SummaryThe molecular and cellular mechanisms responsible for cytotoxic T lymphocyte (CTL)-induced immunopathology are not well defined. Using a model in which hepatitis B surface antigen (HBsAg)-specific CTL cause an acute necroinflammatory liver disease in HBsAg transgenic mice, we demonstrate that class I-restricted disease pathogenesis is an orderly, multistep process that involves direct as well as indirect consequences of CTL activation. It begins (step 1) almost immediately as a direct antigen-specific CTL-target cell interaction that triggers the HBsAgpositive hepatocyte to undergo programmed cell death (apoptosis). It progresses (step 2) within hours to a focal inflammatory response in which antigen-nonspecific lymphocytes and neutrophils amplify the local cytopathic effect of the CTL. The most destructive pathogenetic function of the CTL, however, is to secrete interferon 3' when they encounter antigen in vivo, thereby activating the intrahepatic macrophage and inducing a delayed-type hypersensitivity response (step 3) that destroys the liver and kills the mouse. We propose that the principles illustrated in this study are generally applicable to other models of class I-restricted, CTL-induced immunopathology, and we suggest that they contribute to the immunopathogenesis of viral hepatitis during hepatitis B virus infection in humans.

During hepatitis B virus (HBV) infection, distinct host-virus interactions may establish the patterns of viral clearance and persistence and the extent of virusassoiated pathology. It is generally thought that HBV-speciflc class I-restricted cytotoxic T lymphocytes (CTLs) play a critical role in this process by destroying infected hepatocytes. This cytopathic m nism, however, could be lethal if most of the hepatocytes are Infected. In the current study, we demonstrate that class I-retricted HBV-speclfic CTLs profoundly suppress hepatoceliular HBV gene expression in HBV transgenic mice by a noncytolytic process, the strength ofwhich greatly exceeds the cytopathic effect of the CTLs in magnitude and duration. We also show that the regulatory effect of the CTLs is initially mediated by interferon y and tumor necrosis factor a, is delayed in onset, and becomes independent of these cytokines shortly after it begins. The data indicate that the anti-viral CTL response activates a complex regulatory cascade that inhibits hepatoceflular HBV gene expression without kifling the cell.The extent to which this mechanism contributes to viral clearance or viral persistence during HBV infection rem to be determined.The control of hepatitis B virus (HBV) infection is thought to be mediated by the class I-restricted cytotoxic T-lymphocyte (CTL) response. Patients with acute viral hepatitis, who successfully clear the virus, mount a multispecific polyclonal CTL response to several HBV-encoded antigens, whereas persistently infected patients with chronic hepatitis do not (1). Hepatitis B surface antigen (HBsAg)-specific murine CTL clones cause a necroinflammatory liver disease when they are injected into HBsAg-positive transgenic mice, and the cytopathic effect of the CTLs is largely mediated by the inflammatory cytokines that they release when they are activated by antigen recognition (2, 3). While the data indicate that the CTL response to HBV plays a critical role in viral clearance and disease pathogenesis, the extent to which viral clearance depends on the destruction of infected cells is not known at this time.We have shown that hepatocellular HBV gene expression in transgenic mice is negatively regulated, noncytopathically, by the pharmacological administration of recombinant interleukin (IL) 2 and tumor necrosis factor (TNF) a and that the IL-2 effect is mediated by TNF-a, which inhibits HBV gene expression by a posttranscriptional mechanism in these animals (4-6). We now report that hepatocellular HBV gene expression is profoundly inhibited, noncytopathically, by HBsAg-specific class I-restricted CTLs activated physiologically by antigen recognition in vivo, and we show that the inhibitory effect of the CTLs, which is mediated by TNF-a and interferon y (IFN-y), greatly exceeds their cytopathic effect in magnitude and duration. MATERIALS AND METHODSHBV Transgenic Mice. The two lineages studied were selected because they do not develop evidence of spontaneous liver disease and they display lineage-specific differences...

