The roles of p53 and p21 in normal development and hyperthermia‐induced malformations

BACKGROUND : Hyperthermia (HS) is a well‐studied teratogen that induces serious malformations, including neural tube defects. Our previous studies have shown that HS induces apoptosis by activating the mitochondrial apoptotic pathway. Prior to activation of the mitochondrial apoptotic pathway, HS also activates p53 and its target genes. In the present study, we determine whether p53 and/or p21 play a role as teratogen suppressors or inducers of HS‐induced malformations. METHODS : Pregnant mice carrying all three p53 or p21 genotype embryos were exposed to HS on day 8.5. Subsequently, fetuses were collected on day 15.5, and genotyped. In addition to genotype, we also determined the number of resorptions and dead fetuses as well as the number and types of external malformations. RESULTS : In the absence of HS exposure, fetuses exhibiting exencephaly and spina bifida were observed in approximately 11% of p53 ?/? fetuses, whereas no malformations were observed among p21 ?/? fetuses. Exposure to HS resulted in an increase in exencephaly and polydactyly in fetuses of all three p53 genotypes. However, the incidence of these malformations was statistically significantly higher in p53 ?/? compared to p53 +/? and p53 +/+ fetuses. Exencephaly was the only malformation observed in p21 fetuses exposed to HS, with an approximately 2‐fold increase among p21 +/? and a 3‐fold increase among p21 ?/? compared to p21 +/+ fetuses. CONCLUSIONS : Our study confirms that p53 plays a role in normal development and has shown, for the first time that p53 and p21 function to suppress HS‐induced malformations. Birth Defects Res (Part B) 86:40‐47, 2009. © 2009 Wiley‐Liss, Inc.     

Lymphocyte HSP72 following exercise in hyperthermic runners: The effect of temperature

Heat shock protein 72 (HSP72) is the most inducible HSP, but is not always increased in lymphocytes following exercise. This field study examined whether lymphocyte HSP72 was increased in hyperthermic (T_rec>39.0 °C) male athletes following a 14 km competitive race in cool conditions (ambient temperature 11.2 °C). A comparison was also made between control runners (n=7) and those treated for exertional heat illness (n=9). Lymphocyte HSP72 was not increased in control runners immediately post- compared with pre-race, and there was no difference between both groups of runners. A second study of the race (ambient temperature 14.6 °C) found that lymphocyte HSP72 in control (n=7) and treated (n=9) athletes was higher 2 days post- compared with immediately post-race (p<0.01) and these increases were correlated with post-exercise T_rec (p<0.05).     

Response of Transformed and Normal Mouse Cell Lines to Anti-Melanin Compounds,Hyperthermia, and Radiation

Five cell lines (one parental, two transformed melanin producing, and two transformed non-melanin producing) were evaluated for the responses to 2- and 4-hydroxyanisole (2HA, 4HA) alone or combined with hyperthermia or radiation. All cells exhibited a non-specific toxic response to the two compounds and the effect was exposure time and concentration dependent and was greater for 4HA compared to 2HA. In addition, the two melanin-producing cell lines were more sensitive, demonstrating specific toxicity to such cell lines. The treatment with either 2HA or 4HA combined with heat and radiation resulted mostly in additive or antagonistic effects, except for one combination of 2HA plus radiation in the melanin-producing R25 cells. Thus, while these compounds may be useful in therapy for pigmented melanomas, combined treatment with radiation is not recommended.     

Cytoprotection of vitamin E on hyperthermia-induced damage in bovine mammary epithelial cells

The protective effects of vitamin E (VE) against hyperthermia-induced damage in bovine mammary epithelial cells (BMEC) were studied. The structure of BMEC membrane was damaged by hyperthermia treatment. The VE (25 nmol/ml) efficiently increased cell viability and attenuated morphological damages in hyperthermia-treated BMEC. Compared with the control, VE significantly reduced lactate dehydrogenase leakage and malondialdehyde formation in hyperthermia-treated BMEC. Meanwhile, superoxide dismutase activity was increased significantly in the presence of VE. It is inferred that VE displayed cytoprotective effects on hyperthermia-induced damage in BMEC through increasing intracellular antioxidant levels and decreasing lipid peroxidation.     

