γ射线对人淋巴细胞cx43和ANLN基因转录表达的影响

目的 研究γ射线对人外周血淋巴细胞 cx43和ANLN基因转录表达的影响.方法 对数生长期的淋巴细胞,分别给予1、2、3、4、5、6 Gy的60Coγ射线照射,照射后12 h,以及2 Gy照射后4、8、12、24、36、48、72 h,分别提取总RNA,反转录成cDNA.利用实时荧光定量PCR技术,检测各组cx43和ANLN基因表达改变.结果 人外周血淋巴细胞cx43 mRNA表达水平在2 Gy照射后4、8、12 h明显增高,分别为对照组(未照射组)的6.74、9.06、7.22倍(P＜0.05);24～72 h,其表达水平与对照组相比没有明显变化.1、2、3、4、5、6 Gy剂量照射后12 h,cx43 mRNA表达水平显著增高(P＜0.05).ANLN mRNA表达水平在2 Gy不同时间点及1～5 Gy照射后12 h,表达降低(P＜0.05),6 Gy照射后12 h其表达开始升高,为对照组的6.08倍(P＜0.05).结论 γ射线照射2 Gy不同时间点及不同剂量照射后12 h,cx43基因表达上调,ANLN 基因表达下调.1～3 Gy剂量照射后12 h,cx43 mRNA表达在此范围内有时间和剂量的依赖性.cx43可能会发展为核事故受照射人员的分子生物学剂量标记物.     

pcEgr-hp53稳定转染联合X射线诱导人肺腺癌A549细胞凋亡及相关凋亡蛋白表达变化

目的：探讨辐射诱导表达载体pcEgr-hp53体外稳定转染人肺腺癌A549细胞后联合X射线照射,诱导细胞凋亡的作用及相关凋亡蛋白Bax、Bcl-2和caspase-3蛋白表达的变化。方法：以脂质体介导携有外源野生型p53基因的辐射诱导表达载体pcEgr-hp53和pcDNA3.1,体外转染A549细胞,筛选稳定转染的细胞克隆并扩增培养,所表达的A549-hp53和A549-vect分别接受0、0.5、2和5Gy X射线照射,即8个实验组,采用TUNEL法和流式细胞术检测稳定转染联合辐射对细胞凋亡和Bax、Bcl-2和caspase-3蛋白表达变化的影响。结果：A549-hp53组加不同剂量照射与0 Gy组比较,其百分数均明显增加（0.5-5Gy,P〈0.05-P〈0.01）,Bcl-2蛋白表达在0.5-5Gy时明显下降,而Bax蛋白明显增加（P〈0.05-P〈0.01）,caspase-3蛋白表达在2-5Gy时明显增加（P〈0.01）;A549-hp53照射组不同剂量照射,与A549-vect相应照射剂量组比较,其百分数在0.5Gy时无明显差异,在2-5 Gy时为（P〈0.01）,Bcl-2蛋白表达明显下降（0.5Gy,P〈0.01;2-5Gy,P〈0.05）,Bax和caspase-3蛋白表达0.5-5Gy时明显增加（P〈0.05-P〈0.01）。结论：体外p53基因转染联合X射线照射可诱导肿瘤细胞凋亡明显增多,凋亡相关蛋白Bcl-2表达下调,Bax、caspase-3表达上调,具有显著的肿瘤抑制作用。     

