Colony inhibitory effect of recombinant human tumor necrosis factor (rH-TNF) and/or recombinant human interferon (rH-IFN)-alpha, -beta and -gamma on human lung cancer cell lines

This study was conducted to assess, by continuous exposure, the inhibitory effects of rH-TNF and rH-IFN-alpha, -beta and -gamma, either alone or in combination, on the colony formations of human lung cancer cell lines. The cell lines tested were PC-7 and PC-9 (adenocarcinoma), PC-10 (squamous cell carcinoma) and PC-13 (large cell carcinoma). Additional experiments were performed with L929 (transformed murine fibroblast), because of its known high sensitivity to TNF. rH-TNF inhibited the colony formations of PC-10 and L929 even at a low concentration (10 U/ml). However, PC-7, PC-9 and PC-13 were resistant to rH-TNF. rH-IFN-alpha, -beta and -gamma inhibited the colony formation of PC-10 (less than 50% of control) at the highest concentration tested (10(4) U/ml), but only rH-IFN-beta inhibited that of PC-7. PC-9, PC-13 and L929 were insensitive to all three rH-IFNs, even at the highest concentrations tested. Combination effects of rH-TNF and rH-IFN-alpha or -gamma were synergistic only at the highest concentration combinations tested in PC-9. However, the maximum inhibition of colony formation of PC-7, PC-9 or PC-13 in any concentration combination treatment was less than 50%.     

Synergistic effects of recombinant human tumor necrosis factor and hyperthermia on in vitro cytotoxicity and artificial metastasis

Synergy in cytotoxic effect between recombinant human tumor necrosis factor and hyperthermia (incubation at 38.5 degrees C or 40 degrees C) was observed to occur against L-M (mouse tumorigenic fibroblast) cells and shown to be related to an accelerated turnover rate of recombinant human tumor necrosis factor-receptor complex under elevated temperatures rather than to changes in number of cell receptors or binding strength. However, no synergy in cytotoxic effect was observed to occur against human embryonic lung (HEL) cells. A clearly synergistic inhibition of metastatic tumor growth by combined administration of recombinant human tumor necrosis factor (300 units) and whole-body hyperthermia (40 degrees C, 30 min) was also observed in BALB/c mice previously given injections of 1 x 10(6) Meth-A (MH) cells/mouse via tail vein, neither of which alone resulted in significant inhibition.     

Enhancement of lysosomal enzyme activity by recombinant human tumor necrosis factor and its role in tumor cell killing in vitro.

We investigated the effect of recombinant human tumor necrosis factor (TNF) on the lysosomal enzyme activity of various established cell lines in vitro. Incubation of 1 x 10(6) TNF-sensitive mouse tumorigenic fibroblasts (L-M cells) in the presence of TNF (100 U/ml) for 48 h increased the total (the sum of the enzyme activities in the lysosomes and the cytoplasm) acid phosphatase and beta-glucuronidase activities by 3.7- and 4.2-fold, respectively. The same increase was observed even when 1 U/ml of TNF was added to some cultures and no further augmentation occurred at 10 or 100 U/ml. Measurement of total and free enzyme activities showed that TNF stimulation not only enhanced the total intracellular enzyme activity but also accelerated the conversion into free (cytoplasmic) enzyme activity. Addition of a lysosomotropic agent (methylamine) suppressed both the enhancement of lysosomal enzyme activity and the cytotoxicity of TNF. A similar enhancement of lysosomal enzyme activities was also detected in various TNF-sensitive tumor cell lines, and a strong correlation (acid phosphatase: r = 0.836, beta-glucuronidase: r = 0.910) was observed between the enhancement of enzyme activity and sensitivity to TNF. No such increase was detected in TNF-resistant human diploid cells. These results show that TNF induces the activation and release of lysosomal enzymes in TNF-sensitive cells, and suggest that such events may play an important role in TNF-mediated cytotoxicity.     

