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Background & Aims: We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. The objective of the present study was to evaluate the effect of octadecenyl thiophosphate (OTP), a novel rationally designed, metabolically stabilized LPA mimic, on radiation-induced apoptosis of intestinal epithelial cells in vitro and in vivo. Methods: The receptors and signaling pathways activated by OTP were examined in IEC-6 and RH7777 cell lines and wild-type and LPA1 and LPA2 knockout mice exposed to different apoptotic stimuli. Results: OTP was more efficacious than LPA in reducing gamma irradiation–, camptothecin-, or tumor necrosis factor α/cycloheximide–induced apoptosis and caspase-3-8, and caspase-9 activity in the IEC-6 cell line. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA2 but not LPA1 and LPA3 transfectants. In C57BL/6 and LPA1 knockout mice exposed to 15 Gy gamma irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase-3–positive cells but was ineffective in LPA2 knockout mice. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but was without any effect in LPA2 knockout mice. Intraperitoneally administered OTP reduced death caused by lethal dose (LD)100 /30 radiation by 50%. Conclusions: Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA2 receptor subtype. [Copyright &y& Elsevier]

We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor κB (NF-κB) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90rsk signaling cascade in a p53- independent fashion. In turn, p90rsk interacts with the IκB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-κB activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-κB pathway that opposes the apoptotic response following DNA damage. [ABSTRACT FROM AUTHOR]

Upon engagement of the B Cell Receptor (BCR) of WEHI 231 immature B cells, a drop in c-Myc expression is followed by activation of the cyclin-dependent kinase inhibitor (CKI) p27Kip1, which induces growth arrest and apoptosis. Here, we report inverse patterns of p27 and c-Myc protein expression follow BCR engagement. We present evidence demonstrating, for the first time, that the p27Kip1 gene is a target of transcriptional repression by c-Myc. Specifically, the changes in p27 protein levels correlated with changes in p27 mRNA levels, and gene transcription. Induction of p27 promoter activity followed BCR engagement of WEHI 231 cells, and this induction could be repressed upon co-transfection of a c-Myc expression vector. Inhibition of the TATA-less p27 promoter by c-Myc was also observed in Jurkat T cells, vascular smooth muscle, and Hs578T breast cancer cells, extending the observation beyond immune cells. Consistent with a putative Inr element CCAGACC (where +1 is underlined) at the start site of transcription in the p27 promoter, deletion of Myc homology box II reduced the extent of repression. Furthermore, enhanced repression was observed upon transfection of the c-Myc ‘super-repressor’, with mutation of Phe115 to Leu. The sequences mediating transcriptional activity and c-Myc repression were mapped to bp -20 to +20 of the p27 gene. Finally, binding of Max was shown to facilitate c-Myc binding and repression of p27 promoter activity. Overall, these studies identify the p27 CKI gene as a new target whereby c-Myc can control cell proliferation, survival and neoplastic transformation. Oncogene (2001) 20, 1688–1702. [ABSTRACT FROM AUTHOR]

Abstract: Nuclear-Factor-κB (NF-κΒ can counteract transforming growth factor-β1 (TGF-β1)-induced apoptosis in malignant hepatocytes through up-regulation of its downstream genes, such as X-linked inhibitor of apoptosis protein (XIAP). Reports have demonstrated that TGF-β1 can induce oxidative stress, and c-Jun N-terminal Kinase1 (JNK1) is indispensable for TGF-β1-induced apoptosis pathway, but the relationship between radical oxygen species (ROS) and the activation of JNKs is still unclear. In the present study, we found that ROS can induce JNK activation in TGF-β1 mediated apoptosis in hepatocytes. The inhibitors of hydrogen peroxide and superoxide, which were produced by mitochondria under stress, could inhibit the phosphorylation of c-Jun in XIAP knockdown cells. In conclusion, it is the first time to show that both NF-κB and antioxidants can counteract TGF-β1-induced apoptosis in hepatic cell death through JNK1 pathway. [Copyright &y& Elsevier]

