Depletion of the novel p53-target gene carnitine palmitoyltransferase 1C delays tumor growth in the neurofibromatosis type I tumor model.

Despite the prominent pro-apoptotic role of p53, this protein has also been shown to promote cell survival in response to metabolic stress. However, the specific mechanism by which p53 protects cells from metabolic stress-induced death is unknown. Earlier we reported that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific member of a family of mitochondria-associated enzymes that have a central role in fatty acid metabolism promotes cell survival and tumor growth. Unlike other members of the CPT family, the subcellular localization of CPT1C and its cellular function remains elusive. Here, we report that CPT1C is a novel p53-target gene with a bona fide p53-responsive element within the first intron. CPT1C is upregulated in vitro and in vivo in a p53-dependent manner. Interestingly, expression of CPT1C is induced by metabolic stress factors such as hypoxia and glucose deprivation in a p53 and AMP activated kinase-dependent manner. Furthermore, in a murine tumor model, depletion of Cpt1c leads to delayed tumor development and a striking increase in survival. Taken together, our results indicate that p53 protects cells from metabolic stress via induction of CPT1C and that CPT1C may have a crucial role in carcinogenesis. CPT1C may therefore represent an exciting new therapeutic target for the treatment of hypoxic and otherwise treatment-resistant tumors.

KRAS mutations in primary tumours and post-FOLFOX metastatic lesions in cases of colorectal cancer.

BACKGROUND: KRAS mutations are predictive markers for the efficacy of anti-EGFR antibody therapies in patients with metastatic colorectal cancer. Although the mutational status of KRAS is reportedly highly concordant between primary and metastatic lesions, it is not yet clear whether genotoxic chemotherapies might induce additional mutations. METHODS: A total of 63 lesions (23 baseline primary, 18 metastatic and 24 post-treatment metastatic) from 21 patients who were treated with FOLFOX as adjuvant therapy for stage III/IV colorectal cancer following curative resection were examined. The DNA samples were obtained from formalin-fixed paraffin-embedded specimens, and KRAS, NRAS, BRAF and PIK3CA mutations were evaluated. RESULTS: The numbers of primary lesions with wild-type and mutant KRAS codons 12 and 13 were 8 and 13, respectively. The mutational status of KRAS remained concordant between the primary tumours and the post-FOLFOX metastatic lesions, irrespective of patient background, treatment duration and disease-free survival. Furthermore, the mutational statuses of the other genes evaluated were also concordant between the primary and metastatic lesions. CONCLUSION: Because the mutational statuses of predictive biomarker genes were not altered by FOLFOX therapy, specimens from both primary tumours and post-FOLFOX tumour metastases might serve as valid sources of DNA for known genomic biomarker testing.

KRAS mutations detected by the amplification refractory mutation system-Scorpion assays strongly correlate with therapeutic effect of cetuximab.

BACKGROUND: We aimed to compare the sensitive and quality-controlled KRAS testing with direct sequencing and to assess the impact on decision making of treatment. PATIENTS AND METHODS: We analysed genomic DNA isolated from macrodissected formalin-fixed paraffin-embedded specimens by direct sequencing and an amplification refractory mutation system-Scorpion assay (ARMS/S) method. Cetuximab was administered to patients identified as having wild-type (WT) KRAS using direct sequencing. Therapeutic effects were evaluated according to their KRAS status as determined by ARMS/S. RESULTS: Among the 159 patients, the overall mutation rate was determined to be 37.0% by direct sequencing and 44.0% by ARMS/S. For the patients diagnosed as WT by direct sequencing and treated with cetuximab (n=47), a response rate of 16.0% was observed for 38 ARMS/S WT patients, whereas 9 ARMS/S mutant (MUT) patients failed to respond. The ARMS/S WT patients showed significant improvement in progression-free survival (PFS) and overall survival (OS) compared with ARMS/S MUT patients (PFS median 5.0 vs 1.7 months, hazards ratio (HR)=0.29, P=0.001; OS median 12.1 vs 3.8 months, HR=0.26, P=0.001). CONCLUSION: Sensitive and quality-controlled KRAS testing may provide improved predictive power to determine the efficacy of anti-epidermal growth factor antibodies.

[Expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 mRNA and its alternative splice variants in rats with experimental diabetes mellitus].

