Regulation of scatter factor production via a soluble inducing factor.

Scatter factor (SF) (also known as hepatocyte growth factor [HGF]) is a fibroblast-derived cytokine that stimulates motility, proliferation, and morphogenesis of epithelia. SF may play major roles in development, repair, and carcinogenesis. However, the physiologic signals that regulate its production are not well delineated. We found that various human tumor cell lines that do not produce SF secrete factors that stimulate SF production by fibroblasts, suggesting a paracrine mechanism for regulation of SF production. Conditioned medium from these cell lines contained two distinct scatter factor-inducing factor SF-IF activities: a high molecular weight (> 30 kD), heat sensitive activity and a low molecular weight (< 30 kD) heat stable activity. Further studies revealed that SF-producing fibroblasts also secrete factors that stimulate their own SF production. We characterized the < 30-kD SF-IF activity from ras-3T3 (clone D4), a mouse cell line that overproduces both SF and SF-IF. The < 30-kD filtrate from ras-3T3 conditioned medium induced four- to sixfold increases in expression of SF biologic activity, immunoreactive protein, and mRNA by multiple SF-producing fibroblast lines. Ras-3T3 SF-IF activity was stable to boiling, extremes of pH, and reductive alkylation, but was destroyed by proteases. We purified ras-3T3 SF-IF about 10,000-fold from serum-free conditioned medium by a combination of ultrafiltration, cation exchange chromatography, and reverse phase chromatography. The purified protein exhibited electrophoretic mobility of about 12 kD (reduced) and 14 kD (nonreduced) by SDS-PAGE. The identity of the protein was verified by elution of biologic activity from gel slices. Purified SF-IF stimulated SF production in a physiologic concentration range (about 20-400 pM). Its properties and activities were distinct from those of IL-1 and TNF, two known inducers of SF production. We suggest that SF-IF is a physiologic regulator of SF production.

Effects of mitomycin C alone and in combination with X-rays on EMT6 mouse mammary tumors in vivo.

The effects of mitomycin C alone and in combination with radiation on the cells of EMT6 mouse mammary tumors in BALB/cKaRw mice were examined. At doses near the toxic level, approximately 98% of the tumor cells were killed by a single injection of mitomycin C. Both proliferating and quiescent cells and both hypoxic and aerobic cells were killed by the drug. Cytotoxicity with mitomycin C occurred rapidly and was apparently complete within 30 minutes after injection of the drug . No evidence was found for repair of potentially lethal mitomycin C damage or sublethal mitomycin C damage by the tumor cells. Mitomycin C and radiation in combination produced an additive cytotoxicity; neither agent was found to alter significantly the shape of the dose-response curve for the other agent. The cytotoxicity of mitomycin C and radiation in combination depended on the sequence and timing of the treatments; additive toxicities were obtained when mitomycin C was given just after, just before, or up to 24 hours before irradiation, but the combination was less effective when mitomycin C was given 2-12 hours after irradiation.

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.

Effects of clumps and clusters on survival measurements with clonogenic assays.

The growth of colonies in semisolid medium is receiving widespread attention as a possible test for predicting the clinical response of individual human tumors to specific drugs. One problem encountered in these studies is the difficulty of preparing single-cell suspensions from solid tumors. Often, microscopic examination of the cultures just after plating reveals many clusters or clumps of cells; these are often large enough and numerous enough that some plates are counted or fixed immediately so that this "background" can be subtracted from the "colony" count used to assess cell viability. However, this correction addresses only one of the problems created by the presence of clumps and clusters. It does not eliminate errors and artifacts introduced by multiplicity (multiple clonogenic cells contributing to a colony), abortive clones, and cell volume changes, or by inhomogeneities in the microenvironment, cell metabolism, and drug distribution within clumps. Because of such factors, survival curves determined using suspensions contaminated with clumps and clusters may provide inaccurate assessments of the true drug sensitivity of the individual tumor cells.

Effect of host age on the transplantation, growth, and radiation response of EMT6 tumors.

