Cell Biology and Toxicology, Vol. 9, No. 1, 1993 49

A R S E N I T E I N D U C E D S E N S I T I Z A T I O N A N D S E L F - T O L E R A N C E O F R E U B E R H 3 5 H E P A T O M A C E L L S

FRED A.C. WlEGANT 1, JAN E.M. SOUREN 1, HAN VAN RIJN z, AND ROELAND VAN WIJK 1

1Department of Molecular Cell Biology University of Utrecht

Utrecht, The Netherlands

2Department of Radiation Oncology Free University Hospital

Amsterdam, The Netherlands

Our data show that a short incubation with arsenite (30-300 #M) induces a biphasic change in cellular sensitivity towards a second exposure to arsenite. A transient sensitization was followed by the development of self-tolerance.

Sensitization was measured using the step-down protocol; i.e., application of a high dose of arsenite pretreatment (100 or 300 #M) followed immediately by incubation in a low dose of arsenite (1-30 ltM), with extensive rinsing in between. Whereas no effect of i and 3 #M on cellular survival is observed without pretreatment, a large decrease in cell survival can be established when these low doses of arsenite are applied immediately after a i hr pretreatment with 100 or 300 #M arsenite.

According to the step-down protocol, a high dose of toxic compounds is applied and is followed by prolonged incubation in a lower concentration of the initial toxic compound. This might be a more acctu'ate model for studying the effects of toxic insults on cells and organisms in the manner in which they occur in their natural environment. The level of tolerance was determined by a 1 hr test treatment with 300 #M arsenite applied at different times after pretreatment. Using this fractionated treatment protocol, it was established that tolerance increases with the increasing time intervals between the sodium arsenite treatments, during the 6 hr studied.

These observations suggest that sensitization gradually decreases, whereas tolerance develops. Furthermore, our data indicate that the condition of

1. Address all correspondence to: Dr. F.A.C. Wiegant, Department of Molecular Cell Biology, Padualaan 8, 3584 CH Utrecht, The Netherlands. Tel: 31-30-533972, Fax: 31-30-513655.

2. Key Words: cytotoxicity, sensitization, sodium arsenite, step-down protocol, tolerance.

Cell Biology and Toxicology, Vol. 9, No. 1, pp. 49-59 Copyright �9 1993 Kluwer Academic Publishers

ISSN: 0742-2091

50 Wiegant et al.

pretreatment determines the extent to which the early sensitivity increases, as

well as the development of tolerance later on. A relatively high arsenite

concentration leads to more sensitized cells, which are transformed into more

tolerant cells in comparison with the effect of a lower arsenite concentration.

INTRODUCTION

Mammalian cells respond in a remarkable manner to abrupt changes in environmental conditions. An apparent defense mechanism, commonly referred to as the heat-shock response or stress response, is elicited by a wide variety of physical and chemical agents, including heat shock and exposure to heavy metals and amino acid analogs (Nover, 1984, 1991; Burdon, 1986; Welch, 1990). One aspect of the stress response which has been characterized extensively is a change in sensitivity. This has mostly been studied following heat shock (Hahn and Li, 1990). Depending on the experimental conditions, exposure to heat may lead either to an increase (i.e., thermosensitization) or to a decrease (i.e., thermotolerance) in sensitivity for a second heat treatment. Directly after an initial heat treatment, cells are more thermosensitive in vitro, as well as in vivo (Lindegaard and Overgaard, 1990). This is commonly measured by the step-down protocol, in which the initial heat treatment is immediately followed by a second treatment at a lower hyperthermic temperature. The enhanced thermal sensitivity of cells has been demonstrated by cell survival kinetics (Henle, 1980; Jung, 1982, 1989).

A decreased sensitivity after an initial treatment was observed by using a fractionated treatment protocol; i.e., a short exposure to a high dose, followed by incubation at control temperature (37~ and subsequent testing at increased test temperatures. In this way it has been shown that the thermosensitive phase is followed by a phase in which tolerance is developed (Henle et al., 1978; Li and Hahn, 1980; Mooibroek et al., 1988; Hahn and Li, 1990). A transient thermal resistance, or thermotolerance, can be induced not only by heat; but also by many other seemingly unrelated stimuli. Sodium arsenite is an environmental stressor, which has been studied extensively. Toxic effects of arsenite are mainly exerted through its interaction with sulfhydryl groups in proteins and its interference with DNA replication and repair (Squibb and Fowler, 1983; Vasken Aposhian, 1989).

