Katsuhiro Ogawa

Nasal T-cell lymphoma as a type of so-called "lethal midline granuloma".

Six cases were described in which an initial clinical diagnosis of “rhinitis gangrenosa progressiva” or lethal midline granuloma was made. The histological examinations of their surgical and autopsy specimens proved that their nasologic diseases could all be identified as malignant lymphoma arising from the nasal cavity, showing the general histologic characteristics reported for T‐cell lymphomata derived from peripheral T‐cells. This histologic observation was then confirmed by immunofluorescence studies using various antisera directed toward either human T‐ or B‐cell‐surface antigens. These studies clearly demonstrated that their malignant cells bore human Ly‐l‐like antigen but lacked human TL‐like and la‐like antigens as well as surface‐bound immunoglobulins, indicating their peripheral T‐cell origin. These data may suggest that so‐called “rhinitis gangrenosa progressiva” or lethal midline granuloma contains at least two distinct disease categories, one of which is Wegeners granulomatosis, and the other of which is nasal T‐cell lymphoma as described herein.

PTEN/Akt signaling through epidermal growth factor receptor is prerequisite for angiogenesis by hepatocellular carcinoma cells that is susceptible to inhibition by gefitinib.

Hepatocellular carcinoma (HCC) is one of the most common tumor-related causes of death worldwide for which there is still no satisfactory treatment. We previously reported the antiangiogenic effect of gefitinib, a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has been used successfully to treat lung cancer. In this study, we investigated the effects of gefitinib on tumor-induced angiogenesis by using HCC cell lines (HCC3, CBO12C3, and AD3) in vitro as well as in vivo. Oral administration of gefitinib inhibited angiogenesis induced by HCC3 and CBO12C3, but not by AD3 in the mouse dorsal air sac model. Production of both vascular endothelial growth factor (VEGF) and chemokine C-X-C motif ligand 1 (CXCL1) by EGF-stimulated HCC was more markedly inhibited by gefitinib in HCC3 and CBO12C3 cells than in AD3 cells. EGF stimulated the phosphorylation of EGFR, Akt, and extracellular signal-regulated kinase 1/2 (ERK1/2) in HCC3 and CBO12C3 cells, whereas EGF stimulated phosphorylation of EGFR and ERK1/2, but not Akt in AD3 cells. In fact, Akt was constitutively activated in the absence of EGF in AD3 cells. Gefitinib inhibited Akt phosphorylation in all three cell lines, but it was about five times less effective in AD3 cells. The concentration of PTEN in AD3 cells was about a half that in HCC3 and CBO12C3 cells. Transfection of HCC3 cells with PTEN small interfering RNA reduced their sensitivity to gefitinib in terms of its inhibitory effect on both Akt phosphorylation and the production of VEGF and CXCL1. In conclusion, effect of gefitinib on HCC-induced angiogenesis depends on its inhibition of the production of angiogenic factors, probably involving a PTEN/Akt signaling pathway.

Roles of Stat3 and ERK in G-CSF signaling

G‐CSF specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil‐granulocyte lineage. Although Stat3 was thought to be essential for the transduction of G‐CSF–induced cell proliferation and differentiation signals, mice deficient for Stat3 in hematopoietic cells show neutrocytosis and infiltration of cells into the digestive tract. The number of progenitor cells in the neutrophil lineage is not changed, and G‐CSF–induced proliferation of progenitor cells and prolonged neutrophil survival were observed in Stat3‐deficient mice. In hematopoietic cells from Stat3‐deficient mice, trace levels of SOCS3, a negative regulator of granulopoiesis, were observed, and SOCS3 expression was not induced by G‐CSF stimulation. Stat3‐null bone marrow cells displayed a significant activation of extra‐cellular regulated kinase 1 (ERK1)/ERK2 under basal conditions, and the activation of ERK was enhanced and sustained by G‐CSF stimulation. Furthermore, the augmented proliferation of Stat3‐deficient bone marrow cells in response to G‐CSF was dramatically decreased by addition of a MEK1 inhibitor. These results indicate that Stat3 functions as a negative regulator of G‐CSF signaling by inducing SOCS3 expression and that ERK activation is the major factor responsible for inducing the proliferation of hematopoietic cells in response to G‐CSF.

Activated hepatic stellate cells overexpress p75NTR after partial hepatectomy and undergo apoptosis on nerve growth factor stimulation.

Abstract: Background: Expression of neurotrophins (NTs) and their receptors is increased during hepatic regeneration, but their role is not well understood.

