Tae Jun Park

Constitutive Induction of p-Erk1/2 Accompanied by Reduced Activities of Protein Phosphatases 1 and 2A and MKP3 Due to Reactive Oxygen Species during Cellular Senescence

The mechanism of senescence-associated cytoplasmic induction of p-Erk1/2 (SA-p-Erk1/2) proteins in human diploid fibroblasts was investigated. p-Erk1/2 proteins were efficiently dephosphorylated in vitro by protein phosphatases 1 and 2A (PP1/2A) and MAPK phosphatase 3 (MKP3). Specific activity of PP1/2A and MKP3 activity significantly decreased during cellular senescence, whereas their protein expression levels did not. To investigate possible mechanism of phosphatase inactivation, we measured reactive oxygen species (ROS) generation by fluorescence-activated cell sorting analysis and found it was much higher in mid-old cells than the young cells. Treating the young cells once with 1 mm H2O2 remarkably induced p-Erk1/2 expression; however, it was transient unless repeatedly treated until 72 h. Multiple treatment of the cells with 0.2 mm H2O2 significantly duplicated inactivation of PP1/2A; however, thiol-specific reagents could reverse the PP1/2A activities, suggesting the oxidation of cysteine molecule in PP1/2A by the increased ROS. When the cells were pretreated with 10 mm N-acetyl-l-cysteine for 1 h, Erk1/2 activation was completely blocked. To elucidate which cysteine residue and/or metal ion in PP1/2A was modified by H2O2, electrospray ionization-tandem mass spectrometry analyses were performed with purified PP1C-α and found Cys62-SO3H and Cys105-SO3H, implicating the tertiary structure perturbation. H2O2 inhibited purified PP1C-α activity by both oxidation of Cys residues and metal ion(s), evidenced by dithiothreitol and ascorbate-restoration assay. In summary, SA-p-Erk1/2 was most likely due to the oxidation of PP1/2A, which resulted from the continuous exposure of the cells to vast amounts of ROS generated during cellular senescence by oxidation of Cys62 and Cys105 in PP1C-α and metal ion(s).

Induction of growth inhibition of 293 cells by downregulation of the cyclin E and cyclin-dependent kinase 4 proteins due to overexpression of TIS21.

We earlier reported that TIS21 mRNA expression was markedly decreased in A549 and NCIH69 human lung cancer cells and in thymic carcinoma tissues obtained from transgenic mice containing simian virus 40 large T antigen (J Cancer Res Clin Oncol 121:279–284, 1995). To determine how TIS21 inhibits growth, we made 293 cells that constitutively expressed TIS21 protein. The constitutive TIS21 expresser lines C9 and C11 grew to a lower saturation density than did those in the vector‐transfected clones (V7 and V10) and antisense‐transfected clones (AS1 and AS4), and the size of the C9 and C11 cells increased significantly after transfection with TIS21 cDNA. The serum‐stimulated cell cycle was analyzed by fluorescence‐activated cell sorting after double thymidine treatment; V10 progressed normally through the cell division cycle, but C9 and C11 cells accumulated continuously in G1 phase until 36 h after treatment. On the other hand, the progression of cells that had already entered to S or G2/M phase was not inhibited. When cell‐cycle regulatory proteins were measured, C9 and C11 cells showed significantly reduced synthesis of cyclin E and cyclin‐dependent kinase (cdk) 4 as well as a decrease in cyclin E–associated cdk activity. These observations led us to conclude that TIS21 overexpression in G1 phase decreased the amounts of cyclin E and cdk4, thereby decreasing the activity of cdks at the G1‐S transition. Mol. Carcinog. 23:25–35, 1998.

BTG2 suppresses cancer cell migration through inhibition of Src-FAK signaling by downregulation of reactive oxygen species generation in mitochondria

BTG2 is a tumor suppressor gene. It is frequently downregulated in human cancer tissues, and its loss is associated with cancer cell metastasis, suggesting that the suppression of BTG2 plays a critical role in cancer cell migration and invasion. Here, we report that re-expression of BTG2 decreased cell migration and invasion in A549 and PC3 cancer cells. Furthermore, BTG2 expression was correlated with downregulation of focal adhesion kinase (FAK) Tyr576 and Tyr925 residues phosphorylation, while Tyr397 which is the autophosphorylation site was not influenced by BTG2 expression. c-Src phosphorylation which is the upstream of FAK was not influenced, whereas c-Src kinase activity was significantly decreased by BTG2 expression. BTG2 overexpression increased Src reduction state and inhibited reactive oxygen species (ROS) generation by being localized in mitochondria. Mitochondria-target BTG2 also inhibited cell migration via downregulation of Src-FAK signaling. In conclusion, our study reveals that BTG2 negatively regulated cancer cell migration by inhibiting Src activity through downregulation of ROS generation in mitochondria.

Skp2 enhances polyubiquitination and degradation of TIS21/BTG2/PC3, tumor suppressor protein, at the downstream of FoxM1.

