Biomedical subjects
S Nagamori
Publications and source records attributed to S Nagamori.
Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT.
Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.
Involvement of insulin-like growth factor binding protein-3 in the retinoic acid receptor-alpha-mediated inhibition of hepatocellular carcinoma cell proliferation.
We examined the relationship between the expression of retinoic acid receptor-alpha (RAR-alpha) and upregulation of insulin-like growth factor binding protein-3 (IGFBP-3) in the retinoid-induced inhibition of hepatocellular carcinoma (HCC) cell proliferation. HCC cell lines showed a marked expression of RAR-alpha, whereas the expression levels of RAR-beta and RAR-gamma were relatively lower. An RAR-alpha agonist significantly inhibited the HCC cell proliferation both in vitro and in vivo. The RAR-alpha expression closely related to the upregulation of IGFBP-3 as compared with RAR-beta or RAR-alpha expressions. RAR-alpha agonist would be beneficial to inhibit the growth of HCC.
Developments in bioartificial liver research: concepts, performance, and applications.
As an alternative to liver transplantation, numerous researchers have been working toward the goal of development of a fully functional artificial liver. In recent years, artificial liver support systems have been advocated as interim treatments for patients awaiting hepatocyte replacement therapy or liver transplantation; so-called "bridging" treatments. It is recognized that an effective artificial liver system requires: (1) a viable and highly functional hepatocyte cell line, (2) a suitable bioreactor environment and peripheral control systems, and (3) an effective extracorporeal circulatory system to incorporate an artificial liver system. Conventional systems have, however, suffered from various drawbacks, including incompatibility of cell cultures derived from non-human cells, insufficient cell proliferation, rapid deterioration of cellular function due to an impoverished cellular environment, and lack of system scalability. A newly established artificial liver system overcomes many of these problems and demonstrates a long-term capacity to maintain multiple liver-specific functions, such as protein synthesis, enzyme activity, and drug metabolism, both quantitatively and qualitatively. The present review provides an overview of the concepts underpinning artificial liver systems, the performance of presently available systems and the practical applications of available systems and those in development.
Two SecG molecules present in a single protein translocation machinery are functional even after crosslinking.
SecG, a membrane component of the protein translocation apparatus of Escherichia coli, undergoes membrane topology inversion, which is coupled to the membrane insertion and deinsertion cycle of SecA. Eighteen SecG derivatives possessing a single cysteine residue at various positions were constructed and expressed in a secG null mutant. All the SecG-Cys derivatives retained the SecG function, and stimulated protein translocation both in vivo and in vitro. Inverted membrane vesicles containing a SecG-Cys derivative were labeled with a membrane-permeable or -impermeable sulfhydryl reagent before or after solubilization with a detergent. The accessibility of these reagents to the cysteine residue of each derivative determined the topological arrangement of SecG in the membrane. Derivatives having the cysteine residue in the periplasmic region each existed as a homodimer crosslinked through disulfide bonds, indicating that two SecG molecules closely co-exist in a single translocation machinery. The crosslinking did not abolish the SecG function and the crosslinked SecG dimer underwent topology inversion upon protein translocation.
Nuclear translocation of Fos is stimulated by interaction with Jun through the leucine zipper.
Jun and Fos, b-ZIP transcription factors, form a heterodimer and bind to DNA enhancer elements, thereby regulating the expression of target genes. The present study was undertaken to investigate the molecular mechanism underlying nuclear translocation of the Jun/Fos complex. For this purpose, normal rat kidney cells were microinjected with a DNA expression vector containing wild-type or mutant c- or v-jun together with c- or v-fos, followed by detection of the subcellular localization of Jun or Fos by immunofluorescence staining. The nuclear accumulation of Fos was markedly enhanced by the presence of wildtype Jun, but not by Jun mutants lacking nuclear targeting or zipper dimerization functions, implying that Jun and Fos mutually interact via their leucine zippers and translocate from the cytoplasm to the nucleus using the markedly stronger nuclear localization signal of Jun.
