Wild type p53 gene causes reorganization of cytoskeleton and,therefore, the impaired deformability and difficult migration of murine erythroleukemia cells

We studied the role of p53 gene in the biophysics and biology in murine erythroleukemia cell line (MEL), with the goal of understanding the influence of this tumor suppressor gene on the deformability and metastasis of tumor cells. Experiments were performed on MEL and p53-transfected MEL (MEL-M with mutant p53 gene and MEL-W with wild-type p53 gene). The cell growth curves indicated that the over-expression of wild-type p53 gene significantly suppressed the growth of MEL, with G(0)-G(1) arrest and apoptosis shown by flow cytometric assays. Confocal laser scanning microscopy revealed that the MEL-W had a more compact organization of the F-Actin cytoskeleton than MEL and MEL-M. Fluorescence polarization measurement indicated a higher membrane fluidity of MEL-W than the other two groups. Fourier transform infrared spectroscopy (FT-IR) showed changes in the composition and/or structure of membrane lipids in MEL-W, with decreases in secondary structures of proteins such as alpha-helix, turns and bends and random coil, in comparison to MEL and MEL-M. The osmotic fragility curves indicated that MEL-W was more fragile and micropipette experiments showed that they had increased elasticity and reduced deformability in comparison to MEL and MEL-M. The adhesion assay with the use of the flow chamber revealed a lower adhesion rate of MEL-W to endothelial cells at high shear stress. The present study on the molecular biology with biophysics of MEL cells contributes to our knowledge on the tumor suppressor gene p53.     

Temporal changes in the expression of protein phosphatase 1 and protein phosphatase 2A in proliferating and differentiating murine erythroleukaemia cells

Rhythmic changes in the expression of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) were investigated during hexamethylene bisacetamide (HMBA) induced differentiation of murine erythroleukaemic (MEL) cells. Cell extracts were analysed by SDS-PAGE and western immunoblotting using specific antibodies. An immunospecific band of molecular mass 36 kDa (catalytic subunit) was detected for PP1. For PP2A, two immunospecific bands of 32 kDa (proteolytically cleaved catalytic subunit) and 36 kDa (catalytic subunit) were observed. Comparisons of proliferating and differentiating cells using only one time point showed no significant differences between mean values for the expression of the PP1 or PP2A enzyme proteins. This kind of analysis, implying that HMBA had little effect, proved misleading, as comparisons using multiple time points showed rhythmic patterns of protein expression which were modulated by the differentiating agent. The effects were complex affecting both the frequency and phasing of rhythms. The results add further support for the view that live cells are multi-oscillators and for the concept that differentiation depends on changes in temporal organization of complex autodynamic feedback loops and multiple interactions between control circuits performing in parallel. In particular, modulation of the dynamics of key proteins, such as PP1 and PP2A, may be a possible mechanism for controlling cellular function and reversing transformation in accordance with long standing theoretical and other experimental data.     

Phospholipase C ε‐1 inhibits p53 expression in lung cancer

The pathogenesis of lung cancer is to be further investigated. Recent reports indicate that phospholipase C ε‐1 (PLCE1) is a critical molecule involved in tumour growth. This study aims to investigate the role of PLCE1 in the regulation of apoptosis in lung cancer cells. In this study, the surgically removed non‐small‐cell lung cancer (NSCLC) tissue was collected from 36 patients. Single NSCLC cells were prepared from the tissue, in which immune cells of CD3⁺, CD11c⁺, CD19⁺, CD68⁺ and CD14⁺ were eliminated by magnetic cell sorting. The expression of PLCE1 and p53 was assessed by quantitative real‐time polymerase chain reaction and Western blotting. Apoptosis of NSCLC cells was analysed by flow cytometry. The results showed that, in cultured NSCLC cells, high levels of PLCE1 and low levels p53 were detected; the two molecules showed a negative correlation (p < 0.01). The addition of anti‐PLCE1 antibody increased the expression of p53 in NSCLC cells, which increased the frequency of apoptotic NSCLC cells. We conclude that NSCLC cells express high levels of PLCE1, which suppresses the expression of p53 in NSCLC cells. PLCE1 can be a therapeutic target of NSCLC. Copyright © 2013 John Wiley & Sons, Ltd.     

