[CANCER RESEARCH 55, 5459-5464, November 15, 19951

tosis has been recognized in tumors in vivo (13), the kinetics ofhyperthermia-induced apoptosis in tumor tissues and, even moreimportantly, the relationship of apoptosis to tumor response have notreceived close attention.

It is well known that apoptosis in normal tissues occurs underphysiological conditions (14). Chemotherapeutic drugs and irradiationhave also been shown to induce apoptosis in normal thymocytes (15,16) and in the intestinal mucosal epithelium (17—20).Apoptoticchanges in normal tissues may be related closely to treatment-inducedtoxicity. Therefore, hyperthermia-induced apoptosis in the normaltissues will be important to understand; yet, there are very few studiesthat examine the effect of heat on normal tissues. Allan et a!. described hyperthermia-induced apoptosis in both the small intestine(21) andthetestis(22).To optimizethetherapeuticefficacyof WBHas an anticancer treatment, it may be important to identify the criticalnormal tissue targets of WBH-induced apoptotic cell death and toincrease our understanding of the kinetics of WBH-mediated apoptosis in other normal tissues in vivo.

In these studies, comparing two transplantable tumors with different sensitivities to hyperthermia, we examined the in vivo kinetics ofapoptosis induced by WBH (41.5°Cfor 2 h) histologically and thencorrelated the extent of apoptosis with tumor response. In addition, weexamined the extent and kinetics of WBH-mediated apoptosis innormal tissues of rats.

MATERIALSAND METHODS

Animals. Experiments were performed using female Fischer 344 rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) with body weights ranging fromb40 to 170 g. Rats were fed standard laboratory chow, allowed free access towater, and housed under controlled conditions with a 12-h light/dark cycle. Allrats were allowed a 1-week environmental adaptation period before theirexperimental use.

Tumors. We used two transplantable tumors: a fibrosarcoma (23) and theWard colon carcinoma (24), which were grown and maintained in donor ratsby monthly and bimonthly implantation, respectively. For experiments usingthe fibrosarcoma, viable tumor cells (106), assessed using trypan blue, wereinjected s.c. into the left flank of rats. Tumors were produced in 100% of theinjected rats (25). For experiments using the Ward colon carcinoma, tumorfragments (1 mm3) were implanted s.c. into the left flank of rats. Tumorsoccurred in 100% of the transplanted rats. Treatment was initiated whentumors reached 6—8mm in thickness.

WBH. Using a modification ofthe technique described by Lord et a!. (26),rats were anesthetized with halothane and placed tumor side down on gauzehammocks on the surface of a warm water bath maintained at 41.5°C. Thenecessary anesthetic depth during WBH treatment was maintained by elevatingthe head of the rat out of the water and fitting the nose of the rat into ahalothane anesthetic inhalation mask (27). The ties of the hammocks thatsuspend the rats in the water were pinned to the outside of the water bath sothat the degree of immersion of the rat's body in the water could be controlledby loosening or tightening the ties of the hammock to lower or raise, respectively, the level of the body of the rat in the water (23). In this manner, the core

body temperature of the rat could be regulated easily and maintained at thewater bath temperature of 41 .5°Cfor the duration of the 2-h WBH treatmenttime. The thermostatically controlled circulating water bath was maintained at41.5°C,using a Haake model E 12 circulator and heater as described previ