Invasiveness of hepatocellular carcinoma cell lines: contribution of membrane-type 1 matrix metalloproteinase.
Intrahepatic metastasis is one of the malignant features of hepatocellular carcinoma (HCC). Matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (u-PA)/plasmin, are known to be associated with the invasive properties of various types of tumor cells. In this study, we examined which proteinases play a role in the metastatic invasion of human HCC cell lines. JHH-5 and JHH-6 cells constitutively expressed mRNAs for both membrane-type 1 matrix metalloproteinase (MT1-MMP) and u-PA and invaded through reconstituted MATRIGEL in vitro, whereas JHH-7 cells expressed u-PA mRNA but not MT1-MMP and did not invade. However, hepatocyte growth factor (HGF) induced MT1-MMP expression on the surface of JHH-7 cells and markedly increased invasiveness of JHH-7 in a concentration-dependent manner. Moreover, cleavage activity for pro-MMP-2 was induced in HGF-treated JHH-7 cells. MMP inhibitor, rather than serine proteinase inhibitor, potently inhibited HCC cell invasion. Intrahepatic injection of HCC cell lines into athymic nude mice caused visible intrahepatic metastases in vivo. Moreover, JHH-7 tumors showed expression of MT1-MMP mRNA, while in vitro cultured JHH-7 cells did not. These findings suggest that MT1-MMP plays an important role in the invasive properties of HCC cells, and that HGF modifies the invasive properties of noninvasive HCC cells.
Retinol esterification activity contributes to retinol transport in stellate cells.
The mechanisms of retinol transport and accumulation in hepatic stellate cells (HSC) remain to be elucidated. Our previous studies suggested that retinol esterification activity, particularly lecithin:retinol acyltransferase (LRAT) activity, in liver retinoid metabolism is important to elucidate the relationship between retinol uptake by HSC and the esterification of retinol. In the present study, using a human HSC-like cell line, LI90, we demonstrated that retinol esterification activity of LI90 cells is similar to that of primary cultures of rat HSC and higher than that of a human hepatoma cell line. Further, since progesterone or diphospho-lauroyl-phosphatidylcholine increased retinol esterification activity of LI90 cells, it is likely that LRAT contributes to retinol esterification in LI90. We examined retinol esterification in LI90 cells and clearance of retinol from culture medium. The percentages of both retinol and esterified retinol in LI90 cells increased in a manner dependent on retinol concentration in medium, whereas that of retinol in medium decreased. The percentages of esterified and unesterified retinol in LI90 cells and of retinol in medium were linearly dependent on the logarithm of the initial concentration of retinol in the medium. These results suggest that retinol esterification activity contributes to retinol uptake by HSC and maintenance of non-toxic retinol levels in plasma.
5-fluorouracil and low-dose recombinant interferon-alpha-2a in patients with hormone-refractory adenocarcinoma of the prostate.
BACKGROUND: The effectiveness of a chemotherapy regimen including 5-fluorouracil (5-FU) and recombinant interferon-alpha-2a (rIFN-alpha-2a) was evaluated in hormone-refractory prostate cancer patients. METHODS: Patients received a continuous intravenous infusion of 5-FU at 600 mg/m2/day for 5 days (D1-D5), followed by a bolus injection of 5-FU on D15 and D22. Patients received intramuscular injection of rIFN-alpha-2a at 3 million IU on D1, D3, D5, D15, and D22. This schedule was repeated every 4 weeks. RESULTS: Between 1993 and 1995, 23 patients with hormone refractory prostate cancer were enrolled in this study. Two of five patients with nodal disease exhibited partial responses according to the NPCP criteria. Fourteen of 17 patients with bone disease showed stable disease. Of 21 patients assessible for response, 9 patients had a decrease in the PSA level greater than 50% of baseline. Bone pain disappeared partially or completely in 8 of 14 patients with this symptom at entry. The median overall survival was 18 months. The associate toxicity was well tolerable. CONCLUSIONS: Combination chemotherapy of 5-FU and low dose rIFN-alpha-2a in patients with hormone-refractory prostate cancer proved feasible, and with acceptable toxicity.
Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample.
We constructed a full-length complementary DNA (cDNA) clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood from the carrier was eventually transfused to a patient who later developed typical posttransfusion hepatitis C. It was also shown to be infectious to chimpanzees. We obtained 12 overlapping cDNA fragments altogether, covering the entire HCV genome. By subcloning and sequencing, clones considered to constitute the major population were selected. We could also detect 98 base pairs of extra sequences at the 3' end of the genome. After confirming the overlapping sequences, we combined the fragments to make a full-length cDNA. The HCV population in the donor was heterogeneous, as determined by their nucleotide sequences of the hypervariable region in envelope protein, but a few virus clones were selected in the recipient after transmission. The similar convergence of the virus population was previously observed when the same blood sample was injected into a chimpanzee. Interestingly, virus clones isolated during the acute phase in the recipient and the chimpanzee had sequences in the hypervariable region identical to that of the full-length cDNA clone. The full-length cDNA clone of HCV constructed in this study may originate from infectious virus clones.
Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: ultrafine structure of functionally enhanced hepatocarcinoma cell lines.
With a view to initiating clinical trials, cell morphology and function for a newly developed artificial liver support system employing highly functional human liver cell line, FLC-7, cultured in a radial flow bioreactor were compared to cells grown in a conventional monolayer culture. The radial flow bioreactor consists of a vertically extended cylindrical matrix comprised of porous glass bead microcarriers through which liquid medium flows from the periphery in toward the central axis generating a beneficial concentration gradient of oxygen and nutrients, while preventing excessive shear stresses or buildup of waste products. The three-dimensional culture system supports high-density (1.1 x 10(8) cells/ml-matrix), large scale cultures (4.4 x 10(10) cells/400 ml-bioreactor) with long-term viability. Scanning and transmission electron microscopy (SEM and TEM) revealed that cells cultured in a monolayer system were flattened and extended with numerous cytoplasmic projections. Cells in the three-dimensional culture were spherical and covered with microvillilike processes resembling liver cells in vivo. The cells were solidly attached on the surfaces and within the pores of the microcarriers in highly dense colonies. The spherical cells remained in close contact with adjacent cells, while circulation of liquid medium flowed freely through spaces between cells. FLC-7 cells produced albumin at a rate of 6.41 micrograms/24 h/10(6) cells. Alpha-fetoprotein (AFP) production dropped nearly threefold in comparison to monolayer cultures. Results demonstrated that the new artificial liver support systems (ALSS) provides a superior three-dimensional culture environment that allows cells to perform at naturally functioning levels.
Treatment of metastatic nonseminomatous germ cell tumors of the testis: significance of the international consensus prognostic classification as a prognostic factor-based staging system.
BACKGROUND: We reviewed treatment results in patients with metastatic nonseminomatous germ cell tumors of the testis and examined the significance of the International Consensus Prognostic Classification to make appropriate risk-based decisions concerning induction chemotherapy. METHODS: We divided 37 patients treated with platinum-based combination chemotherapy into good, intermediate, and poor prognostic groups utilizing the International Consensus Prognostic Classification. The data was analyzed for both overall survival and progression-free survival among the 3 prognostic groups. RESULTS: Among the 37 patients, 10 died (8 of progressive disease, 1 of pneumonia during induction chemotherapy and 1 of cyclophosphamide-induced hemorrhagic cardiomyolitis during salvage chemotherapy). The survivors were followed for 6 to 1 84 months from the beginning of induction chemotherapy (median, 80 months). Five of the 37 patients (14%) were classified as having a good prognosis, 1 8 (48%) as intermediate, and 14 (38%) as having a poor prognosis. The patients in the poor prognostic group had a 5-year overall survival of only 40%, while those in the good and intermediate groups had 5-year overall survivals of 100% and 94%, respectively. When we applied the International Consensus Prognostic Classification to patients with advanced disease classified by the Indiana University Staging System, these patients could be clearly divided into good-risk and poor-risk groups. CONCLUSIONS: The International Consensus Prognostic Classification is easily applicable and accurate for risk assessment in patients with metastatic nonseminomatous germ cell tumors of the testis. This classification will now be widely used in general oncology practices and for clinical trials in these patients.
