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Biomedical subjects

K Ohtake

Publications and source records attributed to K Ohtake.

At least 37 records · Page 2Linked to original sources
PubMed1993

Malignant transformation of a mouse liver epithelial cell line by transfection of an activated c-H-ras gene with a point mutation at codon 12.

In order to scrutinize the reason why in mouse liver system only activated H-ras gene with a point mutation at codon 61 but not codon 12 is frequently seen although the latter mutation is highly frequent in methylnitrosourea-induced rat mammary tumors, transforming activity of these two types of mutated H-ras gene was investigated utilizing an immortalized but not fully transformed mouse liver epithelial cell line MLE-10, established in our laboratory. MLE-10 cells were transfected with activated human c-H-ras gene having a point mutation at either codon 12 (PT24) or 61 (PSK2), together with PSV2neo, or with PSV2neo only. G418 resistant colonies, propagated separately, gave rise to 6, 3 and 6 lines respectively. All the PT24 and PSK2-transfected cell lines were growth capable in both soft agar and nude mouse subcutis, with similar growth rate and morphological features whereas none of the cell lines transfected with the PSV2neo only revealed such growth capability. The results thus revealed that c-H-ras with a mutation at codon 12 has oncogenic activity to the mouse hepatocyte, although after immortalization, at the degree similar to the same gene with a mutation at codon 61.

Animals↗
PubMed1993

Integrin VLA-5 negative primary plasma cell leukemia.

We present a case of primary plasma cell leukemia with Bence Jones proteinuria. After combination chemotherapy, leukemic cells and the urinary levels Bence Jones protein were decreased. Small lytic bone lesions were detected only in the skull. Typical plasma cells were rarely seen in peripheral blood on the hyperleukocytic phase, however they were increased in the advanced stages. The most important diagnostic sign was persistent expression of CD38 antigen on leukemic cells throughout the entire course of the illness and these leukemic cells expressed very late antigen-4 (VLA-4) but not VLA-5.

ADP-ribosyl Cyclase↗
PubMed1993

Hepatocyte growth factor transforms immortalized mouse liver epithelial cells.

Transforming activity of hepatocyte growth factor (HGF) was demonstrated utilizing immortalized but not fully transformed mouse hepatocytes (MLE-10). Rat HGF cDNA, expressed under the control of a cytomegalovirus promoter, was transfected together with the neomycin resistance gene (PSV2neo) into MLE-10 cells by the calcium phosphate method, and propagated G418-resistant colonies were harvested colony by colony. After checking for integration and expression of exogenous HGF, five cell lines (MLE-10-HGF-1-5) were established. Three cell lines transfected with the vector only (MLE-10-CMV-1-3) were also established in the same manner. All MLE-10-HGF cell lines grew much faster than the MLE-10-CMV and original MLE-10 cells in culture and produced large colonies in soft agar, which colony production was blocked by the addition of anti-HGF antibody to the agar. After addition of HGF, original and MLE-10-CMV lines produced colonies in soft agar. The high-HGF-production lines (MLE-10-HGF-4 and -5) also gave rise to tumors within 2 weeks when implanted into the nude mice subcutis. In contrast, all MLE-10-CMV and original MLE-10 cells were negative in these growth assays. A rough parallelism between the level of HGF expression and the growth rate in both soft agar and nude mice subcutis was evident among MLE-10-HGF cell lines. Those with higher HGF production tended to grow in a scattered fashion in culture. High-affinity HGF receptor, HGFR/met, was expressed in MLE-10 and all the derived cell lines. Since HGF and/or HGFR/met gene expression is seen in various tumors and the serum HGF level is elevated in patients with hepatic disease, the present results indicate a possible significance of HGF and its receptor system in carcinogenesis, most probably via autocrine and/or paracrine mechanisms.

Animals↗
PubMed1992

Clonal growth of hepatitis B virus-integrated hepatocytes in cirrhotic liver nodules.

A total of 83 cirrhotic nodules (pseudolobules) individually collected from 11 cirrhotic livers of hepatitis B virus carrier patient were analyzed for the frequency and mode of hepatitis B virus integration as well as histological features. Southern blot analysis disclosed discrete bands at higher molecular weight region in 26 of 83 nodules (31.3%), indicating a clonal growth of hepatocytes with viral integration. Considerable variation (0-75%) existed in the positive rates for discrete bands in nodules among livers. Molecular cloning revealed the sequence flanking an integrated viral sequence to be host DNA and thus confirmed true integration. Histological analysis, however, did not reveal any neoplastic-appearing foci of growth within nodules, despite the fact that the detection sensitivity would predict clones of more than 10(5) cells to give rise to clonal integration patterns on Southern blot analysis. The question of whether clonal expansion of hepatocytes reflects any viral integration-associated growth advantage and/or a preneoplastic condition awaits future studies.

