In-Kyung Kim

Prostaglandin A2 and Δ12‐prostaglandin J2 induce apoptosis in L1210 cells

Treatment of L1210 cells with prostaglandin A2 (PGA2) or 9‐deoxy‐Δ9,12‐13,14‐dihydro PGD2 (Δ12‐PGJ2) resulted in significant G2/M arrest and subsequent DNA fragmentation at concentrations that are cytotoxic to the cells. On agarose gel electrophoresis, DNA ladder formation was evident 24 h after the addition of Δ12‐PGJ2 and remained apparent through 72 h, whereas G2/M accumulation was observed 6 h after the treatment. When the morphology of cells was examined by electron microscopy, L1210 cells incubated with a cytotoxic dose of PGA2 or Δ12‐PGJ2 for 24 h showed the characteristic morphological features of apoptosis such as chromatin condensation, nuclear fragmentation and formation of apoptotic body. Cycloheximide blocked the DNA fragmentation and morphological changes induced by Δ12‐PGJ2. Our results suggest that these cyclopentenone PGs caused apoptotic cell death of L1210 cells which is preceded by G2/M accumulation and requires de novo protein synthesis.

Protein kinase C modulates telomerase activity in human cervical cancer cells

Telomerase, a ribonucleoprotein reverse transcriptase that extends telomeres of eukaryotic chromosomes is repressed in normal somatic cells but is activated during development and neoplasia. The regulation mechanism of telomerase activity in cancer cells is not clearly known. In this report, a possible affect of PKC on telomerase activity was examined using HeLa and CUMC-6 cervical cancer cell lines. Exposure of cells to PKC inhibitor, bisindolylmaleimide I and Go6976, and high levels of PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in the inhibition of PKC activity in both cells. Telomerase activities were also inhibited by bisindolyl-maleimide I and Go6976, respectively, in a time-dependent manner. As PKC activity changes in TPA-treated cervical cancer cells, telomerase activities were increased at low dose of TPA and decreased at high dose. The expression levels of human telomerase subunits, human telomerase RNA (hTR) were not influenced by PKC modulating drugs. In contrast, the expression of full-length human telomerase reverse transcriptase (hTERT) was decreased after exposure to bisindolylmaleimide I and Go6976 in a time-dependent manner. hTERT expression was not affected by low dose of TPA. In contrast, high dose of TPA inhibited hTERT expression level. But the expression patterns of β-deletion transcript of hTERT after 72 h of treatment with PKC inhibitors or high dose of TPA exposure were not discernable as compared with those of full-length hTERT transcripts to PKC modulating drugs. These results suggest that PKC-modulating drugs altered telomerase activities by affecting full-length hTERT expression profile in human cervical cancers.

Intracellular glutathione level modulates the induction of apoptosis by Δ12-prostaglandin J2

We studied the effect of intracellular glutathione (GSH), which was known to conjugate readily with an alpha, beta-unsaturated carbonyl of 9-deoxy-delta 9,12-13,14-dihydroPGD2 (delta 12-PGJ2), on the cytotoxicity of delta 12-PGJ2. delta 12-PGJ2 caused DNA fragmentation in human hepatocellular carcinoma Hep 3B cells, which was blocked by cycloheximide (CHX). The delta 12-PGJ2-induced apoptosis was augmented by GSH depletion resulted from pretreatment with buthioninine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. On the contrary, N-acetyl-cysteine (NAC), a precursor of cysteine, elevated the GSH level and protected cells from initiating apoptosis by delta 12-PGJ2. Sodium arsenite, a thiol-reactive agent, also induced apoptosis, which was potentiated or attenuated by BSO or NAC treatment respectively. These results suggest that the apoptosis-inducing activity of delta 12-PGJ2 is due to thiol-reactivity and intracellular GSH modulates the delta 12-PGJ2-induced apoptosis by regulating the accessibility of delta 12-PGJ2 to target proteins containing thiol groups.

Sox-4 is a positive regulator of Hep3B and HepG2 cells' apoptosis induced by prostaglandin (PG)A2 and Δ12-PGJ2

We reported earlier that expression of Sox-4 was found to be elevated during prostaglandin (PG) A2 and Δ12-PGJ2 induced apoptosis in human hepatocarcinoma Hep3B cells. In this study, the role of Sox-4 was examined using human Hep3B and HepG2 cell lines. Sox-4 induction by several apoptotic inducer such as A23187 (Ca2+ ionophore) and etoposide (topoisomerase II inhibitor) and Sox-4 transfection into the cells were able to induce apoptosis as observed by the cellular DNA fragmentation. Antisense oligonucleotide of Sox-4 inhibited the induction of Sox-4 expression and blocked the formation of DNA fragmentation by PGA2 and Δ12-PGJ2 in Hep3B and HepG2 cells. Sox-4-induced apoptosis was accompanied with caspase-1 activation indicating that caspase cascade was involved in this apoptotic pathway. These results indicate that Sox-4 is involved in Hep3B and HepG2 cells apoptosis as an important apoptotic mediator.

Induction of unfolded protein response during neuronal induction of rat bone marrow stromal cells and mouse embryonic stem cells

When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2α phosphorylation. Transcription of two downstream targets of eIF2α, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation.

