Makoto Takahama

Hypomethylation of CpG Sites and c-myc Gene Overexpression in Hepatocellular Carcinomas, but Not Hyperplastic Nodules, Induced by a Choline-deficient L-Amino Acid-defined Diet in Rats

We have investigated aberrant methylation of CpG nucleotides (CpG sites) and gene expression of c‐myc during hepatocarcinogenesis induced by a choline‐deficient, L‐amino acid‐defined (CDAA) diet in rats. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 50 and 75 weeks and then killed. Macroscopically detectable nodules, which were histologically confirmed to be hyperplastic nodules (HNs) or well‐differentiated hepatocellular carcinomas (HCCs), were dissected free from the surrounding tissue. Normal control liver was obtained from 6‐week‐old rats. Methylation of CpG sites of the c‐myc gene was investigated in bisulfite‐treated DNA isolated from normal liver, HNs and HCCs. All 33 cytosines in the 5′‐upstream region of the c‐myc gene were fully methylated in control liver and the 4 HNs. In contrast, these cytosines were completely unmethylated in 5 HCCs. Examination of the c‐myc expression by reverse transcription‐polymerase chain reaction (RT‐PCR) analysis also showed a marked increase as compared to the low levels in normal livers and HNs. These results suggest that hypomethylation of the c‐myc gene might play a critical role in malignant transformation from HN to HCC during CDAA diet‐induced hepatocarcinogenesis in rats.

Frequent mutations of Ki‐ras but no mutations of Ha‐ras and p53 in lung lesions induced by N‐nitrosobis(2‐hydroxypropyl)amine in rats

Point mutations of the Ki‐ras and p53 genes in rat lung lesions induced by N‐nitrosobis(2‐hydroxypropyl)amine (BHP) were investigated by polymerase chain reaction‐single‐strand conformation polymorphism analysis followed by direct sequencing using paraffin‐embedded tissues. Male Wistar rats 6 wk old were given 2000 ppm BHP in drinking water for 15 wk. Another group was given drinking water without BHP. The rats were killed 20–27 wk after the beginning of the experiment. Lung adenomatous and squamous lesions, including carcinomas, were induced. The frequencies of Ki‐ras mutations were 40% (six of 15) in alveolar hyperplasias, 36% (five of 14) in adenomas, 72% (18 of 25) in adenocarcinomas, 20% (three of 15) in squamous metaplasias, 50% (three of six) in squamous cell carcinomas, and 50% (five of 10) in adenosquamous carcinomas. The mutations were all G → A transitions at the second position of codon 12; no other mutations were detected. However, Ha‐ras mutations in exons 1 and 2 and p53 mutations in exons 5, 6, and 7 were not detected in adenocarcinomas and squamous cell carcinomas. These results indicate that Ki‐ras mutation is an early genetic event in some adenomatous and squamous lung carcinogenesis and that Ki‐ras mutations can cause benign lesions to convert to malignant lesions. The results also show that Ha‐ras and p53 mutations are not involved in rat lung carcinogenesis induced by BHP.

Overexpression of matrix metalloproteinase (MMP)-9 correlates with metastatic potency of spontaneous and 4-hydroxyaminoquinoline 1-oxide (4-HAQO)-induced transplantable osteosarcomas in rats

In the present experiment, the expression of matrix metalloproteinase (MMP)-2 and MMP-9, key proteins in the MMP family, and the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, antagonistic proteins against MMP-2 and MMP-9, respectively, were investigated by Northern blot analysis in rat transplantable osteosarcomas with high and low metastatic potencies. Two transplantable osteosarcomas, one induced with the carcinogen, 4-hydroxyaminoquinoline 1-oxide (4-HAQO) (COS, chemical carcinogen-induced osteosarcoma), and the other, a spontaneous lesion (SOS, spontaneous osteosarcoma), were repeatedly transplanted from lung nodules to generate lines with high metastatic potency, C-SLM (chemical carcinogen-induced osteosarcoma, selected lung metastatic lesions) and S-SLM (spontaneous osteosarcoma, selected lung metastatic lesions), respectively. MMP-9 was overexpressed in both S-SLM and C-SLM, and TIMP-2 in the case of S-SLM. Neither MMP-2 nor TIMP-1 was overexpressed in either of the transplantable osteosarcomas with high metastatic potentials. The active form MMP-9, studied by zymography, increased in S-SLM and C-SLM but not in SOS and COS. MMP-9 mRNA expression was highly correlated with the gelatinolytic activity of active form MMP-9 (r = 0.85, P < 0.0001) and with the activation ratio of MMP-9 (r = 0.83, P < 0.0001). However, the active form MMP-2 was not detectable in all cases. These results suggest that overexpression of MMP-9 mRNA is one of the essential factors in the acquisition of metastatic potential in rat transplantable osteosarcomas.

