Yunjin Zang

A new approach utilizing real-time qPCR to detect in vitro base excision repair.

DNA lesions in mammalian cells may be induced by reactive oxygen species, alkylation, and ionizing radiation. This damage can then be repaired via the base excision repair (BER) pathway, which includes single strand break repair (SSBR). Thus, the BER (SSBR) pathway plays a critical role in maintaining genomic integrity, and may help us to better understand the mechanisms of aging, tumor formation, and degenerative diseases. AP site (apurinic/apyrimidinic site) or damaged base excision, nucleotide insertion and ligation are included in the BER (SSBR) pathway, which are facilitated by different enzymes at each step. Most previous in vitro BER studies have used modified radiolabeled (32)P oligonucleotide substrates. Which is a very conventional method for in vitro BER assay. However, the use of radioactive isotope material was limited in various laboratories which are unable to handle radioactive hazard. In this study, we developed a novel technique using real-time quantitative PCR (qPCR) to quantify BER activity in in vitro assays. Single strand breaks, DNA ligase activity, and glycosylase activity were detected to establish the feasibility and advantages of this qPCR technique for in vitro BER profiling.

Curcumin protects against hepatic ischemia/reperfusion induced injury through inhibiting TLR4/NF-κB pathway

The TLR4/NF-κB pathway had important roles in hepatic ischemia/reperfusion (I/R) injury. In this study, we reported a protective effect of curcumin against hepatic I/R injury via TLR4/NF-κB pathway. Curcumin significantly inhibited cell apoptosis, and decreased levels of LDH and production of TNF-a, IL-1b, and IL-6 in the cell supernatant. In addition, curcumin ameliorated elevated TLR4 and NF-κB caused by hypoxia/reoxygenation stimulation in BRL-3A cells. In vivo assays revealed that curcumin reduce levels of ALT and AST, and reversed TLR4/NF-κB signaling pathway caused by hepatic I/R stimulation in liver tissues. These results suggested that curcumin ameliorates hepatic I/R injury, which may be mediated in part via the TLR4/NF-κB signaling pathway.The TLR4/NF-κB pathway had important roles in hepatic ischemia/reperfusion (I/R) injury. In this study, we reported a protective effect of curcumin against hepatic I/R injury via TLR4/NF-κB pathway. Curcumin significantly inhibited cell apoptosis, and decreased levels of LDH and production of TNF-a, IL-1b, and IL-6 in the cell supernatant. In addition, curcumin ameliorated elevated TLR4 and NF-κB caused by hypoxia/reoxygenation stimulation in BRL-3A cells. In vivo assays revealed that curcumin reduce levels of ALT and AST, and reversed TLR4/NF-κB signaling pathway caused by hepatic I/R stimulation in liver tissues. These results suggested that curcumin ameliorates hepatic I/R injury, which may be mediated in part via the TLR4/NF-κB signaling pathway.

ASPP2 involvement in p53-mediated HIV-1 envelope glycoprotein gp120 neurotoxicity in mice cerebrocortical neurons

The mechanisms behind HIV-1-associated neurocognitive disorders are still unclear. Apoptosis-stimulating protein 2 of p53 (ASPP2) is a damage-inducible p53-binding protein that stimulates p53-mediated apoptosis and transactivates proapoptotic and cell cycle regulatory genes. It has been reported that ASPP2 has a specific regulatory function in the death of retinal ganglion cells and the development of Alzheimer’s disease. In this study, we used p53 and ASPP2 knockout mice and primary cerebrocortical neuron culture to analyze the role of the interaction between ASPP2 with p53 in HIV-1 envelope glycoprotein gp120-induced neurotoxicity. The results showed that 10 ng/mL gp120 protein might stimulate p53 overexpression and translocation to the nucleus, and 30 ng/mL gp120 protein could stimulate both p53 and ASPP2 translocation to the nucleus, but only with p53 overexpression. The primary cultured neurons of p53−/−ASPP2+/− mice had a higher survival rate than p53−/− mice under gp120 protein stress. The interaction of ASPP2 with p53 induced by a high dose of gp120 stimulated Bax transcription and contributed to caspase-3 cleavage, and ASPP2-siRNA attenuated gp120 induced neuron death through inhibition of Bax expression. These results suggest that ASPP2 plays an important role in p53-mediated neuronal apoptosis under gp120 stress.

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.

Mitochondrial DNA mutations in blood samples from HIV-1-infected children undergoing long-term antiretroviral therapy

We have analyzed mutations in whole mitochondrial (mt) genomes of blood samples from HIV-1-infected children treated with long-term antiretroviral therapy (ART), who had an excellent virological response. HIV-1-infected children who have undergone ART for 4 y with an excellent virological response (group A; 15 children) and ten healthy children (controls) without HIV-1 infection were enrolled retrospectively. Peripheral blood mononuclear cells (PBMCs) were obtained and mt DNA mutations were studied. The total number of mtDNA mutations in group A was 3 H more than in the controls (59 vs. 19, P<0.001) and the same trend was seen in all mtDNA regions. Among these mtDNA mutations, 140 and 28 mutations were detected in group A and the controls, respectively. The D-loop, CYTB and 12s rRNA were the three most common mutation regions in both groups, with significant differences between the groups observed at nucleotide positions C309CC, T489C CA514deletion, T16249C and G16474GG (D-loop); T14783C, G15043A, G15301A, and A15662G (CYTB); and G709A (12s rRNA). G15043A and A15662G had been associated with mitochondrial diseases. Our findings suggest that mtDNA mutations occur frequently in long-term ART-treated, HIV-1-infected children who have an excellent virological response, although they did not have obvious current symptoms. The CYTB region may play an important role in mtDNA mutation during ART, which might contribute to the development of subsequent mitochondrial diseases.

