Microcalorimetric Study of the Metabolism of U-937 Cells

http://www.paper.edu.cn

Journal of Thermal Biology 27 (2002) 129–135

Microcalorimetric study of the metabolism of U-937 cells undergoing apoptosis induced by the combined treatment of hyperthermia and chemotherapy Liu Yuwena, Wang Cunxina, Zheng Congyib,*, Wu Haixiangb, Wang Zhiyonga, Qu Songshenga

a College of Chemistry and Molecule Science, Wuhan University, Wuhan, 430072, China b College of Life Science, Wuhan University, Wuhan, 430072, China

Received 20 January 2001; received in revised form 7 April 2001; accepted 19 May 2001

Abstract

Hyperthermia is a useful adjunct in cancer therapy, as it can increase the effectiveness and decrease the toxicity of currently available cancer treatments such as chemotherapy and radiation. In this study we determined the power-time curves of U-937 cell line treated by the combination of hyperthermia and Carmofur by using an LKB 2277 Bioactivity Monitor. The maximal thermal power and the heat production were used to evaluate the antitumor effect. Our results show that the combined treatment of hyperthermia and Carmofur had a synergistic antitumor effect, which is consistent with the apoptosis ratio obtained by TUNEL assay. The results also indicate that the metabolic activity of apoptotic cells is lower than that of normal cells. Thus microcalorimetry is a powerful tool in fields of hyperthermia. r 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Microcalorimetry; Hyperthermia; Metabolism; Apoptosis; TUNEL

1. Introduction requires a detailed understanding of the role of exposure conditions such as temperature, length of heating time Hyperthermia has been found to greatly enhance and the dose of antitumor drugs, etc. Microcalorimetry cancer cell killing and potentiate the action of several is a powerful tool to solve these problems. It provides a antitumor agents, including bleomycin (Hahn, 1979), continuous measurement of heat production, thus cyclophosphamide (Urano et al., 1985), mitomycin C indicating useful information about the activity of the (Barlogie et al., 1980), cisplatin (Murthy et al., 1987), total metabolism of the sample under examination in and Adriamycin (Bates and Mackillop, 1986). Its both a qualitative and quantitative way (Karnebogen application enables the use of lower doses of chemother- et al., 1993). apy or radiation, thus decreasing the toxicity of such The various metabolic events occurring within cells cancer treatments. The use of long duration, mild are heat-producing reactions and the amount of heat is temperature hyperthermia in combination with che- related to the sum of process taking place in cells. The motherapy or radiation in the treatment of cancer has maximal thermal power and the total heat production in been developed (Zhu et al., 1995; Wilder et al., 1993; the calorimetric experiments can be used to evaluate the Wiedemann et al., 1993). However, successful applica- metabolic activity of cells, therefore, it can be the tion of this form of hyperthermia in clinical cases foundation for us to study the antitumor eﬀect of combination of hyperthermia and Carmofur. *Corresponding author. fax: +86-27-8788-3833. In a previous publication from this laboratory, E-mail address: [email protected] (Z. Congyi). microcalorimetry was employed to study heat sensitivity

