Mitsuru Nakanishi

8-Hydroxy-2'-deoxy-guanosine is a risk factor for development of hepatocellular carcinoma in patients with chronic hepatitis C virus infection

Background and Aim:  Increased production of reactive oxygen species, which cause oxidative DNA damage, is considered to be related to hepatocarcinogenesis. 8‐Hydroxy‐2′‐deoxy‐guanosine (8‐OHdG) is a useful marker of DNA damage induced by oxidative stress. The aim of this study was to determine whether expression of 8‐OHdG is a risk factor for the development of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV) infection.

ATM-dependent nuclear accumulation of IKK- α plays an important role in the regulation of p73-mediated apoptosis in response to cisplatin

I kappa B kinase (IKK) complex plays an important role in the regulation of signaling pathway that activates nuclear factor–kappa-B (NF-κB). Recently, we reported that cisplatin (CDDP) treatment causes a remarkable nuclear accumulation of IKK-α in association with stabilization and activation of p73. However, underlying mechanisms of CDDP-induced nuclear accumulation of IKK-α are elusive. Here, we found that ataxia–telangiectasia mutated (ATM) is one of upstream mediators of IKK-α during CDDP-induced apoptosis. In response to CDDP, ATM was phosphorylated at Ser-1981, which was accompanied with nuclear accumulation of IKK-α in HepG2 cells, whereas CDDP treatment had undetectable effects on IKK-α in ATM-deficient cells. Indirect immunofluorescence experiments demonstrated that phosphorylated form of ATM colocalizes with nuclear IKK-α in response to CDDP. In vitro kinase assay indicated that ATM phosphorylates IKK-α at Ser-473. Moreover, IKK-α-deficient MEFs displayed CDDP-resistant phenotype as compared with wild-type MEFs. Taken together, our present results suggest that ATM-mediated phosphorylation of nuclear IKK-α, which stabilizes p73, is one of the main apoptotic pathways in response to CDDP.

NFBD1/MDC1 associates with p53 and regulates its function at the crossroad between cell survival and death in response to DNA damage.

NFBD1/MDC1, which belongs to the BRCT superfamily, has an anti-apoptotic activity and contributes to the early cellular responses to DNA damage. Here we found that NFBD1 protects cells from apoptotic cell death by inhibiting phosphorylation of p53 at Ser-15 under steady state as well as early phase of DNA damage, thereby blocking its transcriptional and pro-apoptotic activities. During late phase of DNA damage, a remarkable reduction of NFBD1 was observed in dying but not in surviving A549 cells bearing wild-type p53. Small interference RNA-mediated knockdown of the endogenous NFBD1 resulted in an increase in sensitivity to adriamycin in A549 cells but not in p53-deficient H1299 cells. Immunoprecipitation and luciferase reporter analyses demonstrated that NFBD1 binds to the NH2-terminal region of p53 and strongly inhibits its transcriptional activity. Additionally, BRCT domains, which can interact with p53, reduced the adriamycin-induced phosphorylation levels of p53 at Ser-15 and also suppressed the transcriptional activity of p53. Thus, our present findings strongly suggest that NFBD1 plays an important role in the decision of cell survival and death after DNA damage through the regulation of p53.

Oxidative stress induces p53-dependent apoptosis in hepatoblastoma cell through its nuclear translocation

Hepatoblastoma (HBL) is the most common malignant liver tumor in children. Since tumor suppressor p53 is rarely mutated in HBL, it remains unknown whether p53 could contribute to the hepatocarcinogenesis. In the present study, we have found for the first time that, like neuroblastoma (NBL), wild‐type p53 was abnormally accumulated in the cytoplasm of the human HBL‐derived Huh6 cells. In accordance with this notion, immunohistochemical analysis demonstrated that p53 is largely expressed in cytoplasm of human primary HBLs. In response to the oxidative stress, Huh6 cells underwent apoptotic cell death in association with the nuclear translocation of p53 and the transactivation of its target gene implicated in apoptotic cell death. siRNA‐mediated knockdown of the endogenous p53 conferred the resistance of Huh6 cells to oxidative stress. Intriguingly, histone deacetylase inhibitor (nicotinamide) treatment strongly inhibited the oxidative stress‐induced nuclear translocation of p53 as well as the p53‐dependent apoptosis in Huh6 cells. In contrast to the previous observations, the cytoplasmic anchor protein for p53 termed Parc had undetectable effect on the cytoplasmic retention of p53. Collectively, our present results suggest that the abnormal cytoplasmic localization of p53 might contribute at least in part to the development of HBL.

