Molecular Mechanisms Leading to the Phenotypic Development in Paternal and Maternal Uniparental Disomy for Chromosome 14

Human chromosome 14q32.2 carries a cluster of imprinted genes. They include paternally expressed genes (PEGs) such as DLK1 and RTL1, and maternally expressed genes (MEGs) such as GTL2 (alias, MEG3), RTL1as (RTL1 antisense), and MEG8. Consistent with this, paternal and maternal uniparental disomies for chromosome 14 (upd(14)pat and upd(14)mat) cause distinct phenotypes. In this review, we summarize the current knowledge about the underlying factors for the development of upd(14)pat and upd(14)mat phenotypes. The data available suggest that the DLK1-GTL2 intergenic differentially methylated region (IG-DMR) plays an important role in the maternal to paternal epigenotypic switch, and that excessive RTL1 expression and decreased DLK1 and RTL1 expression play a major role in the development of upd(14)pat-like and upd(14)mat-like phenotypes, respectively.     

Prevention of colonic aberrant crypt foci by dietary feeding of garcinol in male F344 rats

The modifying effects of dietary feeding of a polyisoprenylated benzophenone, garcinol, isolated from Garcinia indica fruit rind on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of garcinol on proliferating cell nuclear antigen (PCNA) index in ACF and activities of detoxifying enzymes of glutathione S-transferase (GST) and quinone reductase (QR) in liver. In addition, we examined the effects of garcinol on 12-O-tetradecanoylphorbol-13-acetate-induced O(2)(-) generation in differentiated human promyelocytic HL-60 cells and lipopolysaccharide (LPS)- and interferon (IFN)-gamma-induced nitric oxide (NO) generation in mouse macrophage RAW 264.7 cells. Western blotting analysis of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression was done in LPS- and IFN-gamma-treated mouse macrophage RAW 264.7 cells. Rats were given subcutaneous injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce ACF. They also received the experimental diet containing 0.01 or 0.05% garcinol for 5 weeks, starting 1 week before the first dosing of AOM. AOM exposure produced 97 +/- 15 ACF/rat at the end of the study (week 5). Dietary administration of garcinol caused significant reduction in the frequency of ACF: 72 +/- 15 (26% reduction, P < 0.01) at a dose of 0.01% and 58 +/- 8 (40% reduction, P < 0.001) at a dose of 0.05%. Garcinol administration significantly lowered PCNA index in ACF. Feeding of garcinol significantly elevated liver GST and QR activities. In addition, garcinol could suppress O(2)(-) and NO generation and expression of iNOS and COX-2 proteins. These findings might suggest possible chemopreventive ability of garcinol, through induction of liver GST and QR, inhibition of O(2)(-) and NO generation and/or suppression of iNOS and COX-2 expression, on colon tumorigenesis.     

The external radiotherapy with three-dimensional conformal boost after the neoadjuvant androgen suppression for patients with locally advanced prostatic carcinoma

PURPOSE: To analyze the results in patients with locally advanced prostatic carcinoma treated by hormonal therapy followed by external radiotherapy using three-dimensional conformal radiation therapy (3D-CRT) boost. METHODS AND MATERIALS: From 1987 to 1995, 46 patients with histologically proven locally advanced adenocarcinoma of the prostate were treated with 3D-CRT at the National Cancer Center Hospital, Tokyo. The neoadjuvant androgen suppression started immediately after the diagnosis followed by radical radiation therapy, according to the prospective protocol. They were treated with photons of 6-14 MV for wide fields and the boost, of which a multiple-leaf collimator of 2-cm width was available. The boosted dose was delivered with the rotational 3D-CRT, after the delivery of whole pelvis 4-field box from a dose of 40-46 Gy up to 66 Gy. The planning target volume encompassed 1 cm outside throughout the clinical target volume, and the prostate and the seminal vesicles were included in the boost field. RESULTS: The 3D-CRT boost treatment completed as planned in all 46 patients. The median follow-up for all the patients was 60 months (range, 5-120 months). Nineteen of 46 patients died. Of these, 11 patients died of the intercurrent diseases. For all 46 patients, the 5- and 8-year overall survival rates were 61.3% and 42.4%, and the 5- and 8-year cause-specific survival rates were 82.4% and 64.4%, respectively. The prostate-specific antigen (PSA) relapse-free rates for 5- and 8-year were 64.6% and 52.5%, and the clinical local control rates for 5 and 8 years were 75.3% and 69.9%, respectively. The preradiation therapy PSA and the Gleason score were the factors that significantly associated with PSA relapse-free survival. Sixteen of 46 patients (35%) showed at least one form of late toxicities. Of these, 3 patients experienced late complications of Grade 3 (urinary, 2, proctitis, 1). CONCLUSION: The treatment results were fairly good and were consistent with those in Western countries, indicating that this study shows the preliminary status of 3D-CRT for the locally advanced prostate cancer in Japan. Preradiation therapy PSA seems to be a significant predictor of PSA relapse-free survival (p = 0.004) after neoadjuvant androgen suppression.     

