SETBP1 mutations drive leukemic transformation in ASXL1-mutated MDS.

Mutations in ASXL1 are frequent in patients with myelodysplastic syndrome (MDS) and are associated with adverse survival, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) is not fully understood. Recently, it has been found that deletion of Asxl1 or expression of C-terminal-truncating ASXL1-MTs inhibit myeloid differentiation and induce MDS-like disease in mice. Here, we find that SET-binding protein 1 (SETBP1) mutations (SETBP1-MT) are enriched among ASXL1-mutated MDS patients and associated with increased incidence of leukemic transformation, as well as shorter survival, suggesting that SETBP1-MT play a critical role in leukemic transformation of MDS. We identify that SETBP1-MT inhibit ubiquitination and subsequent degradation of SETBP1, resulting in increased expression. Expression of SETBP1-MT, in turn, inhibited protein phosphatase 2A activity, leading to Akt activation and enhanced expression of posterior Hoxa genes in ASXL1-mutant cells. Biologically, SETBP1-MT augmented ASXL1-MT-induced differentiation block, inhibited apoptosis and enhanced myeloid colony output. SETBP1-MT collaborated with ASXL1-MT in inducing acute myeloid leukemia in vivo. The combination of ASXL1-MT and SETBP1-MT activated a stem cell signature and repressed the tumor growth factor-ß signaling pathway, in contrast to the ASXL1-MT-induced MDS model. These data reveal that SETBP1-MT are critical drivers of ASXL1-mutated MDS and identify several deregulated pathways as potential therapeutic targets in high-risk MDS.

Pharmacological preconditioning with doxorubicin. Implications of heme oxygenase-1 induction in doxorubicin-induced hepatic injury in rats.

Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide, and iron. HO-1, an inducible form, is thought to contribute to resistance to various types of oxidative stress. Doxorubicin (DOX) produces clinically useful responses in a variety of human cancers. We reported previously that prior administration of DOX ameliorated subsequent hepatic ischemia and reperfusion injury. The aim of this study was to examine whether this pharmacological preconditioning was useful for another type of hepatic injury induced by a non-surgical method. When a high dose of DOX (10 mg/kg body weight) was administered directly to rat liver via the portal vein, serum aspartate transaminase (AST) and alanine transaminase (ALT) levels increased markedly 24 hr after the injection. Under this condition, zinc-protoporphyrin IX, a specific inhibitor of HO-1, caused both serum AST and ALT levels to be elevated further. When a low dose of DOX (5 mg/kg body weight) was administered to rats via the tail vein as pharmacological preconditioning 3 days before the injection of a high dose of DOX via the portal vein, the levels of serum AST and ALT in rats clearly were improved as compared with rats without the preconditioning. Expression of HO-1 in the liver was confirmed 3 days after the administration of a low dose of DOX. In addition, prior administration of zinc-protoporphyrin IX abolished the effect of DOX preconditioning. Immunohistochemical analysis showed that the positive staining of HO-1 protein induced by a low dose of DOX was localized to histiocytes infiltrating periportal areas. These results strongly suggest that pharmacological preconditioning with DOX may generally help to attenuate subsequent oxidant-induced hepatic injury.

Inhibition of expression of ornithine decarboxylase by c-myc antisense oligonucleotide at the promotion stage of lung tumorigenesis in mice.

Regulation of ornithine decarboxylase (ODC) expression at a promotion stage of lung carcinogenesis is a key clue to suppress the cancer. In this study, we investigated that the ODC induction at the promotion stage of lung carcinogenesis in mice could be inhibited through the suppression of the expression of c-myc, a transcription factor for ODC. The treatment with c-myc antisense oligonucleotide decreased the carcinogen-elevated level of pulmonary ODC protein at the promotion stage, but the sense oligonucleotide had no influence on the level. Overall, it is possible that the induction of ODC in the carcinogenic process of lung is regulated at its transcriptional level.

[A case of non-invasive pulmonary aspergillosis that rapidly deteriorated].

