Oxidative damage in murine tumor cells treated in vitro by recombinant human tumor necrosis factor

Treatment of three murine tumor cell lines, L929, P388, and Pan-02, in vitro with recombinant human tumor necrosis factor (rhTNF) produced evidence of oxidative damage as measured by (a) increases in intracellular glutathione levels, (b) the formation of intracellular oxidized glutathione and (c) the formation of thymine glycols in DNA. L929, the most sensitive of the three cell lines to the cytotoxic activity of rhTNF, had the lowest total glutathione content and was observed to have the highest levels of oxidized glutathione and thymine glycol formation. In addition, the radical buffering capacity of these cells was significantly compromised within 7 h of treatment with rhTNF. The P388 and Pan-02 cell lines, with total glutathione levels about 50-fold higher than L929, also showed evidence of oxidative attack, although to a lesser extent than L929. The radical buffering capacity of these cell lines was not altered by rhTNF treatment. A rhTNF-resistant subline of L929 (L929r), produced by successive passaging in vitro in the presence of TNF, increased its glutathione and oxidized glutathione levels in response to a subsequent rhTNF challenge. Meth A, a cell line resistant to rhTNF in vitro but not in vivo, showed no evidence of oxidative damage following rhTNF treatment, despite having a low radical scavenging capacity and a sensitivity to H2O2. The results with Meth A suggest that the interaction of rhTNF with this cell line does not occur in the same manner as the other cell lines, perhaps due to receptor differences or to some type of "uncoupling" of the signal-response network between the TNF receptor and a putative secondary messenger(s). These results are consistent with the hypothesis that: (a) the mechanism of action of rhTNF involves the production of oxidative damage, including damage to the DNA; (b) the sensitivity to rhTNF in vitro is related to the radical scavenging capacity of the cell; and (c) cells can respond to rhTNF challenge by increasing their free radical scavenging capacity.     

Intracellular hydroxyl radical production induced by recombinant human tumor necrosis factor and its implication in the killing of tumor cells in vitro

This study investigated the effect of recombinant human tumor necrosis factor (rhTNF) on hydroxyl radical production by established cell lines in vitro, and its implication in the killing of tumor cells by rhTNF. During incubation of TNF sensitive mouse tumorigenic fibroblast L-M cells (2 X 10(7) cells) in the presence of rhTNF (100 U), hydroxyl radical production as detected by the evolution of methane gas from dimethyl sulfoxide increased gradually, at 18 h reaching 1.8 times that in the absence of rhTNF. This increase was dependent on the concentration of rhTNF and was effectively prevented by the simultaneous addition of anti-rhTNF monoclonal antibody III 2F3, which inhibited both the binding of rhTNF to its receptor and the cytotoxic activity of rhTNF. The addition of iron chelator 2,2'-bipyridine, which inhibits iron-catalized Fenton reaction and so inhibits hydroxyl radical generation, suppressed both the increase of hydroxyl radical production and the cytotoxicity induced by rhTNF. A similar increase in hydroxyl radical production in the presence of rhTNF was also detected with TNF-sensitive human myosarcoma-derived KYM cells, but no such increase was detected with TNF insensitive human embryonic lung fibroblast HEL cells. The results show that rhTNF induces increased hydroxyl radical production in TNF-sensitive cells, and suggest that this plays an important role in the mechanism of tumor cell killing by rhTNF.     

Synergistic antitumor effects of topoisomerase inhibitors and natural cell-mediated cytotoxicity

The mechanism of augmentation of tumor cell killing by immune effector cells and chemotherapeutic drugs was studied. The effect of treating tumor cells with various antineoplastic drugs on their sensitivity to murine natural cell-mediated cytotoxicity in vitro was investigated. Pretreatment with actinomycin D at nontoxic concentrations rendered L929 and WEHI-164 tumor cells more susceptible to killing by mouse spleen lymphocytes in a dose-dependent manner. Similarly, enhancement of L929 tumor cell killing by natural cell-mediated cytotoxicity was observed following treatment of the target cells with the topoisomerase II inhibitors, Adriamycin, amsacrine, bisantrene, etoposide, and teniposide, as well as with topoisomerase I inhibitor, camptothecin. In contrast, drugs which induce their cytotoxic effects by mechanisms that do not involve topoisomerase inhibition such as bleomycin, vinblastine, vincristine, and mitomycin C failed to exhibit synergism with natural cell-mediated cytotoxicity. However, moderate synergy was consistently observed with cis-platinum. The effector cells responsible for the cytotoxicity in the present system are natural cytotoxic cells since they kill WEHI-164 but not YAC cells, are resistant to treatment with anti-asialo-GM1 antibody, and their activity is abolished by anti-tumor necrosis factor antibodies. Indeed, tumor necrosis factor-mediated cytotoxicity of WEHI-164 or L929 was enhanced by treatment of the target cells with topoisomerase II inhibitors. Moreover, WEHI-164 cells selected for tumor necrosis factor resistance were resistant to natural cell-mediated cytotoxicity, and no synergy could be observed with topoisomerase inhibitors.     

