Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

Unusually strong electronic correlation and field-induced ordered phase in YbCo2.

We report the first study of electrical resistivity, magnetization, and specific heat on YbCo2. The measurements on a single-phased sample of YbCo2bring no evidence of magnetic ordering down to 0.3 K in a zero magnetic field. The manifestations of low Kondo temperature are observed. The specific heat value divided by temperature,C/T, keeps increasing logarithmically beyond 7 J/mol K2with decreasing temperature down to 0.3 K without no sign of magnetic ordering, suggesting a very large electronic specific heat. Analysis of the magnetic specific heat indicates that the large portion of the low-temperature specific heat is not explained simply by the low Kondo temperature but is due to the strong intersite magnetic correlation in both the 3dand 4felectrons. Temperature-dependent measurements under static magnetic fields up to 7 T are carried out, which show the evolution of field-induced transition above 2 T. The transition temperature increases with increasing field, pointing to a ferromagnetic character. The extrapolation of the transition temperature to zero field suggests that YbCo2is in the very proximity of the quantum critical point. These results indicate that in the unique case of YbCo2, the itinerant electron magnetism of Co 3d-electrons and the Kondo effect within the vicinity of quantum criticality of Yb 4f-local moments can both play a role.

Establishment of two interleukin 6 (B cell stimulatory factor 2/interferon beta 2)-dependent human bone marrow-derived myeloma cell lines.

Two IL-6-dependent human multiple myeloma cell lines, ILKM2 and ILKM3, were established from the bone marrow of patients with IgG-K multiple myeloma. Both cell lines had the typical morphology and immunocytochemical features of myeloma cells. The surface phenotype of both cell lines was PCA-1+, OKT10+, CD10(J-5)-, CD19(B4)-, CD20(B1)-, CD21(B2)-, and OKIa-1-. A monoclonal cytoplasmic Ig, IgG-K or K L chain, was positive in ILKM2 or ILKM3, respectively. EBV nuclear antigen was negative in both cell lines. They proliferated in the presence of macrophages or macrophage-derived factors (MDF). Among the recombinant cytokines examined, IL-6 most strongly augmented the growth of both cell lines. The anti-IL-6 antibody completely inhibited the IL-6-dependent growth and almost completely inhibited the MDF- or purified MDF-dependent growth of both cell lines, ILKM2 and ILKM3 are now being maintained in the culture medium containing 2 ng/ml rIL-6. These results suggest that IL-6 produced by macrophages may play an important role in the growth of myeloma cells in vivo and that macrophages or IL-6 can be used for establishing human myeloma cell lines.

Endovascular treatment of vertebral artery dissection using stents and coils: its pitfall and technical considerations.

OBJECTIVE: There are various options for the treatment of vertebral artery dissection aneurysms (VADA). Treatment with stents may be an effective method to treat VADA involving the posterior inferior cerebellar artery (PICA) and dissection of the dominant vertebral artery (VA). In this article, our personal experience of the treatment of VADAs by using stents and coils is reported. METHODS: Since 1998, 26 cases of VADA have been treated by endovascular surgery by the first author. Of these cases, 6 cases were treated using stents, 3 of which were treated using stent and coils, 2 patients were treated using double overlapping stents, and the remaining one patient was treated using a single stent. RESULTS: In all patients, dissection aneurysms were successfully covered by stents. There was one complication: an intraprocedural rupture during additional coil insertion without neurological deterioration. Follow-up angiography was performed in all 5 surviving patients except for one patient who died due to the severity of his original subarachnoid hemorrhage (mean duration of follow-up angiography 22.8 months, range 15-57 months). Total or subtotal disappearance of the VADA was achieved in all 5 cases. At one year after the treatment, all 5 surviving patients remained clinically stable without any neurological deficit. CONCLUSIONS: Treatment using stents is an effective alternative for the treatment of VA dissecting aneurysms, especially for lesions of the dominant VA or involving the PICA. However, additional coil insertion should be performed very carefully and may be avoided if stagnation of contrast material is achieved after overlapping stenting.

