Deletion of phospholipase A2 group IVc induces apoptosis in rat mammary tumour cells by the nuclear factor-κB/lipocalin 2 pathway.

Although some forms of phospholipase A2, the initiator of the arachidonic acid cascade, contribute to carcinogenesis in many organs, the contribution of phospholipase A2 group IVc (Pla2g4c) remains to be clarified and the function of the enzyme in cancer development is unknown. The Hirosaki hairless rat (HHR), a mutant rat strain with autosomal recessive inheritance, derived spontaneously from the Sprague-Dawley rat (SDR). The HHRs showed a lower incidence and much smaller volume of mammary tumours induced by 7,12-dimethylbenz[a]anthracene, and a markedly increased number of TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling)-positive apoptotic cells was detected. Array comparative genomic hybridization and PCR analyses revealed the deletion of 50-kb genomic DNA on 1q21, including Pla2g4c, in HHRs. The Pla2g4c gene was expressed in the ductal carcinoma cells and myoepithelial cells in SDRs, but not in HHRs. The direct involvement of Pla2g4c in the prevention of cell death was demonstrated through the inhibition of its expression in rat mammary tumour RMT-1 cells using siRNA. This treatment also induced expression of lipocalin 2 (Lcn2) and other NF-κB (nuclear factor κB)-related genes. siRNA-induced apoptosis was inhibited by Lcn2 repression or NF-κB inhibitors. This is the first report on Pla2g4c gene-deficient rats and their low susceptibility to mammary carcinogenesis by enhancing NF-κB/Lcn2-induced apoptosis.

Establishment of a recessive mutant small-eye rat with lens involution and retinal detachment associated with partial deletion and rearrangement of the Cryba1 gene.

From our stock of SDRs (Sprague-Dawley rats), we established a mutant strain having small opaque eyes and named it HiSER (Hirosaki small-eye rat). The HiSER phenotype is progressive and autosomal recessive. In HiSER eyes, disruption and involution of the lens, thickening of the inner nuclear layer, detachment and aggregation of the retina, rudimentary muscle in the ciliary body and cell infiltration in the vitreous humour were observed. Genetic linkage analysis using crossing with Brown Norway rat suggested that the causative gene(s) is located on chromosome 10. Microarray analysis showed that the expression level of the Cryba1 gene encoding ßA3/A1-crystallin on chromosome 10 was markedly decreased in HiSER eyes. Genomic PCR revealed deletion of a 3.6-kb DNA region encompassing exons 4-6 of the gene in HiSERs. In HiSER eyes, a chimaeric transcript of the gene containing exons 1-3 and an approximately 250-bp sequence originating from the 3'-UTR of the Nufip2 gene, located downstream of the breakpoint in the opposite direction, was present. Whereas the chimaeric transcript was expressed in HiSER eyes, neither normal nor chimaeric ßA3/A1-crystallin proteins were detected by Western blot analysis. Real-time RT (reverse transcription)-PCR analysis revealed that expression level of the Nufip2 gene in the HiSER eye was 40% of that in the SDR eye. These results suggest that the disappearance of the ßA3/A1-crystallin protein and, in addition, down-regulation of the Nufip2 gene as a consequence of gene rearrangement causes the HiSER phenotype.

Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

Naturally occurring regulatory T cells (nTregs), important for immune regulation and the maintenance of self-tolerance, develop in the thymus. The Hirosaki hairless rat (HHR), derived from the Sprague-Dawley rat (SDR), was shown to have decreased peripheral lymphocyte number, small thymus, and leukocyte infiltration in its dermis. In the HHR thymus, the medulla was underdeveloped and nTreg number was decreased. Array comparative genome hybridization revealed the deletion of an NK cell lectin-like receptor gene, Ly49s3, detecting MHC class I molecules on target cells, in the chromosome 4q42 region in HHRs. The gene was expressed in thymic conventional dendritic cells (cDCs) in SDRs, but not in HHRs. When CD4-single-positive or CD4(+)CD8(-)CD25(-) thymocytes were cultured with thymic cDCs, the expression of nTreg marker genes was lower when these cells were from HHRs than from SDRs, suggesting that HHR cDCs are deficient in the ability to induce and maintain nTreg differentiation. Expression of the genes was recovered when Ly49s3 was expressed on HHR thymic cDCs. Expression levels of MHC class II genes, presumably from cDCs, were parallel to those of nTreg marker genes in mixed-cell cultures. However, in the presence of an anti-MHC class I Ab, blocking interaction between Ly49s3 and MHC class I molecules, the expression of the former genes was upregulated, whereas the latter was downregulated. These results suggest that Ly49s3 contributes to nTreg regulation along with MHC class II molecules, whose effects alone are insufficient, and loss of Ly49s3 from thymic cDCs is the reason for the nTreg deficiency in HHRs.