α-FETOPROTEIN AND EARLY HISTOLOGICAL CHANGES OF HEPATIC TISSUE IN DAB-HEPATOCARCINOGENESIS*

It has been generally accepted that the sequential histological changes observed in the hepatic tissue of rats during the course of hepatocarcinogenesis are as follows: Diffuse hyperplasia of hepatocytes appearing in the initial stage, nodular proliferation of the cells in the following stages, and hepatomas in the final stage.’. ’’ During these carcinogenic courses, stepwise alteration of hepatocytes leading to cancer might occur as an expression of preneoplastic transformation. One of the phenotypic changes in preneoplastic stages of hepatocarcinogenesis is the appearance of muscle-type isozyme of aldolase, as in hepatomas.” This type of enzyme was further identified as a fetal one? It is conceivable that isozymes and/or isoproteins in preneoplastic cells as well as in neoplastic cells are of the fetal type. The pattern of serum protein synthesized by hepatocytes is expected to shift from adult to fetal type as the cells undergo cancerization. It was reported that a-fetoprotein (AFP) , one of the fetal serum proteins, was found in a certain period of hepatocarcin~genesis.~-~) The authors have studied hepatic changes of rats administered with 3’-Me-DAB, and obtained the following results: l”-l‘, 2!) ( 1 ) The period of AFP appearance was coincident with that in which a deviation in isozyme pattern of various enzymes of the tissues toward fetal and neonatal types was observed. These enzymes were aldolase, acid phosphatase, and nonspecific esterases. ( 2 ) In the liver, there were a great number of so-called “oval cells,” some of which showed images of differentiation toward hepatocytes. ( 3) Oval-cell proliferation and its differentiation to the hepatocytes may play a principal role in the occurrence of fetal deviation in the function of the hepatic tissue during the early stages of carcinogenesis.

Neoplastic hepatocyte growth associated with cyclin D1 redistribution from the cytoplasm to the nucleus in mouse hepatocarcinogenesis

Cyclin D1 overexpression is a frequent change in hepatocellular carcinomas (HCCs). Our present study demonstrated that cyclin D1 overexpression with abundant cyclin E, cdk4, cdk2, and p27Kip1 (p27) occurred in neoplastic hepatocytes from the early stage of mouse hepatocarcinogenesis. While cyclin D1 expression was mainly found in the cytoplasm of the tumor cells, it shifted to the nucleus in association with cell proliferation after the animals were subjected to a partial hepatectomy (PH), and then returned once more to the cytoplasm when the cells became quiescent. Inhibition of PI3 kinase (PI3K) by Ly294002 in mouse HCC cells in vitro suppressed the nuclear shift of cyclin D1 as well as cell proliferation, while PI3K activation by PTEN suppression failed to induce nuclear shift of cyclin D1, suggesting that PI3K activation is essential but not sufficient for the cyclin D1 nuclear shift. While MEK‐ERK1/2 inhibition by PD98059 and mTOR inhibition by rapamycin affected the cyclin D1 nuclear shift and cell proliferation to a lesser extent, both these inhibitors reduced cyclin D1 levels. Finally, although p27, cdk4 and calmodulin (CaM) were detected in the cyclin D1 immunoprecipitates from both quiescent and proliferating HCC cells, Hsc70 and SSeCKS were detected only in the immunoprecipitate from quiescent cells, and p21Waf1/Cip1 (p21) was detected only in that from proliferating cells, suggesting that the cyclin D1 complex is different in quiescent and proliferating cells. These observations indicate that the nuclear/cytoplasmic localization of cyclin D1 plays an important role in the proliferation/quiescence of neoplastic hepatocytes.

Immunocytochemical localization of BGP in human bones in various developmental stages and pathological conditions

Boneγ-carboxyglutamic acid containing protein (BGP) was isolated from human bone and anti-BGP antibody was produced in rabbits. Localization of BGP was investigated immunocytochemically by light and electron microscopy in human bones in various developmental stages and pathological conditions. In the bones of a 12 week fetus, osteoblasts stained strongly in areas of bone formation. However in bones of late fetal stages and in newborns and adults, BGP was localized predominantly in the osteoid and bone matrix in the ossifying front. Osteoblasts and osteocytes also stained positive, but less dominantly than in the early fetus. Electron microscopy showed that BGP was localized in the ER and Golgi cisternae of osteoblasts and osteocytes, and the collagen fibers of the osteoid and bone matrix. The intensity and distribution of staining were not significantly different in osteoporosis and osteoarthritis. These observations indicate that BGP is synthesized by osteoblasts most actively in early fetal life and is then deposited on collagen fibers of the osteoid and bone matrix.