High density culture of immortalized liver endothelial cells in the radial-flow bioreactor in the development of an artificial liver.
Liver endothelial cells are important components of the tissue along the hepatic sinusoid. They are responsible for microcirculation in the liver and scavenger functions. It would therefore be important to include these cells in any hybrid type of artificial liver in addition to hepatocytes. However, it is difficult to culture these cells in vitro. The development of a liver endothelial cell line, which maintains the characteristics of the primary culture, would thus be of great benefit in the development of an artificial liver. In the present study we established immortalized liver endothelial cells from the liver of an H-2Kb-tsA58 transgenic mouse, which harbors the SV40 TAg gene. Hepatic sinusoidal cells isolated from H-2Kb-tsA58 mouse proliferated in the presence of gamma-interferon at 33 degrees C. Four clones were established, out of which clone M1 had the highest amounts of PGI2 production, as well as plasminogen activator activity and internalized acetylated low density lipoprotein. On culture dishes the M1 cells grew individually and spread. Sieve plates on the cell surface were not readily visible, but small pores were detected under electron microscopic observation. These results suggest that M1 clone cells originated from liver endothelial cells. Moreover it was possible to culture the immortalized liver endothelial cells in a radial-flow bioreactor for 5 days, with a maximum 6-keto prostaglandin F1alpha production of 25 microg per day. This suggests that immortalized liver endothelial cells and a radial-flow bioreactor can prove useful tools in the development an artificial liver.
Persistence of hepatitis C virus RNA in established human hepatocellular carcinoma cell lines.
The persistence of the viral RNA of hepatitis C virus (HCV) was examined in 13 hepatocellular carcinoma (HCC) and two hepatoblastoma cell lines by reverse transcription followed by the polymerase chain reaction (RT-PCR). HCV RNA was detected in three HCC lines (JHH-1, JHH-4, and JHH-6) and negative-strand viral RNA was found in JHH-4, indicating that there is a putative replicative intermediate of HCV in JHH-4 cells. To rule out the possibility of contamination, the partial nucleotide sequences of HCV-specific PCR products of these three cell lines were determined. The clone from JHH-1 belonged to genotype 1 (1a or 1b), and the clones from JHH-4 and JHH-6 belonged to genotype 2b, but their sequences differed from each other. These cell lines may be useful for studies related to HCV.
Mother-to-child transmission of a hepatitis C virus variant with an insertional mutation in its hypervariable region.
BACKGROUND/AIMS: We have analyzed the molecular basis of mother-to-child transmission of hepatitis C virus. METHODS/RESULTS: Healthy pregnant women were screened for anti-HCV antibody and babies born to hepatitis C virus carrier mothers were prospectively investigated. Among the 35 pairs studied, the hepatitis C virus genome was detectable in only one baby, who did not show any significant symptoms of hepatitis. The viral load in the blood of the mother was one of the highest of the 35, and the population of the hepatitis C virus genome was heterogeneous. Furthermore, she was found to have a mixed infection with type 1a and type 1b hepatitis C virus. However, the hepatitis C virus genome obtained from the baby was only from type 1b, less heterogeneous and composed of the clones which were detected in the blood of the mother. The selected hepatitis C virus had a 12-nucleotide insertion in the amino-terminus of the E2 hypervariable region of the genome. CONCLUSIONS: The incidence of mother-to-child transmission of hepatitis C virus from carrier mothers was shown by this prospective study to be low. The presence of selection pressure during transmission was suggested. The biological significance of the virus with 12-nucleotide insertion has to be determined.