Pleiotropic effects of fenretinide in neuroblastoma cell lines and multicellular tumor spheroids

The efficacy and mechanism of action of fenretinide (4-HPR), a vitamin A analogue, was investigated in a panel of six neuroblastoma cell lines and multicellular tumor spheroids. The latter are three dimensional cell aggregates and as such, a model for micrometastases. In all cell lines, the production of reactive oxygen species (ROS) increased with 163-680% after 1 h of treatment with 4-HPR. In addition, a decrease of the mitochondrial membrane potential of 30-75% was observed after 4 h of incubation with 4-HPR. A 6-12-fold difference was observed between the IC50 values for cell proliferation and viability between the most sensitive (IMR32) and most resistant (NASS) cell line towards 4-HPR. Flow cytometric analysis showed an increased amount of apoptotic bodies and no cell-cycle arrest. The antioxidant Trolox completely inhibited the accumulation of 4HPR-induced ROS and prevented the 4HPR-associated cytotoxicity. In all neuroblastoma spheroids, 4-HPR induced a complete cytostasis at clinical relevant concentrations (3-10 microM). Immunohistochemical analysis of 4-HPR-treated spheroids showed a decreased staining for proliferation marker Ki-67 and an increased staining for cleaved-PARP, a marker of apoptosis. Our results suggest that 4-HPR might be a promising agent for the treatment of micrometastases and high-risk neuroblastoma.     

Association of lysosomal activity with sensitivity and resistance to tumor necrosis factor in murine L929 cells

The cytotoxic mechanism of action of tumor necrosis factor (TNF) was examined using murine L929 fibrosarcoma cells in vitro. Two cell lines were evaluated: parental TNF sensitive (L929S) (50% cytotoxic concentration, 2-6 ng/ml); and TNF resistant (L929R) (50% cytotoxic concentration, greater than 10,000 ng/ml). The latter resistant cell line was developed by serial passage in increasing concentrations of recombinant human TNF. Sensitive cells demonstrated cytolytic and cytostatic effects at TNF concentrations between 2 and 6 ng/ml, respectively. However, TNF failed to show any selective depression of RNA, DNA, or protein synthesis or ATP content in these cells until general cell death was apparent, as defined by the cell rounding and lifting off the plastic surface. The cytokine also failed to cause DNA single-strand breaks, as detected by alkaline elution techniques. TNF was also found to be no more active in glutathione-depleted cells than in target cells containing normal glutathione levels. In contrast, various nonspecific lysosomotropic agents such as ammonium chloride and D-saccharic acid lactone led to a marked inhibition of the cytotoxic action of TNF in vitro. Furthermore, significant differences in lysosomal enzyme activity were noted between L929S and L929R cells. The changes in L929R cells involved a 50% reduction in total lysosomal protein levels and a marked depression of beta-glucuronidase activity. In contrast, L929R lysosomal hexosaminidase activity was significantly elevated over the L929S cells. From these studies it is concluded that the antitumor activity of TNF does not involve specific inhibition of macromolecular synthesis, ATP production, or the level of reduced thiols. Instead, TNF cytotoxicity appears to require functional lysosomes, which are altered when TNF resistance develops in vitro.     

Anti-tumour cytotoxin produced by human monocytes: studies on its mode of action.

