UCN-01 suppresses thymidylate synthase gene expression and enhances 5-fluorouracil-induced apoptosis in a sequence-dependent manner.

UCN-01, a protein kinase C/cyclin-dependent kinase inhibitor, suppressed thymidylate synthase (TS) protein expression in a dose-dependent manner with near complete suppression at 1 microM after a 24-h exposure in human gastric cancer cell line SK-GT5. Other protein kinase C/cyclin-dependent kinase inhibitors, including flavopiridol and safingol, had a similar effect on TS protein expression, but to a lesser degree. Moreover, UCN-01 repressed the induction of TS after 5-fluorouracil (FU) exposure by 90-95% and significantly enhanced the induction of apoptosis by FU from 4-8% with either FU or UCN-01 alone to 46+/-1% (P < 0.005 versus either single drug, reverse sequence, or the combination) when UCN-01 was given after FU. The effect of UCN-01 on TS was associated with a dose-dependent suppression of the E2F-1 protein, a transcriptional activator of TS. Northern blot analysis revealed that TS mRNA levels decreased gradually as the concentration of UCN-01 increased, but that E2F-1 mRNA levels remained relatively unchanged. UCN-01 may provide a novel way to enhance cellular sensitivity toward FU by means of suppressing TS expression mediated mainly by down-regulation of E2F-1.

UCN-01 suppresses E2F-1 mediated by ubiquitin-proteasome-dependent degradation.

E2F-1 regulates the transcription of genes required for DNA synthesis. Previously, we have reported that UCN-01 suppresses E2F-1 protein expression without any noticeable effect on its mRNA level in gastric cancer cell line SK-GT5 (Clin. Cancer Res., 4: 2201-2206, 1998). In this study, we investigated the mechanism responsible for the suppression of E2F-1 expression by UCN-01 in SK-GT5 cells. After 24-h exposure to 1 microM UCN-01, E2F-1 protein expression was decreased by >99%. The suppressive effect of UCN-01 could be reversed by ubiquitin-dependent proteasome inhibitors such as calpain inhibitor I and lactacystin. Transfection experiments using expression plasmids encoding full-length E2F-1 or truncated E2F-1 with deletion of the COOH-terminal region (which is required for eliciting ubiquitination and protein degradation) revealed that the expression of truncated E2F-1 was not affected by UCN-01. Other cell-cycle-related and ubiquitin-proteasome-regulated proteins such as p21, p27, and cyclin B1 were not repressed by UCN-01 in E2F-1-overexpressing cells. In vitro-translated, full-length E2F-1 degraded more rapidly upon incubation with extracts from UCN-01-treated cells when compared with truncated E2F-1. Taken together, these data indicate that UCN-01 suppresses E2F-1 protein expression mediated by the ubiquitin-proteasome pathway in a specific manner.

Regulation of folate-binding protein gene expression by DNA methylation in methotrexate-resistant KB cells.

Folate-binding protein (FBP) is responsible for the cellular transport of folate and methotrexate (MTX) in human KB (nasopharyngeal epidermoid carcinoma) cells. The levels of membrane-associated FBP and FBP mRNA are decreased 70-80% in an MTX-resistant KB subline (KB1BT) (Hsueh C-T and Dolnick BJ, Oncol Res 4: 497-505, 1992). Southern blot analysis did not reveal any differences in FBP gene organization or copy number between KB1BT and KB cells. However, there was a 70% decrease in the FBP gene transcription rate and no change in FBP mRNA stability in KB1BT cells. Assessing genomic DNA methylation by MspI and HpaII restriction analysis suggested that the FBP gene in KB1BT cells was more methylated than in KB cells. These alterations in the expression, transcription rate and DNA methylation state of the FBP gene did not change when KB1BT cells were grown in the absence of MTX for 8 months (MTX-free KB1BT). When MTX-free KB1BT cells were exposed to 2.5 microM 5-aza-2'-deoxycytidine for 72 hr, the FBP gene became hypomethylated and the levels of membrane-associated FBP and FBP mRNA increased by 2- to 3-fold. These data indicate that decreased FBP gene expression in KB1BT cells results from increased DNA methylation.

Altered folate-binding protein mRNA stability in KB cells grown in folate-deficient medium.

