents of this class. Previous studies have focused on the enzymes that reductively activate the mitomycins and on the interaction of these drugs with DNA; none of these studies has fully explained the differences in cytotoxicity observed under hypoxic and aerobic conditions. The present investigation demonstrates that the rate of Por uptake is directly correlated with cytotoxicity under both aerobic and hypoxic conditions. Uptake of Por into hypoxic cells is more rapid than into aerobic cells at equal drug concentrations. Hypoxic cells also accumulate drug in concentrations well in excess of those in the extracellular medium; this is apparently a reflection of drug sequestration in these cells. This sequestration of Por, which affects the rate and extent of uptake in hypoxic cells, does not take place in aerobic cells. The failure of aerobic cells to sequester drug is evidenced by the very rapid efflux of Por from these cells upon removal of extracellular Por and by the fact that aerobic cells attain a state of equilibrium between the intracellular and extracellular drug concentrations. The findings demonstrate that differences in the uptake and retention of Por are associated with the preferential toxicity of Por to hypoxic cells.

Modification of the metabolism and cytotoxicity of bioreductive alkylating agents by dicoumarol in aerobic and hypoxic murine tumor cells.

We have demonstrated previously that dicoumarol (DIC) increased the generation of reactive metabolites from mitomycin C (MC) in EMT6 cells under hypoxic conditions in vitro. This increased reaction rate was associated with an increased toxicity of MC to hypoxic EMT6 cells. In contrast, aerobic cells treated with DIC in vitro were protected from MC toxicity. We now demonstrate that DIC sensitizes EMT6 cells to two MC analogues, porfiromycin (POR) and the 7-N-dimethylaminomethylene analogue of mitomycin C (BMY-25282), in hypoxia and protects cells from these agents in air, despite the fact that POR is preferentially toxic to hypoxic cells and BMY-25282 is preferentially toxic to aerobic cells. In contrast, DIC increases menadione cytotoxicity in both air and hypoxia and has no effect on the cytotoxicity of Adriamycin. We have also shown previously that the preferential toxicity of POR to hypoxic cells is associated with an increased rate of drug uptake. In the present study, DIC had no measurable effect on the uptake of [3H]POR but increased the extent of efflux of this agent. MC-induced DNA cross-links, which have been proposed as the lesions responsible for the lethality of MC, are decreased by DIC in air and increased by DIC in hypoxia, in concert with the observed modifications of MC cytotoxicity by DIC. However, in aerobic cells treated with DIC and MC, the decrease in DNA interstrand cross-links is not directly associated with a decrease in cytotoxicity. L1210 cells, which have no measurable quinone reductase activity, demonstrate increased toxicity when treated with DIC and MC in hypoxia, as observed with EMT6 cells. Unlike EMT6 cells, however, L1210 cells are not protected by DIC from MC toxicity in air. Taken together, these findings suggest that DIC is altering the intracellular metabolism of MC and that quinone reductase or another, unidentified, enzyme sensitive to DIC may be involved in activating MC to a toxic product in aerobic EMT6 cells.

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.

Modulation of the cytotoxicity of mitomycin C to EMT6 mouse mammary tumor cells by dicoumarol in vitro.

Aerobic and hypoxic cultures of EMT6 mouse mammary tumor cells were used to study the effects of dicoumarol (DIC) on the cytotoxicity of mitomycin C (MC). DIC protected aerobic cells from MC toxicity, but sensitized hypoxic cells to the cytotoxic actions of this antibiotic. Survival curves for cells treated with 1.5 microM MC +/- 100 microM DIC for different periods of time under aerobic or hypoxic conditions showed that DIC acted as a dose-modifying agent, that is, an agent which changed the slopes, but not the shapes, of the MC survival curves. Experiments that examined the effects of the DIC concentration on the modulation of MC cytotoxicity revealed significant perturbations in MC toxicity with a DIC concentration of 100 microM and increased sensitization/protection with increasing levels of DIC. DIC altered the toxicity of MC only when it was present during exposure of the cells to MC. Treatment with DIC before or after (but not during) MC did not alter the amount of cytotoxicity. Addition of DIC to cell cultures seconds before the addition of MC was as effective as addition of DIC 30 min to 2 h before MC. Taken together, these findings suggest that DIC reversibly inhibits one or more enzymes involved in the activation and inactivation of MC, and that this modulation of the enzymatic processing of MC alters the cytotoxicity of the drug.