We studied the expression mRNA of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) in the rat lung and kidney in experimental diabetes mellitus. For investigation we select two isoforms of PFKFB-2 with different C-terminus. The level of the expression of both PFKFB-2 mRNA isoforms is decreased in the kidney and lung in rats with experimental diabetes mellitus respect to the control animals. Moreover, four new alternative splice variants of PFKFB-2 mRNA were identified in the rat kidney. These splice variants of PFKFB-2 mRNA have different inserts and/or deletions in 6-phosphofructo-2-kinase as well as in fructose-2,6-bisphosphatase part of PFKFB-2. Three alternative splice variants cannot encode active 6-phosphofructo-2-kinase as a result of deletion of two catalytic domains (E and F). They encode fructose-2,6-bisphosphatase. It was shown that these alternative splice variants express in the kidney and lung and that this expression changes in rats with experimental diabetes mellitus with respect to the control animals. The results of this investigation clearly demonstrated that diabetes mellitus significantly affects the expression and alternative splicing of PFKFB-2 in the kidney and lungs and showed the complexity of regulatory mechanisms of glucose metabolism in this disease.

[Complete resection of an advanced mediastinal nonseminomatous germ cell tumor with multiple distant metastases after down-staging by chemotherapy].

A mediastinal nonseminomatous germ cell tumor was completely resected after down-staging by chemotherapy despite the presence of multiple distant metastases. A 22-year-old female was admitted for superior vena cava (SVC) syndrome. Her SVC was obstructed by a large anterior mediastinal tumor; she also exhibited distant metastases on a left rib, in the liver, and multiple in the lung. The blood alpha-fetoprotein (AFP) level was extremely elevated to 57,530 ng/ml. Four courses of BEP therapy [cisplatin (CDDP), bleomycin (BLM), etoposide (VP-16)] and a high dose chemotherapy followed by a peripheral blood stem cell transplantation made the tumor become smaller and effected its down-staging. Residual mediastinal tumor with an intravascular tumor in SVC was completely resected. The SVC was reconstructed by an artificial vessel graft. A mediastinal nonseminomatous germ cell tumor, even though it has multiple distant metastases, can achieve down-staging and complete resection by a chemotherapy based on scientific evidence.

Expression of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase-3 and VEGF mRNA in rat liver, lung and heart: effect of methyl tertbutyl ether.

The main goal of this work was investigation of the effect of methyl tertbutyl ether, ecologically dangerous chemical compound, on the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) and vascular endothelial growth factor (VEGF) mRNA in different rat organs. Expression of PFKFB-3 and VEGF is a hypoxia inducible factor (HIF)-dependent process which significantly increases under hypoxia, in malignant tumors and other pathology. In this study we have shown that PFKFB-3 and VEGF mRNA expression in the liver, lung, and heart changes in rats, treated with methyl tertbutyl ether for two months, in organ-specific manner. Expression of alternative splice variants of PFKFB-3 mRNA as well as VEGF mRNA also changes in organ-specific manner in rats, treated with methyl tertbutyl ether. The effect of methyl tertbutyl ether on the expression of PFKFB-3 and VEGF mRNA and its alternative splice variants is dose-dependent. Results of this investigation clearly demonstrated that methyl tertbutyl ether affects the expression of PFKFB-3, a key regulatory enzyme of glycolysis, as well as VEGF, very important factor of angiogenesis, in an organ-specific and dose-dependent manner.

Expression of mouse 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 mRNA alternative splice variants in hypoxia.

Expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) mRNA alternative splice variants was studied in different mouse tissues in hypoxic conditions in vivo. Significant increase of the expression of PFKFB-3 mRNA was observed in the mouse lungs, testes and brain in hypoxia. Several new alternative splice variants of PFKFB-3 mRNA were identified in the lung, testis, brain and skeletal muscle. They have different length and amino acid sequence of C-terminal regulatory part. However, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase catalytic domains were identical. Moreover, the expression of different alternative splice variants of PFKFB-3 mRNA has shown tissue specificity and different levels of induction in hypoxic conditions in vivo. Results of this investigation indicate a possible role of PFKFB-3 splice isoform in cell adaptation to hypoxic conditions.

Molecular cloning and characterization of cDNAs encoding dopamine receptor-1 and -2 from brain-suboesophageal ganglion of the silkworm, Bombyx mori.

