Inhibition of p53 function prevents renin-angiotensin system activation and stretch-mediated myocyte apoptosis

To determine whether stretch-induced activation of p53 is necessary for the up-regulation of the local renin-angiotensin system and angiotensin II (Ang II)-induced apoptosis, ventricular myocytes were infected with an adenoviral vector carrying mutated p53, Adp53m, before 12 hours of stretch. Noninfected myocytes and myocytes infected with AdLacZ served as controls. Stretching of Adp53m-infected myocytes prevented stimulation of p53 function that conditioned the expression of p53-dependent genes; quantity of angiotensinogen (Aogen), AT(1), and Bax decreased, whereas Bcl-2 increased. Ang II generation was not enhanced by stretch. Conversely, stretch produced opposite changes in noninfected and AdLacZ-infected myocytes: Aogen increased twofold, AT(1) increased 2. 1-fold, Bax increased 2.5-fold, and Ang II increased 2.4-fold. These responses were coupled with 4.5-fold up-regulation of wild-type p53. Stretch elicited comparable adaptations in p53-independent genes, in the presence or absence of mutated p53; renin increased threefold, angiotensin-converting enzyme increased ninefold, and AT(2) increased 1.7-fold. Infection with Adp53m inhibited myocyte apoptosis after stretch. Conversely, stretch increased apoptosis by 6.2-fold in myocytes with elevated endogenous wild-type p53. Thus, a competitor of p53 function interfered with both stretch-induced Ang II formation and apoptosis, indicating that p53 is a major modulator of myocyte renin-angiotensin system and cell survival after mechanical deformation.     

Insulin-like growth factor-1 and post-ischemic brain injury

Insulin-like growth factor-1 (IGF-1) is a naturally occurring neurotrophic factor that plays an important role in promoting cell proliferation and differentiation during normal brain development and maturation. The present review examines recent evidence that endogenous IGF-1 also plays a significant role in recovery from insults such as hypoxia–ischemia and that giving additional exogenous IGF-1 can actively ameliorate damage. It is now well established that neurons and other cell types die many hours or even days after initial injury due to activation of programmed cell death pathways. IGF-1 and its binding proteins and receptors are intensely induced within damaged brain regions following brain injury, suggesting a possible a role for IGF-1 in brain recovery. Exogenous administration of IGF-1 within a few hours after brain injury is now known to be protective in both gray and white matter and leads to improved somatic function. In contrast, pre-treatment is ineffective, likely reflecting limited intracerebral penetration of IGF-1 into the uninjured brain. The neuroprotective effects of IGF-1 are mediated by IGF-1 receptors and its binding proteins and are specific to particular cellular phenotypes and brain regions. The window of opportunity for treatment with IGF-1 is limited to a few hours after normothermic brain injury, reflecting its specific actions on early, intracellular events in the apoptotic cascade. However, injury-associated mild post-hypoxic hypothermia, which delays the development of cell death, can shift and dramatically extend the window of opportunity for delayed treatment with IGF-1. Such a combined approach is likely to be essential for any clinical treatment.     

UBTD1 induces cellular senescence through an UBTD1–Mdm2/p53 positive feedback loop

The tumour suppressor p53 plays an important role in tumourigenesis. Besides inducing apoptosis, it regulates cellular senescence, which constitutes an important barrier to tumourigenesis. The mechanism of regulation of cellular senescence by p53 and its downstream pathway are poorly understood. Here, we report that the ubiquitin domain‐containing 1 (UBTD1) gene, a new downstream target of p53, induces cellular senescence and acts as a novel tumour suppressor by a mechanism that depends on p53. Expression of UBTD1 increased upon cellular senescence induced by serial passageing of cultures, as well as by exposure to DNA‐damageing drugs that induce premature senescence. Over‐expression of UBTD1 induces senescence in human fibroblasts and cancer cells and attenuation of the transformed phenotype in cancer cells. UBTD1 is down‐regulated in gastric and colorectal cancer tissues, and its lower expression correlates with a more aggressive phenotype and worse prognosis. Multivariate analysis revealed that UBTD1 expression was an independent prognostic factor for gastric cancer patients. Furthermore, UBTD1 increased the stability of p53 protein, by promoting the degradation of Mdm2 protein. Importantly, UBTD1 and p53 function mutually depend on each other in regulating cellular senescence and proliferation. Thus, our data suggest that, upon DNA damage, p53 induction by UBTD1 creates a positive feedback mechanism to further increase p53 expression. Our results establish UBTD1 as a regulator of cellular senescence that mediates p53 function, and provide insights into the mechanism of Mdm2 inhibition that impacts p53 dynamics during cellular senescence and tumourigenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