The pulmonary diseases caused by the Aspergillus species include invasive forms, for example, invasive pulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, and non-invasive pulmonary aspergillosis. Though these forms are defined pathologically by the presence of the Aspergillus species that invades the lung tissue, they are used as clinical entities. We report a case of non-invasive pulmonary aspergillosis which, from the clinical data, appeared likely to be misdiagnosed as the chronic invasive form. A 45 year-old man received chemoradiotherapy for lung cancer as well as undergoing an left upper lobectomy. Two weeks after the surgery the patient developed a cough, high fever and chest pain. Chest radiography and chest computed tomography showed a rapidly enlarging cavity with an internal mass and infiltration in the left lower lung field. A transbronchial biopsy specimen of the cavity wall showed fungal hyphae. Bronchial washing culture grew Aspergillus fumigatus. Itraconazole and amphotericin B were administered, but the patient's condition did not improve. A left lower lobectomy was performed. The histologic findings showed that the fungal hyphae were only on the surface of the cavity wall, and were surrounded by necrosis and widespread inflammatory cell infiltration. No fungal invasion of the viable lung tissue was seen. The area of infiltration revealed an organizing pneumonia without Aspergillus or other organisms. Our final diagnosis was non-invasive pulmonary aspergillosis. There has been no recurrence of the lung cancer or of the pulmonary aspergillosis in the three years since surgery. It is reported that non-invasive pulmonary aspergillosis passes through a period so active that it seems to be the invasive form for its entire clinical course. To avoid confusion in diagnosis, establishment of a comprehensive clinical classification of pulmonary aspergillosis will be needed.

Correlation between the expression of methionine adenosyltransferase and the stages of human colorectal carcinoma.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet) from ATP and L-methionine. AdoMet is the major methyl donor in most transmethylation reactions in vivo, and it is also the propylamino donor in the biosynthesis of polyamines. In the present study, we assessed MAT activity in human colons with colorectal carcinoma and the values were compared with those of morphologically normal adjacent mucosa. Higher levels of MAT activity were observed in the colorectal carcinoma than in the normal colon. The ratio of MAT activity in tumor tissue versus normal tissue seemed to be correlated well will the stage of the colorectal tumor. Furthermore, immunoblot analysis showed that the high levels of MAT activity observed in colorectal carcinoma were due to the increased amounts of MAT protein. Immunohistochemical analysis revealed that MAT was most abundant in goblet cells, particularly in granules in the supranuclear area of these cells. In the colorectal carcinoma tissues, MAT was strongly stained in the cancerous cells and localized in granules in the supranuclear region. The results of this preliminary study suggest that determination of the relative ratio of MAT activity in both normal and tumor regions in human colorectal carcinoma could be a clinically useful tool for determining the stage of malignancy of colorectal carcinomas.

Doxorubicin preconditioning: a protection against rat hepatic ischemia-reperfusion injury.

Doxorubicin produces clinically useful responses in a variety of human cancers. However, the toxicity of doxorubicin has limited its usefulness. This side effect is mainly due to the doxorubicin-mediated free radical formation. Administration of doxorubicin (10 mg/kg body weight) to rats intravenously induces heme oxygenase-1 (HO-1) in the liver. The levels of HO-1 protein were first detected at 6 hours and peaked at about 18 to 24 hours after the injection. It is known that HO-1 plays a protective role against the oxidative injury. Therefore, we have examined the protective effect of doxorubicin preconditioning against the hepatic ischemia-reperfusion injury. Partial hepatic ischemia was produced in the left and medium lobes for 45 minutes followed by 120 minutes reperfusion. When low doses of doxorubicin (1 mg/kg body weight) was intravenously administered to rats 2 days before the ischemia, the serum alanine transaminase (ALT) levels in the preconditioning rat were clearly improved compared with those in the rat without preconditioning. Under this situation, zinc-protoporphyrin IX, a specific inhibitor of HO-1, was injected subcutaneously to rats at 3 and 16 hours before the ischemia, the ALT levels were not improved by doxorubicin preconditioning. Histopathologic examination also supported these results. Although the HO-1 protein level was fairly low 2 days after the doxorubicin administration, significant amounts of HO-1 protein were detected. Our results indicated that the induction of HO-1 played a protective role against hepatic ischemia-reperfusion injury and that doxorubicin preconditioning is more clinically useful than other preconditioning methods.