Japanese multicenter phase II study of CHOP followed by radiotherapy in stage I-II, diffuse large B-cell lymphoma of the stomach

CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) followed by radiotherapy is regarded as standard care for localized aggressive lymphoma; however, prospective confirmation of its applicability to localized primary gastric lymphoma is inadequate, and most patients in Japan have been initially treated with gastrectomy. We conducted a multicenter phase II study to evaluate the feasibility and efficacy of the non-surgical treatment. Eligibility criteria required primary gastric diffuse large B-cell lymphoma, stage I-II(1), age 20-75, performance status 0-1 and adequate organ function. Treatment consisted of three cycles of CHOP followed by radiotherapy 40.5 Gy. Fifty-five patients were enrolled between December 1999 and February 2003, and 52 eligible patients were analyzed. Patient characteristics were as follows: median age, 61 years; 28 men, 24 women; 36 with stage I, 16 with stage II(1); 47 with a low International Prognostic Index (IPI) and five with a low-intermediate IPI. All but one patient completed planned treatment. No serious complications including massive hemorrhage or perforation were observed. A complete response was achieved in 48 of the 52 patients (92%, 95% confidence interval: 82-98%) and progressive disease in three. Two patients underwent salvage gastrectomy due to disease persistence or recurrence. With a median follow-up period of 28 months, 2-year progression-free and overall survivals were 88 and 94%, respectively. CHOP followed by radiotherapy is safe and highly effective in localized gastric diffuse large B-cell lymphoma. This organ-preserving treatment should be considered as a very reasonable therapeutic option.     

BMK1 is activated in glomeruli of diabetic rats and in mesangial cells by high glucose conditions

BACKGROUND: High glucose causes renal cell injury through various signal transduction pathways, including mitogen-activated protein (MAP) kinases cascades. Big MAP kinase 1 (BMK1), also known as extracellular signal-regulated kinase 5 (ERK5), is a recently identified MAP kinase family member and was reported to be sensitive to osmotic and oxidative stress. However, the role of BMK1 in diabetic nephropathy has not been elucidated yet. METHODS: We investigated whether BMK1 is activated in the glomeruli of Otsuka Long Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus in comparison with the control Long Evans Tokushima Otsuka (LETO) rats. We also examined the effect of high glucose on BMK1 activity in cultured rat mesangial cells. RESULTS: BMK1 and ERK1/2 but not p38 were activated in the glomeruli of OLETF rats, which showed diabetic nephropathy at 52 weeks of age. High glucose, in addition to a high concentration of raffinose, caused rapid and significant activation of BMK1 in rat mesangial cells. MAP kinase/ERK kinase (MEK) inhibitors, U0126 and PD98059, both inhibited BMK1 activation by high glucose in a concentration-dependent manner. Protein kinase C (PKC) inhibition by GF109203X and PKC down-regulation with long-time phorbol myristate acetate (PMA) treatment both inhibited BMK1 and Src kinase activation. Src kinase inhibitors, herbimycin A and PP2, also inhibited high glucose-induced BMK1 activation. PKC inhibitors, Src inhibitors and MEK inhibitors, all inhibited cell proliferation by high glucose. Finally, transfection of dominant-negative MEK5, which is an upstream regulator of BMK1, abolished the BMK1-mediated rat mesangial cell proliferation stimulated by high glucose. CONCLUSION: In the present study, we demonstrated that high glucose activates BMK1 both in vivo and in vitro. It was suggested that high glucose induces PKC- and c-Src-dependent BMK1 activation. It could not be denied that BMK1 activation is induced through an osmotic stress-sensitive mechanism. BMK1-mediated mesangial cell growth may be involved in the pathogenesis of diabetic nephropathy.     

