Expression of the polyubiquitin gene early in the buprenorphine hydrochloride-induced apoptosis of NG108-15 cells.

To clone genes expressed early in the buprenorphine hydrochloride (Bph)-induced apoptosis of NG108-15 nerve cells, we adopted a previously reported rapid and simple differential display (DD) cloning procedure. Complementary DNA was generated from differentially expressed mRNAs by reverse transcription (RT) using a fully degenerate 6-mer oligonucleotide as the primer. PCR amplification was then conducted using a combination of three arbitrary but defined 10-bp nucleotide primers. The differentially generated DNA fragments were detected by agarose gel electrophoresis, and 9 were excised from the gel and subcloned into a sequencing vector. Three DNAs that were specifically expressed upon Bph-induced apoptosis of NG108-15 cells were sequenced. Their specific expression was then confirmed by reverse-transcription PCR. One was identified as the mouse polyubiquitin gene c, and the others remain unidentified. Northern and western blots indicated the transcription and translation of polyubiquitin early in Bph-induced apoptosis. The polyubiquitination of apoptotic cellular proteins was also confirmed.

[Effect of bcl-2 over-expression on the membrane structure of NG108-15 nerve cells: effectiveness of membrane-structure analysis using FACS for the first screening of apoptosis-inducing drugs].

Bcl-2 is a Bcl-2 family protein that is known to be anti-apoptotic and is predominantly localized to the mitochondria. We previously showed that an analgesic, buprenorphine hydrochloride (Bph), induces apoptosis in the rodent-derived nerve cell line, NG108-15, through the mitochondrial apoptotic route. A Bcl-2-overexpressing strain of NG108-15 cells, Bcl-2 (P2), was established, and the effect of Bcl-2 expression on Bph-induced apoptosis was compared between the mock vector-transfected NG108-15 cells and the Bcl-2 (P2) cells. The Bcl-2 (P2) cells died after treatment with Bph, and we observed all the biological and morphological markers of apoptosis that we tested for. In flow cytometric analysis, a difference in the cell membrane phospholipid flip-flop pattern-a feature of apoptosis- was observed between the NG108-15 cells and the Bcl-2 (P2) cells. Here, we show by flow cytometric analysis that Bcl-2 over-expression may affect the membrane structure of Bcl-2 (P2) cells. An increased fluorescein isothiocyanate (FITC) signal of annexin V-FITC, which typically represents phospholipid flip-flop of the cellular membrane in early apoptosis, was barely detected in the Bcl-2 (P2) cells. Since our previous study reported the localization of over-expressed Bcl-2 protein to the cell membrane of Bcl-2 (P2) cells, together these observations suggest that the Bcl-2 protein may affect the integrity of the structure of the NG108-15 cell membrane.

Over-expressed Bcl-2 cannot suppress apoptosis via the mitochondria in buprenorphine hydrochloride-treated NG108-15 cells.

We previously reported that the morphine alkaloid derivative buprenorphine hydrochloride (Bph) induces rapid apoptosis in NG108-15 nerve cells accompanied by the activation of caspase-3. Here, we found this kind of apoptosis was also accompanied by rapid loss of the mitochondrial membrane potential, followed by the efflux of cytochrome c from the mitochondria to the cytosol and the activation of caspases-9 and -3. Together, these results strongly suggested the Bph death signal was routed through the mitochondrial pathway in NG108-15 cells. In these cells, serum-starvation induces a different apoptosis, which we exploited to investigate Bcl-2's role as an apoptosis inhibitor. We made an NG108-15 transfectant, Bcl-2(P2), that stably expressed human Bcl-2, and used it to test Bcl-2's effect on the serum-starvation-induced apoptosis in NG108-15 cells. Cell viability, DNA-ladder formation, and efflux of cytochrome c from the mitochondria were all detected, showing that the human Bcl-2 functioned normally in the Bcl-2(P2) cells. Although the apoptotic events tested were identical in the parental cells and transformants, Bcl-2 expression completely failed to inhibit Bph-induced apoptosis in the Bcl-2(P2) cells.

Apoptosis-like cell death of human breast cancer cell line MCF-7 induced by buprenorphine hydrochloride.

