Jiming Yin

Cytokines in CSF correlate with HIV-associated neurocognitive disorders in the post-HAART era in China

In the current era of highly active antiretroviral therapy (HAART), the incidence of HIV dementia has declined, but the prevalence of HIV-associated neurocognitive disorder (HAND) remains high. HIV-induced systemic and localized inflammation is considered to be one of the mechanisms of HAND. Changes in cytokine levels in the cerebrospinal fluid (CSF) during HIV infection might help to identify HAND. To investigate whether the cytokine profile of the CSF during HIV infection could be used as a biomarker of HAND, we compared cytokine levels in the CSF of HIV-infected cases with and without neurocognitive impairment. Cytokine concentrations in the CSF were measured by quantification bioassays (Luminex xMAP). HIV-infected cases with neurocognitive impairment demonstrated higher levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, induced protein (IP)-10, and granulocyte colony-stimulating factor (G-CSF) in the CSF than those without neurocognitive impairment (G-CSF (p = 0.0003), IL-8 (p = 0.0046), IP-10 (p < 0.0001), and MCP-1 (p < 0.0001)). There was no significant impact of HAART on cytokine levels in the CSF, except for IP-10, which was higher in HAART-treated patients with impaired cognition (p = 0.0182). Findings from this preliminary study suggest that elevated levels of the cytokines IL-8, MCP-1, G-CSF, and IP-10 in the CSF are associated with neurocognitive impairment in HIV infection, and these cytokines likely represent a biomarker profile for HAND.

Depending on the stage of hepatosteatosis, p53 causes apoptosis primarily through either DRAM‐induced autophagy or BAX

Apoptosis mediated by p53 plays a pathological role in the progression of hepatosteatosis. It is noteworthy that p53 can promote the expression of damage‐regulated autophagy modulator (DRAM), an inducer of autophagy‐mediated apoptosis. However, the relationship between p53‐mediated apoptosis and autophagy in hepatosteatosis remains elusive. This study aimed to examine how p53 orchestrates autophagy and apoptosis to affect hepatosteatosis.

ASPP2 attenuates triglycerides to protect against hepatocyte injury by reducing autophagy in a cell and mouse model of non-alcoholic fatty liver disease.

ASPP2 is a pro‐apoptotic member of the p53 binding protein family. ASPP2 has been shown to inhibit autophagy, which maintains energy balance in nutritional deprivation. We attempted to identify the role of ASPP2 in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD). In a NAFLD cell model, control treated and untreated HepG2 cells were pre‐incubated with GFP‐adenovirus (GFP‐ad) for 12 hrs and then treated with oleic acid (OA) for 24 hrs. In the experimental groups, the HepG2 cells were pre‐treated with ASPP2‐adenovirus (ASPP2‐ad) or ASPP2‐siRNA for 12 hrs and then treated with OA for 24 hrs. BALB/c mice fed a methionine‐ and choline‐deficient (MCD) diet were used to generate a mouse model of NAFLD. The mice with fatty livers in the control group were pre‐treated with injections of GFP‐ad for 10 days. In the experimental group, the mice that had been pre‐treated with ASPP2‐ad were fed an MCD diet for 10 days. ASPP2‐ad or GFP‐ad was administered once every 5 days. Liver tissue from fatty liver patients and healthy controls were used to analyse the role of ASPP2. Autophagy, apoptosis markers and lipid metabolism mediators, were assessed with confocal fluorescence microscopy, immunohistochemistry, western blot and biochemical assays. ASPP2 overexpression decreased the triglyceride content and inhibited autophagy and apoptosis in the HepG2 cells. ASPP2‐ad administration suppressed the MCD diet‐induced autophagy, steatosis and apoptosis and decreased the previously elevated alanine aminotransferase levels. In conclusion, ASPP2 may participate in the lipid metabolism of non‐alcoholic steatohepatitis and attenuate liver failure.

Mitochondrial toxicity studied with the PBMC of children from the Chinese national pediatric highly active antiretroviral therapy cohort.

As the backbone of highly active antiretroviral therapy (HAART), nucleoside reverse transcriptase inhibitors (NRTIs) have effectively improved outcomes for HIV-infected patients. However, long-term treatment with NRTIs can cause a series of pathologies associated with mitochondrial toxicity. To date, the status and mechanism of mitochondrial toxicity induced by NRTIs are still not clear, especially in HIV-infected children. As part of the national pediatric HAART program in China, our study focused on mitochondrial toxicity and its potential mechanism in HIV-1-infected children who were divided into two groups based on their duration of treatment with NRTIs: one group received treatment for less than 36 months and one group was treated for 36 to 72 months. The control group comprised age-matched non-HIV-infected children. Blood lactic acid and ATP levels in peripheral blood mononuclear cells (PBMCs) were measured to evaluate mitochondrial function, and mtDNA copies and mutations in PBMCs were determined for detecting mtDNA lesions. Simultaneously, TK2 and P53R2 gene expression in PBMC was measured. As compared with the control group, blood lactic acid levels in both NRTI treatment groups were significantly higher, whereas ATP levels and mtDNA mutation rates in PBMCs did not differ between the control and the two NRTI treatment groups. Both NRTI treatment groups exhibited significant mtDNA loss. N Moreover, we found that P53R2 mRNA expression and protein levels were significantly reduced in both treatment groups and that TK2 mRNA expression and protein levels were induced in the long-term NRTI treatment group. These results suggest that mitochondrial toxicity occurs in long-term HAART patients and that P53R2 and TK2 levels in PBMCs are useful biomarkers for detecting mitochondrial toxicity in patients on long-term treatment with NRTIs.