130 L. Yuwen et al. / Journal of Thermal Biology 27 (2002) 129–135 of tumor cells combined with other methods (Feng et al., serum (GIBCO Co., U.S.A.) with 100 IU ml1 penicillin 1997). The results demonstrated that temperature has a and 100 mgml1 streptomycin added (pH=7.2–7.3). The remarkably selective inhibitory action against tumor same medium was used for the calorimetric experiments. cells. Carmofur, obtained from Mitsui Seiyaku, Japan, and This paper describes the application of microcalori- purified by recrystallization twice in acetone (analytical metric method in pharmacology and drug effect evalua- pure). TUNEL assays were carried out by using tion. It is a quick, quantitative, inexpensive and versatile ApoAlert DNA Fragmentation Assay Kit (CLON- method to determine the therapeutic effectiveness of TECH Laboratories, Inc., USA). certain treatment. We examined hyperthermia- and chemotherapy-induced apoptosis in a human histiocytic 2.2. Equipments lymphoma cell line, U-937, by means of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Microcalorimeter, LKB 2277 Bioactivity Monitor nick-end-labeling (TUNEL) assay. The observation of (Thermometric AB, Sweden), was used to obtain the apoptotic cell-specific green fluorescent dots confirmed power-time curves. For more details of the performance that apoptosis occurred in the culture process. An LKB and construction of the instrument, see the articles 2277 Bioactivity Monitor was applied to determine the (Suurkuusk and Wadsoo,1982;. Xie et al., 1988). power-time curves of U-937 under the same conditions. Olympus BH-2 Fluorescence Microscope (Olympus The antitumor effect was evaluated by the maximal Inc., Japan). thermal power and the total heat Q released during the measurement period (48 h). The experiments showed that the combined use of carmofur and hyperthermia 2.3. Experimental determination had a synergistic antitumor effect and the metabolic activity of normal cells was higher than that of apoptotic The power-time curves of U-937 cells were recorded cells. The apoptosis ratio obtained in the TUNEL assay using the ampoule method. Cells in exponential growth phase were counted by using a Thoma’s hemocytometer also indicated the synergistic antitumor effect of a 5 1 combination of carmofur and hyperthermia. and the initial cell number was set to 10 cell ml by Carmofur is an orally active derivative of 5-fluorour- adding the medium. The 1 ml suspension of the cells acil (5-FU) and is well known to release 5-FU with different concentrations of carmofur was moved spontaneously as given in the following (Kobari et al., into a 3 ml glass ampoule, filled with 5% CO2, sealed 1981). and then was put into the microcalorimeter with a sealed reference ampoule to monitor the growth of the cell. Each experiment was repeated twice, and the difference of metabolic heat obtained in the same group were less than 5%. The measurement was carried out at 37.1C, which corresponded to the physiological body temperature, and 401C, 431C, i.e. hyperthermia, respectively. Carmo- fur was added into the sample at the beginning of the experiments. For the evaluation of these power-time curves, two parameters were usedFthe maximal thermal power (P.inmW) and the integral of the curve serving as a measure of the total heat released (Q in J).

2. Materials and methods 2.4. Sample preparation for TUNEL assay

2.1. Materials Samples were prepared as described in the ApopAlert DNA fragmentation assay kit user manual. After being The U-937, a kind of human histiocytic lymphoma fixed and permeabilized, samples were incubated for 1 h cell (ATCC CRL-1593) was provided by China Center with 500 U ml1 of terminal deoxynucleotidyl transfer- for Type Culture Collection, Wuhan University. ase incubation buffer and the reaction was terminated by Cultures in exponential growth phase are obtained by transferring samples to buffer containing 300 mM NaCl seeding 105 cell ml1 U-937 cells into a T-25 plastic flask and 30 mM sodium citrate. Samples were then washed in containing 8 ml medium at 371C, 5% CO2 for 2 days. PBS and incubated with PI (propidium iodide). Finally, The medium consists of 90% RPMI 1640 medium samples were viewed under an Olympus BH-2 Fluores- (GIBCO Co., U.S.A.), 10% heat-inactivated fetal calf cence Microscope using a standard fluorescein filter set 中国科技论文在线 http://www.paper.edu.cn

L. Yuwen et al. / Journal of Thermal Biology 27 (2002) 129–135 131

(520720 nm). The amplification ratio was set at 200. presented is also shown in Fig. 1. The initial cell number The photographs are shown in Fig. 5. was 105 ml1 for all experiments. From Fig. 1, we see that the effect of Carmofur on 2.5. Statistical analysis metabolism of U-937 cells was concentration dependent. With the addition of Carmofur, the maximal thermal The statistical comparison of the metabolic data of power, P, decreased (shown in Fig. 2). Values of Pm cells with combined treatments and without treatment which are shown in Table 1 are correlated to the was made using the paired Student’s t-test. A value of concentration of Carmofur, c,as Po0:05 was considered significant. Pm ¼ 13:77 0:5678c and R ¼ 0:9839: ð1Þ Since P ¼ dQ=dt; the area under the curve records the total metabolic heat released during the experimental 3. Results period (48 h). From Fig. 1, the values of metabolic heat which demonstrate the total metabolic activity of 3.1. The power-time curves for U-937 cells with different the culture of U-937 cells are obtained and shown concentrations of carmofur in Table 1. The inhibitory ratio of Carmofur (I1) are also shownÀÁ in Table 1, which was defined as I1 (in The power-time curves obtained when a culture of U- %)= Q0 Q1=Q0 100% where Q0 is the total heat 937 cells were inoculated with carmofur which had released of U-937 cells at 371C without Carmofur, Q1 is 1 concentrations corresponding to 1, 5, 10, 20 mgml at the total heat released with Carmofur. 1 37 C are shown in Fig. 1. The power-time curve from The linear relation between I1 and concentration of the control experiment in which no carmofur was Carmofur is shown in Fig. 3. The I1–c equations