Active NF-κB renders malignant hepatocytes refractory to the growth inhibitory and pro-apoptotic properties of transforming growth factor β1 (TGF-β1). NF-κB counteracts TGF-β1-induced apoptosis through up-regulation of downstream target genes, such as XIAP and Bcl-XL, which in turn inhibit the intrinsic pathway of apoptosis. In addition, induction of NF-κB by TGF-β1 inhibits JNK signaling, thereby attenuating TGF-β1-induced cell death of normal hepatocytes. However, the mechanism involved in the negative cross-talk between the NF-κB and JNK pathways during TGF-β1 signaling has not been determined. In this study, we have identified the XIAP gene as one of the critical mediators of NF-κB-mediated suppression of JNK signaling. We show that NF-κβ plays a role in the up-regulation of XIAP gene expression in response to TGF-β1 treatment and forms a TGF-β1-inducible complex with TAK1. Furthermore, we show that the RING domain of XIAP mediates TAK1 polyubiquitination, which then targets this molecule for proteosomal degradation. Down-regulation of TAK 1 protein expression inhibits TGF-β1 -mediated activation of JNK and apoptosis. Conversely, silencing of XIAP promotes persistent JNK activation and potentiates TGF-β1-induced apoptosis. Collectively, our findings identify a novel mechanism for the regulation of JNK activity by NF-κB during TGF-β1 signaling and raise the possibility that pharmacologic inhibition of the NF-κB/XIAP signaling pathway might selectively abolish the pro-oncogenic activity of TGF-β1 in advanced hepatocellular carcinomas (HCCs) without affecting the proapoptotic effects of TGF-β1 involved in normal liver homeostasis. [ABSTRACT FROM AUTHOR]

Nuclear factor-kappaB (NF-kappaB) plays an important role during liver neoplastic development through transcriptional regulation of prosurvival genes, which then counteract the death-inducing signals elicited by the host immune response. The c-Myc proto-oncogene is frequently deregulated in liver tumors. Furthermore, enforced expression of c-Myc in the liver promotes the development of hepatocellular carcinomas, a process that is accelerated by coexpression with transforming growth factor-alpha (TGF-alpha). TGF-alpha/c-Myc-derived hepatocellular carcinomas display reduced apoptotic levels compared with those of single c-Myc transgenic hepatocellular carcinomas, suggesting that TGF-alpha provides a survival advantage to c-Myc-transformed hepatocytes. Given that TGF-alpha/c-Myc hepatocellular carcinomas display constitutive NF-kappaB activity, here, we have tested the hypothesis that enforced expression of TGF-alpha results in constitutive NF-kappaB activation and enhanced cell survival using TGF-alpha/c-Myc-derived hepatocellular carcinoma cell lines. We show that TGF-alpha induces NF-kappaB through the phosphatidylinositol 3-kinase/Akt axis in these bitransgenic hepatocellular carcinomas. Furthermore, we found that adenovirus-mediated inhibition of NF-kappaB activity impairs the ability of TGF-alpha/c-Myc-derived tumor cells to grow in an anchorage-independent fashion due to sensitization to c-Myc-induced apoptosis. Lastly, we show that NF-kappaB inhibits c-Myc-induced activation of caspase-9 and caspase-3 through up-regulation of the antiapoptotic target genes Bcl-X(L) and X-linked inhibitor of apoptosis (XIAP). Overall, these results underscore a crucial role of NF-kappaB in disabling apoptotic pathways initiated by oncogenic transformation.

