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The objective was to test the hypothesis that wild-type p53-function is required for the enhancement of the cytotoxicity of cis-diammine-dichloroplatinum(II) ...

INT. J. HYPERTHERMIA,

2001,

VOL.

17,

NO.

4, 337 ± 346

Wild-type p53-function is not required for hyperthermia-enhanced cytotoxicity of cisplatin

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C. VAN BREE{{*, N. A. P. FRANKEN{, F. A. M. SNEL{, J. HAVEMAN{ and P. J. M. BAKKER{ { Department of Medical Oncology, Division of Internal Medicine; { Department of Radiotherapy, Academic Medical Centre, University of Amsterdam, Room F0-205, PO Box 22700, 1100 DE Amsterdam, The Netherlands (Received 20 October 2000; accepted 20 March 2001) The objective was to test the hypothesis that wild-type p53-function is required for the enhancement of the cytotoxicity of cis-diammine-dichloroplatinum(II) (cDDP) cytotoxicity by hyperthermia (HT). Human colorectal carcinoma cells (RKO) with wild-type p53-function and transfectants with HPV16-E6 or with a dominant negative mutant p53 were used. Cells were treated with HT (60 min at 418C, 438C, 458C: HT41, HT43, HT45), with various doses of cDDP alone or with a combined treatment, simultaneously applied. Survival was determined by clonogenic assays. Levels and localization of p53 were analysed with immunocytochemistry and Western blotting. The extent of HT41-enhanced cytotoxicity of cDDP was similar in all cell lines studied. Immunocytochemistry of wild-type p53 cells showed that p53 is transferred to the nucleus within 5 h after HT43, whilst after HT41 no signi® cant eŒects were observed. Cell fractionation experiments of wild-type p53 cells showed that, immediately after HT43/ HT45, nuclear p53-levels increased as compared to controls, but could not be extracted from the matrix. The extractability was restored 3± 5 h after treatment. No signi® cant diŒerences in p53-levels were observed after HT41. These results indicate that, although HT43/HT45 might shortly inactivate p53-function, probably by protein aggregation to the nuclear matrix, the HT-enhanced cDDP-cytotoxicity does not depend on p53-function. Key words: Hyperthermia, cisplatin, protein p53, cell death, Western blotting.

1.

Introduction The p53 tumour suppressor gene is mutated in many human cancers1 . Wild-type p53 protein is a transcription factor for a variety of genes involved in the regulation of cell cycle arrest, apoptosis and DNA damage repair. The function of p53 can selectively be inactivated by the expression of the oncoproteins E6 of the high-risk Human papillomaviruses2 . From studies using isogenic cell systems, it has become evident that inactivation of p53-function increases the cytotoxicity of cisplatin (cisdiammine-dichloroplatinum(II) : cDDP 3, 4 ). Treatment of tumours by hyperthermia (HT) enhances the cell killing eŒects of cDDP, both in various animal tumour models5 and in patients with recurrent cervical carcinoma6 . Inhibition of DNA damage repair by HT has been suggested to be the most important mechanism of this interaction in vivo7 . Contradictory ® ndings have been reported with regard to the function of p53 after HT. A transient G1 cell cycle arrest after HT was observed in human ® broblasts, which was mediated by the transfer of p53 to the nucleus, where p53 trans* To whom correspondence should be addressed. e-mail: [email protected] Internationa l Journal of Hyperthermia ISSN 0265± 6736 print/ISSN 1464± 5157 online # 2001 Taylor & Francis Ltd http://www.tandf.co.uk/journals DOI: 10.1080/0265673011005313 7

