Tumors are considered to be possible targets of immunotherapy using stimulated and expanded autologous or allogeneic natural killer (NK) cells mismatched for MHC class I molecules and inhibitory NK receptors. NK cell–based immunoadjuvant therapies are carried out in combination with standard chemotherapeutic protocols. In the presented study, we characterized the effect of 28 frequently used chemotherapeutic agents on the capacity of NK cells to kill target cells. We found that treatment of NK cells with the drugs vinblastine, paclitaxel, docetaxel, cladribine, chlorambucil, bortezomib, and MG-132 effectively inhibited NK cell–mediated killing without affecting the viability of NK cells. On the other hand, the following drugs permitted efficient NK cell–mediated killing even at concentrations comparable with or higher than the maximally achieved therapeutic concentration in vivo in humans: asparaginase, bevacizumab, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea, streptozocin, and 6-mercaptopurine. [Mol Cancer Ther 2007;6(2):644–54]

The goal of immune-based adjuvant therapeutic strategies is to stimulate immune effector cells to kill malignant cells, which may result in a better clinical outcome. Infiltration of solid tumors with inflammatory cells is a well-known phenomenon (13). Several studies showed that the infiltration of natural killer (NK) cells in malignant tumors was associated with a better clinical outcome (4, 5).

NK cells are large granular lymphocytes that are part of the innate immune system (6). Mature NK cells are mostly circulating in the peripheral blood, where they represent ∼10% to 15% of all blood lymphocytes. Phenotypically, NK cells express surface antigen CD56, which has unknown function on NK cells, and lack the typical T-cell antigen CD3 (7). Physiologically, NK cells have direct cytotoxic activity against tumor targets and virus-infected cells (8). NK cells can regulate adaptive immune response by IFN-γ production and can mediate antibody-dependent cellular toxicity through membrane FcγRIII (CD16; ref. 6). These cells have the ability to directly kill target cells and produce immunoregulatory cytokines in transfused cancer patients (912).

The outcome of NK cell-target cell interactions is regulated by a fine integrative balance between inhibitory and activating receptors; the first elicited by the recognition of MHC class I molecules by NK inhibitory receptors (killer immunoglobulin–like receptors, CD94/NKG2A, and immunoglobulin-like transcript) and the latter by NK cell–activating (NKG2D and natural cytotoxicity receptor) receptors that can sense stress inducible glycoprotein (MHC class I chain–related proteins and UL16 binding proteins) expression on tumor- and virus-infected target cells (13). Because NK cells are able to spontaneously lyse tumor cells, considerable effort has been focused on investigating the possible use of NK cells in the treatment of human cancer. Recent understanding of NK cell development and function has permitted the investigation of novel NK cell–based immunotherapeutic modalities. A promising approach is to expand allogeneic NK cell clones in vitro, with typed killer immunoglobulin–like receptor expression, and to transfuse these cells into leukemic patients (1416).

Basse et al. (17) showed that the level of accumulation of NK cells in murine tumor metastases in vivo is dependent on the dose and frequency of interleukin 2 (IL-2) administration. NK cells have also been used in human therapeutic regimens that involved the administration of high or low doses of IL-2, alone or in combination with IL-2–activated blood lymphocytes or purified populations of NK cells, for primary lung and renal cell carcinomas (18, 19). In other studies, extended low-dose IL-2 was used to activate NK cells in patients with solid tumors who received intensive chemotherapy or in patients with acute myeloid leukemia (20, 21). Harker et al. (22) suggested that IL-2–induced lymphokine-activated killer cells may be useful in the treatment of chemotherapy-resistant cancers.

The chemotherapeutic regimens for malignancies restrict the effectivity of the immune system and significantly decrease the level of NK cells (23). The activity of NK cells is also affected by some cytostatic drugs (24). Utsugi et al. (25) observed enhanced NK cell–mediated cytotoxicity following treatment of the target cells with topoisomerase inhibitors. Killing by NK cells requires microtubule integrity, which may be influenced by antimicrotubule agents (26). It is important to consider the drug-induced effects when chemotherapy and NK cell–mediated immunotherapy are planned to be used in parallel.

The aim of this study was to systematically investigate the effect of the most frequently used cytostatic drugs on NK cell–mediated cytotoxicity. We carried out more than 4,000 cell survival assays on single-cell levels using automated laser confocal microscopy to establish the pattern of in vitro effect of 29 different cytotoxic drugs on the efficiency of NK cell–mediated killing.

Isolation and Culturing of Human Polyclonal Peripheral Blood NK Cell Cultures

Polyclonal NK cells were acquired from lymphocyte-enriched buffy coat derived from healthy donor blood (Blodbank, Karolinska Sjukhuset, Stockholm). Peripheral blood mononuclear cells were isolated by centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway). NK cells were isolated from peripheral blood mononuclear cells by negative magnetic bead selection (Stemcell Technologies, Vancouver, Canada) according to the manufacturer's instructions. Purified NK cells were >95% CD3CD56+. NK cells were cultured either overnight or for several weeks in IMDM supplemented with 10% human serum (AB, Blodbank), 2 mmol/L l-glutamine, 1× nonessential amino acids, 1 mmol/L sodium pyruvate, 50 units/mL penicillin-streptomycin, 50 μmol/L 2-mercaptoethanol (media and supplements bought from Life Technologies, Inc., Carlsbad, CA), and 100 units/mL IL-2 (Peprotech, Inc., London, United Kingdom).

Eight primary NK cell isolates were tested in the present study. Five NK cell isolates were restimulated every 3 to 4 days with fresh IL-2–containing media for 1 week and were periodically monitored for the CD3CD56+ phenotype. Three NK cell isolates were stimulated with IL-2 only for 24 h.

Cell Lines and Culture Conditions

Cytotoxic NK tumor cell lines Nishi (27), NKL (28), NK92 (29), and KHYG-1 (30) were cultured in IMDM supplemented with different concentrations of IL-2 (Nishi and NK92, 100 units/mL; NKL, 200 units/mL; KHYG-1, 450 units/mL). K562 cell line was used as target in the in vitro killing assay. These cells were cultured in IMDM (Sigma, Dorset, United Kingdom) supplemented with 10% FCS (Sigma), 100 mmol/L l-glutamine (Sigma), and 80 μg/mL gentamicin (Sigma). Cell suspensions were grown in a humidified incubator at 37°C in an atmosphere containing 5% CO2. Cell counts were adjusted to 1 × 106/mL and the cells were fed twice a week. The absence of Mycoplasma contamination was ensured by regular monitoring with Hoechst 33258 staining.

In vitro Killing Assay (Standard Chromium-51 Release Assay)

Target cells were labeled for 1 h at 37°C, 7.5% CO2 with 100 μCi of Na251CrO4 (Amersham Biosciences, Uppsala, Sweden) added directly to the cell pellet. Radioactively labeled target cells were washed thrice with RPMI, and 15 × 103 cells were plated per well in flat-bottomed 96-well microtiter plates to replicate cell densities in the flat-bottomed microtiter plates. NK cells were subsequently added at 5:1 effector-to-target ratio per well (total volume of 200 μL/well) and incubated for 5 h at 37°C, 5% CO2. Spontaneous release was determined by incubation of labeled target cells with media alone whereas maximal release was determined by lysis of target cells in 10% SDS. Spontaneous release was <15% of the maximal release. One hundred microliters of supernatant were collected for scintillation counting on a γ irradiation counter. Each measurement was done in triplicate and the average of three wells was used for calculation of specific lysis. Specific lysis (killing effectiveness) was calculated as follows: killing effectiveness (%) = [(cpm experimental well − cpm spontaneous release) / (cpm maximal release − cpm spontaneous release)] × 100.