p53 and Mdm2: not all cells are equal

The human TP53 gene is well known as a tumour suppressor. Less well appreciated are the potential roles of p53 in regulating postnatal development and the cell-type specific effects of p53. In this issue of the Journal of Pathology, Lozano and colleagues show that p53 has the ability to block progenitor cell expansion in haematopoiesis and spermatogenesis but not in specific epithelial tissues. These data challenge the view that p53 acts equally in all cell types and have important implications for the applicability of p53 therapeutics in the treatment of human cancers.     

星形细胞瘤中PTEN、Mdm2和p53的表达及其相关性研究

目的: 探讨不同组织病理分级的星形细胞瘤中PTEN、Mdm2和p53的表达水平, 并分析PTEN影响Mdm2、p53表达的信号转导机制。方法: 采用免疫组织化学方法检测68例星形细胞瘤标本中PTEN、Mdm2和p53的表达水平。结果:星形细胞瘤中PTEN、Mdm2、p53的表达水平分别是54.4%(37/68)、41.2%(28/68)、45.6%(31/68)。PTEN阳性标本中Mdm2的表达率(24.3%, 9/37)与PTEN阴性标本中该蛋白的表达率(61.3%, 19/31)相比差异显著, 统计学分析显示PTEN表达与Mdm2表达呈负相关(P＜0.01)。Mdm2表达和p53表达一致符合率达66.2%(45/68), 两者的表达密切相关(P＜0.05)。结论: (1)PTEN、Mdm2和p53表达与星形细胞瘤的组织病理分级相关。(2)抑癌基因PTEN可以下调癌基因Mdm2的表达水平。(3)Mdm2和p53的表达存在一致性。     

Mdm2 gene amplification in gastric cancer correlation with expression of Mdm2 protein and p53 alterations.

Mdm2, localized on chromosome 12, is considered a negative regulator of p53 function and seems to play a role in the pathogenesis of a variety of tumors. The mdm2 amplification in advanced-stage gastric carcinoma has not yet been investigated. Mdm2 amplification was determined in 43 gastric carcinomas, and the genetic results were correlated with mdm2 protein expression, p53 alterations, and clinicopathologic data. The tumors were classified according to Lauren: 20 intestinal-type tumors, 19 tumors of diffuse growth inclusive of a primary small cell carcinoma, and 4 carcinomas with mixed differentiation. Staging was based on the pTNM classification system. Mdm2 and p53 were demonstrated by immunohistology on formalin-fixed and paraffin-embedded tumor tissue. The mdm2 oncogene was amplified by nonradioactive hybridization of tumor DNA with an mdm2 cDNA probe. The Southern blots were evaluated densitometrically. For p53 mutation screening, we analyzed the highly conservative regions of the p53 gene (exons 4 to 8) with the use of the polymerase chain reaction-single-strand conformation polymorphism technique. Polymerase chain reaction products with band shifting were directly sequenced. Mdm2 amplification was demonstrated in 18 tumors (41.8%). The mdm2 gene was amplified more frequently in carcinomas with a diffuse growth pattern. Gastric carcinomas of the intestinal type, however, showed a higher frequency of p53 alterations. There was no statistical significance of the molecular genetic and immunohistologic results of the mdm2/p53 status to staging as well as to age and sex of the patients. The mdm2/p53 pathway is a part of the carcinogenesis of gastric carcinoma. Only approximately 20% of gastric carcinomas failed to show mdm2 and/or p53 alterations. The upregulation of the mdm2 oncogene and the accompanying inactivation of the tumor suppressor gene 53 seem to play a role above all in carcinomas of the diffuse type.     