It is unknown, however, whether exposure to arsenite also leads to (bi-phasic) alterations in the sensitivity to a subsequent treatment with arsenite. In the experiments reported here, we studied the induction by arsenite pretreatment of a sensitization, as well as of a tolerance, to a second arsenite treatment at the level of cellular survival. For this purpose, both the step-down protocol and the fractionated treatment protocol were used. We show that a short incubation with arsenite induces a transient increase in sensitivity to a second arsenite treatment. Low concentrations of arsenite, which alone do not affect survival, are toxic when applied immediately after treatment with a high dose of arsenite. This enhanced sensitivity is temporary and is followed by an enhanced tolerance towards arsenite.

Cell Biology and Toxicology, Vol. 9, No. 1, 1993 51

MATERIALS AND METHODS

Cells and Culture Condition The rat hepatoma cell line Reuber H35 was routinely grown at 37~ as subconfluent monolayer cultures in plastic flasks. Standard growth medium consisted of Leibovitz (L15) medium (Flow Laboratories) supplemented with 10% fetal calf serum (Gibco) at pH 7.4, and with the antibiotics streptomycin sulfate (100 gg/ml) and potassium penicillin G (100 U/ml).

Determination of Cell Survival To establish the effect of treatment on cell survival, cellular viability (in terms of reproductive potential or colony-forming ability) was measured using the clonal-assay method (Schamhart et al., 1984). Using this method, cells which remain viable after treatment are given the opportunity to divide and to form a visible colony during a period of one week. The complete procedure follows.

After arsenite treatment, monolayers of cells were rinsed thoroughly and trypsinized for 3 min (0.05% trypsin and 0.02% EDTA) in phosphate buffered saline (PBS; consisting of 8 g NaC1, 0.2 g KCI, 1.16 g Na2HPO4.2H20, and 0.2 g KH2PO4 per liter, at pH = 7.4). After

trypsinization, the cells were resuspended in 5 ml of L15 medium containing 10% fetal calf serum. Cell number was determined with a particle counter (Analys Instrument AJ134).

Single cells were inoculated in appropriate numbers (usually 800 cells) in TC 25 cm 2 flasks (Greiner) and incubated at 37~ One week later, colony formation was judged after fixation and Giemsa staining. Colony-forming efficiency or plating efficiency (PE) was expressed as the percentage of plated cells growing to a colony during the next 7 days. Plating efficiency of control cultures ranged between 50-60%, as was described previously (Schamhart et at., 1984).

The cytotoxic response of the treated cultures was expressed as the percentage of relative survival; i.e., flae PE of the treated cells divided by the PE of control (untreated) cells. Results of survival experiments were expressed as Do values (Henle, 1978; Jung, 1982). Do is defined

as the time (in minutes) required to reduce cell survival to 1/e times its initial value on the exponential portion of the survival curve. The values were calculated by linear least-squares analysis of the linear portion of the survival curves. In the case of step-down experiments, the sensitizing factor was calculated in terms of the ratio: Do (control)/Do (step-down).

In order to determine a change in sensitivity after treatment, the LD50 value was calculated using linear regression analysis. The LD50 value is defined as the concentration of arsenite required to kill 50% of the remaining population.

In some experiments, as indicated below in the Results section, the pre-plating procedure was as described before (Wiegant et al., 1985). Cells were seeded in 25 cm 2 culture flasks in complete medium. After attachment, 4 hr later, the medium was replaced, in order to expose

52 Wiegant et al.

ceils to arsenite in fresh medium, followed by rinsing and replacing of complete medium. Colony growth was judged 1 week later after fixation and Giemsa staining. Experiments were done with duplicate samples and each experiment was repeated at least once. Results are shown as averages of duplicate experiments.

Step-down Treatment Protocol Step-down treatment consisted of the application of a high dose of sodium arsenite (100 IxM or 300 JAM), followed by incubation in medium containing a lower dose of arsenite (1, 3, 10, or 30 I.tM). Between high and low dose application, cell cultures were rinsed 3 times with phosphate buffered saline.