Human monocytes can be induced to synthesize a cytotoxin which affects certain tumour cell lines. The interaction of monocyte cytotoxin with a susceptible cell line (L929) has been studied to obtain clues to the mode of action of the cytotoxin. The cytotoxin acts directly on the cells rather than on the culture medium and is cytotoxic at higher concentrations and cytostatic at lower concentrations. First signs of cell damage appear about 20 h after contact with the cytotoxin which must be present throughout this period. The cytotoxin probably acts on the cell surface and is more effective at 40 degrees C than at 37 degrees C. For a given amount of cytotoxin the effects are inversely proportional to the target cell concentration. Treatment of the cytotoxin with phenanthroline inhibits cytotoxicity while treatment of the target cells with actinomycin D, but not cycloheximide or puromycin, enhances cytotoxicity. After 24 h cytotoxin treatment the target cells exhibit reduced respiration rate but enhanced glycolysis and glucose uptake suggesting mitochondrial dysfunction. A possible interpretation of these data is that the monocyte cytotoxin is a metalloenzyme which inactivates a cell surface receptor for a nutrient essential for mitochondrial function.     

(E)-5-(2-bromovinyl)-2'-deoxyuridine potentiates ganciclovir-mediated cytotoxicity on herpes simplex virus-thymidine kinase--expressing cells

Tumor cells expressing the thymidine kinase gene of the herpes simplex virus (HSV-tk) are rendered highly susceptible to the cytotoxic effects of different antiherpes drugs. In an attempt to enhance cytotoxicity of this therapeutic approach in glioma and other tumor cell lines transduced with the HSV-tk gene, we evaluated tumor cell killing following co-administration of two different prodrugs metabolized by HSV-tk, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), and ganciclovir (GCV). In 8 of 12 cell lines investigated, addition of BVDU in concentrations showing no cytotoxic effect or only limited cytotoxicity could enhance GCV-mediated cell killing by as much as one order of magnitude. In co-cultures consisting of HSV-tk(+) (9L STK) and HSV-tk(-) (9L wild-type) cells, we also observed potentiation of GCV-mediated cytotoxicity in the presence of BVDU, suggesting strongly enhanced bystander cell killing. BVDU is thought to exert its cytotoxic effect through inhibition of thymidylate synthase activity or by incorporation into replicating DNA. Both effects could be observed in all HSV-tk--expressing cells investigated, including cell lines which did not exhibit cytotoxicity after incubation with BVDU. These findings argue against current concepts of BVDU-mediated cytotoxicity in HSV-tk--expressing cells. Taken together, our data suggest that gene therapy utilizing prodrug activating enzymes may be rendered more effective by simultaneous treatment with two different prodrugs metabolized by the same enzyme.     

In vitro immunization against human tumor cells with tumor cell fractions.

Tumor cell fractions isolated from tumor lines SH-3 (breast carcinoma) and RPMI-7932 (malignant melanoma) by differential centrifugations were capable of transforming lymphocytes into cytotoxic effector cells. Lymphocytes cultured alone in human AB plasma did not become cytotoxic to tumor cells. However, when cultured with tumor cell fractions sedimented at 1000 X g(R1), 20,000 X g(R2), and 100,000 X g(R3), these lymphocytes became markedly cytotoxic to specific tumor targets in a 3.5-hr (51)Cr release assay. R2 fractions were significantly more immunogenic than were R3 fractions (p less than 0.05). Although lymphocytes sensitized with SH-3 tumor cell fractions were cytotoxic to SH-3 tumor cells, they were also cytotoxic to cells from RPMI-7932 and RPMI-8322 (malignant melanoma) tumor lines and vice versa. Cells from tumor lines HT-29 (colon carcinoma) and COLO 110 (ovary carcinoma) were significantly less susceptible to lysis by effector cells generated against SH-3. These immune cells, although capable of killing cells from tumor lines, were not able to lyse cells from autochthonous normal lymphoid lines or normal lymphocytes that have been transformed by phytohemagglutinin. Tumor cell fractions were not immunogenic at low (5- to 20-mul/0.75 ml) concentrations; an increase of 4- to 10- fold in their concentrations was usually followed by a decrease in immunization.     