Porfiromycin as a bioreductive alkylating agent with selective toxicity to hypoxic EMT6 tumor cells in vivo and in vitro.

Hypoxic cells may limit the curability of solid tumors by conventional chemotherapeutic agents and radiotherapy. Agents which are preferentially toxic to cells with low oxygen contents could therefore be useful as adjuncts to the regimens now used to treat these cancers. To date, the best agent of this type that we have tested is porfiromycin. Porfiromycin is similar to mitomycin C in its toxicity to hypoxic EMT6 cells in vitro but has much less toxicity than mitomycin C to well-oxygenated EMT6 cells. EMT6 cell sonicates reduce mitomycin C and porfiromycin to reactive electrophiles at similar rates under hypoxic conditions, a finding that correlates with cytotoxicity, whereas the rate of production of reactive species from both drugs is very slow under aerobic conditions. We also show that porfiromycin is capable of killing hypoxic radiation-resistant cells in solid EMT6 tumors. Appropriate regimens combining porfiromycin (which preferentially kills hypoxic cells) and radiation (which preferentially kills aerated cells) may therefore be especially efficacious for the treatment of solid tumors.

Differentiation of WEHI-3B D+ monomyelocytic leukemia cells by retinoic acid and aclacinomycin A.

WEHI-3B D+ monomyelocytic leukemia cells were induced to differentiate to mature granulocytes when treated with either 30 nM aclacinomycin A or 7 microM retinoic acid. Differentiation was assessed by the appearance of mature granulocytic phenotypes, as measured by the ability to reduce nitro blue tetrazolium, morphological changes, an increase in cell surface Fc receptors, as well as the loss of proliferative capacity. Maximum differentiation occurred 3 days after drug exposure. Analysis of DNA histograms of treated cells indicated that cells accumulated in the G1 phase of the cell cycle after 8 h of exposure to either inducer, with maximum accumulation occurring by 20 h; this arrest was observed prior to the phenotypic appearance of mature cells. The minimum interval of time necessary to commit cells to a differentiation pathway, which was less than one doubling time (9.2 h), closely paralleled the initial accumulation of cells in the G1 phase of the cell cycle. Since drug exposure for more than one cell division was required for maximum differentiation, the observed kinetics of maturation is consistent with a stochastic model. These studies support the idea that this cell line would be a particularly good model for extrapolation of findings with differentiating agents in culture to therapeutic monitoring in animals, since WEHI-3B D+ leukemia cells can be readily propagated in vivo in BALB/c mice.

Enhancement of anthracycline growth inhibition in parent and multidrug-resistant Chinese hamster ovary cells by cyclosporin A and its analogues.