Role of Hyperthermia in Effects of Electroconvulsive Shock on Protein Synthesis in the Rabbit

The effects of electroconvulsive shock (ECS) on rectal temperature (TR) and on protein synthesis in brain and liver were compared in rabbit, rat, and mouse. Protein synthesis status was assessed using an in vitro amino acid incorporation method which provides information equivalent to polyribosome profiles. In the rabbit, TR rose from 39.5 +/- 0.4 degrees C to 40.4 +/- 0.2 degrees C within 10 min following a single ECS, and significant hyperthermia persisted for at least 60 min. This effect was markedly attenuated in animals housed at 4 degrees C. In vitro protein synthesis activities of rabbit brain and liver preparations were significantly reduced following ECS only in those animals whose TR exceeded 40 degrees C. In the rat, ECS gave rise to a significant hyperthermia, but in no case did TR exceed 40 degrees C, and protein synthesis activity of brain supernatants was not affected. In the mouse, ECS reduced TR and had no effect on in vitro protein synthesis activity. These results demonstrate that the unique sensitivity of protein synthesis in rabbit tissues to electroconvulsive shock is a direct consequence of the hyperthermia that arises following ECS in this species.     

Potentiation of the destructive effect of heat (42°C) on synchronized cancer cells in culture by cell specific antiserum

1. 1. A kinetic study was made of the effects of hyperthermia and cell specific antibody on non-synchronized lag phase cultures and synchronized exponentially growing cultures of a malignant cell line. The effects on the degree of cell synchrony were also investigated.

2. 2. Hyperthermic treatment of synchronized SDB monolayer cell populations with maximum replication rate sensitized the cells to subsequent destruction by cell specific antibody.

3. 3. Hyperthermic treatment of SDB monolayer cultures with low replication rate and varying degrees of metabolic activity produced no such sensitization.

4. 4. Hyperthermia was disruptive to a synchrony procedure involving a blockade of DNA synthesis by excess thymidine.

高温对原代支持细胞增殖能力和occludin紧密连接蛋白的影响

目的:研究高温对原代大鼠睾丸支持细胞增殖作用和对紧密连接蛋白的损伤机制.方法:体外分离培养雄性Wistar大鼠睾丸支持细胞(Sertoli cell,SC),免疫组化FasL鉴定.实验分为对照组(36℃)和实验组(37℃、38℃、39℃).在相应的温度下培养5d后,CCK-8法检测SC增殖作用,Western印迹法检测OCLN表达水平.结果:体外培养SC的纯度＞90％,CCK-8结果显示,细胞增殖活性在36℃时最强,37～39℃时逐渐降低;Western印迹显示,36℃时OCLN表达量最高;高于36℃时,OCLN表达量随着温度的增加而降低(P＜0.05).结论:高温影响SC增殖活性,破坏了支持细胞紧密连接蛋白的正常表达和血睾屏障的完整性,从而影响正常精子的形成.     

放疗联合热疗治疗复发性卵巢癌患者的临床疗效及对患者免疫功能的影响

目的:探讨放疗联合热疗治疗复发性卵巢癌患者的临床疗效及对患者免疫功能的影响。方法:回顾性选取2011年1月-2014年5月在我院诊治的复发性卵巢癌患者99例,按治疗方法将患者分为对照组47例和观察组52例。对照组患者给予三维适行放射治疗,观察组在对照组的基础上给予盆腔部位热疗。治疗后,比较两组患者的临床疗效及免疫功能。结果:治疗后,对照组总缓解率为53.19%,观察组总缓解率为78.85%,显著高于对照组(P0.05);治疗后,观察组与对照组外周血中CD3+、CD4+及CD4+/CD8+含量均较治疗前明显升高(P0.05),而CD8+含量则显著降低(P0.05),且观察组患者CD3+、CD4+及CD4+/CD8+显著高于对照组(P0.05),CD8+水平则明显低于对照组(P0.05)。两组患者治疗后骨髓抑制、胃肠道反应及泌尿系统反应的发生率、1年及2年生存率比较均无显著性差异(P0.05),观察组治疗后3年生存率显著高于对照组(P0.05)。结论:与单纯三维适行放射治疗相比,放疗联合热疗能有效提高复发性卵巢癌的临床疗效,显著增强患者免疫功能,延长生存期,且不会增加不良反应。     