Methionine cytotoxicity in the human breast cancer cell line MCF-7

Summary Among the first nutrients to be linked to cancer were methyl group containing nutrients including methionine. Methionine and its metabolic derivatives are essential components in several indispensable biological reactions including protein synthesis, polyamine synthesis, and many transmethylation reactions. The purpose of this study was to determine the extent to which methionine excess affects the proliferation and gene expression of the human breast cancer cell line MCF-7. Cells were first grown in control medium; the medium was then replaced with either control or methionine-supplemented treatment media. We found that 5 and 10 g/L methionine significantly suppressed cell growth on day 1, and no further growth was detected after 3 d of treatment. Cell, proliferation in the methionine treated group was significantly lower than that of the control group. Northern analysis revealed that expression of p53 in methionine-treated MCF-7 cells was approximately 70% lower than that of control cells. p53 is a key cell cycle regulatory, protein that has been implicated in tumorigenesis and cancer progression. Alteration of the p53 tumor suppressor gene is the most common genetic change found in a wide variety of malignancies, including cancer. This study shows that excess methionine (5 g/L) inhibited proliferation of MCF-7 breast cancer cells, and down regulation of p53 is correlated with this inhibition. These findings may aid in the development of nutritional strategies for breast cancer therapy.     

Human herpesvirus 6 open reading frame U14 protein and cellular p53 interact with each other and are contained in the virion

A mass spectroscopic analysis of proteins from human herpesvirus 6 (HHV-6)-infected cells showed that the HHV-6 U14 protein coimmunoprecipitated with the tumor suppressor p53. The binding of U14 to p53 was verified by coimmunoprecipitation experiments in both Molt-3 cells infected with HHV-6 and 293 cells cotransfected with U14 and p53 expression vectors. Indirect immunofluorescence assays (IFAs) showed that by 18 h postinfection (hpi) U14 localized to the dot-like structures observed in both the nucleus and cytoplasm where p53 was partly accumulated. Despite Northern blotting evidence that U14 follows late kinetics, the U14 protein was detected immediately after infection (at 3 hpi) by IFA. In addition, by Western blotting, U14 was detected at 0 hpi or in the presence of cycloheximide which completely abolished the expression of IE1 protein. In addition to U14, p53 was detected at 0 hpi although it was not detected in mock-infected cells. Furthermore, both U14 and p53 were clearly detected in the viral particles by Western blotting and immunoelectron microscopy, supporting the idea that U14 and p53 are incorporated into virions. Our study provides the first evidence of the incorporation of cellular p53 into viral particles and suggests that p53 may play an important role in viral infection.     

EB病毒感染人胎鼻咽上皮细胞逃避老化期过程中抑制p16^INK4a表达

细胞周期调控紊乱是细胞永生化进程中一个重要的分子事件,本文利用Western blotting和S-P法分别检测p16^INK4a、p53、p21^WAF1／CIP1和E2F1的蛋白表达,试图从细胞周期调控的角度,探讨EB病毒诱导人胎鼻咽上皮细胞逃避老化期的分子机制。结果表明,EB病毒通过抑制p16^INK4a表达而阻断p16^INK4a／Rb途径,上调转录因子E2F1,而对p53、p21^WAF1／CIP表达无明显的影响。结果初步揭示,EB病毒介导的p16^INK4a／Rb／E2F1细胞周期调控紊乱参与了人胎鼻咽上皮细胞逃避老化期过程,为进一步探讨鼻咽癌发病机制提供了科学依据。     

Apoptosis resistance in pigmented villonodular synovitis

OBJECTIVE: Pigmented villonodular synovitis (PVNS) is a proliferative lesion originating from synovial tissue with a locally aggressive behaviour. We analysed the pathogenetic role of apoptosis resistance for sustained cell proliferation in PVNS. METHODS: The expression of bcl-2, p53 and Ki-67 was examined in 80 cases of PVNS using immunohistochemistry. In 43 of these cases, DNA content and distribution of cell-cycle phases were investigated by flow cytometry. Additionally, 10 cases of PVNS were analysed by multi-parametric flow cytometry for expression of p53, caspase3, and bcl-2 and by TUNEL to detect DNA fragmentation. RESULTS: No apoptotic cell fractions were detected in any investigated cases. Expression of bcl-2 was found in 84% of cases (up to 6.5% of cells) and was significantly associated with DNA-fragmentation observed by TUNEL (p=0.037). Orthologous p53 expression was observed in 37% of cases. The level of p53 expression correlated with the proliferative activity and the expression of both caspase3 (p=0.017) and bcl-2 (p=0.0013). (No statistically significant correlations between expression of bcl-2, p53, caspase3, DNA fragmentation or proliferative index and age, sex of patients, disease recurrence, growth pattern or size of lesion were found). CONCLUSION: Apoptosis resistance is a critical event in the progression of PVNS and may contribute to the survival of the proliferating synovial cells in PVNS and to the permanent slow progression of these lesions.     