Ebselen inhibits p38 mitogen-activated protein kinase-mediated endothelial cell death by hydrogen peroxide

Ebselen (2-phenyl-1, 2-benzisoselenazol-3[2H]-one) is a seleno-organic compound exhibiting both glutathione peroxidase and antioxidant activity. Although it has been reported that ebselen is effective against hydrogen peroxide (H(2)O(2))-induced cell death in several cell types, its effect on endothelial cell damage has not yet been elucidated. In the present study, we examined the effect of ebselen on H(2)O(2)-induced human umbilical vein endothelial cells (HUVECs) death, and its intracellular mechanism. Our findings showed that pretreatment of HUVECs with ebselen resulted in a significant recovery from H(2)O(2)-induced cell death in a concentration-dependent manner. In addition to the inhibition of lactate dehydrogenase (LDH) leakage, ebselen inhibited H(2)O(2)-induced cytochrome c release and caspase-3 activation and the resultant apoptosis in HUVECs. Moreover, it was observed that H(2)O(2) significantly stimulated activation of mitogen-activated protein (MAP) kinases, i.e., p38 MAP kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2). Ebselen inhibited H(2)O(2)-induced p38 MAP kinase, but not JNK or ERK1/2 activation. Furthermore, SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]-1H-imidazole), a specific p38 MAP kinase inhibitor, inhibited H(2)O(2)-induced p38 MAP kinase phosphorylation, cytochrome c release, caspase-3 activation, as well as cell death in HUVECs. These findings suggest that ebselen attenuates H(2)O(2)-induced endothelial cell death through the inhibition of signaling pathways mediated by p38 MAP kinase, caspase-3, and cytochrome c release. Thus, inhibition of p38 MAP kinase by ebselen may imply its usefulness for prevention and/or treatment of endothelial cell dysfunction, which was suggested to be the first step in the development of atherosclerosis.     

Src and Cas are essentially but differentially involved in angiotensin II-stimulated migration of vascular smooth muscle cells via extracellular signal-regulated kinase 1/2 and c-Jun NH2-terminal kinase activation

Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and migration. Src activity is known to be required for the migration of a number of cell types. p130Cas was reported to be essential for cell migration and actin filament reorganization. Mitogen-activated protein (MAP) kinases were also reported to be critical regulatory factors for growth and migration of VSMC. However, precise intracellular mechanisms involving c-Src, p130Cas, and MAP kinases in Ang II-stimulated migration of VSMC have not been well elucidated. Here we demonstrated that Ang II rapidly and significantly stimulated tyrosine phosphorylation of Src and Cas and their association in rat aortic smooth muscle cells (RASMC). Ang II-stimulated tyrosine phosphorylation of Src and Cas and activation of ERK1/2 and JNK, but not p38, were potently inhibited by Src family tyrosine kinase inhibitors, herbimycin A (HA) and PP2. Ang II-stimulated Src and Cas association, tyrosine phosphorylation of Cas, and activation of ERK1/2 and JNK were suppressed in kinase-inactive Src (KI Src)-overexpressed RASMC. Ang II-stimulated JNK activation but not ERK1/2 activation was blocked in substrate domain-deleted Cas (DeltaSD Cas)-overexpressed RASMC. In addition, HA, PP2, ERK1/2 inhibitor, 2'-amino-3'-methoxyflavone (PD98059) and JNK inhibitor, and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) significantly inhibited Ang II-stimulated migration of RASMC. Ang II-induced colocalization of Src and Cas and migration were inhibited in both KI Src- and DeltaSD Cas-overexpressed RASMC. These findings suggest that Src and Cas are essentially but differentially involved in Ang II-stimulated migration of VSMC through the activation of ERK1/2 and JNK.     

Acceptance of dialysis therapy in elderly patients with chronic renal failure

The aim of the present study is to clarify relevant factors concerning acceptance of dialysis therapy in elderly patients with chronic renal failure. Patients with advanced renal failure aged 60 years and over (152 cases) were investigated. The male/female ratio was 85:67. The age was 76 +/- 7 years (mean +/- standard deviation). The proportion of patients with acceptance of dialysis to patients with non-acceptance was 121:31. In all patients, the cause of renal failure (non-diabetes/diabetes), serum albumin level, comorbid conditions, ambulation, cognitive function, marital status, and presence of younger cohabitants were surveyed. The patients were divided into two groups for each category. Patients were categorized as the young-old (aged 60 to 74 years) and the old-old (aged 75 years and over). Serum albumin level was categorized as either low (less than 3.5 g/dl) or normal (3.5 g/dl and over). The number of patients who accepted dialysis therapy was evaluated for each group. Intergroup comparisons were carried out by the chi 2 test. Statistically significant factors were age (p < 0.0001), serum albumin level (p = 0.016), ambulation (p = 0.011), cognitive function (p < 0.0001), and marital status (p = 0.009). Multivariant logistic regression analysis was also performed using background factors as explanatory variables and acceptance or non-acceptance of dialysis therapy as a dependent variable. The factors presented by the nominal scale were converted to dummy variables. Statistically significant factors were age (p < 0.0001) and cognitive function (p < 0.0001). Serum albumin level, ambulation, and marital status were significant only in the chi 2 test. This could be explained by the close correlations of these factors with age and cognitive function. The old-old category and poor cognitive function were dominant factors with regard to non-acceptance of dialysis therapy.