Differential effects of reticulophagy and mitophagy on nonalcoholic fatty liver disease

Autophagy affects the pathological progression of non-alcoholic fatty liver disease (NAFLD); however, the precise role of autophagy in NAFLD remains unclear. In this study, we want to identify the role of autophagy including reticulophagy and mitophagy in NAFLD pathogenesis. When HepG2 cells were treated with 400 μM oleic acid (OA), increased reticulophagy was induced 8 h after treatment, which correlated with an anti-apoptotic response as shown by the activation of the PI3K/AKT pathway, an increase in BCL-2 expression, and the downregulation of OA-induced lipotoxicity. When treated with OA for 24 h, DRAM expression-dependent mitophagy resulted in increased apoptosis in HepG2 cells. Inhibition of reticulophagy aggravated and increased lipotoxicity-induced apoptosis 8 h after treatment; however, the inhibition of mitophagy decreased hepatocyte apoptosis after 24 h of OA treatment. Results from the analysis of patient liver samples showed that autophagic flux increased in patients with mild or severe NAFL. PI3K/AKT phosphorylation was observed only in samples from patients with low-grade steatosis, whereas DRAM expression was increased in samples from patients with high-grade steatosis. Together, our results demonstrate that reticulophagy and mitophagy are independent, sequential events that influence NAFLD progression, which opens new avenues for investigating new therapeutics in NAFLD.

Exogenous p53 and ASPP2 expression enhances rAdV-TK/ GCV-induced death in hepatocellular carcinoma cells lacking functional p53

Suicide gene therapy using herpes simplex virus-1 thymidine kinase (HSV-TK) in combination with ganciclovir (GCV) has emerged as a potential new method for treating cancer. We hypothesize that the efficacy of HSV-TK/GCV therapy is at least partially dependent on p53 status in hepatocellular carcinoma (HCC) patients. Using recombinant adenoviral vectors (rAdV), TK, p53, and ASPP2 were overexpressed individually and in combination in Hep3B (p53 null) and HepG2 (p53 wild-type) cell lines and in primary HCC tumor cells. p53 overexpression induced death in Hep3B cells, but not HepG2 cells. ASPP2 overexpression increased rAdV-TK/GCV-induced HepG2 cell death by interacting with endogenous p53. Similarly, ASPP2 reduced survival in rAdV-TK/GCV-treated primary HCC cells expressing p53 wild-type but not a p53 R249S mutant. Mutated p53 was unable to bind to ASPP2, suggesting that the increase in rAdV-TK/GCV-induced cell death resulting from ASPP2 overexpression was dependent on its interaction with p53. Additionally, γ-H2AX foci, ATM phosphorylation, Bax, and p21 expression increased in rAdV-TK/GCV-treated HepG2 cells as compared to Hep3B cells. This suggests that the combined use of HSV-TK, GCV, rAdV-p53 and rAdV-ASPP2 may improve therapeutic efficacy in HCC patients lacking functional p53.

The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T

The mitochondrial toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is due to the inhibition of mitochondrial DNA (mtDNA) polymerase γ (pol γ). Previous studies have shown that wild type p53 (wtp53) can interact with pol γ and mtDNA to enhance mitochondrial DNA base excision repair (mtBER) activity and increase the accuracy of DNA synthesis. The N-terminal transactivation domain and central specific DNA-binding domain of p53 play critical roles in the stimulation of BER. In this study, we identified the possible roles of wtp53, Δ40p53 and Δ133p53 in regulating mtDNA pol γ activity in cells with d4T treatment. The results show that Δ40p53 and Δ133p53 can exist in mitochondrial fragments and form polymers with themselves or wtp53. Unlike wtP53, Δ133p53 alone cannot increase DNA pol γ activity. More importantly, we found that Δ133p53 played a negative role in p53 stimulation of DNA pol γ activity when studied in d4T-treated and d4T-untreated mitochondrial extracts. Gel shift data also indicate that Δ40p53 and Δ133p53 cannot interact with APE. Wtp53 and Δ40p53 can act antagonize the effect of d4T inhibition of DNA pol γ activity. However, when wtp53 interacted with Δ133p53, DNA pol γ activity was significantly decreased. Conclusion: Δ133p53 negatively regulates p53’s stimulation of pol γ in the presence and absence of d4T.