Cytotoxicity to tumor cells of monocytes from normal individuals and cancer patients

This study was aimed at characterizing the parameters which regulate human monocyte-mediated cytotoxicity to tumor cells as well as characterizing the target cell specificity, kinetics, etc., of the cytotoxic mechanism. Normal human peripheral blood monocytes were cytotoxic to tumor cells in an in vitro assay, measuring release of [³H]thymidine from human target cells. Monocyte cytolysis was observed with several adherent tumor lines including T24, a bladder cancer line; LR, a melanoma line; and an SV40-transformed W138 fibroblast line, with maximal cytolysis observed at 72 h. Lymphokines were not required to induce and only infrequently enhanced monocyte cytotoxicity. Prolonged exposure of monocytes to lymphokines or in vitro culturing of monocytes prior to lymphokine exposure did not alter the monocytes' response to lymphokine signals with respect to cytotoxicity. Lymphokines induced monocytes to exhibit enhanced spreading, suggesting that monocytes were susceptible to lymphokine signals but that the development of cytolytic function was independent of lymphokines. In contrast to the cytolysis of adherent tumor cells, monocytes were less effective in killing the non-adherent lymphoid target cells K562, Raji, and CEM. Monocytes were selectively cytotoxic to tumor cells and generally did not kill normal human fibroblast cell lines or PHA-stimulated lymphocytes. Monocytes from cancer patients exhibited normal cytotoxicity to several human tumor lines. Plasmas from some cancer patients were inhibitory to cytotoxicity mediated by both autologous monocytes and normal monocytes.     

Potentiation of temozolomide and topotecan growth inhibition and cytotoxicity by novel poly(adenosine diphosphoribose) polymerase inhibitors in a panel of human tumor cell lines.

Potent poly(ADP-ribose) polymerase (PARP) inhibitors have been developed that potentiate the cytotoxicity of ionizing radiation and anticancer drugs. The biological effects of two novel PARP inhibitors, NU1025 (8-hydroxy-2-methylquinazolin-4-[3H]one, Ki = 48 nM) and NU1085 [2-(4-hydroxyphenyl)benzamidazole-4-carboxamide, Ki = 6 nM], in combination with temozolomide (TM) or topotecan (TP) have been studied in 12 human tumor cell lines (lung, colon, ovary, and breast cancer). Cells were treated with increasing concentrations of TM or TP +/- NU1025 (50, 200 microM) or NU1085 (10 microM) for 72 h. The potentiation of growth inhibition by NU1025 and NU1085 varied between the cell lines from 1.5- to 4-fold for TM and 1- to 5-fold for TP and was unaffected by p53 status. Clonogenic assays undertaken in two of the cell lines confirmed that the potentiation of growth inhibition reflected the potentiation of cytotoxicity. NU1025 (50 microM) was about as effective as 10 microM NU1085 at potentiating growth inhibition and cytotoxicity, consistent with the relative potencies of the two molecules as PARP inhibitors. Potentiation of cytotoxicity was obtained at concentrations of NU1025 and NU1085 that were not toxic per se; however, NU1085 alone was 3-fold more cytotoxic (LC50 values ranged from 83 to 94 microM) than NU1025 alone (LC50 > 900 microM). These data demonstrate that PARP inhibitors are effective resistance-modifying agents in human tumor cell lines and have provided a comprehensive assessment protocol for the selection of optimum combinations of anticancer drugs, PARP inhibitors, and cell lines for in vivo studies.     

Mechanism of the cytotoxic effect of tumor necrosis factor

The mechanism of murine tumor necrosis factor (TNF) cytotoxicity against tumor cell lines (L929, HeLa, K562) was investigated. Electron microscopic observation revealed that most of the organellas of L929 cells incubated with partially purified murine TNF underwent almost complete lysis with no drastic disruption of the cytoplasmic membrane, while injection of the TNF into the cytoplasm or nuclei of L929 cells caused no apparent morphological change or growth inhibition. Preincubation of the TNF with tumor cells (L929, HeLa, K562) resulted in a decrease in cytotoxic activity which was proportional to their susceptibility to TNF, thus indicating their absorption of TNF. The susceptibility of L929 tumor cells to TNF was apparently suppressed by treatment with proteases, suggesting the existence of protease-sensitive recognition sites for TNF on the tumor cell.     

Phosphatase inhibitors modulate the growth-regulatory effects of human tumor necrosis factor on tumor and normal cells.

