Expression of the tumor necrosis factor gene in tumor cells correlates with reduced tumorigenicity and reduced invasiveness in vivo

We investigated whether a constitutive production of low amounts of tumor necrosis factor (TNF) by neoplastic cells affects their in vivo tumorigenicity. TNF-resistant derivatives were isolated from the TNF-sensitive murine fibrosarcoma cell lines L929s and WEHI164cl13s, L929r1-type TNF-resistant subclones were found to constitutively produce TNF in vitro, in contrast to non-TNF-producing but TNF-resistant L929r2 and WEHI164cl13r2 cell clones. The TNF-sensitive parental cell lines as well as the L929r2 and WEHI164cl13r2 cell lines similarly induced fast-growing tumors upon s.c. inoculation into nude mice (Swiss-nu/nu). In contrast, the TNF-producing L929r1-type cells showed reduced tumorigenicity and in vivo growth rate, which both inversely correlated with the level of in vitro-produced TNF. Tumor take incidence but not the in vivo growth rate of L929r1-type cells was greatly facilitated by prior whole body gamma-irradiation (350 rads) of the recipient, implying the involvement of host mechanisms at least in the lower take incidence of L929r1 tumors. These host mechanisms, possibly activated by tumor-produced TNF, acted only locally, inasmuch as the growth of an inoculum of L929s cells was not influenced either by a simultaneous distant inoculum of L929r1 cells or by established, distant L929r1 tumors. Efforts to eliminate these host mechanisms by prior local UV irradiation of the skin were unsuccessful. All L929 cell types were found to be similarly susceptible to killing by host cytotoxic effector cells (macrophages and natural and lymphokine-activated killer cells). Histological investigation did not reveal clear differences in tumor-associated inflammatory cells but revealed that tumors induced by L929r1-type cells, in contrast to L929s and L929r2 tumors, did not show invasiveness in host tissues. Moreover, L929r1 tumors were frequently encapsulated, which was never observed for tumors induced by L929s and L929r2 cells. Taken together, our results suggest that tumor-derived TNF locally activates host antitumor activities. Possible effector mechanisms are discussed.     

Relationship between tumour cell morphology, gap junctions and susceptibility to cytolysis by tumour necrosis factor

Tumour necrosis factor (TNF) is directly cytolytic to certain tumour cell lines in vitro, although TNF-resistant variants can be selected from these susceptible lines by exposure to TNF. While studying TNF-susceptible L929 cells and their resistant variant, L929/R, we noted that within L929 colonies the cells were widely spaced whereas they were closely packed in L929/R colonies. L929/R cells also adhered more strongly to plastic and differed from L929 in cell shape. Similar observations were made with TNF susceptible and resistant variants of two other cell lines (RK13 and a plastic adherent U937 subline). The tendency of resistant cells to grow closely together suggests the possibility of inter-cell communication for the TNF resistant state. However, like L929 and U937, L929/R and U937/R did not communicate by gap junctions and we could find no evidence of extracellular mediators of TNF resistance. Rather the differences in colonial morphology, cell shape and plastic adherence may be secondary to an underlying mechanism which defines TNF susceptibility/resistance.     

Transformation is associated with an increase in sensitivity to TNF-mediated lysis as a result of an increase in TNF-induced protein tyrosine phosphatase activity

Using the tumor necrosis factor (TNF)-resistant cell line B/CN and an anchorage-independent variant, 10ME, we have shown a relationship between transformation and sensitivity to TNF. Here, we report a role for protein tyrosine phosphorylation in expression of these phenotypes. Several studies have demonstrated the involvement of protein phosphorylation in the TNF signaling pathways that leads to cell death. We show that TNF treatment of the TNF-sensitive, transformed cells results in a marked increase in protein tyrosine phosphatase (PTP) activity and a decrease in protein tyrosine kinase (PTK) activity. In contrast, TNF treatment of the TNF-resistant, non-transformed parental cells results in a marked increase in PTK activity. Also, the PTP inhibitors vanadate and bromotetramisole decrease TNF lytic activity, indicating that the PTP activity observed is an integral part of the lytic process. Treatment of targets with vanadate prior to TNF exposure had no effect on TNF-mediated lysis. In contrast, the addition of vanadate up to 4 hr after TNF-treatment resulted in a decrease in TNF-mediated lysis. Our findings indicate that the phosphatase activity is induced after TNF binds its receptor. Our data also indicate that the decrease in TNF-mediated lysis caused by PTP inhibitors is not due to the inhibition of the TNF lytic mechanism. Instead, vanadate increases a TNF resistance mechanism; it does so by blocking the PTP-mediated inhibition of the TNF resistance mechanism. Further, the lineage relationship of these cell lines suggests that there is a biochemical relationship between anchorage-independence, tumorigenicity and the protein tyrosine phosphorylation that governs sensitivity to TNF.     