In vitro Killing Assay (Novel Fluorescent Labeled Killing Assay)

The effects of drugs on in vitro killing efficacy of different NK cell lines were assessed using microtiter plates. Twenty-nine drugs were tested, each at four different concentrations in triplicates on flat-bottomed 384-well plates. Each well was loaded with 20-μL cell suspension of NK cells containing 15,000 cells and was incubated for 20 h at 37°C in 5% of CO2. Target (K562) cells were stained with CellTracker green CMFDA (Invitrogen, Sweden). A time-lapse image sequence of NK cell killing of target cells is illustrated in Fig. 1. As a next step, 20 μL of target cells (K562) containing 3,000 cells were seeded on the same plate and were incubated with the NK cells for 5 h (effector-to-target ratio, 5:1). Dead cells were differentially stained red with ethidium bromide. The precise numbers of green target cells and red dead cells were determined by fluorescent detection using a custom-designed automated laser confocal fluorescent microscope (a modified Perkin-Elmer UltraView LCI) at the Karolinska Institute visualization core facility. The images were captured using the computer program QuantCapture 4.0, and the living and dead target cells were identified and individually counted using the computer program CytotoxCount 3.0. The target cells were identified as dead if they were stained red and green at the same time. Both programs were developed at Karolinska Institute visualization core facility using OpenLab Automator programming environment (Improvision). On each plate, eight wells were set up as controls to determine the baseline killing activity. These wells contained NK and K562 cells in culture medium only (without drugs). As additional control, effector NK cells alone were incubated under the same conditions. To monitor the possible short-term effect on the target cells, K562 cells were incubated alone and with all the individual drugs for 5 h. Killing effectiveness of NK cells was calculated for every well using the following formula: [number of dead target cells (both red and green signal in the same cell) / number of all (green) target cells] × 100.

Figure 1.

Time-lapse image sequence of NK cells killing a K562 target cell. Target cells were labeled with CellTracker green (CMFDA; Invitrogen). NK cells were labeled with CellTracker blue (CMAC; Invitrogen). Dead cells were labeled with ethidium bromide (red). Images were taken with an automated laser confocal microscope (a modified Perkin-Elmer UltraView LCI). Arrows, green target cells as they get killed by the surrounding NK cells and turn red. Time scale is given in seconds.

Figure 1.

Time-lapse image sequence of NK cells killing a K562 target cell. Target cells were labeled with CellTracker green (CMFDA; Invitrogen). NK cells were labeled with CellTracker blue (CMAC; Invitrogen). Dead cells were labeled with ethidium bromide (red). Images were taken with an automated laser confocal microscope (a modified Perkin-Elmer UltraView LCI). Arrows, green target cells as they get killed by the surrounding NK cells and turn red. Time scale is given in seconds.

Close modal

To normalize the killing effect values of different NK populations, the following formula was used: (killing effectiveness in the wells with drugs / mean killing effectiveness values of the control wells) × 100%. Mean killing efficacy was determined from the average killing of all NK populations. The red-stained cells that were not green at the same time were considered as dead NK cells. The cytotoxic effect (%) of the drugs on NK cells was calculated with the following formula: (number of dead NK cells in the wells with drugs / number of dead NK cells in the control wells) × 100%.

Drugs

For the in vitro killing test, 29 drugs were used (summarized in Table 1). Twenty-eight of these drugs are regularly used in the treatment of human patients. All the drugs were dissolved in 50% DMSO and printed on 384-well flat-bottomed plates using a high-density metal pin array (50-nL replica volumes) in Biomek 2000 fluid dispenser robot (Beckman, Fullerton, CA). The same robot was used to generate the drug masterplates containing the triplicates of four different drug dilutions using a single-tip automatic dispenser head.

Table 1.