Cyclosporins have been shown to sensitize multidrug-resistant cells to chemotherapeutic agents but, generally, have minimal effect on sensitive lines. We studied the effect of cyclosporin A (CsA) and two nonimmunosuppressive analogues, 11-methyl-leucine- (11-Me-Leu-CsA) and 6-methyl-alanine-cyclosporin A (6-Me-Ala-CsA), on the action of doxorubicin (DOX) and 4'-epidoxorubicin against parent (AuXB1) and multidrug-resistant (CHRC5) Chinese hamster ovary cells. CsA and its two analogues reduced the IC50 values for DOX in sensitive AuXB1 cells from 0.1 to 0.01-0.02 microM. Cyclosporins reduced the IC50 of DOX in resistant CHRC5 cells from 9 to 0.1 (CsA), 0.7 (6-Me-Ala-CsA), and 1.2 microM (11-Me-Leu-CsA). Similar results were seen when cyclosporins were combined with 4'-epidoxorubicin. The cyclosporins alone had no effect at these concentrations (1-2.0 micrograms/ml). Dose-response curves suggested that CsA was a more potent modifying agent than 11-Me-Leu-CsA towards resistant CHRC5 cells. The ability of the cyclosporins to enhance anthracycline growth inhibition in parent AuXB1 cells may be related to an increase in drug uptake and an increase in anthracycline-induced DNA damage. CsA increased DOX accumulation as well as DOX-associated DNA single-strand breaks in AuXB1 cells over those seen in cells exposed to DOX alone. The degree of increase in DNA breaks paralleled the degree of growth inhibition seen in cells exposed to the same concentrations of drugs. In contrast, CsA had no effect on DOX accumulation or DNA single-strand breaks in CHRC5 cells. These findings imply that, in the resistant cell line, the enhanced anthracycline growth inhibition in the presence of CsA is independent of DOX accumulation and single-strand DNA breaks. These studies demonstrate that CsA and two nonimmunosuppressive analogues can sensitize both sensitive and resistant Chinese hamster ovary cells to anthracyclines. Furthermore, the mechanisms underlying this effect may differ between sensitive and multidrug-resistant cells.

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.

Acute in vivo resistance in high-dose therapy.

In the design of sequential high-dose chemotherapy regimens, the selection of antitumor alkylating agents to be included in each intensification and the interval between the intensifications are critical to the design of the therapy. The tumor cell survival assay and tumor growth delay assay using the murine EMT-6 mammary carcinoma were used as a solid tumor model in which to address these issues. Tumor-bearing mice were treated with high-dose melphalan or cyclophosphamide followed 7 or 12 days later by melphalan, cyclophosphamide, thiotepa, or carboplatin. After treatment with melphalan both 7 and 12 days later, the tumor was resistant to each of the four drugs studied. After treatment with cyclophosphamide both 7 and 12 days later, the tumor was resistant to melphalan and thiotepa but was not resistant to cyclophosphamide or carboplatin. To extend the interval between high-dose treatments to 14 and 21 days, after the first intensification the tumor was transferred to second hosts that were either drug-treated or not drug treated. When high-dose melphalan-treated tumors were treated with a second high dose of melphalan, the tumors were very resistant with the 14-day interval and less resistant with the 21-day interval. This small effect was evident in the bone marrow colony-forming unit, granulocyte-macrophage (CFU-GM), except in the hosts pretreated with melphalan. When high-dose cyclophosphamide-treated tumors were treated with a second high dose of cyclophosphamide, drug resistance was observed both with the 14-day and 21-day interval if the host was non-pretreated or was pretreated with melphalan, but not if the host was pretreated with cyclophosphamide. The same was true in the bone marrow CFU-GM. Tumor growth delay studies supported these findings in that treatment with high-dose cyclophosphamide, melphalan, thiotepa, and carboplatin resulted in less than additive tumor growth delay, whereas treatment with high-dose cyclophosphamide prior to treatment with high-dose melphalan, cyclophosphamide, thiotepa, or carboplatin resulted in additivity to greater-than-additive tumor growth delay. High-dose combination regimens required dose reduction of the drugs, which resulted in decreased tumor growth delays.

Porfiromycin as an adjunct to radiotherapy in young and old mice.