X线电离辐射通过上调c-Myc表达促进肺癌A549细胞上皮间质转化

目的:探讨电离辐射对肺癌A549细胞上皮间质转化(EMT)的影响及其可能机制。方法:采用不同剂量(0 Gy、1 Gy、2 Gy、4 Gy和8 Gy)的X射线分别照射肺癌A549细胞不同时间,通过倒置显微镜观察X射线照射12 h、24 h和48 h时肺癌A549细胞形态的改变;Western blot检测X射线照射12 h与24 h时EMT相关蛋白vimentin、N-cadherin及E-cadherin和转录因子c-Myc的表达。结果:照射后肺癌A549细胞轮廓不清,突起增多,边缘不规则且呈煎蛋样塌陷,以8 Gy X射线照射48 h时肺癌A549细胞上皮间质化形态最明显。与0 Gy照射剂量对照组相比,vimentin虽然于4 Gy剂量照射12 h时下调,但是处理24 h时各剂量照射组vimentin均表现为上调,其中2 Gy剂量照射组其上调最明显(P0.01);与0 Gy照射剂量对照组相比,1 Gy、2 Gy及4 Gy照射组照射肺癌A549细胞24 h后其N-cadherin表达上调(P0.05);各照射剂量对肺癌A549细胞E-cadherin表达没有显著影响。照射24 h后肺癌A549细胞c-Myc表达上调,以4 Gy剂量照射组表达差异最明显(P0.01)。结论:X线电离辐射可能通过上调c-Myc表达促进肺癌A549细胞发生上皮间质转化。     

大剂量γ线照射对小鼠免疫功能近期、远期的影响

目的 :观察大剂量γ线照射对小鼠免疫功能近期、远期的影响。方法 :将 2 2 5只清洁级C5 7小鼠 ,随机分为 0、6、9、12、15和 2 0Gy 6个剂量组 ,经γ线全身 1次照射后 ,于照后 1～2 8d和 3～ 12月活杀取材 ,用原位末端标记 (TUNEL)和May Grunwald Giemsa(MGG)染色 ,检测细胞凋亡并观察其与照射剂量的关系。用流式细胞仪检测外周血T细胞亚群的变化。结果 :(1)照后早期 (1～ 14 )d外周血淋巴细胞的凋亡率即出现明显升高 ,随着照射剂量的增加凋亡率的升高更为显著 ;而T细胞亚群经不同剂量照射后持续下降 ,剂量为 2 0Gy时降至最低 ,其中以CD8 T细胞对辐射的敏感性最高 ,因而推测早期的严重损伤是急性辐射免疫损伤的重要特点之一。 (2 )照射后 1月淋巴细胞的凋亡率降低 ,T细胞及其亚群也逐渐恢复。然而直至照后 6～ 12月 ,无论是淋巴细胞的凋亡率还是CD3 T细胞及CD8 T细胞亚群 ,均未恢复到对照的水平 ,提示大剂量辐射对机体免疫系统的损伤呈现较重的远期效应。 (3)本实验还发现 ,12≤Gy照射后 ,外周血淋巴细胞的凋亡率与照射剂量呈明显的剂量效应关系 ,15～ 2 0Gy照射后未观察到明显的量效关系。结论 :照射后早期外周血淋巴细胞的大量凋亡 ,可能是导致T细胞亚群的百分率急剧下降和后期免疫功能受损的重要原     

不同剂量X射线对雄性大鼠精子顶体酶活性的影响

目的:检测不同剂量X-射线对雄性大鼠精子顶体酶的影响,以探讨体外X-射线对大鼠精子顶体酶活性的影响。方法:将10周龄成熟雄性大鼠40只,随机分为4组:2Gy、4Gy、6Gy照射组和0Gy对照组;每组10只。按不同剂量照射,1h后处死大鼠,以改良的Kennedy法分别测定不同剂量X射线作用下的精子顶体酶活性。结果:2Gy、4Gy、6Gy照射组与0Gy对照组相比顶体酶活性显著降低(P<0.05),照射组酶活性较0Gy对照组有显著降低;4Gy、6Gy照射组与2Gy照射组相比较顶体酶活性显著降低(P<0.05);而4Gy照射组与6Gy照射组相比较则无统计学意义。结论:X-射线对大鼠精子顶体酶有明显的抑制作用。     

干扰HTERT降低人乳腺癌MCF-7细胞对γ射线引起的DNA损伤反应

 目的 研究HTERT RNA干扰对人乳腺癌MCF-7细胞γ射线照射引起的DNA损伤反应的影响。方法 通过逆转录病毒为载体的HTERT-siRNA,抑制人乳腺癌MCF-7细胞中端粒酶催化亚单位HTERT表达,实时定量RT-PCR及Western blot确认HTERT表达水平、用137Cs放射源以3 Gy剂量γ射线照射细胞,于照射前以及照射后1、2、4、8和12h收集细胞,采用磷酸化53BP1 抗体进行免疫荧光染色,并于照射前、照射后1、4和12h收集细胞,Western blot检测53BP1蛋白磷酸化水平。结果 HTERT-siRNA处理的细胞HTERT表达水平比对照细胞降低3.20 (2-△△Ct)倍, HTERT蛋白水平降低2.56倍。HTERT-siRNA处理的细胞对γ射线引起的DNA损伤反应显著降低。结论 RNA干扰HTERT表达水平,可以降低人乳腺癌MCF-7细胞对γ射线照射引起的DNA损伤反应。     