AUC values of the used drugs and calculated RAUC values for NK cells

Drug groupsDrug nameBrand name, companyConcentrations with NK cells alone (μg/mL)Concentrations with target cells together (μg/mL)AUC in vivo (μg h/mL)In vivo dose (AUC)ReferencesRAUC for NK cells
Topoisomerase inhibitor effect Anthracyclines Epirubicin Epirubicin Meda, Meda 0.02–2.5 0.01–1.245 2.412 90 mg/m2 Fogli et al. (31) 0.17–20.65 
  Daunorubicin Cerubidin®, Aventis Pharma 0.05–6.25 0.02–3.125 1.2786 1.5 mg/kg Andersson et al. (32) 0.75–97.75 
  Doxorubicin Doxorubicin Teva, Teva 0.02–2.5 0.01–1.245 0.82464 50 mg/m2 Toffoli et al. (33) 0.5–60.4 
 Epipodophyllotoxin Etoposide Sigma 0.244–31.25 0.12–15.625 5.06 100 mg/m2/d Gruber et al. (34) 0.97–123.52 
 Camptothecins Topotecan Hycamtin, GlaxoSmithKline 0.04–5 0.02–2.5 0.0196 1.2 mg/m2/d Gerrits et al. (35) 39.85–5,101 
Microtubule inhibitors Taxanes Paclitaxel Taxol, Orifarm 0.06–7.5 0.03–3.75 13.49 175 mg/m2 Fogli et al. (31) 0.08–11.12 
  Docetaxel Taxotere®, Aventis Pharma 0.4–50 0.2–25 3.326 85 mg/m2 Rischin et al. (36) 2.35–301 
 Vinca alkaloids Vincristine Vincristine Mayne, Mayne Pharma 0.005–0.625 0.003–0.312 0.182 1.32 mg/m2 Desai et al. (37) 0.5–68.67 
  Vinblastine Velbe®, STADApharm 0.001–1.25 0.005–0.625 0.218 1.7 mg/m2 Bates et al. (38) 0.9–114.6 
  Vinorelbine Navelbine®, Pierre Fabre 0.098–12.5 0.05–6.25 0.899 80 mg/m2/wk Freyer et al. (39) 2.17–277.94 
 Platinum analogues Carboplatin Carboplatin Mayne, Mayne Pharma 0.098–12.5 0.05–6.25 348000 360 mg/m2 Ghazal-Aswad et al. (40) 5.6 × 10−6–0.0008 
  Oxaliplatin Eloxatin, Sanofi-Synthelabo 0.05–6.25 0.025–3.125 71.5 130 mg/m2 Graham et al. (41) 0.02–1.75 
Antimetabolites Antifolate Methotrexate Methotrexate Pharmacia, Pfizer 0.244–31.25 0.12–15.625 13200000 12 g/m2 Crews et al. (42) 3.7 × 10−7–4.7 × 10−5 
 Purine antagonists 6-Mercaptopurine Sigma 0.814–104.16 0.4–52.08 0.2587 85 mg/m2 Chan et al. (43) 62.8–8,052.57 
  Cladribine Leustatin, Janssen-Cilag 0.01–1.25 0.005–0.625 0.1541 5 mg/m2 Albertioni et al. (44) 1.26–162.17 
 Pyrimidine antagonists 5-Fluorouracil Fluorouracil Mayne, Mayne Pharma 0.488–62.5 0.244–31.245 11.59 400 mg/m2 Casale et al. (45) 0.85–107.83 
  Cytarabine Cytarabine Pfizer, Pfizer 0.977–125 0.5–62.5 523.4 1 g/m2 Gruber et al. (34) 0.04–4.78 
  Gemcitabine Gemzar, Orifarm 0.586–75 0.3–37.5 9.3 1,000 mg/m2 Fogli et al. (31) 1.25–161.3 
 Antitumor antibiotics Bleomycin Bleomycin Baxter, Baxter 0.293–37.5* 0.15–18.75* 0.089* 8 IU/kg/d Peng et al. (46) 65.5–8,427 
  Dactinomycin Cosmegen®, MSD 0.005–0.625 0.003–0.312 300 1.5 mg/m2 Veal et al. (47) 0.00033–0.0416 
 Protease inhibitors Bortezomib Velcade®, Janssen-Cilag 0.01–1.25 0.001–0.625 0.0438 1.45 mg/m2 Papandreou et al. (48) 4.45–571 
  MG-132 Sigma 0.098–12.5 0.05–6.25     
 Alkylating agents Cyclophosphamide Sendoxan, Baxter 0.4–50 0.2–25 367 50 mg/kg Xie et al. (49) 0.02–2.72 
  Ifosphamide Holoxan®, Baxter 0.4–50 0.2–25 1,827.7 3 g/m2/d Boddy et al. (50) 0.005–0.55 
  Chlorambucil Sigma 0.977–125 0.5–62.5 0.883 0.2 mg/m2 (GlaxoSmithKline Research Triangle Park)§ 22.12–2,831 
  Streptozocin Sigma 0.488–62.5 0.24–31.245     
 Miscellaneous Asparaginase Asparaginase Medac, Medac 0.05–6.25* 0.025–3.125* 0.943* 30.000 IU/m2 Ylikangas et al. (51) 1.04–132.53 
  Hydroxyurea Sigma 0.488–62.5 0.24–31.245 82.49 15 mg/kg Yan et al. (52) 0.12–15.15 
  Bevacizumab Avastin®, Roche 0.244–31.25 0.122–15.625     
Drug groupsDrug nameBrand name, companyConcentrations with NK cells alone (μg/mL)Concentrations with target cells together (μg/mL)AUC in vivo (μg h/mL)In vivo dose (AUC)ReferencesRAUC for NK cells
Topoisomerase inhibitor effect Anthracyclines Epirubicin Epirubicin Meda, Meda 0.02–2.5 0.01–1.245 2.412 90 mg/m2 Fogli et al. (31) 0.17–20.65 
  Daunorubicin Cerubidin®, Aventis Pharma 0.05–6.25 0.02–3.125 1.2786 1.5 mg/kg Andersson et al. (32) 0.75–97.75 
  Doxorubicin Doxorubicin Teva, Teva 0.02–2.5 0.01–1.245 0.82464 50 mg/m2 Toffoli et al. (33) 0.5–60.4 
 Epipodophyllotoxin Etoposide Sigma 0.244–31.25 0.12–15.625 5.06 100 mg/m2/d Gruber et al. (34) 0.97–123.52 
 Camptothecins Topotecan Hycamtin, GlaxoSmithKline 0.04–5 0.02–2.5 0.0196 1.2 mg/m2/d Gerrits et al. (35) 39.85–5,101 
Microtubule inhibitors Taxanes Paclitaxel Taxol, Orifarm 0.06–7.5 0.03–3.75 13.49 175 mg/m2 Fogli et al. (31) 0.08–11.12 
  Docetaxel Taxotere®, Aventis Pharma 0.4–50 0.2–25 3.326 85 mg/m2 Rischin et al. (36) 2.35–301 
 Vinca alkaloids Vincristine Vincristine Mayne, Mayne Pharma 0.005–0.625 0.003–0.312 0.182 1.32 mg/m2 Desai et al. (37) 0.5–68.67 
  Vinblastine Velbe®, STADApharm 0.001–1.25 0.005–0.625 0.218 1.7 mg/m2 Bates et al. (38) 0.9–114.6 
  Vinorelbine Navelbine®, Pierre Fabre 0.098–12.5 0.05–6.25 0.899 80 mg/m2/wk Freyer et al. (39) 2.17–277.94 
 Platinum analogues Carboplatin Carboplatin Mayne, Mayne Pharma 0.098–12.5 0.05–6.25 348000 360 mg/m2 Ghazal-Aswad et al. (40) 5.6 × 10−6–0.0008 
  Oxaliplatin Eloxatin, Sanofi-Synthelabo 0.05–6.25 0.025–3.125 71.5 130 mg/m2 Graham et al. (41) 0.02–1.75 
Antimetabolites Antifolate Methotrexate Methotrexate Pharmacia, Pfizer 0.244–31.25 0.12–15.625 13200000 12 g/m2 Crews et al. (42) 3.7 × 10−7–4.7 × 10−5 
 Purine antagonists 6-Mercaptopurine Sigma 0.814–104.16 0.4–52.08 0.2587 85 mg/m2 Chan et al. (43) 62.8–8,052.57 
  Cladribine Leustatin, Janssen-Cilag 0.01–1.25 0.005–0.625 0.1541 5 mg/m2 Albertioni et al. (44) 1.26–162.17 
 Pyrimidine antagonists 5-Fluorouracil Fluorouracil Mayne, Mayne Pharma 0.488–62.5 0.244–31.245 11.59 400 mg/m2 Casale et al. (45) 0.85–107.83 
  Cytarabine Cytarabine Pfizer, Pfizer 0.977–125 0.5–62.5 523.4 1 g/m2 Gruber et al. (34) 0.04–4.78 
  Gemcitabine Gemzar, Orifarm 0.586–75 0.3–37.5 9.3 1,000 mg/m2 Fogli et al. (31) 1.25–161.3 
 Antitumor antibiotics Bleomycin Bleomycin Baxter, Baxter 0.293–37.5* 0.15–18.75* 0.089* 8 IU/kg/d Peng et al. (46) 65.5–8,427 
  Dactinomycin Cosmegen®, MSD 0.005–0.625 0.003–0.312 300 1.5 mg/m2 Veal et al. (47) 0.00033–0.0416 
 Protease inhibitors Bortezomib Velcade®, Janssen-Cilag 0.01–1.25 0.001–0.625 0.0438 1.45 mg/m2 Papandreou et al. (48) 4.45–571 
  MG-132 Sigma 0.098–12.5 0.05–6.25     
 Alkylating agents Cyclophosphamide Sendoxan, Baxter 0.4–50 0.2–25 367 50 mg/kg Xie et al. (49) 0.02–2.72 
  Ifosphamide Holoxan®, Baxter 0.4–50 0.2–25 1,827.7 3 g/m2/d Boddy et al. (50) 0.005–0.55 
  Chlorambucil Sigma 0.977–125 0.5–62.5 0.883 0.2 mg/m2 (GlaxoSmithKline Research Triangle Park)§ 22.12–2,831 
  Streptozocin Sigma 0.488–62.5 0.24–31.245     
 Miscellaneous Asparaginase Asparaginase Medac, Medac 0.05–6.25* 0.025–3.125* 0.943* 30.000 IU/m2 Ylikangas et al. (51) 1.04–132.53 
  Hydroxyurea Sigma 0.488–62.5 0.24–31.245 82.49 15 mg/kg Yan et al. (52) 0.12–15.15 
  Bevacizumab Avastin®, Roche 0.244–31.25 0.122–15.625     

NOTE: Ratio of in vitro and in vivo AUC levels (RAUC) values were formed to calculate relationship between the in vitro drug concentrations and the clinical doses. In vitro AUC values were determined using the following formula: in vitro used concentration (μg/mL) × 20 h.