Tumor necrosis factor alpha (TNF-alpha) has been shown to inhibit the growth of tumor cells and stimulate the growth of certain normal cells in vitro. The mechanism by which TNF exerts its cell growth-regulatory effects is not known. In this report, we investigated the effects of phosphatase inhibitors on the cell growth-inhibitory effects of TNF on L-929, a highly sensitive murine connective tissue tumor cell line, and on the growth-stimulatory effects of TNF on normal human fibroblasts. The antiproliferative effects of TNF on L-929 cells were inhibited by orthovanadate, an inhibitor of phosphotyrosine phosphatases, in a dose-dependent manner. Okadaic acid, which is a specific inhibitor of phosphoserine- and phosphothreonine-specific phosphatases, also blocked the growth-inhibitory effects of TNF, suggesting that TNF may function through the activation of certain phosphatases. These inhibitors had no effect on TNF receptors. Addition of phosphatase inhibitor, even 12 h after the treatment of cells with TNF, was sufficient to block the antiproliferative effects of the cytokine, suggesting that the inhibitor is acting at a late event in the pathway of action of TNF. Cells were protected by orthovanadate from the cytotoxic effects of TNF even in the presence of actinomycin D or cycloheximide, thus indicating the lack of a requirement for de novo protein synthesis. Orthovanadate altered the cell morphology from flat spindle shapes to rounded ones. Besides anticellular effects, a phosphatase inhibitor also suppressed the proliferative effects of TNF on human fibroblasts. These results thus suggest that phosphatases may be needed for both proliferative and antiproliferative effects of this cytokine. This is the first report to suggest that phosphatases play a role in the growth-regulatory action of TNF.     

Implication of hydroxyl radical production in the killing of tumor cells by recombinant human tumor necrosis factor

We investigated the effect of recombinant human tumor necrosis factor (rhTNF) on hydroxyl radical production by established cell lines in vitro, and its implication in the killing of tumor cells by rhTNF. During incubation of TNF sensitive mouse tumorigenic fibroblast L-M cells in the presence of rhTNF, hydroxyl radical production detected by the evolution of methane gas from dimethyl sulfoxide increased gradually, at 18 hours reached 1.8 times of that in the absence of rhTNF. This increase of hydroxyl radical production was dependent on the concentration of rhTNF. The addition of iron chelator 2,2'-bipyridine which inhibits iron catalized Fenton reaction and inhibits hydroxyl radical generation, suppressed both the increase of hydroxyl radical production and the cytotoxicity induced by rhTNF. The increase of hydroxyl radical production stimulated by rhTNF was also detected in TNF sensitive human myosarcoma derived KYM cells as well as in L-M cells, but no change was detected in TNF insensitive human embryonic lung fibroblast HEL cells. These results suggest that rhTNF induced increase of hydroxyl radical production may play a significant role in the mechanism of tumor cell killing by rhTNF.     

Natural cell-mediated cytotoxicity in mice against non-lymphoid tumor cells and some normal cells

Lymphocytes from normal mice were found to have cell-mediated cytotoxicity, in a short-term ⁵¹Cr release assay, against a variety of non-lymphoid tumor cells as well as against lymphomas. Some of the non-lymphoid tumors were as susceptible to natural cytotoxicity as the standardly used lymphoid lines. Some tissue culture cell lines and in vivo passaged tumor lines were susceptible to lysis, as were some primary virus-induced lymphomas. Tumors which arose in nude mice, which have high levels of natural cytotoxic activity, were all resistant to lysis. In addition to the susceptibility of transformed cells to natural cell-mediated cytotoxicity, some untransformed cultured cells and cells from normal tissues were targets for this mechanism. Low levels of cytotoxicity were seen with normal thymus cells, bone-marrow cells, and short term cultures of macrophages, whereas normal spleen and lymph-node cells were completely resistant to lysis. These results indicate a broader spectrum for mouse natural cell-mediated cytotoxicity reactivity than has been previously recognized.     

Membrane-associated interleukin 1 alpha as a mediator of tumor cell killing by human blood monocytes fixed with paraformaldehyde

Human blood monocytes isolated by centrifugal elutriation from healthy donors were tested for ability to produce membrane-associated antitumor monokine(s) in response to activation stimuli such as various types of interferon (IFN) and/or synthetic desmethyl muramyl dipeptide (norMDP). IFNs (alpha, beta, and gamma) and norMDP rendered blood monocytes cytotoxic to allogeneic A375 melanoma cells, as assayed by measuring release of [125I]iododeoxyuridine in 72 h. When monocytes were treated with any type of IFN for 16 h, and then fixed with paraformaldehyde, they did not show cytotoxicity to A375 cells, but when they were fixed after treatment with norMDP or lipopolysaccharide they showed significant cytotoxicity to A375 melanoma cells. This membrane-associated antitumor monokine induced by the synergistic actions of suboptimal concentrations of IFN-gamma and norMDP, was cytotoxic to HT-29 colon cancer cells as well as A375 melanoma cells, but not to actinomycin D-treated L-929 cells. The fixed monocyte-mediated cytotoxicity against A375 melanoma cells was completely inhibited by a specific anti-interleukin 1 alpha antiserum, but not by a specific anti-interleukin 1 beta antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated interleukin 1 alpha is involved through cell-to-cell contact in the host defense mechanism against cancer.     