Endoscopic ultrasound-guided fine needle aspiration biopsy for splenic tumor: a case series.

Splenic tumors are occasionally found in clinical practice but the diagnosis is often difficult if only serologic and imaging tests are used. Therefore, pathologic sampling is required in such cases. Endoscopic ultrasonography (EUS) provides a good image of the spleen through the gastric wall, and a transgastric EUS-guided fine needle aspiration (EUS-FNA) biopsy may be easier than the percutaneous approach. Furthermore, a large-gauge needle may raise the capability of EUS-FNA for the histopathologic diagnosis. The aim of this study was to evaluate the yield of EUS-FNA using a large-gauge needle for a splenic tumor. Five patients with splenic tumor were subjected to EUS-FNA with a 19-gauge needle to obtain histopathologic materials. A pathologic sample was obtained in all cases, and the diagnoses were lymphoma (n = 2), sarcoidosis (n = 2), and inflammatory pseudotumor (n = 1). EUS-FNA using a 19-gauge needle is safe and useful for the diagnosis of splenic tumors.

Safety studies of LP20 powder produced from heat-killed Lactobacillus plantarum L-137.

The safety of LP20 and its prototype, a powder, with potential use in food, produced from a mixture of dextrin and heat-killed Lactobacillus plantarum L-137, was assessed in an acute study in mice, and in an in vitro bacterial reverse mutation assay, an in vitro chromosome aberration assay, and an in vivo mouse micronucleus assay. LP20 prototype was not acutely toxic when administered to male and female Slc:ICR mice by single gavage at 2000mg/kg bw. Dosing was not associated with mortality, clinical signs, changes in bodyweight, or macroscopic abnormalities. The LD(50) in mice was greater than 2000mg/kg bw. There was no evidence of genotoxicity of LP20 in the Ames assay (0-5000microg/plate) or in the in vitro chromosome aberration assay with Chinese hamster lung fibroblasts (0-5000microg/mL). Administration of two consecutive daily doses of 500, 1000, or 2000mg/kg bw by gavage to male Crlj:CD-1 mice was not associated with an increased incidence of micronuclei and did not alter the ratio of polychromatic to normochromatic erythrocytes. These studies show that LP20 powder is not acutely toxic and is without genotoxic activity both in vitro and in vivo.

Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum.

REASONS FOR PERFORMING STUDY: Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. HYPOTHESIS: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. METHODS: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n=100) and sera (n=100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n=95) and horses (n=50). RESULTS: Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K63 and K238 of the EGF-like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 x biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals. CONCLUSIONS AND POTENTIAL RELEVANCE: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.

The yield of endoscopic ultrasound-guided fine needle aspiration for histological diagnosis in patients suspected of stage I sarcoidosis.

BACKGROUND AND STUDY AIM: Sarcoidosis is a systemic disorder of unknown cause that is characterized by a pathological hallmark, noncaseating granuloma. Bilateral hilar lymphadenopathy (BHL) is a major clinical feature, but it is sometimes difficult to exclude other diseases, especially in cases where there are no pulmonary abnormalities (stage I). Bronchoscopic transbronchial biopsy is currently a popular method by which to obtain pathological material, but its diagnostic power is insignificant. Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), also attempted recently, makes the sampling of pathological material easier and better, but the diagnoses are still based on cytological findings. Our study aimed to evaluate the yield of transesophageal EUS-FNA for histological confirmation of stage I sarcoidosis. METHODS: The study was a prospective comparative study to investigate the diagnostic sensitivities of FNA cytology and FNA histology. Subjects were consecutive patients with BHL without lung lesions on chest radiographs or chest CT who were referred to our hospitals between December 2003 and April 2006. Transesophageal EUS-FNA was performed with 19-gauge needles instead of the conventional 22-gauge needles. RESULTS: Forty-one patients were included in this study, and both histological and cytological materials were obtained successfully by EUS-FNA in all patients. Histopathological examination of the FNA sample showed noncaseating granuloma in 34 (94.4%) of the 36 patients with a final diagnosis of sarcoidosis. In contrast, only 28 of the 36 (77.8%) were diagnosed as having sarcoidosis on the basis of cytological findings. The difference was statistically significant (P = 0.0444). CONCLUSION: FNA histology is better suited than FNA cytology to establishing the diagnosis of stage I sarcoidosis, and EUS-FNA with a 19-gauge needle plays a important role in this process.

