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Transient increase in telomerase activity of proliferating fibroblasts and endothelial cells in granulation tissue of the human skin

Makoto Osanai, Tohru Tamaki, Motoki Yonekawa, Akio Kawamura, Norimasa Sawada

Wound Repair and Regeneration 10 ( 1 ) 59 - 66 2002年

Although granulation tissue formation is an important step for second-intention wound healing, the molecular events underlying this process are still poorly understood. To investigate the role of telomerase in the formation of granulation tissue, we measured the activity of this enzyme and determined the expression and localization of human telomerase reverse transcriptase mRNA using human skin samples. Telomerase activity in the tip of the granulation tissue where fibroblasts actively proliferate was detected at a level 5.6 ± 1.5 times higher than that at the edge of the tissue when using a polymerase chain reaction-based telomeric repeat amplification protocol assay coupled with enzyme-linked immunosorbent assay. This, together with the findings from semiquantitative reverse transcriptase-polymerase chain reaction and in situ hybridization of human telomerase reverse transcriptase, revealed that proliferating cell nuclear antigen-positive fibroblasts and endothelial cells in the progressing granulation tissue showed de novo activation of telomerase with high human telomerase reverse transcriptase mRNA expression. This condition may be a prolongation of cellular replicative capacity taking advantage of the positive regulatory dynamics of cell growth. We conclude that the regulation of telomerase activity may play an important role in granulation tissue formation in wound healing.

DOI PubMed

Unique cellular features in atypical adenomatous hyperplasia of the lung: Ultrastructural evidence of its cytodifferentiation

M Osanai, T Igarashi, Y Yoshida

ULTRASTRUCTURAL PATHOLOGY ( TAYLOR & FRANCIS INC ) 25 ( 5 ) 367 - 373 2001年09月

Atypical adenomatous hyperplasia (AAH) of the lung could be a good material to understand the histogenesis of peripherally occurring, well-differentiated adenocarcinoma. However, its true biological significance remains to be clarified. The authors present the histomorphological studies of this lesion and compare the ultrastructure with that of nonmucinous bronchioloalveolar carcinoma (BAC) to define characteristic features of AAH. Light microscopy showed the well-preserved pulmonary architecture, proliferated neoplastic cells without marked cellular atypia, and no transitional area to obvious adenocarcinoma. Intranuclear inclusion was present in a large number of neoplastic cells. Electron microscopy revealed that cuboidal or low columnar neoplastic cells proliferated actively but were not crowded on slightly thickened fibrous alveolar septa with both Clara-like granules and small lamellar bodies in the cytoplasm resembling that of Clara cell and type 2 pneumocyte. Some of the nuclei had characteristic invaginations of its nuclear membrane. Although the findings appear to be nonspecific for AAH, the authors emphasize that AAH is an alveolar intraepithelial neoplasia that represents a very early stage in the continuous developmental spectrum of adenomatous neoplasia in the bronchioloalveolar region corresponding to dysplasia or intraepithelial neoplasia in other organs, and will give the significance to speculate its histogenesis.

DOI

Adenocarcinoma arising in gastric heterotopic pancreas: Clinicopathological and immunohistochemical study with genetic analysis of a case

M Osanai, N Miyokawa, T Tamaki, M Yonekawa, A Kawamura, N Sawada

PATHOLOGY INTERNATIONAL ( BLACKWELL PUBLISHING ASIA ) 51 ( 7 ) 549 - 554 2001年07月

Heterotopic pancreas in the stomach is a relatively common congenital condition, but the risk of malignant transformation is extremely low. In this study, we describe a case of adenocarcinoma arising from a gastric heterotopic pancreas and we consider its morphological and immunohistochemical features and genetic analysis, in order to examine its histogenesis. This unusual sequela was seen in a 57-year-old woman. Image studies showed a protruding lesion with a central ulcer located in the lesser curvature from the angle to the body of the stomach. A biopsy specimen: confirmed this lesion as adenocarcinoma before total gastrectomy. The tumor showed mixed patterns of solid neoplastic-cell proliferation and moderately differentiated glandular structures, and also showed transitional lesions to obvious malignancy, that is, dysplasia, or adenocarcinoma in situ. Neoplastic cells had positive immunoreactivity for carbohydrate antigen (CA) 19-9. mucin (MUC) 1, and insulin, and the mutant allele-specific amplification method revealed a point mutation at K-ras codon 12 (GGT [Gly]-->GAT [Asp]), which is the most common mutational change observed in patients with pancreatic carcinoma. The features of the present case provide clear evidence that this tumor originated from heterotopic pancreatic tissue rather than from gastric epithelium.