Radiobiological data and measurements with O2 microelectrodes show that EMT6 tumors implanted into aged mice have a higher proportion of radioresistant, hypoxic cells than do tumors implanted into young adult animals; radiation is less effective in killing cells in tumors in old mice than in tumors in young adult mice. The studies reported here examine the effects of porfiromycin (POR), a bioreductive alkylating agent shown previously to be preferentially toxic to hypoxic EMT6 cells in vitro and in solid tumors in young adult mice. POR was effective in attacking the hypoxic cells of tumors in aged mice; regimens combining POR with x-rays overcame the radioresistance of tumors in the old animals. Comparisons of the distribution of 3H-labeled POR in young and old mice showed that tumors in aged mice had a slightly larger proportion of areas with necrotic features, which bound higher levels of tritiated POR than did healthy tumor regions without necrotic features. Studies of histology, lissamine green distributions, binding of tritiated POR, and radiation and POR cytotoxicity suggested that tumors in old mice contained a larger proportion of poorly perfused tumor cells, and that cells in these regions were resistant to radiation and sensitive to POR. Studies of the distribution of POR in normal tissues and of the toxicity of POR to bone marrow progenitor cells (CFU-GM) revealed no differences between young and old animals, showing that the differences observed in tumors reflected differences in the microenvironments within the tumors, rather than differences in the processing of drug in young and old animals.

Induction of the differentiation of HL-60 promyelocytic leukemia cells by palmitoleic and myristoleic acids.

Exposure of HL-60 promyelocytic leukemia cells to palmitoleic or myristoleic acids for 6 days produced both functional and morphological granulocytic maturation. Considerably less or no induction of differentiation occurred with a variety of other fatty acids. Combinations of fatty acids with the granulocytic inducer of maturation, DMSO, did not significantly increase the degree of differentiation of HL-60 cells over that produced by the fatty acids alone. A series of HL-60 cell clones were isolated which differed in sensitivity to the differentiation inducing activities of palmitoleic acid, myristoleic acid, and DMSO. These findings imply that myristoleic acid and palmitoleic acid act to initiate the maturation process by events that are distinct from those produced by DMSO. The capacity of myristoleic and palmitoleic acids to induce leukemic cell differentiation is discussed with respect to protein acylation by fatty acids.

Preclinical studies of porfiromycin as an adjunct to radiotherapy.

The bioreductive alkylating agent porfiromycin (POR) is more toxic to EMT6 cells that are hypoxic at the time of treatment than to aerobic cells. The toxicity of POR to hypoxic EMT6 cells in vitro was similar to that of mitomycin C (MC): the aerobic toxicity of POR was considerably less than that of MC. Treatment of cells in vitro with POR before and during irradiation did not sensitize either hypoxic or aerobic cells to X rays; instead, only additive cytotoxicity was produced. In contrast, treatment of solid EMT6 tumors in vivo with POR plus radiation produced supra-additive cytotoxicity, as assessed by analyses of the complete dose-response curves for the killing of tumor cells by radiation alone or by POR alone. The supra-additivity of the combination regimens appeared to reflect the preferential killing by each agent of those tumor cells which were in an environment conferring resistance to the other agent. In contrast, combinations of POR and X rays produced only additive cytotoxicities to marrow CFU-GM. Supra-additive antineoplastic effects were obtained at doses of POR which produced little hematologic or other host toxicity. The complementary cytotoxicities of radiation and POR to cells in different microenvironments in solid tumors and the absence of a similar effect in normal tissue make optimized regimens combining radiotherapy and POR unusually promising for the treatment of solid tumors.

Modulation of the antineoplastic efficacy of mitomycin C by dicoumarol in vivo.

Dicoumarol (DIC) modulates the intracellular metabolism of mitomycin C (MC) in vitro, increasing the toxicity of MC to hypoxic EMT6 cells and decreasing its toxicity to aerobic cells. The present experiments assessed whether DIC could be used to increase the therapeutic ratio attainable in vivo when MC was used as an adjunct to radiotherapy. Experiments with transplanted EMT6 tumors in mice showed that DIC increased the toxicity of MC to hypoxic tumor cells and increased the antineoplastic efficacy of regimens combining MC with radiation. DIC did not increase the hematologic toxicity of MC, and pretreatment with DIC plus MC did not augment radiation-induced skin reactions. The increase in antineoplastic effect was therefore obtained without a concomitant increase in normal tissue toxicities, and therapeutic gain was obtained.