Regulation of methionine adenosyltransferase activity by the glutathione level in rat liver during ischemia-reperfusion.

Hepatic ischemia was induced by clamping the hepatic artery, portal vein, and bile duct. After 15 min of ischemia, the hepatic glutathione (GSH) content rapidly decreased. On the other hand, after the start of reperfusion, the hepatic GSH levels promptly increased and reached a peak at about 1 h, and thereafter decreased to a minimum level by 2 h. Under such conditions, we examined the changes in the methionine adenosyltransferase (MAT) activity in the liver. Though the time course of MAT activity was somewhat delayed compared with that of the hepatic GSH levels, both patterns were substantially similar during ischemia-reperfusion. In contrast to the changes in the MAT activity during ischemia-reperfusion, the levels of MAT protein were unchanged during these periods. When endogenous antioxidant coenzyme Q(10) (CoQ(10)) was administered to rats prior to ischemia, both the reduction in the MAT activity and hepatic GSH levels induced by ischemia-reperfusion were protected. Our findings suggest that CoQ(10) may posttranslationally regulate the MAT activity via the changes in the GSH level in the liver.

Characterization of CFTR expression in a human pulmonary mucoepidermoid carcinoma cell line, NCI-H292 cells.

The NCI-H292 cell, a human pulmonary mucoepidermoid carcinoma cell line, is commonly used for studying bacterial and viral infections of airway epithelial cells. Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) is the main cause of fetal lung infection in cystic fibrosis patients. In this study, we examined CFTR expression in NCI-H292 cells to determine whether NCI-H292 cells possess sufficient, normally functioning CFTR. The results of RT-PCR and Northern blotting analysis indicated that the CFTR gene expression level was much lower in NCI-H292 cells than in T84 cells. However, Western blotting analysis showed that protein expression in NCI-H292 cells was comparable to that in T84 cells. Furthermore, whole-cell and cell-attached patch clamp electrophysiological techniques indicated that the Cl- current induced by intracellular cAMP elevation in NCI-H292 cells was comparable to that in T84 cells. These findings suggest that NCI-H292 cells with a low level of CFTR gene expression possess enough functional CFTR to show a physiological response.

Activation of extracellular signal-regulated kinase in lung tissues of mice treated with carcinogen.

The activation of extracellular signal-regulated kinase (ERK) in lung tissues of mice, as determined by the appearance of phosphorylated form, was observed on day 30 after urethane injection, and the activation also occurred in urethane-induced lung tumors. Immunohistochemical analysis using anti-phosphorylated ERK antibody indicated that the active form of ERK localized in alveolar epithelial cells. Furthermore, we confirmed by immunoprecipitation and immunoblot analysis that other essential components of the ERK cascade, that is, Ras, Raf and MEK (known as ERK kinase) were activated. These results indicate that the activation of the ERK signal in alveolar epithelial cells at the early stage of urethane-induced lung carcinogenesis is an important factor to develop lung tumors.

Activation of epidermal growth factor receptor in the early phase after renal ischemia-reperfusion in rat.

In order to estimate a regenerative response in the early phase after renal ischemia-reperfusion in rat, we examined the time course of the activation of epidermal growth factor receptor (EGFR) as a response of signal transduction pathway after 45 min ischemia in kidney. The activation of EGFR was observed 5-30 min after the start of reperfusion. Simultaneously, superoxide anion/hydrogen peroxide generated in the mitochondrial fraction was elevated during the same period. On the other hand, the level of EGF decreased in a time-dependent manner. These results suggested that superoxide anion/hydrogen peroxide generated during the ischemia-reperfusion other than EGF could act as an activator for the EGFR. In summary, the activation of EGFR is important as a regenerative response at an early stage after the start of reperfusion in ischemic kidney.

