Different inducibility of radiation- or heat-induced p53 -dependent apoptosis after acute or chronic irradiation in human cultured squamous cell carcinoma cells

Purpose : This study was undertaken to clarify the effects of acute or chronic pre-irradiation on the induction of p53 -dependent apoptosis by X-rays or heat shock. Materials and methods : Having an identical genotype except for p53 -status, the human cultured squamous cell carcinoma cells (SAS) were transfected with a mutant p53 gene (SAS/m p53) or neo alone (SAS/ neo) as a control. After acute X-irradiation (1Gy min -1) , chronic gamma-irradiation (0.001 Gy min -1) or heat shock (44°C), the cells were for the incidence of apoptotic bodies and DNA ladders, cellular levels of p53 and bax, and caspase-3 activity. Results : It was found that (1) a challenge treatment with X-rays (5.0 Gy) or heat shock (30 min) immediately after chronic pre-irradiation (1.5Gy) but not acute pre-irradiation (1.5 Gy) resulted in lower levels of apoptosis than those observed after challenge treatment only in SAS/ neo cells; (2) a challenge treatment-induced apoptosis was observed 48h after cessation of chronic pre-irradiation in SAS/ neo cells; (3) apoptosis was barely increased in SAS/m p53 cells; and (4) the levels of apoptosis-related proteins after challenge treatments were strongly correlated with the above phenomena. Conclusions : Chronic pre-irradiation at a low dose-rate suppressed induction of p53 -dependent apoptosis via bax and caspase-3. These findings suggest that chronic pre-irradiation suppressed p53 function through radiation-induced signalling and/or p53 stability.     

p53-dependent thermal enhancement of cellular sensitivity in human squamous cell carcinomas in relation to LET.

PURPOSE: To investigate the dependence on p53 gene status of the thermal enhancement of cellular sensitivity against different levels of linear energy transfer (LET) from X-rays or carbon-ion (C-) beams. MATERIALS AND METHODS: Two kinds of human squamous cell carcinoma cell lines were used with an identical genotype except for the p53 gene. SAS/mp53 cells were established by transfection with mutated p53 (mp53) gene to SAS cells having functional wild-type p53 (wtp53). As the control, a neo vector was transfected to the SAS cells (SAS/neo cells). Both cells were exposed to X-rays or accelerated C-beams (30-150 KeV microm(-1)) followed by heating at 44 degrees C. Cellular sensitivity was determined by colony-forming activity. Induction of apoptosis was analysed by Hoechst 33342 staining of apoptotic bodies and agarose-gel electrophoresis for the formation of DNA ladders. RESULTS: It was found that (1) there was no significant difference in cellular sensitivity between SAS/neo and SAS/mp53 cells to LET radiation of >30 KeV microm(-1), although the radiosensitivity of SAS/neo cells to X-rays was higher (1.2-fold) than that of SAS/mp53 cells; (2) there was an interactive thermal enhancement of radiosensitivity below an LET of 70 KeV microm(-1) in SAS/neo cells, although only additive thermal enhancement was observed in SAS/mp53 cells through all LET levels examined; (3) low-LET radiation induced apoptosis only in SAS/neo cells; (4) high-LET radiation at an isosurvival dose-induced apoptosis of SAS/neo cells at a higher frequency compared with that with low-LET radiation; (5) high-LET radiation-induced p53-independent apoptosis in SAS/mp53 cells; and (6) thermal enhancement of cellular sensitivity to X-rays was due to induction of p53-dependent apoptosis. CONCLUSIONS: The findings suggest that thermal enhancement of radiosensitivity may result from p53-dependent apoptosis induced by inhibition of p53-dependent cell survival system(s) through either regulation of the cell cycle or induction of DNA repair. It is also suggested that the analysis of p53 gene status of cancer cells may predict response to combined therapies with low-LET radiation and hyperthermia.     

