BrdU Labeling Index Research Articles

Bioassay by intratracheal instillation for detection of lung toxicity due to fine particles in F344 male rats

We have established and documented an in vivo bioassay for detection of hazards with intratracheally instilled fine particles, which can be used for risk assessment of toxicity of materials inhaled into deep lung tissue of humans (Yokohira et al. Establishment of a bioassay system for detection of lung toxicity due to fine particle instillation: sequential histopathological changes with acute and subacute lung damage due to intratracheal instillation of quartz in F344 male rats. J Toxicol Pathol 2005;18:13–8). For validation we here examined toxicity of fine particles from quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black with this bioassay. A total of 108, 10-week-old F344/DuCrj male rats were randomly divided into 8 groups. Groups 1 to 5 underwent intratracheal instillation of the 5 test particles (4 mg/rat) suspended in 0.2 ml vehicle (saline or 10% propylene glycol and 1% sodium carboxymethyl cellulose in saline: PG-CMC) with a specially designed aerolizer, and subgroups of 7 rats were killed on Days 1 and 28 thereafter. Groups 6 and 7 similarly were exposed to saline and PG-CMC, respectively, as vehicle controls, while group 8 was maintained untreated. Using histopathological changes and immunohistochemically assessed bromodeoxyuridine (BrdU) labeling indices, inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-3 (MMP-3) levels as end points, the quartz treated group exhibited high toxicity, while the values for the other particle-treated groups pointed to only slight effects. Although additional efforts are needed to establish advantages and disadvantages with our bioassay, models featuring intratracheal instillation clearly can be useful for detection of acute or subacute lung toxicity due to inhaled fine particles by using histopathological scoring and markers like BrdU and iNOS for screening purposes in short-term studies.

SILENCING OF BASIGIN BY RNA INTERFERENCE REDUCES THE TUMORIGENITY OF PANCREATIC CANCER CELLS

Basigin (Bsg) is released by tumor cells to stimulate MMP synthesis in adjacent mesenchymal cells and has gained growing interest due to its role in cancer cell - host interaction. Because the role of Bsg in cancer cell biology is unknown, we developed an inducible siRNA expression system that is based on the tetracycline repressor and eukaryotic RNA polymerase III promoter H1 in MiaPaCa2 pancreatic cancer cells to investigate the Bsg loss of function phenotye in MiaPaCa2 with respect to invasion and tumorigenity. Wild type MiaPaCa2 strongly express and release Basigin as assessed by rtPCR, western-blotting, immunoassay and fluorescence-microscopy. RNAi reduced the expression of Basigin mRNA as well as cell associated and soluble Bsg protein by more than 90%. In the presence of 10% fetal calf serum Basigin silencing reduced adhesion dependent tumor cell proliferation in culture whereas only a modest reduction of colony formation in soft-agar growth was observed. The reduced growth rate correlated with a reduction of the BrdU labeling index (−30%). Moreover, Basigin knock-out reduced invasiveness of cancer-cells in a boyden chamber assay and in the CAM-Assay. Finally, Basigin silencing reduced the size of subcutaneously induced cancer-cell xenografts in nude-mice grown for 6 weeks by almost 50%. To elucidate the genes involved in the Bsg knock-out phenotype, we employed a whole genome DNA microarry. Using stringent selection criteria we identified 913 genes that were differentially expressed after tetracylcine induced bsg silencing compared to the uninduced counterpart. The differentially regulated genes were significantly overrepresented in Gene Ontology groups linked to regulation of signal transduction, cell-growth and apoptosis. In conclusion we identified the Basigin protein as a major regulator of cell growth and invasion in pancreatic cancer. Moreover, our results imply the therapeutic potential of RNAi in the treatment of pancreatic cancer by targeting overexpressed genes like Basigin.

Neogenesis and proliferation of β-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of β-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a β-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of β-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of β-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted β-cell differentiation (mainly from duct cells) and proliferation of pre-existing β-cells, resulting in an increase of the β-cell mass that improved glucose tolerance in diabetic mice.