Fig. 1. Power-time curves of U-937cells with an initial cell number of 105 cell ml1 in 1 ml RPMI 1640 medium containing 1 10% fetal calf serum at 371C with carmofur (a) 0 mgml ,(b) Fig. 2. Relationship between Pm and the concentration of 1 mgml1, (c) 5 mgml1, (d) 10 mgml1, (e) 20mgml1. Carmofur at 371C.

Table 1 Comparison of metabolic heat production of U-937 cells growing for 48 h at 371Ca c/mgml1 0151020

Pm (mW) 14.6070.70 12.4570.61 11.4170.52 7.0570.34 2.8570.14 Q (J) 2.01370.089 1.78170.080 1.51270.072 0.95870.046 0.43370.032 I1 (in %) 0 11.574.0% 24.973.6% 52.472.3% 78.571.6%

a Q: Total heat released with diﬀerent concentrations of Carmofur at 371C, I1: Inhibitory ratio with diﬀerent concentrations of Carmofur at 371C 中国科技论文在线 http://www.paper.edu.cn

132 L. Yuwen et al. / Journal of Thermal Biology 27 (2002) 129–135 obtained are as follows: Carmofur). As the inhibitory ratio shown in Table 2 and I ¼ 0:05787 þ 0:03843cR¼ 0:9850: ð2Þ Fig. 6, the most effective condition was the combined 1 use of Carmofur with hyperthermia(431C), and the The half-inhibitory concentration IC50 of Carmofur can effects were significantly different (Po0:01) from the be deduced from Eq. (2), which is 11.5 mgml1. control group.

3.2. The power-time curves for U-937 cells at higher 3.3. Result of TUNEL assay temperatures To determine the effect of hyperthermia combined In order to investigate the synergistic antitumor effect with Carmofur on apoptosis of U-937 cells, TUNEL of Carmofur on U-937 cells better at higher tempera- assay was employed on samples cultured for up to 48 h ture, the initial concentration of Carmofur was set to (Fig. 5). Fig. 6 shows the apoptosis percentages of U-937 5 mgml1 of which have an appropriate inhibitory ratio cells which were determined by counting 200–300 cells in (24.9%) on metabolic heat of U-937 cells. The power- 5–6 vision fields. In control sample at 371C (Fig. 5A), time curves for U-937 cells at 371C, 401C and 431C cells exhibited only strong red fluorescence stained by without Carmofur or with 5 mgml1 Carmofur are PI, round in shape and almost of the same size. In shown in Fig. 4a–c respectively. The temperature sample at 401C with Carmofur (Fig. 5B), cells under- sensitivity of U-937 cells can easily be seen from Fig. 4d, going apoptosis exhibited the apoptotic cell-specific in which the power-time curves of U-937 cells without green fluorescence while the normal cells exhibited red Carmofur at 371C, 401C and 431C are put together. fluorescence with the same size as that in Fig. 5A. In The maximal thermal power and total heat released sample at 431C with Carmofur (Fig. 5C), except for the during the measurement period (48 h) obtained are two kinds of fluorescence in Fig. 5B, many smaller red shown in Table. 2. granules can be observed which indicated that some of Define theÀÁ inhibitory ratio of temperature as I2 the cells died through necrosis. (in 100%)= Q0 Q1=Q0 100%; the inhibitory ratio Fig. 7 shows the apoptosis percentage (AP) defined as of carmofurÀÁ combined with hyperthermia as I3 (in AP=(number of apoptotic cells)/(total number of cells). 100%)= Q0 Q2=Q0 100%; where Q0; Q1 and Q2 It should be noted that the AP of U-937 cells treated stand for the total heat released of U-937 cells at 371C with 5 mgml1 Carmofur at 431C was less than that at without Carmofur (Q ¼ 2:013 J), at a certain tempera- 401C, and the AP of U-937 cells at 371C with 40 mgml1 ture without Carmofur and at a certain temperature Carmofur was less than that with 30 mgml1 Carmofur. with 5 mgml1 Carmofur, respectively. In the light of this definition, the inhibitory ratios were obtained and 0 shown in Table 2. Let I3 be the simple summation of the 4. Discussion inhibitory ratio of temperature ðI2Þ and the inhibitory 1 1 ratio of 5 mgml Carmofur at 371C(I1 at 5 mgml 4.1. Synergistic antitumor of hyperthermia and Carmofur in the microcalorimetric study