Expression of NGF in hepatocellular carcinoma cells with its receptors in non-tumor cell components

Nerve growth factor (NGF) is suggested to have a role in tumor progression in addition to its role in differentiation and survival of neuronal cells. We investigated expression of NGF and its receptors, TrkA and p75NTR, in hepatocellular carcinomas (HCCs). Although hepatocytes and hepatic stellate cells (HSCs) showed respectively weak and intense NGF immunostaining in the background livers of patients suffering from liver cirrhosis (LC) or chronic hepatitis (CH), intense staining was demonstrated in HCC cells of 33 of 54 (61.1%) tumors. RT‐PCR detected NGF mRNA in 7 freshly‐isolated HCC samples, and in 2 of 4 cases, in which both background livers and tumors could be analyzed, NGF mRNA was more abundant in the tumors than the background livers. TrkA was detected in the smooth muscle cells of hepatic arteries, but it was negative in tumor cells as well as non‐neoplastic hepatocytes. p75NTR and α‐smooth muscle actin (αSMA) was expressed in HSCs in the background liver and fibroblast‐like cells in stromal septa, whereas HSCs within the HCC tissues were mostly negative for p75NTR but positive for αSMA. This suggests that HSCs in HCC have a different property from those in background livers. Furthermore, the stromal septa contained abundant nerve fibers, which may be related to the increased NGF expression in HCC cells. NGF and its receptors are then thought to have a role in cellular interactions involving HCC cells, HSCs, arterial cells and nerve cells in HCC tissues.

Effects of colchicine on the hepatocellular transport of indocyanine green in the rat

SummaryThe effects of colchicine on plasma elimination and biliary excretion of indocyanine green (ICG) and sulfobromophthalein (BSP) in rats were examined. Elimination of two different doses of ICG (6 mg and 20 mg/kg body weight) from plasma was significantly delayed when rats were treated with colchicine (3 mg/kg body weight) 3 h prior to the administration of the dye. On the other hand, disappearance of BSP (100 mg/kg) from plasma was not influenced by colchicine. The fact that the difference in the ICG elimination from plasma between colchicine-treated and salinetreated rats was minimal in the early period (i.e., 2 min after administration of the dye), but evident after its half-life (i.e., 10 min, when 6 mg/kg body weight of ICG was given), suggested that colchicine mainly affected the hepatocellular transport of ICG rather than the uptake of the dye by hepatocytes. Colchicine also significantly reduced the excretion of ICG (6 mg and 20 mg/kg) into bile but did not alter that of BSP (100 mg and 200 mg/ kg). On the other hand, the same amount of lumicolchicine (3 mg/kg) did not have any effect on the biliary excretion of ICG. These results suggested that ICG is transported through hepatocytes into bile with the aid of the cytoplasmic microtubular system, whereas BSP is handled by hepatocytes in a different way.

Roles of the Pas1 and Par2 genes in determination of the unique, intermediate susceptibility of BALB/cByJ mice to urethane-induction of lung carcinogenesis : Differential effects on tumor multiplicity, size and Kras2 mutations

The C3H/HeJ (C3H), A/J and BALB/cByJ (BALB) mouse strains are respectively resistant, sensitive and intermediate regarding the induction of lung tumors by urethane. The phenotypic difference between C3H and A/J is largely determined by the Pas1 (Pulmonary adenoma susceptibility 1) gene on chromosome 6, the A/J allele of which dominantly increases the tumor burden. We recently found that BALB mice possess a unique lung tumor resistance gene on chromosome 18, designated Par2 (Pulmonary adenoma resistance 2), which partially, but dominantly suppresses the sensitive phenotype of A/J mice (Oncogene 13: 1599 – 1604, 1996). It has, however, remained unclear why BALB mice carrying the Par2 gene are significantly more sensitive to urethane-induced lung carcinogenesis than C3H mice that have no dominant lung tumor resistance genes. In the present study, using (C3H×BALB)F1×C3H backcross mice treated with urethane, we demonstrated that BALB mice possess the disease allele of the Pas1 gene despite their 15-fold more resistance relative to A/J mice (LOD=22.6). The BALB Par2 allele only significantly reduced the mean lung tumor multiplicity (LOD=4.4) in the backcross population carrying the BALB allele of Pas1, indicating that the intermediate BALB phenotype may at least in part be the result of interactions between these two dominant genes. While the BALB Pas1 allele increased both the mean multiplicity and size of lung tumors, the BALB Par2 allele affected only the mean tumor multiplicity, implying that they are involved in different stages of multi-step lung carcinogenesis. In addition, we found that 68% of lung tumors from the BALB Pas1-positive backcross mice contained activating point mutations of the Kras2 oncogene, tightly linked to the Pas1 locus, whereas these genetic alterations were absent in tumors from BALB Pas1-negative mice. The Par2 genotype exhibited no effect on this parameter. Since the activating point mutations were observed exclusively in the BALB allele as already reported with lung tumors in (C57BL/6J×BALB/cJ)F1 mice, BALB Pas1 or possibly Kras2 itself may confer selective growth advantage on the affected urethane-initiated lung lesions.