Effects of a heat shock protein inhibitor KNK437 on heat sensitivity and heat tolerance in human squamous cell carcinoma cell lines differing in p53 status

PURPOSE: The effects of a heat shock protein (hsp) inhibitor KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolactam) were examined on the heat sensitivity and heat tolerance of human cancer cells with special reference to p53 status. MATERIALS AND METHODS: Human squamous cell carcinoma (SAS) and glioblastoma cell lines (A-172) transfected with mutant p53 (mp53) or control neo genes were used. KNK437 was added in culture medium at a final concentration of 50, 100 or 300 microM 1 h before heating (42 degrees C). Surviving fractions of cells were measured by use of a clonogenic assay. Effects of KNK437 on the accumulation of heat shock proteins and DNA binding activity of heat shock factor 1 were examined with Western blot analysis and gel mobility-shift assay, respectively. Heat-induced apoptotic bodies were detected by Hoechst 33342 staining. RESULTS: The mp53-transfected SAS (SAS/mp53) and A-172 (A-172/mp53) cells were more resistant to heat than the neomycin (neo)-transfected SAS (SAS/neo) and A-172 (A-172/neo) cells. The constitutive amount of hsp27 was larger in SAS/mp53 than in SAS/neo cells. Clear differences in the constitutive amounts of hsp40, hsp72 and hsp90 were not observed between SAS/mp53 and SAS/neo cells. KNK437 enhanced the heat sensitivity in SAS/mp53 and A-172/mp53 cells more effectively than in neo control cells. Heat tolerance was suppressed by KNK437 in SAS/mp53 and SAS/neo cells and also in A-172/mp53 and A-172/neo cells. Along with suppression of heat tolerance, KNK437 suppressed heat-induced accumulation of both hsp27 and hsp72. Heat-induced apoptotic bodies were enhanced by KNK437 in SAS/mp53 and SAS/neo cells. CONCLUSION: The results suggest a possible mechanism for the heat sensitivity of SAS cells. Heat sensitivity depends on p53 status regulating the amount of hsp27. Heat tolerance is suppressed by KNK437 through the suppression of heat-induced accumulations of hsp27 and hsp72 and the induction of p53-independent apoptosis.     

Radiation response of apoptosis in C57BL/6N mouse spleen after whole-body irradiation

Purpose : Primary conditioning low dose irradiation suppresses the molecular responses against secondary challenge high dose irradiation; this phenomenon has been termed the radioadaptive response. The mechanism of the radioadaptive response is not yet clear. This study was undertaken to elucidate the radiation response of apoptosis in mouse spleen after whole-body irradiation. Materials and methods : The induction of apoptosis was analysed in the spleens of C57BL/6N mice after chronic irradiation with γ-rays at 1.5 Gy (0.001 Gy/min for 25 h) followed by challenge irradiation with X-rays at 3.0Gy (1 Gy/min). Results : Accumulation of p53 and Bax, and the induction of apoptosis were observed dose-dependently in mouse spleen 12 h after acute irradiation at a high dose-rate. However, it was found that there was significant suppression of the accumulation of p53 and Bax, and induction of apoptosis 12 h after challenge irradiation at 3.0Gy at a high dose-rate following chronic preirradiation at 1.5Gy at a low dose-rate. In addition, the combination of pre-irradiation at 1.5Gy at a high dose-rate and challenge irradiation at 3.0Gy at a high dose-rate could not suppress the accumulation of p53 and Bax or the induction of apoptosis. Conclusions : Chronic pre-irradiation at a low dose-rate suppressed Bax-mediated apoptosis. These findings suggest that the radioadaptive response in mouse spleen may be due to a suppression of p53-mediated apoptosis.     