Adult↗
PubMed1992

Alteration of rat liver proteoglycans during regeneration.

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.

Animals↗
PubMed1992

Frequent spontaneous sister chromatid exchange in hepatocytes of transgenic mice harboring the SV40-T antigen gene.

In order to shed light on the causal mechanisms of hepatocarcinogenesis in the transgenic mouse into which the albumin-promotor-regulated SV40-T antigen gene has been introduced (T+ mouse), and especially on the frequent chromosomal aberrations seen in cultured hepatocytes and hepatocellular neoplasms derived from such animals, the frequency of sister chromatid exchange (SCE) and karyotype abnormalities were investigated in a hepatocyte primary culture system. Cells were obtained through collagenase perfusion from T+ mice at 16-18 days of age, when no morphological changes are apparent, and from nontransgenic littermates, and cultured in the presence of bromodeoxyuridine. SCE was seen in transgenic hepatocytes twice as frequently as in their normal counterparts. No karyotype abnormalities in terms of numerical change or gross aberration were detected at this phase. The results thus suggest mutagenic properties for the T antigen, which may play an important role in hepatocarcinogenesis in this transgenic mouse.

Animals↗
PubMed1992

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration.

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.

Animals↗
PubMed1992

The role of radiotherapy in the management of salivary gland carcinomas.

A retrospective study was performed on 44 patients with carcinoma originating in the major and minor salivary glands to examine the effects of postoperative radiotherapy on locoregional and distant control and survival. 22 patients were treated by surgery alone and the 22 other patients were treated by a combination of surgery and postoperative irradiation. In the surgery group, local recurrence developed in all 8 patients with evidence of residual disease at the surgical margins, whereas local control was achieved in 7 of 15 patients with positive surgical margins in the combination group and the control rate was related to the amount of residual disease. Neck metastasis, which developed in 13 patients (30%), was not affected by the status of surgical margins or by the treatment modality. On the other hand, the incidence of distant metastasis seen in 19 patients (43%) was much higher in patients with positive surgical margins and the development of distant metastasis in these patients was not prevented by postoperative irradiation. The survival rates at 5, 10 and 15 years were 54, 48 and 41%, respectively, for the irradiated patients, whereas the values for the patients treated by surgery alone were 75, 70 and 70%, respectively. The results indicate that postoperative irradiation is effective in controlling local recurrence but not neck and distant metastases. Wide excision with sufficient surgical margins followed by postoperative radiotherapy and systemic chemotherapy are essential to obtain a better prognosis.

Adolescent↗
PubMed1992

[Prognostic significance of echogenic lesion within small hepatocellular carcinoma].

To evaluate prognostic significance of echogenic lesion within small hepatocellular carcinoma (SHCC, less than or equal to 2 cm in diameter), clinical and pathological findings of 32 cases with SHCC containing echogenic lesion (echogenic SHCC) were compared with those of 55 cases with non-echogenic SHCC. Compared with the non-echogenic SHCC group, the frequency of clinical stage I was significantly higher, and there were significantly more cases with solitary tumor relative to cases with multiple tumors in the echogenic SHCC group. Histologically, the incidence of the HCC composed of well-differentiated tumor cells corresponding to Edmondson's grade I was significantly higher in the echogenic SHCC group than in the non-echogenic SHCC group. Although HCCs tended to become progressively less differentiated with increasing tumor sizes in the both groups, the process of cellular change appeared to proceed more slowly in the echogenic SHCC group. Survival rate after tumor detection was 73% at three years, 56% at five years and 48% at seven years and nine years in the echogenic SHCC group, while it was 46% at three years, 42% at five years and 0% at seven years in the non-echogenic SHCC group. The present results showed that the presence of echogenic lesion within SHCC could be useful prognostic indicator.

Aged↗
PubMed1991

The value of single versus multiple sections for detection of lymph node metastasis.