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activated p218 . However, in the same human carcinoma cell line as used in this study, it was shown that HT functionally inactivates p53 by restricting it to the cytoplasm9 . In addition, it was found that the interaction between cDDP and HT depends on p53-function in human glioblastoma cells10 . These ® ndings initiated one to test the hypothesis that HT partly increases the cytotoxicity of cDDP by preventing the nuclear transfer of p53 and, thus, inactivates p53-function. Cytotoxicity and accumulation of p53 were analysed after treatment with cDDP and HT in an isogenic system using the human colorectal carcinoma cell line RKO containing functional p53 and transfectants with HPV16-E6 or with a dominant negative mutant p53. The results indicate that HT increases the cytotoxicity of cDDP, but does not inactivate the function of p53. 2. Materials and methods 2.1. Cell lines The human colorectal carcinoma cell line (RKO, referred to as wt-p53-1) with wild-type p53 alleles and stable transfectants with the pCMVneo expression vector alone (RCNeo, referred to as wt-p53-2), with HPV16-E6 (RC10.1 and RC10.2, referred to as E6-1 and E6-2) or with a dominant negative mutant p53 (substitution of alanine for valine at codon 143, mp53.13, referred to as m-p53) has been described previously2,11 . These cell lines were kindly provided by Dr Kathleen Cho (RKO, RCNeo, RC10.1 and RC10.2) and by Dr Michael Kastan (m-p53). The cells were cultured as monolayers in McCoy’s 5A medium with 25 mm HEPES, supplemented with 10% foetal bovine serum, glutamine, penicillin and streptomycin at 378C in an atmosphere of 5% CO2 in air. For selection, geneticin (200 mg/ml) was added to the complete medium to maintain the transfectants . Loss of G1 arrest and basal and induced levels of p53 after irradiation were routinely checked throughout the study 2,11,12 . In order to obtain exponentially growing cells, 300 000 cells were plated into 60 mm dishes (Corning Costar Europe, Badhoevedorp, The Netherlands) 3 days prior to treatment in complete medium, without geneticin to minimize in¯ uences of geneticin on the outcome of treatment. In this experimental set-up, the cell population doubling time for all clones was 18± 22 h. 2.2. Treatments A stock-solution of cDDP (1 mg/ml sterile saline, Platosin1, Pharmachemie, Haarlem, The Netherlands) was stored at room temperature. Appropriate dilutions were freshly prepared for each experiment. Cells were exposed to cDDP in various concentrations for 70 min without or with simultaneous application of HT, which was performed by placing the dishes in a thermostaticall y regulated waterbath5 . This waterbath was placed in an incubator with the appropriate atmosphere. Ten minutes after placement of the dishes into the waterbath, the desired temperature in the wells was reached, and this was denoted as the starting time of HT. After treatment, the cells were washed twice with fresh medium. 2.3. Clonogenic cell survival Cells were harvested and counted using a Coulter Counter (Coulter Electronics, Luton, UK) and plated in appropriate density in 6-well macroplates (Greiner Labortechnik, Frickenhausen, Germany). After 10± 12 days incubation for colony formation, the colonies were ® xated by 6% glutaraldehyde and stained with 0.05% crystal violet. Colonies of 50 cells or more were scored as originating from a single