RESULTS

Survival Sensitivity and Sensitization Figure 1A shows the survival of H35 cells that were incubated at arsenite concentrations between 0 and 300 ktM for various periods of time. Data were obtained in post-plating experiments. During the 8 hr in which cells were studied, it was found that 1 and 3 I.tM arsenite do not have a significant effect on ceil survival. A small decrease in cell survival was observed in the presence of 10 or 30 ~tM when incubation lasted for more than 4 hr. A large decrease in survival was already observed upon shorter incubation periods in the presence of 100 ptM and 300 ptM arsenite.

Studying the effect of a 1 hr exposure to different concentrations of arsenite revealed that there was no significant decrease in survival when cells were exposed to concentrations up to 100 ~tM arsenite (Figure 1B). After a 1 hr exposure to 3 I.tM arsenite, however, a small but significant 20% increase in plating efficiency was observed (p = .029) (n = 12). An enhanced colony forming efficiency by low doses of arsenite (0.7-3 [tM) has been described previously by Lee et al. (1985) and Barrett et al. (1989) in Syrian hamster embryo cells. Exposure of H35 ceils for 1 hr to arsenite concentrations in excess of 100 ktM resulted in increased cell death. In the presence of 300 ktM for I hr, only 42% of the cell population survived.

Step-down treatment with arsenite revealed an increased sensitivity towards the second arsenite treatment in that portion of the cell population which survived the pretreatment (Figure 2). Step-down treatment consists of an initial treatment at a high concentration (pretreatment), followed by a thorough washing and immediate exposure to a lower arsenite concentration. Without pretreatment, no significant effect of 1 and 3 ktM is observed on cell survival (Figure

Cell Biology and Toxicology, Vol. 9, No. 1, 1993 53

Y 0 0 0

v

0

O)

0

0

0

I - - - - - - I - - - - - - I

0 0 0 0

(%) l~'^!^Jns aA!]-~la~l

v 0 d~

,-' o o 0 ' ~ ~'

.~N~ ~ ~.~ 0 "''I ~ ~,

~ 8

o N

54 Wiegant et al.

, m

100 .E (3 ~ �9 A

10 I I I

0 3 6 9

B 100

50

10 i I 0 3 6 9

40 C

10

5 i i i

0 3 6 9

Time (hours)

FIGURE 2. Survival of H35-cells after pretreatment with 0, 100, or 300 ~tM arsenite for 1 hr (Figures 2A, 2B and 2C, respectively) washed three times and immediately followed by an exposure to lower concentrations of arsenite (0 (O), 1 (A), 3 (O), 10 (A) or 30 (--)) during the indicated period of time. After the experimental condition, cells were rinsed twice, trypsinized, and then replated for colony-forming analysis.

Cell Biology and Toxicology, Vol. 9, No. 1, 1993 55

2A). When, however, these low concentrations are applied immediately after the pretreatment with 100 or 300 gM arsenite, a decrease in cell survival is observed (Figures 2B and 2C,

respectively).

In Table 1, the D O value of survival curves and the sensitizing factors are shown in data

obtained after treatment with various arsenite concentrations in control and in step-down conditions (D o values are from Figure 2). From the sensitizing factor, it can be concluded that

cells are slightly more sensitive following a pretreatment with 300 gM, in comparison with

100 gM. When 3 gM arsenite is applied as post-treatment, a sensitizing factor of 4.57 is observed after 100 gM pretreatment, and a factor of 5.35 is observed after a 300 ~tM

pretreatment. Exposure to 10 or 30 gM arsenite for a period of time greater than 3 hr already affects survival in control conditions. A particular feature is that when these concentrations are

applied after pretreatment, less sensitization is observed, in comparison with exposure to 1 or

3 IIMo

T A B L E 1. Effect o f Pre trea tment with 0, 100, or 300 laM Arseni te for 1 H o u r on the D o - V a l u e s o f Surv iva l C u r v e s as wel l as on Sens i t i za t ion (expressed as sens i t i z ing factor) .

Pretreatment (gM arsenite) Do-value Sensitizing factor

Post treatm. 0 100 300 txM 0 100 300 l, tM 1 9800 1950 i950 1 5.03 5.03 3 4900 960 820 1 4.57 5.35

10 880 635 560 1 1.39 1.57 30 485 530 395 1 0.92 1.23

H35 cells were exposed to low doses of arsenite (0, 1, 3, 10 gM) directly after a pretreatment with 0, 100, or 300 gM arsenite for 1 hr (Do-values are from Figure 2).