致死剂量的γ线照射后小鼠胸腺淋巴细胞的凋亡规律及与bax、bcl-2和Bcl-XL表达的关系

目的：观察致死剂量γ线照射后小鼠胸腺淋巴细胞凋亡特征及与Bax、Bcl-2和Bcl-XL蛋白和mRNA表达的关系，为急性重度以上放射病的治疗提供依据。方法：清洁级C57小鼠250只，随机分为0、6、9、12、15和20 Gy6个剂量组.经γ线全身1次照射后，于照射后1～28d和3～12个月，活杀取胸腺和外周血，用原位末端标记(TUNEL)和麦格-姬姆萨(MGG)染色检测胸腺淋巴细胞的凋亡：用原位杂交和碱性磷酸酶免疫组化染色法，检测Bax、Bcl-2和Bcl-XL mRNA和其蛋白的表达。结果.(1)各剂量组小鼠外周血白细胞在照射后6h，出现一过性的升高，以后迅速下降.6Gy照射后7d降至最低，至照射后28d基本恢复到正常水平：(2)不同剂量的γ线照射后24h，胸腺淋巴细胞的凋亡率迅速升高；在6～12Gy范围内与照射的剂量呈正比，≥15Gy照射后变化不明显。(3)6Gy照射后24h，胸腺淋巴细胞的凋亡率达高峰，而后开始降低；但直至照射后6个月和12个月，凋亡率仍明显高于对照组。(4)6Gy照射后3h，胸腺淋巴细胞中Bax蛋白的表达即出现增加，至24h达峰值，显示出时效关系；而Bcl-2蛋白于照射后3h即明显下降，24h降至最低；Bcl-XL蛋白也在照射后24h降至最低。bax和bel-2 mRNA的检测与蛋白水平的检测一致。结论：经6～12 Gy照射后，淋巴细胞的凋亡与照射剂量成正比，≥15 Gy照射后凋亡率下降，提示≤12 Gy照射后胸腺淋巴细胞的凋亡是其死亡的主要方式。促凋亡蛋白Bax表达的增加及抗凋亡蛋白Bcl-XL表达的下降，与照射剂量和淋巴细胞的凋亡率呈现较好的对应关系，提示它们在致死剂量的照射所致胸腺淋巴细胞凋亡的调控中起着重要作用。     

早期生长反应基因-1的表达与射线诱导胶质母细胞瘤凋亡的相关研究

目的 研究Egr-1基因在胶质母细胞瘤细胞放射前后的表达变化和放射敏感性的研究.方法 培养人恶性神经胶质母细胞瘤(GBM)细胞系T98G,分别提取4Gy剂量射线照射前及照射后不同时间点细胞的总RNA,通过实时定量PCR(qRT-PCR)检测Egr-1表达水平;.收获4Gy剂量射线照射后0、4、8、12、24和36h的细胞,采用细胞增殖抑制实验,及流式细胞术进行细胞周期和细胞凋亡检测.结果 T98G细胞在照射后Egr-1表达水平与对照组比较有显著差异(P =0.017);T98G细胞在射线照射后的生长抑制率与对照组比较有显著差异(P=0.026);射线照射后0、4、8、12 h细胞S期比例逐渐减低,G0/G1期比例逐渐升高,并在照射后12 h达到峰值,G2/M期无明显变化,照射后12、24、36 h细胞S期比例逐渐升高,G0/G1期比例逐渐减低,G2/M期无明显变化,各组之间相比,差异有统计学意义(P=0.035).射线照射后0、4、8、12h细胞凋亡率逐渐升高,并在照射后12 h达到峰值,照射后12、24、36 h细胞凋亡率逐渐降低,各组之间相比,差异有统计学意义(P =0.019).结论 通过射线诱导Egr-1基因表达而引起细胞周期和细胞凋亡的变化;射线诱导的Egr-1基因表达水平与射线诱导的细胞凋亡成正相关.     