The characteristics of EMT6 tumors in young adult (3- to 4-month-old) and aged (20- to 28-month-old) BALB/c KaRw mice were compared. The number of tumor cells implanted s.c. necessary to cause tumors in 50% of the injection sites was lower in aging than in young adult mice (69 and 138 cells for young adult mice versus 8.8 and 16 cells for aging mice in two experiments). The latent period of intradermally implanted tumors was shorter in aging mice than in young animals; however, the growth curves of established tumors were similar. The number and appearance of lung colonies after injection of cells i.v. and the pattern of spontaneous metastases were similar in young and aged animals. The cloning efficiencies (viabilities) of cells suspended from tumors in young adult and aging animals were the same (approximately 30% in both groups). Radiation dose-response curves for the cells of tumors in young and aging mice were different and suggested that the proportion of hypoxic cells was higher in tumors on aging animals. These findings suggest that both immunological and nonimmunological tumor-host interactions differ in young and aged animals and that such factors may influence the natural history of the tumor and the response of the tumor to treatment.

Preferential activation of mitomycin C to cytotoxic metabolites by hypoxic tumor cells.

Mitomycin C, a bioreductive alkylating agent with clinical utility against several human tumors, was found to be selectively toxic at a relatively low concentration (1.5 micro M) to EMT6 tumor cells made chronically hypoxic by preincubation in 95% N2-5% CO2 for 4 hr prior to drug exposure. This selective cytotoxicity correlated well with the preferential activation and metabolism of mitomycin C by sonicated cell preparations. The bioactivation of mitomycin C to an alkylating agent by EMT6 and Sarcoma 180 cell sonicates required hypoxic conditions and a reduced nicotinamide adenine dinucleotide phosphate-generating system. Furthermore, the formation of reactive drug metabolites and the disappearance of mitomycin C from the reaction mixture were inhibited by carbon monoxide. The presence of potassium cyanide in the incubation mixture did not affect either the rate of overall metabolism or the rate of formation of reactive metabolites. A high rate of disappearance of mitomycin C from the medium of intact cultures of EMT6 cells was found only in those cultures which were made chronically hypoxic. These data suggest that bioreductive alkylating agents like mitomycin C have the potential to attack selectively the chemotherapeutically resistant hypoxic cell component of solid tumors. Thus, agents capable of bioreductive alkylation should be useful adjuncts to existing therapeutic regimens which are effective against well-oxygenated cells.

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.

Genetic instability induced by the tumor microenvironment.

The tumor microenvironment is characterized by regions of fluctuating hypoxia, low pH, and nutrient deprivation. To determine the genetic consequences of growth under these conditions, we used a tumorigenic cell line carrying a recoverable, chromosomally based lambda phage shuttle vector designed to report mutations without the need for genetic selection of mutant cells. The cells were grown in parallel either in culture or as tumors in nude mice. The frequency of mutations arising in cells within the tumors was found to be 5-fold higher than that in otherwise identical cells grown in culture. A distinct pattern of mutation was also seen, with significantly more deletions and transversions in the tumors than in the cell cultures. Furthermore, exposure of the cultured cells to hypoxia produced an elevated mutation frequency and a mutation pattern similar to that seen in the tumors. These results indicate that the conditions within solid tumors are mutagenic and suggest that a fundamental mechanism of tumor progression in vivo is genetic instability induced by the tumor microenvironment.

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.

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.

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.

Diminished DNA repair and elevated mutagenesis in mammalian cells exposed to hypoxia and low pH.

The tumor microenvironment is characterized by regions of fluctuating and chronic hypoxia, low pH, and nutrient deprivation. It has been proposed that this unique tissue environment itself may constitute a major cause of the genetic instability seen in cancer. To investigate possible mechanisms by which the tumor microenvironment might contribute to genetic instability, we asked whether the conditions found in solid tumors could influence cellular repair of DNA damage. Using an assay for repair based on host cell reactivation of UV-damaged plasmid DNA, cells exposed to hypoxia and low pH were found to have a diminished capacity for DNA repair compared with control cells grown under standard culture conditions. In addition, cells cultured under hypoxia at pH 6.5 immediately after UV irradiation had elevated levels of induced mutagenesis compared with those maintained in standard growth conditions. Taken together, the results suggest that cellular repair functions may be impaired under the conditions of the tumor microenvironment, causing hypermutability to DNA damage. This alteration in repair capacity may constitute an important mechanism underlying the genetic instability of cancer cells in vivo.