It has been reported that hyperthermia can greatly enhance cancer cell killing effect of other treatments such as chemotherapy (Kowal and Bertino, 1979) and radiation (Chung et al., 1998). Here we show that the combined use of hyperthermia and carmofur had a synergistic antitumor effect. Fig. 1 shows that the more the Carmofur added, the lower the power-time curves were. This indicates that Carmofur changed the metabolic mechanism of U-937 cell. Figs. 2 and 3 indicate that the relation between inhibitory effect on the metabolic heat and the concentration of Carmofur is approximately linear. At 371C, 401C and 431C, the power-time curves of U-937 cells in the presence of Carmofur are all below that without Carmofur (Fig. 4a–c). The temperature sensitivity of U-937 cells is shown in Fig. 4d. Analysis of the metabolic heat of the cells growing for 48 h (Table 1) Fig. 3. Relationship between inhibitory ratio and the concen- indicates that the combined use of Carmofur and tration of Carmofur at 371C. hyperthermia has a synergetic effect. The inhibitory 中国科技论文在线 http://www.paper.edu.cn

L. Yuwen et al. / Journal of Thermal Biology 27 (2002) 129–135 133

Fig. 4. (a) Power-time curves of U-937 cells at 371C: a. control; b. 5 mgml1 Carmofur. (b) Power-time curves of U-937 cells at 401C: a. control; b. 5 mgml1 Carmofur. (c) Power-time curves of U-937 cells at 431C: a. control; b. 5 mgml1 Carmofur. (d) Power-time curves of U-937 cells without Carmofur at: A. 371C; B. 401C; 431C.

Table 2 Comparison of metabolic heat production of U-937 cells growing for 48 ha

T/1C Pm, 1/mW Pm, 2/mW Q1/J Q2/J I2 (%) I3 (%) I3 (%) 37 14.6070.70 11.4170.52 2.01370.089 1.51270.072 0 24.973.6 24.973.6 40 11.670.60 7.170.36 1.73070.085 0.87070.041 14.174.2 56.872.0 39.074.2 43 9.370.45 5.670.30 1.33970.065 0.60770.029 33.973.2 72.471.4 58.873.6

a 1 Pm;1: Maximal thermal power without Carmofur, Pm, 2: Maximal thermal power with 5 mgml Carmofur, Q1: Total heat released 1 without Carmofur, Q2: Total heat released with 5 mgml Carmofur, I2: Inhibitory ratio of temperature without Carmofur, I3: 0 Inhibitory ratio of combined treatment, I3: Inhibitory ratio without Carmofur at certain temperature plus that of at 371C with Carmofur.

ratio of the combined treatment (I3) is much more than the cytotoxicity of Carmofur. We believe that this 0 the simple summation (I3) of the inhibitory ratio of is because: (1) Hyperthermia appears to modify 1 temperature (I2) and that of 5 mgml Carmofur at 371C several physical properties of the membrane, especially 1 (I1 at 5 mgml Carmofur). The results of microcalori- permeability and ﬂuidity. Passive intake of small metric study indicated that hyperthermia can potentiate molecules across the cell membrane increase with rising 中国科技论文在线 http://www.paper.edu.cn

134 L. Yuwen et al. / Journal of Thermal Biology 27 (2002) 129–135

Fig. 5. Fluorescence microscope detecting of apoptosis cells with TLNEL in U-937 cells ( 200) (A). U-937 cells at 371C without carmofur (B). U-937 cells at 401C with 5 mgml1 Carmofur. (C). U-937 cells at 431C with 5 mgml1 Carmofur (a). normal cell (negative) (b). apoptosis cell (positive) (c). necrotic cell.

hyperthermia can potentially promote free radical formation which have been demonstrated to be media- tors of cellular injury (Shawn et al., 1998). (3) Higher temperatures can weaken the capability of self-repair systems in cells by stimulating the production of interferon (antiviral and antitumor proteins) (Chang and Wu, 1991). (4) Hyperthermia can induce cell apoptosis through the dysfunction of mitochondria (Yuen et al., 2000). It is obvious that the combined use of hyperthermia and chemotherapy can reduce the dose of drugs used in chemotherapy from our study, which may reduce the side eﬀects of the drugs on patients. Carmofur is clinically useful in thermochemotherapy.