Potentiation of topoisomerase inhibitor-induced DNA strand breakage and cytotoxicity by tumor necrosis factor: enhancement of topoisomerase activity as a mechanism of potentiation

A combination of tumor necrosis factor (TNF) and the topoisomerase I inhibitor, camptothecin, or the topoisomerase II inhibitors, teniposide and amsacrine, produced dose-dependent synergistic cytotoxicity against the murine L929 fibrosarcoma cells. Similar synergy was not observed with a combination of TNF and bleomycin. To define the role of TNF in the augmentation of tumor cell killing by topoisomerase I or II inhibitors, the effect of TNF on the production of enzyme-linked DNA strand breaks induced in cells by topoisomerase inhibitors was investigated. L929 cells incubated for 1 h with the topoisomerase inhibitors contained protein-linked strand breaks. In contrast, TNF alone did not induce DNA strand breakage. However, when cells were incubated simultaneously with TNF and camptothecin, amsacrine, Adriamycin, actinomycin D, teniposide, or etoposide, increased numbers of strand breaks were produced. Preincubation of the cells with TNF for 30 min or 3 h before the addition of camptothecin or etoposide resulted in no more strand breaks than that observed in cells incubated with the drugs alone. TNF treatment of L929 cells produced a rapid and transient increase in specific activity of extractable topoisomerases I and II. These increases were maximum at 2-5 min of TNF treatment and by 30 min the activities of extractable enzymes were equal to or less than those detected in extracts from untreated cell controls. The transient nature of the increase in extractable topoisomerase activity may explain the kinetics and significance of the order of addition of TNF and inhibitors for maximal synergistic activity. These data are consistent also with a role for topoisomerase-linked DNA lesions in the TNF-mediated potentiation of killing of L929 cells by topoisomerase inhibitors.     

The development of human tumor-cell resistance to TNF-alpha does not confer resistance to cytokine-induced cellular cytotoxic mechanisms.

We have derived a TNF-alpha-resistant clone (RA-I) from the parental TNF-sensitive human breast-adenocarcinoma cell line (MCF-7). The acquisition of TNF-resistance was not associated with endogenous TNF production or with differential levels of TNF receptors since both MCF-7 and RA-I display comparable TNF-receptor expression. We have investigated the relationship between acquisition of resistance to TNF and susceptibility to lysis by cytokine-activated effectors. Experiments were performed using human peripheral-blood monocytes stimulated with IL-2, IFN or GM-CSF, and lymphokine-activated killer cells as effector cytotoxic cells. Our data indicate that both TNF-resistant (RA-I) and TNF-sensitive (MCF-7) cells were killed by IL-2-activated monocytes. Incubation of monocytes with IFN also resulted in the activation of their tumoricidal activity against MCF-7 and RA-I. When stimulated monocytes were pre-incubated in the presence of a TNF-specific neutralizing monoclonal antibody, prior to co-culture with target cells, no effect on their lytic capacity was observed. Thus, the monocyte killing does not appear to involve the membrane form of TNF. These observations suggest that, in our experimental system, IL-2 and IFN are able to induce non-TNF-mediated mechanisms of cytotoxicity by monocytes. Experiments performed using GM-CSF and LPS for monocyte stimulation indicate that, although both reagents were efficient in inducing the membrane form of TNF on monocytes, they did not enhance the cell-killing capacity towards MCF-7 and RA-I targets. Furthermore, using IL-2-stimulated LGL as effector cells, we show in this study that the TNF-resistant clone RA-I was as sensitive as MCF-7 to human LAK cells.     

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.     

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.     

