Overexpression of midkine in pancreatic duct adenocarcinomas induced by N-Nitrosobis(2-oxopropyl)amine in hamsters and their cell lines.

The expression of midkine (MK) was investigated in pancreatic ductal hyperplasias, atypical hyperplasias and adenocarcinomas induced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters, and in hamster ductal adenocarcinoma cell lines (HPD-1NR, -2NR and -3NR). MK mRNA was clearly overexpressed in invasive pancreatic duct adenocarcinomas (PCs) and the three cell lines as assessed by northern blot analysis, and MK protein expression increased from ductal hyperplasia through atypical hyperplasias, intraductal carcinomas and invasive PCs by immunohistochemistry. The extent of overexpression of MK mRNA in PCs was almost the same as in hamster whole embryonic tissue. MK is reported to be a retinoid-responsive gene, but MK mRNA expression was not affected by treatment with all-trans retinoic acid (tRA) or N-(4-hydroxyphenyl)retinamide (4-HPR) in HPD-1NR cells. The results thus suggest that MK expression is involved in the development and progression of pancreatic ductal adenocarcinomas induced by BOP in hamsters, with loss of upregulation by retinoic acid.

Analysis of prognostic and survival factors related to treatment of low-grade astrocytomas in adults.

Prognostic factors for low-grade astrocytomas have been proposed, but optimal treatment remains controversial. Eighty-eight consecutive adult patients with supratentorial low-grade astrocytomas were retrospectively reviewed to determine specific factors influencing outcome. All underwent craniotomy (43 radical resections, 45 nonradical resections). Sex, age at diagnosis, preoperative Karnofsky performance status (KPS), tumor location, estimated extent of resection, radiation, chemotherapy, histological type, p53 status, MIB-1 staining and the apoptotic index were assessed as parameters for prognostic significance. KPS (p = 0.03), tumor location (p < 0.001), extent of surgical resection (p < 0.001) and radiotherapy (p = 0.01) were significantly associated with longer survival rates by univariate analysis. Multivariate analysis also showed a significant correlation between radiation therapy after surgical removal and survival time (p < 0.001). p53 status was not of importance in determining the necessity for radiotherapy. Radical surgical removal is the most important factor in the management of low-grade astrocytomas. Radiation therapy appears to be effective in improving the prognosis regardless of the extent of surgical resection or the p53 status.

Inhibitory effects of combined administration of antibiotics and anti-inflammatory drugs on lung tumor development initiated by N-nitrosobis(2-hydroxypropyl)amine in rats.

The effects of antibiotics and anti-inflammatory drugs on the promotion stage of lung carcinogenesis initiated with N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats were investigated in two experiments with a similar protocol. In experiment 1, rats received tap water containing 2000 p.p.m. BHP for 12 weeks followed by basal diet or basal diet containing 0.02% erythromycin (EM), 0. 04% ampicillin (ABPC), 1.5% sho-saiko-to, 0.02% EM plus 1.5% sho-saiko-to or 0.04% ABPC plus 1.5% sho-saiko-to for 8 weeks after BHP administration. The development of adenocarcinomas (AC), squamous cell carcinomas (SqC) and adenosquamous carcinomas (ASqC) was completely inhibited in rats given ABPC plus sho-saiko-to and the numbers of lung lesions including alveolar hyperplasias, adenomas and carcinomas were decreased in rats given EM plus sho-saiko-to or ABPC plus sho-saiko-to. Neutrophil and macrophage infiltration into alveolar spaces of the lung were also markedly suppressed. In experiment 2, rats received BHP in the same manner as in experiment 1 and basal diet or basal diet containing 0.04% ABPC, 0.006% piroxicam, 0.04% ABPC plus 0.006% piroxicam and 0.04% ABPC plus 0.75% ougon for 8 weeks. The incidence and number of carcinomas, including ACs, SqCs and ASqCs were decreased in rats given ABPC plus piroxicam or ABPC plus ougon. Bacteria, mainly Escherichia coli, were detected in broncho-alveolar lavage of rats receiving BHP. The results suggest that chronic inflammation might be involved in the progression of lung carcinogenesis by BHP in rats and its suppression may therefore be useful as a chemopreventive strategy in lung cancer clinics.

Expression of matrix metalloproteinase 2 (MMP-2), membrane-type 1 MMP and tissue inhibitor of metalloproteinase 2 and activation of proMMP-2 in pancreatic duct adenocarcinomas in hamsters treated with N-nitrosobis(2-oxopropyl)amine.