Long-term Follow-up of Laparoscope-Assisted Living Donor Hepatectomy.

BACKGROUND: We have introduced and performed laparoscope-assisted surgery in living donor hepatectomy. The objective of this study was to investigate the long-term results of laparoscope-assisted living donor hepatectomy. METHODS: From 2006 to 2016, laparoscope-assisted living donor hepatectomy was performed in 11 patients (laparoscopic group), and conventional open living donor hepatectomy was performed in 40 patients (conventional group). Intraoperative and postoperative complications were evaluated according to the Clavien-Dindo classification and analyzed in the laparoscopic group for comparison with the conventional group. RESULTS: The median postoperative follow-up period was 88 months (range, 58-120 months) in the laparoscopic group. One donor in the conventional group died from a motor vehicle crash 16 months after surgery. All others were alive and returned to their preoperative activity level. Regarding intraoperative and early (≤90 days after surgery) postoperative complications, 1 patient (1/11, 9%) showed biliary fistula (Grade IIIa) in the laparoscopic group. In the conventional group, 6 patients (6/40, 15%) showed surgical complications of Grade I in 2 patients and Grade II in 4 patients. Regarding late (>90 days after surgery) postoperative complications, biliary stricture was observed in 1 patient of the laparoscopic group; this patient developed hepatolithiasis 6 years after surgery, and endoscopic lithotomy and extracorporeal shockwave lithotripsy were performed, resulting in successful treatment. Late complications were not observed in the conventional group. CONCLUSION: One donor in the laparoscopic group showed Grade IIIa late complications. The introduction of laparoscopic surgery to living donor hepatectomy should be performed carefully.

Clonality analysis of lymphoproliferative disorders in patients with Sjögren's syndrome.

The aim of this study was to clarify the nature of the clonal lymphocyte infiltration in Sjögren's syndrome (SS) patients associated with lymphoproliferative disorders. We examined B cell clonality in lymphoproliferative tissues from six primary SS patients associated with lymphoproliferative disorders or lymphoma by cloning and sequencing of the gene rearrangement of the immunoglobulin heavy chain complementarity determining region 3 (IgVH-CDR3). Three patients with sequential observation showed progressional clonal expansion with the presence of the same subclone in different tissues during the course of disease. Among them, one patient developed mucosa-associated lymphoid tissue (MALT) lymphoma in glandular parotid. The other three SS patients concomitant with malignant B cells lymphomas showed different clonal expansion of B cells between nodal sites and salivary glands. The cloanality analysis indicated that monoclonal B cell population could spread from one glandular site to another site during the course of SS, suggesting that the malignant clone may arise from the general abnormal microenvironment, not restricted to the glandular tissue, in some SS patients.

Pseudomyxoma peritonei and mucinous pyometral fluid arising from an ovarian borderline mucinous tumor: case report.

An extremely rare case of a pseudomyxoma peritonei (PMP) and mucinous pyometral fluid, possibly arising from an ovarian borderline mucinous tumor is reported. A 68-year-old Japanese patient received an expolatory laparatomy under a working diagnosis of a PMP, left ovarian cystic tumor and an umbilical hernia. Surgery and platinum-based chemotherapy induced a 15-month disease-free condition.

Primary lymphoma of the uterine corpus: an unusual location for a common disease--case report.

A case of a primary uterine corpus lymphoma in a 75-year-old woman is described. Immunohistochemical studies showed diffuse large B-cell type one. Primary lymphoma of the uterine corpus is considered to be an unusual location for a common disease.

Prognostic heterogeneity of the seventh edition of UICC Stage III gallbladder carcinoma: Which patients benefit from surgical resection?

