Masaaki Miyakoshi

Hypoxia-Independent Overexpression of Hypoxia-Inducible Factor 1α as an Early Change in Mouse Hepatocarcinogenesis

Hypoxia-inducible factor 1 (HIF-1) is involved in tumor progression/metastasis and activated in various cancers. Here we show that HIF-1alpha, which plays a major role in HIF-1 activation, is overexpressed in preneoplastic hepatocytic lesions from a very early stage during hepatocarcinogenesis in mice and man. Transcriptional targets of HIF-1, such as vascular endothelial growth factor, glut-1, c-met, and insulin-like growth factor II (IGF-II), were also overexpressed in mouse lesions. Oxygen tension within the lesions was not different from that of the normal hepatic tissues, indicating that HIF-1alpha expression was independent of hypoxia. On the other hand, Akt, the pathway of which can up-regulate HIF-1alpha expression, was activated in the mouse lesions, whereas HIF-1alpha was markedly down-regulated in the mouse hepatocellular carcinoma (HCC) cell lines after treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1alpha expression is dependent on PI3K/Akt signaling. Conversely, HIF-1alpha knockdown by short interfering RNA in the HCC cell line resulted in decreased expression of activated Akt together with the HIF-1 target genes, indicating that Akt activation is reversely dependent on HIF-1 activation. Treating the HCC cells with IGF-II or epidermal growth factor (EGF) up-regulated both phospho-Akt and HIF-1alpha, whereas inhibition of IGF-II or EGF signaling down-regulated them both, suggesting that IGF-II and EGF can, at least in part, mediate the activation of Akt and HIF-1alpha. However, Akt was not activated by IGF-II or EGF in the HIF-1alpha knockdown cells, indicating that expression of the HIF-1 target genes is necessary for the Akt activation. These findings suggest that the reciprocal activation of PI3K/Akt signaling and HIF-1alpha may be important in the progression of hepatocarcinogenesis.

Effects of Bone Marrow and Hepatocyte Transplantation on Liver Injury

BACKGROUND The therapeutic effects of bone marrow and hepatocyte transplantation were investigated regarding the treatment of retrorsine-partial hepatectomy-induced liver injury. METHODS Analbuminemic F344alb rats were given two doses of retrorsine 2 wk apart, followed 4 wk later by transplantation with F344 rat bone marrow cells or hepatocytes immediately after a two-thirds hepatectomy. The survival rate, liver regeneration rate, liver functions, albumin-positive hepatocytes, and normal albumin gene sequences in the liver and serum albumin levels were investigated in the recipients. RESULTS Although 65% retrorsine/partial hepatectomy-treated F344alb died between 1 and 11 d after the partial hepatectomy, only 27.5% of the animals died following bone marrow transplantation, and 50% with hepatocyte transplantation. Both bone marrow and hepatocyte transplantation ameliorated acute liver injury after a partial hepatectomy. Bone marrow transplantation yielded a very small increase in the number of albumin-positive hepatocytes in the liver, while hepatocyte transplantation resulted in massive replacement of the liver tissues by the donor hepatocytes associated with an elevation of serum albumin after an extended time. CONCLUSIONS Both bone marrow and hepatocyte transplantation could prevent acute hepatic injury, conceivably due to a paracrine mechanism.

Serine 727 phosphorylation of STAT3: an early change in mouse hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine.

STAT3 activation is involved in development and progression of hepatocellular carcinoma (HCC). We investigated STAT3 activation during hepatocarcinogenesis induced by neonatal diethylnitrosamine (DEN) treatment in mice. Nuclear accumulation and phosphorylation of STAT3 were detected in altered hepatocyte foci in the early stages as well as adenomas and HCCs in the late stages. Although total STAT3 levels were the same between the hepatic lesions and normal livers, S727‐phosphorylated STAT3 was enhanced in adenomas and HCCs, whereas Y705‐phosphorylated STAT3 was detected mainly in HCCs. In mouse HCC cell lines, although both S727 and Y705 remained un‐ or hypophosphorylated under serum‐free conditions, fetal bovine serum (FBS) induced strong S727/weak Y705 phosphorylation, STAT3 nuclear accumulation and cell proliferation, whereas IL‐6 treatment without FBS caused Y705 phosphorylation without S727 phosphorylation, STAT3 nuclear accumulation or cell proliferation. When HCCs were simultaneously treated with FBS/IL‐6, selective suppression of S727 phosphorylation by an MEK inhibitor prevented STAT3 nuclear accumulation and cell proliferation. Furthermore, an S727 phosphorylation‐deficient STAT3 mutant (S727A) had a diminished capacity to accumulate in the nucleus when compared with wild‐type (WT) or the phosphorylation‐mimic mutant (S727D) following treatment with FBS/IL‐6. After treatment with FBS/IL‐6, the cells expressing the S727A mutant proliferated more slowly than those expressing WT or S727D mutant. In contrast, suppression of Y705 phosphorylation by a JAK inhibitor in the FBS/IL‐6 treated cells did not affect STAT3 nuclear accumulation or cell proliferation. Taken together, these data demonstrate that STAT3 activation, mainly through S727 phosphorylation, contributes to the DEN‐induced hepatocarcinogenesis at the earliest stages.

