CP-115,953 Stimulates Cytokine Production by Lymphocytes

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1995, p. 476–483 0066-4804/95/$04.0010 Copyright q 1995, American Society for Microbiology

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CP-115,953 Stimulates Cytokine Production by Lymphocytes KRISTIAN RIESBECK*

AND

ARNE FORSGREN

Department of Medical Microbiology, Lund University, Malmo ¨ General Hospital, S-214 01 Malmo ¨, Sweden Received 14 June 1994/Returned for modification 12 September 1994/Accepted 28 November 1994

The cytotoxic quinolone CP-115,953 specifically exerts its inhibitory effect upon eukaryotic topoisomerase II. CP-115,953 stimulates DNA cleavage mediated by topoisomerase II with a potency approximately 600 times greater than that of ciprofloxacin, a quinolone antibacterial agent that currently is in clinical use. Because ciprofloxacin has been reported to strongly enhance interleukin-2 production, we considered it important to study the effect of CP-115,953 on interleukin-2 and gamma interferon (IFN-g) mRNA and protein expression in mitogen-stimulated human peripheral blood lymphocytes. For comparison, novobiocin and the antineoplastic drug etoposide were also included in the study. CP-115,953 (25 mM) enhanced interleukin-2 mRNA levels up to 8-fold and IFN-g mRNA concentrations up to 6.5-fold. In contrast, ciprofloxacin (282 mM) induced mRNAs for interleukin-2 and IFN-g up to 20-fold and 7.8-fold, respectively. However, CP-115,953 showed more prolonged kinetics of IFN-g mRNA production than ciprofloxacin. At high concentrations ($141 mM), ciprofloxacin was a greater inducer of interleukin-2 production and exhibited a higher level of stimulatory action than CP-115,953 on IFN-g synthesis. At low concentrations, however, CP-115,953 (#25 mM) was more potent than ciprofloxacin in inducing interleukin-2 and IFN-g synthesis. Etoposide or novobiocin did not influence cytokine mRNA expression. Thus, among the topoisomerase II inhibitors tested, fluoroquinolones are unique in stimulating cytokine synthesis in lymphocyte cultures. CP-115,953 exhibits reduced cytotoxic effects in etoposide-resistant CHO cells, suggesting that topoisomerase II is the putative target for CP-115,953. This has also been confirmed in Saccharomyces cerevisiae with temperature-sensitive mutants (13). Thus, CP-115,953 converts the enzyme into a cellular poison comparable with other topoisomerase II-targeted antineoplastic drugs such as etoposide (an epipodophyllotoxin; VP-16). CP-115,953 stimulates DNA cleavage mediated by topoisomerase II from calf thymus with a potency that is approximately 600-fold greater than that of the antimicrobial agent ciprofloxacin (6-fluoro-7-[19-piperazinyl]-1-cyclopropyl1,4-dihydro-4-quinoline-3-carboxylic acid) (Fig. 1) (2) and 50fold greater than that of the antineoplastic drug etoposide (12). Although CP-115,953 is considerably more potent than etoposide in enhancing topoisomerase II-mediated cleavage, CP115,953 is equipotent to etoposide in promoting cell death (12). In addition to their antibacterial effects, recent investigations have demonstrated that fluoroquinolones (e.g., ciprofloxacin, ofloxacin, and norfloxacin) can also interfere with the human immune system in numerous ways (5, 15, 36, 44). Ciprofloxacin induces hyperproduction of interleukin-2 (IL-2) (35 to 282 mM ciprofloxacin) and gamma interferon (IFN-g) (141 to 282 mM) in cultures of mitogen-stimulated lymphocytes (5, 36–39). In previous studies, we showed that this superinduction was due to accumulation of IL-2 and IFN-g mRNAs and increased gene transcription (38, 39). The concentrations of the transcriptional regulation factors NFAT-1 (nuclear factor of activated T cells) and AP-1 (activator protein 1) were found to be increased by ciprofloxacin (282 mM). It was also demonstrated that ciprofloxacin (141 to 282 mM) increases mRNA levels for an array of other cytokines, including IL-1a, IL-3, IL-4, granulocyte macrophage colony-stimulating factor, tumor necrosis factor alpha, and lymphotoxin (39). Moreover, ciprofloxacin (45 mg/kg of body weight per 24 h) enhances repopulation of murine hematopoietic organs in sublethally irradiated mice, most likely by stimulating IL-3 and granulocyte-macrophage colony-stimulating factor synthesis (28). For comparison with the in vitro studies, a clinically relevant dose