The analgesic buprenorphine hydrochloride (Bph) induced apoptosis-like cell death in the caspase-3-deficient human breast cancer cell line, MCF-7. This apoptosis-like cell death activated key molecules in the mitochondrial apoptotic pathway: cytochrome c, caspase-9, caspase-7, and caspase-6. Bph caused the release of fluorescent protein from the mitochondria of MCF-7 cells transfected with the pDsRed2-Mito-vector in a time-dependent manner, suggesting disruption of the mitochondrial membrane. Zn(2+) as high as 2 mM did not inhibit the DNase that took part in this apoptosis. Thus, this unidentified DNase might resemble other DNases involved in apoptosis-like cell death whose activity is not inhibited by zinc ion.

Evaluation of cell death caused by CDF (cyclophosphamide, doxorubicin, 5-fluorouracil) multi-drug administration in the human breast cancer cell line MCF-7.

We evaluated the features of cell death induced by CDF (cyclophosphamide [CPA], doxorubicin [DOX], 5-fluorouracil [5-FU]) multi-drug administration in vitro using the human breast cancer cell line MCF-7. Used individually, DOX and 5-FU induced 60% cell death in MCF-7 cells, at 5 microg/ml and 25 microg/ml, respectively, by the 4th day following drug treatment. CPA was the least cytotoxic of the 3 drugs, causing only 20% cell death, even at the high concentration of 500 microg/ml. Treating cells with a mixture of all three anticancer drugs resulted in 60% cell death, on the second and third day following drug treatment. The nature of the cytotoxicity of CPA, DOX, and 5-FU was investigated, because these drugs are sometimes used to induce apoptosis. Biochemical analysis showed faint DNA fragmentation in the case of DOX or all three drugs, but not for treatment with CPA or 5-FU. In contrast, the morphological apoptotic feature of a condensed nucleus was observed only for CPA and 5-FU. Flow cytometric data agreed with the morphological results in that the FACS cytogram for DOX and for all three drugs was different from that for CPA or 5-FU given alone. These observations suggested that the cell death induced by these anticancer drugs in the human breast cancer cell line MCF-7 is a mixture of apoptotic and non-apoptotic, but it becomes completely non-apoptotic in the case of multi-drug administration.

Expression of transcriptional repressor NC2alpha during Xenopus early embryogenesis.

To study the mechanisms behind the suppression of gene expression in the early phase of Xenopus development (cleavage stage), we conducted in silico cloning of the Xenopus transcriptional repressor NC2alpha. A search of the GenBank EST database using human NC2alpha as a probe identified Xenopus mitotic phosphoprotein 30 (xMP30) as a prime candidate for Xenopus NC2alpha (xNC2alpha). Full-length cDNA sequencing showed that xNC2alpha/xMP30 had a 68.9% identity at the amino acid level with its human counterpart. Northern blotting showed that xNC2alpha existed abundantly as a maternal mRNA. After the fertilization, the expression of xNC2alpha rapidly increased and reached a maximum 3 h before midblastula transition (MBT). Then its expression gradually decreased toward the early neurula stage. The expression profile of xNC2alpha mRNA is compatible with that of xNC2beta, which is the other component of the Xenopus NC2 transcriptional repressor.

Genomic cloning of Xenopus TFIIS (TCEA1) and identification of its transcription start site.

We previously cloned the cDNA of the transcription factor TFIIS (SII) from Xenopus laevis and showed that its expression was not constant during Xenopus development. To investigate its regulation, we cloned the genomic DNA of Xenopus TFIIS, focusing on the 5'-promoter region. Here, we present the Xenopus TFIIS genomic sequence (-1730 to +214) and transcription start site (cap site). We define the position of the primary cap site as the adenine located 142 bp upstream from the adenine of the ATG (Met) codon. Another putative start-site region, where 13 transcriptional start sites are clustered within 12 bp, was mapped about 100 bp downstream of the primary cap site. Although a computer search found putative trans-element binding sites proximal to two Xenopus TFIIS transcription start sites, we could not identify typical "TATA" or "CAATT" boxes upstream of the primary cap site, probably owing to TFIIS's character as a "house keeping gene".