Apoptotic protease activating factor 1 (Apaf-1)-independent cell death suppression by Bcl-2.

Reportedly, antiapoptotic Bcl-2 family proteins suppress apoptosis by binding to and inhibiting members of the CED-4 family of caspase activators. To explore this question, we used embryonic stem (ES) cells in which one (-/+) or both (-/-) copies of the gene encoding apoptotic protease activating factor 1 (Apaf-1), a CED-4 homologue, were disrupted by homologous recombination. Stable clones of heterozygous (-/+) and homozygous (-/-) Apaf-1 knockout ES cells that overexpressed Bcl-2 were generated. Withdrawal of serum growth factors or stimulation of heterozygous ES cells with staurosporine (STS), ultraviolet (UV)B irradiation, etoposide (VP16), or cisplatin induced apoptosis followed by cell death (determined by failure to exclude propidium iodide dye). These cell death stimuli also induced activation of several types of caspases and loss of mitochondrial membrane potential (DeltaPsi) in heterozygous (+/-) Apaf-1 knockout ES cells. In addition, overexpression of Bcl-2 protected against these events in Apaf-1-expressing ES cells. In contrast, STS, UVB, and VP16 induced little or no caspase activation and apoptosis in homozygous (-/-) Apaf-1 knockout ES cells. Nevertheless, Apaf-1-deficient ES cells subjected to these cell death stimuli or deprived of growth factors did eventually die through a nonapoptotic mechanism associated with loss of DeltaPsi. Moreover, Bcl-2 overprotection preserved DeltaPsi, reduced the percentage of Apaf-1(-/)- ES cells undergoing cell death, and increased clonigenic survival. The extent of Bcl-2-mediated cytoprotection was not significantly different for heterozygous (-/+) versus homozygous (-/-) Apaf-1 knockout cells. Furthermore, although Bcl-2 could be readily coimmunoprecipitated with Bax, associations with Apaf-1 were undetectable under conditions where Apaf-1 interactions with procaspase-9 were observed. We conclude that Bcl-2 has cytoprotective functions independent of Apaf-1, preserving mitochondrial function through a caspase-independent mechanism.

Characteristics of light intensity enhancement of a silver nanoprism with rounded corners.

We have fabricated silver nanoprisms of 100-600 nm side length by focussed ion beam lithography and measured the light intensity scattering spectra using dark-field microscopy. Two resonance peaks due to localized surface plasmon excitation were observed in the spectra and their central frequency shown to depend on the prism size. The near-field electromagnetic intensity distribution with TE-polarized light at the vacuum wavelength of 632.8 nm was measured. We have obtained a much lower light intensity enhancement than previously numerically predicated. However, scattering spectra obtained numerically, taking into account roundness of the prism corners, agree well with experimental ones. At the same time, the numerically determined field distribution was different to the near-field intensity obtained experimentally. Our results suggest the particular shape of the corner region of the prism is a key factor for obtaining a large light intensity enhancement and shaping the local field distribution.

Blood pressure regulates platelet-derived growth factor A-chain gene expression in vascular smooth muscle cells in vivo. An autocrine mechanism promoting hypertensive vascular hypertrophy.

To clarify the role of PDGF A-chain in hypertensive vascular hypertrophy of spontaneously hypertensive rats (SHRs), we studied levels of PDGF A-chain gene expression and transcription factors related to the gene in vascular smooth muscle cells (VSMCs) of SHRs in vivo. RNase protection assay and in situ hybridization showed that PDGF A-chain mRNA levels in VSMCs of SHRs were twofold higher than in those of normotensive Wistar-Kyoto rats. Gel retardation assays showed that levels of Sp1 and AP-2 in VSMCs of SHRs were twofold more abundant than in those of Wistar-Kyoto rats. Treatment with four pharmacologically different species of antihypertensive drugs for 2 wk decreased the levels of both PDGF A-chain mRNA and Sp1, but not AP-2 level in VSMCs of SHRs with regression of aortic hypertrophy, indicating that increases in levels of both PDGF A-chain mRNA and Sp1 in VSMCs of SHRs were associated with high blood pressure. These results suggest that high blood pressure is a stimulus which upregulates PDGF A-chain gene expression in VSMCs of SHRs, resulting in an autocrine enhancement in hypertensive vascular hypertrophy, and that the activation of the gene may be mediated through increases in Sp1 in these cells.