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clonogenic cell. Surviving fractions were calculated by dividing the plating e ciency of treated cells by that of control cells (mean control plating e ciency § SE: wt-p53-1: 0.49 § 0.10; wt-p53-2: 0.40 § 0.09; E6-1: 0.48 § 0.08; E6-2: 0.42 § 0.06; m-p53: 0.50 § 0.06). Survival after combined treatment was also corrected for the cytotoxicity of HT41 alone (mean surviving fraction § SE: wt-p53-1: 0.41 § 0.07; wt-p53-2: 0.28 § 0.07; E6-1: 0.46 § 0.08; E6-2: 0.51 § 0.13; m-p53: 0.43 § 0.16). 2.4. Immunocytochemistry Nuclear accumulation of p53 was detected by an indirect immunostaining method, as described earlier8 . Cells (300 000) were cultured on cover slips (15 mm) in 24-wells plates for 2 days to ensure exponential growth. After HT, cells were incubated for various periods at 378C. Cells were ® xed with ice-cold methanol:aceton (1:1) for 2 min at 208C and air-dried. For immunostaining, the cover slips were washed four times with PBS and stained for 60 min at room temperature with mAb DO-7 (1:400 diluted in PBS). After washing with PBS, the cover slips were incubated for 60 min at room temperature with a biotin-conjugated anti-mouse IgG (1:100 diluted in PBS, Jackson Immunoresearch Laboratories, West Grove, PA, USA). After washing with PBS, the cover slips were incubated with ¯ uorescein-conjugate d streptavidin (1:100 diluted in PBS, Jackson Immunoresearch Laboratories, West Grove, PA, USA). After counterstaining with DAPI (5 mg/ml PBS, 5 min at room temperature), the cover slips were washed with PBS and were embedded in antifadesolution (Vectashield, Vector laboratories, Burlingame, CA, USA). Slides were examined using a ¯ uorescence microscope (Ortholux II, Leica, Wetzlar, Germany) under blue light excitation (495 nm) and emission ® lters (519 nm) to detect ¯ uorescein and under UV light excitation (372 nm) and emission ® lters (456 nm) to detect DAPI. Pictures were taken using a CCD-camera (Lambert instruments, Leutingerwolde, The Netherlands). 2.5. Cell fractionation and Western blotting Cells were fractionated according to the following method recommended by R. L. Warters (University of Utah): cells were harvested, washed in ice-cold phosphate buŒered saline (PBS) and pelletted. The cells were resuspended in Eppendorf tubes in ice-cold hypotonic buŒer (10 mm HEPES pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm DTT, 0.5 mm PMSF, 50 mg/ml antipain, 10 mg/ml leupeptin, 10 mg/ml pepstatin A, 0.5 mm AEBSF, 2 mg/ml aprotinin). After 15 min swelling on ice, the cells were lysed by adding NP-40 to an end-concentration of 0.5% and were centrifuged for 2 min at maximum speed (15 800 g) at 48C in an Eppendorf centrifuge. The supernatant was collected as the cytoplasmic fraction; nuclei were still intact as evaluated by phase-contrast microscopy. The pellet was resuspended in ice-cold extraction buŒer (20 mm HEPES pH 7.9, 0.4 m NaCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm DTT, 1.0 mm PMSF, 50 mg/ml antipain, 10 mg/ml leupeptin, 10 mg/ml pepstatin A, 1.0 mm AEBSF, 2 mg/ml aprotinin) and mixed vigorously for 30 min at 48C. After centrifugation for 5 min at maximum speed at 48C, the supernatant was collected as the nuclear extract. The remaining pellet was considered to represent the nuclear matrix. Samples were stored at 708C. Protein samples were resolved by 12% SDS-polyacrylamid e gel electrophoresis and transferred to nitro-cellulose membranes using standard techniques. Equal protein loading was checked by staining the membranes with Ponceau Red. Immunodetection of p53 was performed with mAb DO-7 (DAKO, Glostrup, Denmark), a peroxidase-conjugate d secondary anti-mouse

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3. Results 3.1. Sensitivity to cDDP and HT The sensitivity to cDDP, with or without HT41, for cells with functional and abrogated p53 is shown in ® gure 1. The p53-inactivate d cell lines E6-1 and E6-2 are a factor 1.4 and 1.5, respectively, more sensitive to cDDP, as compared to the wildtype p53 cell lines (table 1). The sensitivity of the dominant-negativ e mutant p53 cell line m-p53 to cDDP was not signi® cantly diŒerent from the wild-type p53 cell lines. HT-induced enhancement was obtained in all cell lines and examples for a wild-type p53 cell line and a p53-inactivate d cell line are shown (® gure 1, right panel). The thermal enhancement of the cytotoxicity of cDDP ranged from 2.2± 3.5, but was not signi® cantly diŒerent between wild-type p53 and p53-inactivate d cells (table 1). 3.2. Immunocytochemistr y of p53 after HT As p53-status did not in¯ uence HT-enhanced cytotoxicity of cDDP, one investigated whether p53 was transferred to the nucleus of wild-type p53 cells after HT at 418C and higher temperatures, as reported earlier8,9 .