In order to better understand the relationship between dopamine and the release of diapause hormone into the blood, we cloned and characterized cDNAs encoding Bombyx mori dopamine receptor-1 and -2 (BmDopR1 and 2) from the pupal brain-suboesophageal ganglion. BmDopR1 and 2 had high similarities to group 1 (Drosophila melanogaster DOP1 and Apis mellifera DOP1) and group 2 (D. melanogaster DopR99B, A. mellifera DOP2 and Papilio xuthus DOP1), respectively. When BmDopR1 and 2 were expressed in human embryonic kidney (HEK) cells, they responded to dopamine by increasing intracellular cAMP levels, thus indicating the presence of D1-like receptors. There were no clear differences in BmDopR1 and 2 mRNA levels between brain-suboesophageal ganglion complexes of diapause and nondiapause egg producers during pupal-adult development. BmDopR1 and 2 mRNAs were concentrated in the mushroom body calyx rather than in the suboesophageal ganglion. Taking into account the results of earlier experiments on excised regions corresponding to mushroom bodies, BmDopR1 and 2 in the mushroom body apparently play a role in the release of diapause hormone.

[Comparison of the images in virtual bronchoscopy under different conditions].

OBJECTIVE: We report herein the comparison of the virtual bronchoscopy (VB) images which were constructed with 2 different computed tomography (CT) scanners combined with 3 different applications in 2 healthy adult volunteers. METHODS: CT scanners were multi-detector row CT (MDCT) [64 detectors] and MDCT (16 detectors). Applications, by which VB images were made, were Leonardo (Leo), Ziostation (Zio), and Plus XNVZ2 (Plus). The image quality was evaluated by 3 expert bronchoscopists. RESULTS: The change of the threshold value was necessary in Leo for practical use in subsegmental bronchi and more distal area, but unnecessary in Plus or Zio. When Plus was used, the VB images from the data obtained with MDCT (16 detectors) and MDCT (64 detectors) had almost equal quality. CONCLUSIONS: Although the process to construct VB images was different in each application, it was regarded that Plus was not inferior to Zio or Leo in VB image quality.

Alternative splice variants of rat 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase-4 mRNA.

Expression of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-4 (PFKFB-4) mRNA was studied in different rat organs. Several new unique alternative splice variants of PFKFB-4 mRNA were identified. They have deletions or inserts in fructose-2,6-bisphosphatase region as well as different length and amino acid sequence of C-terminal part. However, 6-phosphofructo-2-kinase catalytic domains were identical in all variants. Moreover, the expression of different alternative splice variants of PFKFB-4 mRNA has shown tissue specificity. Expression of both alternative splice variants of PFKFB-4 mRNA significantly changed in rats treated by methyl tretbutyl ether, ecologically dangerous chemical compound. Results of this investigation indicate a possible role of PFKFB-4 splice isoforms in cell-specific regulation of glycolysis and demonstrate the sensitivity of the regulation of alternative splicing to the action of toxic chemical compounds, in particular methyl tertbutyl ether.

Irradiated pancreatic cancer cells undergo both apoptosis and necrosis, and could be phagocytized by dendritic cells.

The interaction of immature dendritic cells (DC) with irradiated pancreatic cancer cells was examined. Flow cytometric analysis using annexin V and propidium iodide revealed that ionizing radiation (25-35 Gy X-ray) induced both apoptosis and necrosis in pancreatic cancer cell lines. After irradiation, PK-1 and Panc-1 cells were likely to undergo necrosis, whereas MIAPaCa-2 cells underwent apoptosis. When DiO-stained immature DCs were co-incubated with DiI-stained irradiated MIAPaCa-2, it was observed under fluorescent microscopy that DCs phagocytized dead tumor cells as early as 4 h after co-incubation. The DCs' phagocytosis of irradiated tumor cells was also confirmed by flow cytometry. These results suggest that irradiated pancreatic cancer cells, which undergo both apoptosis and necrosis, could be a good source of tumor-associated antigens for cross-presentation by DCs.

Hepatitis C virus core protein enhances the activation of the transcription factor, Elk1, in response to mitogenic stimuli.

Mitogen-activated protein kinase (MAPK) pathways play key roles in cell proliferation, transformation of mammalian cells, and the stress response. We and other investigators showed that hepatitis C virus (HCV) core protein has an oncogenic potential, but its mechanism has remained unknown. We previously demonstrated that the MAPK-extra-cellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway and its downstream target, the serum response element (SRE), is activated in BALB/3T3 cells producing HCV core protein. To elucidate the precise mechanism by which HCV core protein activates the MEK-ERK pathway, we transiently expressed HCV core protein in several cell lines and studied the signal transduction of the pathway, using Gal4-Elk1 luciferase assay, in vitro kinas assay of MAPK, and Western blotting analysis. We discovered that, in the presence of mitogenic signal, HCV core protein enhanced Elk1 activation working downstream of MEK without affecting ERK activity and Elk1 phosphorylation. Our data suggest that HCV core protein may activate Elk1 through a pathway alternative to the typical phosphorylation cascade. These findings might give new insights into the role of HCV in hepatocarcinogenesis.