BACKGROUND: This study sought to evaluate the prognostic heterogeneity of Stage III (Union for International Cancer Control, seventh edition) gallbladder carcinoma. METHODS: Of 175 patients enrolled with gallbladder carcinoma who underwent radical resection, 22 were classified with Stage IIIA disease (T3N0M0) and 46 with Stage IIIB disease (T2N1M0 [n = 23] and T3N1M0 [n = 23]). The median number of retrieved lymph nodes per patient was 18. RESULTS: This staging system failed to stratify outcomes between Stages IIIA and IIIB; survival after resection was better for patients with Stage IIIB disease than for patients with Stage IIIA disease, with 5-year survival of 54.9% and 41.0%, respectively (p = 0.366). Multivariate analysis for patients with Stage III disease revealed independently better survival for patients with T2N1M0 than for patients with T3N0M0 (p = 0.016) or T3N1M0 (p = 0.001), with 5-year survival of 77.0%, 41.0%, and 31.0%, respectively. When N1 status was subdivided according to the number of positive nodes, 5-year survival in patients with T2M0 with 1-2 positive nodes, T2M0 with ≥3 positive nodes, T3M0 with 1-2 positive nodes, and T3M0 with ≥3 positive nodes was 83.3%, 50.0%, 45.8%, and 0%, respectively (p < 0.001). CONCLUSIONS: The prognosis of T2N1M0 disease was better than that of T3N0/1M0 disease, suggesting that not all node-positive patients will have uniformly poor outcomes after resection of gallbladder carcinoma. T2M0 with 1-2 positive nodes leads to a favorable outcome after resection, whereas T3M0 with ≥3 positive nodes indicates a dismal prognosis.

p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells.

Temozolomide (TMZ) is a DNA-methylating agent that has recently been introduced into Phase II and III trials for the treatment of gliomas. TMZ produces O6-methylguanine in DNA, which mispairs with thymine during the next cycle of DNA replication. Subsequent futile cycles of DNA mismatch repair can lead to a p53-associated apoptotic cell death, although this mechanism has been described mostly in hematopoietic neoplasms. We studied the action of TMZ in gliomas and the role p53 might play by using U87 glioma cells that were either p53-wild-type or p53-deficient (by virtue of expression of the viral oncoprotein E6). LN-Z308 cells, in which p53 gene is deleted, were also used. p53-proficient U87 MG cells underwent a prolonged, p53- and p21(Waf1/Cip1)-associated G2-M arrest beginning 2 days after TMZ treatment. Although very few of these cells underwent apoptosis, most underwent senescence over a 10-day period. p53-deficient (E6-transfected U87 and LN-Z308) cells similarly underwent G2-M arrest in response to TMZ, but this arrest was accompanied by only minor changes in p53 or p21(Waf1/Cip1) and was reversed within 7 days of TMZ treatment in association with the appearance of cells with either 8n or subG1 DNA content. These results suggest that glioma cells respond to TMZ by undergoing G2-M arrest. p53 is not necessary for this G2-M arrest to occur but is important in the duration of G2-M arrest and in the ultimate fate of TMZ-treated cells. Therefore, the integrity of the G2-M cell cycle checkpoint may be important in the cytotoxicity of TMZ in glioma cells.

Abrogation of the Chk1-mediated G(2) checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells.