10 0

Surviving fraction

10 0

Surviving fraction

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IgG (DAKO, Glostrup, Denmark) and enhanced chemoluminescence (Amersham Pharmacia Biotech, Uppsala, Sweden). To verify the method of the cell fractionation experiments after HT, the response after ionizing radiation was evaluated. Basal levels of p53 were very high in m-p53 cells and undetectable in E6-1 and E6-2 cells as compared to the wild-type p53 cells in the cytoplasm, in the nuclear extract and in the nuclear matrix. Four hours after irradiation with 4 Gy of ®-rays, an increase in p53-levels was found in the cytoplasm, in the nuclear extract and in the nuclear matrix of wild-type p53 cells, which was not observed in the E6-1, E62 and in m-p53 cells.

10 -1

10 -1

wt-p53-1 wt-p53-2 E6-1 E6-2 mp53.13

10 -2

10 -3

0

2

10 -2

wt-p53-1 E6-1 wt-p53-1+HT41

4

6

[cDDP], mg/ml

8

10

10 -3

E6-1+HT41 0

2

4

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[cDDP], mg/ml

Figure 1. Sensitivity to treatment with cisplatin alone (cDDP, left panel) and to simultaneously combined treatment with hyperthermia (HT41: 60 min at 418C, right panel) for cells with functional (wt-p53-1 and wt-p53-2) and abrogated p53 by transfection of HPV-E6 (E6-1 and E6-2) or by a dominant-negative mutant p53 (m-p53). Surviving fraction is plotted as a function of cDDP-dose in mg/ml. For clarity, only the results with wt-p53-1 and E6-1 cells are given in the right panel. Mean values with standard errors of at least three separate experiments are shown.

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Wild-type p53-functio n Table 1. Cell line

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wt-p53-1 wt-p53-2 E6-1 E6-2 m-p53

IC10 and TER in cells with wild-type or abrogated p53-function. IC10 7.03 § 0.31 7.08 § 0.56* 5.18 § 0.32** 4.80 § 1.04 6.35 § 0.57

IC10 with HT41

TER

2.86 § 0.23 2.67 § 0.26 1.48 § 0.14 1.87 § 0.45 2.95 § 0.21

2.5 § 0.2 2.7 § 0.3 3.5 § 0.4 2.6 § 0.8 2.2 § 0.2

IC10 is the concentration cDDP in mg/ml needed to decrease cell survival to 10% . TER is the thermal enhancement ratio of the IC10 without and with HT at 418C. Values are expressed as the mean SE of at least three separate experiments. Inactivation of p53-function leads to a signi® cant increase in sensitivity to cDDP (a factor 1.4 § 0.1 for E6-1 (* p 5 0.024) and 1.5 § 0.3 (** p 5 0.029) for E6-2 as compared to the transfection control wt-p53-2).

The number of p53-positive nuclei increased in wild-type p53 cells 5 h after HT43 (wt-p53-1 cells in ® gure 2, similar results were obtained for wt-p53-2 cells). However, no clear increase in the number of p53-positive nuclei was observed after HT41. In order to be able to detect changes in p53-levels immediately after HT, the m-p53 cells that express high levels of dominant-negativ e p53 were analysed. The nuclear p53levels were not signi® cantly changed after HT43, but after HT45 the nuclear ¯ uorescence appeared to decline. No ¯ uorescence was observed in p53-inactivate d E6-1 or E6-2 cells with or without HT.

Figure 2. Nuclear presence of p53 in wild-type p53 cells (wt-p53-1) and in dominant-negative mutant p53 cells (m-p53) in control cells or in hyperthermia-treated cells (HT43/HT45: 60 min. at 438C or at 458C) at 0 and 5 hours after treatment. Nuclear counterstaining with DAPI of control m-p53 cells is shown for comparison (top right ® gure). Detection of p53 was performed by an indirect immuno¯ uorescent staining using an anti-p53 mouse monoclonal antibody (DO-7, DAKO).

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C

0

HT41 1 3

5

C

0

HT43 1 3

5

cytoplasm nuclear extract

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nuclear matrix Figure 3. Levels of p53 in the cytoplasm, in the nuclear extract or in the nuclear matrix of wild-type p53 cells (wt-p53-1) in control cells (C) or in hyperthermia-treated cells (HT41/ HT43: 60 min at 418C or 438C) at 0, 1, 3 and 5 h after treatment.