*

For bleomycin and asparaginase, the concentrations are in international units per milliliter.

Not used in the clinical practice.

No data are available.

§

GlaxoSmithKline Research Triangle Park N. Prescribing Information Leukeran. Available from: http://us.gsk.com/products/assets/us_leukeran.pdf.

The highest drug concentration was selected as the maximum drug concentration physiochemically achievable at 600× dilution (50 nL of drug in a 30-μL assay volume). These concentrations are in a comparable range with the in vivo plasma concentrations in patients (3152).5

5

GlaxoSmithKline Research Triangle Park N. Prescribing Information Leukeran. Available from: http://us.gsk.com/products/assets/us_leukeran.pdf.

The starting concentrations of the dilution series (1×, 5×, 25×, 125×) for the individual drugs were initially determined based on the solubility of the different agents. The fifth to sixth rows of Table 1 represent the drug concentrations that were presented in the wells with NK cells alone and after increasing the volumes with 20-μL cell suspension of target cells.

Testing the drug plates on a large number of in vitro tumor cell lines and primary tumors, it was possible to find sensitive cell lines for each individual drugs, showing that all the drugs on the plate were active (data not shown).

A way to calculate a relationship between the in vitro drug concentrations and the in vivo ones is to use the area under the curve (AUC) values of the individual drugs. For this comparison, ratio of area under the curve (RAUC) values were determined by the following formula: in vitro used concentration (μg/mL) × 20 h/ AUC (μg h/mL) in vivo. A RAUC value >1 indicates that the in vitro used dose is higher than the one used in clinical practice. If this value is 1, it means that our in vitro dose corresponds to the clinical dose. In vivo AUC levels, clinical doses, and calculated RAUC values of the individual drugs from the literature are summarized in Table 1. RAUC values are also shown in Fig. 2.

Figure 2.

RAUC values for NK cells (data are also represented in Table 1). To calculate relationship between the in vitro used drug concentrations and the in vivo used values, RAUC values were determined by the following formula: in vitro used concentration (μg/mL) × 20 h / AUC (μg h/mL) in vivo. RAUC > 1, the in vitro dose is higher than the clinical dose. RAUC < 1, the in vitro dose is lower than the clinical dose. RAUC = 1, the in vitro dose is corresponding to the clinical dose.

Figure 2.

RAUC values for NK cells (data are also represented in Table 1). To calculate relationship between the in vitro used drug concentrations and the in vivo used values, RAUC values were determined by the following formula: in vitro used concentration (μg/mL) × 20 h / AUC (μg h/mL) in vivo. RAUC > 1, the in vitro dose is higher than the clinical dose. RAUC < 1, the in vitro dose is lower than the clinical dose. RAUC = 1, the in vitro dose is corresponding to the clinical dose.

Close modal

Comparison of the Novel In vitro Killing Assay to the Standard Chromium-51 Release Assay

The two methods were compared with each other in the absence of drugs under the same conditions (incubation time, effector-to-target ratio, flat-bottomed plates instead of V-bottomed plates). The results of our single-cell fluorescence method correlate closely to the standard chromium-51 release assay on different NK cell lines. Figure 3 shows the in vitro killing capacity of a NK tumor cell line (NKL) and two NK cell clones tested with both methods.

Figure 3.

Comparing of the fluorescent in vitro killing assay to the standard chromium-51 release assay. NKL cell line and two IL-2–activated primary NK cell polyclonal preparations were incubated together with K562 target cells for 5 h. Effector-to-target ratio was always 5:1. KE, killing effectiveness.

Figure 3.

Comparing of the fluorescent in vitro killing assay to the standard chromium-51 release assay. NKL cell line and two IL-2–activated primary NK cell polyclonal preparations were incubated together with K562 target cells for 5 h. Effector-to-target ratio was always 5:1. KE, killing effectiveness.

Close modal

Effect of Drugs on the Killing Capacity of Different NK Cells

The eight different primary NK cell polyclonal preparations from healthy donors and four NK tumor cell lines that were tested with the novel fluorescent method in the present study represent a variety of cells with different origin and in vitro history. The investigation included separated NK cells from healthy donors with 1 week of in vitro culturing together with IL-2 along with freshly separated NK cells cultured with IL-2 only for 24 h. NK tumor cell lines showed relatively low killing efficiency (≤10%) in our novel experimental model system. Microscopy evaluation of killing activity requires the use of flat-bottomed plate instead of conventional V-bottomed plates. Many of the NK tumor lines showed low spontaneous mobility and required high-level cellular crowding to be able to kill effectively. Control killing effectiveness values for all investigated NK cell lines can be seen in Table 2.

Table 2.

Killing effectiveness of different NK cell lines

KE (%)SD
NK cells treated with IL-2 for 1 wk   
    Clone 1 27.93 5.01 
    Clone 2 64.16 8.49 
    Clone 3 77.47 14.27 
    Clone 4 20.92 2.5 
    Clone 5 24.61 4.92 
NK cells treated with IL-2 for 1 d   
    Clone 1 55.99 4.67 
    Clone 2 41.05 3.56 
    Clone 3 31.83 7.01 
NK tumor cell lines   
    Nishi 5.9 2.53 
    NKL 8.55 1.54 
    NK92 10.05 4.48 
    KHYG-1 9.94 3.06 
KE (%)SD
NK cells treated with IL-2 for 1 wk   
    Clone 1 27.93 5.01 
    Clone 2 64.16 8.49 
    Clone 3 77.47 14.27 
    Clone 4 20.92 2.5 
    Clone 5 24.61 4.92 
NK cells treated with IL-2 for 1 d   
    Clone 1 55.99 4.67 
    Clone 2 41.05 3.56 
    Clone 3 31.83 7.01 
NK tumor cell lines   
    Nishi 5.9 2.53 
    NKL 8.55 1.54 
    NK92 10.05 4.48 
    KHYG-1 9.94 3.06 

Abbreviation: KE, killing effectiveness.

The NK cells of different origins showed a very similar killing efficacy pattern in the presence of most of the drugs. This phenomenon is illustrated in Fig. 4, summarizing the killing effectiveness of all NK cell populations in the presence of 29 drugs at four different concentrations. Each individual thin curve represents the average of three independent measurements. The thick broken line is the mean of all curves, the mean killing efficacy. The curves labeled by black spots are NK cell isolates, treated with IL-2 only for 1 day. The gray shaded area marks the SD. Figure 5 illustrates the cytotoxic effect (%) of all of the drugs on NK cells, as shown in a similar way in Fig. 4. All the mean killing efficacy values are represented in Table 3. Importantly, none of the drugs influenced the viability of the K562 target cells during the 5-h incubation (data not shown). The number of dead NK cells in the drug-treated cultures that received IL-2 only for 1 day was higher than those in the drug-treated cultures that received IL-2 for 1 week. None of the NK populations showed dose-dependent increase in the number of dead cells. Consequently, the dose-dependent decrease in the killing effect of NK cells in case of certain drugs cannot be simply explained by drug-induced NK cell death (Fig. 5).