Proliferation and differentiation of duct epithelial cells after partial pancreatectomy in rats

The proliferation and differentiation of pancreatic duct epithelial cells in remnant pancreas during regeneration after partial pancreatectomy in rats were studied, and the source of pancreatic stem cells was characterized. Partial (90 %) pancreatectomy was performed on 4- to 5-week-old Sprague-Dawley rats, and different duct epithelial cells and acinar cells were detected by immunohistrochemical stain method and scored using 5-bromo-2'-deoxyuridine (BrdU) labeling index (LI) at various time points after partial pancreatectomy. It was found that at 24 h after partial pancreatectomy proliferation started in the main, large and small duct cells, and persisted in small duct cells to day 5. There was significant difference between the experimental group and the control group (P<0.001). Acinar cells positive for BrdU were greatly increased and reached the peak LI on day 5. The destroyed lobular architecture almost totally recovered on day 7, and the newly islet cells appeared around the pancreatic ducts. These results suggest that regeneration after partial pancreatectomy is involved in proliferation and differentiation of pancreatic stem cells, and pancreatic stem cells may locate in the pancreatic ductules.

Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation

We recently demonstrated that deficiency in endothelial nitric oxide synthase (eNOS) results in congenital septal defects and postnatal heart failure. The aim of this study was to investigate the role of eNOS in cardiomyocyte proliferation and maturation during postnatal development. Cultured eNOS knockout (eNOS(-/-)) cardiomyocytes displayed fewer cells and lower bromodeoxyuridine (BrdU) incorporation in vitro compared with wild-type (WT) cardiomyocytes (P < 0.05). Treatment with the nitric oxide (NO) donor diethylenetriamine NONOate increased BrdU incorporation and cell counts in eNOS(-/-) cardiomyocytes (P < 0.05). Inhibition of nitric oxide synthase activity using N(G)-nitro-L-arginine methyl ester decreased the level of BrdU incorporation and cell counts in WT cardiomyocytes (P < 0.05). Vascular endothelial growth factor (VEGF) increased the level of BrdU incorporation in cultured WT cardiomyocytes in a dose- and time-dependent manner (P < 0.05). Conversely, VEGF did not alter BrdU incorporation in eNOS(-/-) cardiomyocytes (P = not significant). Furthermore, deficiency in eNOS significantly decreased BrdU labeling indexes in neonatal hearts in vivo. Although WT hearts displayed a rapid decrease in atrial natriuretic peptide (ANP) expression in the first week of neonatal life, ANP expression in eNOS(-/-) hearts remain elevated. Our study demonstrated that NO production from eNOS is necessary for postnatal cardiomyocyte proliferation and maturation, suggesting that eNOS plays an important role during postnatal heart development.

In VitroCulture of Mesenchymal Lineage Cells Established from the Colonial Tunicate Botryllus primigenus

Body trunks were isolated from juvenile zooids of the Japanese colonial tunicate Botryllus primigenus and cultured in vitro to establish tissue-specific cell lines. Epidermal cells from some explants spread and formed a flat sheet consisting of vacuolated cells. They then dissociated into single cells, and their growth stopped within two weeks. Continuously proliferating cells were established from four explants. After the 20th implantation, nuclear and mitochondrial DNAs were extracted from these cells. The nucleotide sequences of proliferating cell nuclear antigen (PCNA) and mitochondrial large ribosomal RNA (mtlrRNA) completely matched the PCNA and mtlrRNA taken from living colonies of B. primigenus; this shows that the four independently proliferating cells were indeed of the Botryllus origin. One cell line (Bp0306E10) comprised round-shaped cells with a diameter of 8-10 microm. These cells have been cultured in vitro with a doubling time of approximately 24 hours since June, 2003. The BrdU labeling index was approximately 2%. Monoclonal antibodies raised against the cultured cells recognized a 28 kDa polypeptide and stained free mesenchymal cells in vivo. G418-resistant subclonal cells could be established by introducing a tunicate retrotransposon loaded with the neomycin resistance gene into the cells by electroporation. This study is the first to succeed in producing a sustainable cell culture of Botryllus.