3.3. Cell fractionation and Western blotting of p53 after HT In order to investigate the localization of p53 after HT, cell fractionation studies were performed in which the levels of p53 were evaluated by Western blotting. The changes in levels of p53 in the cytoplasm, in the nuclear extract and in the remaining nuclear matrix are shown of wt-p53-1 cells (® gures 3 and 5) and m-p53 cells (® gure 4). Only immediately after HT41 (® gure 3, left panel), a slight increase in p53 levels was found in wild-type p53 cells, which was returned to control levels 1 h after HT41. Immediately after HT43 (® gure 3, right panel), a marked decrease in p53-levels in the nuclear extracts was observed, which was restored after 1 h. In the cytoplasm, clearly elevated levels of p53 were found after 3± 5 h. In the nuclear matrix fraction, p53levels were increased from 0 to 5 h after HT43. In m-p53 cells, no changes were found using HT41 and HT43 in all cell fractions (data not shown). Immediately after HT45 (® gure 4), p53-levels were signi® cantly reduced in cytoplasm and nuclear extract as compared to control m-p53 cells, whilst the level of p53 in the nuclear matrix fraction increased. Again, the extractability of p53 from heated nuclei was restored at 5 h after treatment. In ® gure 5, it is demonstrated that the behaviour of p53 after combined treatment is dominated by the eŒect of HT. A decreased p53-level in the nuclear extract as compared to control was observed immediately after HT43 alone, as well as after

C

HT45 0 5

cytoplasm

nuclear extract nuclear matrix Figure 4. Levels of p53 in the cytoplasm, in the nuclear extract or in the nuclear matrix of dominant-negative mutant p53 cells (m-p53) in control cells (C), or in hyperthermiatreated cells (HT45: 60 min at 458C) at 0 and 5 h after treatment.

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C

cDDP 0 5

HT43 0

5

cDDP+HT43 0 5

cytoplasm nuclear extract

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nuclear matrix Figure 5. Levels of p53 in the cytoplasm, in the nuclear extract or in the nuclear matrix of wild-type p53 cells (wt-p53-1) in control cells (C), or in cells treated with cDDP alone, with HT alone (HT43: 60 min at 438C), or with combined treatment (cDDP1 HT43) at 0 and 5 h after treatment.

combined treatment. An increased p53-level in the nuclear extract was found 5 h after both HT43 alone and combined treatment. Increased p53-levels in the nuclear matrix fraction were found immediately and 5 h after both treatments. In ® gure 5, it is also shown that an increased p53-level is observed 5 h after cDDP alone in the cytoplasm and in the nuclear extract, but not in the nuclear matrix fraction. 4.

Discussion To determine the role of p53 in HT-enhanced cytotoxicity of cDDP, a wellestablished isogenic cell system of human colorectal carcinoma cell line RKO that harbours wild-type p53-function, was used. Throughout this study, the cell system was regularly checked for the response to ionizing radiation with regard to accumulation of p53 and the G1 cell cycle arrest2,11,12 . The results con® rm that inactivation of p53-function by transfection of HPV-E6 enhances the cytotoxicity of cDDP. However, the enhancement factor of 1.4 determined at the 10% survival level is markedly lower than that of ¹3 derived from the IC50-level, as reported by Fan et al.3 for the same isogenic cell system as used in these experiments. One determined survival with clonogenic assays after a 70 min exposure to cDDP in order to be able to combine cDDP-treatment simultaneously with HT. According to the method described by Fan et al.3 , one has also analysed the eŒects of cDDP alone using a proliferation assay. The proliferation data con® rmed the results of the clonogenic assay with respect to the increased sensitivity to cDDP when p53 was inactivated by transfection of HPV-E6 (enhancement factor of 1.4). Also in contrast to Fan et al.3 , the dominant-negative mutant p53 clone m-p53 responded in this experiment similar to cDDP-treatment as the wt-p53 clones. Possibly, the long-term culturing of the m-p53 cells has altered some features of the cells which may be related to the `gain of function’ properties that have been described for mutant p5313 . In other isogenic cell systems, functional inactivation of p53 also enhances the cytotoxicity to cDDP and UV-irradiation4,14,15. Both induce DNA damage that is repaired by the nucleotide excision repair pathway which is modulated by p5314,16-19 . Because of the high cytotoxicity of HT alone at higher temperatures20 , one could only investigate the sensitivity to combined cDDP and HT at a moderate heat dose of 60 min at 418C, which reduced survival of the cell lines studied to 30± 50% . The data indicate that HT enhanced the cytotoxicity of cDDP, irrespective of p53-func-