Independent regulation of invasion and anchorage-independent growth by different autophosphorylation sites of the macrophage colony-stimulating factor 1 receptor.

Invasion of tissue by macrophages and implantation into the uterine wall by placental trophoblasts are known to be regulated by the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R, the product of the c-fms proto-oncogene). Recently, the clinical importance of CSF-1 and CSF-1R in invasive breast carcinoma has been recognized, but the significance of coexpression of CSF-1 and CSF-1R in mammary epithelial cell invasion has not been explored. In the present study, we investigated the invasive potential of a noninvasive, CSF-1R-negative, mouse mammary epithelial cell line (HC11) expressing a high level of CSF-1, which was stably transfected with the mouse wild-type CSF-1R. Compared with parental cells, transfected cells expressing a wild-type CSF-1R invaded 100-fold more efficiently through a barrier of reconstituted basement membrane (Matrigel) and formed colonies in soft agar, whereas the cellular growth rate was only slightly increased. Analysis of cell-conditioned medium by zymography and quantitative enzyme activity assays showed that clones transfected with a wild-type CSF-1R expressed significantly higher levels of urokinase-type plasminogen activator than did untransfected clones. Furthermore, after injection into the tail veins of BALB/c mice, CSF-1R-expressing clones also produced a 10-fold higher incidence of lung tumors than the parental cell line. We also analyzed HC11 clones transfected with CSF-1R mutated at two major autophosphorylation sites (Tyr-->Phe807 and Tyr-->Phe721). Mutation at Tyr807 eradicated the stimulatory effect of Fms expression on the invasive ability of HC11 cells and substantially reduced the metastatic potential of the transfected clones but did not alter the Fms-induced anchorage-independent growth in soft agar. In contrast, mutation at Tyr721 of Fms had no effect on invasion as measured in the in vitro assay but markedly abolished Fms-induced colony formation in soft agar and eradicated the metastatic potential of the transfected clones. Our results suggest that expression of CSF-1R can facilitate cellular invasion and anchorage-independent growth in mammary epithelial cells, and these two processes are independently regulated by separate phosphotyrosine sites of CSF-1R.

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.

Reversal of mitomycin C resistance by overexpression of bioreductive enzymes in Chinese hamster ovary cells.

The clinical utility of antineoplastic agents is limited by the development of drug resistance by tumors. Mitomycin C (MC) is a bacterial product that must be enzymatically reduced to exert anticancer activity. We have demonstrated that expression of the bacterial MC resistance-associated (MCRA) protein in Chinese hamster ovary (CHO) cells confers profound resistance to this antibiotic under aerobic conditions, but not under hypoxia. MCRA produces resistance to MC by redox cycling of the activated hydroquinone intermediate back to the prodrug form. A CHO cell line developed by stepwise exposure to increasing concentrations of MC likewise expressed high level resistance to MC in air, but not under hypoxia. The overexpression of DT-diaphorase and NADPH:cytochrome c (P-450) reductase, two enzymes known to activate MC, restored sensitivity to MC in both MCRA-transfected and drug-selected cell lines. The level of sensitization was proportional to the quantity of enzyme activity expressed, supporting the concept that the levels of these two activating enzymes are important for sensitivity to MC. The findings of resistance to MC in air but not under hypoxic conditions and of restoration of sensitivity to MC by increasing levels of DT-diaphorase activity, properties not adequately explained by other resistance mechanisms (i.e., decreases in MC activation, repair of DNA lesions, and/or drug efflux), support the hypothesis that a functional mammalian homologue of MCRA may be involved in producing resistance to MC.