5459

ABSTRACT

Apoptosis in tumor and normal tissues was examined in rats treatedwith whole-body hyperthermia (WBH; 41.5°Cfor 2 h). WBH alone produced 0.5 day of tumor growth delay (TGD) in a fibrosarcoma and 5.8days of TGD in the Ward colon carcinoma. This difference in WBHinduced TGD indicates that the fibrosarcoma is relatively resistant toWBH, whereas the Ward colon carcinoma is relatively heat sensitive. Aquantitative histological assay for apoptosis demonstrated that the extentof apoptosis in the fibrosarcoma reached a maximum level of 19% 4 hafter WBH and returned to the control level by 24 h. In contrast, WBHinduced apoptosis with a peak value of 43% at 8 h in the Ward coloncarcinoma, and the apoptotic level remained elevated above the controllevel until 48 h after WBH. Within normal tissues, the spleen and thelymph nodes showed WBH-induced apoptosis; however, the highest levelof WBH-induced apoptosis as well as the most prolonged increase inapoptotic levels occurred in the thymus. The WBH-induced apoptosis inthe thymus remained elevated above the control level until 48 h afterWBH. Within the entire gastrointestinal tract, the small intestine was themost sensitive to WBH. Apoptotic cells were observed in the small bowelmucosa following WBH exposure. We also noted a minor WBH-lnducedincrease in the apoptotic level in the bone marrow. Except for the case ofthe thymus, increased apoptotic levels In the normal tissues declined afterpeak levels at 4 h, and apoptosis above control levels was not seen beyond12 h following WBH. Thus, within the normal tissues, WBH-inducedapoptosis declined to basal levels within 12—48h. These data indicate thatboth the extent and the kinetics of WBH-induced apoptosis differ betweenthe two tumors and, meaningfully, between tumor and normal tissues. Theextent and duration of apoptosis seem to correlate with tumor response toWBH.

INTRODUCTION

Apoptotic cell death is a process distinguishable from necrotic celldeath. Apoptosis occurs in both physiological and pathological conditions and plays an important role in the regulation of tissue development (1—3).Apoptosis occurs in tumor cells in response to therapeutic stimuli such as cytotoxic drugs and radiation (4—9).Hyperthermia also activates the process of apoptosis in tumors (4, 5,10, 11). Harmon et a!. (11) demonstrated that the type of cell deathchanged from apoptosis to necrosis when the thermal dose wasincreased. It is generally believed that mild forms of injury induceapoptosis, whereas more severe forms of insult result in necrosis (1,

12). Thus, the mode of cell death following hyperthermia is dependenton the severity of the heat stress. WBH5 at moderate temperatureelevations induces apoptosis in tumors. Although heat-induced apop

Received 8/24/95; accepted 9/14/95.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by National Cancer Institute Grants R01-CA-43O90 and R01-CA-41581.

2 On leave from Department of Surgery II, Faculty of Medicine, Kyushu University,

Fukuoka 812, Japan.3 On leave from First Department of Surgery, Faculty of Medicine, Tottori University,

Yonago 683, Japan.4 To whom requests for reprints should be addressed, at Division of Oncology, The

University of Texas Medical School, P.O. Box 20708, Houston, TX 77225.5 The abbreviations used are: WBH, whole-body hyperthermia; YSI, Yellow Springs

Instrument Co.; TGD, tumor growth delay; H & E, hematoxylin and eosin.

Apoptosis in Tumors and Normal Tissues Induced by Whole Body Hyperthermia

in Rats1

Yoshihisa Sakaguchi,2 L. Clifton Stephens, Masato Makino,3 Tetsuya Kaneko,3 Frederick R. Strebel,Lynn L Danhauser, Gaye N. Jenkins, and Joan M. C@Bull4Division of Oncolog v. Department of Internal Medicine, University of Texas Medical School [V. S., M. M., T. K.. F. R. S.. L L D., G. N. J.. I. M. C. B.]. Houston, Texas 77225,and Section of Veterinary Pathology, M. D. Anderson Cancer Center IL C. S.], Houston, Texas 77030

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Table 1 Antitumor effects ofWBH in the fibrosarcoma and the Ward coloncarcinomaTumorTreatmentTG@

(days)TGDb(days)Fibrosarcoma

ward coloncarcinomaControlWBH

ControlWBH7.0

±0.7'7.5 ±0.7

8.9 ±1.8147@35d0.5 5@8d

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Fig. 1. Photomicrographs of H & E-stained sections of the fibrosarcoma (X400). A, theuntreated tumor is composed of sheets of highly pleomorphic cells. B, the treated tumor4 h after WBH. There are widely scattered apoptotic cells in the tumor. Arrows point toapoptotic cells.