Radiation response of apoptosis in C57BL/6N mouse spleen after whole-body irradiation

PURPOSE: Primary conditioning low dose irradiation suppresses the molecular responses against secondary challenge high dose irradiation; this phenomenon has been termed the radioadaptive response. The mechanism of the radioadaptive response is not yet clear. This study was undertaken to elucidate the radiation response of apoptosis in mouse spleen after whole-body irradiation. MATERIALS AND METHODS: The induction of apoptosis was analysed in the spleens of C57BL/6N mice after chronic irradiation with gamma-rays at 1.5 Gy (0.001 Gy/min for 25 h) followed by challenge irradiation with X-rays at 3.0Gy (1 Gy/min). RESULTS: Accumulation of p53 and Bax, and the induction of apoptosis were observed dose-dependently in mouse spleen 12 h after acute irradiation at a high dose-rate. However, it was found that there was significant suppression of the accumulation of p53 and Bax, and induction of apoptosis 12 h after challenge irradiation at 3.0Gy at a high dose-rate following chronic preirradiation at 1.5Gy at a low dose-rate. In addition, the combination of pre-irradiation at 1.5Gy at a high dose-rate and challenge irradiation at 3.0Gy at a high dose-rate could not suppress the accumulation of p53 and Bax or the induction of apoptosis. CONCLUSIONS: Chronic pre-irradiation at a low dose-rate suppressed Bax-mediated apoptosis. These findings suggest that the radioadaptive response in mouse spleen may be due to a suppression of p53-mediated apoptosis.     

WAF1 accumulation by carbon-ion beam and alpha-particle irradiation in human glioblastoma cultured cells

PURPOSE: There have been no reports about the effects of heavy-ion beams on the expression of the WAF1 gene, although ionizing radiation such as y-rays and X-rays is well known to induce WAF1 (p21/CIP1/sdi1) gene expression in a p53-dependent manner. In the present study, it was examined whether WAF1 accumulation was induced after carbon-ion (C-) beam or alpha-particle irradiation in four glioblastoma cell lines. MATERIALS AND METHODS: A colony assay for radiosensitivity and Western blot analysis of WAF1 were applied to two human glioblastoma cell lines, A-172 bearing wild-type p53 (wtp53) and T98G bearing mutated p53 (mp53). A-172/neo and A-172/mp53 were transfected with a control vector (containing only a neo selection marker) and a mp53 expression vector respectively. RESULTS: The amount of WAF1 increased markedly after X-ray irradiation in A-172 and A-172/neo cells but not in T98G and A-172/mp53 cells. The level of WAF1 reached a plateau at 3-10 h after X-ray irradiation at 5 Gy in A-172 and A-172/neo cells. Likewise, the levels of WAF1 in A-172 and A-172/neo cells reached a plateau at 3-10 h and 6-24 h after C-beam (3.0 Gy) and alpha-particle (4.5 Gy) irradiation respectively. The amount of WAF1 increased markedly in a dose-dependent manner 10 h after X-ray, C-beam or alpha-particle irradiation in A-172 and A-172/neo cells but not in T98G or A-172/mp53 cells. In addition, cell survival assay showed that these cell lines were most sensitive to C-beams, less sensitive to alpha-particles and least sensitive to X-rays at 10% survival. There was no difference in sensitivity among these cell lines against C-beam and alpha-particle irradiation whereas wtp53 cells (A-172 and A-172/neo) were more sensitive to X-rays than mp53 cells (A-172/mp53 and T98G). CONCLUSIONS: These results indicate that C-beams and alpha-particles induce p53-dependent WAF1 accumulation as well as is the case with X-rays, suggesting that WAF1 protein accumulation may not contribute to cell killing.     

p53 -Dependent thermal enhancement of cellular sensitivity in human squamous cell carcinomas in relation to LET