다층 배양된 암세포에서 파크리탁셀의 항증식효과 분석

Human solid tumors exhibit a multicellular resistance (MCR) resulting from limited drug penetration and decreased sensitivity of tumor cells when interacting with their microenvironments. Multicellular cultures represent solid tumor condition in vivo and provide clinically relevant data. There is little data on antitumor effect of paclitaxel (PTX) in multicellular cultures although its MCR has been demonstrated. In the present study, we evaluated antiproliferative effects of PTX in multicellular layers (MCL) of DLD-1 human colorectal carcinoma cells. BrdU labeling index (LI), thickness of MCL, cell cycle distribution and cellular uptake of calcein were measured before and after exposure to PTX at 0.1 to 50 for 24, 48 and 72 hrs. BrdU LI and thickness of MCL showed a concentration- and time-dependent decrease and the changes in both parameters were similar, i.e., 34.2% and 40.6% decrease in BrdU LI and thickness, respectively, when exposed to for 72 hr. The DLD-1 cells grown in MCL showed increase in and resistance to cell cycle arrest and apoptosis compared to monolayers. Calcein uptake in MCL did not change upon PTX exposure, indicating technical problems in multicellular system. Overall, these data indicate that antitumor activity of PTX may be limited in human solid tumors (a multicellular system) and MCL may be an appropriate model to study further pharmacodynamics of PTX.

Etiology of bromate-induced cancer and possible modes of action-studies in Japan

Renal cell tumors were significantly increased in male and female rats given potassium bromate at 250 and 500 mg/L in drinking water. In at least one other study renal cell tumors were produced in male rats at 125 mg/L. Among male mice given 750 mg/L of potassium bromate, there were no significant differences in renal cell tumors between treated and control groups after 88 weeks on test. In oxidative DNA damage tests 8-oxodeoxyguanosine (8-oxodG also referred to as 8-OH-dG) was induced in DNA in the male rat kidney in 1 week, and in females after 3 weeks at 500 mg/L, and also in both male and female rats at 250 mg/L, but not at 125 mg/L. DNA adducts are considered to be an initial step in the carcinogenesis process, however, the administered doses are not always sufficient to cause mutations, possibly due to DNA repair. In the two-step rat renal carcinogenesis model using N-ethyl- N-hydroxyethylnitrosamine (EHEN) as initiator, promotion activity by potassium bromate was measured using the BrdU labeling index. The promoting activity of bromate in male rats was much greater and extended to doses as low as 60 mg/L in male rats, whereas in females the response was limited to 250 and 500 mg/L. Therefore, it was concluded that the mechanisms contributing to cancer in the male rat were more complex than in the female rat. The accumulation of α 2μ-globulin in the kidneys of male rats exposed to potassium bromate probably accounts for the greater labeling index in the male rat relative to the female rat. Accumulation of α 2μ-globulin as a result of treatment with chemicals is unique to the male rat and does contribute to carcinogenic responses. Neither humans nor female rats display this response. Nevertheless, bromate must be considered carcinogenic because of the response of the female rats. The better correlation between 8-oxodG formation and tumor response indicates that dose–response information from the female rat would be much more relevant to human risk assessment. The fact that an elevation of BrdU-LI in the kidney of the female rat is consistent with the possibility that cell proliferation observed in female rats resulted from oxidative stress and/or cytotoxic responses in the kidney. Therefore, oxidative stress is most likely the mechanism of interest for cancer risk in humans.