Apoptosis in the Tumor Tissues. Apoptosis in the two tumorsinduced by WBH was examined histopathologically between 4 and 48h after WBH. The transplanted fibrosarcoma grows as a noncircumscribed s.c. mass with invasion of surrounding tissues. The tumor iscomposed of sheets of highly pleomorphic cells (Fig. lÀ).The tumorcells have large irregular shaped nuclei that have vesiculated chromatin and large prominent nucleoli. Fig. lB shows the tumor 4 h after theWBH treatment. The tumor has widely scattered apoptotic cells. Thetransplanted Ward colon carcinoma grows as a multilobulated nonencapsulated mass, which compresses and invades the surroundingtissues. The tumor is composed of variable-size disorganized clumpsof cells (Fig. 14). The tumor cells have moderate to abundant cytoplasm and ovoid nuclei that contain single or multiple large nucleoli.The tumor contains a few cells that show the features of apoptosis.Fig. 2B shows the tumor 4 h after the WBH treatment. The tumorcontains widespread extensive apoptosis accompanied by coagulativenecrosis. Destruction of the tumor is extensive.

We examined the kinetics of apoptosis induced by WBH. Fig. 3shows the difference in the kinetics of apoptosis comparing the twotumors.

We observed: (a) the timing of the induction of a peak level ofapoptosis following WBH treatment was similar for both tumors.WBH induced a fairly rapid increase in apoptosis such that thepercentage of apoptosis was greatest at 4 h after WBH for thefibrosarcoma and at 4 to 8 h after WBH for the Ward colon tumor;

ously (23). Both the water bath temperature and the rat core body temperaturewere monitored continuously to an accuracy of 0.01°Cby YSI Nol 402thermistor small animal rectal probes connected to a YSI model 4002 12-channel switch box and a YSI model 49TA digital display telethermometer(YSI, Yellow Springs, OH; Ref. 25). The probes were calibrated against amercury thermometer (Ertco; American Society of Testing and Materialsstandard MC) certified by the National Institute of Standards and Technology.Body temperature was recorded every S mm. An average of 30 mm was

required for the rectal temperature to first reach 41.5°C.Throughout the WBHtreatment, both the water bath and the rat core body temperature were maintamed for 2 h at a temperature of 41.5 ±0.1°C.General anesthesia using 1%halothane in pure oxygen was administered for all WBH treatments. Thisanesthesia affects neither tumor growth nor normal tissue toxicity (27, 28).

Antitumor Effects of WBH. The antitumor effects of WBH were determined by an in vivo TGD assay, as described previously (28). Briefly, tumorsize was measured by using a vernier caliper to determine three perpendiculardiameters (d), and the tumor volume (v) was calculated by using the formula:v (dl X t12 X d3)/2.

Tumor size was measured every 2 days in the fibrosarcoma or every 3 daysin the Ward colon carcinoma. These time intervals used to monitor tumor sizeare based on previously established growth curves of the two tumors. TheWard colon carcinoma is a much slower-growing tumor compared with thefibrosarcoma.

TOD was calculated as the difference between control and treated rats in thetumor growth time to reach bO times the initial treatment volume for thefibrosarcoma or 3 times the initial treatment volume for the Ward coloncarcinoma, respectively. Each group consisted of five to six rats.

Histopathological Studies ofApoptosis. Separate tumor-bearing rats wereused for the histopathological examination of apoptosis induced by WBH. Todetermine the extent of apoptosis in tumors and normal tissues, rats wereeuthanized at indicated times after WBH. Tissues were fixed in 10% buffered

formalin (jH 6.9—7.1).From each paraffin-embedded sample, 4-gxm-thicksections were prepared and stained with H & E for light microscopic evaluation. Each group making up time points after WBH consisted of three rats.

The quantitative assay for apoptosis in tumor tissue was performed using anuclear aberration assay as described previously (8, 9). Briefly, five fields ofnonnecrotic areas were selected in each specimen, and 100 nuclei in each fieldwere categorized as normal, mitotic, or aberrant. The aberrations were characterized by overall shrinkage and homogeneous dark basophilia. Infrequently,several small apoptotic fragments were encountered in close proximity. On thebasis of size and clustering, such fragments were considered to represent theremains of a single cell and were counted as one apoptotic nucleus. The levelsof apoptosis in normal tissues were scored on a graded scale of 0—4:0, none;1, modest; 2, mild; 3, moderate; and 4, severe. All histopathological examinations were performed in a blinded fashion by the same veterinary pathologist(L C. S.).