Sensitivity of human KB cells expressing platelet-derived endothelial cell growth factor to pyrimidine antimetabolites.

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism. However, little is known about its physiological functions. We previously purified dThdPase from human placenta, isolated a complementary DNA clone for this enzyme, and sequenced it. There was complete sequence identity between 120 amino acids of human dThdPase and the sequence of platelet-derived endothelial cell growth factor (PD-ECGF). Human KB epidermal carcinoma cells transfected with platelet-derived endothelial cell growth factor complementary DNA expressed a 55-kDa protein that was detected with anti-dThdPase antibody and the cell lysate had dThdPase activity. The sensitivity of transfected cells to the antimetabolites was compared with that of untransfected KB cells. The sensitivity of the transfected cells to Doxifluridine (5'-deoxy-5-fluorouridine) was higher than that of untransfected KB cells. Transfected cells were also more sensitive to Tegafur than untransfected KB cells. These results demonstrate that dThdPase is involved in the activation of these anticancer agents. Since many cancer tissues contain high dThdPase activity compared with normal tissues, these transfected and untransfected KB cells are useful for studying the role of dThdPase in the activation of pyrimidine antimetabolites and also in angiogenesis.

Predictive value of Ki-67 and argyrophilic nucleolar organizer region staining for lymph node metastasis in gastric cancer.

Proliferative activities in 91 primary gastric carcinomas and 36 corresponding metastatic perigastric lymph nodes were investigated using Ki-67 labeling percentage and an argyrophilic nucleolar organizer region (AgNOR) count. Tumors with a high proliferative activity often metastasized to lymph nodes, and the proliferative activities of the primary lesion and the perigastric lymph node metastases were similar. A significant correlation was recognized between the Ki-67 labeling percentage and the AgNOR count (r = 0.744; P less than 0.001). The Ki-67 labeling percentage and AgNOR count proved to be useful predictors of nodal metastasis regardless of tumor size, depth of invasion, and histological type. Even when tumors are smaller (less than 7 cm) or the stage of the disease is early (pT1, 2), the formation of metastasis increased with an increased Ki-67 labeling percentage or AgNOR count. The combination analysis of depth of invasion with Ki-67 labeling percentage or AgNOR count gives a more precise prediction of nodal metastasis, compared with histological analysis alone.

Role of thymidine phosphorylase activity in the angiogenic effect of platelet derived endothelial cell growth factor/thymidine phosphorylase.

Human thymidine phosphorylase (dThdPase) has been reported to be identical with the platelet-derived endothelial cell growth factor (PD-ECGF). To investigate whether the dThdPase activity of PD-ECGF/dThdPase is indispensable to its angiogenic activity, three PD-ECGF/dThdPase mutants, K115E (Lys-115-->Glu), L148R (Leu-148-->Arg) and R202S (Arg-202-->Ser) were made by site-directed mutagenesis. Although the expression level of the three mutant PD-ECGF/dThdPases in the COS-7 cells was similar to that of wild-type PD-ECGF/dThdPase, dThdPase activity was not detected in the COS-7 cells transfected with the mutant PD-ECGF/dThdPase cDNA. The lysates of COS-7 cells transfected with the wild-type PD-ECGF/dThdPase cDNA had angiogenic activity, but those transfected with the mutant PD-ECGF/dThdPase cDNAs did not. An inhibitor of dThdPase, 6-amino-5-chlorouracil, inhibited the angiogenic activity of purified dThdPase. These findings indicate that dThdPase activity is indispensable to the angiogenic activity of PD-ECGF/dThdPase.

Consistency of DNA ploidy between primary and recurrent gastric carcinomas.