Dihydromyricetin Reduced Bcl-2 Expression via p53 in Human Hepatoma HepG2 Cells

Dihydromyricetin (DHM) is a major active ingredient of flavonoids compounds. It exhibited anticancer activity and induced apoptosis in human hepatocellular carcinoma HepG2 cells according to our previous data. In this study, we investigated whether p53 is involved in DHM-triggered viability inhibition and apoptosis induction in cancer cells. MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay was employed to evaluate the viability of HepG2 cells after DHM treatment. Meanwhile, p53 small interfering RNA (siRNA) was adopted to silence p53 expression. Protein level of p53 and Bax/Bcl-2 were evaluated by western blot analysis. Cell counting assay showed that DHM inhibited HepG2 cell growth effectively in a time- and dose-dependent manner. P53 expression was significantly increased after DHM treatment, whereas Bcl-2 was reduced potently. Furthermore, after co-treatment with Pifithrin-α (PFT-α, p53 inhibitor), Bcl-2 expression was reversed. The expression of Bax was no significant change, which was also observed after p53 silence. These findings defined and supported a novel function that DHM could induce human hepatocellular carcinoma HepG2 cells apoptosis by up-regulating Bax/Bcl-2 expression via p53 signal pathway.     

Deregulated lncRNA expression profile in the mouse lung adenocarcinomas with KRAS‐G12D mutation and P53 knockout

Recent studies have demonstrated that aberrant long non‐coding RNAs (lncRNAs) expression are suggested to be closely associated with multiple human diseases, lung cancer included. However, the roles of lncRNAs in lung cancer are not well understood. In this study, we used microarrays to investigate the aberrantly expressed lncRNAs in the mouse lung adenocarcinoma with P53 knockout and the Kras^G12D mutation. Results revealed that 6424 lncRNAs were differentially expressed (≥ 2‐fold change, P < .05). Two hundred and ten lncRNAs showed more than 8‐fold change and conserved across human and were further analysed in the primary mouse lung adenocarcinoma KP cells, which were isolated from the p53 knockout and the Kras^G12D mutation mice. Among all the 210 lncRNAs, 11 lncRNAs' expression was regulated by P53, 33 lncRNAs by KRAS and 13 lncRNAs by hypoxia in the primary KP cells, respectively. NONMMUT015812, which was remarkably up‐regulated in the mouse lung adenocarcinoma and negatively regulated by the P53 re‐expression, was detected to analyse its cellular function. Results showed that knockdown of NONMMUT015812 by shRNAs decreased proliferation and migration abilities of KP cells. Among those aberrantly expressed lncRNAs in the mouse lung adenocarcinoma, NONMMUT015812 was a potential oncogene.     

A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia.

Cell cycle checkpoints can enhance cell survival and limit mutagenic events following DNA damage. Primary murine fibroblasts became deficient in a G1 checkpoint activated by ionizing radiation (IR) when both wild-type p53 alleles were disrupted. In addition, cells from patients with the radiosensitive, cancer-prone disease ataxia-telangiectasia (AT) lacked the IR-induced increase in p53 protein levels seen in normal cells. Finally, IR induction of the human GADD45 gene, an induction that is also defective in AT cells, was dependent on wild-type p53 function. Wild-type but not mutant p53 bound strongly to a conserved element in the GADD45 gene, and a p53-containing nuclear factor, which bound this element, was detected in extracts from irradiated cells. Thus, we identified three participants (AT gene(s), p53, and GADD45) in a signal transduction pathway that controls cell cycle arrest following DNA damage; abnormalities in this pathway probably contribute to tumor development.