Chemotherapeutic attack of hypoxic tumor cells by the bioreductive alkylating agent mitomycin C.

Since the cure of solid tumors is limited by the presence of cells with low oxygen contents, we have approached the development of treatment regimens and of new drugs for these tumors by investigating agents which are preferentially bioactivated under hypoxia. Major emphasis has been directed at studying the mode of action of the mitomycin antibiotics, as bioreductive alkylating agents. Using primarily the EMT6 mouse mammary carcinoma as a solid tumor model, we have found that mitomycin C and porfiromycin are preferentially toxic to cells with low oxygen contents. The mitomycin analog BMY-25282 is more toxic to hypoxic cells than are mitomycin C and porfiromycin; however, unlike these antibiotics, BMY-25282 is preferentially toxic to well-oxygenated cells. With these three mitomycins, we have observed a correlation between cytotoxicity to hypoxic cells, the rate of generation of reactive products, and the redox potentials of the drugs. Investigations of the enzymes in EMT6 cells that could possibly activate mitomycin C have revealed that cytochrome P-450 and xanthine oxidase are not present in measurable quantities and therefore are not responsible for activation of mitomycin C. Activities representative of NADPH-cytochrome c reductase and DT-diaphorase are present in these neoplastic cells. Comparison of these enzymatic activities in EMT6, CHO, and V79 cells with the rate of generation of reactive products under hypoxia shows a direct correlation between these two parameters, but there is no quantitative correlation between these two parameters and the amount of cytotoxicity. Use of purified NADPH-cytochrome c reductase and inhibitors of this enzyme demonstrated that NADPH-cytochrome c reductase can activate mitomycin C, but that it is probably not the only enzyme participating in this bioactivation in EMT6 cells. The DT-diaphorase inhibitor dicoumarol was employed to show that this enzyme is not involved in the activation of mitomycin C to a cytotoxic agent. Instead, DT-diaphorase appears to metabolize mitomycin C to a nontoxic product. This property has been exploited to develop a new treatment regimen for solid tumors. Using X-rays to eliminate well oxygenated cells of a solid tumor implant of the EMT6 carcinoma, we have found that the combination of dicoumarol plus mitomycin C is more toxic to hypoxic tumor cells in vivo than mitomycin C alone. Furthermore, knowledge of the biochemical mechanism of mitomycin C activation permits a prediction of which tumors can best be treated with this combination of drugs by measuring enzymatic activities in biopsy specimens.

Preferential kill of hypoxic EMT6 mammary tumor cells by the bioreductive alkylating agent porfiromycin.

Hypoxic cells in solid tumors represent a therapeutically resistant population that limits the curability of many solid tumors by irradiation and by most chemotherapeutic agents. The oxygen deficit, however, creates an environment conducive to reductive processes; this results in a major exploitable difference between normal and neoplastic tissues. The mitomycin antibiotics can be reductively activated by a number of oxidoreductases, in a process required for the production of their therapeutic effects. Preferential activation of these drugs under hypoxia and greater toxicity to oxygen-deficient cells than to their oxygenated counterparts are obtained in most instances. The demonstration that mitomycin C and porfiromycin, used to kill the hypoxic fraction, in combination with irradiation, to eradicate the oxygenated portion of the tumor, produced enhanced cytodestructive effects on solid tumors in animals has led to the clinical evaluation of the mitomycins in combination with radiation therapy in patients with head and neck cancer. The findings from these clinical trials have demonstrated the value of directing a concerted therapeutic attack on the hypoxic fraction of solid tumors as an approach toward enhancing the curability of localized neoplasms by irradiation.

Lisofylline as a modifier of radiation therapy.