Purpose : To investigate the dependence on p53 gene status of the thermal enhancement of cellular sensitivity against different levels of linear energy transfer (LET) from X-rays or carbon-ion (C-) beams. Materials and methods : Two kinds of human squamous cell carcinoma cell lines were used with an identical genotype except for the p53 gene. SAS/m p53 cells were established by transfection with mutated p53 (m p53) gene to SAS cells having functional wild-type p53 (wtp53). As the control, a neo vector was transfected to the SAS cells (SAS/ neo cells). Both cells were exposed to X-rays or accelerated C-beams (30-150 KeV w m -1) followed by heating at 44°;C. Cellular sensitivity was determined by colony-forming activity. Induction of apoptosis was analysed by Hoechst 33342 staining of apoptotic bodies and agarose-gel electrophoresis for the formation of DNA ladders. Results : It was found that (1) there was no significant difference in cellular sensitivity between SAS/ neo and SAS/m p53 cells to LET radiation of >30 KeV w m -1, although the radiosensitivity of SAS/ neo cells to X-rays was higher (1.2-fold) than that of SAS/m p53 cells; (2) there was an interactive thermal enhancement of radiosensitivity below an LET of 70 KeV w m -1 in SAS/ neo cells, although only additive thermal enhancement was observed in SAS/m p53 cells through all LET levels examined; (3) low-LET radiation induced apoptosis only in SAS/ neo cells; (4) high-LET radiation at an isosurvival dose-induced apoptosis of SAS/ neo cells at a higher frequency compared with that with low-LET radiation; (5) high-LET radiation-induced p53-independent apoptosis in SAS/m p53 cells; and (6) thermal enhancement of cellular sensitivity to X-rays was due to induction of p53-dependent apoptosis. Conclusions : The findings suggest that thermal enhancement of radiosensitivity may result from p53-dependent apoptosis induced by inhibition of p53-dependent cell survival system(s) through either regulation of the cell cycle or induction of DNA repair. It is also suggested that the analysis of p53 gene status of cancer cells may predict response to combined therapies with low-LET radiation and hyperthermia.     

Analysis of death pattern in cancer cells by using different kinds of LET radiation

We investigated the death pattern of cancer cells by using different kinds of linear energy transfer (LET) radiation. We used two human squamous cell carcinoma cell lines with an identical genotype except for the p53 gene. SAS/mp53 cells were established by transfection with the mp53 gene to SAS cells having functional p53 (wtp53). As the control, a neovector was transfected to the SAS cells (SAS/neo cells). Both types of cells were exposed to X-rays (1.5 KeV/micron) or accelerated C-beams (30-100 KeV/micron). The frequency of cell death (apoptosis and necrosis) was measured by acridine orange/ethidium bromide(AO/EB) double staining for fluorescence microscopy. We found that (1) low-LET radiation induced apoptosis only in SAS/neo cells; (2) high-LET radiation at an iso-survival dose induced apoptosis not but necrosis in SAS/neo cells at a higher frequency; (3) high-LET radiation induced p53-independent apoptosis even in SAS/mp53 cells. Our findings suggest that high-LET radiotherapy is expected to (1) have validity in its application to patients carrying mutated p53 cancer cells and (2) reduce injury to adjacent normal tissue for high-frequency-induced apoptosis without inflammatory response. We propose that elucidation of p53-independent apoptosis-related genes might provide new insights into radiotherapy for cancer.     

WAF1 accumulation by carbon-ion beam and alpha-particle irradiation in human glioblastoma cultured cells

Purpose : There have been no reports about the effects of heavy-ion beams on the expression of the WAF1 gene, although ionizing radiation such as gamma-rays and X-rays is well known to induce WAF1 (p21/CIP1/sdi1) gene expression in a p53 -dependent manner. In the present study, it was examined whether WAF1 accumulation was induced after carbon-ion (C-) beam or alpha-particle irradiation in four glioblastoma cell lines. Materials and methods : A colony assay for radiosensitivity and Western blot analysis of WAF1 were applied to two human glioblastoma cell lines, A-172 bearing wild-type p53 (wt p53) and T98G bearing mutated p53 (m p53) . A-172/neo and A-172/mp53 were transfected with a control vector (containing only a neo selection marker) and a m p53 expression vector respectively. Results : The amount of WAF1 increased markedly after X-ray irradiation in A-172 and A-172/neo cells but not in T98G and A-172/mp53 cells. The level of WAF1 reached a plateau at 3-10 h after X-ray irradiation at 5 Gy in A-172 and A-172/neo cells. Likewise, the levels of WAF1 in A-172 and A-172/neo cells reached a plateau at 3-10 h and 6-24 h after C-beam (3.0Gy) and alpha-particle (4.5 Gy) irradiation respectively. The amount of WAF1 increased markedly in a dose-dependent manner 10 h after X-ray, C-beam or alpha-particle irradiation in A-172 and A172/neo cells but not in T98G or A-172/mp53 cells. In addition, cell survival assay showed that these cell lines were most sensitive to C-beams, less sensitive to alpha-particles and least sensitive to X-rays at 10% survival. There was no difference in sensitivity among these cell lines against C-beam and alpha-particle irradiation whereas wt p53 cells (A-172 and A-172/neo) were more sensitive to X-rays than m p53 cells (A-172/mp53 and T98G). Conclusions : These results indicate that C-beams and alpha-particles induce p53 -dependent WAF1 accumulation as well as is the case with X-rays, suggesting that WAF1 protein accumulation may not contribute to cell killing.     