TIS21(/BTG2/PC3) has been shown to work as a pan-cell cycle inhibitor and a negative regulator of cyclin B1/cdk1 and forkhead box M1 (FoxM1). Moreover, loss of TIS21 expression has been suggested as an early event in carcinogenesis of thymus, prostate, kidney, and liver. However, there is no report yet what regulates the in vivo stability of TIS21 protein. Here, TIS21 was found to be a target of ubiquitin ligase, S phase kinase associated protein 2 (Skp2), the expression of which was regulated by FoxM1. Leucine rich repeat (LRR) domain of Skp2 could bind to TIS21 C-terminus and facilitated TIS21 degradation via ubiquitin-proteasome pathway. Skp2 without LRR and C-terminus deleted TIS21 (TIS21DeltaC) failed to interact with each other, and failure of their interaction prolonged half-life of TIS21 protein. Furthermore, in vivo function of TIS21, inhibition of cell growth, was regulated by expressions of Skp2 and FoxM1; It was significantly enhanced by knock down of Skp2 expression in the TIS21 adenovirus infected cells, whereas it was significantly ameliorated by co-expression of FoxM1 with TIS21. These data indicate that TIS21 is a novel target of SCF-Skp2 ubiquitin ligase, which is regulated by expression of FoxM1.

Gankyrin is frequently overexpressed in breast cancer and is associated with ErbB2 expression.

Gankyrin is a subunit of the 26S proteasome, and has been known to degrade p53 and retinoblastoma protein and promote the tumorigenicity and metastasis in some malignancies. However, the role of gankyrin in breast cancer has not been explored. In this study, we investigated the expression of gankyrin in breast cancer and evaluated its effect on breast cancer. Representative cancer tissues including normal breasts from 60 patients with breast cancer were stained immunohistochemically for gankyrin, estrogen receptor, progesterone receptor, and ErbB2. We evaluated the relationship between gankyrin expression and clinicopathologic parameters or prognostic markers. We also attempted to clarify the mechanism of gankyrin involved in breast carcinogenesis by using MCF7 breast cancer cells. Gankyrin was weakly expressed in normal breast epithelial cells, however, tumor regions of 37/60 (61.7%) cases showed an overexpression of gankyrin. Gankyrin overexpression was associated with extensive intraductal carcinoma (p=0.014) and ErbB2 positivity (p=0.031) in invasive ductal carcinoma. In MCF7 breast cancer cells, downregulation of gankyrin was associated with a reduction of cell proliferation and tumorigenicity. In conclusion, gankyrin was identified in normal breasts and overexpressed in invasive breast cancers. The overexpression of gankyrin was associated with extensive intraductal carcinoma and ErbB2 expression in breast cancer.

TIS21(/BTG2/PC3) accelerates the repair of DNA double strand breaks by enhancing Mre11 methylation and blocking damage signal transfer to the Chk2(T68)-p53(S20) pathway.

DNA double strand breaks (DSBs) occur more frequently in TIS21(-/-) mouse embryo fibroblasts than that in wild type MEFs (wt-MEFs). Therefore, the role TIS21 plays in the DNA damage response was investigated. Adenoviral transduction of Huh7 tumor cells with the TIS21 gene accelerated the repair of DSBs induced by etoposide treatment as evaluated by clearance of γH2AX foci and the Comet assay. TIS21 increased methylation of Mre11 and protein arginine methyltransferase 1 (PRMT1) activity, leading to Mre11 activation in vitro and in vivo, as determined by immunoprecipitation and radiolabeling analyses. When downstream DNA damage response mediators were evaluated in various human cancer cells lines, TIS21 was found to strongly inhibit Chk2(T68) and p53(S20) phosphorylation by p-ATM(S1981) but not p53(S15). The loss of Chk2 activation after etoposide treatment reduced apoptosis in the cells by downregulating the expression of E2F1 and Bax. These data suggest that TIS21 regulates DSB repair and apoptosis. Expression of TIS21 promoted the repair of DSBs and reduced apoptosis by blocking the damage signal from p-ATM(S1981) to Chk2(T68)-p53(S20)via the activation of Mre11 and PRMT1.

Regulation of selection of liver nodules initiated with N-nitrosodiethylamine and promoted with nodularin injections in Fischer 344 male rats by reciprocal expression of transforming growth factor-β1 and its receptors

To investigate how glutathione‐S‐transferase placental form (GST‐P)+ hyperplastic nodules (HNs) are selected and to determine the driving force for progression or regression of HNs, changes in transforming growth factor‐β1 (TGF‐β) and its receptors were examined during hepatocarcinogenesis initiated by N‐nitrosodiethylamine (DEN) and promoted by nodularin. The induction of TGF‐β1 expression in the GST‐P+ HNs was dependent on nodularin injections for 10 wk, which started the third week after DEN initiation. The kinetics of TGF‐β1 induction during carcinogenesis were quite different from that of simple regeneration after partial hepatectomy (PH): hepatocytes initiated with DEN alone induced TGF‐β1 expression for 24 d, and subsequent stimulation by PH on the fourteenth day after DEN initiation super‐induced TGF‐β1 mRNA (50 times that of the control level), as opposed to a transient expression for less than 5 d by PH alone. GST‐P+ HNs did not express TGF‐β receptors I (RI) and II (RII) during the early stage of carcinogenesis, whereas the surrounding hepatocytes strongly expressed both of these receptors. On cessation of nodularin injection, however, the expression of RI and RII in the HNs changed significantly: RII+ nodules appeared, and the number and area of RII+/− nodules were significantly increased at 10 wk after the cessation. These findings indicate that induction of TGF‐β expression in GST‐P+ HNs might be a strong selection pressure that allows outgrowth of RII− nodules during liver carcinogenesis. Mol. Carcinog. 26:83–92, 1999.