p53基因表达在辐射诱导骨髓细胞凋亡和修复中作用研究

目的 探讨p53基因表达在辐射导的骨髓细胞凋亡和修复中的改变与作用。方法 经^60Coγ射线以5．5Gy全身辐射78只LACA小鼠。于照后4wk内分批活杀，将骨髓分别进行HE染色和超薄切片，并进行P53蛋白LSAB免疫组化染色和突变型p53(mtp53)cDNA-DNA原位杂交。结果 照射后小鼠骨髓出现明显损伤及损伤后重建现象；照射后6h,造血细胞凋亡显著，并出现凋亡的典型改变；P53蛋白于造血细胞凋亡及修复时均明显增多，而mtp53DNA仅于修复期呈阳性。结论 p53基因表达与了辐射诱导的骨髓细胞凋亡和凋亡后的修复过程。     

低水平辐射对大鼠血浆和某些腺体中cAMP和cGMP水平的影响

本研究分二批进行,第一批用成年雄性大鼠每天接受X射线全身照射50mGy/次和100mGy/次,剂量率为15mGy/分,6次/周,共照5周(30次),累积剂量分别为1.5Gy和3.0Gy。受照大鼠睾丸重量在停照后4周内呈持续性减轻,以照射剂量大,停照时间长者,减轻最明显(相当于对照43.9%)。睾丸中cAMP含量于停照后1周升高,以100mGy组明显(P<0.01),停照后2周仍显著高于对照(P<0.05),停照后4周回降到正常较低水平。而血浆和肾上腺中cAMP和胸上腺中cGMP含量无明显变化。第二批实验动物条件与第一批相同,每天接受16.3mGy,~(60)Coγ射线全身照射,剂量率为45.3pGy/分,6次/周,每次照射6小时,在照射6、10、14、18、22周,累积剂量分别为0.59、0.98、1.37、1.76、2.15Gy时与相应对照组比较,垂体、睾丸中cAMP含量及睾丸重量无显著差别,证实慢性小剂量全身照射对睾丸组织的损伤主要取决于剂量率大小,剂量率愈大损伤愈明显。     

Temporal events in skin injury and the early adaptive responses in ultraviolet-irradiated mouse skin

We examined the effects of ultraviolet (UV) radiation on the time course for induction of sunburn (apoptotic) cells and expression of proteins known to be associated with growth arrest and apoptosis in SKH-hr1 mouse skin. Mice were irradiated with a single dose (2.5 kJ/m(2)) of UV from Kodacel-filtered (290-400 nm) FS40 sunlamps and the skin tissues were analyzed at various times after irradiation for the presence of apoptotic cells and expression of p53, p21(Waf-1/Cip1), bcl-2, bax, and proliferating cell nuclear antigen. The results indicated that p53 expression was induced early in the epidermis, reaching maximum levels 12 hours after irradiaton, and p21(Waf-1/Cip1) expression in the epidermis peaked at 24 hours after irradiation. In contrast, UV radiation induced high levels of bax at 24 to 72 hours after irradiation with a concomitant decrease in bcl-2 expression. Coinciding with these changes, apoptotic cells began to appear 6 hours after irradiation and reached a maximum at 24 hours after irradiation. Interestingly, proliferating cell nuclear antigen expression, which was initially confined to the basal layer, became dispersed throughout the basal and suprabasal layers of the skin at 48 hours and paralleled marked hyperplasia. These results suggest that UV irradiation of mouse skin induces apoptosis mediated by the p53/p21/bax/bcl-2 pathway and that the dead cells are replaced by hyperproliferative cells, leading to epidermal hyperplasia. This implies that UV-induced apoptosis and hyperplasia are closely linked and tightly regulated and that dysregulation of these two events may lead to skin cancer development.     