Efficacy of therapy for advanced hepatocellular carcinoma: intra-arterial 5-fluorouracil and subcutaneous interferon with image-guided radiation.

Background and Aim:  To evaluate the efficacy of intra‐arterial 5‐fluorouracil (5‐FU) and subcutaneous interferon (IFN) combined with image‐guided radiation therapy (IGRT) in advanced hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT).

Identification of protein kinase A catalytic subunit β as a novel binding partner of p73 and regulation of p73 function

Post-translational modifications play a crucial role in regulation of the protein stability and pro-apoptotic function of p53 as well as its close relative p73. Using a yeast two-hybrid screening based on the Sos recruitment system, we identified protein kinase A catalytic subunit β (PKA-Cβ) as a novel binding partner of p73. Co-immunoprecipitation and glutathione S-transferase pull-down assays revealed that p73α associated with PKA-Cβ in mammalian cells and that their interaction was mediated by both the N- and C-terminal regions of p73α. In contrast, p53 failed to bind to PKA-Cβ. In vitro phosphorylation assay demonstrated that glutathione S-transferase-p73α-(1–130), which has one putative PKA phosphorylation site, was phosphorylated by PKA. Enforced expression of PKA-Cβ resulted in significant inhibition of the transactivation function and pro-apoptotic activity of p73α, whereas a kinase-deficient mutant of PKA-Cβ had no detectable effect. Consistent with this notion, treatment with H-89 (an ATP analog that functions as a PKA inhibitor) reversed the dibutyryl cAMP-mediated inhibition of p73α. Of particular interest, PKA-Cβ facilitated the intramolecular interaction of p73α, thereby masking the N-terminal transactivation domain with the C-terminal inhibitory domain. Thus, our findings indicate a PKA-Cβ-mediated inhibitory mechanism of p73 function.

NF-κB regulates the stability and activity of p73 by inducing its proteolytic degradation through a ubiquitin-dependent proteasome pathway

Nuclear factor kappa B (NF-κB), which exists as heterodimeric complexes composed of p50 and p65, has been shown to play an important role in cell survival processes. In the present study, we found for the first time that NF-κB has an ability to induce the ubiquitin-dependent proteasomal degradation of proapoptotic p73α. The activation of NF-κB in tumor necrosis factor α (TNF-α)-stimulated H1299 cells resulted in a significant reduction in the amounts of the endogenous p73α. Consistent with these results, TNF-α-mediated downregulation of p73α was observed in wild-type (WT) mouse embryonic fibroblasts (MEFs) but not in p65-deficient MEFs. Ectopic expression of NF-κB decreased a half-life of p73α by increasing its ubiquitination levels, and thereby inhibiting the transcriptional activity as well as proapoptotic function of p73α, whereas NF-κB had undetectable effects on p53. Immunoprecipitation experiments demonstrated that, under our experimental conditions, NF-κB does not bind to p73α in mammalian cultured cells. In contrast to WT p65, the COOH-terminal deletion mutant of p65 (p65ΔC) failed to reduce the expression levels of p73α, suggesting that NF-κB-mediated proteolytic degradation of p73α requires the transcriptional activity of NF-κB. Taken together, our present results imply that NF-κB-mediated degradation of proapoptotic p73 is a novel inhibitory mechanism of p73 that regulates cell survival and death.