Pleiotrophin inhibits transforming growth factor β1-induced apoptosis in hepatoma cell lines

Pleiotrophin (PTN) is a hepatocyte growth factor and considered to play roles in liver fibrogenesis and hepatocarcinogenesis. In this study we examined the mechanism of the action of PTN in these pathological processes. First, we confirmed that hepatic stellate cells (HSCs) and Kupffer cells, and also later hepatocytes in hyperplastic nodules increased PTN mRNA expressions during carbon tetrachloride‐induced liver fibrosis. Then, the relationship between PTN and transforming growth factor β1 (TGFβ1), a known potent pro‐fibrogenetic cytokine, in carcinogenesis was investigated using hepatoma cell lines. Huh‐7 human hepatoma cells weakly expressed PTN, but HepG2 human hepatoma cells and FaO rat hepatoma cells did not. Recombinant (r) TGFβ1 induced the cultured Huh‐7 cells to undergo apoptosis, which was inhibited by rPTN. Huh‐7 cells became resistant to TGFβ1‐, but not mitomycin C‐induced apoptosis when transfected with PTN gene, indicating the specificity of the PTN anti‐apoptotic activity. Poly ADP ribose polymerase, procaspase‐8 and procaspase‐3 were not cleaved in the TGFβ1‐reluctant cells. The TGFβ1‐induced caspase‐3 activation was also suppressed in Huh‐7 and FaO cells both transduced with PTN gene‐bearing adenoviruses. In summary, PTN was expressed in HSCs, Kupffer cells, and hepatocytes in fibrotic liver. We propose that PTN specifically antagonizes the TGFβ1 activity during liver fibrosis.

TSH signaling overcomes B-RafV600E-induced senescence in papillary thyroid carcinogenesis through regulation of DUSP6.

B-RafV600E oncogene mutation occurs most commonly in papillary thyroid carcinoma (PTC) and is associated with tumor initiation. However, a genetic modification by B-RafV600E in thyrocytes results in oncogene-induced senescence (OIS). In the present study, we explored the factors involved in the senescence overcome program in PTC. First of all, we observed down-regulation of p-extracellular signal-regulated kinases 1/2 and up-regulation of dual specific phosphatase 6 (DUSP6) in the PTC with B-RafV600E mutation. DUSP6 overexpression in vitro induced extracellular signal-regulated kinases 1/2 dephosphorylation and inhibited B-RafV600E–induced senescence in thyrocytes. Although DUSP6 protein was degraded by B-RafV600E–induced reactive oxygen species (ROS), thyroid-stimulating hormone (TSH) stabilized DUSP6 protein by increasing Mn superoxide dismutase expression and inhibited B-RafV600E–induced senescence. Although serum TSH was not increased, its receptor was markedly upregulated in PTC with B-RafV600E. Furthermore, TSH together with DUSP6 reactivated Ras signaling, resulted in activation of Ras/AKT/glycogen synthase kinase 3β, and stabilized c-Myc protein by inhibiting its degradation. These observations led us to conclude that increased TSH signaling overcomes OIS and is essential for B-RafV600E–induced papillary thyroid carcinogenesis.

Differential expression of O6-methylguanine-DNA methyltransferase during diethylnitrosamine-induced carcinogenesis and liver regeneration in Sprague-Dawley male rats.

Abstract Differential expression of DNA-O6MeG: protein-l-cysteine S-methyltransferase (MGMT) acti- vity and posttranslational modification of the pro- tein during liver regeneration and carcinogenesis were compared in Sprague-Dawley male rats after partial hepatectomy and/or single i.p injection of diethylnitrosamine (DEN, 200 mg/kg). Regenerating hepatocytes after partial hepatectomy induced MGMT transiently within 3 days; however, the induction of MGMT was persistent for 2 weeks after DEN injection, and the combined treatment of DEN and partial hepatectomy maintained the elevated MGMT level for up to 4 weeks. The increased activity was transcriptionally regulated, when analyzed by Northern blot hybridization. The major active form of MGMT protein in the partially hepatectomized or DEN-treated rats was a 26-kDa or 24-kDa species respectively, which was confirmed by Western blot analysis and gel slice assay. The biological significance of the differential induction of MGMT during partial hepatectomy or DEN-induced carcinogenesis is not obvious; however, further studies on possible posttranslational modifications of MGMT protein might shed some light on the functional aspect of MGMT induction.