RESULTS

Antitumor Effects. Table 1 shows the TGDs for WBH againstfibrosarcoma and Ward colon carcinoma. The fibrosarcoma used inthis study is resistant to WBH and showed only a minor and nonsignificant TGD of 0.5 day. In contrast, the Ward colon carcinoma isrelatively sensitive to WBH and showed a significant TGD of 5.8 days(P < 0.05 compared with control).

aTumorgrowthtime(TGT)wasthetimeforthetumortoreach10timesand3timestreatment volume in the fibrosarcoma and the Ward colon carcinoma, respectively.

b TGD was calculated as the difference in TGT between control and treated rats.CMean ±SD.d@ < o.os compared with control.

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Fig. 2. Photomicrographs of H & E-stained sections of the Ward colon carcinoma(X400). A, the untreated tumor is composed of disorganized clumps of cells of variablesize. There are a few spontaneous apoptotic cells. B, the treated tumor 4 h after WBH. Thetumor contains widespread, extensive numbers of apoptotic cells, accompanied by coagulative necrosis. Arrows point to apoptotic cells.

(b) the magnitude of the peak level of apoptosis was significantlygreater for the Ward colon tumor (39 and 43% at 4 and 8 h, respectively, after WBH) in comparison to the fibrosarcoma (19% at 4 hafter WBH; P < 0.05); and (c) although apoptosis was rapidly induced

in both the Ward colon carcinoma and the fibrosarcoma, the duration

of increased apoptosis due to WBH treatment was considerably longerfor the Ward colon tumor in comparison to the fibrosarcoma. In theWard colon tumor, the peak level of apoptosis of about 40% wasmaintained for at least 4 h (at 4 and 8 h after WBH), followed by arelatively gradual decline in the percentage of apoptosis to pretreatment levels by 48 h after WBH. In contrast, the peak level of 19%apoptosis at 4 h after WBH in the fibrosarcoma was of a much shorter

duration, declining rapidly to 8% by 6 h after WBH and then further

decreasing gradually to pretreatment levels by 24 h after WBH.

Apoptosis in the Normal Tissues. Apoptosis in the normal tissuesinduced by WBH was examined histologically from 4 to 48 h afterWBH. Fig. 4 shows the microscopic features of normal tissues exammed 4 h after WBH. We observed the greatest increase in apoptosisfollowing WBH exposure in the thymus. All lobules have clumps ofapoptotic cells throughout the cortex with a lesser level of apoptosis

in the medulla (Fig. 4B). WBH-induced cellular loss by apoptosis wasalso observed in the white pulp of spleen. Most of the splenicperiarteriolar lymphoid sheaths and the marginal zones containclumps of apoptotic cells (Fig. 4D). Changes in the incidence ofapoptosis following WBH were also observed in the mediastinal

lymph nodes, mesenteric lymph nodes, and gut-associated lymphoidtissues. In the gastrointestinal tract, apoptosis was prominent in thesmall intestine. Apoptotic cells were observed in the epithelium andthe lamina propria of the mucosal villus zone (Fig. 4F) in specimenscollected at 4 and 8 h after the beginning the 2-h WBH treatment at41.5°C.These apoptotic bodies seemed to be of lymphoid origin. Atthese same time points following WBH treatment, but to a lesserdegree, apoptosis was also observed in the crypt epithelium. Apoptosis was insignificant in the large intestine, and the stomach did notdemonstrate any apoptosis following WBH exposure.

In the bone marrow, there were increased numbers of apoptoticcells among the developing myeloid elements associated with a generalized marrow hypocellularity after WBH (Fig. 4H). Throughout thetime period of examination, we observed only negligible to nonexistent WBH-mediated apoptosis in the heart, lung, kidney, liver, pancreas, salivary glands, adrenal gland, ovary, and uterus.

