Yosuke Ohtake

Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.

Critical role of farnesoid X receptor for hepatocellular carcinoma cell proliferation

Farnesoid X receptor (FXR), a pivotal factor maintaining bile acid homeostasis, has been recently shown to be a critical factor required for liver regeneration. The elucidation of the mechanism how FXR controls the proliferation of hepatocellular carcinoma cells is useful to establish the therapy for liver cancer. Here, we show that FXR plays a crucial role in the proliferation of human hepatocellular carcinoma cell line, HepG2, Huh7 and HLE. The treatment of HepG2 with FXR siRNA elevates the level of p16/INK4a expression resulting in the inhibition of cell proliferation. By contrast, FXR activation reduces p16/INK4a expression and stimulates the cell proliferation. The ectopic expression of the active form of Ras that causes strong activation of extracellular signal-regulated kinase (ERK) leads to the decrease in FXR expression, suggesting that FXR expression is negatively regulated via Ras/ERK pathway. The elevation of p16/INK4a expression and the inhibition of cell proliferation by FXR knockdown are also observed in Huh7 and HLE. In this study, we have suggested a novel mechanism by which hepatocellular carcinoma cell proliferation is regulated: FXR stimulates cell proliferation by suppressing the p16/INK4a expression, whereas Ras/ERK pathway down-regulates the FXR expression, leading to the suppressed cell proliferation in hepatocellular carcinoma cell lines.

Effects of polyamines on histone polymerization.

It is generally accepted that the nucleosome structure is not static, and that alternative conformations are adopted in response to several stimuli associated with the different functions. Histones are substrates for transglutaminase (TGase), and polymerized histone and polyamine binding histone have been suggested to play important roles in nucleus. We examined whether histone polymerization catalyzed by TGase was influenced by polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM). PUT inhibited histone polymerization, and SPD slightly prevented it. However, SPM slightly enhanced histone polymerization. These results indicate that the nuclear accumulation of the polyamines may play an important role in nuclear remodeling by histone modification. We speculate that histone cross-linking by TGase may be involved in the chromatin structure. Also, we propose that histone cross-linking by TGase may be responsible for the changes in DNA function such as transcription and replication and that TGase may be involved in cell growth and differentiation.

Transglutaminase down-regulates the dimerization of epidermal growth factor receptor in rat perivenous and periportal hepatocytes

Objective:  Recently, we found that transglutaminase 2 (TG2) might be involved in the difference in proliferative capacities between periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) through down‐regulation of high‐affinity epidermal growth factor receptor (EGFR). However, it is uncertain whether this high‐affinity EGFR contributes to the hepatocyte growth signalling pathway. Here, we have investigated the influence of TG2 on EGF‐induced EGFR dimerization and its phosphorylation, which are important steps in the hepatocyte proliferative/growth signalling pathway, in PPH and PVH.

Transglutaminase differentially regulates growth signalling in rat perivenous and periportal hepatocytes.

Abstract.   The influence of transglutaminase 2 (TG2) activity on the proliferative effect of epidermal growth factor (EGF) and on EGF receptor affinity in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) has been investigated using a primary culture system. PPH and PVH subpopulations have been isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [3H] thymidine incorporation into hepatocytes. The assay for binding of [125I] EGF to cultured hepatocytes was analysed by Scatchard plot analysis. Pretreatment with the TG2 inhibitor monodansylcadaverine (MDC) greatly increased EGF‐induced DNA synthesis in both PPH and PVH. Furthermore, [125I] EGF binding studies in PVH treated with MDC indicated that high‐affinity EGF receptor expression was markedly up‐regulated, whereas in PPH, there was no significant effect. Treatment with retinoic acid (RA), an inducer of TG2 expression, significantly decreased EGF‐induced DNA synthesis in both PPH and PVH. Binding studies in the presence of RA revealed that the high‐affinity EGF receptor was down‐regulated and completely absent in both PPH and PVH. These results suggest that TG2 was involved in the differential growth capacities of PPH and PVH through down‐regulation of high‐affinity EGF receptors.