Folate-binding protein (FBP), a high-affinity folate receptor, is responsible for cellular accumulation of folate and folate analogs such as methotrexate in human KB (nasopharyngeal carcinoma) cells. Both FBP and FBP mRNA increase 3- to 5-fold when KB cells are grown in folate-deficient (less than 10 nM folate) medium (KB-FD), compared with growth in standard folate-replete medium containing at least 2 microM folate (KB-FR). The possible mechanisms of enhanced FBP gene expression in KB-FD were examined in this study. Southern blot analysis revealed no significant change in the FBP gene organization or copy number in the KB-FD DNA. While hypomethylation of the FBP gene was observed in KB-FD DNA, relative to KB-FR DNA, exposure of KB-FR to the DNA methylation inhibitors did not result in elevated FBP mRNA levels. The transcriptional rate of the FBP gene was the same in KB-FR and KB-FD. RNA half-life studies indicated that the half-life of FBP mRNA in KB-FD was increased approximately 2.5-fold, compared with KB-FR. Thus, the increase in the steady-state levels of FBP mRNA in KB-FD can be attributed partly to increased FBP mRNA stability.

Selective inhibition of cyclooxygenase-2 enhances mitomycin-C-induced apoptosis.

PURPOSE: Cyclooxygenase-2 (COX-2) is involved in antiapoptosis signaling, and its induction may require activation of protein kinase C (PKC). Safingol (SAF), a PKC inhibitor, has been shown to enhance apoptosis induced by mitomycin-C (MMC) in human gastric cancer MKN-74 cells. The aim of this study was to identify the role of COX-2 in MMC-induced apoptosis in MKN-74 cells. METHODS: Protein expression of COX-2 and Bcl-2 and activation of PKCalpha were examined by Western blot analysis. Apoptosis induction was examined by staining with bisbenzimide trihydrochloride (Hoechst-33258) of condensed chromatin, which characterizes the cells undergoing apoptosis. COX-2 mRNA levels were examined by Northern blot analysis. RESULTS: After exposure for 1-2 h to 1 microg/ml MMC, upregulation of COX-2 and Bcl-2 protein expression was noted. The activation of PKCalpha occurred within 1 h of MMC exposure, and temporally preceded the induction of COX-2. Similar results were observed in cells exposed to the PKC activator, 3-phorbol 12-myristate 13-acetate. Cotreatment with SAF and MMC abolished the induction of COX-2 by MMC. Furthermore, NS-398, a selective COX-2 inhibitor, significantly enhanced MMC-induced apoptosis by fivefold from 4 +/- 2% (MMC alone) to 20 +/- 2% (MMC plus NS-398). There was no discernible change in COX-2 mRNA levels after a 2-h exposure to MMC but a twofold increase after a 24-h exposure. CONCLUSIONS: MMC upregulates COX-2 expression, which appears to be an antiapoptotic signal downstream of PKC. Selective inhibition of COX-2 can therefore provide a novel way to enhance MMC-induced apoptosis independent of inhibiting PKC.

rTS gene expression is associated with altered cell sensitivity to thymidylate synthase inhibitors.

rTS is a recently discovered gene, phylogenetically conserved and found to be expressed in a wide variety of cell lines. rTS has also been found to be overexpressed in two cell lines resistant to FU and to MTX. The MTX-resistant cell line was found to have a high degree of cross resistance to several TS inhibitors. An apparent paradox to this correlation of rTS overexpression and resistance to TS inhibitors is the observation that expression of transfected rTS alpha results in enhanced sensitivity of cells to the TS inhibitor prodrug TFT and a modest increase in resistance to FUdR. Since immunoprecipitation of TS leads to the co-immunoprecipitation of two proteins within the expected molecular weight range of the two rTS proteins, it may be that both proteins bind to TS in vivo and modify its activity. Preliminary data substantiate this conclusion. It is conceivable that the ratio of the two rTS proteins associated with TS in vivo may differentially alter TS activity depending upon their stoichiometry or possibly posttranslational modification. Thus it may be possible for rTS to confer greater sensitivity to one pyrimidine analog (e.g., TFT) which is a product analog but to increase resistance or have a minor effect on a substrate analog (e.g., FdUMP) by stabilizing different conformations of TS. The structure of the rTS proteins suggests they are expected to have catalytic activity which involves proton abstraction from an alpha-carbon of a carboxyl group. Whether this enzyme activity is functional and related to pyrimidine metabolism awaits further study.

Folate-binding protein mRNA is decreased in methotrexate-resistant KB cells.