DNA damage regulated autophagy modulator 1 recovers the function of apoptosis‑stimulating of p53 protein 2 on inducing apoptotic cell death in Huh7.5 cells

Overexpression of apoptosis-stimulating of p53 protein 2 (ASPP2) can induce apoptotic cell death in hepatoma cells, which contributes to a killing effect of ASPP2 on treating hepatocellular carcinoma (HCC). In the present study, ASPP2 overexpression failed to induce apoptotic cell death in the HCC Huh7.5 cell line, but promoted autophagy development by inhibiting AKT/mTOR pathway. Inhibition of autophagy using 3-methyladenosine recovered the function of ASPP2 on inducing apoptotic cell death, indicating that ASPP2-induced autophagy has an anti-apoptotic role in Huh7.5 cells. A previous study demonstrated that ASPP2-induced autophagy could induce apoptosis in a CHOP- and DRAM-dependent manner, in which CHOP is involved in the initiation of autophagy and DRAM allows autophagy to induce apoptosis. In the present study, CHOP and DRAM were not involved in ASPP2-induced autophagy; however, the induction of DRAM overexpression recovered the apoptosis-inducing function of ASPP2, indicating that DRAM overexpression switches the role of ASPP2-induced autophagy from anti-apoptotic to pro-apoptotic in Huh7.5 cells. Thus, in combination with DRAM, ASPP2 may better perform its pro-apoptotic role by preventing the occurrence of anti-apoptotic autophagy.

Amphiregulin impairs apoptosis-stimulating protein 2 of p53 overexpression–induced apoptosis in hepatoma cells

Overexpression of apoptosis-stimulating protein 2 of p53 (ASPP2) induces apoptotic cell death in hepatoma cells (e.g. HepG2 cells) by enhancing the transactivation activity of p53, but long-term ASPP2 overexpression fails to induce more apoptosis since activation of the epidermal growth factor/epidermal growth factor receptor/SOS1 pathway impairs the pro-apoptotic role of ASPP2. In this study, in recombinant adenovirus-ASPP2-infected HepG2 cells, ASPP2 overexpression induces amphiregulin expression in a p53-dependent manner. Although amphiregulin initially contributes to ASPP2-induced apoptosis, it eventually impairs the pro-apoptotic function of ASPP2 by activating the epidermal growth factor/epidermal growth factor receptor/SOS1 pathway, leading to apoptosis resistance. Moreover, blocking soluble amphiregulin with a neutralizing antibody also significantly increased apoptotic cell death of HepG2 cells due to treatment with methyl methanesulfonate, cisplatin, or a recombinant p53 adenovirus, suggesting that the function of amphiregulin involved in inhibiting apoptosis may be a common mechanism by which hepatoma cells escape from stimulus-induced apoptosis. Thus, our data elucidate an apoptosis-evasion mechanism in hepatocellular carcinoma and have potential implications for hepatocellular carcinoma therapy.

Δ40p53 is involved in the inactivation of autophagy and contributes to inhibition of cell death in HCT116-Δ40p53 cells

Δ40p53 is an isoform of wild-type p53 (wtp53). Here, we assessed whether Δ40p53 has the same functions as wild-type p53 in the regulation of cell death and autophagy. First, we used HCT116 (p53+/+) and H1299 (p53-free) cells to produce two cell lines (HCT116-Δ40p53 and H1299-Δ40p53) that express exogenous Δ40p53 but not wtp53. By using these cell lines, we determined that Δ40p53 inhibited starvation-induced autophagy, as does wtp53. This inhibition arises from both Δ40p53 and wtp53 having 3′-5′ exonuclease activity, which reduces the levels of double-stranded RNA (dsRNA) and then inhibits PKR/eIF2α-induced autophagy in cells exposed to starvation. Like wtp53, the translocation of Δ40p53 to the nucleus increased in cells in response to Methyl methane sulfonate (MMS) treatment-induced DNA damage. Previous studies have shown that nuclear wtp53 can induce DRAM expression and DRAM-induced autophagy in cells in response to DNA damage, thereby contributing to apoptotic cell death as DRAM-induced autophagy is a pro-apoptotic factor. Here, nuclear Δ40p53 did not individually induce DRAM-induced autophagy and cell death in response to DNA damage. However, nuclear Δ40p53 inhibited wtp53-induced DRAM expression and cell death. Thus, Δ40p53 and wtp53 have 3′-5′ exonuclease activity and inhibit starvation-induced autophagy in the cytoplasm; however, nuclear Δ40p53 inhibits wtp53-induced cell death by impairing the transactivation activity of wtp53. Because wtp53 inhibits tumor and viral infection by inhibiting autophagy and promoting degradation of viral dsRNA, it is reasonable to believe that Δ40p53 has the similar functions. A deeper study of these functions of Δ40p53 is needed in the future.