DOI PubMed

Adenocarcinoma arising in gastric heterotopic pancreas: Clinicopathological and immunohistochemical study with genetic analysis of a case

M Osanai, N Miyokawa, T Tamaki, M Yonekawa, A Kawamura, N Sawada

PATHOLOGY INTERNATIONAL ( BLACKWELL PUBLISHING ASIA ) 51 ( 7 ) 549 - 554 2001年07月

DOI

Occludin and claudin-1 concentrate in the midbody of immortalized mouse hepatocytes during cell division

T Kojima, Y Kokai, H Chiba, M Osanai, K Kuwahara, M Mori, Y Mochizuki, N Sawada

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY ( HISTOCHEMICAL SOC INC ) 49 ( 3 ) 333 - 339 2001年03月

It has been believed that epithelial cells maintain tight junctions at all times, including during cell division, to provide a continuous epithelial seal. However, changes in localization of integral tight junction proteins during cell division have not been examined. In this study, using SV40-immortalized mouse hepatocytes transfected with human Cx32 cDNA, in which tight junction strands and the endogenous tight junction proteins occludin, claudin-1, ZO-1, and ZO-2 were induced, we examined changes in localization of the tight junction proteins at all stages of cell division. All tight junction proteins were present between mitotic cells and neighboring cells throughout cell division. in late telophase, the integral tight junction proteins occludin and claudin-1, but not the cytoplasmic proteins ZO-1 and ZO-2, were concentrated in the midbody between the daughter cells and were observed at cell borders between the daugher and neighboring cells. These results indicate that the integral tight junction proteins are regulated in a different manner from the cytoplasmic proteins ZO-1 and ZO-2 during cytokinesis.

Retinoid X receptor alpha and retinoic acid receptor gamma mediate expression of genes encoding tight-junction proteins and barrier function in F9 cells during visceral endodermal differentiation

H Kubota, H Chiba, Y Takakuwa, M Osanai, H Tobioka, GI Kohama, M Mori, N Sawada

EXPERIMENTAL CELL RESEARCH ( ACADEMIC PRESS INC ) 263 ( 1 ) 163 - 172 2001年02月

Retinoids are critical for differentiation of columnar epithelial cells and for preventing metaplasia of these cells into stratified squamous epithelial cells, in which tight junctions (TJs) are essentially absent. This implies that retinoids might play important roles in regulating the structures and functions of TJs of columnar epithelium. F9 murine embryonal carcinoma cells differentiate into epithelial cells resembling visceral endoderm bearing TJs, when grown in suspension as aggregates in the presence of retinoic acid (RA). We show that RA induces the TJ structure and expression of several TJ-associated molecules, such as ZO-1, occludin, claudin-6, and claudin-7, as well as a barrier function in the genetically engineered cell line F9: rtTA:Cre-ERT L32T2, which allows sophisticated genetic manipulations simply by addition of ligands (H. Chiba et at, 2000, Exp. Cell Res. 260, 334-339). Interestingly, our data indicate that a barrier for small substances is generated after that for large ones during de novo formation of TJs, We also compared the RA-induced expression of TJ components and barrier function in RXR alpha (-/-)-RAR gamma (-/-) F9 cells with those in wild-type cells and show that the retinoid signals for transduction of these events are mediated by specific RXR-RAR pairs. (C) 2001 Academic Press.

DOI PubMed CiNii

Expression of GFR alpha-1, receptor for GDNF, in rat brain capillary during postnatal development of the BBB

H Utsumi, H Chiba, Y Kamimura, M Osanai, Y Igarashi, H Tobioka, M Mori, N Sawada

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY ( AMER PHYSIOLOGICAL SOC ) 279 ( 2 ) C361 - C368 2000年08月