Resistance to tumor necrosis factor-induced apoptosis in mouse fibroblasts is a genetically dominant phenotype

Although many tumor cells are sensitive to tumor necrosis factor (TNF)-induced cell death, most normal cells are resistant. To determine whether the sensitive phenotype or the resistant phenotype is genetically dominant, we constructed somatic cell hybrids of TNF-resistant (TNP) C3H mouse 10T1/2 fibroblasts and Ha-ms-transformed TNF-sensitive (TNFs) 10T-EJ cells and then tested the sensitivity of those hybrids to TNF-induced cell death. All somatic cell hybrid cell lines tested were resistant to TNF-induced cell death. The TNFr 10T1/2 cells, however, exhibited sensitivity to TNF-induced cell death in the presence of cycloheximide (CHX), whereas TNFs 10T-EJ cells did not show any further increase in sensitivity to TNF-induced cell death in the presence of CHX. In addition, the killing of 1OT1/2 cells by TNF in the presence of CHX involved apoptosis. These results demonstrate that resistance to TNF-induced apoptosis is a genetically dominant phenotype and that certain protein(s) constitutively expressed or induced by TNF in resistant cells may confer protection against TNF-induced apoptosis.     

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.     

Sensitivity of resistant human tumor cell lines to tumor necrosis factor and adriamycin used in combination: correlation between down-regulation of tumor necrosis factor-messenger RNA induction and overcoming resistance.

A number of human tumor cell lines of various histological origin were examined for their sensitivity and resistance to tumor necrosis factor-alpha (TNF) and Adriamycin (ADR). Six ovarian lines, and one each of a renal, lung, and B-cell line, were tested for putative mechanisms of resistance to these agents. Cytotoxicity resulting from TNF or ADR showed no overall correlation in these lines. The combination of TNF and ADR produced enhanced cytotoxicity against these tumor lines and furthermore resulted in overcoming the resistance of TNF or ADR alone or in combination. A proposed mechanism of TNF resistance in tumor cells is the endogenous production of TNF mRNA and protein. There was a positive correlation between resistance to TNF and the constitutive production of TNF mRNA and protein. The TNF-resistant lines that did not constitutively produce TNF mRNA and protein and the three TNF-sensitive tumor lines exhibited up-regulation of their TNF mRNA in the presence of TNF or phorbol myristate acetate/ionophore, but did not secrete any detectable protein. Due to the enhanced cytotoxicity seen with the combination of TNF and ADR, the effect of this combination on the level of TNF mRNA was examined. ADR alone reduced the constitutive level of TNF mRNA and in combination with TNF reduced the level of induction produced by TNF. This down-regulation of TNF mRNA by ADR may play a role in the enhanced cytotoxicity seen with the combination of these 2 agents.     

Caffeine potentiates the lethality of tumour necrosis factor in cancer cells.

In this study we have investigated the interaction of caffeine, a prototypic methylxanthine, and TNF on the induction of cell death in mouse and human cell lines during progression from G1 to successive phases of the cell cycle. Exposure of cells to TNF (0.1-100 ng ml-1) as single agent for 48 h caused low or no lethality. The rates of cell death increased significantly when cells cultured with TNF for 24 h were exposed to caffeine (2.5-20 mM). The magnitude of the enhancement by caffeine was TNF and caffeine dose-dependent. The most effective response to this combination was observed in the mouse cell lines, WEHI and L929, followed by the human cell lines, HeLa, A375 and MCF-7, respectively. In L929 cells, TNF treatment did not inhibit DNA synthesis during the first S phase of the cell cycle (20-24 h), but it did block the progress toward a second S phase, indicating the cells were arrested at G2 phase or mitosis. Caffeine had great enhancer effect on L929 cells exposed to TNF for 24 h, but the effect was reduced in cells with either less than 24 h or greater than 28 h of exposure. L929 cells stimulated with TNF died via apoptosis, as judged by both morphological criteria and the occurrence of internucleosomal DNA cleavage. Exposure of TNF-treated cells to caffeine caused a greater increase in the proportion of apoptotic cells as well as the extent of internucleosomal DNA fragmentation.     