This study was undertaken to determine the value of serial sectioning of lymph nodes as an aid in optimally examining cervical specimens for metastasis. The presence or absence of lymph node metastasis in 802 lymph nodes obtained from 51 consecutive neck dissection specimens were initially determined by the routine method, examination of one section from each node. This examination revealed 40 lymph nodes with metastatic involvement. Further study of the 716 lymph nodes that were initially interpreted as free of tumor by serial sectioning added only two positive nodes. Thus, serial sectioning did not contribute significantly to the detection of lymph node metastasis.

Humans↗
PubMed1991

Low frequency of ras activation in 2-acetylaminofluorene- and 3'-methyl-4-(dimethylamino)azobenzene-induced rat hepatocellular carcinomas.

Activation of ras protooncogenes in 11 2-acetylaminofluorene (AAF)-induced hepatocellular carcinomas (HCCs) or liver cell lines and 9 3'-methyl-(dimethylamino)azobenzene (3'-Me-DAB)-induced HCCs in rats was examined using the NIH3T3 cell transfection assay and oligonucleotide hybridization analysis. Only one cell line established from a AAF-treated rat liver demonstrated transforming activity with a point mutation and ATTA transversion at the second position of H-ras codon 61. The rates of ras activation were thus very low for both AAF- and 3'-Me-DAB-induced rat HCCs, the results thus extending and confirming the findings indicating that ras activation in rat HCCs induced by various type of carcinogens is infrequent.

2-Acetylaminofluorene↗
PubMed1991

In vitro progression-associated c-H-ras activation in neoplastic hepatocyte lines established from SV40-T antigen gene-harboring transgenic mice.

In order to investigate the molecular mechanisms of multistep hepatocarcinogenesis in SV40-T antigen gene-harboring transgenic mice, 9 hepatocellular carcinoma (HCC) lines and 10 "preneoplastic" hepatocyte lines were established from the animals and their biological and molecular changes during culture were investigated. Three of the 9 HCC lines showed progression during culture in terms of growth rate and growth capability in soft agar and in nude mice. This progression was associated with the appearance of activated c-H-ras oncogene. Including these 3 lines, H-ras activation was observed in a total of 7 of the 9 HCC lines (78%), whereas it was found only in 1 of 10 (10%) "preneoplastic" hepatocyte lines. These data thus indicate that H-ras activation may be an event occurring at a relatively late stage of hepatocarcinogenesis in this transgenic mouse system and that it may serve towards completion of the carcinogenic process together with the T-antigen.

Animals↗
PubMed1991

c-Ki-ras point mutations in ductectatic-type mucinous cystic neoplasms of the pancreas.

Ductectatic-type mucinous cystic neoplasms of the pancreas constitute a recently recognized new human pancreatic tumor entity. Examination for the presence of point mutations at codon 12 of K-ras by oligonucleotide hybridization in 5 adenomas and 3 carcinomas revealed alteration in 3 and 2, respectively. In 4 of these positive cases, the transition was GGT----GAT (Gly----Asp) with the remaining one, found in a cancer, being GGT----GTT (Gly----Val). In two carcinoma cases, the same point mutation was detected both in the carcinoma area and in a coexisting adenoma component. Thus K-ras point mutation appears to be associated with this particular type of neoplasm in the same manner as observed for typical exocrine pancreas carcinomas. Our study also indicates the possible existence of an adenoma-carcinoma sequence in the evolution of this type of neoplasm and we suggest that K-ras activation may be an important event in the phase of adenoma development.

Adenoma↗
PubMed1991

Hepatocarcinogenesis in transgenic mice carrying albumin-promoted SV40 T antigen gene.

We have developed transgenic mice that inherit albumin promoter-regulated simian virus 40 (SV40) large T antigen gene, expressed specifically in hepatocytes. These mice all develop multifocal hepatocellular carcinomas at around 5 months and die of liver insufficiency by 7 months. Sequential morphological observation of hepatocarcinogenesis revealed 5 distinct stages: (I) newborn to 2 weeks of age, neither recognizable histological changes nor cellular replication in spite of T antigen expression; (II) between 3 and 7 weeks, diffuse cytomegalic change of hepatocytes with numerous abnormal mitoses, usually resulting in cell death; (III) from 7 weeks onwards, quasi-regenerative small hepatocyte foci with a decreased tendency for cytomegaly in spite of T antigen expression, rapidly replacing the hepatic tissue; (IV) 11 weeks of age and thereafter, neoplastic foci and nodules with enzymatic alteration; (V) 20 weeks of age and thereafter, gross hepatocellular carcinomas with occasional pulmonary metastases. Considerable variation existed both in morphological and enzymatic features and T antigen expression among neoplastic lesions, including carcinomas. Thus, these transgenic mice clearly show a multistep process in hepatocarcinogenesis with remarkable synchrony and provide a promising model for analyzing the essential events of carcinogenesis at different stages.