Low p38 MAPK and JNK activation in cultured hepatocytes of DRH rats; a strain highly resistant to hepatocarcinogenesis

DRH rats are a hepatocarcinogenesis‐resistant strain isolated from hepatocarcinogenesis‐sensitive Donryu rats, and the liver of DRH shows less histological damage and fewer/smaller neoplastic hepatic lesions by the treatment with hepatocarcinogens. To investigate the mechanism of the resistance, the properties of hepatocytes of DRH and Donryu were compared. In primary culture, DRH hepatocytes exhibited higher proliferation and less apoptosis than Donryu hepatocytes in the presence of EGF and insulin. However, such difference was not correlated to the degree of DNA damage associated with cell culture or cell cycle checkpoint function. Although the mitogen‐activated protein kinases [EGF receptor (EGFR) and extracellular signal regulating kinases (ERK1/2)] were activated to the same degree, the stress‐activated protein kinases [p38 mitogen‐activated protein kinase (p38) and c‐jun N‐terminal kinase (JNK)] were activated to a lesser degree in the DRH hepatocytes. Treatment with 2‐acetylaminofluorene (2‐AAF) in vivo also resulted in less JNK and p38 activation in the DRH livers. Furthermore, apoptosis signal‐regulating kinase 1 (ASK1) was inhibited by the lysate from the DRH but not by the Donryu hepatocytes. The low activation of the stress‐activated protein kinases may be linked to the resistance to cellular stress, which may underlie the hepatocarcinogenesis‐resistance in DRH rats.

Different growth capacity between infant and adult mouse hepatocytes in vitro correlates to the cyclin D1 level without relation to oxidative DNA damage

Abstract: Background: Proliferating capacity of hepatocytes is rapidly decreased during growth into maturity, but its exact reason(s) are not well known.

Unique Properties of Hepatocarcinogenesis-Resistant DRH Rat Hepatocytes Linked or Not Linked to the Drh1 Locus on Rat Chromosome 1

Hepatocarcinogenesis-resistant DRH rats exhibit few and small preneoplastic hepatocytic lesions during hepatocarcinogenesis, of which traits have been assigned to two major chromosomal regions, Drh1 and Drh2. In this study, hepatocytes from DRH.F344-Drh1, a congenic strain in which the Drh1 chromosomal region was replaced with that of F344 rats, were compared to hepatocytes from Donryu (original strain), DRH, and F344 rats. Although DRH hepatocytes exhibited low proliferation and p38 dephosphorylation after lead nitrate (LN) treatment despite cytokine and Cox2 activation, DRH.F344-Drh1 hepatocytes exhibited high responses, as did Donryu and F344 hepatocytes. Moreover, although DRH hepatocytes were resistant to hepatotoxins, DRH.F344-Drh1 hepatocytes were as sensitive to hepatotoxins as Donryu and F344 hepatocytes. However, DRH.F344-Drh1 hepatocytes like DRH hepatocytes proliferated at lower rates in vitro and contained smaller nuclei than Donryu and F344 hepatocytes. Thus, low responses to LN and resistance to hepatotoxins in DRH hepatocytes were linked to the Drh1 locus, while low proliferation in vitro and small nuclear size were not linked to the Drh1 locus.

Abstract LB-427: Unique properties of hepatocarcinogenesis-resistant DRH rat hepatocytes linked or not linked to the Drh1 locus on rat chromosome 1

DRH rats were established by the selective mating of Donryu rat progeny that exhibited resistance to hepatocarcinogenesis. DRH rats show smaller and fewer preneoplastic hepatocytic lesions than ordinary rats after treatment with hepatocarcinogenic regimens, and these traits have been assigned to two major genetic loci, Drh1 on chromosome 1 and Drh2 on chromosome 4. The DRH hepatocytes have the unique properties such as low proliferation after lead nitrate (LN) treatment, resistance to hepatotoxic chemicals and small cell size. If some of these properties are linked to the Drh1 or Drh2 loci, the properties may be related to the mechanism(s) of the hepatocarcinogenesis-resistance in DRH rats. In this study, to explore what properties are linked to the Drh1 locus, we compared hepatocytes of DRH.F344-Drh1 rats, a congenic DRH strain in which the DRH chromosomal region including the Drh1 locus was substituted by that of F344 rats, were compared with hepatocytes of Donryu, DRH and F344 rats. After LN treatment, although DRH hepatocytes proliferated at low rate, DRH.F344-Drh1 hepatocytes proliferated at high rate comparable to Donryu and F344 hepatocytes. In addition, the DRH.F344-Drh1 livers showed high p38 MAPK dephosphorylation after LN treatment like Donryu and F344 livers, while such p38 MAPK dephosphrylation was not seen in the DRH liver. However, increase of TNF-α, IL-6 and Cox2 mRNA and NFκB/Stat3 activation in the liver was almost at the same degree in Donryu, DRH and DRH.F344-Drh1 rats, suggesting that the low responses to LN in the DRH hepatocytes were not dependent on low cytokine/Cox2 activation but rather due to the inherent properties of DRH hepatocytes. After 200 mg/Kg diethylnitrosamine (DEN) or 5 ml/Kg CCl4 treatment, severe hepatic injury occurred in DRH.F344-Drh-1 rats like Donryu and F344 rats, while its degree was much less in DRH. On the other hand, the DRH.F344-Drh1 as well as DRH hepatocytes exhibited lower proliferation after the EGF or HGF treatment in vitro than Donryu and F344 hepatocytes. Furthermore, the nuclear size of DRH.F344-Drh1 hepatocytes was comparable to that of DRH hepatocytes, which was smaller than those of Donryu and F344 hepatocytes. These findings indicate that the Drh1 locus is linked to the low responses to LN and hepatotoxin resistance in DRH hepatocytes, but not to low proliferation in vitro and small nuclear size. (Supported by the grants from the Japanese Ministry of Education, Science, Culture and Sports.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-427. doi:10.1158/1538-7445.AM2011-LB-427