Temozolomide (TMZ) produces O(6)-methylguanine in DNA, which in turn mispairs with thymine, triggering futile DNA mismatch repair (MMR) and ultimately cell death. We found previously that in p53-proficient human glioma cells, TMZ-induced futile DNA MMR resulted not in apoptosis but rather in prolonged, p53- and p21-associated G(2)-M arrest and senescence. Additionally, p53-deficient cells were relatively more TMZ resistant than p53-deficient glioma cells, which underwent only transient G(2)-M arrest before death by mitotic catastrophe. These results suggested that prolonged G(2)-M arrest might protect cells from TMZ-induced cytotoxicity. In the present study, we therefore focused on the mechanism by which TMZ induces G(2)-M arrest and on whether inhibition of such G(2)-M arrest might sensitize glioma cells to TMZ-induced toxicity. U87MG glioma cells treated with TMZ underwent G(2)-M arrest associated with Chk1 activation and phosphorylation of both cdc25C and cdc2. These TMZ-induced effects were inhibited by the Chk1 kinase inhibitor UCN-01. Although not in itself toxic, UCN-01 increased the cytotoxicity of TMZ 5-fold, primarily by inhibiting cellular senescence and increasing the percentage of cells bypassing G(2)-M arrest and undergoing mitotic catastrophe. In addition to enhancing TMZ-induced cytotoxicity in p53-proficient cells, UCN-01 also blocked TMZ-induced Chk1 activation and transient G(2)-M arrest in p53-deficient U87MG-E6 cells and similarly enhanced TMZ-induced mitotic catastrophe and cell death. Taken together, these results indicate that Chk1 links TMZ-induced MMR to G(2)-M arrest. Furthermore, inhibition of the cytoprotective G(2) arrest pathway sensitizes cells to TMZ-induced cytotoxicity and may represent a novel, mechanism-based means of increasing TMZ efficacy in both p53 wild-type and p53 mutant glioma cells.

Modulation of experimental colon tumorigenesis by types and amounts of dietary fatty acids.

Epidemiological studies and laboratory animal model assays suggest that a high intake of dietary fat promotes colorectal cancer. Several in vivo and in vitro studies support the hypothesis that omega-6 fatty acids promote colon tumorigenesis, whereas omega-3 fatty acids lack promoting activity. Fat intake in the United States traditionally includes high amounts (30% of total caloric intake) of saturated fat rather than omega-6 fatty acids. Therefore, the present study was designed to compare the modulatory effects of a high-fat diet containing mixed lipids (HFML), a diet rich in saturated fatty acids (the average American diet), a diet with fish oil (HFFO) that is rich in omega-3 fatty acids, and a low-fat corn oil diet (LFCO) on the formation of chemically induced colonic aberrant crypt foci (ACF) and tumors, cyclooxygenase (COX)-2 activity, and apoptosis during experimental colon carcinogenesis. At 5 weeks of age, groups of male F344 rats were fed a 5% corn oil diet (LFCO). At 7 weeks of age, rats intended for carcinogen treatment received s.c. injections of azoxymethane at a dose level of 15 mg/kg of body weight once weekly for 2 weeks. Beginning 1 day after the carcinogen treatment, groups of rats were then maintained on experimental diets containing 20% HFML or 20% HFFO. Rats were killed at 8, 23, or 38 weeks after azoxymethane treatment. Colonic ACF and tumors were evaluated histopathologically, and apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated nick end labeling method. Colonic mucosae and tumor samples harvested at week 38 were analyzed for COX-2 synthetic activity and expression. The rats fed the HFML diet showed significantly increased total colonic ACF (P < 0.001-0.0001) with a multiplicity of > or = 4 aberrant crypts/focus (P < 0.0001) compared with the effects of the HFFO or LFCO diets at week 8, 23, and 38. Interestingly, there was a 2- to 3-fold increase (> or = 4) in multicrypt foci in rats given the HFML diet as compared with such foci in rats fed the HFFO or LFCO diets. By week 23, the HFML diet had significantly increased the incidence of colonic tumors (30-60%) and their multiplicity (100-141%) when compared with the effects of the LFCO or HFFO diets. At week 38, the HFML diet had induced 100% colon tumor incidence and a 4-fold multiplicity of adenocarcinomas compared with the LFCO and HFFO diets. At weeks 23 and 38, a significantly lower percentage of apoptotic colonic epithelial cells were observed in the tumors of animals fed the HFML diet as compared with those fed the HFFO diet. The HFML diet caused significantly increased levels of COX-2 activity in colon tumors (P < 0.05-0.01), and these tumors had enhanced levels of COX-2 expression as compared with those in assays with LFCO or HFFO diets. These observations demonstrate for the first time that HFML diets containing high levels of saturated fatty acids (such as those in Western diets) promote colon carcinogenesis. Although the mechanisms involved in colon tumor promotion by a HFML diet are not fully known, our results indicate that the modulation of eicosanoid production via the influence on COX activity and the suppression of apoptosis may play a key role in HFML diet-induced colon tumorigenesis.