DNA topoisomerases are a class of enzymes that primarily alter DNA conformation through a concerted breaking and rejoining of DNA strands, thereby controlling the topological state of DNA (3, 4, 49). Topoisomerase II is not only crucially involved in DNA replication but also participates in transcription, DNA repair, and recombination (18). Nucleotide sequencing of genes encoding the topoisomerase enzymes from eukaryotic and prokaryotic cells shows all type II topoisomerases to be structurally and phylogenetically related. Eukaryotic DNA topoisomerase II corresponds to the bacterial DNA gyrase and is a ubiquitous ATP-dependent type II topoisomerase (4). Eukaryotic topoisomerase II is the primary cellular target for several clinically important antitumor agents (e.g., etoposide) (7, 46), while the prokaryotic counterpart is the target for fluoroquinolone antibacterial drugs (24). The lethal target of fluoroquinolone antibiotics in bacteria is the association between DNA and DNA gyrase (24, 34). Fluoroquinolones in clinical use have, in general, been found to be only weak inhibitors of the eukaryotic topoisomerase II in in vitro assays exploring relaxation or catenation of supercoiled double-stranded DNA (19, 25, 26). However, a number of new fluoroquinolone derivatives with enhanced activity against eukaryotic topoisomerase II exhibited by their strong inhibitory effects on eukaryotic DNA replication have been characterized, and their structure-activity relationships have been determined (3, 9, 12, 13, 17, 19, 29, 43, 47, 52). CP-115,953 (6,8difluoro-7-[49-hydroxyphenyl]-1-cyclopropyl-4-quinolone-3-carboxylic acid) is one of the most investigated quinolones with an increased effect upon eukaryotic topoisomerases (Fig. 1). CP115,953 enhances enzyme-mediated DNA cleavage in the presence of topoisomerase II from Drosophila melanogaster, Chinese hamster ovary (CHO) cells, or calf thymus (12, 42). Findings obtained with an unwinding assay using topoisomerase I proved CP-115,953 to be nonintercalative in nature (42).

* Corresponding author. Mailing address: Department of Medical Microbiology, Lund University, Malmo ¨ General Hospital, S-214 01 Malmo ¨, Sweden. Phone: 46-40-331340. Fax: 46-40-336234. 476

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floxacin was more active. However, etoposide or novobiocin did not stimulate cytokine secretion by mitogen-stimulated peripheral blood lymphocytes. MATERIALS AND METHODS FIG. 1. The fluoroquinolones CP-115,953 and ciprofloxacin.

of ciprofloxacin (750 mg, i.e., '10 mg/kg/12 h) administered orally produces a peak plasma drug concentration of 25 to 50 mM. The aim of the present report was to investigate whether CP115,953 influences T-lymphocyte functions. We also wanted to compare the effect of CP-115,953 with those of ciprofloxacin and the coumarin-based drug novobiocin (48). In addition, the clinically important antitumor agent etoposide was included in our experimental analyses. Evidence is presented that CP-115,953 stimulates mitogen-activated human peripheral blood lymphocytes to secrete more IL-2 and IFN-g than untreated control cells. The effect of CP-115,953 was confirmed by Northern (RNA) blots; i.e., cytokine mRNA levels were also upregulated in the presence of CP-115,953. Despite this apparently stimulatory effect on IL-2 and IFN-g production by CP115,953, cell proliferation was inhibited as measured by [3H]thymidine incorporation and survival data. At low concentrations, CP-115,953 was a better inducer of IL-2 production than ciprofloxacin, while at high concentrations cipro-