Colonial morphology of tumour cells and susceptibility to cytolysis by tumour necrosis factor. The role of cellular fibronectin deposition in the extracellular matrix

The tumour cell lines U937A and L929 form large, loosely packed colonies in vitro and can be killed by the cytokine tumour necrosis factor (TNF). In contrast, their TNF-resistant mutants U937A/R and L929/R form tightly packed colonies. Since cells which form loose colonies have increased metastatic potential it is important to understand the factors governing colonial morphology. To this end, we have compared the extracellular matrices (ECMs) of the 'loose' lines, U937A and L929 with their 'tight' mutants. By immunofluorescence, a polyvalent anti-U937A serum revealed a fibrillar network in the ECMs of the 'loose' lines which was absent in the 'tight'. On Western blotting of ECMs the antiserum detected an additional 300 kDa protein in the 'loose' lines which was subsequently shown to be cellular fibronectin. The four lines secreted comparable amounts of fibronectin and this was qualitatively indistinguishable between 'loose' and 'tight' cells by peptide mapping or lectin binding. It is concluded that the differences in colonial morphology are due to the 'tight' mutants' inability to incorporate fibronectin into the ECM.     

Murine tumor necrosis-inducing factor: purification and effects on myelomonocytic leukemia cells

The tumor necrosis-inducing factor (TNF) found in sera of Corynebacterium parvum-treated, endotoxin-stressed BALB/C and outbred albino CD-1 mice has been purified to a single band of protein by polyacrylamide gel electrophoresis after identification and removal of contaminating albumin and transferrin. This purified TNF has a molecular weight of 140,000, is glycoprotein in nature, and migrates on free electrophoresis as an alpha 2-globulin. TNF activity was continuously monitored during purification by bioassay in vitro (tumor cell lysis) and was confirmed by demonstration of induction of tumor necrosis in vivo. A single target tumor cell line, murine myelomonocytic leukemia (WEHI/3), was used in both assays. In the in vivo assay, controls were heat-inactivated samples of TNF. As additional controls, a line of TNF-resistant WEHI/3 cells was used in the in vitro assay. Results from in vivo radiolabeling of TNF-sensitive and TNF-resistant cells indicated a difference between their cytoplasmic peptide profiles. Optimal TNF production was not altered in C. parvum-endotoxin-treated mice by treatment with silica, a substance that is specifically toxic for macrophages. Exposure of mice to 650 rad whole-body radiation, which is not markedly damaging to macrophage elements in the reticuloendothelial system, completely abrogated the ability of the mice to produce TNF after C. parvum-endotoxin treatment. These findings suggest that in the sera of C. parvum-endotoxin-treated mice the protein that induces necrosis in tumors may not be of macrophage origin.     

Resistance to cytolysis by tumor necrosis factor alpha in malignant gynecological cell lines is associated with the expression of protein(s) that prevent the activation of phospholipase A2 by tumor necrosis factor alpha.

Although there are a limited number of cell lines that are sensitive to cytolysis by tumor necrosis factor alpha (TNF alpha), the vast majority are resistant. The analysis of TNF alpha-sensitive cells has shown that phospholipase A2 is activated by TNF alpha in these cells and that the activity of phospholipase A2 is required for their cytolysis. Many cell lines that are resistant to TNF alpha-mediated cytolysis are dependent on the maintenance of protein synthesis for their resistance. We have recently shown that this is also true for TNF alpha-resistant cell lines derived from cervical (ME-180 and SiHa) and ovarian (SK-OV-3 and OVCAR-3) carcinomas, in that they are sensitive to cytolysis by TNF alpha only in the presence of protein synthesis inhibitors. Here we show that the TNF alpha-mediated cytolysis of these resistant cell lines in the presence of the protein synthesis inhibitor emetine is similar to that of sensitive cells, in that cytolysis is inhibited by the inhibitors of phospholipase A2. The measurement of the release of radiolabeled material from cervical and ovarian carcinoma cell lines prelabeled with [3H]arachidonic acid showed that not only was phospholipase A2 required for the cytolysis of these cells by TNF alpha in the presence of protein synthesis inhibitors, but more importantly, phospholipase A2 was not activated by TNF alpha unless protein synthesis was inhibited. These results indicate that a protein synthesis-dependent resistance mechanism expressed by these cell lines blocks TNF alpha-mediated cytolysis by preventing the activation of phospholipase A2 by TNF alpha.     