Proliferative response of hairy cells to B cell growth factor (BCGF): in vivo inhibition by interferon-alpha and in vitro effects of interferon-alpha, -beta, and -gamma

Hairy cell leukemia (HCL) is a pre-plasma B cell tumor which responds to interferon (IFN)-alpha therapy. In vitro, B cell growth factor (BCGF) can induce proliferation of hairy cells. We have investigated the effect of in vitro and in vivo treatments with different recombinant IFN on the capacity of hairy cells to proliferate in response to human BCGF. In vitro treatment of leukemic cells from HCL patients with recombinant IFN-alpha-2 (5/5 cases) or IFN-beta (4/5 cases) resulted in a marked inhibition of the BCGF-dependent response. This suppressive effect was obtained with IFN concentrations of 1000, 100 IU/ml, and even occasionally 10 IU/ml. In contrast, no such inhibition was observed with IFN-gamma, despite the presence of specific IFN-gamma receptors on hairy cells at densities similar to receptors for IFN-alpha/beta. The IFN-alpha-induced suppression of the proliferative response of hairy cells to BCGF was also observed in vivo in two patients within 6-12 hr after administration of single doses of IFN-alpha. When hairy cells were maintained in culture for 1 week, they recovered their capacity to be stimulated by BCGF. This reversion was also shown in vivo in hairy cells isolated 1 week after IFN administration. Since in vivo growth of hairy cells could possibly result from the autocrine secretion of BCGF, we propose that the therapeutic effect of IFN-alpha on HCL may be due in part to an inhibition of such autocrine loop.     

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.     

In vitro growth inhibition of cisplatin-resistant human lung cancer cell lines by recombinant human tumor necrosis factor and/or recombinant human interferon-gamma by virtue of collateral sensitivity

The colony-inhibitory effects of recombinant human tumor necrosis factor (rH-TNF) and recombinant human interferon-gamma (rH-IFN-gamma) were evaluated in four human lung cancer cell lines and their cisplatin-resistant sublines. The cell lines tested were PC-7 and PC-9 (adenocarcinoma), H69 and N231 (small cell lung cancer) and four cisplatin-resistant sublines, PC-7/1.0, PC-9/0.5, H69/0.2 and N231/0.2, which were 20.0, 7.1, 4.8 and 8.4 fold resistant to cisplatin, respectively, compared to the respective parental cell line in terms of IC50 in a soft agar colony assay. All parental cell lines were resistant to rH-TNF and rH-IFN-gamma, alone or in combination. However, two resistant sublines showed sensitivity to rH-TNF and rH-IFN-gamma. Colony formation by PC-9/0.5 was significantly inhibited, in the absence or presence of cisplatin, by 10(2) U/ml of rH-TNF (less than 50% of control) and the inhibition was synergistic with that produced by 10(3) or 10(4) U/ml of rH-IFN-gamma. RH-IFN-gamma inhibited the colony formation of H69/0.2 only at the highest concentration tested (10(4) U/ml) (less than 50% of control) and the combined effect with rH-TNF was additive. These results suggest that rH-TNF and rH-IFN-gamma may have some potential in overcoming cisplatin resistance by virtue of collateral sensitivity.     

Induction of synthesis of tumor necrosis factor in human and murine cell lines by exogenous recombinant human tumor necrosis factor

Treatment of sensitive human myosarcoma cells (KYM-S) with exogenous tumor necrosis factor (r-TNF) resulted in the production of TNF by the cells. The newly synthesized cellular TNF was identified immunologically on Western blots and as a single 1.8-kilobase band on Northern blots. TNF synthesis began within 2 h of administration of the exogenous TNF in a dose-dependent manner. r-TNF also induced TNF synthesis in mouse tumorigenic fibroblasts (L-M). Resistant sublines of these cells as well as TNF nonsensitive human diploid fibroblasts possessed TNF mRNA without pretreatment, indicating an inverse correlation between levels of TNF expressed and sensitivity to the cytotoxic effects of exogenous TNF. It is conceivable that the newly synthesized cellular TNF functions in some protective manner to block cytolytic effects of exogenous TNF.     

Effect of recombinant human tumor necrosis factor on human nasopharyngeal carcinoma cell line in vitro

The cytostatic and cytotoxic effects of recombinant human tumor necrosis factor alpha (rHuTNF alpha) on a cultured nasopharyngeal carcinoma (NPC) cell line were studied. rHuTNF alpha inhibited tritiated thymidine ([3H]TdR) and [14C]leucine incorporation by NPC cells. rHuTNF alpha also inhibited the growth of NPC cells as determined by microphotometry. Cytotoxic effect of rHuTNF alpha was observed 2 days after incubation. Pulse hyperthermia or continuous hyperthermia in combination with rHuTNF alpha treatment was markedly cytotoxic for NPC cells in vitro. Addition of actinomycin D, cycloheximide, puromycin or vincristine sulfate to NPC cells also potentiated the cytotoxic effect of rHuTNF alpha.     

