p53-independent apoptosis in UV-irradiated mouse skin: possible inhibition by 50 Hz magnetic fields

Our recent results suggest that 50 Hz magnetic fields (MF) enhance ultraviolet (UV)-induced tumorigenesis in mouse skin. The aim of the present experiment was to study suppression of apoptosis as a possible mechanism for MF effects on skin tumorigenesis. Another aim was to test the importance of a UV and MF exposure schedule, particularly the role of MF exposure prior to UV irradiation. Female mice were exposed to a UV dose of 2 human MED and to 100 microT MF of 50 Hz, using the following exposure schedules: group 1 sham MF 24 h, UV 1 h, sham MF 24 h; group 2 sham MF 24 h, UV 1 h, MF 24 h; group 3 MF 24 h, UV 1 h, MF 24 h. Lamps emitting simulated solar radiation (SSR) were used for UV irradiation. Skin samples were analysed for apoptosis, expression of the p53 gene, activity of the enzyme ornithine decarboxylase (ODC) and polyamine concentrations. A significantly (p = 0.017) lower number of apoptotic cells was measured in group 2 compared to group 1. A similar but not statistically significant (p = 0.064) decrease was also detected in group 3. No p53 expression was detected in any sample. The levels of ODC and putrescine did not differ significantly between the UV-only and UV and MF-exposed groups. Spermidine and spermine levels were significantly (p = 0.014 and 0.014, respectively) lower in group 3 than in group 1, but no decrease was observed in group 2. Our findings suggest that SSR induces p53-independent apoptosis in mouse skin and that the apoptotic response may be inhibited by exposure to MF. The exposure schedule did not alter the MF effect. The results do not support a causal role for polyamines in MF effects on apoptosis.     

p53-independent induction of GADD45 and GADD153 in mouse lungs exposed to hyperoxia

Previous studies have shown that lungs of adult mice exposed to >95% oxygen have increased terminal deoxyribonucleotidyltransferase dUTP nick end-label staining and accumulate p53, the expression of which increases in cells exposed to DNA-damaging agents. The present study was designed to determine whether hyperoxia also increased expression of the growth arrest and DNA damage (GADD) gene 45 and GADD153, which are induced by genotoxic stress through p53-dependent and -independent pathways. GADD proteins have been shown to inhibit proliferation and stimulate DNA repair and/or apoptosis. GADD45 and GADD153 mRNAs were not detected in lungs exposed to room air but were detected after 48 and 72 h of exposure to hyperoxia. In situ hybridization and immunohistochemistry revealed that hyperoxia increased GADD45 and GADD153 expression in the bronchiolar epithelium and GADD45 expression predominantly in alveolar cells that were morphologically consistent with type II cells. Hyperoxia also increased GADD expression in p53-deficient mice. Terminal deoxyribonucleotidyltransferase dUTP nick end-label staining of lung cells from p53 wild-type and p53-null mice exposed to hyperoxia for 48 h revealed that hyperoxia-induced DNA fragmentation was not modified by p53 deficiency. These studies are consistent with the hypothesis that hyperoxia-induced DNA fragmentation is associated with the expression of GADD genes that may participate in DNA repair and/or apoptosis.     

Selective COX-2 inhibitor (celecoxib) decreases cellular growth in prostate cancer cell lines independent of p53