Abstract 789: Adenovirus p53 enhances the antitumor effect of adenovirus thymidine kinase/ganciclovir on Hep3B cells by apoptosis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background and Aims: Although Thymidine Kinase/Ganciclovir(TK/GCV) is a promising tumor-targeted chemotherapy by DNA damage, it is reported that tumor cells having wild-type p53 are more sensitive to chemotherapeutic agents. Furthermore some evidence have suggested approximately half of all cancers possess inactivated p53. Numerous studies have shown the key role of p53 as a tumor suppressor is blocking cell cycle progression and/or inducing apoptosis in response to high level of DNA damage. Given that TK/GCV results in DNA damage, exogenous wild-type p53 should enhance efficacy of adenovirus (adv)-mediated antitumor effect of TK/GCV on p53 −/- tumor cells. Thus, this article aims to determine whether p53 −/- cancer cells are sensitive at TK/GCV, whether coexpression of p53 and TK/GCV can actively kill p53−/- tumor cells and the main mechanisms responsible for TK/GCV tumor cell death. Methods:Hep3B cells (p53−/−) and HepG2(p53+/+) cells were infected with adv-p53 and adv-TK one day prior to GCV. The cellular growth inhibition was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and immunofluorescent staining (PI and Calcein AM). Apoptosis and cell cycle arrest were measured using annexin V-FITC /PI double staining and PI by flow cytometry. p53,PUMA and p21 mRNA expression were analyzed using real-time quantitative PCR . The protein levels of p53,PUMA and p21 were detected by Western blotting. Results:The MTT results showed that cell viability (%) of HepG2 cell (p53+/+) treated with adv-TK(107/ml)/GCV(10ug/ml) were lower than Hep3B (p53−/−)(p<0.01). Growth inhibition of Hep3B treated with adv-p53 in combination with adv-TK/GCV was higher than adv-p53 and adv-TK/GCV alone (p<0.01). Annexin V-FITC/PI double staining and propidium iodide (PI) staining demonstrates that Hep3B cell treated with adv-p53 and adv-TK/GCV had significantly increased apoptosis compared to adv-p53 (p<0.01) and adv-TK/GCV(p<0.05) alone. The expression level of p53,p21 and PUMA mRNA and protein was significantly higher in Hep3B cell treated with adv-p53 and adv-TK/GCV compared to adv-p53 and adv-TK/GCV alone in a time-dependent manner. Conclusions:These results suggest that the p53−/- cancer cell is not sensitive to TK/GCV. p53 −/- tumor cells were actively killed by a combination adv- p53 with TK/GCV. p53-mediated apoptosis is the main mechanisms responsible for TK/GCV kill tumor cells. Citation Format: Kai Liu, Xiuhong Liu, Tao Wen, Feng Ren, Jiming Yin, Daojie Liu, Huiguo Ding, Ning Li, Dexi Chen. Adenovirus p53 enhances the antitumor effect of adenovirus thymidine kinase/ganciclovir on Hep3B cells by apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2013-789 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

糖原合成酶激酶-3β在D-GalN/LPS诱导的小鼠急性肝衰竭中的作用

目的：研究细胞信号分子糖原合成酶激酶-3β（glycogen synthase kinase-3β,GSK-3β）在D-氨基半乳糖/脂多糖联合注射诱导小鼠重型肝炎肝衰竭中的作用.方法：以C57BL/6小鼠为研究对象,腹腔注射D-氨基半乳糖/脂多糖建立小鼠重型肝炎肝衰竭模型.动物实验分组：对照组,重型肝炎肝衰竭模型组,SB216763干预组（建模前2h腹腔注射）,SB216763治疗组（建模后2h腹腔注射）.Western blot检测肝脏组织GSK-3β磷酸化水平,检测血清转氨酶（alanine aminotransferase,ALT）、天门冬酸氨基转移酶（aspartate aminotransferase,AST）评价肝脏功能,观察肝脏组织病理变化评价肝脏损伤情况,实时荧光定量PCR法检测肝脏细胞炎症因子基因表达,并检测凋亡相关蛋白Caspase 3的活性表达.多组样本均数的两两比较采用One-way ANOVA分析（方差齐者用LSD-t检验,方差不齐者用Games-Howell法）,P〈0.05有统计学意义.结果：Western blot结果显示,GSK-3β在急性肝衰竭过程中磷酸化水平先降低（活性升高）后再次升高;抑制GSK-3β活性,无论是干预还是治疗都改善肝脏功能（血清ALT、AST水平明显下降,肝组织病理损伤明显改善）,并且抑制炎症反应[抑制促炎因子肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）、白介素6（Interleukin-6,IL-6）、IL-1β表达,促进抗炎因子IL-10高表达],并可降低凋亡相关蛋白Caspase 3表达.结论：在D-氨基半乳糖/脂多糖诱导小鼠急性肝衰竭过程中GSK-3β被激活,抑制GSK-3β活性通过降低炎症反应和肝细胞凋亡从而改善肝损伤.因此,对信号分子GSK-3β活性进行干预有可能为重型肝炎肝衰竭的治疗提供一个新的靶点.