Eleven patients with gastric carcinoma, who had undergone re-resection of the lesion due to recurrence in the remnant stomach, were the subjects of a cytophotometric DNA analytical study. The objective was to determine whether or not the DNA cytogenic profile of cancer cells would be consistent during the growth of the carcinoma. The DNA distribution patterns were grouped into types I, II, and III, according to the proportion of aneuploid cell population. Ten of 11 had the same DNA distribution patterns in the primary and recurrent lesions, while in the other one patient with type III in the primary lesion, type II was evident at the time of recurrence. The DNA cytogenic profile of cancer cells is thus a valid cell marker of a given tumor and should be applicable for determining the natural history of gastric carcinoma.

High expression of ganglioside alpha-2,8-sialyltransferase (GD3 synthase) gene in adult T-cell leukemia cells unrelated to the gene expression of human T-lymphotropic virus type I.

Expression of ganglioside alpha-2,8-sialyltransferase (GD3 synthetase; EC 2.4.99.8) gene and gangliosides in human leukemia cells were analyzed. Flow cytometric analysis of various types of leukemia cells using monoclonal antibodies revealed that GD3 was expressed at a moderate level on adult T-cell leukemia (ATL) cells and weakly on T-cell acute lymphocytic leukemia cells. Accordingly, high levels of GD3 synthase mRNA in ATL and low levels in T-cell acute lymphocytic leukemia were demonstrated by semiquantitative reverse transcription-PCR analysis. High-level expression of the GD3 synthase gene in ATL cells was detected in the uncultured state, and was not changed during short-term culture in vitro. This was in contrast with the rapid up-regulation of GM2/GD2 synthase gene, which was observed within 24 h after start of culture after the emergence of human T-lymphotropic virus type I (HTLV-I) gene expression in cultured normal T lymphocytes with or without HTLV-I p40tax protein, GD3 synthase gene was almost equally expressed, whereas GM2/GD2 synthase gene was much more strongly expressed in the p40tax-positive T cells. These findings indicated that the GD3 synthase gene was highly expressed in ATL cells unrelated to HTLV-I genome expression.

Progesterone and its metabolites: the potent inhibitors of the transporting activity of P-glycoprotein in the adrenal gland.

P-glycoprotein (P-gp) is a transmembrane glycoprotein responsible for the multidrug resistant (MDR) phenotype in various cancer cells. It has been shown that P-gp transports various kinds of anti-cancer agents as well as hydrophobic chemicals. Although P-gp is also expressed in normal human tissues, such as liver, kidney, and adrenal gland, its function and transporting substrates in these tissues are still unknown. In previous work, we demonstrated that some compounds in human plasma modulate the transporting activity of P-gp. We also found that P-gp is expressed at a high level in the bovine adrenal gland and that this tissue contains large amount of compounds which inhibit the transporting activity of P-gp. We purified such compounds from the adrenal gland by monitoring the ability to enhance the accumulation of [3H]vincristine in MDR cells. Two major compounds were purified and identified as progesterone and pregnenolone by nuclear magnetic resonance (NMR) analysis. Progesterone was the most potent and abundant compound that inhibited the transporting activity of P-gp among the compounds extracted from bovine adrenal gland with methanol. We also found that six authentic progesterone metabolites in the 5 beta-metabolic pathway but none in the 5 alpha-metabolic pathway were able to enhance the accumulation of [3H]vincristine in MDR cells and to inhibit [3H]azidopine photolabeling of P-gp in the adrenal gland. These results indicate that some progesterone metabolites can interact with P-gp and that stereoisomerism around carbon 5 of the progesterone metabolites is important for them to be recognized by P-gp.

Motility related protein 1 (MRP1/CD9) expression in colon cancer.