Preventive potential of wheat bran fractions against experimental colon carcinogenesis: implications for human colon cancer prevention.

Epidemiological studies suggest an inverse relationship between the intake of dietary fiber, particularly fiber from cereal grains, and colon cancer risk. Animal model assays have demonstrated that the protective effects of dietary fiber on colon cancer development depend on the nature and source of the fiber. Wheat bran (WB) appears to inhibit colon tumorigenesis more consistently than do oat bran or corn bran. This study was designed to determine whether specific WB fractions such as WB fiber, WB lipids, or phytic acid differentially affect colon carcinogenesis in a well-established colon cancer model. In addition, the modulating effect of specific fractions of WB on the activities of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-1 and COX-2 enzymes were assessed in colon tumors as those have been shown to play a role in tumor progression. At 5 weeks of age, groups of male F344 rats were assigned to one of six diets: a high-fat diet containing 10% WB (control diet) and experimental high-fat diets containing 10% dephytinized WB (WB-P), 10% defatted WB (WB-F), 10% dephytinized and defatted WB (WB-PF), 10% WB-PF fortified with 2% bran oil and/or with 0.4% phytate. At 7 weeks of age, all eats except those in the vehicle-treated groups were given two weekly s.c. injections of azoxymethane (AOM) at a dose rate of 15 mg/kg body weight/week. They continued to receive their respective diets until 50 weeks after carcinogen treatment and were then killed. Colon tumors were analyzed for iNOS, COX-1, and COX-2 expression and enzymatic activities. Colon tumors were evaluated histopathologically and classified as adenomas and adenocarcinomas. We found that removal of phytic acid (WB-P) or lipids (WB-F) from WB had no significant effect on colon tumor incidence (% animals with tumors) or multiplicity (tumors/ animal), whereas removal of both phytate and lipids from WB (WB-PF) significantly increased colon tumor multiplicity and volume. Interestingly, WB-PF fortified with excess bran oil or with bran oil plus phytate significantly inhibited colon tumor incidence, multiplicity, and volume; but supplementation of WB-PF with phytate alone had no significant effect on colon tumorigenesis in rats suggesting that lipid fraction of WB possesses tumor-inhibitory properties. Moreover, feeding WB-PF diet significantly increased iNOS, total COX and COX-2 enzyme activities, and iNOS protein expression in colon tumors as compared with wheat bran control diet. Feeding the WB-PF that was fortified with excess bran oil alone or with bran oil plus phytate significantly suppressed the activities of iNOS and COX-2 as well as the expression of iNOS and COX-2 in colon tumors compared with that in rats fed the WB diet or WB-PF diet. The study demonstrates for the first time that the lipid fraction of wheat bran has strong colon tumor inhibitor properties. The exact mechanism(s) by which the lipid fraction of WB inhibits colon carcinogenesis in addition to alteration of iNOS and COX activities remains to be elucidated. Additional studies are warranted to identify biologically active constituents of lipid fraction of WB and their relative role in colon tumor inhibition.

Frequent beta-catenin gene mutations and accumulations of the protein in the putative preneoplastic lesions lacking macroscopic aberrant crypt foci appearance, in rat colon carcinogenesis.