Effects of p53 status and wortmannin treatment on potentially lethal damage repair,with emphasis on the response of intratumor quiescent cells

PURPOSE: To examine the effects of p53 status and wortmannin treatment on potentially lethal damage repair, referring to the response of intratumor quiescent cells. METHODS: Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were injected subcutaneously into both hind legs of Balb/cA nude mice. Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. The mice then received gamma-rays with or without subsequent wortmannin administration. Right after or 24 h after gamma-ray irradiation alone or 24 h after wortmannin administration following irradiation, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling [quiescent (Q) cells] was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. RESULTS: On the whole, larger values of MN frequency and surviving fraction were observed in SAS/mp53 cells than in SAS/neo cells, and Q cells showed lower MN frequencies than total cells. Without wortmannin, SAS/neo tumor cells, especially Q cells within SAS/neo tumors, showed large potentially lethal damage repair (PLDR) capacities, compared with total or Q tumor cells within SAS/mp53 tumors that showed little PLDR capacity. Wortmannin treatment inhibited the PLDR in SAS/neo tumors very effectively, but showed no apparent effect on either total or Q tumor cells within SAS/mp53 tumors. CONCLUSION: PLDR in vivo was thought to be a p53-dependent event whether in total or Q tumor cell populations.     

Radiation-induced apoptosis in the scid mouse spleen after low dose-rate irradiation

PURPOSE: To elucidate the process of radioadaptation, the role of DNA-PK activity was examined using the scid mouse defective in DNA-PKcs. MATERIALS AND METHODS: The induction of apoptosis in the spleens of the C.B-17 Icr scid mouse and the parental mouse was studied after chronic irradiation with gamma-rays at 1.5 Gy (0.001 Gy min(-1) for 25 h) followed by challenge irradiation with X-rays at 3.0 Gy (1.0 Gy min(-1) for 3 min). RESULTS: When the wild-type mouse was previously exposed to chronic irradiation (1.5 Gy) at a low dose-rate (0.001 Gy min(-1)), apoptosis induced by acute irradiation (3.0 Gy, 1.0 Gy min(-1)) was significantly suppressed, especially in the splenic white pulp. There was no change by acute irradiation after chronic irradiation in the scid mouse, although an effect was detected in the spleen after acute irradiation alone. CONCLUSIONS: These data suggest that DNA-PK activity might play a major role in the radioadaptive response following pre-irradiation at a low dose-rate.     

Radiation-induced apoptosis in the scid mouse spleen after low dose-rate irradiation

Purpose : To elucidate the process of radioadaptation, the role of DNA-PK activity was examined using the scid mouse defective in DNA-PKcs. Materials and methods : The induction of apoptosis in the spleens of the C.B-17 Icr scid mouse and the parental mouse was studied after chronic irradiation with γ-rays at 1.5 Gy (0.001Gy min -1 for 25 h) followed by challenge irradiation with X-rays at 3.0 Gy (1.0 Gy min -1 for 3 min). Results : When the wild-type mouse was previously exposed to chronic irradiation (1.5Gy) at a low dose-rate (0.001 Gy min -1) , apoptosis induced by acute irradiation (3.0 Gy, 1.0Gy min -1) was significantly suppressed, especially in the splenic white pulp. There was no change by acute irradiation after chronic irradiation in the scid mouse, although an effect was detected in the spleen after acute irradiation alone. Conclusions : These data suggest that DNA-PK activity might play a major role in the radioadaptive response following pre-irradiation at a low dose-rate.     