Fig. 5 shows the kinetics of apoptosis in the thymus, spleen, smallintestine, and bone marrow. In the thymus, the level of apoptosis wasgreatest at 8 h after WBH. Thereafter, the cortex became thin withhypocellularity associated with a decrease in apoptotic cells. Althoughapoptotic cells were seen in some areas even 24 h after WBH, thethymus was atrophic without any apoptotic cells at 48 h, suggestingthat cell loss was complete. Apoptosis in the spleen, in contrast,peaked 4 h after WBH, and then levels declined rapidly to pretreatment levels by 12 h after WBH. At 12 h, the spleen showed hypocellularity resulting from cell loss by apoptosis, but apoptotic cellswere not seen. Thereafter, overall increased numbers of lymphoidcells in the lymphoid tissue with plentiful mitotic figures were observed. The peak of apoptosis in the small intestine also occurred 4 hafter WBH. Only a few apoptotic cells remained 4 h later and themucosa reverted completely to normal 12 h after WBH. The bonemarrow showed minimal apoptotic changes. Although there was anobservable increase in the number of apoptotic cells at 4 h, theapoptotic cells disappeared almost completely by 8 h after WBH. Atthat time, hyperplasia occurred with increased numbers of erythroidand myeloid cells accompanied by numerous megakaryocytes.

DISCUSSION

Tumors consist of cell populations in which cell gain and cell lossoccur simultaneously. The balance between cell gain and cell loss

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Fig. 3. Kinetics of apoptosis in tumor tissues induced by WBH. Rats bearing fibrosarcoma (0) or Ward colon carcinoma (•)were euthanized at the indicated times after thebeginning of'WBH treatment, and apoptosis was quantified as described in “MaterialsandMethods.―The control values for tumor apoptosis are indicated at the zero time point onthe X axis. The error bars indicate the SD for the mean value at each data point.

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Fig. 4. Photomicrographs of H & E-stained sections of the normal tissues (X400). Treated rats were euthanized 4 h after the beginning of WBH. The photomicrographs are ofrepresentative animals from the control and treated groups. A, thymus of a control rat. B, thymus of a treated rat. There are numerous apoptotic cells seen throughout the cortex. C,spleen of a control rat. D, Spleen of a treated rat. The white pulp contains widespread apoptotic celis, especially in the periarteriolar lymphoid sheaths. E, small intestine of a controlrat. F, small intestine of a treated rat. The lamina propria of the villi contains apoptotic cells. G, bone marrow of a control rat. H, bone marrow of a treated rat. There are scatteredapoptotic cells. Arrows point to apoptotic cells.

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APOPTOSIS INDUCED BY HYPERThERMIA

correlate with the sensitivity of each tumor to WBH-induced cellkilling, as measured by TGD.

In the Ward colon carcinoma treated with WBH, necrosis wasobserved, as well as apoptosis. Increasing the dose of stimuli maycause a switch from cell death by apoptosis to cell death by necrosis(1 1, 12). Both necrosis and apoptosis, however, can occur simultaneously in the tissues through independent processes. Why cells enterthe different paths of cell death in response to hyperthermia is notclear. Possibly the induction of necrosis is partially due to an indirecteffect of hyperthermia altering tumor microcirculation in solid tumors,in addition to direct heat-induced cytotoxicity. The antitumor effect ofWBH, of course, is an outcome of cell loss through at least these twoprocesses of apoptosis and necrosis.

Apoptosis is an essential process for living tissues to maintaintheir architecture and functions. Large numbers of cells can bedeleted physiologically from living normal tissues by apoptosisunder a variety of physiological situations as a normal biologicalresponse, which is activated when a multicellular organ requiresthe deletion of specific cells (14). Apoptosis also occurs in normaltissues in response to cytotoxic modalities such as radiation, chemotherapeutic drugs, and hyperthermia (17—22).In this study, wedemonstrated and characterized the occurrence of WBH-inducedapoptosis in normal tissues.