Abstract

Celecoxib is a clinically available COX-2 inhibitor that has been reported to have antineoplastic activity. It has been proposed as a preventative agent for several types of early neoplastic lesions. Earlier studies have shown that sensitivity of prostatic carcinoma (PCa) to celecoxib is associated with apoptosis; however, these studies have not demonstrated adequately whether this effect is dependent on p53 status. We studied the relation between sensitivity to celecoxib and the phenotypic p53 status of PCa cells lines, LNCaP (wild type p53), PC3 (null p53) and DU145 (mutated p53). Cellular growth was assessed at 24, 48, 72 and 96 h after celecoxib treatment at concentrations of 0, 10, 30, 50, 70 and 100 μM using an MTT assay. Cellular proliferation (Ki-67 expression) was determined by immunocytochemistry. Phenotypic expression of p53 was analyzed by western blotting. The effects of celecoxib on cellular growth and its association with p53 were assessed after down-regulation of p53 using synthetic interfering RNAs (siRNA) in LNCaP cells. Expression of p53 and COX-2 at mRNA levels was assessed by quantitative real time polymerase reaction (qRT-PCR). We found that celecoxib inhibited cellular growth and proliferation in a dose-dependent manner in all three cell lines; LNCaP cells with a native p53 were the most sensitive to celecoxib. We observed a down- regulation effect on p53 in LNCaP cells exposed to ≥ 30 μM celecoxib for 72 h, but found no significant changes in the p53 levels of DU145 cells, which have a mutated p53. Reduced COX-2 expression was found with decreased p53 in LNCaP and PC-3 cells that were exposed to ≥ 20 μM of celecoxib for 72 h, but COX-2 expression was increased in DU145 cells. All three cell lines demonstrated pan-cytotoxicity when exposed to 100 μM celecoxib. When p53 expression was inhibited using siRNA in LNCaP cells, the inhibitory effects on cellular growth usually exerted by celecoxib were not changed significantly. Celecoxib reduces the growth of prostate cancer cell lines in part by decreasing proliferation, which suggests that the inhibition of growth of LNCaP cells by celecoxib is independent of normal levels of native p53.     

Interactions between Cells with Distinct Mutations in c-MYC and Pten in Prostate Cancer

In human somatic tumorigenesis, mutations are thought to arise sporadically in individual cells surrounded by unaffected cells. This contrasts with most current transgenic models where mutations are induced synchronously in entire cell populations. Here we have modeled sporadic oncogene activation using a transgenic mouse in which c-MYC is focally activated in prostate luminal epithelial cells. Focal c-MYC expression resulted in mild pathology, but prostate-specific deletion of a single allele of the Pten tumor suppressor gene cooperated with c-MYC to induce high grade prostatic intraepithelial neoplasia (HGPIN)/cancer lesions. These lesions were in all cases associated with loss of Pten protein expression from the wild type allele. In the prostates of mice with concurrent homozygous deletion of Pten and focal c-MYC activation, double mutant (i.e. c-MYC+;Pten-null) cells were of higher grade and proliferated faster than single mutant (Pten-null) cells within the same glands. Consequently, double mutant cells outcompeted single mutant cells despite the presence of increased rates of apoptosis in the former. The p53 pathway was activated in Pten-deficient prostate cells and tissues, but c-MYC expression shifted the p53 response from senescence to apoptosis by repressing the p53 target gene p21^Cip1. We conclude that c-MYC overexpression and Pten deficiency cooperate to promote prostate tumorigenesis, but a p53-dependent apoptotic response may present a barrier to further progression. Our results highlight the utility of inducing mutations focally to model the competitive interactions between cell populations with distinct genetic alterations during tumorigenesis.     

Apoptosis of fetal testicular cells is regulated by both p53-dependent and independent mechanisms

A portion of fetal germ cells undergoes apoptosis in the physiological context, but the molecular mechanisms of their apoptosis are largely unknown. Because p53 tumor suppressor gene product promotes apoptosis in various types of cells, we have investigated the expression of p53 in fetal gonads and examined the influence of loss of p53 function in fetal gonad cells using mice deficient in the p53 gene. We found that the expression of p53 protein in fetal testis was induced after 15.5 dpc (days post coitum), while the expression was not detected in fetal ovary. The number of apoptotic cells found in the seminiferous tubules of fetal testes was not significantly different between p53-deficient and wild-type mice until 16.5 dpc. At 17.5 dpc, however, more apoptotic cells were observed in wild-type testes than in the p53-deficient mice. In contrast, a similar number of apoptotic cells was found in fetal ovaries throughout these developmental stages. These observations indicate that p53 promotes apoptosis of fetal testicular cells after 16.5 dpc.     

Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats.

Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone.     

p53 is involved in inducing testicular apoptosis in mice by the altered redox status following tertiary butyl hydroperoxide treatment

In testis, seminiferous epithelium is one of the most productive self-renewing systems in which apoptosis is an important phenomenon. Alteration in the cellular redox status has several detrimental effects on the cells, one of which is increased rate of apoptotic signals disturbing the natural balance. Since apoptotic responses to various therapeutic agents and toxicants follow diverse molecular mechanisms, therefore, the present study was designed to explore apoptosis in testes under the effect of oxidative stress. Tertiary butyl hydroperoxide (tBHP) was used to induce oxidative stress in mice. It was found that ROS production in testes by tBHP resulted in increased apoptosis. The apoptosis was evident from TUNEL staining in Zenker-fixed paraffin-embedded testicular sections of tBHP treated mice testis and DNA fragmentation analysis. Increased mRNA and protein expression of p53 in testis were observed by using RT-PCR and ELISA techniques, respectively. This indicates that p53 expression is linked to ROS generation in mice testes. The functional status of p53 was also assessed by upregulation of cyclin dependent kinase inhibitor, p21. Thus tBHP induced oxidative stress subject testicular cells to apoptosis which seems to involve p53.     

Prostate-specific expression of p53(R172L) differentially regulates p21, Bax, and mdm2 to inhibit prostate cancer progression and prolong survival

Loss of heterozygosity or mutation at the p53 tumor suppressor gene locus is frequently associated with advanced human prostate cancer. Hence, replacement p53 gene therapy may prove to be efficacious for this disease. While many mutations result in p53 molecules with oncogenic properties, other variants may possess wild-type properties with increased tumor suppressor activity. We have chosen to investigate the activity of a naturally occurring variant p53 molecule, p53(R172L), carrying an arginine-to-leucine mutation at codon 172. We demonstrate that p53(R172L) can differentially activate expression of genes involved in cell cycle control and apoptosis in vitro. Transgenic mice expressing a subphysiological level of a p53(R172L) minigene (PB-p53(R172L)) in the prostate epithelium were generated and bred to the transgenic adenocarcinoma mouse prostate (TRAMP) model of prostate cancer. While PB-p53(R172L) transgenic mice developed normally with no detectable prostate gland phenotype, we observed a significant increase in the apoptotic index in the prostate glands of TRAMP x PB-p53(R172L) F1 mice. We noted an increase in the expression of Bax in the bigenic mice concomitant with the reduced incidence and rate of tumor growth and increased survival. While low-level expression of the p53(R172L) variant had no obvious influence on normal prostate tissue, it was able to significantly inhibit prostate cancer progression in the context of a genetically predisposed model system. This suggests that additional tumor-related events specifically influence the ability of the variant p53(R172L) molecule to inhibit tumor growth. These studies support gene therapy strategies employing specific p53 variants.     

Effects of 50‐Hz magnetic field exposure on hormone secretion and apoptosis‐related gene expression in human first trimester villous trophoblasts in vitro

Evidence from epidemiological and animal studies showed that exposure to extremely low frequency magnetic fields (ELF‐MF) could produce deleterious effects on reproduction. In order to investigate the possible mechanism of MF exposure on reproductive effects, first trimester human chorionic villi at 8–10 weeks' gestation were obtained, and trophoblasts were isolated, cultured, and exposed to a 50‐Hz MF for different durations. The human chorionic gonadotropin (hCG) and progesterone in the culture medium was measured by electrochemiluminescence immunoassay. The mRNA levels of apoptosis‐related genes bcl‐2, bax, caspase‐3, p53, and fas in trophoblasts were analyzed using real‐time RT‐PCR. The results showed that exposure of trophoblasts to MF at 0.2 mT for 72 h did not affect secretion of hCG and progesterone from these cells. There was also no significant change in secretion of these hormones when trophoblasts were exposed to a 0.4 mT MF for 48 h. However, MF significantly inhibited hCG and progesterone secretion of trophoblasts after exposure for 72 h at 0.4 mT. Results of apoptosis‐related gene expression analysis showed that, within 72 h of exposure at 0.4 mT, there was no significant difference between MF exposure and control on the expression pattern of each gene. Based on results of the present experiment, it is suggested that exposure to MF for a longer duration (72 h) could inhibit secretion of hCG and progesterone by human first trimester villous trophoblasts, however, the effect might not be related to trophoblast apoptosis. Bioelectromagnetics 31:566–572, 2010. © 2010 Wiley‐Liss, Inc.     