Evaluation of Cell Proliferation in Canine Tumors by the Bromodeoxyuridine Labeling Method, Immunostaining of Ki-67 Antigen and Proliferating Cell Nuclear Antigen

Cell proliferation activity and correlation between the LI (labeling index) of BrdU (5-bromo-2'-deoxy-uridine) and the expression of the Ki-67 antigen or PCNA (proliferating cell nuclear antigen) were evaluated by immunohistochemistry in 18 canine spontaneous neoplasms. Spearman's rank correlation coefficient and linear regression analysis revealed a significant correlation between the BrdU LI and Ki-67 LI, BrdU LI and PCNA LI, and BrdU LI and MI (mitotic index) in each case [rs = 0.917, P < 0.01 (BrdU LI vs. Ki-67 LI), rs = 0.736, P < 0.01 (BrdU LI vs. PCNA LI), rs = 0.748, P < 0.01 (BrdU LI vs. MI) by Spearman's rank correlation coefficient; and r = 0.918, P < 0.01 (BrdU LI vs. Ki-67 LI), r = 0.626, P < 0.01 (BrdU LI vs. PCNA LI), r = 0.763, P < 0.01 (BrdU LI vs. MI) by linear regression analysis]. Ki-67 LIs were more closely correlated with BrdU LIs than PCNA LIs or MIs, and the Ki-67 antigen is useful as an endogenous proliferation marker in canine neoplasms.

Biphasic Increase of Epithelial DNA Synthesis in the Urinary Bladder of Rats Treated with a Tumor Promoter, Sodium L-ascorbate

Sequential changes in the levels of DNA synthesis in urinary bladder epithelium following administration of a rat tumor promoter, sodium L-ascorbate (Na-AsA) for 8 weeks were examined. Male 6-week-old F344 rats were allocated into 4 groups, and given powdered basal diet containing Na-AsA at concentrations of 0 (control), 1.25, 2.5 or 5.0%. The 5-bromo-2'-deoxyuridine (BrdU) labeling index in bladder epithelium in the treated groups increased at week 1, but returned to low levels between weeks 2 and 6, and increased at week 8 once again, essentially with dose dependency. The epithelium of the urinary bladder appeared normal at weeks 1 and 2, but slight simple hyperplasia was observed at weeks 4, 6 and 8 with a dose-dependent response. Our results demonstrate that the early DNA synthesis induced in the rat bladder epithelium by Na-AsA is biphasic in nature.

Regenerative capacity of individual liver lobesin the microsurgical mouse model

In the development of microsurgical mouse models of hepatic regeneration and repair, lobe-specific regenerative responses were observed. We therefore determined the hepatic regenerative capacity of individual mouse liver lobes. In mice, 26, 60, 75, and 83% of total liver mass was resected. Bromo-deoxyuridine (BrdU) was injected prior to liver harvest and the BrdU labeling index determined in all remaining individual liver lobes. BrdU-positive nuclei were seen in all liver lobes after the 26 and 60% resection, but significantly fewer were detected in the caudate lobe. In the 75% group, equally distributed positive nuclei were found. However, BrdU labeling was scant in the 83% group. In microsurgical mouse liver-regeneration models, the average hepatic response depends on amount of liver tissue resected and on the remaining liver lobe. BrdU incorporation can vary significantly among individual lobes. The lobe-specific differences observed may prove valuable in further investigations of hepatic regeneration and repair.

Reciprocal Interaction between Megakaryocytes and Myeloma Cells Results in Increased Survival and Proliferation of Myeloma Cells.