Comparison of recombinant tumor necrosis factor and the monocyte-derived cytotoxic factor involved in monocyte-mediated cytotoxicity

A direct comparison of recombinant tumor necrosis factor (rTNF) and the monocyte-derived cytotoxic factor (CF) which is involved in monocyte-mediated cytotoxicity revealed immunological, physiochemical, and biological similarities, indicating that TNF is an effector molecule in monocyte-mediated cytotoxicity. Neutralizing antiserum raised against rTNF completely inhibited the ability of CF-containing monocyte supernatants to induce cytolysis and cell death of sensitive target cells and, conversely, antiserum raised against purified CF completely inhibited the cytotoxic activity of rTNF. Both CF and rTNF have an apparent isoelectric point of 5.8-5.9 as determined by chromatofocusing, and a molecular weight of about 40,000 as determined by gel filtration. Moreover, when present in monocyte supernatants with a total protein concentration of about 1 mg/ml and 0.1% sodium dodecyl sulfate, both CF and rTNF migrated with a molecular weight of about 35,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pure rTNF, however, migrated with a molecular weight of 17,000, suggesting that the relative amount of sodium dodecyl sulfate to protein is critical for dissociating the apparent dimeric structure of TNF. CF and rTNF were also similar with respect to their ability to kill various types of target cells the sensitivity of which to TNF differ, and the dose-response curves of cytotoxicity obtained with CF-containing monocyte supernatants and rTNF were similar. As is the case with anti-CF serum, anti-rTNF serum inhibited drug-dependent cellular cytotoxicity and cytolysis mediated by both freshly isolated monocytes and in vitro cultured unactivated and lymphokine-lipopolysaccharide activated monocytes, indicating that TNF is an effector molecule in both drug-dependent cellular cytotoxicity and "classical" monocyte-mediated cytotoxicity.     

Human tumor xenografts treated with recombinant human tumor necrosis factor alone or in combination with interferons

We have studied the activity of recombinant human tumor necrosis factor (rHuTNF) on six different human tumor xenografts derived from primary breast and bowel tumors and maintained by passage in nude mice. When 5 micrograms rHuTNF was given daily intratumorally to mice with established (approximately, 0.5 cm) tumors, total tumor regression was observed by 3-4 weeks in three of six xenograft lines. In a further two lines tumor stasis or significant slowing of growth was seen. This antitumor action was not accompanied by any consistent macroscopic change in the tumor such as necrosis, but histological examination revealed tumor cell degeneration and a large peritumoral infiltration of host inflammatory cells after 4-7 days therapy. In contrast to these data, little effect was seen when the same dose of rHuTNF was administered i.p. to nude mice bearing these tumors. In only two of six lines was any significant slowing of tumor growth seen. A 5-fold increase in the i.p. dose resulted in improved activity on only one of two xenograft lines tested. Efficacy of the i.p. rHuTNF dose could, however, be enhanced by simultaneous administration of human interferon, alpha or gamma. No obvious signs of toxicity were observed at all rHuTNF doses administered and weights of control and treated mice at the end of the experiments were comparable.     

Cachectic effects of recombinant human tumor necrosis factor in rats

Treatment of rats with either intermittent bolus i.v. injections or continuous i.v. infusions of the same sublethal daily dose of tumor necrosis factor (TNF) results in decreased food intake and decreased nitrogen balance compared to saline-treated control rats. After 4 days of treatment, rats treated with intermittent bolus doses of TNF develop tolerance to the nutritional effects and consume normal amounts of food and have nitrogen balance similar to those of saline treated rats. Rats receiving the continuous infusion of TNF do not. Rats treated with both routes of TNF lose more weight than pair fed rats who eat the same mean amount as the continuous TNF treated group. In addition, 56% of rats receiving continuous infusion TNF die during the 8-day experimental period while rats receiving either intermittent bolus TNF or similar food intake (pair fed) do not. Body composition studies of rats that completed the 8 days of treatment indicate that rats receiving either continuous infusion or intermittent bolus TNF have increased percentages of body water and reduced percentages of body solid compared to saline treated control rats. Rats pair fed to the food intake of continuous TNF treated rats also had increased percentages of body water and reduced percentages of body solid, but changes were significantly less than those observed in continuous TNF infused rats. Continuous TNF infusion reduced total body nitrogen and potassium while pair feeding did not reduce potassium and reduced nitrogen to a lesser degree. Pair feeding and continuous TNF infusion reduced total body fat to a similar extent. Twice a day administration of TNF resulted in lesser changes in carcass water, solid, nitrogen, lipid, and potassium than continuous infusion of the same dose of TNF. The results indicate that continuous infusion of TNF can produce anorexia, weight loss, edema, loss of body protein, lipid and cell mass, and lethality which is markedly ameliorated with bolus doses of TNF. The findings are consistent with the hypothesis that slow continuous secretion of sublethal amounts of TNF may mediate cancer cachexia.     