5-Fluorouracil (5-FU) induced apoptosis in gastric cancer cell lines: role of the p53 gene

We examined chemosensitivity to 5-fluorouracil (5-FU) in four human gastric cancer cell lines, by analyzing the expression of p53 and its related genes. Treatment with 1mM 5-FU induced variable degrees of apoptosis in the cultured cells. The apoptotic indices 72 h after treatment were approximately 14% in MKN-74 (wild-type p53 gene), 12% in MKN-45 (wild-type), 3% in MKN-28 (mutated) and 0.5% in KATO-III cells (deleted), respectively. On the other hand, 50 M 5-FU had little effect on the induction of apoptosis in MKN-74 cells, the value being approximately 2% after 72 h. Induction of P53 expression was noted 3 h after initiating the treatment, followed by the induction of P21/Waf1 after 6 h in both MKN-74 and MKN-45 cells. The same expression mode was noted in MKN-74 treated with 50 M 5-FU. Conversely, the level of P53 expression was constant in MKN-28 cells and absent in KATO-III cells, in which P21/Waf1 had never been induced. The Bax/Bcl-2 expression ratio was gradually elevated for up to 72 h in MKN-74 and MKN-45 cells treated with 1mM 5-FU; in contrast, it was unchanged in MKN-28 and KATO-III cells, and MKN-74 treated with 50 M 5-FU. These results might indicate that (1) 1mM 5-FU induces apoptosis in cultured gastric cancer cells carrying the wild-type p53 gene, but not those carrying the mutated type or a gene deletion, and (2) the elevated Bax/Bcl-2 expression ratio plays a more crucial role than the higher expression of P21/Waf1 in the induction of p53- gene dependent apoptosis.     

Apoptotic rate and metallothionein levels in the tissues of cadmium-and copper-exposed rats

It is well known that cadmium (Cd) has toxic and carcinogenic effects in rodents and humans, but the effects of Cd on apoptosis are still not clear. Although some studies have shown that Cd has apoptotic potential, other studies have shown that Cd can be antiapoptotic. Parameters such as sensitivity of the exposed organism or cells and the exposure conditions should be important in delineating the effect of Cd on apoptosis. In the present study, we aimed to determine the apoptotic index (AI) of Sprague-Dawley rat tissues that are loaded at a lower Cd concentration than the critical concentration (50 microg/g) for its toxic effects. Metallothionein (MT) levels of tissues were also determined and the experiments repeated with copper (Cu)-exposed rats. We detected decreases in the apoptotic index in liver and lung tissues of Cd-exposed groups accompanied with an increase in MT levels. Also, decreases of AI were detected in the liver tissues of Cu-exposed groups. These findings indicate that Cd can suppress apoptosis in vivo. The possible role of MT expression on the suppression of apoptosis and the importance of free-Cd ion concentration on switching antiapoptotic effects to proapoptotic effects are also discussed.     

Phosphorylation and gene expression of p53 are not affected in human cells exposed to 2.1425 GHz band CW or W-CDMA modulated radiation allocated to mobile radio base stations

A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields induce apoptosis or other cellular stress response that activate p53 or the p53-signaling pathway. First, we evaluated the response of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and wideband code division multiple access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced apoptosis or any signs of stress. Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80, 250, and 800 mW/kg, and CW radiation at 80 mW/kg for 24 or 48 h. Human IMR-90 fibroblasts from fetal lungs were exposed to both W-CDMA and CW radiation at a SAR of 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the percentage of apoptotic cells were observed between the test groups exposed to RF signals and the sham-exposed negative controls, as evaluated by the Annexin V affinity assay. No significant differences in expression levels of phosphorylated p53 at serine 15 or total p53 were observed between the test groups and the negative controls by the bead-based multiplex assay. Moreover, microarray hybridization and real-time RT-PCR analysis showed no noticeable differences in gene expression of the subsequent downstream targets of p53 signaling involved in apoptosis between the test groups and the negative controls. Our results confirm that exposure to low-level RF signals up to 800 mW/kg does not induce p53-dependent apoptosis, DNA damage, or other stress response in human cells.     