In order to assess the significance of changes in metalloproteinase activity in pancreatic carcinogenesis, the expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9, respectively), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, and membrane-type 1 MMP (MT1-MMP) and MT2-MMP in ductal lesions in a rapid-production model for pancreatic duct carcinomas (PCs) in hamsters initiated with N-nitrosobis(2-oxopropyl)amine (BOP) and in subcutaneous transplantable tumors of hamster pancreatic duct carcinoma (HPDs) was investigated. Northern analysis revealed MMP-2, MMP-9, TIMP-2 and MT1-MMP mRNAs to be overexpressed in PCs. Immunohistochemically, elevated levels of MMP-2 were apparent in early duct epithelial hyperplasias and staining increased from atypical hyperplasias to carcinomas. Gelatin zymography demonstrated clear activation of proMMP-2 but not proMMP-9 in both of primary and HPD tumors, the MT1-MMP mRNA level and proMMP-2 activation being significantly correlated (r = 0.893, P < 0.001). In our rapid production model, 0.1 and 0.2% OPB-3206, an inhibitor of MMPs, given in the diet after two cycles of augmentation pressures for 48 days decreased the incidence and number of carcinomas. Gelatin zymography demonstrated that OPB-3206 inhibited activation of proMMP-2 in pancreatic cancer tissues. These results indicate that overexpression of MMP-2, TIMP-2 and MT1-MMP, and cell surface activation of proMMP-2 by MT1-MMP, are involved in the development of PCs, and that MMP-2 expression at the protein level appears in the early phase of pancreatic duct carcinogenesis. OPB-3206 may be a candidate chemopreventive agent for pancreatic ductal adenocarcinomas.

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.

Overexpression of midkine in lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine in rats and its increase with progression.

The expression of midkine (MK) in lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was examined. The animals were administered 2000 p.p.m. of BHP in their drinking water for 12 weeks, then maintained without further treatment until being killed 20-28 weeks after the beginning of the experiment. MK mRNA expression of adenocarcinomas and squamous cell carcinomas assessed by means of the reverse transcriptase-polymerase chain reaction and northern blot analysis was significantly higher than in rat embryonic tissues (positive controls) and contrasted strongly with the lack in normal lungs. MK protein was detected immunohistochemically in 58.3% of alveolar hyperplasias, 92.3% of adenomas and 100% of adenocarcinomas and squamous cell carcinomas. The extent of staining significantly increased along with malignant progression in adenomatous (pre-)neoplastic lesions and tended to become more pronounced with malignant progression in squamous lesions. The results suggest that MK may play some essential roles in the development and progression of lung tumors induced by BHP in rats.

Increased telomerase activity in intrahepatic cholangiocellular carcinomas induced by N-nitrosobis(2-oxopropyl)amine in hamsters.

Telomerase activities in intrahepatic cholangiocarcinomas induced by N-nitrosobis(2-oxopropyl)amine (BOP) in female hamsters were determined using a telomeric repeat amplification protocol (TRAP) assay followed by densitometric quantification. Each determination was repeated to confirm the results and telomerase activity was also detected by gel electrophoresis. An increase was evident in all of 10 cholangiocarcinomas examined, with levels ranging from 2.48 to 4.40 times the normal liver value by densitometric quantification. This finding of a consistent increase suggests that telomerase activation is involved in the development of intrahepatic cholangiocarcinomas and immortalization of cancer cells.

Increased telomerase activity and absence of p53 mutation in oligoastrocytomas induced by N-ethyl-N-nitrosourea in rats.

The question of whether the changes in telomerase activity and/or the alteration of the p53 gene are involved in the development of oligo-astrocytomas induced by N-ethyl-N-nitrosourea (ENU) in rats was addressed. Telomerase activity levels of oligo-astrocytomas, including early neoplastic lesions, were significantly increased as compared to the normal controls, correlating with the degree of malignancy. In contrast, no mutations of p53 exons 5-7 were found in early neoplastic lesions or oligo-astrocytomas. These results indicate that the activation of telomerase occurs during astrocytoma carcinogenesis and contributes to the development of brain tumors, but the alterations of p53, at least on exons 5-7, may not be involved in this process.

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.

Genetic variations in recurrent astrocytic tumors detected by restriction landmark genomic scanning.

The genetic mechanisms associated with recurrence of advanced astrocytic tumors are poorly understood. We therefore analyzed 24 biopsy specimens from 12 patients with a recurrent astrocytic tumor by a two-dimensional gel electrophoresis method, termed restriction landmark genomic scanning (RLGS). Four spot changes were commonly present in the primary astrocytomas, indicating that the corresponding gene alterations were early events in the development of this tumor type. Altered spots were more frequent and of different distribution in recurrent tumors than in the primary astrocytomas. In particular, specifically increased intensity for spots on chromosomes 9-12 and 18 were observed in the secondary tumors, suggesting a relation with recurrence. The same spot changes observed in primary tumors were also found in the respective secondary lesions but with strikingly different densities in some cases, suggesting increased genetic instability. The altered segments provide important candidate regions for the search for genes involved in events leading to progression and more malignant recurrent tumors.