Drugs. Preservative-free CP-115,953 and ciprofloxacin were kindly donated by Pfizer (Groton, Conn.) and by Bayer (Wuppertal, Germany), respectively. Etoposide (injection substance; 20 mg/ml) was kindly provided by Bristol-Myers Squibb (Bromma, Sweden). Novobiocin was purchased from Sigma (St. Louis, Mo.) and diluted in water (100 mg/ml). CP-115,953 was dissolved as a 25 mM solution in 0.1 M NaOH, diluted to 5 mM stocks with 10 mM Tris-HCl (pH 8.0), and stored at 2708C (43). Ciprofloxacin and phytohemagglutinin (PHA) (Wellcome, Dartford, England) were dissolved in RPMI 1640 (Gibco, Paisley, Scotland). PHA was used at a final concentration of 1 mg/ml. Cell cultures. Human peripheral blood lymphocytes were isolated from buffy coats with citrate or from heparinized blood from healthy donors by centrifugation on a step gradient of Ficoll-Isopaque (Lymphoprep; Pharmacia, Uppsala, Sweden) (38). Triplicate lymphocyte samples were incubated in microtiter plates (106/ml) at 378C in a humidified 5% CO2 atmosphere in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, and gentamicin (12 mg/ml) (38). Thymidine incorporation, cell number determination, and cytokine analysis. To quantify DNA synthesis, peripheral blood lymphocytes were pulsed with [methyl-3H]thymidine (1 mCi; TRK 120; specific activity, 5 Ci/mmol; Amersham, Buckinghamshire, England) during the last 18 h of incubation. Radioactivity was measured in a scintillation counter. For cell number determination, viable lymphocytes were counted in a Bu ¨rker chamber with trypan blue (Sigma). The sensitivity of lymphocytes was defined as the drug concentration yielding a 50% decrease in growth (EC50). The growth percentage (EC) was defined as follows: EC 5 100 (number of drug-treated cells on the final day of the growth curve 2 initial cell number) (number of cells in the control well on the final day 2 initial cell number). IL-2 biological activity in supernatants was analyzed by the IL-2dependent stimulation of proliferation of the murine cytokine T-lymphocyte cell

FIG. 2. Influence of CP-115,953 on lymphocyte survival in PHA-stimulated peripheral blood lymphocytes. Ciprofloxacin is included for comparison. Peripheral blood lymphocytes (106/ml) were stimulated with PHA (1 mg/ml) and incubated for 48 to 96 h in the presence of CP-115,953 (A to C) or ciprofloxacin (D to F) at the indicated concentrations (0.78 to 100 and 35 to 282 mM for CP-115,953 and ciprofloxacin, respectively) added at initiation of cultures. The means of results from five (CP-115,953) or four (ciprofloxacin) different blood donors are shown. At 96 h, (2.1 6 0.8) 3 106 cells per ml were detected in control cultures. Relative survival is expressed as the percentage of the initial value, which is designated as 100% at the onset of cultures. Error bars indicate standard errors.

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FIG. 3. Influence of CP-115,953 compared with ciprofloxacin on [3H]thymidine incorporation in PHA-stimulated peripheral blood lymphocytes. Lymphocytes (106/ml) were stimulated with PHA (1 mg/ml) and incubated in the presence of CP-115,953 (A to C) or ciprofloxacin (D to F) at the indicated concentrations supplemented at culture initiation. The means of results from six (CP-115,953) or four (ciprofloxacin) different blood donors are shown. Lymphocytes were pulsed with [3H]thymidine during the last 18 h of incubation at the indicated times (48 to 96 h). [3H]thymidine uptake is expressed as a percentage of that of controls at every time point. [3H]thymidine incorporation in control cells after 96 h of incubation was 128,526 6 55,083 dpm. Error bars indicate standard errors.