It is well known that the blood-brain barrier (BBB) matures at similar to 2 wk after birth in the rat. Recently, we showed that glial cell line-derived neurotrophic factor (GDNF) enhances the barrier function of porcine endothelial cells forming the BBB in culture. In the present study, we examined the relation between permeability of the BBB, using Evans blue as a tracer, and expression of the GDNF family receptor (GFR alpha-1) during postnatal development of the BBB. Morphometric analysis showed that exudation of Evans blue from capillaries of the cerebral cortex progressively decreased until postnatal day 21. Inversely, immunohistochemical examinations showed expression of GFR alpha-1 in the capillaries at postnatal day 3 and expression that reached the same levels as observed in adult rats by postnatal day 10. However, c-ret, which is thought to mediate a signal evoked by binding of GDNF to GFR alpha-1, was not expressed in the capillaries of the brain cortex in 3-mo-old rats. On the other hand, the tight junction proteins occludin and ZO-1 appeared to be fully expressed at birth. The reciprocal relation between GFR alpha-1 expression and the permeability of the BBB strongly suggests active participation of GDNF in postnatal development of the BBB, although the mechanism(s) involved is still veiled.

Gain of chromosomes 15 and 19 is frequent in both mouse hepatocellular carcinoma cell lines and primary tumors, but loss of chromosomes 4 and 12 is detected only in the cell lines

K Ogawa, M Osanai, M Obata, K Ishizaki, K Kamiya

CARCINOGENESIS ( OXFORD UNIV PRESS ) 20 ( 11 ) 2083 - 2088 1999年11月

Chromosomal alterations were investigated in hepatocellular carcinoma cell lines, primary tumors and liver epithelial cell lines derived from normal livers of C57BL/6J x C3H/HeJ F-1 and C3H/HeJ x C57BL/6J F-1 mice. In the primary tumors, non-random gain of chromosomes 15 and 19 was found in seven and five of 14 hepatocellular carcinomas, respectively. On the other hand, in the cases of both liver epithelial and hepatocellular carcinoma cell lines, frequent changes were loss of chromosomes 4 (4/9 cell lines) and 12 (3/9) as well as gain of chromosomes 15 (5/9) and 19 (4/9), These results indicate that the chromosomal gain is associated with both in vivo carcinogenesis and establishment of cell lines, while the loss is specific for the latter. PCR analysis using polymorphic microsatellite DNA markers revealed that the loss of chromosome 12 as well as chromosome 4 was much more frequent for the C57BL/6J hepatocarcinogenesis-resistant rather than the susceptible C3H/HeJ strain.

DOI PubMed CiNii

Morphology, proliferation and apoptosis of mouse liver epithelial cells cultured as spheroids

GH Lee, M Osanai, Y Tokusashi

JAPANESE JOURNAL OF CANCER RESEARCH ( ELSEVIER SCI IRELAND LTD ) 90 ( 10 ) 1109 - 1116 1999年10月

The MLEC10 is an epithelial cell line derived from an untreated, normal C3H/HeN mouse liver We previously demonstrated that tumorigenic variants from this cell line produced moderately differentiated hepatocellular carcinomas in nude mice, However, it has remained unclear whether the parental MLEC10 cells represent immortalized hepatocytes or so-called oval cells, both of which may serve as precursors for hepatocellular neoplasms. In this study, we performed 3-dimensional, spheroid culture of the MLEC10 cells in order to facilitate histological assessment of their lineage. Spheroidal aggregates were formalin-fixed and embedded in paraffin for routine light-microscopic observation of hematoxylin and eosin-stained sections. Histopathologically, the MLEC10 cells were indistinguishable from immature hepatocytes and distinct from oval cells. At the electron-microscopic level, their hepatocytic nature was evidenced by bile canaliculus structures and glycogen storage. Intriguingly, the spheroids contained fragmentary material reminiscent of Councilman bodies, implying apoptosis of the hepatocytes. Although the cells significantly proliferated during the first three days of culture, apoptotic death then resulted in a 75% decrease in viable cell number. Thereafter, both apoptosis and cell division appeared silent, the numbers being unchanged. Expression of the p53 tumor suppressor gene became gradually elevated, correlating positively with growth arrest, but negatively with apoptosis, suggesting that the cell death occurred independently of p53, Our results indicate that at least some liver epithelial cell lines derived from untreated murine livers exhibit a hepatocytic morphology in spheroid culture. Also, the present culture system provides a useful tool for investigating biological phenomena, e.g. apoptosis, specifically involving liver cells, under 3-dimensional conditions.