Activating mutations in the beta-catenin gene is thought to be responsible for the excessive beta-catenin signaling involved in the majority of carcinogen-induced colonic carcinomas. To determine whether beta-catenin signaling is involved in the initial stage of colon carcinogenesis, mutational analysis of the gene and immunohistochemistry for beta-catenin protein were performed in the early appearing lesions, including aberrant crypt foci (ACF), of colonic mucosa in rats given azoxymethane. Male F344 rats received s.c. injections of azoxymethane at a dose of 15 mg/kg body weight, once weekly for 3 weeks, and they were sacrificed 10 weeks after the carcinogen treatment. The colonic mucosa was examined in en face preparations and in serial sections after the observation in whole mount preparations. Microscopical observations in the cross sections have shown two populations of histologically altered crypts. The first type had a macroscopic feature resembling ACF [histologically altered crypts with ACF appearance (HACAs)]. The second type of altered crypts did not have the ACF-like appearance and could not be clearly distinguished from adjacent normal crypts in whole mount preparations [histologically altered crypts with macroscopically normal-like appearance (HACNs)]. The beta-catenin gene mutations were recognized in 10 of 15 HACNs (67%) and 3 of 15 HACAs (20%). Frequent immunoreactivity of beta-catenin protein was seen in the cytoplasm of HACNs (13 of 15 cases), whereas apparent accumulation was not confirmed in any HACAs analyzed. These results suggest that (a) there are two types of putative preneoplastic lesions in cancer-predisposed colonic mucosa, and beta-catenin signaling may contribute to the initial stage of colon carcinogenesis; and (b) HACNs are more likely to be direct precursors of colon tumors than HACAs in the rat colon carcinogenesis.

Chemoprevention of azoxymethane-induced colon carcinogenesis by dietary feeding of S-methyl methane thiosulfonate in male F344 rats.

Modifying effects of dietary exposure of S-methyl methane thiosulfonate (MMTS) isolated from cauliflower Brassica oleracea L. var. botrytis on rat colon carcinogenesis induced by azoxymethane (AOM) and on the expression of cell proliferation biomarkers were investigated in two experiments. In experiment 1, male F344 rats were given three s.c. injections of AOM (15 mg/kg body weight) and fed 100 ppm MMTS for 5 weeks, starting 1 week before the first dose of AOM. The frequency of colonic aberrant crypt foci was determined at 5 weeks after the start. Feeding of 100 ppm MMTS for 5 weeks significantly decreased the number of aberrant crypt foci/colon. Colonic mucosal ornithine decarboxylase activity and the number of silver-stained nucleolar organizer regions per nucleus in colonic epithelium were significantly decreased by MMTS treatment compared with those of AOM alone. In experiment 2, effects of dietary feeding of MMTS at two doses (20 and 100 ppm) during the postinitiation phase on intestinal tumorigenesis initiated with AOM were investigated by using a long-term experiments in male F344 rats. Incidence of intestinal neoplasms of rats fed MMTS-containing diets after AOM exposure were reduced in a dose-dependent manner. Feeding of MMTS during the postinitiation phase decreased the number of aberrant crypt foci/colon, colonic ornithine decarboxylase activity, 5-bromodeoxyuridine-labeling index in colonic epithelium, and polyamine level in blood compared with those of AOM alone. These results suggest that MMTS might be a possible chemopreventive agent for intestinal neoplasia.

Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma.

The formation of human malignant gliomas is thought to involve the accumulation of multiple genetic alterations. To define the function of specific alterations in glioma formation, we serially introduced genetic alterations functionally equivalent to those noted in human malignant gliomas into normal human astrocytes (NHAs). We then monitored the ability of each of these alterations to contribute to the growth of otherwise genetically stable NHAs into intracranial malignant gliomas. Using this model, we show that expression of human telomerase catalytic component (hTERT), but not E7-mediated inactivation of pRb or E6/E7-mediated inactivation of p53/pRb, was sufficient to initiate the tumorigenic process by circumventing cellular senescence in astrocytes. hTERT expression, even in combination with inactivation of p53/pRb, did not transform astrocytes. These alterations together, however, cooperated with ras pathway activation (initiated by expression of mutant H-Ras), but not with phosphatidylinositol 3-kinase pathway activation (initiated by expression of myristoylated Akt) or epidermal growth factor receptor activation, to allow for the formation of intracranial tumors strongly resembling p53/pRb pathway-deficient, telomerase-positive, ras-activated human grade III anaplastic astrocytomas. These results identify four pathways as key in the development of human anaplastic astrocytomas.