Immunomodulatory properties of recombinant murine and human tumor necrosis factor

Tumor necrosis factor has traditionally been thought to have direct cytostatic and cytotoxic properties with little or no direct immunomodulatory activity. We report here that tumor necrosis factor is able to activate macrophages both in vitro and in vivo and can increase a mixed lymphocyte response and act as an adjuvant for both T- and B-cells in vivo. Adjuvant activity in T-cells occurred in conjunction with the administration of a suboptimal syngeneic tumor cell vaccine. In addition, tumor necrosis factor demonstrated a potent dose-dependent effect on bone marrow stem cell number, dramatically depressing cellularity and thus total stem cell number. An appreciable interval is required for recovery from such stem cell depletion. Therefore, the study of the therapeutic activity of tumor necrosis factor must include a consideration of its immunomodulatory properties.     

Phase I study of recombinant human tumor necrosis factor

Summary A phase I clinical and pharmacokinetic study of recombinant human tumor necrosis factor (rH-TNF) was conducted in a single dose schedule in 33 patients with advanced cancer. rH-TNF was given by i.v. infusion over 30 min with a starting dose of 1x10⁵ units/m². The dose was escalated up to 16x10⁵ units/m² according to the modified Fibonacci scheme. Toxic effects were similar but not identical to those reported with interferons and interleukin-2, and included fever, rigors, nausea and vomiting and anorexia in a non-dose-dependent manner, and hypotension, leukocytosis, thrombocytopenia and transient elevation of transaminases (SGOT and SGPT) in an approximately dose-dependent manner. DIC syndrome was observed in one patient who had received 16x10⁵ units/m². The dose-limiting toxicities were hypotension, thrombocytopenia and hepatotoxicity, and the maximum tolerated dose in a single i.v. infusion of rH-TNF appeared to be 12x10⁵ units/m² when thrombocytopenia and elevation of SGOT and SGPT were taken as the dose-limiting toxicities. However, if hypotension was included, the maximum safely tolerated dose appeared to be 5x10⁵ units/m².     

Effect of recombinant human tumor necrosis factor on tumor and spleen in mice

The ability of recombinant human tumor necrosis factor (rH-TNF-alpha) to induce regression of sarcoma 180 in vivo was evaluated. The tumor was cured by TNF in the course of 4 weeks. TNF inhibited proliferation of sarcoma 180 cells in vitro, which suggests a direct effect of TNF on tumor cells in vivo. In parallel to the TNF effect on tumor growth, some cell parameters in spleen were investigated. Activation of splenic macrophages was enhanced in vitro. This result suggests that macrophages may participate in the host defense against the tumor. In the first phase of therapy, TNF did not affect the proliferation of splenocytes but increased the transition of G0 into G1 cells. Furthermore, TNF normalized the tumor-induced increase of null cells in tumor-bearing mice. All parameters investigated in spleen reached normal values at the time of tumor regression. Our results suggest that various mechanisms may be involved in TNF-induced regression of sarcoma 180.     

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.     

Cures and partial regression of murine and human tumors by recombinant human tumor necrosis factor

We tested the effect of recombinant human tumor necrosis factor (TNF) on the growth of the murine methylcholanthrene induced fibrosarcoma and the human ovarian carcinoma (NIH:OVCAR-3) in mice. The mice received multiple doses (25-250 micrograms/kg) of TNF starting 7-10 days after s.c. transplantation of tumors when they were easily palpable. TNF was administered i.v. every other day for a total of 6 injections per mouse, or i.p. daily for 7 days. Complete tumor regression was observed in the methylcholanthrene induced tumor bearing mice in 90% of the mice treated with TNF (100 micrograms/kg), 67% treated with TNF (50 micrograms/kg), and 34% treated with TNF (25 micrograms/kg). Tumors which did not completely regress were growth retarded during the course of TNF treatment. All mice given the highest TNF dose are still alive and tumor free (currently over 400 days), whereas the median survival of control mice was 28-39 days. Partial regression was observed in 100% of mice bearing the ovarian carcinoma treated i.p. with 250 micrograms/kg. Injections of TNF i.v. resulted in higher percentage of cures than i.p. injections at similar dose levels. These results suggest that tumor necrosis factor represents a likely potent drug against solid tumors and that the method of administration is critical in optimizing its use in cancer.     