Megakaryocytes are often expanded in bone marrow (BM) areas infiltrated with myeloma (MM) cells while thrombocytopenia is uncommon in untreated MM patients. The aim of this study was to investigate the reciprocal interaction between primary megakaryocytes and MM cells. Cultures of megakaryocytes and their precursors were prepared by incubating mobilized peripheral blood in IMDM media supplemented with BSA, thrombopoeitin (TPO), IL-6 and IL-3. Following removal of all non-adherent cells, the remaining adherent megakaryocytes (>90%) highly expressed c-MPL, CD41a and factor VIII, and had various degree of ploidy. We initially demonstrated that purified MM cell-conditioned media from 7 patients increased migration of megakaryocyte precursors across a 8 μM pore size membrane 3.6±0.9 fold (p=0.001), an effect that was inhibited by anti-CXCR4 neutralizing antibody (p<0.003). To study whether TPO is produced in myelomatous BM, mesenchymal stem cells (MSCs) were cultured on the backside of 1 μM pore-size transwell inserts' membranes while MM cells were incubated in the upper chamber of the inserts. Histologic examination demonstrated that the MSC's cytoplasmic villi pass through the membrane pores, allowing contact with tumor cells. Using qRT-PCR, we demonstrated upregulation of TPO expression by 5±3 and 3±1 folds in co-cultured MSCs and MM cells, respectively. Co-culture of megakaryocyte precursors with CD138-selected MM cells stimulated their differentiation into mature megakaryocytes as determined by flow cytometry DNA content. This suggests that megakaryopoiesis and platelet maintenance are locally and systemically regulated in MM. To study the effect of megakaryocytes on tumor cells, purified MM cells from 14 patients were co-cultured with megakaryocytes in media containing FBS and TPO at an estimated ratio of 1000:1 for 5–7 days. Myeloma cells did not firmly adhere to megakaryocytes and were easily removed by gentle pipetting. Survival and proliferation of MM cells were determined by trypan blue exclusion, annexin V/PI flow cytometry, MTT assay and BrdU labeling index. MM cells in co-cultures with megakaryocytes had more viable cells (225,000±22,000 vs. 482,000±46,000, p<0.0001), higher MTT OD reading (0.4±0.1 vs. 1.1±0.2, p<0.03), fewer annexin V positive cells (79%±6 vs. 33%±5, p<0.0008) and higher labeling index (2.1±0.5 vs. 6.7±1.3, p<0.01) compared to MM cells cultured alone. Culture of MM cells in transwell inserts and megakaryocytes on the bottom of wells (non-contact conditions), partially supported survival and proliferation of MM cells, indicating that both soluble factors and physical contact are involved in MM-megakaryocyte interactions. Co-culture of MM cells from 5 patients with either megakaryocytes or osteoclasts resulted in similar stimulatory effects on MM cells. Since bortezomib is increasingly used for MM treatment and known to induce thrombocytopenia in patients with MM we evaluated its effect on isolated megakaryocytes using MTT assay. Bortezomib at concentrations of 0.2–1 nM effectively inhibited growth of megakaryocytes and MM cell lines, CAG and ARP-1, but not MSCs, suggesting an additional mechanism for its anti-MM response. We conclude that megakaryocytes are an important microenvironmental component directly supporting MM growth. Therapeutic interventions that interfere with MM-megakaryocyte interaction may help control MM.

Evidence for Ribosomal Insufficiency Affecting Non-Erythroid Cells in RPS19 and Non-RPS19 Mutated DBA.