Regenerative response in acute renal failure due to vitamin E deficiency and glutathione depletion in rats.

In this study, we investigated some factors contributing to renal regeneration after acute renal failure (ARF) induced by vitamin E (VE) deficiency and glutathione (GSH) depletion. Acute renal failure was induced by feeding rats a vitamin E-deficient diet for 6 weeks and then injecting buthionine sulfoximine (BSO), a glutathione-depleting agent. The level of hepatocyte growth factor (HGF), a renotropic factor for regeneration in the kidney, showed a transient increase at 5 hr after the BSO treatment. Subsequently, renal ornithine decarboxylase (ODC) activity, a marker of G1 phase, and labeling index (LI) of proliferating cell nuclear antigen (PCNA), a marker of DNA synthesis (S phase), reached peaks at 10 and 53 hr after the injection, respectively. Thus, it appears that the increase in ornithine decarboxylase activity and subsequent elevation in proliferating cell nuclear antigen labeling index following the increase in the hepatocyte growth factor level in the kidneys are closely related to the renal regenerative response after acute renal failure.

The inhibitory effect of vitamin E on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA injury and the fixation of the DNA injury in mouse lungs.

In order to estimate the effect of vitamin E on DNA injury and K-ras point mutation at an early stage of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)-induced lung tumorigenesis in mice, the present study was carried out. Presupplement with vitamin E about 15 times more than control for a week significantly inhibited NNK-induced O6-methylguanine formation in the lungs of mice at 4 and 168 h after the injection. At 30 days after the NNK injection. the activation of K-ras oncogene with a 12th codon GC-->AT transition was detected in 56% of lung samples tested by mutant-allele-specific amplification. Vitamin E supplement reduced the frequency of the mutation to 30%. These results suggest that vitamin E suppresses NNK-induced DNA injury and subsequent fixation of the injury during the initiation and post-initiation phases of the lung tumorigenesis in mice.

The repetitive activation of extracellular signal-regulated kinase is required for renal regeneration in rat.

In this study, we investigated the activation of p42 extracellular signal-regulated kinase (ERK2) during renal regeneration after HgCl2-induced acute renal failure (ARF) in rat. ERK2 activation was observed at 5 and 29 hr after HgCl2 injection, respectively. The tyrosine phosphorylation of hepatocyte growth factor receptor (c-MET) occurred between 2.5 and 5 hr after the treatment. On the other hand, the phosphorylation of epidermal growth factor receptor (EGFR) was transiently observed at 29 hr after the injection. The peak of ornithine decarboxylase activity as a marker of G1 phase was at 10 hr, and subsequently the labeling index of proliferating cell nuclear antigen as a marker of S phase increased at 53 hr. These results indicate that the repetitive activation of ERK2 related to the phosphorylation of c-MET and EGFR is required for the renal regeneration in HgCl2-induced ARF of rat.

Primary bronchopulmonary fibrosarcoma: report of a case.

We herein report a case of primary bronchopulmonary fibrosarcoma in a 70-year-old man. The patient was referred to our hospital for investigation of hemosputum and an abnormal shadow. On admission, chest radiograph and computed tomography scan showed a mass lesion in right S3 and an infiltrative shadow in the right upper lobe. Transbronchial biopsy specimens showed findings of malignancy, and adenocarcinoma was suspected. A right pneumonectomy was performed, and pathologic examination confirmed a diagnosis of fibrosarcoma. The patient had an uneventful recovery and no sign of recurrence has been found in the year since his operation, although strict follow-up is essential.

Regulation and immunohistochemical analysis of stress protein heme oxygenase-1 in rat kidney with myoglobinuric acute renal failure.