Expression of a resistance mechanism in ovarian and cervical carcinoma cells prevents their lysis by gamma-interferon

Despite extensive evidence that recombinant human gamma-interferon (IFN-gamma) exerts antiproliferative effects on a variety of cancer cell lines, IFN-gamma has not been shown to lyse cells in vitro. In order to determine whether some cancer cells might actively resist lysis by IFN-gamma, we examined eight arbitrarily selected cell lines derived from gynecological malignancies (ME-180, MS751, HT-3, SiHa, and C-33A human cervical carcinoma lines; Caov-3, SK-OV-3, and NIH:OVCAR-3 human ovarian carcinoma cell lines) for lysis by IFN-gamma. In a 24-h assay involving release of 51Cr from cells, none of these cell lines was lysed by IFN-gamma, either alone or in combination with actinomycin-D or emetine, two inhibitors of protein synthesis. However, pretreatment of cells with 100 units/ml of IFN-gamma for 24 h, followed by inhibition of protein synthesis, led to significantly increased lysis of the cell lines ME-180, MS751, and Caov-3. These results indicate that IFN-gamma induces a lytic mechanism in some cancer cells that is opposed by a protein synthesis-dependent resistance mechanism. This suggests that a combination therapy involving IFN-gamma and inhibitors of protein synthesis may be useful in the treatment of some cancers.     

Interleukin 1, interleukin 6, tumor necrosis factor, and transforming growth factor beta increase cell resistance to tumor necrosis factor cytotoxicity by growth arrest in the G1 phase of the cell cycle

During infection, inflammation, immune responses, and neoplastic growth, various cytokines are produced affecting both susceptibility to and protection from cellular death. We have studied the protective effect of pretreatment of the L929 fibroblast cell line with interleukin 1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha (TNF-alpha), or transforming growth factor beta 1 (TGF-beta) on subsequent TNF/actinomycin D-induced cytotoxicity. The protective effects of these cytokines on TNF cytotoxicity were time and concentration dependent. TGF-beta was the most effective cytokine, followed by TNF, IL-1 beta, and IL-6. Activators of protein kinase C also afforded protection, and TGF-beta acted synergistically with either phorbol 12-myristate 13-acetate or the calcium ionophore A-23187. TGF-beta-induced protection against TNF was observed in cells subjected to prolonged treatment with phorbol 12-myristate 13-acetate. Cells pretreated with prostaglandin E2 or cholera toxin amplified the sensitivity to TNF and inhibited TGF-beta-mediated resistance, whereas indomethacin enhanced the protective effect of TGF-beta. Cells cultured in the presence of IL-1 beta, IL-6, TNF-alpha, or TGF-beta for 6 h inhibited DNA synthesis, and this was associated with concomitant growth arrest in the G1 phase of the cell cycle. On the other hand, prostaglandin E2 or cholera toxin stimulated the progression of cells from G1 toward G2 + M which was associated with increased TNF sensitivity. We conclude that these cytokines protect against death by arresting growth in the G1 phase of the cell cycle.     

Cyclooxygenase-2 (COX-2) inhibitors sensitize tumor cells specifically to death receptor-induced apoptosis independently of COX-2 inhibition

Cyclooxygenase-2 (COX-2) is involved in diverse processes such as inflammation, carcinogenesis and apoptosis. As COX-2 inhibitors interfere with these processes, inhibition of COX-2 has been suggested as a promising anticancer treatment. However, the role of COX-2 in modulation of apoptosis as well as the death pathways affected by COX-2 inhibitors are poorly characterized. Here we demonstrate that the selective COX-2 inhibitors NS-398 and nimesulide increased TNF sensitivity of TNF-resistant HeLa H21 and TNF-sensitive HeLa D98 cells, although this cytokine induced significant COX-2 activity, as judged by prostaglandin E(2) (PGE(2)) production, only in H21 cells. TNF did also not induce PGE(2) production in MCF-7/casp-3 cells stably expressing COX-2; however, nimesulide strongly enhanced TNF-induced apoptosis in these cells. Furthermore, COX-2 activity in HeLa H21 cells could be inhibited by NS-398 concentrations that were 10 000-fold lower compared to those required for the induction of cell death. Most intriguingly, sensibilization to apoptosis was specifically observed in response to activation of death receptors. Not only TNF-induced cell death but also apoptosis triggered by the CD95 and TRAIL receptors was enhanced by nimesulide. In contrast, apoptosis induced by the anticancer drugs doxorubicine and etoposide that target the mitochondrial death pathway remained unaffected. Together, our data suggest that COX-2 inhibitors overcome apoptosis resistance and selectively sensitize tumor cells to the extrinsic death receptor-induced apoptotic pathway independently of COX-2.     