Mitochondrial and intracellular free-calcium regulation of radiation-induced apoptosis in human leukemic cells.

PURPOSE: To investigate the mechanisms and pathways of X-ray apoptosis in Molt-4 cells, focusing on mitochondrial and cytosolic Ca2+ ([Ca2+]i) regulation. MATERIALS AND METHODS: X-irradiated Molt-4 cells and cell extract (CE) were used to analyse: (1) induced apoptosis (Giemsa stain), (2) p53, Bcl-2 and Bax expressions (immunoblot), (3) mitochondrial potential deltapsi(m) and (4) [Ca2+]i (flow cytometry), (5) caspase-3 activity, and (6) roles of [Ca2+]- and caspase-3-mediated pathways by inhibiting either or both pathways for induced apoptosis. RESULTS: Molt-4 cells were sensitive to apoptosis since 5 Gy induced 57 and 94% apoptosis at 6 and 24 h. After 5Gy, p53 was accumulated that upregulated Bax but which repressed Bcl-2 with time, resulting in a 7-fold increase in Bax/Bxl-2 at 6 h. Predominant Bax reduced deltapsi(m), and low-deltapsi(m) cells increased 45 min earlier than apoptosis after 5 Gy. Caspase-3 was activated in apoptotic CE. The caspase-3 inhibitor Ac-DEVD-CHO inhibited apoptosis and DNA-ladder formation by approximately 50%, suggesting a approximately 50% role of caspase-3-activated DNase (CAD). [Ca2+]i was increased after 5 Gy. [Ca2+]i-chelating BAPTA-AM (5 microM) and/or DNase gamma-inhibiting Zn2+ (0.5 mM) inhibited approximately 50% of induced apoptosis and DNA-laddering, indicating a 50% participation of Ca2+/Mg2+-dependent DNase gamma. CONCLUSIONS: The p53-Bax-mitochondria-caspase-3-CAD pathway and the [Ca+2]i-mediated DNase gamma pathway were involved in the regulation of X-ray apoptosis in sensitive Molt-4 cells.     

Restoration by glycerol of p53-dependent apoptosis in cells bearing the mutant p53 gene.

PURPOSE: Effective heat-induced cell death in cultured cells bearing a mutant p53 (mp53) gene was sought by glycerol treatment which led to conformational change from mp53 to wild-type p53 (wtp53) in p53-null murine fibroblasts transfected with mp53. MATERIALS AND METHODS: Heat sensitivity was measured using a colony-forming assay. For heat-induced apoptosis, gel electrophoresis was applied to detect DNA fragmentation and Hoechst33342 staining for apoptotic bodies. Glycerol (0.6 M) was applied to the cultured cells 48 h before heating at 44 degrees C in a water bath. RESULTS: Wtp53 transfectants (MT158/wtp53-1) were sensitive to heat stress compared with mp53 transfectants (MT158/mp53-2 cells), and the combined treatment with glycerol enhanced cell killing only in the MT158/mp53-2 cells. After glycerol pretreatment for 48 h, the subsequent heat treatment enhanced DNA fragmentation and apoptosis in MT158/mp53-2 cells, while DNA fragmentation and apoptotic bodies were not enhanced with heat treatment alone in these cells. In contrast, DNA fragmentation or apoptotic bodies were clearly observed in MT158/wtp53-1 cells 3-24h after heat treatment. Treatment with glycerol alone did not induce apoptosis in the transfectants. CONCLUSIONS: Glycerol appears to function as a chemical chaperone that restores mp53 to wtp53 function.     