A putative binding protein for lipophilic substances related to butterfly oviposition.

A unique protein of 23 kDa (Jf23) was found in the tarsus of the female swallowtail butterfly, Atrophaneura alcinous. Jf23 has 38% identity with a bilin-binding protein, which was found in the cabbage butterfly, Pieris brassicae, and which has two consensus sequences in common with the members of the lipocalin family, suggesting that it is a binding protein for lipophilic ligands. Western blot analysis showed that Jf23 was expressed only in the female, and not in the male. Electrophysiological response of the female tarsi was stimulated by methanolic extract of their host plant, Dutchman's pipe (Aristolochia debilis). The stimulated response was depressed by the presence of Jf23 antiserum. These results suggest that Jf23 is one of the chemosensory signaling proteins, which plays one or more roles in female butterfly oviposition.

Hepatitis C virus core protein regulates cell growth and signal transduction pathway transmitting growth stimuli.

To investigate the transforming potential of hepatitis C virus (HCV), HCV core protein was produced in BALB/3T3 A31-I-1 cells. The cells expressing HCV core gene cooperatively with the v-H-ras gene showed loss of contact inhibition, morphological alterations, and anchorage-independent and serum-independent growth. The cells producing HCV core protein showed enhanced growth against stimulus of growth factor. In addition, antisense oligodeoxynucleotides against mRNA encoding HCV core protein suppressed the growth of HCV core-producing cells. Furthermore, HCV core protein activated mitogen-activated protein kinase and serum response element, which respond to growth stimuli. From these results, we concluded that HCV core protein is involved in the acquisition of cell growth advantage.

Specific interaction of polypyrimidine tract-binding protein with the extreme 3'-terminal structure of the hepatitis C virus genome, the 3'X.

We previously identified a highly conserved 98-nucleotide (nt) sequence, the 3'X, as the extreme 3'-terminal structure of the hepatitis C virus (HCV) genome (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215:744-749, 1995). Since the 3' end of positive-strand viral RNA is the initiation site of RNA replication, the 3'X should contribute to HCV negative-strand RNA synthesis. Cellular factors may also be involved in this replication mechanism, since several cellular proteins have been shown to interact with the 3'-end regions of other viral genomes. In this study, we found that both 38- and 57-kDa proteins in the human hepatocyte line PH5CH bound specifically to the 3'-end structure of HCV positive-strand RNA by a UV-induced cross-linking assay. The 57-kDa protein (p57), which had higher affinities to RNA probes, recognized a 26-nt sequence including the 5'-terminal 19 nt of the 3'X and 7 flanking nt, designated the transitional region. This sequence contains pyrimidine-rich motifs and shows similarity to the consensus binding sequence of the polypyrimidine tract-binding protein (PTB), which has been implicated in alternative pre-mRNA splicing and cap-independent translation. We found that this 3'X-binding p57 is identical to PTB. The 3'X-binding p57 was immunoprecipitated by anti-PTB antibody, and recombinant PTB bound to the 3'X RNA. In addition, p57 bound solely to the 3'-end region of positive-strand RNA, not to this region of negative-strand RNA. We suggest that 3'X-PTB interaction is involved in the specific initiation of HCV genome replication.

Prognostic relevance of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1 and PAI-2 in gastric cancer.

Expression of urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) was evaluated in 125 surgically resected gastric cancers by immunohistochemical analysis. Tissue was stained immunohistochemically with a monoclonal antibody against human uPA and monoclonal antibodies against human PAI-1 and PAI-2. In addition, DNA ploidy patterns were determined by cytofluorometer after staining with propidium iodide. We found that 82 (66%) of the 125 gastric cancers expressed uPA as diffuse cytoplasmic staining, as intensely outlined luminal borders. PAI-1 expression was observed in 62 (50%) of 125 gastric cancer as a fine, diffuse and granular pattern in the cytoplasm. PAI-2 expression was observed in 65 (52%) of the 125 gastric cancers as a diffuse cytoplasmic staining. uPA-positive tumours showed a higher incidence of infiltration, lymph node metastasis and peritoneal dissemination that uPA-negative ones. Patients with uPA-positive tumours proved to have a significantly poorer prognosis than those with negative ones. PAI-1-negative tumours showed a higher incidence of liver metastasis and carried a poorer prognosis than PAI-1-positive ones. There was no significant correlation between uPA or PAI-1 expression and DNA ploidy patterns. Conversely, there was no significant relationship between PAI-2 expression and clinicopathological parameters and prognosis. According to the expression of uPA and PAI-1 status, groups of 19 uPA(-)/PAI-1(-), 44 uPA(+)/PAI-1(-), 23 uPA(-)/PAI-1(+) and 39 uPA(+)/PAI-1(+) were subdivided. Tumours with uPA(+)/PAI-1(-) had a significantly higher incidence of liver metastasis, lymph node metastasis and serosal invasion than the other groups of tumours. Patients with uPA(+)/PAI-1(-) tumours had a significantly poorer prognosis than those with uPA(-)/PAI-1(+) tumours. These results indicate that uPA expression is a useful biological prognostic indicator, and that uPA expression is a useful biological prognostic indicator, and that uPA and PAI-1 may play an important part in the tumour progression and metastasis in gastric cancer.

