The variation in Fas localization and the changes in Fas expression level upon stimulation with growth factors.

Although Fas (APO-1/CD95) is well known as a death receptor, its stimulation occasionally fails to induce apoptosis in malignant cells. On the contrary, Fas is reported to advance the cell cycle in cancer cells. Therefore, we investigated roles of Fas in cell growth and apoptosis using human lung cancer cell lines. Fas was localized in the cytoplasm in exponentially growing cells, whereas only confluent cells expressed Fas on the cell membrane. A stimulation of confluent cells by either of EGF, IGF-I or VEGF induced once a decrease in Fas expression level and its sequential recovery. Fas expression levels in confluent cells were negatively correlated with cell doubling times (r=0.757, p=0.0088). Fas remained on the cell membrane of IgM-treated cells even after the growth factor stimulation, leading to apoptosis with abnormal mitosis, whereas the same stimulation induced Fas internalization in IgG(1)-treated cells. From these results, we suggest that Fas remaining on the cell membrane amplifies to induce apoptosis. Conversely, Fas internalization may enable cancer cells to escape from apoptosis. Our results suggest that growth factor may contribute to the resistance of cancer cells to Fas-mediated apoptosis in an autocrine or paracrine fashion.

Chromosomal circularization in Streptomyces griseus by nonhomologous recombination of deletion ends.

Streptomyces linear chromosomes frequently cause deletions at both ends spontaneously or by various mutagenic treatments, and concomitantly display dynamic structural changes such as circularization and arm replacement. We have cloned and sequenced the fusion junctions of circularized chromosomes in two deletion mutants of Streptomyces griseus. No homology and a 1-bp overlap were found between the deletion ends of the mutant chromosomes. Taking this together with previous results, we concluded that chromosomal circularization in Streptomyces occurs by nonhomologous recombination between deletion ends.

Intracellular conversion of irinotecan to its active form, SN-38, by native carboxylesterase in human non-small cell lung cancer.

The anticancer agent irinotecan (CPT-11) is a prodrug converted to its active form, SN-38, by human carboxylesterase (hCE) and the SN-38 is further metabolized to its inactive form, SN-38G. We investigated the expression of hCE in human lung cancer cells as well as the ability of these cells to convert CPT-11 to SN-38 using surgically resected tumor samples and cultured cell lines. SN-38 was 40- to 3,000-fold more toxic to lung cancer cell lines than CPT-11, which acted more time-dependently than SN-38. Although human lung cancer cells expressed hCE in the cytoplasm, hCE expression levels in cancer cells were not correlated with their drug sensitivities. Although intracellular CPT-11 and SN-38 levels continuously increased within 60 min of CPT-11 exposure, SN-38 levels in cells exposed to SN-38 decreased. Cells with the ability to metabolize SN-38 to SN-38G were more resistant to extracellular SN-38 than cells lacking the ability. Of 25 squamous cell carcinomas, 15 were strongly positive for hCE and six were negative. Of 25 adenocarcinomas, four were strongly positive for hCE and 16 were positive, while five were negative. Thus, 70% of non-small cell lung cancers expressed hCE. From these results, we conclude that human lung cancer cells expressed the enzyme which can convert CPT-11 to SN-38 and that intracellular SN-38 converted from CPT-11 may act as a chemotherapeutic agent together with SN-38 absorbed from the outside and augment the dose intensity of SN-38. Therefore, to assess the effects of CPT-11 prior to chemotherapy, it is important to check if lung cancer cells express hCE.

An association of Bcl-2 phosphorylation and Bax localization with their functions after hyperthermia and paclitaxel treatment.