Long-term follow-up of chronic hepatitis B after the emergence of mutations in the hepatitis B virus polymerase region

Summary.  Treatment of chronic hepatitis B has been greatly improved by the use of lamivudine, but mutations occur in the polymerase region of hepatitis B virus (HBV) and lamivudine‐resistant mutants frequently develop. The emergence of lamivudine‐resistant strains of HBV is a problem for treating chronic hepatitis B using lamivudine. We observed biochemical and virological changes in 15 patients with chronic hepatitis B for a median period of 29 months (range: 4–42 months) after the emergence of lamivudine‐resistant mutants of HBV. Patterns of mutation of the polymerase gene were examined by sequencing the LLAQ motif in domain B and the YMDD motif in domain C. Exacerbation of liver dysfunction occurred in 14 (93.3%) of the 15 patients at a median of 4 months after the emergence of mutations. However, exacerbation of liver dysfunction was observed only in four patients (26.7%) at the time of appearance of the first mutations and in 80.0% of the patients at the time of appearance of the second mutations. Increase in serum alanine aminotransferase (ALT) levels was significantly greater at the time of appearance of second mutations (P = 0.0096). In most cases, wild‐type HBV was mutated with the substitution of only rtM204I at first, and rtL180M/M204I mutations and then rtL180M/M204V mutations subsequently appeared. Further mutations of the polymerase region caused clinical deterioration. Thus as mutations emerge in the polymerase region, the clinical outcome deteriorates. Thus, monitoring the patterns of mutation of the polymerase gene is useful when using lamivudine for treating HBV.

Heat shock factor 1 accelerates hepatocellular carcinoma development by activating nuclear factor-κB/mitogen-activated protein kinase

Heat shock factor 1 (HSF1), a major transactivator of stress responses, has been implicated in carcinogenesis in various organs. However, little is known about the biological functions of HSF1 in the development of hepatocellular carcinoma (HCC). To clarify the functional role of HSF1 in HCC, we established HSF1-knockdown (HSF1 KD) KYN2 HCC cells by stably expressing either small hairpin RNA (shRNA) against HSF1 (i.e. HSF1 KD) or control shRNA (HSF1 control). Tumorigenicity was significantly reduced in orthotopic mice with HSF1 KD cells compared with those with HSF1 control cells. Reduced tumorigenesis in HSF1 KD cells appeared attributable to increased apoptosis and decreased proliferation. Tumor necrosis factor-α-induced apoptosis was increased in HSF1 KD cells and HSF1(-/-) mouse hepatocytes compared with controls. Decreased expression of IκB kinase γ, a positive regulator of nuclear factor-κB, was also observed in HSF1 KD cells and HSF1(-/-) mouse hepatocytes. Furthermore, expression of bcl-2-associated athanogene domain 3 (BAG3) was dramatically reduced in HSF1 KD cells and HSF1(-/-) mouse hepatocytes. We also found that epidermal growth factor-stimulated mitogen-activated protein kinase signaling was impaired in HSF1 KD cells. Clinicopathological analysis demonstrated frequent overexpression of HSF1 in human HCCs. Significant correlations between HSF1 and BAG3 protein levels and prognosis were also observed. In summary, these results identify a mechanistic link between HSF1 and liver tumorigenesis and may provide as a potential molecular target for the development of anti-HCC therapies.

Sensitive assay for quantification of hepatitis B virus mutants by use of a minor groove binder probe and peptide nucleic acids.

ABSTRACT Lamivudine is the first nucleoside analogue that was shown to have a potent effect on hepatitis B virus (HBV). However, the emergence of mutants resistant or cross-resistant to nucleoside/nucleotide analogues remains a serious problem. Several assays for the detection and quantification of antiviral-resistant mutants have been reported, but it has been difficult to measure the amounts of mutants accurately, especially when the target strain is a minor component of the mixed population. It has been shown that accurate measurement of a minor strain is difficult as long as a matching reaction with a single probe is included in the assay. We developed a new method for the quantification of lamivudine-resistant strains in a mixed-virus population by real-time PCR using minor groove binder probes and peptide nucleic acids, and we achieved a wide and measurable range, from 3 to 10 log10 copies/ml, and high sensitivity, with a discriminative limit of 0.01% of the predominant strain. The clinical significance of measuring substitutions not only of M204 but also of L180 residues of HBV polymerase was demonstrated by this method. This assay increases the versatility of a sensitive method for the quantification of a single-nucleotide mutation in a heterogeneous population.