Physiological apoptosis in normal tissues occurs particularly in

tissues with a high cellular turnover (14), because cell death must keeppace with cell production in systems with extensive tissue renewal toprevent tissue expansion. Wyllie (30) suggested that cells in thehigh-turnover state are primed or programmed for apoptosis and, thus,

are killed by means of apoptosis in response to a variety of lethalstimuli. We showed that WBH also induced apoptosis in organs withrapidly renewing cell populations such as lymphoid tissues, intestine(in particular, cells of lymphoid origin in the lamina propria of themucosa), and bone marrow. Lymphoid tissues, especially the thymus,were most sensitive to WBH-induced cell death by apoptosis. Weobserved hypocellularity in the thymus during the time period afterWBH when the levels of apoptosis were decreasing, and this wasfollowed by cellular hyperplasia. The apoptotic change in bone marrow following WBH treatment was relatively mild, but bone marrowhyperplasia was recognized soon after the deletion of apoptotic bod

ies. Apoptosis is a physiological response to remove damaged cellsfrom tissues and may play an important role for maintenance of organstructure and/or functions altered by drug or heat treatment.

In contrast, the mucosa of the intestine returned to its normalcellular status after WBH exposure without secondary mucosal hyperplasia. Also, the extent of apoptosis occurring in the small intestineafter WBH treatment was less than that observed in the thymus or thespleen. Apoptotic cells in the mucosa are phagocytosed rapidly byneighboring healthy enterocytes or extruded into the lumen soon aftertheir formation (17, 18). Potten (31) analyzed apoptosis induced byirradiation and chemotherapeutic drugs in the intestine and suggestedthat apoptosis in the intestine may protect the surrounding uninjuredintestinal cells against drug-induced toxicity. Because cell death viaapoptosis is a process in which cells die singly and are rapidlyeliminated without causing any inflammatory damage to the surrounding tissue, the intestinal tissue is able to maintain its overall cellulararchitecture and continue to function. Therefore, apoptosis in thenormal tissues could be considered an event initiated by both extrinsicand intrinsic forces and may exist as a mechanism of cell deletion thatprotects neighboring cells from damage (14). In organ systems sensitive to WBH-induced damage, a large number of cells that areactually damaged by WBH seem to be removed by apoptosis within12—48h, which may allow the organ system to continue its normalfunction.

I

Fig. 5. Kinetics of apoptosis in normal tissues induced by WBH. Rats were euthanizedat the indicated times after the beginning of WBH, and apoptosis in the thymus (A), spleen(L@@),small intestine (•),and bone marrow (0) was scored on a graded scale of 0—4: 0,none; 1, modest; 2, mild; 3, moderate; and 4, severe. The error bars indicate the SD forthe mean value at each data point.

determines whether a tumor mass grows or regresses (29). Loss ofcells in a tissue results largely from cell death through apoptosisand/or necrosis. Both apoptosis and necrosis occur spontaneouslywithin tumors (30). Cell death, however, is also the end result ofcytotoxicity induced by therapeutic modalities that produce antitumoreffects.

Hyperthermia induces apoptosis in tumor cells in vitro (4, 5, 10,11). Harmon et a!. (13) quantified the incidence of apoptosis in fourin vivo murine tumors and showed that the ability of hyperthermia toinduce apoptosis varied from tumor to tumor. In that study, however,the level of apoptosis was examined only at one time point (4 h aftertreatment), and the correlation between the extent of apoptosis and thetumor response was not discussed. Takano et a!. (10) examinedheat-induced apoptosis using three cell lines in vitro and demonstratedthat the kinetics of apoptosis differed between those cell lines. Thesefindings indicate the need to evaluate both the extent and the kineticsof apoptosis in tumors in vivo. In this study, we measured WBHinduced apoptosis in two tumors, one of which is relatively resistantand the other relatively sensitive to WBH (41.5°Cfor 2 h). We founddifferences between the two tumors in both the extent and the kineticsof apoptosis following treatment. The differing levels of apoptosiscorrelated with the differing WBH-induced response seen in the twotumors.

Spontaneous apoptosis occurred in both tumors, but the level washigher in the heat-sensitive Ward colon carcinoma than in the heatresistant fibrosarcoma. Moreover, the kinetics of WBH-induced apoptosis was different when comparing these two tumors. WBH induced apoptosis rapidly, and a marked increase of apoptotic cells wasobserved in both tumors 4 h after the beginning of WBH. The extent

of apoptosis, however, was greater in the Ward colon carcinoma(39%) compared with the fibrosarcoma (19%). In the fibrosarcoma,the number of apoptotic cells decreased rapidly (by 8 h followingWBH), and meaningful increases in apoptotic cell numbers could nolonger be observed by 24 h. The elimination of apoptotic cells occursby phagocytosis of neighboring cells and macrophages (1). On theother hand, in the Ward colon carcinoma, the percent of apoptoticcells remained at a peak level (43%) at 8 h and then decreased moregradually to the pretreatment level 48 h after WBH. Thus, there wasconsiderable difference in the extent of WBH-mediated apoptosisbetween these two tumors in terms of both the peak values and overallkinetics of apoptosis. In addition, the extent of apoptosis seemed to