4.2. Synergistic apoptosis of U-937 cells treated by the Fig. 6. Inhibitory ratio at diﬀerent temperatures with or combination of hyperthermia and Carmofur in TUNEL assay without Carmofur I2: Inhibitory ratio without carmofur, I3: Inhibitory ratio in the presence of 5 mgml1 Carmofur. In the antitumor action on U-937 cells, apoptosis plays a very important role. The synergistic apoptosis of U-937 cells treated by the combination of hyperthermia and carmofur is obvious from Fig. 6. The apoptosis percentage (AP) at 401C (85%) and 431C (65%) with 5 mgml1 Carmofur are much larger than the simple sum of AP at 401C (3%) or 431C (9%) without Carmofur and AP at 371C with 5 mgml1 Carmofur. It should be noted that the AP at 431C (65%) with Carmofur is less than that at 401C (85%) with Carmofur. We believe that cells died through necrosis more than apoptosis at 431C because of the more severe culture conditions. The pervasive granules in Fig. 5C proved this for they were from necrotic cells. We can conclude that the synergistic antitumor eﬀect of the combined treatment of hyperthermia and Carmofur was achieved by inducing the apoptosis of U-937 cells.

Fig. 7. Apoptosis percentage vs. concentration of Carmofur. 4.3. Relation between metabolic activity and apoptosis temperatures thus the antitumor drugs can enter into the Apoptosis is a physiological or ‘programmed’ form cellular matrix more easily (Culver, 1982; Bates and of cell death, distinct from ‘accidental’ cell death Mackillop, 1986; Kong et al., 2000). (2) A number of or necrosis. A large number of biochemical changes biochemical and physiological events associated with have been observed in cells undergoing apoptosis 中国科技论文在线 http://www.paper.edu.cn