Immunohistochemical expression of Mdm2 and p53 in penile verrucous carcinoma

Verrucous carcinoma (VC) of the penis is an uncommon squamous tumor that pursues a biologically indolent course. Unlike conventional squamous cell carcinoma (SCC) of the penis, pathogenic roles for human papillomavirus (HPV) infection and p53 mutation have not been reported in VC. We compared the immunohistochemical expression of Mdm2 and p53 in 7 cases of VC and 7 cases of SCC. The Mdm2 gene product preferentially labeled the perinuclear membrane in the granular layer of VC tumor cells, whereas SCC cases showed weak, focal, cytoplasmic staining for Mdm2. The mean labeling index for Mdm2 was higher in VC compared to SCC [79.3 (SE +/- 7.2) in VC vs 18.3 (SE +/- 2.4) in SCC, p < 0.001]. In SCC cases, the normal surrounding skin showed mild granular-layer staining and dysplastic foci that failed to stain with Mdm2 antibody. Weak p53 immunolabeling was seen within nuclei of scattered tumor cells in the cases of VC, whereas the SCC cases showed strong nuclear staining of p53 throughout the tumors. The mean labeling index for p53 was lower in VC compared to SCC [24.8 (SE +/- 3.9) in VC vs 64.7 (SE +/- 9.0) in SCC, p < 0.01]. In SCC cases, the normal surrounding skin showed moderate staining for p53, preferentially confined to the basal layer. Dysplastic foci in the cases of SCC showed increased p53 labeling. In summary, immunohistochemical analysis showed significantly different levels of expression of Mdm2 and p53 in penile VC vs SCC. Overexpression of the Mdm2 gene product may be important in the pathogenesis of VC. Since Mdm2 is a negative regulator of p53, overexpression of Mdm2 may explain why p53 is down-regulated and, therefore, permissive to oncogenic transformation.     

Insulin-like growth factor-1 and lean body mass in HIV-infected children

OBJECTIVES: To describe insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-1-binding protein-1 (IGFBP-1) and IGFBP-3 in HIV+ children before and after initiating or changing antiretroviral therapy and to evaluate association of growth and body composition to growth factors at baseline and over time. METHODS: Ninety-seven prepubertal HIV+ children aged 1 month to younger than 13 years were observed over 48 weeks after beginning or changing antiretroviral therapy. Serum IGF-1, IGFBP-1, and IGFBP-3 were measured and compared with age- and sex-specific norms. Anthropometric measures were compared as follows: subjects vs matched children from (a) the National Health and Nutrition Examination Survey to generate z scores and (b) HIV-exposed, uninfected children from Women and Infants Transmission Study; and subjects with normal vs abnormal IGF-1 and IGFBP concentrations at baseline. Anthropometric changes were compared for children whose IGF-1 level normalized vs remaining subjects. Multivariate analysis adjusting for sex, race, and baseline age evaluated associations between anthropometry and IGF-1 and IGFBP concentrations. RESULTS: In multivariate analysis, lower baseline IGF-1 and IGFBP-3 were associated with lower mean weight, height, mid-arm muscle circumference, and mid-thigh circumference z scores. Twenty-four percent of children had a low IGF-1 level at baseline, 50% of whom normalized IGF-1 on study. Children whose IGF-1 normalized had greater increases in mean mid-arm muscle circumference z score (1.00 vs -0.03, P = 0.029), but a trend toward lesser mean height increase (P = 0.082) than remaining subjects. Likewise, in comparison to controls from Women and Infants Transmission Study, mean mid-arm muscle circumference also increased more in children whose IGF-1 normalized (P = 0.024) but mean height changed less (P = 0.003). Fifty-five percent of children had elevated IGFBP-1 at baseline, 69% of whom normalized. CONCLUSIONS: IGF-1 increases and IGFBP-1 decreases in HIV-infected children upon initiation or change in antiretroviral therapy. Improved muscle mass, but not linear growth, is associated with normalized IGF-1 concentration. These findings suggest that IGF-1 may merit evaluation as a potential therapeutic strategy to improve lean body mass in HIV-infected children.     

