Yasuhito Ohkubo

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.

Analysis of Common Deletion (CD) and a novel deletion of mitochondrial DNA induced by ionizing radiation

Purpose: In order to identify supportive evidence of radiation exposure to cells, we analyzed the relationship between exposure to ionizing radiation and the induction of deletions in mitochondrial DNA (mtDNA). Materials and methods: Using human hepatoblastoma cell line, HepG2 and its derivatives, HepG2-A, -89 and -400, established after long term exposure to X-ray, mtDNA deletions were analyzed by polymerase chain reaction (PCR) and real-time PCR after cells were subjected to radiation and genotoxic treatments. Results: Common Deletion (CD), the most extensively studied deletion of mtDNA, was induced within 24 h after exposure to 5 Gray (Gy) of X-rays and was associated with replication of mtDNA. CD became undetectable several days after the exposure due to the death of cells containing mitochondria within which CD had been induced. Furthermore, we found a novel mtDNA deletion that consisted of a 4934 base-pair deletion (4934del) between nucleotide position 8435 and 13,368. A lower dose of ionizing radiation was required to induce the 4934del than for CD and this was independent of the quality of radiation used and was not induced by treatments with hydrogen peroxide (H2O2) and other genotoxic reagents including bleomycin. Conclusion: CD is induced by ionizing radiation, however, the amount of CD detected at a certain point in time after radiation exposure is dependent on the initial frequency of CD induced and the death rate of cells with mtDNA containing CD. The novel mtDNA deletion found in this study, therefore, will be used to determine whether cells were exposed to ionizing radiation.

Clinically relevant radioresistant cells efficiently repair DNA double‐strand breaks induced by X‐rays

Radiotherapy is one of the major therapeutic modalities for eradicating malignant tumors. However, the existence of radioresistant cells remains one of the most critical obstacles in radiotherapy and radiochemotherapy. Standard radiotherapy for tumor treatment consists of approximately 2 Gy once a day, 5 days a week, over a period of 5–8 weeks. To understand the characteristics of radioresistant cells and to develop more effective radiotherapy, we established a novel radioresistant cell line, HepG2‐8960‐R with clinical relevance from parental HepG2 cells by long‐term fractionated exposure to 2 Gy of X‐rays. HepG2‐8960‐R cells continued to proliferate with daily exposure to 2 Gy X‐rays for more than 30 days, while all parental HepG2 cells ceased. After exposure to fractionated 2 Gy X‐rays, induction frequencies of micronuclei and remaining foci of γ‐H2AX in HepG2‐8960‐R were less than those in HepG2. Flow cytometric analysis revealed that the proportion of cells in S‐ and G2/M‐phase of the cell cycle was higher in HepG2‐8960‐R than in HepG2. These suggest that the response of clinically relevant radioresistant (CRR) cells to fractionated radiation is not merely an accumulated response to each fractionated radiation. This is the first report on the establishment of a CRR cell line from an isogenic parental cell line. (Cancer Sci 2009; 100: 747–752)

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.

Targeting of tumor endothelial cells combining 2 Gy/day of X-ray with Everolimus is the effective modality for overcoming clinically relevant radioresistant tumors.

Radiotherapy is widely used to treat cancer because it has the advantage of physically and functionally conserving the affected organ. To improve radiotherapy and investigate the molecular mechanisms of cellular radioresistance, we established a clinically relevant radioresistant (CRR) cell line, SAS‐R, from SAS cells. SAS‐R cells continue to proliferate when exposed to fractionated radiation (FR) of 2 Gy/day for more than 30 days in vitro. A xenograft tumor model of SAS‐R was also resistant to 2 Gy/day of X‐rays for 30 days. The density of blood vessels in SAS‐R tumors was higher than in SAS tumors. Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, sensitized microvascular endothelial cells to radiation, but failed to radiosensitize SAS and SAS‐R cells in vitro. Everolimus with FR markedly reduced SAS and SAS‐R tumor volumes. Additionally, the apoptosis of endothelial cells (ECs) increased in SAS‐R tumor tissues when both Everolimus and radiation were administered. Both CD34‐positive and tomato lectin‐positive blood vessel densities in SAS‐R tumor tissues decreased remarkably after the Everolimus and radiation treatment. Everolimus‐induced apoptosis of vascular ECs in response to radiation was also followed by thrombus formation that leads to tumor necrosis. We conclude that FR combined with Everolimus may be an effective modality to overcome radioresistant tumors via targeting tumor ECs.

Guanine nucleotide-binding protein 1 is one of the key molecules contributing to cancer cell radioresistance

Standard fractionated radiotherapy for the treatment of cancer consists of daily irradiation of 2‐Gy X‐rays, 5 days a week for 5–8 weeks. To understand the characteristics of radioresistant cancer cells and to develop more effective radiotherapy, we established a series of novel, clinically relevant radioresistant (CRR) cells that continue to proliferate with 2‐Gy X‐ray exposure every 24 h for more than 30 days in vitro. We studied three human and one murine cell line, and their CRR derivatives. Guanine nucleotide‐binding protein 1 (GBP1) gene expression was higher in all CRR cells than their corresponding parental cells. GBP1 knockdown by siRNA cancelled radioresistance of CRR cells in vitro and in xenotransplanted tumor tissues in nude mice. The clinical relevance of GBP1 was immunohistochemically assessed in 45 cases of head and neck cancer tissues. Patients with GBP1‐positive cancer tended to show poorer response to radiotherapy. We recently reported that low dose long‐term fractionated radiation concentrates cancer stem cells (CSCs). Immunofluorescence staining of GBP1 was stronger in CRR cells than in corresponding parental cells. The frequency of Oct4‐positive CSCs was higher in CRR cells than in parental cells, however, was not as common as GBP1‐positive cells. GBP1‐positive cells were radioresistant, but radioresistant cells were not necessarily CSCs. We concluded that GBP1 overexpression is necessary for the radioresistant phenotype in CRR cells, and that targeting GBP1‐positive cancer cells is a more efficient method in conquering cancer than targeting CSCs.

The effect of FeCl3 on the accumulation of gallium-67 into inflammatory and normal tissues

The effect of FeCl3 on the uptake of67Ga by inflammatory and normal tissues was studied to clarify the role of transferrin in67Ga uptake by inflammatory tissue. The administration of FeCl3 5 min before the injection of67Ga decreased the uptake of67Ga by liver and spleen, but had little effect on the uptake of67Ga by the inflammatory tissue. These results suggest that67Ga is taken up by normal tissues in a transferrin-bound form but in an unbound form by inflammatory tissue. On the other hand, when FeCl3 was simultaneously injected with67Ga, the uptake of67Ga by liver and spleen was markedly increased but the uptake by inflammatory tissue was decreased.

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.