It is important to detect genes that may be good prognostic markers for colon cancer patients. With this in mind, we identified the motility related protein-1 (MRP1/CD9) gene in human colon tissues. The aim of this study was to clarify the significance of MRP1/CD9 gene expression in human colon cancers. We performed the differential mRNA display technique between tumor/normal paired samples of the colon and identified MRP1/CD9. Eighty-two surgical specimens of primary colorectal cancer were analyzed by means of reverse transcription-PCR for the MRP1/CD9 gene. Its expression status and clinicopathological variables were analyzed univariately and multivariately. The MRP1/ CD9 mRNA expression was positive in 56 cases and negative in 26 cases. The MRP1/CD9 negative cases showed a significantly higher frequency of venous-vessel invasion and liver metastasis, or a worse prognosis than the MRP1/CD9 positive cases (P < 0.05). Multivariate analysis with the Cox regression model disclosed that MRP1/CD9 expression was an independent prognostic factor distinct from the lymph node status. The findings imply that the study of MRP1/CD9 expression may be useful for predicting prognosis of patients with colorectal cancer.

A case of esophageal small cell carcinoma with multiple liver metastases responding to chemotherapy with irinotecan plus cisplatin.

We report a case of small cell esophageal carcinoma (SCEC) with multiple liver metastases treated with some success by chemotherapy with irinotecan (CPT-11) plus cisplatin (CDDP). Radiologic and endoscopic examination of a 75-year-old man with multiple liver tumors disclosed a 4.0-cm type 2 tumor in the middle third of the esophagus. An endoscopically obtained biopsy specimen was diagnosed as undifferentiated small cell carcinoma. Multiple liver metastases were confirmed but lymph node metastases and distant metastases other than those in the liver were not detected. After six courses of chemotherapy with CPT-11 plus CDDP, the primary lesion showed complete response and liver metastases showed partial response. However, because all lesions almost immediately relapsed or progressed, arterial infusion chemotherapy for liver metastases and radiation for the primary lesion were given as second-line treatment. The primary lesion showed complete response with radiation. Arterial infusion chemotherapy prevented the progression of liver metastases once, but the patient died of liver failure at last. No distant lesions including metastatic lymph nodes were confirmed over the course of his illness, and the patient survived for a year after first diagnosis. Although the prognosis of SCEC is quite unfavorable due to highly aggressive behavior, a better prognosis is possible with effective chemotherapy and second-line treatment is important in improving prognosis.

Effects of digoxin, propranolol, and verapamil on exercise in patients with chronic isolated atrial fibrillation.

STUDY OBJECTIVE: The aim was to evaluate the effects of digoxin, propranolol, and verapamil on exercise in patients with chronic isolated atrial fibrillation. DESIGN: Patients with chronic isolated atrial fibrillation underwent maximal exercise testing before and after the administration of digoxin, propranolol, or verapamil. Heart rate, oxygen uptake and oxygen pulse were observed at rest, at gas exchange anaerobic threshold, and at peak exercise. SUBJECTS: The subjects were 10 patients (aged 48-78 years, mean age 60, SD 9, years) with chronic isolated atrial fibrillation. MEASUREMENTS AND MAIN RESULTS: During exercise without medication, the heart rate was 85 (SD 8) beats.min-1 at rest, 127(19) at the level of anaerobic threshold, and 175(17) at peak exercise. With digoxin, heart rate was reduced to 75(9) beats.min-1 at rest (control v digoxin, p less than 0.01). However, reduction of heart rate was not seen at anaerobic threshold or at peak exercise. With propranolol, heart rate was 63(7) beats.min-1 at rest, 99(16) at anaerobic threshold, and 138(28) at peak exercise (control v propranolol, all p less than 0.01). Heart rate with verapamil was 70(13) beats.min-1 at rest, 107(30) at anaerobic threshold, and 138(28) at peak exercise (control v verapamil, p less than 0.05 at rest and at anaerobic threshold, p less than 0.01 at peak exercise. Neither digoxin, nor propranolol, nor verapamil changed the oxygen uptake during exercise. Without medication, oxygen pulse was 6.5(2.0) ml.beat-1 at anaerobic threshold and 7.7(2.1) ml.beat-1 at peak exercise. With digoxin, the change of oxygen pulse, versus without medication, was not significant at rest or at anaerobic threshold but was increased at peak exercise, at 8.3(2.1) v 7.7(2.1) ml.beat-1, p less than 0.05. With propranolol, oxygen pulse was increased to 8.2(1.9) ml.beat-1 at anaerobic threshold and 9.2(2.3) ml.beat-1 at peak exercise (control v propranolol, both p less than 0.01). With verapamil, oxygen pulse was increased to 8.7(1.8) ml.beat-1 at anaerobic threshold and 10.0(2.1) ml.beat-1 at peak exercise (control v verapamil, both p less than 0.01). CONCLUSIONS: Digoxin was effective in reducing heart rate at rest, but failed to reduce it during exercise. Propranolol and verapamil reduced heart rate at all levels of exercise as well as at rest. Oxygen uptake during exercise (total exercise capacity) was not reduced with propranolol or verapamil; this was thought to have been accomplished by an increased oxygen pulse.