Figure 4.

Killing effectiveness of NK cells tested with the fluorescent killing assay. The figure summarizes the killing effectiveness of all NK cell populations in the presence of 29 drugs at four different concentrations. Thin curves, average of three independent measurements. Thick broken line, mean of all curves, the mean killing efficacy (MKE). Curves with black spots, NK cell isolates that were treated with IL-2 only for 1 d. Gray shaded area, SD. A, effective drugs (lowest mean killing efficacy, <60%). B, marginally effective drugs (lowest mean killing efficacy, 60–70%). C, noneffective drugs (lowest mean killing efficacy, >70%). X axis, increasing concentrations of the drug. Y axis, killing effectiveness of NK cells 0% to 120%.

Figure 4.

Killing effectiveness of NK cells tested with the fluorescent killing assay. The figure summarizes the killing effectiveness of all NK cell populations in the presence of 29 drugs at four different concentrations. Thin curves, average of three independent measurements. Thick broken line, mean of all curves, the mean killing efficacy (MKE). Curves with black spots, NK cell isolates that were treated with IL-2 only for 1 d. Gray shaded area, SD. A, effective drugs (lowest mean killing efficacy, <60%). B, marginally effective drugs (lowest mean killing efficacy, 60–70%). C, noneffective drugs (lowest mean killing efficacy, >70%). X axis, increasing concentrations of the drug. Y axis, killing effectiveness of NK cells 0% to 120%.

Close modal
Figure 5.

Cytotoxic effect on NK cells tested with the fluorescent killing assay. Thin curves, average of three independent measurements. Curves with black spots, NK cell isolates treated with IL-2 only for 1 d. The cytotoxic effect (%) of the drugs on NK cells was calculated using the following formula: (number of dead NK cells in the wells with drugs / number of dead NK cells in the control wells) × 100%. [Cytotoxic effect (%) of >100% means more dead NK cells.] A, effective drugs. B, marginally effective drugs. C, noneffective drugs. X axis, increasing concentrations of the drug. Y axis, cytotoxic effect of the drug compared with the control 0% to 500% (control means NK cell survival without the drug, which is 100%).

Figure 5.

Cytotoxic effect on NK cells tested with the fluorescent killing assay. Thin curves, average of three independent measurements. Curves with black spots, NK cell isolates treated with IL-2 only for 1 d. The cytotoxic effect (%) of the drugs on NK cells was calculated using the following formula: (number of dead NK cells in the wells with drugs / number of dead NK cells in the control wells) × 100%. [Cytotoxic effect (%) of >100% means more dead NK cells.] A, effective drugs. B, marginally effective drugs. C, noneffective drugs. X axis, increasing concentrations of the drug. Y axis, cytotoxic effect of the drug compared with the control 0% to 500% (control means NK cell survival without the drug, which is 100%).

Close modal
Table 3.

Mean killing effectiveness of NK cells in the presence of different drugs

125× dilution25× dilution5× dilutionHighest concentration125× dilution25× dilution5× dilutionHighest concentration
Epirubicin     Paclitaxel     
    MKE (%) 98.52 87.28 85.24 79.66     MKE (%) 89.43 85.03 77.36 54.97 
    SD 11.45 20.67 22.28 21.91     SD 9.87 16.00 19.37 24.69 
Daunorubicin     Docetaxel     
    MKE (%) 92.29 87.61 83.01 68.43     MKE (%) 68.49 60.11 51.97 39.85 
    SD 18.22 22.09 24.92 30.94     SD 19.35 24.56 18.40 16.46 
Doxorubicin     Vincristine     
    MKE (%) 84.77 81.47 78.30 75.29     MKE (%) 85.61 82.92 72.76 69.32 
    SD 16.55 17.25 18.15 30.55     SD 20.38 19.56 18.43 20.86 
Etoposide     Vinblastine     
    MKE (%) 97.15 101.54 90.41 85.49     MKE (%) 94.98 95.51 83.78 58.15 
    SD 13.66 11.38 19.18 20.52     SD 9.66 12.13 18.38 16.69 
Topotecan     Vinorelbine     
    MKE (%) 81.25 79.76 72.78 69.60     MKE (%) 92.91 93.43 82.14 79.76 
    SD 14.95 23.82 21.74 28.46     SD 22.12 21.90 21.63 21.96 
Methotrexate     Carboplatin     
    MKE (%) 101.79 95.65 85.15 80.89     MKE (%) 90.87 89.36 85.42 80.62 
    SD 14.39 11.99 23.38 18.75     SD 23.03 25.56 34.35 25.91 
6-Mercaptopurine     Oxaliplatin     
    MKE (%) 99.16 103.02 95.28 92.25     MKE (%) 71.79 72.72 62.47 65.78 
    SD 14.60 12.08 14.46 17.24     SD 18.56 20.61 19.54 18.20 
Cladribine     Bleomycin     
    MKE (%) 89.87 81.83 56.65 47.66     MKE (%) 85.54 76.67 70.38 74.41 
    SD 12.89 14.85 12.82 18.89     SD 17.85 16.97 13.54 19.01 
Fluorouracil     Dactinomycin     
    MKE (%) 96.24 100.34 92.61 87.67     MKE (%) 95.06 87.14 75.95 67.18 
    SD 15.90 18.31 15.49 25.05     SD 17.25 17.06 21.22 23.69 
Cytarabine     Bortezomib     
    MKE (%) 87.38 89.48 76.69 68.42     MKE (%) 97.76 94.86 88.10 56.40 
    SD 15.52 11.12 20.15 28.22     SD 22.20 14.83 29.71 25.26 
Gemcitabine     MG-132     
    MKE (%) 83.92 74.97 60.22 58.12     MKE (%) 94.95 85.47 64.43 15.79 
    SD 17.96 19.41 19.41 25.37     SD 18.81 20.96 17.03 6.67 
Cyclophosphamide     Asparaginase     
    MKE (%) 100.39 95.74 91.91 93.40     MKE (%) 93.40 94.73 81.18 87.03 
    SD 6.79 19.12 17.13 18.21     SD 22.02 24.74 19.73 18.42 
Ifosphamide     Hydroxyurea     
    MKE (%) 99.42 95.93 94.26 83.80     MKE (%) 90.27 94.93 84.75 86.42 
    SD 7.06 9.50 15.95 16.78     SD 7.51 10.20 12.82 22.98 
Chlorambucil     Bevacizumab     
    MKE (%) 84.79 83.86 19.81 13.14     MKE (%) 86.96 80.60 75.82 74.41 
    SD 17.64 22.51 6.37 6.67     SD 15.19 14.59 18.07 18.46 
Streptozocin          
    MKE (%) 96.02 99.77 95.52 93.34      
    SD 16.86 17.94 19.45 19.09      
125× dilution25× dilution5× dilutionHighest concentration125× dilution25× dilution5× dilutionHighest concentration
Epirubicin     Paclitaxel     
    MKE (%) 98.52 87.28 85.24 79.66     MKE (%) 89.43 85.03 77.36 54.97 
    SD 11.45 20.67 22.28 21.91     SD 9.87 16.00 19.37 24.69 
Daunorubicin     Docetaxel     
    MKE (%) 92.29 87.61 83.01 68.43     MKE (%) 68.49 60.11 51.97 39.85 
    SD 18.22 22.09 24.92 30.94     SD 19.35 24.56 18.40 16.46 
Doxorubicin     Vincristine     
    MKE (%) 84.77 81.47 78.30 75.29     MKE (%) 85.61 82.92 72.76 69.32 
    SD 16.55 17.25 18.15 30.55     SD 20.38 19.56 18.43 20.86 
Etoposide     Vinblastine     
    MKE (%) 97.15 101.54 90.41 85.49     MKE (%) 94.98 95.51 83.78 58.15 
    SD 13.66 11.38 19.18 20.52     SD 9.66 12.13 18.38 16.69 
Topotecan     Vinorelbine     
    MKE (%) 81.25 79.76 72.78 69.60     MKE (%) 92.91 93.43 82.14 79.76 
    SD 14.95 23.82 21.74 28.46     SD 22.12 21.90 21.63 21.96 
Methotrexate     Carboplatin     
    MKE (%) 101.79 95.65 85.15 80.89     MKE (%) 90.87 89.36 85.42 80.62 
    SD 14.39 11.99 23.38 18.75     SD 23.03 25.56 34.35 25.91 
6-Mercaptopurine     Oxaliplatin     
    MKE (%) 99.16 103.02 95.28 92.25     MKE (%) 71.79 72.72 62.47 65.78 
    SD 14.60 12.08 14.46 17.24     SD 18.56 20.61 19.54 18.20 
Cladribine     Bleomycin     
    MKE (%) 89.87 81.83 56.65 47.66     MKE (%) 85.54 76.67 70.38 74.41 
    SD 12.89 14.85 12.82 18.89     SD 17.85 16.97 13.54 19.01 
Fluorouracil     Dactinomycin     
    MKE (%) 96.24 100.34 92.61 87.67     MKE (%) 95.06 87.14 75.95 67.18 
    SD 15.90 18.31 15.49 25.05     SD 17.25 17.06 21.22 23.69 
Cytarabine     Bortezomib     
    MKE (%) 87.38 89.48 76.69 68.42     MKE (%) 97.76 94.86 88.10 56.40 
    SD 15.52 11.12 20.15 28.22     SD 22.20 14.83 29.71 25.26 
Gemcitabine     MG-132     
    MKE (%) 83.92 74.97 60.22 58.12     MKE (%) 94.95 85.47 64.43 15.79 
    SD 17.96 19.41 19.41 25.37     SD 18.81 20.96 17.03 6.67 
Cyclophosphamide     Asparaginase     
    MKE (%) 100.39 95.74 91.91 93.40     MKE (%) 93.40 94.73 81.18 87.03 
    SD 6.79 19.12 17.13 18.21     SD 22.02 24.74 19.73 18.42 
Ifosphamide     Hydroxyurea     
    MKE (%) 99.42 95.93 94.26 83.80     MKE (%) 90.27 94.93 84.75 86.42 
    SD 7.06 9.50 15.95 16.78     SD 7.51 10.20 12.82 22.98 
Chlorambucil     Bevacizumab     
    MKE (%) 84.79 83.86 19.81 13.14     MKE (%) 86.96 80.60 75.82 74.41 
    SD 17.64 22.51 6.37 6.67     SD 15.19 14.59 18.07 18.46 
Streptozocin          
    MKE (%) 96.02 99.77 95.52 93.34      
    SD 16.86 17.94 19.45 19.09      