Nuclear factor-kappaB (NF-kappaB) plays a critical role during fetal liver development and hepatic oncogenesis. Here, we have assessed whether NF-kappaB activity is required for murine hepatocellular carcinoma cell survival. We show that adenoviral-mediated inhibition of inhibitor of NF-kappaB kinase-beta (IKK-2) activity in hepatocellular carcinomas derived from transforming growth factor (TGF)-alpha/c-myc bitransgenic mice leads to inhibition of NF-kappaB and promotes tumor necrosis factor (TNF)-alpha-mediated cell death of malignant hepatocytes but not the surrounding peritumorous tissue. Induction of apoptosis is accompanied by inhibition of Bcl-X(L) and XIAP, two pro-survival NF-kappaB target genes. In addition, we have identified the alpha-fetoprotein (AFP) as a novel downstream target of NF-kappaB. We show that repression of IKK-2 activity in hepatocellular carcinomas promotes down-regulation of AFP gene expression. Likewise, genetic disruption of the RelA subunit results in reduced AFP gene expression during embryonic liver development, at a time in which fetal hepatocytes are sensitized to TNF-alpha-mediated cell killing. In this regard, we show that AFP inhibits TNF-alpha-induced cell death of murine hepatocellular carcinomas through association with TNF-alpha and inhibition of TNFRI signaling. Thus, NF-kappaB-mediated regulation of AFP gene expression during liver tumor formation and embryonic development of the liver constitutes a potential novel mechanism used by malignant and fetal hepatocytes to evade immune surveillance.

Nuclear factor kappaB (NF-kappaB) has been implicated in inducible chemoresistance against anthracyclines. In an effort to improve the cytotoxicity of anthracyclines while reducing their cardiotoxic effects, we have developed a novel class of extranuclear-localizing 14-O-acylanthracyclines that bind to the phorbol ester/diacylglycerol-binding C1b domain of conventional and novel protein kinase C (PKC) isoforms, thereby promoting an apoptotic response. Because PKCs have been shown to be involved in NF-kappaB activation, in this report, we determined the mechanism of NF-kappaB activation by N-benzyladriamycin-14-valerate (AD 198) and N-benzyladriamycin-14-pivalate (AD 445), two novel 14-O-acylanthracylines. We show that the induction of NF-kappaB activity in response to drug treatment relies on the activation of PKC-delta and NF-kappaB-activating kinase (NAK), independent of ataxia telengectasia mutated and p53 activities. In turn, NAK activates the IKK complex through phosphorylation of the IKK-2 subunit. We find that neither NF-kappaB activation nor ectopic expression of Bcl-X(L) confers protection from AD 198-induced cell killing. Overall, our data indicate that activation of novel PKC isoforms by cytoplasmic-targeted 14-O-acylanthracyclines promotes an apoptotic response independent of DNA damage, which is unimpeded by inducible activation of NF-kappaB.

We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor kappaB (NF-kappaB) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90(rsk) signaling cascade in a p53-independent fashion. In turn, p90(rsk) interacts with the IkappaB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-kappaB activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-kappaB pathway that opposes the apoptotic response following DNA damage.

Nuclear factor kappaB (NF-kappaB) is an antiapoptotic factor involved in development, regeneration, and neoplastic progression of the liver. Previously, we have shown that stabilization of inhibitor kappaB (IkappaB)-alpha protein following treatment of hepatocytes with transforming growth factor (TGF)-beta1 promoted NF-kappaB repression, which then permitted induction of AP-1/SMAD-mediated liver cell death. Because basal IkappaB-alpha protein turnover is regulated by protein kinase CK2, here we have elucidated the regulation of CK2 kinase activity and its role in control of NF-kappaB levels following treatment with TGF-beta1. We show that both messenger RNA (mRNA) and protein levels of the CK2alpha catalytic subunit are down-regulated following TGF-beta1 stimulation in murine hepatocyte cells. The ensuing inhibition of CK2 kinase activity promotes stabilization of IkappaB protein, which is followed by the shutoff of constitutive NF-kappaB activity and induction of apoptosis. Ectopic expression of CK2alpha inhibits TGF-beta1-induced apoptosis through sustained activation of NF-kappaB. Conversely, expression of a kinase-dead mutant of CK2alpha potentiates TGF-beta1 cell killing. Importantly, we show that hepatocellular carcinomas (HCCs) derived from TGF-beta1 transgenic mice and human HCC cell lines display enhanced CK2 IkappaB kinase activity that contributes in part to an elevated NF-kappaB activity in vivo. In conclusion, inhibition of CK2 expression levels by TGF-beta1 is crucial for the induction of apoptosis of hepatocytes. Circumvention of this process by up-regulation of CK2 activity in transformed cells may contribute to the promotion of TGF-beta1-induced liver carcinogenesis.