Norepinephrine modulates the zonally different hepatocyte proliferation through the regulation of transglutaminase activity

A neurotransmitter, norepinephrine (NE), amplifies the mitogenic effect of epidermal growth factor (EGF) in the liver by acting on the alpha(1)-adrenergic receptor coupled with G protein, Galpha(h). However, the molecular mechanism is not well understood. Galpha(h) is known as a transglutaminase 2 (TG2), a cross-linking enzyme implicated in hepatocyte proliferation. We investigated the effect of NE on EGF-induced cell proliferation and TG2 activity using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in proliferative capacity. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated by the digitonin-collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, EGF receptor (EGFR) dimerization and phosphorylation, and TG2 activity were measured. NE enhanced EGF-induced DNA synthesis, EGF-induced EGFR dimerization, and its phosphorylation in PVH but not in PPH. [(3)H]NE binding studies indicated that PVH was found to have a greater affinity and number of receptors than PPH. Furthermore, NE treatment decreased TG2 activity and increased phospholipase C activity in PVH although TG2 level showed no change. These results suggest that NE-induced amplification of EGF-induced DNA synthesis especially in PVH is caused by upregulation of EGFR activation through the switching of function from TG2 to Galpha(h).

Inhibitory and promotive effects of polyamines on transglutaminase-induced protein polymerization.

Transglutaminase (TGase) has been reported to be involved in the regulation of cell growth. We examined the effects of polyamines on TGase activity. The polymerization of casein was inhibited by putrescine (PUT) and spermidine (SPD). On the other hand, polymerization of N,N-dimethylcasein was increased by spermine (SPM) and SPD. These results suggested polyamines played two distinct roles as inhibitor and promoter for TGase-catalyzed protein polymerization.

The uptake of 111In in the liver and bone marrow of partially hepatectomized and venesectioned rats

Indium-111 ((111)In) has a strong binding affinity for transferrin (Tf), and the (111)In-Tf complex binds to Tf receptor in various tissues. In partial hepatectomy (PH), a part of blood in circulation is lost along with removed liver tissues; consequently, the number of blood cells and the amount of Tf in circulation decreases. These decreases should greatly affect the uptake of (111)In in the liver and bone marrow. In order to investigate this effect, we compared the uptake in partially hepatectomized rats with that in venesectioned rats, in which only the volume of blood in circulation had been decreased. Our data show that fewer blood cells and smaller amount of Tf in circulation due to venesection increased the uptake of (111)In in bone marrow, but not in the liver, whereas PH increased the uptake of (111)In in both bone marrow and liver. The higher bone marrow uptake of (111)In must be related to the increase of the hematopoietic function resulted from the smaller amount of blood; the higher uptake in liver may be closely related to liver regeneration.

Involvement of retinoic acid-induced transglutaminase activity in zonal differences of hepatocyte proliferation after partial hepatectomy.

Background:  The authors have recently demonstrated that there is inverse correlation between transglutaminase (TGase) activity and DNA synthesis in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) at 1 day after partial hepatectomy in rats. In order to ensure the involvement of TGase in the differential growth capacities between periportal and perivenous regions of regenerating liver, the aim of this study was to investigate the effect of retinoic acid, an inducer of TGase expression, on zonal differences of hepatocyte proliferation between PPH and PVH isolated from regenerating rat liver.

Farnesoid X receptor knockdown provides significant growth inhibition in hepatocellular carcinoma cells while it does not interfere with the proliferation of primary human hepatocyte-derived cells

Identification of substances with specific toxicity for carcinoma cells promises to facilitate the development of cancer chemotherapeutics that cause minimal side effects. Here, we show that knockdown of the farnesoid X receptor (FXR) effectively suppresses the proliferation of human hepatocellular carcinoma cell lines HepG2 and HLE accompanied by elevated expression of cyclin-dependent kinase (CDK) inhibitor p16/INK4a and p21/Cip1 proteins. On the other hand, the growth of the primary human hepatocyte-derived cell line Fa2N-4 is not affected by the treatment with FXR siRNA irrespective of marked increases in the mRNAs of p16/INK4a and p21/Cip1. Surprisingly, the expression levels of p16/INK and p21/Cip1 proteins are left unchanged in Fa2N-4 cells that are subjected to the FXR siRNA treatment. Since the expression levels of these CDK inhibitor proteins in FXR-knockdown Fa2N-4 cells were elevated in the presence of proteasomal inhibitor MG132, these CDK inhibitors may be subjected to the proteasomal degradation, thereby counteracting the increased expression of their cognate mRNAs, therefore similar levels of p16 and p21 proteins were observed in control and FXR-knockdown Fa2N-4 cells. These results suggest that FXR-knockdown is effective for inhibiting the proliferation of hepatocellular carcinoma cells, not interfering with the regulatory mechanism of normal hepatocyte growth.