Activation of calcium signaling by hepatitis B virus-X protein in liver cells.

Hepatitis B virus x gene product (HBx) is known to be a transactivator of transcriptional elements that regulate the expression of a variety of genes associated with the growth, differentiation, survival and the apoptosis of cells. However, the exact mechanism of the activation and inhibition of cellular events by HBx remains uncertain. The present study was designed to measure the effect of HBx, on the signal transduction pathways associated with intracellular Ca(2+)mobilization following HBx transfection in the stable Chang liver cells (CHL-X). Enhanced cell proliferation by HBx in CHL-X was confirmed by MTT assay and by the immunodetection of PCNA. The transactivation of AP-1 by HBx induced in CHL-X was inhibited by cyclosporin A (CsA), a mitochondrial Ca(2+)channel blocker and by BAPTA-AM, a cytosolic Ca(2+)blocker. Activation of the SAPK/JNK signaling pathway by HBx was evidenced by the increased phosphorylations of c-Jun (Ser63) and of JNK (Thr183/Tyr185). Increased phospho-Erk/Erk and phospho-Raf1/Raf in HBx-induced CHL-X indicated that HBx might stimulate the MAPK pathway. PI3K activity and cytosolic free Ca(2+)levels were elevated in HBx-induced CHL-X. These results imply that HBx transactivates both JNK and MAPK signal transduction pathways in association with the mobilization of cytosolic Ca(2+).

Induction of p53 and apoptosis by δ 12-PGJ2 in human hepatocarcinoma SK-HEP-1 cells

Human hepatocarcinoma cells (SK‐HEP‐1) were induced to die through apoptosis by treatment with Δ 12‐prostaglandin (PG)J2, as characterized by the appearance of a typical DNA ladder. The induction of apoptosis by Δ 12‐PGJ2 was specifically blocked by cycloheximide (CHX). Western analysis using anti‐p53 or anti‐WAF1 monoclonal antibodies demonstrated that these two protein levels were increased 3 h after Δ 12‐PGJ2 treatment, and accumulated for up to 12 h. The induction of p53 protein seemed to be dependent on the increase of p53 mRNA level, which was inhibited by CHX treatment. However, delayed addition of CHX after Δ 12‐PGJ2 treatment failed to affect both p53 mRNA levels and DNA fragmentation following Δ 12‐PGJ2 treatment, indicating that the inhibition of p53 synthesis may contribute to the protective effect of CHX against Δ 12‐PGJ2‐mediated cytotoxicity. Therefore, our results suggest that the initial events caused by Δ 12‐PGJ2, leading ultimately to SK‐HEP‐1 cell death, involve a certain process required for p53 induction. However, the finding that Δ 12‐PGJ2 is also active against Hep 3B cells which are devoid of a functional p53 indicates that p53 may not be the critical requirement for inducing apoptosis by Δ 12‐PGJ2.

Activation of caspase-8 in 3-deazaadenosine-induced apoptosis of U-937 cells occurs downstream of caspase-3 and caspase-9 without Fas receptor-ligand interaction

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.

A novel function of haptoglobin : haptoglobin-haemoglobin complex induces apoptosis of hepatocarcinomatous Hep 3B cells

Haptoglobin-haemoglobin complex (Cx) showed a cytotoxic effect on the growth of Hep 3B (human hepatocellular carcinoma) cells, dose dependently. The antiproliferative effect of Cx on the multiplication of Hep 3B cells was augmented by the presence of prostaglandin (PG) D2. Antihuman Hb IgG abolished the effect of Cx, dose-dependently, which indicates that the antiproliferative effect of Cx really is exerted by Cx. Hep 3B cells treated with Cx showed the characteristic biochemical changes of apoptosis, such as DNA fragmentation which was blocked by pretreatment with cycloheximide, and the increase of transglutaminase expression. Thus, the antiproliferative effect of Cx against Hep 3B cells occurs via the typical apoptotic pathway.

Active adenosine transporter-mediated adenosine entrance into HL-60 cells leads to the induction of apoptosis through down-regulation of c-Myc

Although adenosine (Ado) is being recently recognized as a potent inducer of apoptosis, molecular mechanism of apoptosis by Ado remains to be elucidated. In this study we observed that c-Myc was rapidly down-regulated in the apoptosis in human promyelocytic leukemia HL-60 cells treated with Ado. To establish the molecular and biochemical mechanisms of apoptosis, we tested the specific effects of several antagonists of Ado receptors or inhibitors of Ado transporter on the induction of apoptosis. Treatment of dipyridamole (DPD), an Ado transport inhibitor, effectively suppressed both c-Myc reduction and DNA fragmentation, suggesting that the induction of apoptosis and down-regulation of c-Myc is mediated by active Ado transporter. It was another evidence supporting the entrance of Ado into cells undergoing apoptosis that Ado cytotoxicity was potentiated by a addition of methylation cycle intermediates. These results suggest that the active Ado transporter-mediated Ado entrance into HL-60 cells leads to the induction of apoptosis through down-regulation of c-Myc.