Intraocular hemorrhage developing during interferon therapy.
A 43-year-old man with chronic active hepatitis C was treated with interferon-beta (IFN-beta) at a dosage of 6 x 10(6) IU/day for a planned 6-week period. Ocular hyperemia, ophthalmalgia, and increased intraocular pressure in the right eye developed 20 days after the start of treatment. Intraocular pressure remained high, even after discontinuation of IFN therapy, laser therapy, and iridectomy. Two days later, the right eye was removed because perforation had occurred. The ocular symptoms that developed in this case were thought to have been caused and exacerbated by IFN administration.
[Treatment of advanced testis cancer with a high dose chemotherapy regimen (PVeBV)].
BACKGROUND: The prognosis of patients with testis cancer classified as being in the advanced extent according to the Indiana University staging system is still poor even when treated with cisplatin based chemotherapy. METHODS: Attempting to increase the efficacy of chemotherapy in this high risk group, we have adopted PVeBV chemotherapy (high dose CDDP+VBL+VP-16+BLM) for recent 8 patients with such advanced conditions. In this study, we analized the treatment outcome of those patients retrospectively. RESULTS: Two patients died during the first course of PVeBV chemotherapy due to cancer progression, while 6 patients treated with 3 to 4 cycles of PVeBv were eligible and assessable for response, survival, and toxicity. Five of those 6 achieved pathological CR (pCR) following surgical resection of residual masses after 3 cycles of PVeBV. The other case was saved by salvage chemotherapy with autologous BMT. All 6 patients were long-term disease free survivors in median follow up of 46 months. With the rG-CSF application and vigorous hydration, acute phase toxic effects (myelosuppression, pulmonary fibrosis and nephrotoxicity) were manageable in this intensive regimen. Long term toxic effects such as peripheral neuropathy and ototoxicity were also tolerable and quality of life in such advanced cancer patients was preserved well. CONCLUSION: To improve a cure rate of high risk testis cancer, the dose escalation of induction chemotherapy should be considered.
[Tumor size and DNA ploidy changes in renal cell carcinomas--flow cytometric analysis of DNA content in renal cell carcinomas associated with von Hippel-Lindau disease].
BACKGROUND: Renal cell carcinomas (RCCs) develop in 8-63% of von Hippel-Lindau disease (VHL) patients, and loss of 3p segments, chromosome aberrations found in 90% of sporadic RCCs, has also been observed in RCCs associated with VHL. In fact, comparative analysis showed that the chromosome aberrations in RCCs associated with VHL are similar to those found in sporadic RCCs. VHL patients have the whole spectrum of tumors from small early lesions to large ones in the same kidney, providing a unique opportunity to analyze tumors in different stages of development. Subsequently deoxyribonucleic acid (DNA) content in RCCs of VHL patients was examined and correlated to their tumor size to gain some insight in the progression of sporadic RCCs. METHODS: From 1988 to 1991, we have experienced 6 cases of RCCs associated with VHL who underwent partial or radical nephrectomy. A total number of 52 paraffin-embedded samples from 33 RCCs from 6 patients with VHL was analyzed by flow cytometry. RESULTS: The sizes of tumors ranged from 0.2 to 8.2 cm. DNA aneuploid patterns demonstrated in none of 9 tumors less than 1.6 cm, 4 of 14 tumors (29%) as large as 1.6 to 2.5 cm, and 5 of 10 tumors (50%) larger than 2.5 cm (p < 0.05). Twelve tumors less than 1.8 cm showed DNA diploid, so the smallest size of aneuploid tumors was 1.8 cm. CONCLUSION: These data suggest that DNA ploidy change (diploid to aneuploid) in RCCs probably takes place as tumors grow approximately 1.8 cm in size.