Frequent alterations of cell-cycle regulators in astrocytic tumors as detected by molecular genetic and immunohistochemical analyses.

Alterations of CDKN2A, RB, and cyclin D1 genes and expression of their products in astrocytic tumors were studied using a combination of molecular genetic and immunohistochemical assays. In addition, the association of gene status with clinical outcome was evaluated. Alterations of CDKN2A and RB gene in 30 lesions were analyzed by single-strand conformation polymorphism of polymerase chain reaction (PCR-SSCP), direct sequencing, and Western blotting. Methylation of the CDKN2A promoter was detected by methylation-specific PCR. Immunohistochemistry was applied to determine the expression of gene products in tumors from 94 patients for whom clinical outcome was also evaluated. Analyses of the CDKN2A gene revealed 12 homozygous or hemizygous deletions, one mutation in exon 1, and three methylations in the promoter. Expression of p 16 protein was not detected in 18 of 30 cases. RB mutations leading to loss of expression of the pRb were found in four (13%) cases, and six were immunohistochemically negative for this protein. Overexpression of cyclin D1 was obtained in 51 (54%) of 94 cases. Patients with pRb-negative tumors had a significantly greater risk of earlier death than those with p16 and cyclin D1 alterations, Both p16 and pRb immunohistochemistry provides useful complementary information and may provide valuable predictive information in screening. The biological consequences of deregulating individual components along cell control pathways are unequal, perhaps reflecting their hierarchical roles in the G1 checkpoint.

[Ruptured traumatic aneurysms of the peripheral anterior cerebral artery: study of delayed hemorrhage after closed head injury].

We report three cases of ruptured traumatic aneurysms of the peripheral anterior cerebral artery after closed head injury. These cases were all young men with closed head injury due to traffic accidents. Consciousness level on admission was coma in all three cases. Case 1 was a 19-year-old man with interhemispheric hematoma on initial CT, then 7 days later his consciousness cleared. However, 14 days later he suddenly lapsed into a deep coma with a severe frontal hemorrhage. Case 2 was a 13-year-old boy. Plain skull films demonstrated a frontal depressed fracture, but CT scan showed no bleeding. Four days later his consciousness cleared but 11 days after trauma, he lapsed into a deep coma with a frontal hemorrhage. Case 3 was a 22-year-old man. Initial CT showed a slight ventricular hemorrhage. Fourteen days later, his consciousness had almost cleared, but then he lapsed into a deep coma with a large frontal hemorrhage 11 weeks after the trauma. These patients all died within a few days after intracranial bleeding. All patients underwent cerebral angiography but none of them showed filling defect. Autopsy was performed and ruptured aneurysms were found on the distal anterior cerebral artery that had no relation to the branch of bifurcation. Histological examination demonstrated a lack of elastic lamina and media in all of these three cases, so each of them was a victim of so-called false aneurysm. Twenty reported cases of ruptured traumatic aneurysms of the peripheral cerebral artery with delayed hemorrhage after closed head injury were reviewed. Factors in the traumatic aneurysm showed no relation to the duration of disturbed consciousness. Within one month, delayed hemorrhage due to ruptured traumatic aneurysm occurred. None of the delayed hemorrhages involved subarachnoid hemorrhage. Subdural hematoma was seen in the distal middle cerebral artery and frontal hemorrhage was found in the distal anterior cerebral artery. We consider that frontal hemorrhage is a predictive finding for the type of delayed hemorrhage due to traumatic aneurysm in the distal anterior cerebral artery.

[The mechanism of cardiorespiratory arrest due to subarachnoid hemorrhage].

This report describes the clinical course of patients with sudden cardiorespiratory arrest (CRA) due to subarachnoid hemorrhage (SAH). We have seen fifteen patients of SAH that presented initially as CRA. All of them were diagnosed as SAH by CT scan. The patients were divided into two groups; one group (early DOA group) included 11 patients, who had been recognized as CRA within 60 minutes from the onset of SAH, the other group (late DOA group) consisted of 4 patients, who developed CRA more than 60 minutes after the initial onset. The major mechanism leading to delayed CRA in the late DOA group appeared to have been from brain stem herniation, but another mechanism appeared to be involved in sudden CRA in the early DOA group. Sixty percent of our patients with CRA due to SAH had a low serum potassium concentration, though hypokalemia was observed in only 4 out of 100 patients with CRA due to diseases other than SAH. These facts suggest that sympathetic hyperstimulation might result not only from stress but also from a disorder of the central autonomic nervous system. We speculate that the mechanism leading to early CRA after SAH appears to result from a disorder of the central autonomic nerve system.