Status of p53, p21, mdm2, pRb proteins, and DNA methylation in gonocytes of control and gamma-irradiated rats during testicular development

In fetal and newborn rat testes, gonocytes, which stop cycling for about 8 days, become highly radiosensitive. The presence of p53, p21, mdm2, and pRb, which are involved in cell cycle, apoptosis control, or both, were studied by immunohistochemistry to determine if their expression is related to this radiosensitivity. A strong cytoplasmic expression of p53 and p21 was detected. Cytoplasmic expression of p53 occurred only in arrested gonocytes, whereas that of p21 was observed before and after the block. P21 was found to colocalize with mitochondria. No expression of mdm2 was detected and pRb was present only when the gonocytes started cycling again. In animals exposed to 1.5 Gy of gamma-irradiation at Day 19 postcoitum, p53 expression was prolonged in time, whereas no change was observed in p21 amounts and localization, compared with controls. Using antibodies against 5-methyl cytosine, it was shown that gonocyte DNA passed from a hypomethylated to a methylated status 1 day after gonocytes stopped cycling. A prolonged survival of gonocytes after exposure to radiation was followed by their progressive apoptosis, which finally involved the entire gonocyte population between Days 6 and 12 postpartum. The elevated but delayed sensitivity of gonocytes to genotoxic stress may be related to the unusual expression of p53 and p21, which may itself be related to the large DNA methylation changes.     

Gene expression changes accompanying p53 activity during estrogen treatment of osteoblasts

Estrogen is known to be anabolic for bone and we have used estrogen treatment as a paradigm to understand how p53 may affect osteoblast differentiation. In previous studies we have shown estrogen treatment to increase p53 functional activity in osteoblasts. Estrogen has been suggested to inhibit apoptosis in osteoblasts. Since the significance of a p53 increase during estrogen treatment is not apparent, we investigated the environment within osteoblasts after treatment with estrogen. We observed two peaks of p53 activity during continuous treatment of 17-[beta]-estradiol (E2) for 72h. The gene expression profile of different cell cycle regulators and apoptosis related genes at different times during treatment with 17-[beta]-estradiol were tested using gene arrays. There was an early increase in expression of several genes involved in apoptosis. This was followed by changes in expression of several genes involved in cell survival and stress response. The second peak of activity was associated with increase in expression of cell cycle regulators. Our results suggest that p53 activity may be a result of activation of several signaling pathways involving apoptosis, cell survival and cell cycle arrest. P53 may have a role in integrating these responses, which eventually results in cell cycle arrest and expression of differentiation markers.     

胚胎期接触双酚A对雄鼠睾丸内PCNA和p53表达的影响

目的研究胚胎期接触双酚A(Bisphenol A,BPA)对雄性胎鼠睾丸发育的影响及睾丸内增殖细胞核抗原(Proliferating Cell Nuclear Antigen,PCNA)和p53表达的影响。方法母鼠怀孕后第2天对其灌服双酚A(剂量5,50,100 mg/ml/day),一直持续分娩,F1代雄性小鼠饲养至75日龄,观察BPA对仔鼠成年后睾丸结构和PCNA和p53表达的影响。结果发现BPA处理组睾丸发育受到抑制,曲细精管直径和管腔直径变小(P0.05),管腔内出现大量胞质残余体,部分管腔出现潴留管腔液,支持细胞生长受到抑制,生精细胞排列紊乱,细胞质出现空泡化。免疫组织化学结果显示在BPA处理组,PCNA除了在精原细胞大量表达外,在初级精母细胞中表达量明显升高(P0.05,P0.01),免疫荧光结果显示胚胎期接触BPA导致成年后睾丸内p53蛋白表达量显著升高(P0.05,P0.01)。结论胚胎期接触BPA对雄鼠睾丸发育有着长期的不良影响,可能源于BPA引起细胞的异常增殖和凋亡。