line CTLL-2 as previously described (36). IFN-g was measured with a commercial enzyme-linked immunoassay (Genzyme, Boston, Mass.). RNA isolation, Northern blots, and cDNA probes. Total RNA was prepared according to the method of Chomczynski and Sacchi (8) with slight modifications as previously reported (38). A total of 20 3 106 to 25 3 106 cells were used for RNA preparation. Before further manipulations, RNA (0.5 mg) was examined on ethidium bromide-stained agarose gels to check that every sample was of comparable concentration and purity. For Northern blots, RNA (10 to 20 mg) was loaded onto formaldehyde-agarose gels and blotted to nylon filters (Hybond-N1; Amersham) as described by the manufacturer. The IL-2-specific probe containing the entire coding region, the IFN-g probe, and the triosphosphate isomerase (TPI) cDNA have been described earlier (38). DNA fragments were labeled with [a-32P]dCTP (specific activity, 3,000 Ci/mmol; PB 10205; Amersham) by random priming (Amersham). Free nucleotides were separated on spin columns (Costar, Cambridge, Mass.) containing Sephadex G-50 (fine; Pharmacia). Filters were hybridized according to standard protocols and exposed for 24 to 72 h to preflashed X-ray film (XAR-5; Kodak, Rochester, N.Y.) at 2708C by using intensifying screens. Autoradiographs were quantified by scanning laser densitometry. Statistics. Student’s t test for paired data was used for statistical calculations; P values of less than 0.05 were considered statistically significant.

RESULTS Effects of CP-115,953 on lymphocyte proliferation and thymidine incorporation. To investigate the cell inhibitory effects of CP-115,953, peripheral blood lymphocytes were stimulated with PHA and incubated in the presence or absence of CP115,953. Lymphocytes were stained with trypan blue after 48 to 96 h of incubation, and cell survival was determined (Fig. 2). In control cultures without CP-115,953, twice as many lymphocytes were found at 96 h of culture than at culture initiation. However, lymphocytes did not divide at 3.1 mM CP-115,953; i.e., at this concentration, the same number of cells were de-

tected as at the onset of the cultures. Less than 35% of the initially added cells were viable at 100 mM (96 h; Fig. 2C) compared with controls. The quinolone antibacterial agent ciprofloxacin did not significantly inhibit lymphocyte proliferation at 35 to 70 mM (Fig. 2F). At the highest ciprofloxacin concentration used (282 mM), .60% of the cells remained viable after 96 h. For comparison, the sensitivity of peripheral blood lymphocytes yielding an EC50 for the two quinolones was calculated from experiments outlined in Fig. 2C and F. At 96 h of incubation, the EC50s for CP-115,953 and ciprofloxacin were '3.1 and '282 mM, respectively. CP-115,953 ($25 mM) inhibited [3H]thymidine incorporation 89% or more in lymphocyte cultures incubated for 48 to 96 h compared with controls (100%) without any drug (Fig. 3). Thymidine uptake at 96 h (Fig. 3C) paralleled the cell survival observations (Fig. 2C). Stimulated peripheral blood lymphocytes incubated in the presence of CP-115,953 at 3.1 mM incorporated 28% [3H]thymidine compared with controls at 96 h. In contrast to the inhibition by CP-115,953, ciprofloxacin (35 to 141 mM) increased thymidine incorporation 1.5- to 2.5-fold at 96 h of incubation (Fig. 3F) (5, 15). Ciprofloxacin at 282 mM completely inhibited thymidine uptake. CP-115,953 stimulated IL-2 and IFN-g production by activated peripheral blood lymphocytes. CP-115,953 (25 mM) increased IL-2 production 2.7-fold at 24 h and 7-fold at 48 h of incubation of PHA-stimulated peripheral blood lymphocytes (Fig. 4A and B). A dose-dependent response of CP-115,953 (0.78 to 25 mM) was clearly manifest at 48 h (Fig. 4B). As much as 45 U of IL-2 per ml was detected in supernatants from