Intramuscular injection of hypertonic glycerol solution to rats results in acute renal injury. In this model, the proximal tubules are characteristically damaged. After glycerol injection renal glutathione (GSH) levels drastically decreased. On the other hand, stress protein heme oxygenase-1 (HO-1) was induced. When N-acetyl cysteine was administered to rats before 1 h glycerol injection, renal function was obviously improved. In this condition, the renal GSH content were sustained in the normal levels and HO-1 protein levels were decreased compared with those of glycerol-treated rats. Induction of HO-1 was accompanied by reduced renal GSH content. In addition, to investigate whether the location of HO-1 protein induced by glycerol injection is restricted to injured region or not in the kidney, we determined the localization of HO-1 protein using immunohistochemical staining. HO-1 protein was identified in the epithelia of the distal tubules, Henle's loop and collecting ducts, but not in the injured proximal tubules.

Differential expression of S-adenosylmethionine synthetase isozymes in different cell types of rat liver.

Mammalian S-adenosylmethionine (AdoMet) synthetase exists as two isozymes, liver-type and nonhepatic-type enzymes, which are the products of two different genes. It is known that the liver-type isozyme is only expressed in adult liver. Whereas, the nonhepatic-type isozyme is widely distributed in various tissues. In addition to the liver-type isozyme, a minor amount of the nonhepatic-type isozyme is also detected in adult liver. To investigate the distribution of these two isozymes in the liver in detail, the localization of these two isozymes was examined in each cell type of liver using a combination of cell fractionation technique and Western blot analysis. In the parenchymal cells, the liver-type isozyme protein was predominantly expressed, and a small amount of the nonhepatic-type isozyme protein was also detected. On the other hand, in the stellate cells the nonhepatic-type isozyme protein was exclusively or only expressed. Interestingly, a large amount of both isozymes were present in endothelial and Kupffer cell fraction. Using both antibodies to anti-rat nonhepatic-type and liver-type isozymes, respectively, immunohistochemical analysis clearly confirmed these results. In addition, in cultured hepatocellular carcinoma cells (FAA-HTC1), the nonhepatic-type isozyme protein only was detected, and the liver-type isozyme protein completely disappeared. This result indicates that the changes in the isozyme expression is regulated within the parenchymal cells. Administration of hepatotoxic drug carbon tetrachloride (CCl4) to rats resulted in about 40% to 50% reduction of enzyme activity in parenchymal cells and stellate cells compared with those of control rats. However, enzyme activity in endothelial and Kupffer cell fraction was not changed.

Structure of the rat methionine adenosyltransferase 2A gene and its promoter.

In this study, to understand the regulation of methionine adenosyltransferase (MAT) gene expression, we isolated the rat MAT2A gene encoding MAT alpha2, the catalytic subunit of non-hepatic-type enzyme MAT II and characterized its structural organization and 5'-flanking region. The gene spans approximately 7 kbp and consists of nine exons interrupted by eight introns. The transcription initiation site, as demonstrated by primer extension analysis, is located 123 bp upstream of the translation start codon. Comparison of the structural organization of the rat MAT2A gene to that of the mouse MAT1A gene encoding MAT alpha1, the subunit of liver-type enzymes MAT I and III, shows that the exon structure of two genes is very similar and the insertion sites of all corresponding introns are identical. A canonical TATA box and a GC box, the potential Sp1-binding site, are found 32 bp and 70 bp upstream of the transcription initiation site, respectively. The 5'-flanking region also contains potential recognition sites for various transcription factors including AP-1, AP-2 and NF-IL6 (C/EBPbeta), and a large G+C-rich domain with the characteristics of a CpG island. The 5'-flanking sequence of the rat MAT2A gene has no significant similarity with those of the MAT1A genes. Transient transfection experiments using a luciferase reporter gene showed that the first 820-bp sequence of the 5'-flanking region directed high levels of luciferase activity in cultured rat kidney fibroblast (NRK-49F) and hepatocellular carcinoma (FAA-HTC1) cells, but not in primary rat hepatocytes. Deletion analysis suggested that the first 343 bp of the 5'-flanking region contained cell-type-specific promoter elements of this gene.