Applying low-intensity pulsed ultrasounds (LIPUS) to a zoledronate-associated atypical femoral shaft fracture without cessation of zoledronate therapy for 3 years follow up: a case report.

Reports are increasing regarding atypical femoral fractures (AFFs) caused by minor trauma in patients using bisphosphonates (BPs) for long periods. Patients with malignant skeletal metastases potentially are at greater risk for these AFFs, especially considering the high dose and the duration of treatment with BPs. We evaluated a case of atypical femoral shaft fracture treated with an intramedullary nail in a patient treated for five years with zoledronate who had breast cancer with metastases to bone. Although bone union was achieved without cessation of zoledronate therapy by applying low-intensity pulsed ultrasounds (LIPUS), the remodeling phase of the fracture healing process was delayed. For BPs-associated AFFs, LIPUS is an alternative to parathyroid hormone (PTH) analogs such as teriparatide that are contraindicated in patients with malignant skeletal metastases. LIPUS is an effective treatment for fracture healing and may avoid the necessity to discontinue BP therapy.

Decrease of hepatic stellate cells in rats with enhanced sensitivity to clofibrate-induced hepatocarcinogenesis.

To examine the possible involvement of nonparenchymal cells in the development of preneoplastic hepatic lesions induced by clofibrate (CF), alterations of these cells were investigated immunohistochemically in glutathione S-transferase M1 gene polymorphic rats (KS and NC types) with different cancer susceptibilities. After CF administration for 8 weeks, α-smooth muscle actin (α-SMA)-positive hepatic stellate cells (HSC) were markedly decreased in sensitive KS-type rats, but not in the NC-type rats. Kupffer cells were decreased with similar extents between them. The sinusoidal endothelial cells were not changed in either type. The other markers for HSC, vimentin and CRBP1, also confirmed the decrease of HSC in the KS type. The decrease of HSC was not observed at 4 weeks of CF administration. Preneoplastic peroxisomal bifunctional enzyme-negative foci were detected in the KS-type rats at 8 weeks of CF administration, but not at 4 weeks. Human HSC were cultured in the presence of clofibric acid and expression of most HSC marker genes, such as vimentin and α-SMA (ACTA2), evaluated by a microarray, was not altered by the treatment, suggesting that HSC loss in the KS-type rats was not due to the direct toxic effect of CF. The expression levels of most HSC marker genes were low in both control and CF-treated rat livers. A possible link between HSC loss and the development of preneoplastic hepatic foci is discussed.

Sustained repression and translocation of Ntcp and expression of Mrp4 for cholestasis after rat 90% partial hepatectomy.

BACKGROUND & AIMS: To clarify the mechanism of persistent cholestasis after massive hepatectomy, the relationship between such cholestasis and the expression and localization of organic anion transporters for bile acids was examined in a rat model. METHODS: Male Sprague-Dawley rats were subjected to 90% hepatectomy, and tissues were harvested at 0, 1, 3, and 7 days for microarray analysis, quantitative real-time polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry to examine the expression of multidrug resistance protein 4 (Mrp4), bile salt export pump (Bsep), and sodium-dependent taurocholate cotransporting polypeptide (Ntcp). RESULTS: Persistently elevated levels of serum bile acids were observed at days 3 and 7. RT-PCR and Western blotting indicated that the expression of Mrp4, a bile acid export pump located in the basolateral membrane, was increased at day 3. The expression of Ntcp, a transporter used to uptake bile acids from the sinusoids, was significantly decreased throughout the period. The levels of Bsep, an export pump localized to the canalicular membrane, were unchanged. Immunohistochemistry revealed the localization of Mrp4 and Bsep in the basolateral and canalicular membranes, respectively. On the other hand, at days 3 and 7, Ntcp was localized in the cytoplasm and was hardly detected in the basolateral membrane. CONCLUSIONS: These results suggested that the sustained repression and translocation of Ntcp and the expression of Mrp4 at the basolateral membrane seem to be responsible for the high blood bile acids levels after massive hepatectomy.