Temporal production of CCL28 corresponds to eosinophil accumulation and airway hyperreactivity in allergic airway inflammation

CCL28 is a recently identified chemokine ligand for CCR10 and CCR3 that has been identified in mucosal epithelial surfaces in diverse tissues. CCL28-mediated eosinophil chemotaxis and peroxidase release were inhibited by preincubation of cells with anti-CCR3. CCL28 was constitutively expressed in lung tissue collected from nonsensitized control mice but increased levels were found in mice sensitized and rechallenged with cockroach antigen (CRA). CCL28 levels peaked in the lungs 24 hours after intratracheal challenge with CRA, whereas eotaxin expression peaked at 8 hours. Increased expression of CCR3 but not CCR10 could be detected during the induction of the CRA-induced pulmonary inflammation. To investigate the role of CCL28 in allergic airway responses, mice were treated with CCL28 antiserum 1 hour before receiving the final CRA challenge. The level of airway hyperresponsiveness in mice treated with anti-CCL28 was significantly reduced at 24 hours, but not 8 hours, compared to mice receiving control serum. This reduction was not related to decreased Th2 cytokine, chemokine, or leukotriene levels at 24 hours although peribronchial eosinophilia was significantly reduced. Thus, CCL28 appears to play a role in regulating eosinophil recruitment to peribronchial regions of the lung possibly by coordinated temporal production with eotaxin.     

Ongoing activation of p53 pathway responses is a long-term consequence of radiation exposure in vivo and associates with altered macrophage activities

The major adverse consequences of radiation exposure, including the initiation of leukaemia and other malignancies, are generally attributed to effects in the cell nucleus at the time of irradiation. However, genomic damage as a longer term consequence of radiation exposure has more recently been demonstrated due to untargeted radiation effects including delayed chromosomal instability and bystander effects. These processes, mainly studied in vitro, are characterized by un-irradiated cells demonstrating effects as though they themselves had been irradiated and have been associated with altered oxidative processes. To investigate the potential for these untargeted effects of radiation to produce delayed damaging events in vivo, we studied a well-characterized model of radiation-induced acute myeloid leukaemia in CBA/Ca mice. Haemopoietic tissues of irradiated CBA/Ca mice exhibit enhanced levels of p53 stabilization, increased levels of p21(waf1), and increased amounts of apoptosis, as expected, in the first few hours post-irradiation, but also at much later times: weeks and months after the initial exposure. Because these responses are seen in cells that were not themselves directly irradiated but are the descendants of irradiated cells, the data are consistent with an initial radiation exposure leading to persistently increased levels of ongoing DNA damage, analogous to radiation-induced chromosomal instability. To investigate the potential source of ongoing oxidative processes, we show increased levels of 3-nitrotyrosine, a marker of damaging nitrogen/oxygen species in macrophages. Not all animals show increased oxidative activity or p53 responses as long-term consequences of irradiation, but increased levels of p53, p21, and apoptosis are directly correlated with increased 3-nitrotyrosine in individual mice post-irradiation. The data implicate persistent activation of inflammatory-type responses in irradiated tissues as a contributory bystander mechanism for causing delayed DNA damage.     

Delayed Resolution of Lung Inflammation in Il-1rn-/- Mice Reflects Elevated IL-17A/Granulocyte Colony-Stimulating Factor Expression

IL-1 has been associated with acute lung injury (ALI) in both humans and animal models, but further investigation of the precise mechanisms involved is needed, and may identify novel therapeutic targets. To discover the IL-1 mediators essential to the initiation and resolution phases of acute lung inflammation, knockout mice (with targeted deletions for either the IL-1 receptor-1, i.e., Il-1r1(-/-), or the IL-1 receptor antagonist, i.e., Il-1rn(-/-)) were exposed to aerosolized LPS, and indices of lung and systemic inflammation were examined over the subsequent 48 hours. The resultant cell counts, histology, protein, and RNA expression of key cytokines were measured. Il-1r1(-/-) mice exhibited decreased neutrophil influx, particularly at 4 and 48 hours after exposure to LPS, as well as reduced bronchoalveolar lavage (BAL) expression of chemokines and granulocyte colony-stimulating factor (G-CSF). On the contrary, Il-1rn(-/-) mice demonstrated increased BAL neutrophil counts, increased BAL total protein, and greater evidence of histologic injury, all most notably 2 days after LPS exposure. Il-1rn(-/-) mice also exhibited higher peripheral neutrophil counts and greater numbers of granulocyte receptor-1 cells in their bone marrow, potentially reflecting their elevated plasma G-CSF concentrations. Furthermore, IL-17A expression was increased in the BAL and lungs of Il-1rn(-/-) mice after exposure to LPS, likely because of increased numbers of γδ T cells in the Il-1rn(-/-) lungs. Blockade with IL-17A monoclonal antibody before LPS exposure decreased the resultant BAL neutrophil counts and lung G-CSF expression in Il-1rn(-/-) mice, 48 hours after exposure to LPS. In conclusion, Il-1rn(-/-) mice exhibit delayed resolution in acute lung inflammation after exposure to LPS, a process that appears to be mediated via the G-CSF/IL-17A axis.     