0 12 24 30 48

Twv* aft8f tPe be@irnsng of WBH Oi)

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Topoisomerase Il-reactive chemotherapeutic drugs induce apoptosis in thymocytes.Cancer Res., 51: 1078—1085,1991.

16. Storey, M. D., Stephens, L. C., Tomasovic, S. P., and Meyn, R. E. A role for calciumin regulation of apoptosis in rat thymocytes irradiated in vitro. Int. J. Radait. Biol., 61:243—251,1992.

17. Searle, J., Lawson, T. A., Abbott, P. J., Harmon, B., and Kerr, J. F. R. An electronmicroscope study of the mode of cell death induced by cancer-chemotherapeuticagents in populations of proliferating normal and neoplastic cells. J. Pathol., 116:129—138,1975.

18. Anilkumar, T. V., Sarraf, C. E., Hunt, T., and Alison, M. R. The nature of cytotoxicdrugs: induced cell death in murine intestinal crypts. Br. J. Cancer, 65: 552—558,1992.

19. Lee, F. D. Importance of apoptosis in the histopathology of drug related lesions in thelarge intestine. J. Clin. Pathol. (Land.), 46: 118—122,1993.

20. Duncan, A. M. V., Heddle, J. A., and Blakey, D. H. Mechanism of induction ofnuclear anomalies by y-radiationin the colonic epithelium of the mouse. Cancer Rca.,45: 250—252,1985.

21. Allan, D. J., and Harmon, B. V. The morphological categorization of cell deathinduced by mild hyperthermia and comparison with death induced by ionizingradiation and cytotoxic drugs. Scanning Electron Microsc., 3: 1121—1133, 1986.

22. Allan, D. J., Gobe, G. C., and Harmon, B. V. Sertoli cell death by apoptosis in theimmature rat testis following X-irradiation. Scanning Microsc., 2: 503—512,1988.

23. Wondergem, J., Bulger, R., Strebel, F., Newman, R., Travis, E., Stephens, L., andBull, J. Effect of cis-diamminedichloroplatinum (II) combined with whole bodyhyperthermia on renal injury. Cancer Res., 48: 440—446, 1988.

24. Danhauser, L L, and Rustum, Y. M. Effect of thymidine on the toxicity, antitumoractivity, and metabolism of 1-B-o-arabinofuranosylcytosine in rats bearing a chemically induced colon carcinoma. Cancer Res., 40: 1274—1280, 1980.

25. Baba, H., Stephens, L. C., Strebel, F. R., Siddik, Z. H., Newman, R. A., Ohno, S., and

Bull, J. M. C. Protective effect of ICRF-187 against normal tissue injury induced byadriamycin in combination with whole-body hyperthermia. Cancer Rca., 51: 3559—3567,1991.

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27. Wondergem, J., Strebel, F., Siddik, Z., Newman, R., and Bull, J. The effects ofanesthetics on cisplatinum-induced toxicity at normal temperatures and during wholebody hyperthermia: the influence of NaCI concentration of the vehicle. tnt. J.Hyperthermia, 4: 643—654, 1988.

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5464

We describe the extent and kinetic features of WBH-induced apoptosis in tumor and normal tissues in vivo. Our fmdings suggest thatapoptosis may play a pivotal role in the outcome of therapy. Indeed,the induction of apoptosis by heat, radiation, or chemotherapy mayforeordain both tumor and normal tissue response to the treatment.

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1995;55:5459-5464. Cancer Res   Yoshihisa Sakaguchi, L. Clifton Stephens, Masato Makino, et al.   Body Hyperthermia in RatsApoptosis in Tumors and Normal Tissues Induced by Whole

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