Survival--Tolerance In order to study the effect of fractionated exposure on the development of tolerance in H35 cells, cultures were pre-treated for 1 hr with medium containing 0, 30, 100, or 300 ~M arsenite, rinsed, and subsequently incubated in arsenite-free medium. The arsenite sensitivity of

the cell population which survived the pretreatment was determined at different times following this first treatment, by exposure of cultures to a test-treatment of 300 gM arsenite for 1 hr. As shown in Figure 3, the sensitivity to a 1 hr, 300 gM test-treatment is highest if this test-treatment is applied immediately after the pretreatment. However, with increasing

time of incubation in arsenite-free medium, the sensitivity to this test-treatment decreases. From 3 hr after pretreatment, ceils show a decreased sensitivity, as compared to control cultures. These data indicate the development of arsenite-induced arsenite tolerance (self- tolerance). Since the level of self-tolerance obtained after a treatment with 300 ~tM is higher than after a treatment with 100 gM or 30 I.tM, it is suggested that the development of arsenite self-tolerance is a function of the pretreatment.

100

56 Wiegant et al.

> = m

+.a

n

tin,

W v W

1 0 I I I

0 3 6 9 Time (hours)

FIGURE 3. Kinetics of development of arsenite-induced self-tolerance. After pretreatment with 0 (O), 30 (<~), 100 (~) or 300 (11) I.tM arsenite during 1 hr, ceils were rinsed three times and reincubated in arsenite-free medium during the indicated time. Then the cytotoxic effect was determined of a test-treatment with 300 I.tM arsenite during 1 hr. After this test treatment, cells were rinsed 2-3 times, trypsinized, and replated for colony-forming analyses. Data were corrected for the effect of pretreatment and are presented as the surviving fraction of pre-treated cells.

Survival---Sensitization and Tolerance In order to study the transition of arsenite sensitization to arsenite tolerance in more detail, cell cultures were pretreated with 0, 100, or 300 mM arsenite for 1 hr. After incubating for specific time intervals in arsenite-free medium, these pretreated cultures were exposed for 1 hr to a range of arsenite concentrations (including 3, 10, 30, 50, 100, 175, 300, and 600 pM), in

order to be able to determine the LD50 value. When pretreatment was followed within half an hour by a second exposure, a sensitization was observed (Figure 4). This sensitization is expressed as a decrease in the LD50 value; apparently less arsenite is required to kill 50% of the cell population. Maximal sensitization was observed 1 hr after pretreatment, whereas sensitization disappeared 3 hr after pretreatment. At that time, the LD50-value was more or less similar to the value of non-pretreated cells. Six hr after arsenite treatment, ceils are

significantly more tolerant. It can be observed that the level of sensitization and, later, the degree of tolerance depends on the arsenite concenlration applied during prelreatment (Figure 4). In comparison with the effect of 100 laM arsenite, pretreatment with 300 pM arsenite

Cell Biology and Toxicology, t/ol. 9, No. 1, 1993 57

causes a greater sensitization and afterwards a higher level of tolerance of the cells against an otherwise lethal concentration of arsenite.

600

:: i r 500

.-i-,

~400 I

300 c O ~" 200

100 _.J

0 I I I f I I I I I l I I

0.5 1 3 6

Time(hours)

FIGURE 4. The concentration of sodium arsenite required to kill 50% of the cell population (LD50) at various times after pretreatment during 1 hr with arsenite (0 (e), 30 (~), 100 ([=1) or 300 gM (11)). After pretreatment, ceils were rinsed three times and incubated in arsenite-free medium for the indicated time (0-6 hr). The medium was then replaced by serum-containing medium with various arsenite concentrations (0, 30, 50, 100, 175, 300, or 600 gM) for 1 hr. After rinsing, cells were further incubated for colony formation. In order to determine the LD50-value, linear regressioll analysis was used.

D I S C U S S I O N

In this study it was shown that exposure of H35 cells to arsenite concentrations of 30, 100, or

300 gM for 1 hr rapidly changes the sensitivity towards a second arsenite treatment. After a

transient increase in sensitivity, a period is observed in which tolerance develops. According to

previous definitions, "tolerance" is described as a stress-induced increase in cellular resistance towards a lethal (heat) stress-treatment, whereas "sensitization" is defined as an increase in cellular susceptibility for a (heat) stress treatment (Jung, 1982).