Loss of Fas-ligand expression in mouse keratinocytes during UV carcinogenesis

Skin cells containing excessive ultraviolet (UV) radiation-induced DNA damage are eliminated by apoptosis that involves the p53 pathway and Fas/Fas-Ligand (Fas-L) interactions. To determine whether dysregulation of apoptosis plays a role in skin cancer development through disruption of Fas/Fas-L interactions, hairless SKH-hr1 mice were exposed to chronic UV irradiation from Kodacel-filtered FS40 lamps for 30 weeks. Their skin was analyzed for the presence of sunburn cells (apoptotic keratinocytes) and for Fas and Fas-L expression at various time points. A dramatic decrease in the numbers of morphologically identified sunburn cells and TUNEL-positive cells was detected as early as 1 week after chronic UV exposure began. After 4 weeks of chronic UV exposure, these cells were barely detectable. This defect in apoptosis was paralleled by an initial decrease in Fas-L expression during the first week of chronic UV irradiation and a complete loss of expression after 4 weeks. Fas expression, however, increased during the course of chronic UV exposure. p53 mutations were detected in the UV-irradiated epidermis as early as 1 week after irradiation began and continued to accumulate with further UV exposure. Mice exposed to chronic UV began to develop skin tumors after approximately 8 weeks, and all mice had multiple skin tumors by 24 weeks. Most of the tumors expressed Fas but not Fas-L. We conclude that chronic UV exposure may induce a loss of Fas-L expression and a gain in p53 mutations, leading to dysregulation of apoptosis, expansion of mutated keratinocytes, and initiation of skin cancer.     

Lung injury via oxidative stress in mice induced by inhalation exposure to rocket kerosene

Rocket kerosene (RK) is a new rocket propellant. Toxicity occurs if a high level of RK is inhaled. To study the toxicity of RK in lung and the mechanisms of RK-induced lung jury, a total of 72 male ICR mice (1.5 months, adult) were randomly assigned to the RK exposure group (RKEG) and normal control group (NCG). Mice were whole-body exposed to room air or aerosol of 18000 mg/m³ RK for 4 hours. Histopathological analysis was performed to evaluate the pulmonary lesions. Oxidative stress was assessed by assay of MDA, SOD, GSH-PX and TAOC. Inflammatory response was estimated by detecting inflammatory cell counts, TNF-α and IL-6 protein levels in serum. The results showed that after 2 to 6 hours of RK exposure, pulmonary vascular dilatation, congestion and edematous widening of the alveolar septum were noted. After 12 to 24 hours post-exposure, diffuse hemorrhage in alveolar space were found, along with the progressive pulmonary vascular dilatation and edematous widening of alveolar septum. During 3 to 7 days of RK-exposure, inflammatory cells were scattered in the lung tissue. The pathological alterations of the lung were alleviated after 14 days post-exposure, and showed significant improvement after 21 days post-exposure. After 30 days of RK exposure, the pathological changes in the lung tissue were nearly recovered except the local thickening of the alveolar wall. Compared with NCG, RK inhalation produced a significant increase of MDA levels and a significant decrease of SOD, GSH-Px and TAOC activity in the lung after 2 hours post-exposure (P < 0.05). There were significant increases of TNF-α and IL-6 protein levels in serum of mice in RKEG after 2, 6 and 12 hours and 1, 4 and 7 days post-exposure compared with NCG (P < 0.05). TNF-α protein levels had a sharp increase after 4 days of exposure. IL-6 protein level was increased at early phase of experiment and then gradually decreased along with the prolonged course of exposure. Considering that the RK-induced lung injury was through the oxidative stress, inhibition of oxidative stress after RK exposure may be urgently needed.     