Adducts of mitomycin C and DNA in EMT6 mouse mammary tumor cells: effects of hypoxia and dicumarol on adduct patterns.

6-CH3-3H-Mitomycin C (MC) was used to identify MC-DNA adducts formed in EMT6 mouse mammary tumor cells. DNA was isolated from cells treated with 3H-MC. The DNA was enzymatically digested, and the digest was analyzed for 3H-labeled adducts by high performance liquid chromatography. All four major adducts previously isolated and characterized in cell-free systems were detected: two different monoadducts and two bisadducts forming DNA-interstrand and DNA-intrastrand cross-links, respectively. No MC-DNA adducts other than the DNA interstrand cross-link had been shown previously to be formed in living cells. A MC-deoxyguanosine adduct of unknown structure was also detected in DNA from EMT6 cells; this adduct was also formed with purified EMT6 DNA. High performance liquid chromatography analysis was further applied to study the relationship between DNA adducts and cytotoxicity. The number of adducts increased with the concentration of MC in both aerobic and hypoxic cells. At a constant drug level, more adducts were observed in cells treated under hypoxic conditions than in cells treated aerobically; at 2 microM MC, 4.8 x 10(-7) and 3.1 x 10(-7) adducts/nucleotide were observed under hypoxic and aerobic conditions, respectively. The increased adduct frequency under hypoxia correlates with the known increased cytotoxicity of MC to EMT6 cells under hypoxic conditions. In addition, a higher ratio of cross-linked adducts to monoadducts was observed in hypoxic cells. The high performance liquid chromatography techniques were also used to examine the effects of dicumarol (DIC) on adduct patterns in cells treated simultaneously with 3H-MC. The MC-DNA adduct frequencies in DIC-treated cells were increased 1.5-fold under hypoxia and decreased 1.6-fold under aerobic conditions from those observed without DIC. This finding correlates with the known DIC-induced increase and decrease in the cytotoxicity of MC in hypoxic and aerobic EMT6 cells, respectively. The monoadduct resulting from monofunctionally activated MC was suppressed by DIC under both hypoxic and aerobic conditions. In addition, DIC induced the selective formation of an unknown DNA-associated radiolabeled substance in hypoxic cells; this is hypothesized to be a cytotoxic DNA lesion produced by a DIC-stimulated oxido-reductase. The methodology developed to measure MC adduct patterns may be useful as an indicator of distinct enzymatic activation processes for this drug.

Scatter factor protects epithelial and carcinoma cells against apoptosis induced by DNA-damaging agents.

Scatter factor (SF) (hepatocyte growth factor) is a cytokine that may play a role in human breast cancer invasiveness and angiogenesis. We now report that SF can block the induction of apoptosis by various DNA damaging-agents, including cytotoxic agents used in breast cancer therapy. SF protected MDA-MB-453 human breast cancer cells, EMT6 mouse mammary tumor cells and MDCK renal epithelial cells against apoptosis induced by adriamycin (ADR), X-rays, ultraviolet radiation, and other agents. Protection was observed in assays of DNA fragmentation, cell viability (MTT), and clonogenic survival. Protection of MDA-MB-453 cells against ADR was dose- and time-dependent; maximal protection required pre-incubation with 75-100 ng/ml of SF for 48 h or more. Protection required functional SF receptor (c-Met), but was not dependent on p53. Western blotting analysis revealed that pre-treatment of MDA-MB-453 cells with SF inhibited the ADR-induced decreases in the levels of Bcl-XL, an anti-apoptotic protein related to Bcl-2; and the dose-response and time course characteristics for SF-mediated increases in the Bcl-XL protein levels of ADR-treated cells were consistent with the degrees of protection against apoptosis observed under the same conditions. Furthermore, Bcl-XL levels were not down-regulated by ADR in MDA-MB-231 breast cancer cells, consistent with the finding that SF failed to protect these cells against ADR, despite the fact that they contain functional c-Met receptor. In contrast to Bcl-XL, SF blocked ADR-induced increases in c-Myc and inhibited the expression of p21WAF1/CIP1 and of the BRCA1 protein in MDA-MB-453 cells. However, SF did not cause significant changes in the cell cycle distribution of ADR-treated cells. These findings suggest that SF-mediated protection of human breast cancer cells may involve inhibition of one or more pathways required for the activation of apoptosis and may particularly target the anti-apoptotic mitochondrial membrane pore-forming protein Bcl-XL as a component of the protective mechanism. By implication, the accumulation of SF within human breast cancers may contribute to the development of a radio- or chemoresistant phenotype.