Glutathione S-transferase A4 is a positive marker for rat hepatic foci induced by clofibrate and genotoxic carcinogens.

Peroxisome proliferators (PP), including clofibrate (CF), are non-genotoxic rodent carcinogens, and oxidative DNA damages are suggested as a causative event for carcinogenesis. Gene expression profiles differ between hepatic lesions induced by PP and genotoxic carcinogens. Our previous study revealed that expression of L-bifunctional enzyme (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, BE) was repressed in preneoplastic lesions induced by PP, whereas it was enhanced in the surrounding tissues. In the present study, we immunohistochemically examined expression of the specific glutathione S-transferase (GST) form, GST-A4, which detoxifies 4-hydroxy-alkenal, the end-product of lipid peroxides, and nuclear factor-erythroid 2-related factor 2 (Nrf2), a transcription factor for many genes encoding drug-metabolizing enzymes and defending enzymes against oxidative stress, during rat hepatocarcinogenesis induced by CF and genotoxic carcinogens. GST-A4 and Nrf2 were not expressed in BE-negative foci at 8 weeks of CF administration, but were expressed in the foci at 60 weeks. GST-A4-positive foci appeared at later stages than BE-negative foci, but its localization was coincidental with that of the latter foci. The areas of GST-A4-positive foci were larger than those of BE-negative foci without GST-A4 expression. Most GST-A4-positive foci were also positive for Nrf2. In rat livers induced by genotoxic carcinogens, GST-P-negative foci as well as GST-P-positive foci were demonstrated. GST-A4 and Nrf2 were expressed in GST-P-negative foci, whereas they were not expressed in most GST-P-positive foci. Thus, GST-A4-positive foci developed in rat livers by CF and genotoxic carcinogen administration, indicating that the enzyme is a positive marker for hepatic foci induced by these different carcinogens.

Histone acetylation and steroid receptor coactivator expression during clofibrate-induced rat hepatocarcinogenesis.

Peroxisome proliferators (PPs), non-genotoxic rodent carcinogens, cause the induction of the peroxisomal fatty acid beta-oxidation system, including bifunctional enzyme (BE) and 3-ketoacyl-CoA thiolase (TH), in the liver. GST M1 gene is polymorphic in Sprague-Dawley rats, NC- and KS-type. The KS-type rats showed enhanced susceptibility to ethyl-alpha-chlorophenoxyisobutyrate (clofibrate, CF), one of the PPs. The degree of BE induction was higher in the KS-type and preneoplastic foci developed after 6-8 weeks of treatment, whereas no foci developed in the NC-type. In the preset study, factors involved in different BE inducibility were investigated. There were no differences in hepatic peroxisome proliferator-activated receptor (PPAR) alpha levels between them. Among various coactivators for PPARalpha, only steroid receptor coactivator (SRC)-3 level was higher in the KS-type. To investigate the association between PPARalpha and SRC-3 or other proteins, nuclear extracts from CF-treated livers were applied to a PPARalpha column. In the KS-type, 110, 72, and 42 kDa proteins were bound and these were identified as SRC-3, BE, and TH, respectively. EMSA supported the binding of these proteins to PPARalpha associated to the BE enhancer in CF-treated KS-type, but not in the NC-type. Histone H3 acetylation was increased 11-fold in the KS-type by CF treatment but not in the NC-type. As BE and TH are responsible for acetyl-CoA production and SRC-3 possesses a histone acetyltransferase activity, these results suggest that enhanced BE induction in the KS-type livers is due to acetylation-mediated transcriptional activation and epigenetic mechanisms might be involved in CF-induced rat hepatocarcinogenesis.