Restoration by glycerol of p53-dependent apoptosis in cells bearing the mutant p53 gene

Purpose: Effective heat-induced cell death in cultured cells bearing a mutant p53 (mp53) gene was sought by glycerol treatment which led to conformational change from mp53 to wild-type p53 (wtp53) in p53-null murine fibroblasts transfected with mp53. Materials and methods: Heat sensitivity was measured using a colony-forming assay. For heat-induced apoptosis, gel electrophoresis was applied to detect DNA fragmentation and Hoechst33342 staining for apoptotic bodies. Glycerol (0.6 m) was applied to the cultured cells 48 h before heating at 44 C in a water bath. Results: Wtp53 transfectants (MT158/wtp53-1) were sensitive to heat stress compared with mp53 transfectants (MT158/mp53-2 cells), and the combined treatment with glycerol enhanced cell killing only in the MT158/mp53-2 cells. After glycerol pretreatment for 48h, the subsequent heat treatment enhanced DNA fragmentation and apoptosis in MT158/mp53-2 cells, while DNA fragmentation and apoptotic bodies were not enhanced with heat treatment alone in these cells. In contrast, DNA fragmentation or apoptotic bodies were clearly observed in MT158/wtp53-1 cells 3-24h after heat treatment. Treatment with glycerol alone did not induce apoptosis in the transfectants. Conclusions: Glycerol appears to function as a chemical chaperone that restores mp53 to wtp53 function.     

低剂量辐射诱导免疫适应性反应的剂量效应

本实验采用Ｘ射线全身照射，观察辐射诱导免疫适应性反应的预照射剂量（Ｄ１剂量）及其后损伤性剂量（Ｄ２剂量）的剂量范围．结果表明：Ｄ１在２５ｍＧｙ～１００ｍＧｙ范围内（剂量率为１２．５ｍＧｙ／ｍｉｎ），Ｄ２在１．０～１．５Ｇｙ范围内（剂量率为０．３３Ｇｙ／ｍｉｎ），Ｄ１与Ｄ２的时间间隔６小时，可诱导昆明小鼠胸腺细胞自发增殖力及丝裂原（ＣｏｎＡ和ＬＰＳ）刺激的脾细胞增殖反应等免疫适应性反应。     

Effects of a heat shock protein inhibitor KNK437 on heat sensitivity and heat tolerance in human squamous cell carcinoma cell lines differing in p53 status

Purpose: The effects of a heat shock protein (hsp) inhibitor KNK437 (N‐formyl‐3,4‐methylenedioxy‐benzylidene‐γ‐butyrolactam) were examined on the heat sensitivity and heat tolerance of human cancer cells with special reference to p53 status.

Materials and methods: Human squamous cell carcinoma (SAS) and glioblastoma cell lines (A‐172) transfected with mutant p53 (mp53) or control neo genes were used. KNK437 was added in culture medium at a final concentration of 50, 100 or 300?µM 1?h before heating (42°C). Surviving fractions of cells were measured by use of a clonogenic assay. Effects of KNK437 on the accumulation of heat shock proteins and DNA binding activity of heat shock factor 1 were examined with Western blot analysis and gel mobility‐shift assay, respectively. Heat‐induced apoptotic bodies were detected by Hoechst 33342 staining.

Results: The mp53‐transfected SAS (SAS/mp53) and A‐172 (A‐172/mp53) cells were more resistant to heat than the neomycin (neo)‐transfected SAS (SAS/neo) and A‐172 (A‐172/neo) cells. The constitutive amount of hsp27 was larger in SAS/mp53 than in SAS/neo cells. Clear differences in the constitutive amounts of hsp40, hsp72 and hsp90 were not observed between SAS/mp53 and SAS/neo cells. KNK437 enhanced the heat sensitivity in SAS/mp53 and A‐172/mp53 cells more effectively than in neo control cells. Heat tolerance was suppressed by KNK437 in SAS/mp53 and SAS/neo cells and also in A‐172/mp53 and A‐172/neo cells. Along with suppression of heat tolerance, KNK437 suppressed heat‐induced accumulation of both hsp27 and hsp72. Heat‐induced apoptotic bodies were enhanced by KNK437 in SAS/mp53 and SAS/neo cells.