Lisofylline is a new methylxanthine. The potential of lisofylline to enhance the response to ionizing radiation of the EMT-6 murine mammary carcinoma in vitro and in vivo was assessed and compared with pentoxifylline. Addition of lisofylline (100 microM) or pentoxifylline (100 microM) from the time of radiation exposure and throughout the time of colony formation did not alter the response of normally oxygenated EMT-6 cells to gamma-radiation delivered at 0.76 Gy/min or 12.3 Gy/min. The dose modifying factor for hypoxic EMT-6 cells by lisofylline or pentoxifylline and radiation delivered at 0.76 Gy/min was 2.3. Higher dose rate radiation (12.3 Gy/min) was more cytotoxic toward hypoxic EMT-6 cells than lower dose rate radiation. The dose modifying factor produced by lisofylline in the high dose rate radiation setting was 1.2. In vivo, using the tumor cell survival assay, lisofylline decreased the shoulder of the radiation survival curve (0.76 Gy/min) by a factor of 1.7 +/- 0.2 while pentoxifylline did not. In the tumor growth delay assay, administration of multiple doses of lisofylline or pentoxifylline along with single dose radiation (0.76 Gy/min) was 1.3, while the radiation dose modifying factor for lisofylline or pentoxifylline administered by continuous infusion was 1.5. Overall, lisofylline was a more effective modifier of gamma-radiation therapy than pentoxifylline, and further investigation of lisofylline in this setting is warranted.

Modulation of the cytotoxicity of porfiromycin by dicoumarol in vitro and in vivo.

The effects of dicoumarol (DIC) on the cytotoxicity of porfiromycin (POR) were studied in vitro using EMT6 mammary tumor cells in monolayer cultures and in vivo using solid EMT6 tumors and bone marrow stem cells. In vitro, POR was more toxic to hypoxic EMT6 cells than to aerobic cells. Exposure of aerobic cultures to DIC protected against POR; in contrast, DIC sensitized hypoxic cells to POR. Treatment of mice with DIC produced a slight increase in the toxicity of POR to cells in solid tumors. The toxicity of POR to marrow stem cells (CFU-GM and CFU-MK) was not altered by DIC. Pretreatment of mice with DIC therefore produced a small improvement in the therapeutic ratio.

Transforming growth factor-beta 1 overexpression produces drug resistance in vivo: reversal by decorin.

The gene for transforming growth factor-beta 1 (TGF-beta 1) was transfected into the murine EMT-6/Parent mammary carcinoma tumor line to form the EMT6/PRK5 beta 1E tumor line. In monolayer culture the EMT-6/PRK5 beta 1E tumor line secretes about 15-times as much TGF-beta 1 into the medium as the EMT-6/Parent line. There was no difference in the response of these two cell lines to 4-hydroperoxycyclophosphamide, cisplatin, melphalan or thiotepa in monolayer culture. When the EMT-6/PRK5 beta 1E cells were grown as a solid tumor in Balb/C mice, plasma levels of TGF-beta 1 were about 5-fold higher than in animals bearing the EMT-6/Parent tumor. The EMT-6/PRK5 beta 1E tumor was markedly resistant to a dosage range of cyclophosphamide, cisplatin, melphalan and thiotepa compared with the EMT-6/Parent tumor. The bone marrow CFU-GM from the animals bearing the EMT-6/PRK5 beta 1E tumor were spared from the cytotoxicity of the drugs compared with the bone marrow CFU-GM from animals bearing the EMT-6/Parent tumor. Administration of decorin, a naturally occurring inhibitor of TGF-beta 1, to animals bearing the EMT-6/PRK5 beta 1E tumor prior to treatment of the animals with the antitumor alkylating agents restored drug sensitivity to the tumor and to the bone marrow CFU-GM. Administration of decorin prior to the antitumor alkylating agents produced very little or no increase in the response of the EMT6/Parent tumor or the bone marrow CFU-GM from those animals. The EMT6/PRK5 beta 1E tumor model allows the effect of secretion of TGF-beta 1 on therapeutic resistance to be assessed directly compared with the EMT-6/Parent tumor. In vivo resistance occurred in the presence of high levels of TGF-beta 1 and was reversed by the TGF-beta 1 inhibitor decorin.