Synergistic cytotoxic effects of recombinant human tumor necrosis factor, interferons, and heat-stress

A synergistic increase in the cytotoxic effects of recombinant human tumor necrosis factor (TNF-alpha), interferons (IFN-alpha, IFN-beta, and IFN-gamma) and heat-stress was demonstrated in vitro. The toxicity of these agents was assessed in the human cervical carcinoma HeLa cell line: the toxic effect was greatly increased when cells pretreated with IFNs or TNF were submitted to a 1-h heat-shock at 45 degrees C. Moreover if the heat-stress followed simultaneous treatment with both cytokines, a synergistic effect between these treatments could be observed. The same observations were made for two other transformed cell lines: the oral epidermoid carcinoma KB cells and the hepatocarcinoma PLC/PRF/5 cells. In contrast, the survival of normal cells (normal foetal lung MRC5 cells and foreskin F 7000 fibroblasts) was only slightly decreased by such treatments. These results suggest that combining a heat-stress with cytokines treatment might be one way of enhancing the sensitivity of cancer cells to the growth inhibitory effects of the individual cytokines.     

Distinction of partially purified human natural killer cytotoxic factor from recombinant human tumor necrosis factor and recombinant human lymphotoxin

Natural killer cell cytotoxic factor (NKCF), a cytotoxic factor contributing to human natural killer cell-mediated cytotoxicity, was generated from lymphocyte-conditioned medium using various stimuli. Crude NKCF activity was concentrated, and partially purified by ammonium sulfate precipitation and gel filtration. NKCF activities eluted as two molecular weight peaks, corresponding to Mr 33,000-43,000 (pool I) and approximately Mr 5,000 (pool II). The cytotoxic activity and target specificity of the partially purified NKCFs were found to be different from both recombinant human TNF and recombinant human lymphotoxin. In the NKCF assay, up to 10(6) units/ml of TNF and lymphotoxin had virtually no effect, whereas both NKCFs lysed 22% (range 17-33%) of the NK-sensitive target K562. In contrast, TNF and lymphotoxin were active in a standard assay against the sensitive murine L929 fibroblast cell line in all concentrations tested (10(-1)-10(6) units/ml). In addition, the effect of these cytotoxic factors in a short-term (4-h) chromium-release assay using peripheral blood mononuclear cells as effector cells was tested: only NKCF (pool I), but not TNF, lymphotoxin, or low molecular weight NKCF (pool II), enhanced NK and lymphokine-activated killer cell cytolysis, both against the NK-sensitive target K562 and the NK-resistant melanoma cell line SK-MEL 30. Results were not affected in the presence of neutralizing antibodies against TNF. NKCF could, therefore, be distinguished from TNF and lymphotoxin with respect to their biological activities.     

Enhancement of cisplatin cytotoxicity by terbium in cisplatin-resistant MDA/CH human breast cancer cells

Purpose: The development of cisplatin resistance is a major problem in the treatment of cancer patients with cisplatin chemotherapy. The membrane binding of terbium (Tb³⁺) has been shown to increase the cellular accumulation of cisplatin in breast cancer cells. Therefore, the ability of Tb³⁺ to modulate the cytotoxicity of cisplatin was investigated in cisplatin-sensitive (MDA) and cisplatin-resistant (MDA/CH) MDA-MB-231 human breast cancer cells. Methods: The cytotoxic parameters of cisplatin were determined using live cell microfluorometry and median effect analysis. Results: MDA/CH cells (IC₅₀ = 142 ± 9 μM) were found to be approximately 3.3-fold more resistant to cisplatin than MDA cells (IC₅₀ = 43.5 ± 3.0 μM). In both cell lines, the IC₅₀ value for cisplatin was reduced two-fold in the presence of 80 μM Tb³⁺, thus indicating that the cytotoxicity of cisplatin is increased by Tb³⁺. The cytotoxic activity of cisplatin alone was observed to be 5.7 and 1.6 times more potent than that of Tb³⁺ alone in MDA and MDA/CH cells, respectively. Combination index analyses revealed that the interaction between cisplatin and Tb³⁺ was only synergistic at very low indices of cell death in MDA cells. However, in MDA/CH cells, the two drugs were synergistic up to intermediate levels of cell death. Conclusions: Our results suggest that the enhancement of cisplatin cytotoxicity by Tb³⁺ is more effective in cisplatin-resistant MDA/CH cells than in cisplatin-sensitive MDA cells. Therefore, terbium is potentially useful in cisplatin combination therapy for breast cancer patients, especially for those patients who have developed resistance to the drug. Received: 16 December 1998 / Accepted: 19 January 1999