热疗联合奥沙利铂对SW480细胞增殖和凋亡的影响

目的:观察奥沙利铂联合热疗对人结肠癌细胞SW480增殖及凋亡的影响,确定联合用药的效果,为临床方案提供参考。方法:采用MTT(四唑盐)法检测热疗、奥沙利铂及联合用药对细胞增殖的影响;瑞士吉姆萨染色法观察细胞形态;流式细胞仪检测细胞凋亡和周期;Western blot检测Bax、Bcl-2以及Caspase8蛋白表达量变化;q PCR检测Bax、Bcl-2以及Caspase8 m RNA的积累。结果:热疗联合奥沙利铂可以显著抑制细胞增殖,与对照组相比,热疗组、化疗组、联合组细胞凋亡率分别为16.2%、20.5%和36.1%,具有显著性差异(P0.01);细胞学形态中,热疗组细胞发生皱缩,化疗组细胞膜破裂;化疗将细胞阻滞在G2/M期,热疗和联合组将细胞阻滞S期;Western blot和qPCR显示Bax/Bcl-2比值上升,Caspase8表达量增加,联合组三种蛋白的表达量均与对照组具有显著性差异(P0.01)。结论:热疗联合奥沙利铂可以显著促进细胞凋亡,提高治疗效果,为结肠癌的治疗提供参考。     

Thermal boost combined with interstitial brachytherapy in breast conserving therapy – Assessment of early toxicity

Background

Hyperthermia (HT) causes a direct damage to cancerous cells and/or sensitize them to radiotherapy with usually minimal injury to normal tissues. Adjuvant HT is probably one of the most effective radiation sensitizers known and works best when delivered simultaneously with radiation. In breast conserving therapy, irradiation has to minimize the risk of local relapse within the treated breast, especially in an area of a tumor bed. Brachytherapy boost reduces 5-year local recurrence rate to mean 5,5%, so there still some place for further improvement. The investigated therapeutic option is an adjuvant single session of local HT (thermal boost) preceding standard CT-based multicatheter interstitial HDR brachytherapy boost in order to increase the probability of local cure.

Aim

To report the short-term results in regard to early toxicity of high-dose-rate (HDR) brachytherapy (BT) boost with or without interstitial microwave hyperthermia (MV HT) for early breast cancer patients treated with breast conserving therapy (BCT).

Materials and methods

Between February 2006 and December 2007, 57 stage IA–IIIA breast cancer patients received a 10 Gy HDR BT boost after conservative surgery and 42.5–50 Gy whole breast irradiation (WBI) ± adjuvant chemotherapy. 32 patients (56.1%) were treated with additional pre-BT single session of interstitial MW HT to a tumor bed (multi-catheter technique). Reference temperature was 43 °C and therapeutic time (TT) was 1 h. Incidence, severity and duration of radiodermatitis, skin oedema and skin erythema in groups with (I) or without HT (II) were assessed, significant p-value ≤ 0.05.

Results

Median follow-up was 40 months. Local control was 100% and distant metastasis free survival was 91.1%. HT sessions (median): reference temperature 42.2 °C, therapeutic time (TT) 61.4 min, total thermal dose 42 min and a gap between HT and BT 30 min. Radiodermatitis grades I and II occurred in 24 and 6 patients, respectively, differences between groups I and II were not significant. Skin oedema and erythema occurred in 48 (85.7%) and 36 (64.3%) cases, respectively, and were equally distributed between the groups. The incidence and duration of skin oedema differed between the subgroups treated with different fractionation protocols of WBI, p = 0.006. Skin oedema was present up to 12 months. No difference in pattern of oedema regression between groups I and II was observed, p = 0.933.

Conclusion

Additional thermal boost preceding standard HDR BT boost has a potential of further improvement in breast cancer local control in BCT. Pre-BT hyperthermia did not increase early toxicity in patients treated with BCT and was well tolerated. All side effects of combined treatment were transient and were present for up to 12 months. The increase in incidence of skin oedema was related to hypofractionated protocols of WBI. The study has to be randomized and continued on a larger group of breast cancer patients to verify the potential of local control improvement and to assess the profile of late toxicity.