Involvement of ETS1 in thioredoxin-binding protein 2 transcription induced by a synthetic retinoid CD437 in human osteosarcoma cells.

CD437, a synthetic retinoid, has a potent antitumor activity, in which an RAR-independent mechanism may be involved. Our previous study showed that CD437 transcriptionally upregulates the expression of thioredoxin-binding protein 2 (TBP2), leading to c-Jun N-terminal kinase 1 (JNK1)-mediated apoptosis. In the present study, we addressed the mechanism, by which CD437 induces TBP2 mRNA expression. CD437 efficiently caused the cell death of human osteosarcoma cells via apoptosis. CD437 also induced JNK1 activation through the upregulation of TBP2 mRNA, in consistent with our previous observation. A luciferase reporter assay for TBP2 promoter activation suggested that CD437-regulated TBP2 mRNA transcription requires the region between -400 and -300, which contains multiple possible ETS-binding sites. Finally, we demonstrated CD437-dependent recruitment of ETS1 transcription factor to this region by chromatin immunoprecipitation assay. These data suggest that ETS1 is involved in CD437-induced TBP2 mRNA expression in human osteosarcoma MG-63 cells.

Ferritin expression in rat hepatocytes and Kupffer cells after lead nitrate treatment.

Lead nitrate induces hepatocyte proliferation and subsequent apoptosis in rat livers. Iron is a constituent of heme and is also required for cell proliferation. In this study, the expression of ferritin light-chain (FTL), the major iron storage protein, was investigated in rat livers after a single intravenous injection of lead nitrate. Western blotting and immunohistochemistry revealed that FTL was increased in hepatocytes around the central veins and strongly expressed in nonparenchymal cells. Some FTL-positive nonparenchymal cells were identified as Kupffer cells that were positive for CD68. FTL-positive Kupffer cells occupied about 60% of CD68-positive cells in the periportal and perivenous areas. The relationships between FTL expression and apoptosis induction or the engulfment of apoptotic cells were examined. TUNEL-positive cells were increased in the treatment group, and enhanced expression of milk fat globule EGF-like 8 was demonstrated in some Kupffer cells and hepatocytes, indicating enhanced apoptosis induction and phagocytosis of apoptotic cells. FTL-positive Kupffer cells were not detected without lead nitrate treatment or in rat livers treated with clofibrate, which induces hepatocyte proliferation but not apoptosis. These results suggest that FTL expression in Kupffer cells after lead treatment is dependent on phagocytosis of apoptotic cells.

Delay in hepatocyte proliferation and prostaglandin D2 synthase expression for cholestasis due to endotoxin during partial hepatectomy in rats.

Infection is a frequent complication of liver transplantation or partial hepatectomy (PH) and sometimes results in cholestasis. We examined factors involved in infection­induced cholestasis after PH, employing a rat PH model and lipopolysaccharide (LPS) as a bacterial toxin. Male Sprague­Dawley rats were subjected to 70% PH and/or LPS injection, and tissues were harvested at 0, 24, 72 and 168 h. Gene expression was analyzed by microarray analysis and reverse transcription­quantitative polymerase chain reaction, and protein levels and localization were analyzed by western blotting and immunohistochemistry, respectively. Plasma bile acid levels were significantly higher in the LPS + PH group than in the PH group. Ribonucleotide reductase regulatory subunit M2 and proliferating cell nuclear antigen peaked at 24 and 72 h in the PH group and LPS + PH group, respectively, indicating a delay in cell proliferation in the latter group. The sodium­dependent taurocholate cotransporting polypeptide and organic­anion­transporting polypeptide 1a1 and 1a2 were reduced in the PH group at 24 h, and were not further decreased in the LPS + PH group. Chemokine ligand 9 (Cxcl9), a chemokine involved in M2 macrophage polarization, increased after 24 h in the LPS and the LPS + PH groups. The number and shape of Cxcl9­positive cells were similar to CD163­positive cells, suggesting that such cells produced the chemokine. Hematopoietic prostaglandin D2 synthase (Ptgds2) was only detected in hepatocytes of the LPS + PH group exhibiting a delay in cell proliferation. Thus, Kupffer cells activated with LPS were suggested to be responsible for a delay in hepatocyte proliferation after PH.