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FIG. 4. Effects of CP-115,953 and ciprofloxacin on IL-2 production. Supernatants from PHA-stimulated peripheral blood lymphocyte cultures (106/ml) were analyzed for IL-2 biological activity after 24 to 72 h of incubation with CP-115,953 (A to C) or ciprofloxacin (D to F). Note that the scale of the y axes in panels D to F is logarithmic. Results for samples from four different blood donors are shown. Error bars indicated standard errors. *, P ,0.05; **, P ,0.01; ***, P ,0.001.

lymphocytes incubated in the presence of CP-115,953 (25 mM; 48 h) compared with 6.4 U/ml in controls. For comparison, in separate experiments lymphocytes treated with ciprofloxacin (282 mM; 48 h) produced more than 500 U of IL-2 per ml (Fig. 4E). IFN-g production in the presence of CP-115,953 was consistent with IL-2 secretion (Fig. 5A). CP-115,953 at 25 mM increased IFN-g production (18 to 48 h) up to 1.4-fold. In contrast, 50 mM CP-115,953 inhibited IFN-g production approximately 50%. Lymphocytes incubated in the presence of CP-115,953 at 6.25 mM demonstrated slower kinetics of IFN-g synthesis but reached the same IFN-g concentrations as those in control cultures after 48 h of incubation. In these experiments, ciprofloxacin (282 mM) increased IFN-g levels 2.2- and 3.7-fold at 18 and 24 h, respectively, but not at all at 48 h compared with untreated controls (Fig. 5B). Enhanced cytokine mRNA levels in the presence of CP115,953. The increased cytokine production by CP-115,953 was confirmed when IL-2 and IFN-g mRNA concentrations were analyzed by the Northern blot technique. CP-115,953, ranging from 6.25 to 100 mM, increased cytokine mRNA levels in PHA-stimulated peripheral blood lymphocytes for 24 to 72 h (data not shown). However, cells from some blood donors were more sensitive to the cytotoxic effect of CP-115,953 at the highest concentrations tested (50 and 100 mM), resulting in unmeasurable (degraded) mRNAs. By means of data collected from the cytokine measurements (Fig. 4 and 5), CP-115,953 concentrations of 6.25 and 25 mM were selected for analysis of IL-2 and IFN-g mRNAs after 24 to 72 h of incubation (Fig. 6). In addition, ciprofloxacin (70 and 282 mM) was included for comparison. The relative intensities of X-ray films are pre-

sented in Fig. 7. It is clearly demonstrated that CP-115,953 (25 mM) enhanced IL-2 mRNA levels 5.8-fold at 48 h and 8.3-fold at 72 h of incubation, compared with untreated lymphocytes. Ciprofloxacin (70 mM) was a weaker IL-2 mRNA stimulatory agent than CP-115,953 (25 mM). CP-115,953 or ciprofloxacin did not affect the control mRNA, i.e., the ‘‘housekeeping’’ gene product TPI. CP-115,953 was a slightly better inducer of IFN-g mRNA than was ciprofloxacin (48 and 72 h of incubation); e.g., at 48 h, CP-115,953 (25 mM) enhanced IFN-g mRNA levels 3.2-fold compared with a 1.4-fold increase in the presence of ciprofloxacin (282 mM) (Fig. 6 and 7). In a few experiments, depending on the blood donor, ciprofloxacin was as good as CP-115,953 in stimulating IFN-g mRNA expression (data not shown and references 38 and 39). However, in general, CP115,953 showed a prolonged kinetics of IFN-g mRNA expression compared with ciprofloxacin. Etoposide and novobicin did not affect cytokine mRNA concentrations. To examine whether other topoisomerase II inhibitors affect cytokine mRNAs in peripheral blood lymphocytes, experiments were performed with the well-characterized anticancer drug etoposide (5). Interestingly, etoposide did not increase IL-2 mRNA levels at concentrations of 1 to 500 mM (Fig. 8). In cells exposed to etoposide at 100 and 500 mM, IL-2 steady-state mRNA concentrations were decreased. At higher etoposide concentrations (.500 mM), it was not possible to measure cytokine mRNAs because of cytotoxic effects on lymphocytes and consequently unstable rRNAs. In contrast to etoposide, which stabilizes the covalent enzyme-DNA cleavage complexes, novobiocin inhibits type II topoisomerase activity by inhibiting the ATP hydrolysis reaction that is essential for