Insulin-like growth factor-1 fails to enhance central nervous system myelin repair during autoimmune demyelination

Previous studies have shown that insulin-like growth factor-1 (IGF-1) has beneficial effects, both clinically and histopathologically, on experimental autoimmune encephalomyelitis (EAE), although results vary depending on species and treatment regimen. The present study investigated whether IGF-1, delivered at different time points during the acute and chronic phases of adoptively transferred EAE in SJL mice, had the ability to affect or enhance myelin regeneration. Central nervous system tissue sampled at different stages of treatment was subjected to detailed neuropathological, immunocytochemical and molecular analysis. The results revealed some transient clinical amelioration and low level remyelination after IGF-1 administration during the acute phase of EAE. However, central nervous system tissue from acute phase treated animals sampled at chronic time points and from animals given IGF-1 during the chronic phase revealed no enhancing effect on remyelination in comparison to vehicle-treated controls. Examination of oligodendrocyte progenitor populations also revealed no differences between IGF-1- and vehicle-treated groups. At the cytokine level, the immunomodulatory molecules TGF-beta2 and TGF-beta3 displayed significant decreases that may have contributed to the transient nature of the effect of IGF-1 on EAE. Together with evidence from previous studies, it appears doubtful that IGF-1 is a good candidate for treatment in multiple sclerosis, for which EAE serves as a major model.     

Bcl-2 and p53 protein expression, apoptosis, and p53 mutation in human epithelial ovarian cancers

Bcl-2 and p53 gene products have been both linked to cell death by apoptosis. In the present study, we examined the relationship of Bcl-2 and p53 protein expression, p53 mutation and apoptosis in normal human ovaries and different types of human ovarian epithelial tumors by immunohistochemical localization, in situ terminal transferase-mediated dUTP nick end labeling and polymerase chain reaction-single strand conformation polymorphism. It was found that Bcl-2 expressed strongly in the surface epithelium of normal ovaries and benign and borderline ovarian tumors but weakly in the malignant tumors. On the contrary, strong protein expression of p53 was found in 54% (25/46) of the malignant epithelial tumors examined but similar expression of p53 was not observed in borderline and benign tumors and normal ovarian surface epithelium. A significant inverse correlation between Bcl-2 and p53 expression was found in the malignant ovarian tumors examined. p53 gene mutation at exons 5-11 was however not a pre-requisite for p53 expression in both borderline and malignant tumors. Apoptotic activities, as reflected by apoptotic indices, were low in normal ovarian surface epithelium and benign tumors but were increased in borderline and malignant tumors, with the highest average apoptotic index found in grade III malignant tumors. Statistical analyses showed a positive correlation between apoptosis and p53 expression, but similar correlation was not found between apoptosis and Bcl-2 expression. Our results also indicate that although expression of Bcl-2 is important during ovarian carcinogenesis, the Bcl-2 protein may have other roles to play apart from being a modulator of apoptosis in human ovarian epithelial cancers.     

大鼠髁突软骨细胞凋亡与胰岛素样生长因子1的影响

背景：胰岛素样生长因子1参与了髁突软骨生长与改建,是软骨发育关键因子。 目的：探索胰岛素样生长因子1对体外培养大鼠髁突软骨细胞凋亡的作用,以及对凋亡相关因子Bcl-2和Bax mRNA及蛋白表达变化的影响。 方法：体外培养并鉴定出生后1,28 d大鼠髁突软骨细胞后,将每个年龄组的髁突软骨细胞分别分为实验组和对照组。饥饿培养24 h后,实验组加入100 μg/L重组大鼠胰岛素样生长因子1细胞因子孵育48 h,对照组正常培养。 结果与结论：与对照组相比,实验组加入重组胰岛素样生长因子1后,髁突软骨细胞数量增多,增殖速度显著增加(P < 0.05)。实时PCR和Western blot检测显示,加入重组胰岛素样生长因子1培养48 h后,各组髁突软骨细胞中bcl-2 mRNA和蛋白表达增加,bax mRNA和蛋白表达减少(P < 0.05)。 提示胰岛素样生长因子1可以促进新出生及青春期大鼠髁突软骨细胞增殖,抑制其凋亡,并可能通过Bcl-2和Bax介导抑制凋亡。     