NOTE: Data were also used for preparing Fig. 4. MKE (%), mean killing effectiveness.

Identification of Highly Effective and Noneffective Drugs

We found that the most effective drugs that inhibited NK cell–mediated killing were chlorambucil, MG-132, docetaxel, cladribine, paclitaxel, bortezomib, gemcitabine, and vinblastine (Fig. 4). For MG-132, no RAUC value could be defined because this drug has not been clinically used. Oxaliplatin, dactinomycin, cytarabine, daunorubicin, vincristine, and topotecan were only marginally effective.

Most NK cell lines were not affected by bevacizumab, bleomycin, doxorubicin, epirubicin, vinorelbine, carboplatin, methotrexate, ifosphamide, etoposide, hydroxyurea, asparaginase, 5-fluorouracil, 6-mercaptopurine, streptozocin, and cyclophosphamide.

In our experimental model, the killing effectiveness of NK cells was tested in the presence of different cytostatic drugs. Previous studies investigated NK cell–mediated cytotoxicity following treatment of target cells with certain cytotoxic drugs (25) or treatment of NK cells with a few chemotherapeutic agents before the NK cell killing assays (24, 26). No systematic study has been carried out to investigate the effect of a large number of different drugs at different concentrations.

In our experimental setup, the NK cells were preincubated alone for 20 h with the drugs to test the drug effect only on the NK cells. This was followed by coincubation with the target cells for 5 h. No drug-induced cytotoxicity was detectable on target cells during this incubation, although other effects on target cells, which influence NK cell killing, cannot be excluded.

The presented data suggest that IL-2–activated NK cell lines share a common profile of cytotoxic drug sensitivity. This profile does not change with different lengths of in vitro culturing with IL-2.

Topotecan, 6-mercaptopurine, and bleomycin did not inhibit NK cell killing effectively even at very high AUC levels. Almost no effect could be seen in case of carboplatin, methotrexate, and dactinomycin, although this might be explained by the relatively low range of RAUC levels achieved in the microcultures.

Although chlorambucil efficiently inhibited the killing effect of NK cells, other alkylating agents like cyclophosphamide and ifosphamide did not affect NK cell killing. This might be explained by the fact that both of the latter compounds are prodrugs that have to be converted into active metabolites by the liver in vivo.

The effectiveness of chlorambucil may also be explained by the high in vitro RAUC levels (Fig. 2; Table 1). The in vitro doses (in vitro AUC levels) of the other effective drugs were in close vicinity to the clinical doses.

Unlike Reiter et al. (53) who found no inhibition of NK cell killing ability when effector cells (NK cells) alone were treated with cladribine, this agent was the only drug among the base analogues that significantly decreased the killing effect of IL-2–activated NK cells in our experimental setup.

Our data also showed that NK cells are particularly sensitive to antimicrotubule drugs and proteosome inhibitors. The dependence of NK cell–mediated killing on microtubule integrity is well known (24, 26). Here we show that different microtubule inhibitors have a variable effect on NK cell–mediated killing. Vinorelbine and vincristine had much less effect on NK cell–mediated killing than vinblastine, docetaxel, or paclitaxel.

Several studies showed that the chymotryptic activity of proteasomes in NK cells might play a role in their cell-mediated cytotoxicity (5456). Kitson et al. (54) reported that synthetic proteasome inhibitors like CEP-1508, CEP-1612, and CEP-3117 can inhibit the rat NK proteasome in a dose-dependent manner and found a 50% inhibition of IL-2–activated NK cell–mediated cytotoxicity. Lu et al. (57) pointed out that MG-132 could reduce NK cell–mediated cytotoxicity of rat NK cells, but this was due to the apoptosis-inducing property of the drug. Here, we show that proteosome inhibitors severely compromise the killing efficiency of human NK cells even in the absence of apoptosis induction.