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FIG. 5. Effect of CP-115,593 compared with ciprofloxacin on IFN-g production. Peripheral blood lymphocytes (106/ml) were incubated with CP-115,953 (6.25 [å], 25 [■], or 50 [h] mM) (A) or ciprofloxacin (35 [å], 141 [h], or 282 [■] mM) (B) at initiation of cultures and stimulated in the presence of PHA (1 mg/ml). At the indicated times (18 to 48 h), supernatants were analyzed for IFN-g by enzyme-linked immunosorbent assay. Results are from experiments with four different blood donors. IFN-g concentrations are given as a percentage of those of drug-free controls (E [100%]) at every time. Mean (6 standard errors) IFN-g concentrations (pg/ml) in controls were 1,591 6 264 (18 h), 2,375 6 441 (24 h), and 5,175 6 414 (48 h). *, P ,0.05; **, P ,0.01; ***, P ,0.001.

enzyme function (48). However, as with etoposide, novobiocin (0.1 to 250 mM) did not increase cytokine mRNA levels compared with those in control cells without drug (not shown). IL-2 production and IFN-g production were (as expected) not influenced by either etoposide or novobiocin. DISCUSSION In this report, we chose to study the effects of CP-115,953 on T-lymphocyte functions. CP-115,953 at 3.1 mM inhibited cell cycle progression and [3H]thymidine incorporation in PHAstimulated peripheral blood lymphocytes (Fig. 2A to C and 3A to C). This is in contrast to ciprofloxacin and other antibacte-

ANTIMICROB. AGENTS CHEMOTHER.

rial fluoroquinolones which at concentrations of #141 mM increased [3H]thymidine uptake two- to threefold compared with untreated control cells (Fig. 3E to F) (5). For comparison, ciprofloxacin at higher concentrations inhibits lymphocyte cell cycle progression (.70 mM) (Fig. 2E to F) and thymidine incorporation (.141 mM) (15). CP-115,953 has been shown to exert great cytotoxicity towards mammalian cells (12). The EC50 for CHO cells is 9 mM for CP-115,953, while only 20% of CHO cells are killed by ciprofloxacin at 500 mM. Furthermore, CP-115,953 shows an EC50 of 14 mM toward human A-431 carcinoma cells (12), while the inhibitory effects of ciprofloxacin on proliferation of Lewis lung carcinoma, a murine bladder carcinoma cell line, is considerably lower, i.e., EC50 of '100 mM (53). In the present study, the EC50s for peripheral blood lymphocytes incubated for 96 h were 3.1 and 282 mM for CP-115,953 and ciprofloxacin, respectively (Fig. 2C and F). Despite the cytotoxic effects, CP-115,953 enhanced IL-2 and IFN-g secretion by PHA-stimulated human peripheral blood lymphocytes. CP-115,953 did not, however, increase IL-2 mRNA and protein concentrations to the same levels as ciprofloxacin (Fig. 4 and 6) (38). This could be explained by the difference between the two fluoroquinolones regarding their cell inhibitory capacity and effects on topoisomerases. CP115,953 and ciprofloxacin stimulate bacterial gyrase-mediated DNA cleavage at minimal effective concentrations of 0.14 and 0.60 mM, respectively (3). Corresponding values for eukaryotic topoisomerase II isolated from calf thymus are 0.3 mM for CP-115,953 and 171 mM for ciprofloxacin (12). However, maximally enhanced IL-2 production by CP-115,953 and ciprofloxacin is achieved at 25 and 282 mM, respectively (Fig. 4 and 7) (29). Thus, a correlation between topoisomerase II inhibition and IL-2 production does not exist. Superinduction of IL-2 and IFN-g by CP-115,953 and ciprofloxacin may suggest that a DNA damage response has been elicited. This is partly supported by findings showing increased concentrations of the transcriptional regulation factors