Polo-like kinase 1 (Plk1) has an important role in the regulation of M phase of the cell cycle. In addition to its cell cycle-regulatory function, Plk1 has a potential role in tumorigenesis. Here we found for the first time that Plk1 physically binds to the tumor suppressor p53 in mammalian cultured cells, and inhibits its transactivation activity as well as its pro-apoptotic function. During the cisplatin-induced apoptosis in human neuroblastoma SH-SY5Y cells, the expression level of Plk1 was significantly decreased both at mRNA and protein levels, whereas cisplatin treatment caused a remarkable stabilization of p53. Systematic immunoprecipitation analyses using a series of deletion mutants of p53 revealed that a sequence-specific DNA-binding region of p53 is required and sufficient for the physical interaction with Plk1. The ectopically overexpressed Plk1 was co-localized with the endogenous p53 in mammalian cell nucleus, as shown by confocal laser microscopy. Expression of exogenous Plk1 and p53 in p53-deficient lung carcinoma H1299 cells greatly decreased the p53-mediated transcription from the p53-responsive p21 WAF1 , MDM2, and BAX promoters, whereas the kinase-deficient mutant form of Plk1 failed to reduce the transcriptional activity of p53. Consistent with the luciferase reporter analysis, Plk1 had an ability to block the p53-dependent induction of the endogenous p21 WAF1 . In addition, Plk1 inhibited the pro-apoptotic function of p53 in H1299 cells. Intriguingly, Plk1-mediated repression of p53 was attenuated with ATM. Thus, our present findings strongly suggest that p53 is a critical target of Plk1, and its function is abrogated through the physical interaction with Plk1.

Tumor suppressor p53-dependent stress response pathways play an important role in cell fate determination. In this study, we have found that glucose depletion promotes the phosphorylation of AMP-activated protein kinase catalytic subunit ␣ (AMPK␣) in association with a significant up-regulation of p53, thereby inducing p53-dependent apoptosis in vivo and in vitro. Thymocytes prepared from glucose-depleted wild-type mice but not from p53-deficient mice underwent apoptosis, which was accompanied by a remarkable phosphorylation of AMPK␣ and a significant induction of p53 as well as pro-apoptotic Bax. Similar results were also obtained in human osteosarcoma-derived U2OS cells bearing wild-type p53 following glucose starvation. Of note, glucose deprivation led to a significant accumulation of p53 phosphorylated at Ser-46, but not at Ser-15 and Ser-20, and a transcriptional induction of p53 as well as proapoptotic p53 AIP1. Small interference RNA-mediated knockdown of p53 caused an inhibition of apoptosis following glucose depletion. Additionally, apoptosis triggered by glucose deprivation was markedly impaired by small interference RNA-mediated depletion of AMPK␣. Under our experimental conditions, down-regulation of AMPK␣ caused an attenuation of p53 accumulation and its phosphorylation at Ser-46. In support of these observations, enforced expression of AMPK␣ led to apoptosis and resulted in an induction of p53 at protein and mRNA levels. Furthermore, p53 promoter region responded to AMPK␣ and glucose deprivation as judged by luciferase reporter assay. Taken together, our present findings suggest that AMPK-dependent transcriptional induction and phosphorylation of p53 at Ser-46 play a crucial role in the induction of apoptosis under carbon source depletion. AMP-activated protein kinase (AMPK)3 was originally identified as an enzyme that has an ability to inhibit hydroxymethylglutaryl-CoA reductase (1) and also regulate acetyl-CoA carboxylase by reversible phosphorylation (2). Subsequent studies demonstrated that AMPK is widely expressed and exists as a heterotrimeric complex, which consists of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥). The mammalian genome contains seven AMPK genes encoding two ␣ (␣1 and ␣2), two ␤ (␤1 and ␤2), and three ␥ (␥1, ␥2, and ␥3) isoforms (3-5). The catalytic ␣ subunit is composed of three functional domains, including an NH 2 -terminal Ser/Thr protein kinase domain, a central auto-inhibitory region, and a COOH-terminal regulatory subunit-binding domain. AMPK acts as an intracellular energy sensor by monitoring cellular energy levels. For example, AMPK becomes activated by the tumor suppressor LKB1 complex-mediated phosphorylation at Thr-172 in response to certain energy-depleting stresses such as glucose deprivation, hypoxia, and oxidative stress, which increase the intracellular AMP:ATP ratio (6 -10). AMPK can also be activated allosterically in the AMP:ATP ratio (11). Upon activation, AMPK down-regulates the ATP consuming metabolic pathways and activates the energy-g...