Bortezomib is a clinically used reversible inhibitor of the chymotrypsin-like activity of the 26S proteasome. A study showed that bortezomib can sensitize tumor cells to the lytic effects of dendritic cell–activated immune effector cells like NK cells or CD8+ lymphocytes (58), but no data have been published on the direct inhibition effect of bortezomib on NK cell–mediated cytotoxicity.

Our results suggest that chemotherapy protocols that include proteosome inhibitors or antimicrotubule drugs may interfere with NK cell–based immunotherapy, if applied simultaneously.

Based on the almost complete absence of inhibitory effect on NK cell–mediated killing even at high RAUC levels (concentrations that are directly comparable with maximally achieved in vivo therapeutic doses), we suggest that the following drugs may be effectively combined with adjuvant NK cell–based immunotherapy: asparaginase, bevacizumab, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea, streptozocin, and 6-mercaptopurine.

Several anticancer drugs have been reported to induce the expression of ligands for activating NK cell receptors on the tumor cell membrane potentially increasing NK cell–mediated cytotoxicity against tumor cells. This information, in combination with the data presented here, could potentially be used in the future to design new NK cell–based adjuvant immunotherapy protocols.

Grant support: The Swedish Cancer Society, the Swedish Research Council, and the Integrative Recognition in the Immune System Center.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: L. Markasz and G. Stuber contributed equally to this work.