Phenomenologically, arsenite pretreatment sensitizes ceils to subsequent exposure to less

lethal arsenite treatment. In this respect, it has been shown that in the time-period of enhanced

sensitivity, lower concentrations of arsenite are required to induce 50% cell death. The

increased sensitivity is lost when cells are incubated in arsenite-free medium for 3-4 hr. After that period, the development of a transient tolerance towards a second arsenite exposure is observed.

58 Wiegant et al.

Furthermore, it was observed that pretreatment with a higher arsenite dose resulted in a larger sensitizing effect towards sub-toxic arsenite concentrations, whereas later in time, a higher level of tolerance was observed. Apparently, the strength of the initial arsenite treatment not only determines in part the sensitivity of cells to a subsequent arsenite treatment, but also the extent to which tolerance can be developed in the remaining cell population.

Our results can be compared with analogous findings following heat shock, applied according to a step-down protocol (Jung, 1982, 1989; Lindegaard and Overgaard, 1990). The sensitizing effect of heat treatment with respect to cellular survival was not only shown to depend on pretreatment temperature, but also on the step-down temperature. A higher pretreatment temperature resulted in a larger step-down effect (Jung, 1982; Lindegaard and Overgaard, 1990; Schamhart et at., 1992). Furthermore, a maximal sensitizing effect of step-down heating was observed when the step-down treatment was given immediately after the initial treatment (Henle, 1980; Jung, 1982). When an increasing period of time at 37~ is allowed between pretreatment and step-down treatment, a development of thermotolerance is observed (Jung, 1982; Dickomey et al., 1984). With respect to the development of sensitization and tolerance induced by heat or arsenite, the development of arsenite-induced sensitization and tolerance, as reported in this study, appears to require more time than the development of thermosensiti- zation and of thermotolerance (Schamhart et al., 1992) and, furthermore, seems to be of a lower level than heat-induced thermotolerance. The transition of triggered (and more sensitive) cells into (thermo)tolerant cells forms part of the model proposed by Li and Hahn (1980).

The induction and development of self-tolerance towards a previously applied stressor has also been described for some non-heat stressors. An induction of self-tolerance by the heavy metal cadmium was described by Cervera (1985). With respect to arsenite self-tolerance, Lee et al. (1989) established an arsenic-resistant CHO cell-clone by progressively increasing the concentration of sodium arsenite in culture medium. The resistant CHO cells were able to proliferate in the presence of 30 t.tM arsenite. Self-tolerance induced by arsenite was also mentioned by Lee and Hahn (1988), although no data were shown.

Since the enhanced synthesis of heat shock proteins (hsps) has been related to the (thermo)tolerant state, we are currently studying the relationship between the development of self-tolerance and the induction and enhanced synthesis of hsps.

Studying the effects of toxic compounds applied according to the step-down protocol may give more insight in understanding the reactions of cells and organisms to sudden physical or chemical insults which are followed by prolonged incubations in decreasing concentrations of the initial toxic compound, such as often occurs in their natural environment.

REFERENCES

BARRETT, J.C., LAMB, P.W., WANG, T.C., and LEE, T.C. (1989). "Mechanisms of arsenic- induced cell transformation," Biol. Trace Element Res. 21: 421-429.

BURDON, R.H. (1986). "Heat shock and the heat shock proteins." Biochem. J. 240: 313-324.

Cell Biology and Toxicology, Vol. 9, No. 1, 1993 59

CERVERA, J. (1985). "Induction of self tolerance and enhanced stress protein synthesis in L-132 cells by cadmium chloride and by hyperthermia." Cell Biol. Int. Rep. 9: 131-141.

DICKOMEY, E., EICKHOFF, J., and JUNG, H. (1984). "Thermotolerance and thermosensitization in CHO and R1H cells: A comparative study." Int. J. Radiat. Biol. 46: 181-192.

HAHN, G.M. and LI, G.C. (1990). "Thermotolerance, thermoresistance and thermosensitization." In: Stress Proteins in Biology and Medicine (R.I. Morimoto, A. Tissibres, and C. Georgopoulos, eds.). Cold Spring Harbor Laboratory Press. pp. 79-100.