Chemotherapy as an adjunct to radiation in the treatment of squamous cell carcinoma of the head and neck: results of the Yale Mitomycin Randomized Trials.

PURPOSE: Two consecutive randomized trials were run at our institution using the bioreductive alkylating agent mitomycin as an adjunct to radiation therapy in an effort to improve outcome in patients with squamous cell carcinoma of the head and neck. METHODS: Between 1980 and 1992, two consecutive randomized trials using mitomycin (trial 1) and mitomycin with dicumarol (trial 2) as an adjunct to radiation therapy in patients with squamous cell carcinoma of the head and neck were conducted at our institution. The patients were stratified by intent of therapy, extent of disease, and primary tumor site. Within each strata, patients were randomized to receive radiation therapy with or without mitomycin (trial 1) or mitomycin/dicumarol (trial 2). RESULTS: A total of 203 patients were enrolled onto both trials, 195 of whom were eligible for analysis. Patients were equally balanced with respect to sex, age, extent of disease, primary site, radiation dose, and total duration of radiation treatment. Hematologic toxicities were more frequently noted in the drug-treated arms, but were acceptable with no drug-related treatment deaths. Nonhematologic toxicities were acceptable and not significantly different between the two arms. As of September 1995, with a median follow-up of 138 months, a statistically significant benefit occurred in the mitomycin arms with respect to cause-specific survival (0.74 +/- 0.05 v 0.51 +/- 0.05; P = .005), local recurrence-free survival (0.85 +/- 0.04 v 0.66 +/- 0.05; P = .002), and local regional recurrence-free survival (0.76 +/- 0.05 v 0.54 +/- 0.05; P = .003). No statistically significant difference in overall survival was obtained (0.48 +/- 0.05 mitomycin arms v 0.42 +/- 0.05 radiation alone). CONCLUSION: The bioreductive alkylating agent mitomycin is a safe and effective adjunct to radiation therapy in the treatment of squamous cell carcinoma of the head and neck. The statistically and clinically significant improvement in local regional relapse and cause-specific survival obtained support the use of mitomycin as an adjunct to radiation therapy in the management of squamous cell carcinoma of the head and neck.

Effects of mitomycin C and porfiromycin on exponentially growing and plateau phase cultures.

Laboratory studies and clinical trials are exploring the use of hypoxia-directed cytotoxic agents as adjuncts to radiotherapy. Because hypoxia and the microenvironmental inadequacies associated with hypoxia in solid tumours inhibit cell proliferation, an essential requirement for the successful use of hypoxia-directed drugs in cancer therapy is that these drugs be toxic to quiescent tumour cells, as well as tumour cells progressing rapidly through the cell cycle. The experiments reported here compared the cytotoxicities of mitomycin C and porfiromycin to exponentially growing and plateau phase cultures of EMT6 mouse mammary tumour cells. The proliferative status of the cultures did not influence the cytotoxicity of mitomycin C under either aerobic or hypoxic conditions, or the cytotoxicity of porfiromycin in air. Exponentially growing cultures were slightly more sensitive than plateau phase cultures to porfiromycin in hypoxia, but the difference between the sensitivities of proliferating and quiescent cells was much smaller than the difference between aerobic and hypoxic cells. No evidence for repair of potentially lethal damage was found after treatment with porfiromycin in air or in hypoxia; this is in agreement with previous findings for mitomycin C. Mitomycin C and porfiromycin therefore exhibit the toxicity to quiescent cells needed for effective use as hypoxia-directed drugs for the treatment of solid tumours.