1
Menon AG, Morreau H, Tollenaar RA, et al. Down-regulation of HLA-A expression correlates with a better prognosis in colorectal cancer patients.
Lab Invest
2002
;
82
:
1725
–33.
2
Jackson PA, Green MA, Marks CG, King RJ, Hubbard R, Cook MG. Lymphocyte subset infiltration patterns and HLA antigen status in colorectal carcinomas and adenomas.
Gut
1996
;
38
:
85
–9.
3
Dolcetti R, Viel A, Doglioni C, et al. High prevalence of activated intraepithelial cytotoxic T lymphocytes and increased neoplastic cell apoptosis in colorectal carcinomas with microsatellite instability.
Am J Pathol
1999
;
154
:
1805
–13.
4
Coca S, Perez-Piqueras J, Martinez D, et al. The prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma.
Cancer
1997
;
79
:
2320
–8.
5
Ishigami S, Natsugoe S, Tokuda K, et al. Prognostic value of intratumoral natural killer cells in gastric carcinoma.
Cancer
2000
;
88
:
577
–83.
6
Trinchieri G. Biology of natural killer cells.
Adv Immunol
1989
;
47
:
187
–376.
7
Robertson MJ, Ritz J. Biology and clinical relevance of human natural killer cells.
Blood
1990
;
76
:
2421
–38.
8
Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer.
Nat Rev Immunol
2001
;
1
:
41
–9.
9
Kiessling R, Klein E, Pross H, Wigzell H. “Natural” killer cells in the mouse. II. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Characteristics of the killer cell.
Eur J Immunol
1975
;
5
:
117
–21.
10
Davies SM, Ruggieri L, DeFor T, et al. Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Killer immunoglobulin-like receptor.
Blood
2002
;
100
:
3825
–7.
11
Miller JS, Soignier Y, Panoskaltsis-Mortari A, et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer.
Blood
2005
;
105
:
3051
–7.
12
Ruggeri L, Mancusi A, Perruccio K, Burchielli E, Martelli MF, Velardi A. Natural killer cell alloreactivity for leukemia therapy.
J Immunother
2005
;
28
:
175
–82.
13
Lanier LL. NK cell recognition.
Annu Rev Immunol
2005
;
23
:
225
–74.
14
Caligiuri MA, Velardi A, Scheinberg DA, Borrello IM. Immunotherapeutic approaches for hematologic malignancies.
Hematology Am Soc Hematol Educ Program
2004
;
1
:
337
–53.
15
VanDeusen JB, Caligiuri MA. New developments in anti-tumor efficacy and malignant transformation of human natural killer cells.
Curr Opin Hematol
2003
;
10
:
55
–9.
16
Klingemann HG. Cellular therapy of cancer with natural killer cells: will it ever work?
J Hematother Stem Cell Res
2001
;
10
:
23
–6.
17
Basse PH, Goldfarb RH, Herberman RB, Hokland ME. Accumulation of adoptively transferred A-NK cells in murine metastases: kinetics and role of interleukin-2.
In Vivo
1994
;
8
:
17
–24.
18
Rosenberg SA, Lotze MT, Muul LM, et al. Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer.
N Engl J Med
1985
;
313
:
1485
–92.
19
Kimura H, Yamaguchi Y. A phase III randomized study of interleukin-2 lymphokine-activated killer cell immunotherapy combined with chemotherapy or radiotherapy after curative or noncurative resection of primary lung carcinoma.
Cancer
1997
;
80
:
42
–9.
20
Farag SS, George SL, Lee EJ, et al. Postremission therapy with low-dose interleukin 2 with or without intermediate pulse dose interleukin 2 therapy is well tolerated in elderly patients with acute myeloid leukemia: Cancer and Leukemia Group B study 9420.
Clin Cancer Res
2002
;
8
:
2812
–9.
21
Meropol NJ, Barresi GM, Fehniger TA, Hitt J, Franklin M, Caligiuri MA. Evaluation of natural killer cell expansion and activation in vivo with daily subcutaneous low-dose interleukin-2 plus periodic intermediate-dose pulsing.
Cancer Immunol Immunother
1998
;
46
:
318
–26.
22
Harker WG, Tom C, McGregor JR, Slade L, Samlowski WE. Human tumor cell line resistance to chemotherapeutic agents does not predict resistance to natural killer or lymphokine-activated killer cell-mediated cytolysis.
Cancer Res
1990
;
50
:
5931
–6.
23
Komada Y, Zhang SL, Zhou YW, et al. Cellular immunosuppression in children with acute lymphoblastic leukemia: effect of consolidation chemotherapy.
Cancer Immunol Immunother
1992
;
35
:
271
–6.
24
Ujhazy P, Babusikova O. NK-cell activity affected by some cytostatic drugs and their additives.
Neoplasma
1991
;
38
:
303
–12.
25
Utsugi T, Demuth S, Hanna N. Synergistic antitumor effects of topoisomerase inhibitors and natural cell-mediated cytotoxicity.
Cancer Res
1989
;
49
:
1429
–33.
26
Katz P, Zaytoun AM, Lee JH, Jr. Mechanisms of human cell-mediated cytotoxicity. III. Dependence of natural killing on microtubule and microfilament integrity.
J Immunol
1982
;
129
:
2816
–25.
27
Cerboni C, Mousavi-Jazi M, Wakiguchi H, Carbone E, Karre K, Soderstrom K. Synergistic effect of IFN-γ and human cytomegalovirus protein UL40 in the HLA-E-dependent protection from NK cell-mediated cytotoxicity.
Eur J Immunol
2001
;
31
:
2926
–35.
28
Cerboni C, Achour A, Warnmark A, et al. Spontaneous mutations in the human CMV HLA class I homologue UL18 affect its binding to the inhibitory receptor LIR-1/ILT2/CD85j.
Eur J Immunol
2006
;
36
:
732
–41.
29
Standeven LJ, Carlin LM, Borszcz P, Davis DM, Burshtyn DN. The actin cytoskeleton controls the efficiency of killer Ig-like receptor accumulation at inhibitory NK cell immune synapses.
J Immunol
2004
;
173
:
5617
–25.
30
Suck G, Branch DR, Smyth MJ, et al. KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity.
Exp Hematol
2005
;
33
:
1160
–71.
31
Fogli S, Danesi R, Gennari A, Donati S, Conte PF, Del Tacca M. Gemcitabine, epirubicin and paclitaxel: pharmacokinetic and pharmacodynamic interactions in advanced breast cancer.
Ann Oncol
2002
;
13
:
919
–27.
32
Andersson B, Andersson I, Beran M, Ehrsson H, Eksborg S. Liquid chromatographic monitoring of daunorubicin and daunorubicinol in plasma from leukemic patients treated with daunorubicin or the daunorubicin-DNA complex.
Cancer Chemother Pharmacol
1979
;
2
:
15
–7.
33
Toffoli G, Corona G, Cattarossi G, et al. Effect of highly active antiretroviral therapy (HAART) on pharmacokinetics and pharmacodynamics of doxorubicin in patients with HIV-associated non-Hodgkin's lymphoma.
Ann Oncol
2004
;
15
:
1805
–9.
34
Gruber A, Liliemark E, Tidefelt U, et al. Pharmacokinetics of mitoxantrone, etoposide and cytosine arabinoside in leukemic cells during treatment of acute myelogenous leukemia-relationship to treatment outcome and bone marrow toxicity.
Leuk Res
1995
;
19
:
757
–61.
35
Gerrits CJ, Schellens JH, Burris H, et al. A comparison of clinical pharmacodynamics of different administration schedules of oral topotecan (Hycamtin).
Clin Cancer Res
1999
;
5
:
69
–75.
36
Rischin D, Ackland SP, Smith J, et al. Phase I and pharmacokinetic study of docetaxel in combination with epirubicin and cyclophosphamide in advanced cancer: dose escalation possible with granulocyte colony-stimulating factor, but not with prophylactic antibiotics.
Ann Oncol
2002
;
13
:
1810
–8.
37
Desai ZR, Van den Berg HW, Bridges JM, Shanks RG. Can severe vincristine neurotoxicity be prevented?
Cancer Chemother Pharmacol
1982
;
8
:
211
–4.
38
Bates SE, Bakke S, Kang M, et al. A phase I/II study of infusional vinblastine with the P-glycoprotein antagonist valspodar (PSC 833) in renal cell carcinoma.
Clin Cancer Res
2004
;
10
:
4724
–33.
39
Freyer G, Delozier T, Lichinister M, et al. Phase II study of oral vinorelbine in first-line advanced breast cancer chemotherapy.
J Clin Oncol
2003
;
21
:
35
–40.
40
Ghazal-Aswad S, Calvert AH, Newell DR. A single-sample assay for the estimation of the area under the free carboplatin plasma concentration versus time curve.
Cancer Chemother Pharmacol
1996
;
37
:
429
–34.
41
Graham MA, Lockwood GF, Greenslade D, Brienza S, Bayssas M, Gamelin E. Clinical pharmacokinetics of oxaliplatin: a critical review.
Clin Cancer Res
2000
;
6
:
1205
–18.
42
Crews KR, Liu T, Rodriguez-Galindo C, et al. High-dose methotrexate pharmacokinetics and outcome of children and young adults with osteosarcoma.
Cancer
2004
;
100
:
1724
–33.
43
Chan GL, Erdmann GR, Gruber SA, et al. Pharmacokinetics of 6-thiouric acid and 6-mercaptopurine in renal allograft recipients after oral administration of azathioprine.
Eur J Clin Pharmacol
1989
;
36
:
265
–71.
44
Albertioni F, Lindemalm S, Reichelova V, et al. Pharmacokinetics of cladribine in plasma and its 5′-monophosphate and 5′-triphosphate in leukemic cells of patients with chronic lymphocytic leukemia.
Clin Cancer Res
1998
;
4
:
653
–8.
45
Casale F, Canaparo R, Serpe L, et al. Plasma concentrations of 5-fluorouracil and its metabolites in colon cancer patients.
Pharmacol Res
2004
;
50
:
173
–9.
46
Peng YM, Alberts DS, Chen HS, Mason N, Moon TE. Antitumour activity and plasma kinetics of bleomycin by continuous and intermittent administration.
Br J Cancer
1980
;
41
:
644
–7.
47
Veal GJ, Cole M, Errington J, et al. Pharmacokinetics of dactinomycin in a pediatric patient population: a United Kingdom Children's Cancer Study Group Study.
Clin Cancer Res
2005
;
11
:
5893
–9.
48
Papandreou CN, Daliani DD, Nix D, et al. Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer.
J Clin Oncol
2004
;
22
:
2108
–21.
49
Xie H, Griskevicius L, Stahle L, et al. Pharmacogenetics of cyclophosphamide in patients with hematological malignancies.
Eur J Pharm Sci
2006
;
27
:
54
–61.
50
Boddy AV, Yule SM, Wyllie R, Price L, Pearson AD, Idle JR. Intrasubject variation in children of ifosfamide pharmacokinetics and metabolism during repeated administration.
Cancer Chemother Pharmacol
1996
;
38
:
147
–54.
51
Ylikangas P, Mononen I. Serious neutropenia in ALL patients treated with l-asparaginase may be avoided by therapeutic monitoring of the enzyme activity in the circulation.
Ther Drug Monit
2002
;
24
:
502
–6.
52
Yan JH, Ataga K, Kaul S, et al. The influence of renal function on hydroxyurea pharmacokinetics in adults with sickle cell disease.
J Clin Pharmacol
2005
;
45
:
434
–45.
53
Reiter Z, Tomson S, Ozes ON, Taylor MW. Combination treatment of 2-chlorodeoxyadenosine and type I interferon on hairy cell leukemia-like cells: cytotoxic effect and MHC-unrestricted killer cell regulation.
Blood
1993
;
81
:
1699
–708.
54
Kitson RP, Miller CA, Goldfarb RH. Non-granular proteolytic enzymes of rat interleukin-2-activated natural killer cells. III. Enhancement of A-NKP 1, 2, and 3 proteolytic activities (cleaving after Arg, Phe and Pro) in response to interleukin-2.
Nat Immun
1995
;
14
:
286
–94.
55
Wasserman K, Kitson RP, Rivett AJ, et al. Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. II. Purification and identification of rat A-NKP 1 and A-NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex.
J Cell Biochem
1994
;
55
:
133
–45.
56
Goldfarb RH, Wasserman K, Herberman RB, Kitson RP. Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. I. Subcellular localization and functional role.
J Immunol
1992
;
149
:
2061
–8.
57
Lu M, Kitson RP, Xue Y, Goldfarb RH. Activation of multiple caspases and modification of cell surface fas (CD95) in proteasome inhibitor-induced apoptosis of rat natural killer cells.
J Cell Biochem
2003
;
88
:
482
–92.
58
Schumacher LY, Vo DD, Garban HJ, et al. Immunosensitization of tumor cells to dendritic cell-activated immune responses with the proteasome inhibitor bortezomib (PS-341, Velcade).
J Immunol
2006
;
176
:
4757
–65.