Cryofiltration: A new approach for removal of anti-blood group antibody

M Takahashi, T Tamaki, M Tanaka, M Katori, N Yokota, Y Takamine, M Osanai, M Naitoh, M Yasuhara, K Kukita, J Meguro, M Yonekawa, A Kawamura

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC ) 30 ( 7 ) 3275 - 3276 1998年11月

DOI

Mechanism of the paradoxical, inhibitory effect of phenobarbital on hepatocarcinogenesis initiated in infant B6C3F(1) mice with diethylnitrosamine

GH Lee, T Ooasa, M Osanai

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 58 ( 8 ) 1665 - 1669 1998年04月

Phenobarbital (PB), a classical rodent hepatopromoter, remarkably enhances hepatocarcinogenesis initiated by diethylnitrosamine (DEN) in adult B6C3F(1), mice. However, it is also known to strongly inhibit liver tumor development in the B6C3F(1) mice initiated with DEN in their infancy. The present study aimed to elucidate the unknown biological mechanisms for this paradoxical, inhibitory effect of PB on B6C3F(1) mouse hepatocarcinogenesis. Male 12-day-old infant B6C3F(1) mice were injected i.p. with DEN and, at 6 weeks of age, divided into PB-treated (PB+ group) and untreated (PB-group) animals. At 24 weeks, PB treatment was ceased for half of the PB+ animals (PB+/- group) and started for half of the PB-animals (PB-/+ group), Finally, all mice were sacrificed at 36 weeks and examined for the development of liver tumors. The mean multiplicity of gross tumors in the PB+ group was only one-fifteenth of that for the PB-group. PB-/+ animals developed fewer than half of the tumors found in PB-mice, indicating that the PB effect depends solely on the treatment duration, rather than the animal age, The effect was proven to be reversible, because the mean tumor multiplicity for the PB+/- group was seven times larger than that for the PB+ group. Stereological analysis revealed the mean volume of hepatocellular proliferative lesions in the PB-animals to be 7.7- and 4.1-fold the values for the PB+ and PB-/+ groups, respectively. The mean proliferating cell nuclear antigen-labeling indices for hepatocellular adenomas in PB+ and PB-/+ animals were also one-third of that for tumors in PB-animals, whereas no significant differences were observed with regard to the mean apoptotic index. In conclusion the inhibitory effect of PB seemed to be primarily caused by the suppression of tumor tell proliferation. Irrespective of the group, most lesions observed were basophilic hepatocellular adenomas or foci, positive for Bcl-2 oncoprotein, They were thus distinct from the eosinophilic Bcl-2- lesions that predominate with PB promotion after the initiation of adult B6C3F(1) mice. This age-dependent nature of initiation, together with the differential responses of Bcl-2+ and Bcl-2- lesions, may be responsible for the apparently contradictory outcomes of PB treatment in infant and adult B6C3F(1) mice.

Cryofiltration apheresis for major ABO-incompatible kidney transplantation

Tohru Tamaki, Mitsuko Tanaka, Masamichi Katori, Makoto Osanai, Mitsuo Yasuhara, Jun-Ichi Meguro, Kazutaka Kukita, Motoki Yonekawa, Akio Kawamura

Therapeutic Apheresis ( Blackwell Publishing Inc. ) 2 ( 4 ) 308 - 310 1998年

The barrier of ABO-incompatible kidney transplantation is the presence of anti-A and anti-B antibodies in the recipient's circulating blood. Double filtration plasmapheresis (DFPP) is usually used to eliminate those antibodies. We tried cryofiltration apheresis (CRYO) in 2 recipients. Patient 1 was a 45-year-old male with B, Rh(+). The titers of IgM anti-A antibody were only reduced from x64 to x32 by the end of 3 sessions of standard CRYO. Renal allografting was not performed. Case 2 was a 29-year-old male with B, Rh(+). CRYO was introduced for 3 sessions. The initial IgM and IgG titers were x128 and negative, respectively. The standard CRYO system was modified by temperature, treated volume, and filter pore size. The IgM anti-A antibody titer was markedly reduced to x2 after the final session of CRYO. The donor was a 56-year-old father with A, Rh(+). Tacrolimus, azathioprine, methylprednisolone, and antilymphocyte globulin were used as the introductory immunosuppression therapy.