[Correlation between the expression of urokinase-type plasminogen activator, E-cadherin and malignancy in gastric cancer].

We aim to elucidate the correlation between clinicopathological parameters and uPA and ECD expression in gastric cancer. Some 125 patients with primary gastric cancer, who were treated at the Second Department of Surgery, Kanazawa University, between 1988 and 1993, were enrolled in this study. The expression of uPA and ECD was evaluated by immunohistochemical staining using a anti-uPA and an anti-ECD monoclonal antibody. The nuclear DNA contents were measured by flow cytometry. Among 125 tumors, 42 (34%) were found to have preserved ECD expression (ECD (+)), and 83 (66%) reduced ECD expression (ECD (-)). uPA immunoreactivity was observed in 82 (65%) of 125 tumors. According to the expression of uPA and ECD status, groups of 22 uPA (-)/ECD (+), 21 uPA (+)/ECD (+), 17 uPA (-)/ECD (-) and 65 uPA (+)/ECD (-) were identified. There was a significant correlation between uPA (+)/ECD (-) status and depth of invasion, liver metastasis, peritoneal dissemination, lymph node metastasis, and venous invasion. Patients with uPA (+)/ECD (-) tumors showed the poorest prognosis and the highest rate of recurrence, as compared with the other groups of patients. No significant correlations were found between uPA (+)/ECD (-) status and DNA ploidy patterns, and histological type. Immunohistochemical analysis of the combination of uPA and ECD expression could be a useful method for the evaluation of metastasis and prognosis in gastric cancer patients. Our results indicate that uPA may have an important role in cancer infiltration and ECD in cancer infiltration and metastasis.

Decreased E-cadherin expression correlates with poor survival in patients with gastric cancer.

E-cadherin, Ca(2+)-dependent intracellular adhesion molecule, is known to be an invasion suppressor gene. To elucidate the correlation between E-cadherin expression and invasion or metastasis in gastric cancer, we examined E-cadherin tissue status immunohistochemically. Ninety-eight primary gastric cancer, prepared by AMeX method, were retrospectively analyzed with anti-E-cadherin monoclonal antibody. In normal gastric epithelium, E-cadherin is expressed homogeneously with a typical membranous staining at cell-cell borders. Decreased and heterogeneous expression is found in 70 of 98 tumours. Tumours with decreased E-cadherin expression had a tendency to infiltrate more deeply in stomach wall, and metastasize in lymph nodes or peritoneal surface. More importantly, decreased E-cadherin expression correlates with shorter survival (z = 3.98, P = 0.00086). These results may indicate that E-cadherin tissue status is a powerful prognostic indicator in gastric cancer. The high malignant potential of tumours with decreased E-cadherin expression may be associated with high potential of lymph node metastasis and peritoneal dissemination.

Prognostic value of urokinase-type plasminogen activators in gastric-cancer.

To elucidate the relation between urokinase-type plasminogen activator (U-PA) and metastasis in gastric cancer, we examined U-PA tissue status immunohistochemically. Ninety-eight primary gastric cancer, prepared by AMeX method, were analyzed with anti-U-PA and anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody. In addition, DNA ploidy patterns were determined by cytofluorometer after staining with propidium iodide. U-PA immunoreactivity can be observed as diffuse cytoplasmic staining, as intensely outlined luminal borders or in desquameted cells in the lumens. U-PA expression-positive tumors showed higher incidence of serosal invasion, lymph node involvement or larger tumors than did U-PA negative ones. There was no correlation between U-PA tissue status and PCNA labeling rates or DNA ploidy patterns. Patients with a U-PA positive tumor survived significantly shorter than those with U-PA negative ones. These results may indicate that U-PA tissue status is a useful biological prognostic indicator in gastric cancer. The high malignant potential of U-PA positive tumors may be associated with a rapid infiltrating capacity through gastric wall but not with a high proliferative activity.