Development of glutathione S-transferase-P-negative foci accompanying nuclear factor-erythroid 2-related factor 2 expression during early stage of rat hepatocarcinogenesis.

Glutathione S-transferase P (GST-P), a marker for rat hepatic preneoplastic lesions, is suggested to bind to Jun N-terminal kinase (JNK) to repress stress response, and GST-P gene expression is regulated by a transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2). In this study, we examined by immunohistochemistry whether JNK2, p38 mitogen-activated protein kinase, and Nrf2 were expressed in GST-P-positive foci induced by the Solt-Farber protocol. At 2 weeks after partial hepatectomy, all GST-P-positive foci were negative for p38, and 86.4 +/- 5.6% and 64.7 +/- 6.3% of GST-P-positive foci were negative for JNK2 and Nrf2, respectively. Western blot analysis showed decreased p38 mitogen-activated protein kinase and JNK2 expression in livers treated with the protocol. In immunohistochemistry, besides GST-P-positive foci, GST-P-negative foci were detected as p38-negative foci in the surrounding tissues positive for p38. In contrast to GST-P-positive foci, most GST-P-negative foci showed enhanced Nrf2 expression. The number of GST-P-negative foci was 76 +/- 18/10 mm(2) of liver section at 2 weeks, but was undetectable at 1 week. The area of GST-P-negative foci was 0.09 +/- 0.05 mm(2), smaller than that of GST-P-positive ones (0.29 +/- 0.23). After treatment with carbon tetrachloride, small vacuoles due to liver injury were frequently observed inside GST-P-negative foci but less frequently in GST-P-positive foci. However, this treatment resulted in expression of JNK2, p38, and Nrf2 in both foci. These results showed development of GST-P-negative foci during the early stage of hepatocarcinogenesis and suggested that Nrf2 is not responsible for GST-P expression in rat hepatic preneoplastic foci.

Clofibric acid, a peroxisome proliferator-activated receptor alpha ligand, inhibits growth of human ovarian cancer.

Recent reports have shown that peroxisome proliferator-activated receptor (PPAR)alpha ligands reduce growth of some types of malignant tumors and prevent carcinogenesis. In this study, we investigated the inhibitory effect of clofibric acid (CA), a ligand for PPARalpha on growth of ovarian malignancy, in in vivo and in vitro experiments using OVCAR-3 and DISS cells derived from human ovarian cancer and aimed to elucidate the molecular mechanism of its antitumor effect. CA treatment significantly suppressed the growth of OVCAR-3 tumors xenotransplanted s.c. and significantly prolonged the survival of mice with malignant ascites derived from DISS cells as compared with control. CA also dose-dependently inhibited cell proliferation of cultured cell lines. CA treatment increased the expression of carbonyl reductase (CR), which promotes the conversion of prostaglandin E(2) (PGE(2)) to PGF(2alpha), in implanted OVCAR-3 tumors as well as cultured cells. CA treatment decreased PGE(2) level as well as vascular endothelial growth factor (VEGF) amount in both of OVCAR-3-tumor and DISS-derived ascites. Reduced microvessel density and induced apoptosis were found in solid OVCAR-3 tumors treated by CA. Transfection of CR expression vector into mouse ovarian cancer cells showed significant reduction of PGE(2) level as well as VEGF expression. These results indicate that CA produces potent antitumor effects against ovarian cancer in conjunction with a reduction of angiogenesis and induction of apoptosis. We conclude that CA could be an effective agent in ovarian cancer and should be tested alone and in combination with other anticancer drugs.

Hair keratin KRT81 is expressed in normal and breast cancer cells and contributes to their invasiveness.