Folate-binding protein (FBP), a high-affinity folate receptor expressed in certain malignant and normal cell lines and tissues, is responsible for cellular transport of folate and structurally related antifolates such as methotrexate in human KB (nasopharyngeal carcinoma) cells. We have developed a sensitive polymerase chain reaction (PCR) assay to quantitate folate-binding protein mRNA levels. This assay was tested in KB and methotrexate (MTX)-resistant KB (KB1BT) cell lines. Based on assay results, we conclude there are 245 molecules of folate-binding protein (FBP) mRNA per KB cell, and there is an approximately 3-fold decrease in FBP mRNA levels per KB1BT cell. The data obtained by quantitative PCR correlate well with northern blot analysis of mRNA levels and membrane-associated folate-binding protein (M-FBP) levels. Moreover, when KB1BT cells were grown in the absence of MTX for 8 months, the levels of FBP and M-FBP mRNA did not change, and there was a substantial decrease in MTX uptake, compared with KB cells.

Augmentation of the immune response of the murine liver by levamisole.

Murine hepatic nonparenchymal cells (NPC) were studied following in vivo treatment with levamisole. This agent was found to increase the cytolytic action of these cells against YAC-1 and P815 target cells. An increase in the cytostatic activity against liver-derived murine colon adenocarcinoma 38 tumor cells was also observed. Treatment with levamisole also augmented the proliferation of the hepatic NPC. Supernatants generated by these cells contained an agent capable of stimulating the proliferation of bone marrow cells from the same mice. The effect of levamisole on different subsets of NPC derived from the liver in this model is discussed.

[The employment of married women in Taiwan: its patterns and causes].

"This research attempts to explore the modes of women's job careers in Taiwan. Using the 1991 wave of 'Taiwanese Social Change Surveys' data, we adopt the information regarding married female respondents' job history since their marriage and found four major modes of job careers in terms of family life cycles: never-stop, stop-after-marriage, stop-after-birth, and never on job.... We further explore the explanatory factors (including individuals' traits, family background factors, and current family statuses) with multi-nominal logistic models." (SUMMARY IN ENG)

Genetic analysis of the nitrogen fixation system in Klebsiella pneumoniae.

Fine structure mapping of nif mutations of Klebsiella pneumoniae was accomplished by means of Pl-transductional crosses and the plasmid R144 drd mediated conjugations. The physical distance between nif mutations based on the percentage of co-transduction with hisD of the nif mutations was estimated. The maximal distance between two mutations was calculated about 3 Kb, and the average distance between different nif mutations was about 1 to 2 Kb. So no "silent region" was shown within the nif cluster nearby the histidine operon. Several hisD-unlinked nif mutants were isolated and investigated genetically and biochemically. They all differed from the glutamineless mutants, one of these mutants was tentatively assigned as a sort of N-assimilation mutant with little activity of glutamate synthetase. It differed from the known N-assimilation mutants in its absence of nitrogenase activity. Since the wild type hisD-linked nif genes carried by the plasmid RP4 failed to complement the defects of the hisD-unlinked nif genes in the recipient cells but they were effective to facilitate E. coli in acquiring the ability to fix nitrogen, which indicates that the hisD-unlinked nif genes necessary for the functioning of the hisD-linked nif genes are present in E. coli.

Low infectious morbidity in patients with heavily pretreated hematological malignancies receiving autologous peripheral blood stem cell transplantation without antimicrobial prophylaxis.

The use of prophylactic antimicrobials during autologous peripheral blood stem cell transplantation (APBSCT) remains controversial. A prospective study was therefore conducted to examine whether the use of prophylactic antimicrobials is necessary. In this study, all the antimicrobials were given therapeutically rather than prophylactically. Twenty-three consecutive patients with heavily pretreated hematological malignancies were enrolled. All of the 23 patients had at least one episode of fever during APBSCT and most were fever of unknown origin (78.3%). Clinically or microbiologically documented infections occurred in only five patients (21.7%). These included bacteremias (three patients), perianal abscess (one patient), and catheter-related phlebitis (one patient). No death, invasive fungal infection, or serious adverse events occurred. The medium duration of fever, intravenous antimicrobial therapy, and hospital stay after transplantation were 5, 10, and 17 days, respectively. In conclusion, without using prophylactic antimicrobials, the infectious morbidity during APBSCT is low even in patients with heavily pretreated hematological malignancies.