L. Yuwen et al. / Journal of Thermal Biology 27 (2002) 129–135 135

(Okada, 1970). The induction of this process requires and non-tumorous tissue samples. Thermochim. Acta. 229, RNA and protein synthesis, and was proved to be an 147–155. active metabolic process by Sellins and Cohen (1987) Kowal, C.D., Bertino, J.R., 1979. Possible benefits of using protein and RNA synthesis inhibitors. Although hyperthermia to chemotherapy. Cancer Res. 39, 2285–2289. apoptosis process involves many biochemical changes Kobari, T., Icuro, Y., Ujiie, A., Namekawa, H., 1981. Metabolism of 1-Hexylcarbamoy1-5-fluorouracil(HCFU), related to the expression of gene and synthesis of protein a new antitumor agent, in rats, rabbits and dogs. and RNA, the apoptotic cell is in an abnormal state Xenobiotica 2, 57–62. after all. Many biochemical reactions indispensable to Kong, G., Braun, R.D., Dewhirst, M.W., 2000. Regular the normal cell growth were slowed or blocked. Kunio et articlesFexperimental therapeuticsFhyperthermia enables al. proved that the impairment of adenosine tripho- tumor-specific nanoparticle delivery: effect of particle size. sphate synthesis may play an important role in Cancer research. 60, 4440–4445. hyperthermic cell killing (Shinohara et al., 1993). The Murthy, M.S., Rao, L.N., Khandkar, J.D., Scanlon, E.F., 1987. biochemical reactions occurring in normal cells will Enhanced therapeutic efficiency of cisplatin by combination inevitably be interrupted in apoptotic cells. Thus the with dietyldithiocarbamate and hyperthermia in a mouse metabolic activity of apoptotic cells was lower than that model. Cancer Res. 47, 774–779. Okada, S., 1970. Radiation Biochemistry. Vol, 1. Cells. of normal cells, while there is no beat produced by Academic Press, New York. pp. 247–307. necrotic cells. Our study confirmed the difference Sellins, K.S., Cohen, J.J., 1987. Gene induction by y-irradiation between the metabolic activity of apoptotic cells and leads to DNA fragmentation in lymphocytes. J. Immuno. that of normal cells and necrotic cells. 139, 3199–3206. Shawn, W.F., Pope, L.M., Garry, R.B., 1998. Increased fux of free radicals in cells subjected to hyperthermia: detection by Acknowledgements electron paramagnetic resonance spin trapping. FEBS Letters. 431, 285–286. This project is supported by National Natural Shinohara, K., Kawakami, N., Ito, A., Kugotani, M., Sciences Foundation of China (No. 29773033, No. Nakano, H., Ishiwata, J., Matsuda, T., 1993. The role of ATP metabolism in hyperthermic cell killing. In: 29873036, No. 30070200). Tadayoshi, M. (Ed.), Cancer Treatment By Hyperthermia, Radiation & Drugs. Burgess Science Press, Great Britain, pp. 79–91. References Suurkuusk, J., Wadsoo,I.,. 1982. A multichannel microcalorimetry system. Chem. Scr. 20, 155–163. Barlogie, B., Corry, P.M., Drewinko, B., 1980. In Vitro Urano, M., Kim, M.S., Kahn, J., Kenton, L.A., Li, M.L., 1985. Thermochemotherapy of Human Colon Cancer Cells with Effect of thermochemotherapy (combined cyclophospha- cis-Dichlorodianunineplatinum(II) and Mitomycin C. mide and hyperthermia) given at various temperatures Cancer Res. 40, 1165–1168. with or without glucose administration on a murine Bates, D.A., Mackillop, W.J., 1986. Hyperthermia, adriamycin fibrosarcoma. Cancer Res. 45, 4162–4166. transport, and cytotoxicity in drug-sensitive and -resistant Wiedemann, G.J., Siemens, H.J., Mentzel, M., Biersack, A., chinese hamster ovary cells. Cancer Res. 46, 5477–5481. Woossmann,W.,. Knocks, D., Weiss, C., Wagner, T., 1993. Chang, C.C., Wu, J.M., 1991. Modulation of antiviral activity Effects of temperature on the therapeutic efficacy of interferon and 20,50-oligoadenylate synthetase gene and pharmacokinetics of ifosfamide. Cancer Res. 53, expression by mild hyperthermia (39.51C) in cultured 4268–4272. human cells. J Biological Chem. 266, 4605–4612. Wilder, R.B., Langmuir, V.K., Mendonca, H.L., Goris, M.L., Chung, D.H., Zhang, F., Chen, F., McLaughlin, W.P., Ljung- Kiox, S.J., 1993. Local hyperthermia and SR 4233 enhance

man, M., 1998. Butyrate attenuates BCLXL expression in the antitumor effects of radioimmunotherapy in nude mice human fibroblasts and acts in synergy with ionizing with human colonic adenocarcinoma xenografts. Cancer radiation to induce apoptosis. Radiat. Res. 149, 187–194. Res. 53, 3022–3027. Culver, G., 1982. Temperature acclimatisation and specific Xie, C.L., Tang, H.K., Song, Z.H., Qu, S.S., 1988. Micro- cellular components in the regulation of thermal sensitivity calorimetric study of bacterial growth. Thermochim. Acta. of mammalian cells. Nat. Cancer Inst. Monographs 61, 123, 33–41. 99–101. Yuen, W.F., Fung, K.P., Lee, C.Y., Choy, Y.M., Kong, S.K., Feng, Y., Luo, Z.F., Qu, S.S., Zheng, C.Y, Xu, H., 1997. Study Ko, S., Kwok, T.T., 2000. Hyperthermia and tumour on the thermosensitivity of tumor cells by microcalorimetry. necrosis factorFan induced apoptosis via mitochondrial Thermochim. Acta. 303, 203–207. damage. Life Sciences 67, 725–732. Hahn, G.M., 1979. Potential for Therapy of Drugs and Zhu, W.G., Shigetoshi, A., Shinobu, K., Ryoji, A., Katsumasa, Hyperthermia. Cancer Res. 39, 2264–2268. N., Hiroshi, S., 1995. Enhancement of hyperthermic Karnebogen, M., Singer, D., Kallerhoff, M., Ringert, R.H., killing in L5178Y cells by protease inhibitors. Cancer Res. 1993. Microcalorimetric investigations on isolated tumorous 55, 739–742.