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tion. This is in agreement with the transient nuclear transfer of p53 in primary human ® broblasts after treatment for 45 min at 438C, which was accompanied by an increased expression of p21 and a G1 cell cycle arrest8 . Indirect evidence for an intact function of p53 after HT alone was demonstrated by a higher induction of apoptosis in wild-type p53 cells, as compared to p53-inactivate d cells20 . This was also shown for transfection of wt-p53 into ® broblasts of p53 knockout mice21 . For the combination of a temperature of 448C for 15 min and cDDP studied in two human glioblastoma cell lines with diŒerent p53-status , it was reported that an interactive HT-enhancement of cDDP cytotoxicity only occurred in wild-type p53 cells10 . In addition, the suppression of heat-induced hsp72 accumulation by cDDP-treatment was found to be dependent on p53-status10 . On the other hand, treatment for 60 min at 438C was reported to restrict p53 in the cytoplasm, accompanied by a decrease in nuclear p53, which resulted in the loss of DNA binding ability in wild-type p53 RKO cells9 . In order to clarify the contradiction in the literature with respect to the function of p53 after HT at temperatures of 438C and higher, experiments were performed to determine what happens with p53 after heat shock with diŒerent temperatures. The immunocytochemistry data clearly show nuclear accumulation of p53 within 5 h after 60 min at 438C in wt-p53 cells, similar to the ® ndings in primary human ® broblasts 8 . The decrease of the supposed transient nuclear transfer after this treatment could not be studied because of the massive induction of apoptosis at later time-points 20 . One could not detect a nuclear accumulation of p53 after 60 min at 418C using immunocytochemistry in wt-p53 cells. In m-p53 cells, 60 min at 418C or at 438C did not lead to signi® cant changes in nuclear accumulation of p53. However, 60 min at 458C appeared to decrease nuclear ¯ uorescence, although it should be noted that only a few cells could be evaluated after this severe heat shock. In order to explore what happens with p53 after HT at diŒerent temperatures, the levels of p53 in several cellular fractions after treatment were determined using Western blotting. As expected, no restriction of p53 to the cytoplasm was observed after HT41 in wild-type p53 cells. Perhaps a short-lived accumulation of p53 is present in the cytoplasm and in the nuclear extract during or immediately after HT41, which may be the result of an increased half-life of p53 due to binding to hsp709 . No signi® cant changes in p53-levels were noted during the ® rst 5 h after HT41 in the nuclear matrix. In agreement with the ® ndings of Graeber et al.9 , immediately after HT43 the p53-level is clearly decreased in the nuclear extract as compared to controls. However, it is shown here that the p53 is not restricted to the cytoplasm, but remains tightly bound to the nuclear matrix. P53 can be found in the nuclear extract after 1± 5 h after HT43, whilst the p53-levels in the nuclear matrix are still elevated as compared to controls. To con® rm that after higher heat doses p53 is bound to the nuclear matrix, which prevents nuclear extraction of p53, the p53-levels were also measured in m-p53 cells. Since the decrease of p53 in the nuclear extract of m-p53 immediately after HT43 was not convincing, one also analysed the eŒects of HT45. Next to a marked decrease found in the cytoplasm, probably the result of protein degradation, a signi® cant decrease was observed in the nuclear extract immediately after HT45, which was again accompanied by an increase in p53-level in the nuclear matrix. The p53-levels appeared to recover in cellular fractions as indicated by the 5h time-point, even after this severe treatment. These changes in p53-levels are likely to be physiological, because they were measured within 5 h after treatment. This