K562 erythroleukemic cells are equipped with multiple mechanisms of resistance to lysis by complement

Resistance of tumor cells to lysis by complement is generally attributed to several protective mechanisms. The relative impact of these mechanisms in the same tumor cell, however, has not been assessed yet. We have analyzed the interaction of the human erythroleukemia tumor cell line K562 with human complement. K562 cells express the membrane complement regulatory proteins CD59, CD55 and CD46. As shown here for the first time, K562 also spontaneously release the soluble regulators C1 inhibitor, factor H, and soluble CD59. Complement resistance of K562 cells is augmented upon treatment with PMA, TNF or even with sublytic complement. Unlike TNF and sublytic complement, PMA enhanced the expression of membrane-bound CD55 and CD59 and led to increased secretion of soluble CD59. In addition, we show that complement-resistant K562 cells express a membrane-associated proteolytic activity, higher than the complement-sensitive K562/S cells. Treatment of complement-resistant K562 cells with serine protease inhibitors enhance their sensitivity to complement-mediated lysis. Inhibitors of protein kinase C (PKC) also sensitize K562 cells to complement lysis, implicating PKC-mediated signaling in cell resistance to complement. Neutralization of the CD55 and CD59 but not of CD46 regulatory activity with specific antibodies significantly increases complement-mediated K562 cell lysis. Treatment of K562 cells with a mixture of inhibitory reagents results in a significant additive enhancing effect on complement-mediated lysis of K562. In conclusion, K562 cells resist a complement attack by concomitantly using multiple molecular evasion strategies. Future attempts in antibody-based tumor therapy should include strategies to interfere with those resistance mechanisms.     

Cycloheximide-induced modulation of TNF-mediated cytotoxicity in sensitive and resistant ovarian tumor cells

The mechanism of sensitivity and resistance of various ovarian carcinoma lines to recombinant tumor necrosis factor (rTNF)-mediated cytotoxicity has been investigated using a 24-h 51Cr-release assay. The cell line PA-1 is sensitive to TNF in a dose-dependent manner, whereas the cell line SKOV-3 is resistant to TNF even at high concentrations. The simultaneous addition of TNF and cycloheximide (CHX) in the assay converted the resistant SKOV-3 line into a sensitive line, but no detectable change was observed with PA-1. rTNF inhibited DNA, RNA, and protein synthesis of the sensitive PA-1 line, whereas it had no effect on SKOV-3. This finding was not due to differences in the expression of TNF receptors, as both cell lines expressed equivalent numbers of receptors. The addition of CHX to TNF resulted in suppression of DNA, RNA, and protein synthesis in both the sensitive and the resistant cell lines. Pretreatment of the cell line with TNF for 3 h and subsequent washing resulted in significant cytotoxicity of the sensitive PA-1 line and some cytotoxicity against SKOV-3. However, if the cells were pretreated with CHX for 3 h followed by rTNF for 24 h, a significant decrease in cytotoxicity was observed in both cell lines. Under these conditions, there was no significant inhibition of DNA, RNA, or protein synthesis. Pretreatment of cells for 24 h with TNF and 24 h with CHX resulted in augmentation of the cytotoxicity of PA-1 and SKOV-3, whereas pretreatment for 24 h with CHX followed by 24 h with TNF resulted in no cytotoxicity. Cells pretreated with CHX for 24 h showed poor binding of [125]I-TNF and poor internalization, whereas cells pretreated for 24 h with TNF showed marked enhancement of internalization. The sensitivity of freshly derived ovarian carcinoma lines to TNF and CHX demonstrated that TNF-resistant cells became more sensitive if treated with CHX. These results demonstrate the potential use of metabolic inhibitors in increasing the sensitivity of fresh ovarian tumor cells to TNF.