Animals↗
PubMed1991

Changes of lung surfactant and pressure-volume curve in bleomycin-induced pulmonary fibrosis.

We investigated whether alveolar surface force increased and participated in the lung pressure-volume relationship in bleomycin-induced pulmonary fibrosis in hamsters and, if so, whether lung surfactant was hampered in the lungs. On the air-filled pressure-volume curve, decreases of lung volume from control level were significantly higher at 3-8 cmH2O pressure on day 10 than on day 30. Because the change of lung tissue elasticity evaluated from the saline-filled pressure-volume curve was equal for the 2 days, the higher decrease of air volume on day 10 was due primarily to contribution of alveolar surface force. Pressure differences between deflation limbs of air-filled and saline-filled pressure-volume curves, which represented net alveolar surface force, were significantly higher at any lung volume between 50 and 90% total lung capacity on day 10, but almost no significance was observed on day 30. Phospholipid concentration in bronchoalveolar lavage fluid significantly decreased on day 10 but had improved by day 30. Analysis of phospholipid species in purified lung surfactant showed decreased fractions of disaturated phosphatidylcholine and phosphatidylglycerol on day 10. Surface-active properties of the surfactant, measured by a modified Wilhelmy balance, were remarkably hampered on day 10, but most of them had improved by day 30. We consider that the quantitative and functional abnormalities of lung surfactant have a part in the aggravation of lung mechanics in the acute phase of pulmonary fibrosis.

Animals↗
PubMed1991

Multistep hepatocarcinogenesis in transgenic mice harboring SV40 T-antigen gene.

We have developed transgenic mice that inherit albumin promoter-regulated simian virus 40 (SV40) large T-antigen gene, expressed specifically in hepatocytes. These mice all develop multifocal hepatocellular carcinomas (HCCs) at around 5 months and die of liver insufficiency by 7 months in remarkable synchrony. The liver tissue appears normal in the initial 3 weeks, and thereafter rapid cytomegalic degeneration of original hepatocytes, proliferation of quasi-regenerative hepatocytes, neoplastic cell foci, nodules and finally HCCs develop in sequence. Considerable variation existed both in morphological and enzymatic features and in T-antigen expression among neoplastic lesions, including carcinomas. Oligonucleotide hybridization studies revealed activating point mutations of the c-H-ras oncogene in 40% (10/25) of tumors obtained at around 6 months. The positive signals were, however, considerably weaker in half of them suggesting that such tumors comprised cells both with and without ras mutation. Sequential observation of culture cell lines established from tumors also revealed the appearance of activated c-H-ras with time, which was associated with biological progression. Thus, in this model system, ras activation may be an event occurring in a relatively late phase of carcinogenesis associated with progression of tumors. Karyotype analysis of cell lines revealed remarkable chromosomal instability. In studies of sister chromatid exchange in hepatocytes, twice as frequent occurrence was demonstrated in transgenic mice as in their counterpart hepatocytes. Thus T-antigen may be contributing as a mutagen to the hepatocarcinogenesis, in addition to its cytotoxic and growth modifying activities.

Animals↗
PubMed1991

[Evaluation of the dose of endotoxin to produce endotoxin shock models in dogs].

Intravenous bolus administration of purified endotoxin (ET) is one of the most popular methods of producing experimental ET shock. In an attempt to evaluate the dose of ET in these experimental models, the kinetics of ET was assessed in various doses (1-0.002 mg.kg-1), and dose response study was performed, measuring chronological changes in hemodynamic (mean arterial pressure & cardiac output) and hematological (leucocyte & platelet counts) parameters. It was demonstrated that plasma ET concentrations were sustained extraordinarily high (greater than 5000 pg.ml-1) in doses of 0.2-1 mg.kg-1 during the observation period of several hours, while they were measurable in doses of 0.02 and 0.002 mg.kg-1, showing a biphasic decrease pattern after ET administration. In particular, plasma ET levels of low dose group (0.002 mg.kg-1) were similar to those of clinical gram negative septic patients. We conclude that the empirically used ET doses of more than 1 mg.kg-1 produce extremely severe endotoxemia and that milder degree of ET shock models produced by low dose of ET (0.002 mg.kg-1) would be more applicable for a pathophysiological study of ET shock.

Animals↗