DOI PubMed

Immunomodulation in transplant patients by cryofiltration

Akio Kawamura, Makoto Osanai, Motoki Yonekawa

Therapeutic Apheresis ( Blackwell Publishing Inc. ) 2 ( 3 ) 205 - 209 1998年

書評論文，書評，文献紹介等

Cryofiltration was applied to major ABO incompatible bone marrow transplant (BMT), renal transplant, and chronic renal rejection cases. The anti-ABO titers IgM and IgG pretreatment by cryofiltration were x64 and x8 in a major ABO incompatible BMT patient. After treatments, they were decreased to x16 and x2, respectively. BMT was performed successfully. In a case of ABO major incompatible renal transplant, anti-A-IgM before cryofiltration treatment was x128, and CH50 was 26.5. Three treatments were performed. As a result, the anti-A-IgM and CH50 levels were decreased to x4 and under 10. After renal transplantation, antibodies appeared again, but because the creatinine level was normal, cryofiltration was not performed again. Five cases of chronic rejection of renal transplants were treated by cryofiltration. They remained hemodialysis (HD) free for an average of 17.4 months, and the longest duration was 32 months. Cryofiltration is a good treatment for extracorporeal immunomodulation in transplantation.

DOI PubMed

Basic clinical study of an easy and effective leukocytapheresis by the use of nonwoven polyester filter

Akio Kawamura, Makoto Osanai, Masamichi Katori, Motoki Yonekawa, Masao Saitoh

Therapeutic Apheresis ( Blackwell Publishing Inc. ) 2 ( 4 ) 292 - 296 1998年

Leukocytapheresis (LCAP) is widely used for the treatment of immunological diseases. We studied a new treatment of LCAP using a nonwoven polyester filter. In a basic study, 30-70% of the lymphocytes were adsorbed. Also, 30-68% of the lymphocyte subsets were removed. This method was applied to 2 patients with corticosteroid-resistant active ulerative colitis. Erosion, edema, bleeding, ulcer formation, and stenosis of the colon were almost completely repaired after 6 LCAP treatments. LCAP using a nonwoven polyester filter will be a very useful treatment for immunological diseases and extracorporeal immunomodulation.

DOI PubMed

Phenobarbital causes apoptosis in conditionally immortalized mouse hepatocytes depending on deregulated c-myc expression: Characterization of an unexpected effect

M Osanai, K Ogawa, GH Lee

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 57 ( 14 ) 2896 - 2903 1997年07月

The CHST8 mouse hepatocyte cell line, conditionally immortalized with the temperature-sensitive SV40 large T antigen gene, rapidly proliferates at 33 degrees C with active expression of the c-myc proto-oncogene but, due to the heat-labile nature of the mutant T antigens, becomes growth arrested and morphologically senescent at 39 degrees C; this is accompanied by the disappearance of c-myc transcripts. In a previous study, we transfected the CHST8 cells at 33 degrees C with an activated c-H-ras or a c-myc, both of which are frequently involved in mouse hepatocarcinogenesis in vivo. When the temperature was shifted to 39 degrees C, cells with only one of the exogenous oncogenes did not escape from the senescence, but those containing both exhibited an immortal phenotype. In the present study, using this in vitro model of hepatocarcinogenesis, we demonstrated that phenobarbital, a tumor promoter of rodent hepatocarcinogenesis, triggers remarkable apoptosis specifically in the c-myc-transfected CHST8 cells at 39 degrees C, which show abundant c-myc expression despite growth arrest, Dissociation of p53 proteins from degrading T antigens followed by a phenobarbital and c-myc-dependent, 15-fold induction of Bax protein, known to activate the apoptotic pathway downstream of p53, occurred in association with this phenomenon. The effects of phenobarbital and c-myc in increasing Bax on shifting the temperature from 33 degrees C to 39 degrees C were additive, with both having similar degrees of influence on the protein level. Interestingly, subsequent introduction of an activated c-H-ras oncogene into the c-myc-transfected CHST8 cells resulted not only in escape from the growth arrest at 39 degrees C hut also in complete inhibition of the phenobarbital-inducible apoptosis along with de novo induction of the Bax antagonist, Bcl-2. These findings strongly suggest that the phenobarbital-inducible apoptosis is mediated by Bax. Although it is a common notion that phenobarbital promotes liver tumor development through suppression of apoptosis, our results, together with the known fact that phenobarbital occasionally inhibits hepatocarcinogenesis in mice, indicate a problematic complexity in its biological activities.

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