The pathophysiology of Diamond Blackfan Anemia (DBA) is not confined to erythropoiesis. Associated physical anomalies and growth retardation are common, and there is an increased risk of hemopoietic and non-hemopoietic cancers. Heterozygous mutations affecting RPS19, encoding ribosomal protein S19, are present in 26% of cases of DBA. The indistinguishable clinical phenotype of DBA patients with and without RPS19 mutations predicts that all DBA genes share a common function, or interdependence in a common pathway. If the known ribosomal role of RPS19 underlies the pathogenesis of DBA, as opposed to an as yet unknown erythroid specific extraribosomal role, then every cell type should be affected, by analogy with the Bst mouse, another mammalian example of a naturally occurring ribosomal protein haploinsufficiency. In addition, cells from DBA patients with and without RPS19 mutations should be similarly affected. EBV lymphoblastoid cell lines (LCLs) generated from peripheral blood lymphocytes from DBA patients were studied as an easily accessible source of non-erythroid DBA cells to test these predictions. As RPS19 haploinsufficiency may not affect ribosomal abundance in the steady state, cells were subjected to serum deprivation and refeeding to induce translational stress. Under these conditions, 6 DBA LCLs, including 2 with RPS19 mutations, showed impaired proliferation in comparison with control LCLs. A similar phenotype could be induced in control LCLs by low dose rapamycin or cycloheximide. On FACS analysis, two subpopulations of EBV LCLs can be distinguished. Staining with Pyronin Y or FITC showed the larger cells to have a tight RNA and total protein content, with no difference between normal and DBA LCLs, and unaffected by treatment with rapamycin or cycloheximide. The other population of smaller cells had a lower, more variable RNA and protein content. DBA LCLs or rapamycin treated control LCLs had a relatively lower proportion of the larger, actively proliferating cells. These results would be consistent with exit from cell cycle of DBA LCLs in response to translational stress. To determine whether the actively proliferating subpopulation of cells showed altered cell cycle kinetics, the cumulative entry into S phase of cells on refeeding was determined by the addition of BrdU at the time of serum refeeding. FACS analysis showed BrdU positive cells to be contained exclusively within the population of larger LCLs in control and DBA. The BrdU labeling index was reduced in DBA LCLs, and exacerbated by rapamycin. A reduction was also observed in rapamycin treated control LCLs. We have thus demonstrated an impaired growth phenotype and alteration in cell cycle kinetics in non-erythroid cells derived from patients with DBA with and without RPS19 mutations, supporting the hypothesis that ribosomal pathophysiology is the underlying common denominator in DBA. As there was no difference in the 28S:18S ratio in total cellular RNA extracted from control or 5 DBA LCLs, pulse-chase labeling with 3H-uridine was used to study nascent rRNA as an indicator of ribosomal biogenesis. Interestingly, in comparison with a control LCL, there was an apparent relative reduction in 18S nascent rRNA in one DBA LCL and in 28S rRNA in another, both without detectable RPS19 mutations, confirmed in repeat independent experiments. Further investigations are in progress to determine whether these findings reflect an epiphenomenon in the context of an immortalised cell line, or truly reflect abnormal ribosomal biogenesis in DBA.

Altered localization and expression of tight-junction proteins in a rat model with chronic acid reflux esophagitis

The esophageal tight junction is responsible for the paracellular sealing of the epithelium. Alteration of the expression of tight-junction proteins plays crucial roles in the pathogenesis of some human diseases. The aim of this study was to investigate the distribution and expression pattern of tight-junction proteins in the esophageal mucosa of control rats and rats with reflux esophagitis. Chronic acid reflux esophagitis was experimentally induced by operation in rats. The animals were killed on days 7 and 14 after the operation. The thickness of the mucosa and the 5-bromo-2-deoxyuridine (BrdU) labeling index were assessed. The expression pattern of the tight-junction proteins claudin 1-4 and occludin in the esophageal mucosa was investigated by immunofluorescence staining and Western blotting in the controls and esophagitis rats. In the esophagitis model, the thickness and BrdU labeling index increased with time. In control rats, claudin-1, -3, and -4 were localized on the cellular membranes of esophageal epithelial cells, mainly in the spinous and granular layers, while claudin-2 was not detected in any layer. Occludin was seen on the cellular membranes in all esophageal mucosal layers. In the esophagitis rats, the expression of claudin-1 was increased both in the plasma membrane and in the cytoplasm around the erosion in the spinous and granular layers. The expression of claudin-4 and occludin shifted to the cytoplasm from the plasma membrane in the spinous and granular layers. In contrast, the expression of claudin-3 was decreased in the spinous and granular layers. The localization and the expression patterns of tight-junction proteins were different in the controls and the rat esophagitis model. The expression of claudin-3 in the esophageal mucosa was decreased, while that of claudin-1 was increased. It is postulated that these alterations in tight-junction proteins most likely increase the permeability of the esophageal the epithelium, thereby impairing the defense mechanism of this epithelium.