Conclusion: The results suggest a possible mechanism for the heat sensitivity of SAS cells. Heat sensitivity depends on p53 status regulating the amount of hsp27. Heat tolerance is suppressed by KNK437 through the suppression of heat‐induced accumulations of hsp27 and hsp72 and the induction of p53‐independent apoptosis.     

0.075 Gy X射线诱导EL-4细胞凋亡及细胞周期的适应性反应

观察低剂量Ｘ射线照射能否诱导ＥＬ－４细胞凋亡及细胞周期的适应性反应。方法采用流式细胞术（ＦＣＭ）检测细胞凋亡及细胞周期。结果单纯２ＧｙＸ射线照射后１２小时ＥＬ－４细胞凋亡显著增多，并伴有明显Ｇ１阻滞；０．０７５Ｇｙ预照射可明显减轻间隔６小时后２Ｇｙ攻击剂量照射所致的细胞凋亡和Ｇ１阻滞。结论０．０７５ＧｙＸ射线离体照射可诱导ＥＬ－４细胞凋亡及Ｇ１阻滞的适应性反应     

电离辐射诱导EL-4细胞Caspase-3、P53蛋白表达及其生物学作用

目的研究电离辐射对EL-4细胞中Caspase-3和P53蛋白表达的影响，及其对辐射诱导细胞凋亡及多倍体细胞的作用。方法采用单克隆抗体免疫荧光标记及流式细胞术检测蛋白表达的变化，采用PI荧光标记及流式细胞术检测细胞凋亡及多倍体细胞的变化。结果实验表明，4．0GyX射线照射后8及12h，EL-4细胞中Caspase-3蛋白表达与对照组比较，差异有统计学意义（P〈0．05）；照射后2、4、8、12及24h，P53蛋白表达与对照组比较，差异有统计学意义（P〈0．05或P〈0．01）。实验还表明，4．0Gy照射后2、4、8、12、24、48及72h，EL-4细胞凋亡与对照组比较，差异有统计学意义（P〈0．05～P〈0．001）；而照射后2～48h，多倍体细胞数与对照组比较则未见明显变化。结论电离辐射可诱导EL-4细胞Caspase-3及P53蛋白表达增高，可能在辐射诱导细胞凋亡中起重要作用，而辐射诱导多倍体细胞的分子通路则可能是P53非依赖性的。     

Threshold effect for teratogenic risk of radiation depends on dose-rate and p53-dependent apoptosis

PURPOSE: To obtain evidence that the p53 gene is indispensable for reduction of high teratogenic risk of radiation at a high dose-rate to zero risk by lowering the dose-rate. MATERIALS AND METHODS: Wild-type p53(+/+), heterozygous p53(+/-) and null p53(-/-) mice were exposed to gamma-rays at high or low dose-rates during days 9.5-10.5 of gestation. The incidence of malformations and prenatal deaths was studied. Frequencies of cells dying by apoptosis were measured during or after protracted irradiation. RESULTS: After irradiation with 2 Gy, the frequency of apoptotic cells increased to 20% for p53(+/+) mice and did not increase at all for p53(-/-) mice. For p53(+/+) mice, 2 Gy y-rays induced 70% malformations when given at 1.06 Gy/min, but no malformations above the control when given at 1.2 mGy/min. In contrast, after irradiation of p53(-/-) foetuses with 2 Gy at 1.2mGy/min, the incidence of malformations increased 12% above control levels. CONCLUSION: Foetal irradiation with 2 Gy at 1.2 mGy/min was not teratogenic for p53(+/+) mice but teratogenic for p53(-/-) mice. This indicates that the p53 gene is indispensable for a threshold effect in the risk of radiation at low doses or dose-rates.