FIG. 6. IL-2 and IFN-g mRNA levels in lymphocytes incubated with CP-115,953 or ciprofloxacin. Peripheral blood lymphocytes (106/ml) were incubated in the presence of ciprofloxacin (Cipro; 70 or 282 mM) or CP-115,953 (CP-115; 6.2 or 25 mM) for 24 to 72 h. Total RNA was isolated and Northern blots were performed. The resulting filters were hybridized with specific cDNA probes for IL-2, IFN-g, and TPI. The data presented are from a single donor but are representative of the results of four independent experiments.

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FIG. 7. Relative cytokine mRNA levels in lymphocytes incubated in the presence of CP-115,953 (CP-115) or ciprofloxacin (Cipro). Scanning densitometry data were obtained from Northern blots shown in Fig. 6. Control cell cultures without any addition of drugs are indicated by a C. Values above the columns show relative intensities, which are expressed as arbitrary units; i.e., controls (24 to 72 h) were set to 100 arbitrary units for the different cytokine mRNAs. All values for IL-2 or IFN-g were divided with the corresponding values for TPI.

NFAT-1 and AP-1 (c-Fos and c-Jun) in the presence of ciprofloxacin (37, 39). In stimulated Jurkat and EL-4 cells, human and murine T-cell lymphoma cell lines, respectively, AP-1 and NFAT-1 are detected 15 min earlier in cells treated with ciprofloxacin than in drug-free controls. The mammalian DNA damage response, analogous to the bacterial SOS response, is considered to be a global response to DNA damage, and the trigger has been suggested to be either the DNA damage itself or a by-product of DNA damage (21, 22). Numerous mammalian genes or gene products, including the proto-oncogenes (e.g., c-fos and c-jun), collagenase, metallothioneins, IL-1, and tumor necrosis factor alpha have been identified during a DNA damage response (11, 14, 21, 23, 30). Other DNA-damaging agents besides topoisomerase inhibitors (i.e., ionizing and UV irradiation, alkylating agents, and antitumor quinones) are able to induce similar cellular events (35). The most widely investigated DNA damage response is the UV response, which initially may be mediated by oxidative stress (11, 27). Interestingly, redox activation of AP-1 DNA binding activity is mediated by an enzyme that also possesses endonuclease DNA repair activity (51), suggesting a link between transcription factor regulation, oxidative signalling, and DNA repair processes in eukaryotes. In addition to fluoroquinolones, bleomycin, adriamycin, and neothramycin have all been reported to potentiate IL-2 production by stimulated lymphocytes in vitro (1, 20). In vitro stimulation of splenocytes obtained from rats administered bleomycin or adriamycin in vivo also generates higher IL-2

concentrations than are found in control cells from untreated animals (20, 33). However, IL-2 enhancement in the presence of these drugs is weak, with only 1.5- to 3-fold increases being observed compared with the 70- to 80-fold upregulated IL-2 levels in the presence of fluoroquinolones such as ciprofloxacin (Fig. 4) (36). In contrast to CP-115,953, which increased IL-2 mRNA concentrations up to 8.3-fold (Fig. 6 and 7), etoposide did not affect IL-2 mRNA concentrations in peripheral blood lymphocytes (Fig. 8). Because DNA topoisomerases are involved in transcription, the differences between the effects of CP-115,953

FIG. 8. The topoisomerase II inhibitor etoposide does not affect IL-2 mRNA levels in lymphocytes. Peripheral blood lymphocytes (106/ml) were stimulated with PHA and incubated in the presence of etoposide (0 to 500 mM) at initiation of cultures. After 30 h of incubation, RNA was isolated and Northern blots were performed and subsequently hybridized with IL-2 and TPI cDNAs. Two more experiments were performed with similar results.