Novel muteins of human tumor necrosis factor with potent antitumor activity and less lethal toxicity in mice

Eight muteins of recombinant human tumor necrosis factor-α (rhTNF; SSSRTP … ²⁹RR … ¹⁵⁵L), in which ²⁹Arg was replaced by another amino acid, were prepared and their anti-tumor effects in BALB/c mice bearing Meth A fibrosarcoma were evaluated. The therapeutic indices, which mark the extent of the therapeutically effective dose, of V29 (²⁹Arg ± Val) and D29 (± Asp) were 3.5 and 3.2, respectively, whereas that of rhTNF was 1.4. Clearly, the therapeutically effective range of these muteins was extended along with a decrease in lethal toxicity. V29 did not produce hypotension in the rat system, but D29 did. In addition, V29 showed potent anti-tumor activity (Tumor Volume Inhibition Rate = 81% on day 15 after implantation) in 3 consecutive injection schedules despite the decreases in toxicity compared with rhTNF. The relative receptor binding constant was determined using HEp-2 cells (expressing mainly 55-kDa-TNF receptor; p55R) and HL60 cells (expressing mainly 75-kDa-TNF receptor; p75R), and revealed that the reduced toxicity of V29 in mice was due to the reduced binding to p55R (34% of rhTNF). On the other hand, the ratio of the constants HEp-2/HL60 of V29 was 11 in comparison with the value of 1.0 for rhTNF, suggesting that this mutein binds preferentially to p55R. The biological activities in human cell lines (HEp-2 and HL60 cells) correlated well with the binding activities to each receptor in vitro. Therefore, the much lower toxicity and the potent anti-tumor activity of this mutein suggest that V29 merits further investigation in pre-clinical and clinical trials.     

Potentiation of vincristine and Adriamycin effects in human hemopoietic tumor cell lines by calcium antagonists and calmodulin inhibitors

verapamil, a calcium-influx blocker, enhanced the cytotoxicity of vincristine (VCR) in vitro 6- to 12-fold in eight human hemopoietic tumor cell lines established from acute lymphatic leukemia, acute myelogenous leukemia, and Burkitt's lymphoma. Great enhancement of VCR cytotoxicity was obtained in a VCR-resistant subline of K562 myelogenous leukemia. A maximum of approximate 100-fold increase in VCR cytotoxicity occurred. Heterogeneity in VCR sensitivity (80-fold difference in sensitivity) was observed in vitro among these human tumor cells. BALL and Daudi cells of B-cell type were more susceptible to VCR. At 6.6 or 20 microM of verapamil, the values for the concentration of drug required for 50% inhibition of cell growth for each cell line fell into a rather narrow range, and heterogeneity in VCR sensitivity among cell lines was circumvented in vitro. Verapamil also enhanced the cytotoxicity of Adriamycin, although the extent of enhancement was considerably small. Enhancement of VCR cytotoxicity also occurred with other calcium antagonists and calmodulin inhibitors. At maximum effective concentration of these reagents, a 3- to 5-fold increase in VCR cytotoxicity occurred in K562 cells. In VCR-resistant K562 cells, a more prominent enhancement (20- to 45-fold) was observed with these reagents. VCR resistance was circumvented in vitro. The mechanism of enhancement of VCR cytotoxicity was explained by the enhanced accumulation of VCR in K562, especially in resistant cells.     