T cell receptor-mediated stimulation of mouse thymocytes induces up-regulation of the GM2/GD2 synthase gene.

cDNA clones of the mouse GM2/GD2 synthase (EC 2.4.1.92) gene were isolated, and their analyses revealed that the protein has a type II transmembrane structure with 533 amino acids, which was very similar to the human homolog except for the mRNA size. The mRNA level in thymocytes dramatically increased after treatment with anti-CD3 monoclonal antibody, whereas it was not elevated when treated with prostaglandin E2. In situ hybridization showed an elevation of mRNA levels in medullar thymocytes, suggesting that T cell receptor-mediated signaling induces up-regulation of the GM2/GD2 synthase gene in mature thymocytes.

Reversal of MRP-mediated vincristine resistance in KB cells by buthionine sulfoximine in combination with PAK-104P.

The mechanism of multidrug resistance protein (MRP)-mediated multidrug resistance (MDR) is still unclear. MRP reportedly transports some GSH conjugates. Recently, we demonstrated that a pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4 -(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P), that reversed P-glycoprotein (P-gp)-mediated MDR directly interacted with MRP and completely reversed the vincristine (VCR) resistance in MRP-mediated MDR C-A120 cells. We investigated the reversing effect of PAK-104P in C-A120 cells in combination with buthionine sulfoximine (BSO), another MDR-reversing agent with a different reversing mechanism. In immunoblots, MRP was overexpressed in C-A120 cells. The level of ATP-dependent [3H]VCR uptake was high in membrane vesicles from KB-C2 cells, but low in those from C-A120 and parental KB-3-1 cells. The sensitivity to VCR of C-A120 cells, but not of KB-C2 cells, was considerably increased by 100 microM BSO. VCR accumulation in C-A120 cells, but not in KB-C2 cells, was also enhanced by BSO. BSO did not inhibit ATP-dependent [3H]LTC4 uptake in C-A120 vesicles. The combination of BSO with PAK-104P at their low concentrations resulted in complete reversal of VCR resistance in C-A120 cells. These findings suggested that BSO might not directly interact with MRP and reversed resistance in MRP-mediated MDR cells by reducing the intracellular glutathione (GSH) level that was needed for the transport of drugs by MRP and suggested a role for the combination of drug resistance-modulating agents with different reversing mechanisms in the reversal of MRP-mediated MDR.

Increased sensitivity to vincristine of MDR cells by the leukotriene D4 receptor antagonist, ONO-1078.

The leukotriene D4 (LTD4) receptor antagonist, 4-oxo-8-[p-(4-phenylbutyloxy)benzoylamino]-2-(tetrazol-5-yl) -4H-1-benzopyran hemihydrate (ONO-1078) is used for the treatment of allergic asthma and other immediate hypersensitivity diseases. We examined the effect of ONO-1078 on the sensitivity to vincristine (VCR) of MRP overexpressing multidrug-resistant CV60 and its parental drug-sensitive KB-3-1 cell lines. The sensitivity to VCR of KB-3-1 and CV60 cells was increased 13- and 15-fold, respectively, by ONO-1078 at the maximum non-toxic concentration (100 microM). The VCR sensitivity of multidrug-resistant KB-C2 cells that overexpressed P-gp was increased 2.6-fold by ONO-1078. The accumulation of VCR in KB-3-1, CV60 and KB-C2 cells was significantly increased by ONO-1078. The efflux of VCR from KB-3-1 cells was not inhibited, but that from CV60 cells was enhanced compared with that from KB-3-1 cells and was partially inhibited by ONO-1078. ONO-1078 competitively inhibited the ATP-dependent [3H]LTC4 uptake in membrane vesicles isolated from CV60 cells. These findings suggest that ONO-1078 inhibits the transporting activity of MRP and that ONO-1078 increases the sensitivity to VCR of KB-3-1 cells by increasing the VCR uptake in the cells.