HENLE, K.J., KARAMUZ, J.E., and LEEPER, D.B. (1978). "Induction of thermotolerance in Chinese hamster ovary cells by high (45 ~ or low (40 ~ hyperthermia." Cancer Res. 38: 570- 574.

HENLE, K.J. (1980). "Sensitization to hyperthermia below 43~ induced in Chinese hamster ovary cells by step-down heating." J. Natl. Cancer Inst. 64: 1479-1483.

JUNG, H. (1982). "Interaction of thermotolerance and thermo-sensitization induced in CHO cells by combined hyperthermic treatment at 40~ and 43~ '' Radiation Research 91: 433-446.

JUNG, H. (1989). "Step-down heating of CHO cells at 37.5-39~ ' ' Int. J. Hyperthermia 5: 665- 673.

LEE, K.J. and HAHN, G.M. (1988). "Abnormal proteins as the trigger for the induction of stress responses: heat, diamide, and sodium arsenite." J. Cell. Physiol. 136: 411-420.

LEE, T.C., OSHIMURA, M., and BARRETT, J.C. (1985). "Comparison of arsenic-induced cell transformation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture." Carcinogenesis 6: 1421-1426.

LEE, T.C., WEI, M.L., CHANG, W.J., HO, I.C., LO, J.F., JAN, K.Y., and HUANG, H. (1989). "Elevation of glutathione levels and glutathione S-transferase activity in arsenic resistant Chinese Hamster Ovary cells." In Vitro Cellular and Developm. Biol. 25: 442-448.

LI, G.C. and HAHN, G.M. (1980). "A proposed operational model of thermotolerance based on effects of nutrients and the initial treatment temperature." Cancer Res. 40: 4501-4508.

LINDEGAARD, J.C. and OVERGAARD, J. (1990). "Step-down heating in a C3H mammary carcinoma in vivo: effects of varying the time and temperature of the sensitizing treatment." Int. J. Hyperthermia 6: 607-617.

MOOIBROEK, J., DIKOMEY, E., ZYWIETZ, F,, and JUNG, H. (1988). "Thermotolerance kinetics and growtll rate changes in the R1H tumour heated at 43~ ' ' Int. J. Hyperthennia 4: 677-686.

NOVER, L. (1984). Heat Shock Response of Eukaryotic cells. Springer Verlag, Berlin. NOVER, L. (1991). Heat Shock Response (L. Nover, ed.). CRC Press, Boca Raton, FL. SCHAMHART, D.H.J., VAN WALRAVEN, H.S., WIEGANT, F.A.C., LINNEMANS, W.A.M., VAN

RUN, J., VAN DEN BERG, J., and VAN WIJK, R. (1984). "Thermotolerance in cultured hepatoma ceils: cell viability, cell morphology, protein synthesis and heat-shock proteins." Radiation Research 98: 82-95.

SCHAMHART, D.H.J., ZOUTEWELLE, G., VAN AKEN, J., and VAN WIJK, R. (1992). "Effects on the expression of heat shock proteins by step-down heathlg and hyperthermia in rat hepatoma cells with a different degree of heat sensitivity." Int. J. Hyperthermia 8: 701-716.

SQUIBB, K.S. and FOWLER, B.A. (1983). "Toxicity of arsenic and its compounds." In: Biological and Environmental Effects of Arsenic. (B.A. Fowler, ed.). Elsevier Science Publishers, Amsterdam. pp. 234-269.

VASKEN APOSHIAN, H. (1989). "Biochemical toxicology of arsenic." In: Reviews in Biochemical Toxicology. (E. Hodgson, J.R. Bend, and R.M. Philpot, eds.). Vol 10. Elsevier, New York. pp. 265-299.

WELCH, W.J. (1990). "The mammalian stress response: cell physiology a,ld biochemistry of stress proteins." In: Stress Proteins in Biology and Medicine (R.I. Morimoto, A. Tissi~res, and C. Georgopoulos, eds.). Cold Spring Harbor Laboratory Press. pp. 223-278.

WIEGANT, F.A.C., TUYL, M., and LINNEMANS, W.A.M. (1985). "Cahnodulin-inhibitors prevent heat-induced cytoskeletal reorganization and potentiate layperthermic cell killing." hat. J. Hyperthermia 1: 157-169.

received: 9/21/92

accepted: 10/29/92