Abstract 1478: Role of Stat3 Ser727 phosphorylation in mouse hepatocarcinogenesis

Inappropriate activation of signal transducer and activator of transcription 3 (Stat3) occurs with extreme frequency in a wide variety of cancers including HCC, and constitutive Stat3 activation in hepatocytes was demonstrated to accelerate hepatocarcinogenesis, while repression of Stat3 activation suppresses hepatocarcinogenesis. Diverse factors such as cytokines, growth factors and oncoproteins can activate Stat3, where phosphorylation of the conserved tyrosine residue at the C-terminus (Y705) results in the Stat3 homodimer or the heterodimer with Stat1, and phosphorylation of the serine residue on the transactivation domain (S727) contributes to its maximal transcriptional activity. The activated Stat dimers bind to specific DNA-response elements in the promoter of target genes and thereby induce unique gene expression programs cooperating with other transcription factors and cofactors. We investigated Stat3 activation in mouse hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine (DEN) and found that Stat3 phosphorylation occurred by two steps, where S727 phosphorylation was seen in the preneoplastic stage, whereas Y705 phosphorylation was further associated with malignant progression. In mouse HCC cells, serum treatment induced Ser727 phosphorylation via MAPK activation, associating with cell proliferation and Stat3 nuclear translocation, whereas IL-6 induced Tyr705 phosphorylation via JAK activation without stimulating cell proliferation and the Stat3 nuclear translation. On the other hand, the Ser727 phosphorylation dead Stat3 (S727A), in which the serine residue was replaced by alanine, reduced the cell proliferation and Stat3 nuclear translocation, while the Ser727-phosphorylation mimic Stat3 (S727D), in which the serine residue was replaced by aspartic acid, enhanced the cell proliferation and Stat3 nuclear translocation. To further investigate the role of Stat3 Ser727 phosphorylation, the Stat3 knockdown HCC cell lines were established by introducing the Stat3 siRNA into the mouse HCC cells. These cell lines showed retarded growth and less attachment to the substrate forming spheroidal aggregates, whereas the parent cells showed rapid proliferation and attached to the substrate with extended morphology. cDNA array analysis revealed that the Stat3 knockdown cells showed variety of gene expression changes relating to cell survival and growth, and RT-PCR demonstrated that the expression of EGFR, FGFR and E-cadherin was markedly reduced in the Stat3 knockdown cells. Our findings indicate that the Ser727-phosphorylated Stat3 plays an important role from the early stage in mouse hepatocarcinogenesis, presumably by controlling cellular growth and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1478.

F344 rat liver nonparenchymal cell transplantation can increase the number of albumin-positive hepatocytes in the liver following hematopoietic reconstitution in irradiated analbuminemic rats.

The adult liver contains hematopoietic stem cells that can reconstitute the bone marrow. We tested whether bone marrow cells (BMCs) derived from liver nonparenchymal cells (LNPCs) can increase the number of hepatocytes within livers. LNPCs from Fischer 344 rats (F344) were infused into the penile veins of F344 congenic Nagase’s analbuminenic rats (F344alb) immediately after whole-body irradiation, and the recipients were sacrificed 8 weeks later. Eleven of 15 (73.3%) F344alb that received the LNPC transplantation after irradiation survived, while only 1 of 8 (12.5%) F344alb that received irradiation alone was alive after 8 weeks. Normal albumin gene sequences were detected by PCR in BMCs of the recipient F344alb that received LNPC transplantation after irradiation, indicating that F344alb bone marrow was reconstituted by F344 LNPCs. Although single or pairs of albumin-positive (Alb+) hepatocytes were seen in the liver of untreated F344alb and those with irradiation or LNPC transplantation alone, clusters consisting of >3 Alb+ hepatocytes were detected in the livers of F344alb with the LNPC or BMC transplantation after irradiation together with single or double Alb+ cells. Normal albumin gene sequences were detected by PCR in the DNA isolated from such Alb+ hepatocyte clusters microdissected from the immunostained sections. The data indicate that BMCs derived from F344 LNPCs could increase the number Alb+ hepatocytes within the F344alb liver.