Keratins are fibrous proteins. Hair keratins constitute hard structures such as the hair and nails, and cytokeratins have been used as markers of breast carcinoma. However, the expression and function of full-size hair keratin genes have not been previously demonstrated in breast cancer. We investigated the expression of the hair keratin, KRT81, and its function in human breast cancer and normal mammary epithelial cells. Western blotting showed full size 55-kDa KRT81 expression in the human breast cancer cell lines, MCF7, SKBR3 and MDA-MB-231, normal human mammary epithelial cells (HMEC), and non-neoplastic cells (MCF10A). Reverse transcription-polymerase chain reaction revealed that the full size KRT81, including its 5' region is expressed in breast cells. Immunohistochemical and immunofluorescence analyses showed that KRT81 was located in the cytoplasm. To investigate the function of KRT81, we knocked down KRT81 by siRNA in MCF10A cells. Microarray analysis revealed that the expression of genes related to invasion such as matrix metallopeptidase (MMP)9 was decreased. In KRT81-knockdown MDA-MB231 cells, zymography revealed a decrease in MMP9 activity, while scratch and invasion assays revealed that KRT81-knockdown decreased cell migration and invasion abilities. This is the first study showing that full size KRT81 is expressed in normal breast epithelial cells and breast cancer cells. Moreover, our results indicate that KRT81 contributes to the migration and invasion of breast cancer cells.

Fatty acid synthase-positive hepatocytes and subsequent steatosis in rat livers by irinotecan.

Using a rat model, we investigated factors contributing to the pathogenesis of irinotecan-associated fatty liver disease. Male Sprague-Dawley rats were administered 200 mg/kg irinotecan by intraperitoneal injection on days 1-4, but not on days 5-7. This schedule was repeated 3 times. Rats were sacrificed 4, 18 and 25 days after the last injection, and liver steatosis was evaluated by hematoxylin and eosin (H&E) staining, microarray analysis and immunohistochemistry. Panacinar intrahepatocyte vacuoles were absent on days 4 and 25, but present on day 18, and this alteration was more prominent around the bile ducts than the central veins. Microarray analysis showed that the expression of genes involved in the synthesis of cholesterol and fatty acids was upregulated on day 4. Immunohistochemistry detected fatty acid synthase (Fasn)-strongly positive hepatocytes as well as the activation of liver progenitor cells on day 4, whereas intracellular vacuoles were evident in carbonic anhydrase 3 (CA3)-positive hepatocytes on day 18. Thus, irinotecan-induced liver steatosis was preceded by Fasn-strongly-positive hepatocytes and liver progenitor cell activation. The magnitude of the decrease in the number of Fasn-strongly positive hepatocytes between days 4 and 18 was similar to that of the increase in the number of CA3-positive hepatocytes accompanying vacuoles.

Expression of MRP2 and MRP3 during liver regeneration after 90% partial hepatectomy in rats.

BACKGROUND: Small-for-size grafts often cause persistent conjugated hyperbilirubinemia in the recipient after adult-to-adult living donor liver transplantation, but the cause has not yet been clarified. In physiologic status, bilirubin is excreted from hepatocytes to the bile canaliculus by means of multidrug resistance protein (MRP) 2 and, in particular circumstances, by means of MRP3 to the sinusoidal space. The aim of this study was to research whether there is any change in bilirubin excretion pattern during liver regeneration with reference to expression of MRP2 and MRP3. METHODS: Sprague-Dawley rats underwent sham operation (n=37), 70% hepatectomy (n=38), or 90% hepatectomy (n=37). The degree of liver regeneration, total and direct bilirubin, protein synthesis, and interleukin (IL)-6 were serially assessed. Expression of MRP2 and MRP3 were semiquantified by Western blotting. RESULTS: The proliferating cell nuclear antigen labeling index indicated rapid liver regeneration after 70% and 90% hepatectomy. Serum levels of total and direct bilirubin increased significantly (P<0.05), and conjugated hyperbilirubinemia was proved only in the 90% hepatectomy group. Coagulation factor VII dipped but increased as early as 12 to 24 hr postoperatively in both hepatectomy groups. Plasma IL-6 levels were significantly increased in the 90% hepatectomy group (P<0.05). Expression of MRP2 was decreased and MRP3 was expressed at 36 and 72 hr postoperatively in the 90% hepatectomy group, whereas no change was observed in MRP expression in the 70% hepatectomy group. CONCLUSIONS: During liver regeneration after critical hepatectomy such as 90% hepatectomy, decrease of MRP2 and expression of MRP3 may play an important role in postoperative hyperbilirubinemia.