Suppressive effect of rice extract on Helicobacter pylori infection in a Mongolian gerbil model

Rice extract has been shown to protect gastric mucosa from stress-induced damage. In this study, the antibiotic effect and the anti-inflammatory effect of orally administered aqueous rice extract on Helicobacter pylori infection and H. pylori-induced gastritis, respectively, in Mongolian gerbils were investigated. Fifty specific-pathogen-free male Mongolian gerbils, seven weeks old, were divided into four groups: uninfected, untreated animals (group A); uninfected, rice extract-treated animals (group B); H. pylori-infected, untreated animals (group C); and H. pylori-infected, rice extract-treated animals (group D). Group C and D animals were killed 12 weeks after H. pylori infection (i.e., at 19 weeks of age) and group A and B animals were also killed at age 19 weeks. The stomachs were removed for histopathological examination with hematoxylin-and-eosin staining and anti-5'-bromo-2'-deoxyuridine (BrdU) immunostaining, and to determine the bacterial burden. Serum anti-H. pylori antibody titers were also tested. In groups A and B, the gastric mucosa showed no inflammatory cell infiltration and a few BrdU-reactive cells. Group C animals developed marked chronic active gastritis in the gastric mucosa, and BrdU-labeled cells in the gastric mucosa markedly increased in number. In group D animals, a significant reduction occurred in the degree of neutrophilic polymorphonuclear cell infiltration into the gastric mucosa, in the BrdU-labeling indices of gastric epithelial cells, and in anti-H. pylori antibody titers in the serum (P < 0.01), compared with although H. pylori was not completely eradicated. The rice extract was effective in suppressing inflammation and epithelial cell proliferation in the gastric mucosa in H. pylori-infected Mongolian gerbils. The rice extract has potential to exhibit a protective effect on H. pylori-related gastric mucosal diseases.

Suppression of azoxymethane‐induced colon cancer development in rats by a prostaglandin E receptor EP1‐selective antagonist

Prostaglandin E(2) is involved in colon carcinogenesis through its binding to the PGE(2) receptor subtypes EP(1), EP(2), EP(3) and EP(4). We have demonstrated that administration of ONO-8711, an EP(1)-selective antagonist, suppresses development of AOM-induced ACF in C57BL/6 mice and F344 rats. ONO-8711 also reduced the numbers of intestinal polyps in Min mice. In the present study, we investigated the long-term effects of ONO-8711 on colon cancer development in rats treated with AOM. Male F344 rats were injected subcutaneously with AOM (15 mg/kg body weight) once a week for the first 2 weeks to develop colon cancer. Administration of 400 or 800 p.p.m. ONO-8711 in their diets for 32 weeks reduced the incidence, multiplicity and volume of colon carcinomas. The incidence of colon adenocarcinomas in AOM-treated rats was 97, 83 and 76% (P < 0.05) in the 0, 400 and 800 p.p.m. of ONO-8711 groups, respectively. The multiplicity of adenocarcinomas was also decreased significantly, being 3.31 +/- 0.33, 2.34 +/- 0.27 (P < 0.05) and 2.06 +/- 0.34 (P < 0.01) with 0, 400 and 800 p.p.m. of ONO-8711, respectively. Moreover, treatment with 800 p.p.m. ONO-8711 reduced the mean volume of adenocarcinomas to 49% (P < 0.05) of the value for the AOM treatment alone. Furthermore, the BrdU labeling index was decreased significantly in colon cancer cells by 800 p.p.m. ONO-8711. These results confirm that EP(1) is involved in colon carcinogenesis and that EP(1)-selective antagonists might be promising candidates for colon cancer chemopreventive agents.

Impaired proliferation of non-parenchymal cells participates in an impairment of liver regeneration in db/db mice

In this study, we examined the possibility that impaired proliferation of non-parenchymal cells affects in an impairment of liver regeneration in db/db mice, which are congenitally deficient in receptors for leptin. Liver regeneration after a two thirds partial hepatectomy (2/3 PH) was impaired in 10-week-old female db/db mice. The proliferation of both hepatocytes and non-parenchymal cells estimated from a bromodeoxyuridine (BrdU) labeling index was suppressed, and the protein expression of vascular endothelial growth factor was blocked in db/db mice. Although the extent of fatty change and the level of epidermal growth factor receptor protein expression in the liver were improved in 5-week-old db/db mice, the regeneration of liver was impaired after 2/3 PH in both 5- and 10-week-old db/db mice. These results suggested that suppressed proliferation of non-parenchymal cells contributes to the impairment of liver regeneration in db/db mice. As leptin has also the angiogenic effect, the angiogenic inhibitor FR-118487 was administered to ICR mice to examine liver regeneration after 2/3 PH, and the rate of regeneration was affected. In conclusion, it is suggested that the suppressed proliferation of non-parenchymal cells contributes to the impairment of liver regeneration probably through a disrupted angiogenesis in db/db mice.