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proceeds the induction of apoptosis, which is known to be dependent on p53-function and occurs 6± 24 h after HT20,21. However, clonogenic survival assayed beyond six cell divisions could not be determined for HT43 and HT45. The aggregation of proteins after heat shock at temperatures above 438C has been shown earlier22 . This nuclear protein aggregation may also explain why the HT43-induced increase of p53 in the nuclear extract could not be demonstrated using immunocytochemistry. The epitope of the antibody was probably masked immediately after treatment, which was restored at later time-points. In conclusion, the results indicate that the behaviour of p53 after HT depends on the temperature applied. The sticking of p53 to the nuclear matrix at high temperature may inhibit p53-function. However, it is shown that it is quickly restored. The enhancement of cDDP-cytotoxicity by HT does not require p53-function. Acknowledgements The authors wish to thank Professor D. GonzaÂlez Gonza lez for his continuous interest in this study. The Maurits & Anna de Kock foundation is acknowledged for sponsoring laboratory equipment. References 1. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991; 253: 49± 53. 2. Kessis TD, Slebos RJ, Nelson WG, Kastan, MB, Plunkett BS, Han SM, Lorincz AT, Hedrick L, Cho KR. Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage. Proc Natl Acad Sci USA 1993; 90: 3988± 92. 3. Fan S, Smith ML, Rivet DJ, Duba D, Zhan Q, Kohn KW, Fornace AJ Jr, O’ Connor PM. Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline. Cancer Res 1995; 55: 1649± 54. 4. Hawkins DS, Demers GW, Galloway DA. Inactivation of p53 enhances sensitivity to multiple chemotherapeutic agents. Cancer Res 1996; 56: 892± 8. 5. van Bree C, Rietbroek RC, Schopman EM, Kipp JB, Bakker PJ. Local hyperthermia enhances the eŒect of cis-diamminedichloro-platinum(II) on nonirradiated and preirradiated rat solid tumors. Int J Radiat Oncol Biol Phys 1996; 36: 135± 40. 6. Rietbroek RC, Schilthuis MS, Bakker PJ, van Dijk JD, Postma AJ, Gonzalez Gonzalez D, Bakker AJ, van der Velden J, Helmerhorst TJ, Veenhof CH. Phase II trial of weekly locoregional hyperthermia and cisplatin in patients with a previously irradiated recurrent carcinoma of the uterine cervix. Cancer 1997; 79: 935± 43. 7. Dahl O. Mechanisms of thermal enhancement of chemotherapeutic cytotoxicity. In: Urano M, Douple E, eds. Hyperthermia and Oncology. Utrecht: VSP, 1994: 9± 28. 8. Nitta M, Okamura H, Aizawa S, Yamaizumi M. Heat shock induces transient p53dependent cell cycle arrest at G1/S. Oncogene 1997; 15: 561± 8. 9. Graeber TG, Peterson JF, Tsai M, Monica K, Fornace AJ Jr, Giaccia AJ. Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by lowoxygen conditions is independent of p53 status. Mol Cell Biol 1994; 14: 6264± 77. 10. Matsumoto H, Hayashi S, Shioura H, Ohtsubo T, Kitai R, Ohnishi K, Hayashi N, Ohnishi T, Kano E. Suppression of heat-induced HSF activation by CDDP in human glioblastoma cells. Int J Radiat Oncol Biol Phys 1998; 41: 915± 20. 11. Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci USA 1992; 89: 7491± 5. 12. van Bree C, Savonije JH, Franken NA, Haveman J, Bakker PJ. The eŒect of p53-function on the sensitivity to paclitaxel with or without hyperthermia in human colorectal carcinoma cells. Int J Oncol 2000; 16: 739± 44. 13. Dittmer D, Pati S, Zambetti G, Chu S, Teresky AK, Moore M, Finlay C, Levine AJ. Gain of function mutations in p53. Nature Genetics 1993; 4: 42± 6.

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