Inhibition of 3T3-L1 adipocyte differentiation by 6-ethoxyzolamide: repressed peroxisome proliferator-activated receptor gamma mRNA and enhanced CCAAT/enhancer binding protein beta mRNA levels.

The effects of 6-ethoxyzolamide (ETZ), a carbonic anhydrase (CA) inhibitor, on differentiation of the mouse preadipocyte cell line 3T3-L1 were examined by quantitative image analysis of intracellular fat storage. For adipocyte differentiation, postconfluent cells were treated with a ligand of the peroxisome proliferator-activated receptor gamma (PPARgamma), troglitazone (TRG), and dexamethasone for 2 days. Differentiated cells showed weak fat staining at day 4 which increased thereafter, correlating with CAIII expression. ETZ treatment for 10 days at a 200microM concentration reduced both the percentage of differentiated adipocytes storing fat and the amount of fat stored in individual cells. These findings were also supported by the results of fluorescence activated cell sorting analysis. Despite their light fat staining, however, CAIII was not expressed in ETZ-treated cells. Furthermore, ETZ delayed the clonal expansion of cells, an early event preceding differentiation. Northern blot analysis revealed a high CCAAT/enhancer binding protein (C/EBP) beta mRNA level and low PPARgamma mRNA in ETZ-treated cells. Thus, increased C/EBPbeta mRNA did not lead to enhanced PPARgamma expression in this case. Another CA inhibitor acetazolamide did not inhibit adipocyte differentiation, although the drug exhibited a similar inhibition pattern for CA activity as ETZ. These results suggested that inhibitory effects of ETZ on adipocyte differentiation were not due to inhibition of CA activity but rather due to altered levels of the transcription factors.

Induction of differentiation and peroxisome proliferator-activated receptor gamma expression in colon cancer cell lines by troglitazone.

PURPOSE: We investigated the relationship between the effects of troglitazone (TGZ) on cellular growth, differentiation and apoptosis induction, and the induction of peroxisome proliferator-activated receptor (PPAR) gamma in three human colon cancer cell lines, HCT-15, DLD-1and LoVo. METHODS: Viable cell number was evaluated by the Alamar blue assay and apoptotic cell death by TUNEL methods. Expression of PPARgamma mRNA and protein was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The differentiation markers of colonic mucosa, villin and MUC2 mRNAs, were analyzed by real-time RT-PCR. RESULTS: HCT-15 and DLD-1 cells proliferated rapidly while LoVo cells grew slowly. TGZ dose-dependently inhibited the proliferation of all the cell lines, and also induced apoptotic cell death. High expression of PPARgamma mRNA and protein was demonstrated in DLD-1 and LoVo cells before TGZ treatment. After the treatment, PPARgamma mRNA and protein levels were increased in HCT-15 and LoVo cells. Villin and MUC2 mRNAs were increased by TGZ treatment in HCT-15 cells while villin mRNA was repressed in LoVo cells. Changes in expression of PPARgamma, villin or MUC2 mRNAs were not observed in DLD-1 cells. CONCLUSIONS: These results suggest that PPARgamma levels are not correlated with the rates of cell proliferation. Differentiation induction by TGZ was only observed in the cell lines with enhanced PPARgamma expression.

Kinetic-controlled hydrolysis of Leu-Val-Val-hemorphin-7 catalyzed by angiotensin-converting enzyme from rat brain.

Leu-Val-Val-hemorphin-7 (LVV-H7, LVVYPWTQRY), an opioid peptide, was found to be hydrolyzed sequentially by rat brain angiotensin-converting enzyme (ACE) in three steps through dipeptidyl carboxypeptidase activity. The kinetic constants evaluated were in order of: k(1) (0.19 min(-1))>>k(2) (0.0008 min(-1)) approximately k(3) (0.0006 min(-1)) in 10 mM NaCl at pH 7.5 giving rise to LVV-H5 almost quantitatively. The decapeptide was noted to be hydrolyzed 164- and 346-fold more efficiently than angiotensin I (Ang I) in k(cat) and kcat/Km values, respectively, at their optimal conditions. The kinetic-controlled preferential action of the brain enzyme on LVV-H7 is suggestive of its multiple roles in vivo.