Bcl-2 family gene expression during severe hyperoxia induced lung injury

Exposure of the lung to severe hyperoxia induces terminal transferase dUTP end-labeling (TUNEL) indicative of DNA damage or apoptosis and increases expression of the tumor suppressor p53 and of members of the Bcl-2 gene family. Because cell survival and apoptosis are regulated, in part, by the relative abundance of proteins of the Bcl-2 family, we hypothesized that lung cells dying during exposure would show increased expression of pro-apoptotic members, such as Bax, whereas surviving cells would have increased expression of anti-apoptotic members, such as Bcl-X(L). The hypothesis is tested in the current study by determining which Bcl-2 genes are regulated by hyperoxia, with specific focus on correlating expression of Bax and Bcl-X(L) with morphologic evidence of apoptosis or necrosis. Adult mice exposed to greater than 95% oxygen concentrations for 48 to 88 hours had increased whole-lung mRNA levels of Bax and Bcl-X(L), no change in Bak, Bad, or Bcl-2, and decreased levels of Bcl-w and Bfl-1. In situ hybridization revealed that hyperoxia induced Bax and Bcl-X(L) mRNA in uniform and overlapping patterns of expression throughout terminal bronchioles and parenchyma, coinciding with TUNEL staining. Electron microscopy and DNA electrophoresis, however, suggested relatively little classical apoptosis. Unexpectedly, Western analysis demonstrated increased Bcl-X(L), but not Bax, protein in response to hyperoxia. Bax and Bfl-1 were not altered by hyperoxia in p53 null mice; however, oxygen toxicity was not lessened by p53 deficiency. These findings suggest that oxygen-induced lung injury does not depend on the relative expression of these Bcl-2 members.     

p53 regulates thymic Notch1 activation

Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP)/CD8+ single positive (SP) transition, but regulation of Notchactivation is not well understood. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. We hypothesize that p53 regulates Notch activity during T cell development. We found that Notch1 expression and activation were negatively regulated by p53in several thymoma lines. Additionally, NIC was elevated in Trp53(-/-) thymocytes as compared to Trp53(+/+) thymocytes. To determine if elevated Notch1 activation in Trp53(-/-) thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP/CD8+ SP T cell ratio was decreased in Trp53(-/-) splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8+SP T cells in Trp53(-/-) mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, we suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation.     

Glutathione reductase targeted to type II cells does not protect mice from hyperoxic lung injury

Exposure of the lung epithelium to reactive oxygen species without adequate antioxidant defenses leads to airway inflammation, and may contribute to lung injury. Glutathione peroxidase catalyzes the reduction of peroxides by oxidation of glutathione (GSH) to glutathione disulfide (GSSG), which can in turn be reduced by glutathione reductase (GR). Increased levels of GSSG have been shown to correlate negatively with outcome after oxidant exposure, and increased GR activity has been protective against hyperoxia in lung epithelial cells in vitro. We tested the hypothesis that increased GR expression targeted to type II alveolar epithelial cells would improve outcome in hyperoxia-induced lung injury. Human GR with a mitochondrial targeting sequence was targeted to mouse type II cells using the SPC promoter. Two transgenic lines were identified, with Line 2 having higher lung GR activities than Line 1. Both transgenic lines had lower lung GSSG levels and higher GSH/GSSG ratios than wild-type. Six-week-old wild-type and transgenic mice were exposed to greater than 95% O2 or room air (RA) for 84 hours. After exposure, Line 2 mice had higher right lung/body weight ratios and lavage protein concentrations than wild-type mice, and both lines 1 and 2 had lower GSSG levels than wild-type mice. These findings suggest that GSSG accumulation in the lung may not play a significant role in the development of hyperoxic lung injury, or that compensatory responses to unregulated GR expression render animals more susceptible to hyperoxic lung injury.