胰岛素样生长因子-1通过细胞外信号调节激酶1/2通路下调转录因子基本转录元件结合蛋白表达抗心肌细胞凋亡

目的 探讨胰岛素样生长因子-1(IGF-1)对大鼠心肌细胞凋亡保护作用的基因调控机制。方法 体外培养新生大鼠心肌细胞,10nmol/L IGF-1刺激的同时,分别加入磷脂酰肌醇-3激酶（PI3K）、细胞外信号调节激酶（ERK）1/2和Raf-1 3条通路抑制剂（20μmol/L）,通过RT-PCR及Western blotting方法观察IGF-1调节基本转录元件结合蛋白（BTEB）的基因表达及其通路调控。100μmol/L H2O2处理诱导心肌细胞凋亡,通过DNA梯度分析、Annexin V-FITC/PI双染色法、Caspase-3活性测定、Hoechest33258染色法观察用BTEB特异性siRNA人为下调BTEB基因表达后对心肌细胞凋亡的影响。结果 大鼠心肌细胞经IGF-1刺激60min后,BTEB mRNA和蛋白表达均明显下降;与对照组相比,加入ERK1/2通路抑制剂PD98059组BTEB的mRNA和蛋白表达均明显增高（P＜0.01）;H2O2诱导的大鼠心肌细胞于下调BTEB表达后,DNA片段化改善,心肌细胞凋亡率下降（P＜0.05）,Caspase-3活性降低（P＜0.05）,凋亡小体减少,与IGF-1的抗心肌细胞凋亡效果相似。
结论 IGF-1可以通过ERK1/2通路下调转录因子BTEB基因表达而发挥抗心肌细胞凋亡的作用。     

Immunoreactivity of p53, Mdm2, p21(WAF1/CIP1) Bcl-2, and Bax in soft tissue sarcomas: correlation with histologic grade.

Tumor growth depends on 2 distinctive pathways: cell proliferation and apoptosis. The p53 pathway is an important regulator of the cell cycle as it triggers growth arrest or leads to apoptosis in response to cellular stress and therefore is commonly targeted during tumorigenesis. Apoptosis is also controlled by the Bcl-2 family, which includes proapoptotic and antiapoptotic proteins. The aim of this study was to investigate the expression of proteins that are involved in the p53 pathway and apoptosis in different types of soft tissue sarcomas and to correlate the expression of these proteins with the histologic grade of sarcoma cases. One hundred fifty-two cases of different types of soft tissue sarcomas were analyzed. The cases consisted of 54 low-grade, 40 intermediate-grade, and 58 high-grade sarcomas. Immunohistochemical stains for p21(WAF1/CIP1), p53, Mdm2, Bcl-2, and Bax proteins were carried out on tissue microarrays. Nuclear reactivity for p53 was detected in 49 cases (32.2%). Overexpression of Mdm2 was found in 18 cases (11.8%) and p21(WAF1/CIP1) immunostaining was seen in 28 tumors (18.4%). p53 and p21(WAF1/CIP1) expression correlated with the tumor grade (low grade, 5.6% and 3.7%; intermediate grade, 22.5% and 20%; high grade, 63.8% and 31%, respectively). Expression of Bax protein was a common finding in soft tissue sarcoma cases. It was detected in 141 cases (92.8%). Bcl-2 was identified in 59 tumors (38.8%) and was more prevalent in high-grade sarcomas (low grade, 25.9%; intermediate grade, 32.5%; high grade, 55.2%). It was concluded that alterations in the p53 pathway and genes that regulate apoptosis are common events in soft tissue sarcomas. The expression of p53, p21(WAF1/CIP1), and Bcl-2 is closely associated with the histologic grade of the tumor, and therefore these proteins may be used as prognostic markers.     

53BP2 induces apoptosis through the mitochondrial death pathway

The p53 binding protein 2 (53BP2) has been identified as the interacting protein to p53, Bcl-2, and p65 subunit of nuclear factor kappaB (NF-kappaB). The TP53BP2 gene encodes two splicing variants, 53BP2S and 53BP2L, previously known as apoptosis stimulating protein 2 of p53 (ASPP2). We found that these 53BP2 proteins are located predominantly in the cytoplasm and induce apoptosis as demonstrated by cleavage of poly ADP ribose polymerase (PARP) and annexin V staining. Furthermore, we demonstrate that 53BP2 is located in the mitochondria and induces apoptosis associated with depression of the mitochondrial trans-membrane potential (DeltaPsim) and activation of caspase-9. From these findings we conclude that 53BP2 induces apoptosis through the mitochondrial death pathway.