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and etoposide on cytokine expression might be explained by their different effects on religation of DNA prior to and after strand passage (42). CP-115,953 only slightly affects DNA religation, while etoposide inhibits the rate of DNA religation approximately eightfold. Although CP-115,953 is more potent than etoposide in stimulating topoisomerase II-mediated DNA cleavage, etoposide is more potent in inhibiting enzyme-catalyzed DNA relaxation (12). The data mentioned above are supported by findings in recent papers showing that etoposide can displace other DNA topoisomerase inhibitors from the enzyme-DNA complex (10, 41). Furthermore, topoisomerase II inhibitors, i.e., quinolones and epipodophyllotoxins, have been shown to interact directly with the DNA bases at the cleavage site, placing the inhibitor binding site precisely at the site of DNA cleavage (16). Interestingly, the preferred bases on the 59 side of each cleaved phosphodiester bond are highly specific to the inhibitor used in the cleavage reaction. Thus, the specific DNA binding sites of various topoisomerase II inhibitors may explain the different effects on IL-2 mRNA expression by etoposide and CP-115,953. IL-2 mRNA and protein levels were unaffected in the presence of etoposide (Fig. 8). However, induction of vimentin mRNA and protein synthesis has been reported in the presence of etoposide or 49-(9-acridinylamino)methanesulfon-maniside (m-AMSA), another topoisomerase II inhibitor (40). Activation of the gadd153 promoter has also been clearly demonstrated in the presence of etoposide and other DNA-damaging agents (31). Inhibition of topoisomerase II by teniposide (VM-26) appears to lead to transcription of heat shock genes both in HeLa cells and in Drosophila cells (45). A noteworthy finding is that the topoisomerase II gene itself belongs to the heat shock genes and is consequently induced by exposure of cells to a temperature of 428C (32). A topoisomerase II that inhibits RNA polymerase II-directed transcriptional activity in vitro has been purified from HeLa cell nuclear extracts (6). Thus, in addition to triggering the DNA damage response, DNA-damaging agents may directly inhibit topoisomerase II and thus facilitate transcription. Direct interference with topoisomerase II might be one mechanism by which CP-115,953 and ciprofloxacin stimulate cytokine transcription. Interestingly, novobiocin did not enhance IL-2 mRNA levels in PHA-stimulated lymphocytes compared with control cells. Novobiocin, which inhibits the ATPase subunit of the topoisomerase II prior to the enzyme’s interaction with DNA (48), does not elicit a DNA damage response, a finding analogous to the failure of bacteria to induce their SOS response in the presence of novobiocin. However, this may partly be explained by the ability of novobiocin to inhibit RNA polymerase II, as has been reported in experiments with the mouse metallothionein I gene (50). In conclusion, CP-115,953 strongly inhibited lymphocyte proliferation in vitro. In contrast to the cytotoxicity, IL-2 production and IFN-g production were increased in the presence of CP-115,953. The promising stimulatory effect on T-lymphocyte cytokines, in combination with a cell inhibitory activity, by CP-115,953 warrants further investigations. ACKNOWLEDGMENTS The excellent technical assistance of Agnethe Henriksson is greatly appreciated. We also thank S. B. Kadin, Pfizer, Inc., for providing us with CP-115,953. This investigation was supported in part by grants from the Anna and Edwin Berger Foundation, the Cancer Foundation at Malmo ¨ General Hospital, the Greta and Johan Kock Foundation, the Swedish Medical Research Council, the Medical Faculty at Lund ¨ sterlund Foundation. University, and the O

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