The effect of larval age on developmental changes in the polyp prepattern of a hydrozoan planula

The larvae of many marine organisms including hydrozoans are lecithotrophic and will not feed until after metamorphosis. In hydrozoans the aboral region of the planula becomes the holdfast and stolon, while the oral region becomes the stalk and hydranth that grows out of the holdfast following metamorphosis. If metamorphosis is delayed, the portion of the planula allocated to form holdfast and stolon shrinks and the region that forms the hydranth increases in size. Planulae also have the ability to regenerate their polyp prepattern. When the aboral region of the planula that does not normally form a hydranth is isolated and metamorphosis is delayed, it acquires the capacity to form a hydranth from the holdfast. A relatively high proportion of entodermal cells of young planulae engage in DNA synthesis (BrdU labeling index); as planulae age, the labeling index falls close to zero. When the polyp prepattern is modified during planula regeneration, entodermal cells are induced to engage in DNA synthesis. If DNA synthesis is inhibited in planulae, the polyp prepattern changes during regeneration and age-related developmental changes in planula are inhibited, suggesting that DNA synthesis is a necessary part of the pattern respecification process.

Histopathological Analyses of Proliferative Potentials of Intracranial Meningiomas Using Bromodeoxyuridine and MIB-1 Immunohistochemistry

The proliferative potentials of intracranial meningiomas, as represented by bromodeoxyuridine (BrdU) labeling index (LI) and MIB-1 staining index (SI), were examined, and their correlation with recurrence and regrowth of meningiomas was reviewed. The mean BrdU LI of recurrent meningiomas was reported to be significantly higher than that of non-recurrent meningiomas. BrdU LI can predict the time to re-operation. Many studies have been conducted to examine the relationship between MIB-1 SI and clinical outcome of meningiomas, and most of them showed a significant relationship. Time interval to next recurrence in recurrent meningiomas is associated with MIB-1 SI, and several investigators have shown a cut-off point for MIB-1 SI that can predict future recurrence. BrdU LI, MIB-1 SI, and tumor doubling time of intracranial meningiomas have a mutual relationship. While many other biological or genetic variables can affect the proliferation of tumors, nevertheless BrdU LI and MIB-1 SI are closely correlated with clinical recurrence or regrowth of meningiomas.

Minoxidil, a KATP channel opener, accelerates DNA synthesis following partial hepatectomy in rats

A large number of studies have reported the action of K(ATP) channel openers in accelerating the proliferation of hepatocytes and many other cell types in vitro. Few studies, however, have examined the proliferative effect of K(ATP) channel openers in vivo. The aim of this study was to determine whether the K(ATP) channel opener minoxidil accelerates liver regeneration after partial hepatectomy (PH) in vivo. Male Wistar rats underwent a 70% partial hepatectomy (PH) after receiving a subcutaneous injection of minoxidil (0.01 mg/kg or 0.03 mg/kg). Some of the rats were intravenously treated with 5-hydroxydecanoic acid (5-HD, 10 mg/kg) just before the minoxidil injection. Seventy-two hours after PH, DNA synthesis was immunohistochemically assessed by bromodeoxyuridine (BrdU) incorporation into the nuclei. Minoxidil induced significant and dose-dependent increase in the BrdU labeling index after PH, and 5-HD reversed this minoxidil-induced change. Minoxidil did not significantly affect the changes in liver weight and liver function after PH. The hepatic levels of prealbumin decreased by about 60% after PH and minoxidil inhibited the decrease. In conclusion, the K(ATP) channel opener minoxidil enhanced DNA synthesis after PH without affecting the liver function.