Clinical significance of pyrimidine nucleoside phosphorylase in colorectal carcinoma.

The expression of pyrimidine nucleoside phosphorylase (PyNPase) mRNA in tumor (T) and normal (N) biopsy specimens obtained from 41 cases of colorectal carcinoma was examined by the reverse transcriptase/polymerase chain reaction (RT-PCR). The higher T/N ratio of the expression of PyNPase mRNA correlated significantly with the presence of lymph vessel invasion (p=0.039), the positive lymph node metastasis (p=0.014) and the advanced stage of the disease (p=0.014). There was a significant con-elation between the results determined by enzyme activity and those determined by RT-PCR (p=0.005). The findings suggested that the determination of PyNPase mRNA by RT-PCR may give useful information on tumor aggressiveness of colorectal carcinoma and this method can be used instead of enzyme activity.

Expression of insulin-like growth factor 2 mRNA in human gastric cancer.

Insulin-like growth factor 2 (IGF2) stimulates cell proliferation and development in normal human growth. In several human cancers, the IGF2 gene is overexpressed and is thus considered to be a growth factor for tumors mediated through both the paracrine and autocrine pathways. However, the significance of IGF2 mRNA expression in gastric cancer has yet to be clarified. We semi-quantitatively measured the expression of IGF2 mRNA in 57 Japanese cases of gastric cancer by means of the reverse transcription polymerase chain reaction and also analyzed the relation between the IGF2 expression status and other clinicopathologic factors. We also performed immunohistochemical staining for IGF2. In 41 of 57 cases (72%), the expression of IGF2 mRNA was greater in tumor tissue (T) than in normal tissue (N). The average tumor/normal (T/N) expression ratio of IGF2 mRNA corrected for that of control gene mRNA was 1.42, while ranging from 0.36 to 3.65. The T/N ratio of infiltrative-type cancers was greater than that of expanding-type cancers (p<0.05). The cases with lymphatic permeation showed a greater T/N ratio than those without lymphatic permeation in expanding-type cancers (p<0. 05). Immunohistochemical staining revealed IGF2 to be detected in cancer cells themselves, especially at the margin of the cancer tissue. The IGF2 gene may thus play an important role in lymph vessel permeation especially in expanding-type gastric cancers.

Expression of ornithine decarboxylase mRNA and c-myc mRNA in breast tumours.

We examined the expression of ornithine decarboxylase (ODC) mRNA in 53 female cases of breast cancer by a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to determine the clinicopathologic significance of its expression. A significantly higher expression of ODC mRNA was, observed in younger patients than in older patients. The patients with a larger sized tumour possessed a significantly higher expression of ODC mRNA. In addition, the cases with a poor prognosis showed significantly higher expression of ODC. Previous studies have reported in vivo and in vitro correlation between the expression of ODC and c-myc genes in human carcinomas. We disclosed a significant correlation between these genes in primary breast carcinomas. We conclude that the expression of ODC may potentially be a new biological marker for breast carcinoma.

Clinical significance of integrin alpha 6 mRNA expression in gastric carcinoma.

Integrin alpha 6 is believed to be involved in malignant biological development. High tumor/normal (T/N) ratio of integrin alpha 6 was observed in intestinal type tumors in gastric carcinoma. In an immunohistochemical study, an intense and continuous staining was seen at the basement layer in the intestinal type tumors. Concerning the variant, there was a significant correlation between the expression ratio of variant B/variant A and the depth of tumor invasion. In conclusion, i) integrin alpha 6 expression was intimately correlated with histologic differentiation, and ii) the overexpression of variant B may be correlated with tumor invasion of gastric carcinoma.