Apoptosis is induced by many kinds of therapy-related inducers, such as hyperthermia and chemotherapeutic agents. However, differences in apoptotic pathways between these inducers remain unclear, although knowing the differences is important to map out a therapeutic strategy. Therefore, we focused on the localization and phosphorylation of Bcl-2 and Bax, key mediators of the apoptotic pathway, after hyperthermia and paclitaxel treatment of PC-10 squamous cell carcinoma cells that excessively expressed Bcl-2 and Bax in the cytoplasm. Paclitaxel treatment markedly induced qualitative changes in Bcl-2, whereas hyperthermia did only quantitative changes in Bax. The levels of Bax increased gradually with the duration of hyperthermia, whereas Bcl-2 levels slightly decreased. On the other hand, paclitaxel treatment induced dose- and time-dependent phosphorylation of Bcl-2. Interestingly, phosphorylated Bcl-2 was observed in the specific subcellular sites, mitochondria- and lysosome-rich fractions. Both treatments disturbed the heterodimerization of Bax with Bcl-2. Hyperthermia, but not paclitaxel treatment, induced a gradual Bax translocation from the cytoplasm to the nucleus. Although both treatments induced a prominent cell cycle disturbance in the G2M phase, paclitaxel treatment induced typical apoptosis, and hyperthermia hardly induced apoptosis. Our results suggest that the subcellular redistribution of Bax and the phosphorylation of Bcl-2 depend on the type of apoptosis inducers, such as hyperthermia and paclitaxel, and Bcl-2 has a central role in the decision of apoptotic outcome. Our data may afford new insights in apoptosis from the aspect of an association of Bcl-2 phosphorylation with intracellular Bax localization.

Bax, Bcl-2, and p53 expression in endometrial cancer.

BACKGROUND: It has not been fully clarified whether alteration of Bax and other apoptosis-relating proteins of Bcl-2 and p53 is involved in endometrial carcinogenesis. METHODS: A total of 56 frozen tissues, which included 14 normal endometria, 13 endometrial hyperplasias (10 without atypia and 3 with atypia), and 29 endometrial carcinomas, were examined for the expression of Bax, Bcl-2, and p53 using immunohistochemistry. For Bax-negative cases, PCR-direct sequencing was performed for the bax gene. For cases with p53 overexpression, mutational analysis was performed for the p53 gene using a yeast functional assay and sequencing. RESULTS: Both Bax and Bcl-2 were distinctly expressed in the normal proliferative phase endometrium. A decreased Bcl-2/Bax ratio in the secretory phase endometrial gland cells due to suppressed Bcl-2 expression was observed. Bax expression was positive in all 13 endometrial hyperplasias, while it was absent in 6 of 29 endometrial carcinomas (20.7%). Negative Bax expression in endometrial carcinoma was not related to tumor stage, histologic subtype, or other histopathologic prognostic factors. Bax expression showed no relationship to either p53 overexpression or Bcl-2 expression. In the DNA of 6 Bax-negative cases, we found a frameshift insertion mutation at codon 58 (AAG to CAAG) in the BH3 domain despite the absence of mutation in the (G)8 tract, suggesting that this codon may be another preferred target for bax mutation other than the (G)8 tract. Mutational analysis was available for 7 of 10 cases with p53 overexpression, in which 5 cases were found to have a missense mutation and 2 cases had no mutation of the p53 gene. At least 10 of 29 (34.5%) cases of endometrial carcinoma were associated with sequence-verified mutation in the bax gene and/or p53 gene. CONCLUSIONS: The bax gene frameshift mutation appears to cause a loss of Bax expression in endometrial carcinoma. Codon 58 may be a preferred target of bax gene mutation in endometrial carcinomas. The bax gene mutation seems to occur in the early stage of the genesis of a subset of endometrial carcinomas.

Central bronchopleural fistulas closed by bronchoscopic injection of absolute ethanol.

Five consecutive bronchopleural fistulas (BPFs) were successfully treated by injecting absolute ethanol directly into the submucosal layer of the fistula under flexible bronchoscopic observation. No complications occurred as a result of this treatment. Our nonsurgical treatment may be very useful to reduce the costs of and duration of hospitalization and to improve the patient's quality of life. This is the first report of the bronchoscopic closure of BPFs by injecting absolute ethanol, and we would recommend this treatment as a first-line therapy for patients with a postoperative central BPF with an orifice that is < 3 mm in diameter.