Expression of vascular endothelial growth factor and its receptors during lung carcinogenesis by N‐nitrosobis(2‐hydroxypropyl)amine in rats

The expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), VEGFR‐1/Flt‐1 and VEGFR‐2/Flk‐1, was investigated by immunohistochemical and northern blot analysis during lung carcinogenesis by N‐nitrosobis(2‐hydroxypropyl)amine (BHP) in male Wistar rats. After BHP was given in the drinking water for 12 wk, the rats were maintained without further treatment until they were killed at 20–28 wk. Immunohistochemical studies revealed VEGF expression in almost all malignancies, the reaction being strongly positive in most adenocarcinomas (15 of 18; 83.3%) and squamous cell carcinomas (four of five; 80.0%), but less so in a total of 120 adenomas and 136 alveolar hyperplasias. In addition, VEGF mRNA and VEGFR mRNAs were found to be overexpressed in most adenocarcinomas and squamous cell carcinomas as well as in one to three of the five adenomas tested. The results indicated that VEGF and VEGFRs play important roles in the acquisition of malignant potential by preneoplastic lung lesions induced by BHP in rats. Moreover, overexpression of VEGF was related to upregulation of VEGFR‐1/Flt‐1 and VEGFR‐2/Flk‐1 expression in malignant and premalignant lung lesions. Mol. Carcinog. 24:287–293, 1999.

Jugular bulb venous oxygen saturation during one-lung ventilation under sevoflurane- or propofol-based anesthesia for lung surgery.

OBJECTIVE During one-lung ventilation (OLV), systemic oxygenation can be compromised. In such a scenario, if anesthetic techniques were used that adversely affected cerebral oxygen balance, the risk for impaired cerebral oxygen balance may be increased. In this study, jugular bulb venous oxygen saturation (SjO(2)) during OLV under sevoflurane- or propofol-based anesthesia for lung surgery was investigated. DESIGN Prospective clinical study. SETTING University hospital. PARTICIPANTS Fifty-two adult patients scheduled for elective thoracic procedures in the lateral position. INTERVENTIONS Patients were randomly allocated to either the sevoflurane or propofol group (n = 26). General anesthesia was maintained with sevoflurane or propofol combined with epidural anesthesia. MEASUREMENTS AND MAIN RESULTS Arterial and jugular bulb blood samples were measured before OLV, 15 minutes after OLV, 30 minutes after OLV, and 15 minutes after the termination of OLV. SjO(2) values in both sevoflurane and propofol groups significantly declined during OLV (p < 0.05). SjO(2) values in the sevoflurane group were higher than in the propofol group, although SaO(2) values were similar (p < 0.05). Regarding the incidence of SjO(2) <50% (cerebral oxygen desaturation), there were significant differences between the sevoflurane group and the propofol group during both normally ventilated conditions (0% v 7.7%, p < 0.05, relative risk [RR]: not applicable) and OLV (1.9% v 26.9%, p < 0.05, RR = 14; 95% confidence interval [CI] 1.91-103). Significant increase in the incidence of SjO(2) <50% during OLV was also observed only in the propofol group (from 7.7% to 26.9%, p < 0.05, RR = 3.5; 95% CI 1.29-12.4). CONCLUSION Cerebral oxygen desaturation was more frequently detected during OLV under propofol- versus sevoflurane-based anesthesia. Cerebral oxygen balance during OLV for lung surgery was less impaired under sevoflurane-based anesthesia compared with propofol; however, the clinical outcome or implications for cognitive function need to be determined.

Tracheal allotransplantation maintaining cartilage viability with long-term cryopreserved allografts