Differential oxygen radical susceptibility of adriamycin-sensitive and -resistant MCF-7 human breast tumor cells

Recent evidence supports the concept that Adriamycin cytotoxicity may be mediated by drug semiquinone free radical and oxyradical generation. We tested this hypothesis further by exposing drug-sensitive (WT) and 500-fold Adriamycin-resistant MCF-7 human breast tumor cells (ADRR) to exogenous superoxide- and hydrogen peroxide-generating systems and subsequently monitored cell proliferation as a measure of cytotoxicity. The ADRR tumor cells tolerated a 4-fold greater exposure than sensitive cells to superoxide generated by the xanthine/xanthine oxidase system. Likewise, exposure to hydrogen peroxide produced by the action of glucose oxidase on glucose revealed a 4-fold diminished susceptibility of the drug-resistant cells to this reduced form of oxygen. Similar results were obtained by the direct application of hydrogen peroxide to cells. For both cell lines, cytotoxicity was dependent upon the magnitude and the duration of reactive oxygen exposure. When WT and ADRR cells were cultured under hyperoxia (95% O2:5% CO2), in order to stimulate the intracellular production of oxyradicals, proliferation was inhibited to a greater extent in the drug-sensitive cell line. Additionally, hyperoxia potentiated the cytotoxicity of Adriamycin to both sensitive and drug-resistant cells, but the effect depended upon the concentration of the drug. Under hyperoxic conditions, Adriamycin caused oxygen radical-dependent cytotoxicity to the WT tumor cells at clinically relevant drug concentrations as low as 2 to 3 nM. With ADRR tumor cells, hyperoxia increased the cytotoxicity of Adriamycin at concentrations above 5 microM. Paradoxically, both the WT and the ADRR tumor cells were equally susceptible to the cytotoxic effects of gamma irradiation. It is known that the Adriamycin-resistant MCF-7 cells greatly overexpress glutathione peroxidase and glutathione transferase activities; however, other biochemical defenses against reactive drug intermediates and oxygen radicals have been reported to be similar in the two cell lines. We have reexamined those observations in this report. The resistance of ADRR breast tumor cells to Adriamycin appears to be associated with a developed tolerance to superoxide, most likely because of a twofold increase in superoxide dismutase activity, and a decreased susceptibility to hydrogen peroxide, most likely because of 12-fold augmented selenium-dependent glutathione peroxidase activity. Acting in concert, these two enzymes would decrease the formation of hydroxyl radical from reduced molecular oxygen intermediates.(ABSTRACT TRUNCATED AT 400 WORDS)     

An analysis of variables affecting the measurement of tumor immunity in vitro with 125I-iododeoxyuridine-labelled target cells. Studies of immunity to primary Moloney sarcomas

Variables affecting the measurement of immunity to adherent tumor cells in vitro have been analyzed using the immune response to primary Moloney sarcomas in BALB/c mice as a model system. IUdR, a gamma-emitting isotope which is incorporated into the DNA of dividing tumor cells, was used to quantitate the number of adherent tumor cells in 0.2 ml cultures. Lymphoid cell-mediated killing of tumor cells was measured by labelling tumor cells before they were cultured with effector cells (cytotoxicity test), and lymphoid cell-mediated inhibition of tumor-cell growth was measured by labelling those tumor cells surviving interactions with effector cells (cytostasis test). Addition of lymphoid cells to adherent tumor cells initiates complex interactions which have non-specific (i.e., unrelated to recognition of tumor antigens) and specific (i.e., related to recognition of tumor antigens) effects on the survival and growth of tumor cells. The magnitude of these effects, as measured in the cytotoxicity or cytostasis tests, depends upon the number, kind, and state of activation and/or sensitization of effector cells and upon the number and susceptibility of tumor cells. Effector cells from control mice have only non-specific effects whereas those from MSV-immunized mice have both non-specific and specific effects. Lymph-node cells (LNC) from normal control mice non-specifically kill IUdR labelled tumor cells in the cytotoxicity test, whereas killing and growth-promoting influences are both present in the cytostasis test. Non-specific cytotoxic effects also were mediated by LNC from MSV-immunized mice, and these effects were of greater magnitude than those mediated by LNC from normal control mice. Our present and previous experiments suggest that non-specific cytotoxicity of LNC is largely due to macrophages. In contrast, non-specific promotion of tumor-cell growth in the cytostasis test is apparently mediated by lymphocytes. Specific cytotoxicity or cytostasis was obtained only when non-adherent LNC (NALNC) containing 99.8% lymphocytes and less than 0.2% macrophages were employed as effector cells. Although both cytotoxicity and cytostasis tests seem to give similar results, we conclude, for a number of reasons, that the cytotoxicity test is preferable for measuring cell-mediated immune responses to adherent tumor cells. Our findings and conclusions should be generally applicable to studies of animal and human tumors.