BACKGROUND Cartilage viability of a cryopreserved tracheal allograft seems to affect graft function and durability. We previously reported the influence of warm ischemia and cryopreservation on cartilage viability of tracheal allografts. For the clinical application of tracheal allotransplantation, it is essential to preserve grafts for a long time. In this study, we assessed cartilage viability of tracheal allografts after long-term cryopreservation in transplantation models. METHODS The tracheas were harvested from Lewis rats. The grafts were frozen to -80 degrees C in a programmable freezer immediately after being harvested and were then stored in liquid nitrogen (-196 degrees C) for different lengths of preservation (1, 2, 6, 9, 12, 18, and 24 months; n for each group = 8). Cartilage viability was evaluated by estimating proteoglycan synthesis. After harvest or thawing of the tracheas, the cartilage was labeled with 4 muCi/mL of Na2 35SO4. Specimens were then hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by a liquid scintillation counter. 35Sulfur incorporation before and after cryopreservation was examined in each group. Tracheal allotransplantation was performed using Lewis rats as donors and Brown Norway rats as recipients. RESULTS The average 35S incorporation in the cartilage before cryopreservation was 224 +/- 17 disintegrations per minute per milligram of tissue protein. The average 35S incorporation in the cartilage after cryopreservation decreased to 67% to 76% compared with that before cryopreservation. There were no significant differences among the groups in 35S incorporations after cryopreservation. Histologic examination after transplantation revealed normal tracheal cartilage in all groups. CONCLUSIONS The viability of tracheal cartilage after cryopreservation decreased to 67% to 76%. There were no significant differences in viability of cartilage among the tracheas after different lengths of cryopreservation. Tracheal allotransplantation after long-term cryopreservation can be safely performed in the rat model.

Induction of telomerase activity during regeneration after partial hepatectomy in the rat

The regulation of telomerase activity during regeneration induced by two-thirds partial hepatectomy (PH) was investigated in 6-week-old male F344 rats. Groups of animals were serially sacrificed 0, 6, 16, 24, 36 and 72 h after the operation and telomerase activity was determined by the telomeric repeat amplification protocol (TRAP) assay followed by densitometric quantification. DNA synthesis was immunohistochemically quantified in terms of bromodeoxyuridine (BrdU) incorporation. The expression levels of telomerase RNA were examined by Northern blot analysis. Telomerase activity was increased significantly from 6 to 36 h but had decreased to close to the normal levels after 72 h. DNA synthesis reached a maximum 24 h after PH. However, the expression levels of telomerase RNA did not change during regeneration. The results suggest that telomerase is actively regulated by unknown mechanisms throughout the cell cycle in regenerating rat hepatocytes.

Increased Telomerase Activity in Hyperplastic Nodules and Hepatocellular Carcinomas Induced by a Choline-deficient L-Amino Acid-defined Diet in Rats

Activation of telomerase has been reported in several human cancers, including hepatocellular carcinomas (HCCs). We investigated telomerase activity during hepatocarcinogenesis induced by a choline‐deficient L‐amino acid‐defined (CDAA) diet in rats. Male F344 rats were given a CDAA diet or a choline‐supplemented L‐amino acid‐defined (CSAA) diet from 6 weeks of age for 75 weeks, and subgroups were killed 10 weeks, 50 weeks and 75 weeks after the beginning of the experiment. Hyperplastic nodules and HCCs were noted in rats fed a CDAA diet for 50 weeks and 75 weeks, respectively. Normal control liver specimens were obtained from 6‐week‐old rats. Telomerase activity was assessed by using a telomeric repeat amplification protocol (TRAP). Normal liver and background parenchyma of rats fed either of the diets for 10 weeks or 50 weeks showed weak telomerase activity. In contrast, markedly increased levels were demonstrated in hyperplastic nodules and HCCs. These results suggest that increased telomerase activity may be a biological feature of preneoplastic lesions that evolve to HCCs in rat liver.

Increased expression of cyclooxygenase-2 protein in rat lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine

Expression of cyclooxygenase (COX)-2 protein in preneoplastic and neoplastic lung lesions induced by the administration of 2000 ppm of N-nitrosobis(2-hydroxypropyl)amine (BHP) in the drinking water to Wistar male rats, was examined immunohistochemically. The majority of alveolar/bronchiolar adenomas (ADs) and all adenocarcinomas (ADCs) examined, stained positive or strongly positive for COX-2. In contrast, only a minority of alveolar/bronchiolar hyperplasias demonstrated immunoreactivity and half of the squamous cell carcinomas examined, were only weakly positive. Western blotting analysis also revealed expression of COX-2 protein in the resected ADs and ADCs. These results clearly indicate up-regulated expression of COX-2 in lung neoplastic lesions, particularly ADs and ADCs, induced by BHP in rats.

Successful surgical treatment of pulmonary artery aneurysm in Behçet's syndrome

We report herein an uncommon clinical case of pulmonary artery aneurysm in Behçets syndrome, for which only a few reports have been published in the literature to date. A 68-year-old Japanese male, who was referred for recurrent fever of unknown origin, anemia and pulmonary nodular opacities, was treated by right lower lobectomy followed by postoperative oral administration of colchicine and corticosteroid. Postoperative pathological examination confirmed a diagnosis of pulmonary artery aneurysm accompanied by vasculitis and thrombi. Clinical and radiographic feature are presented herein.