[Effects of As2O3 and all-trans retinoic acid on the growth of HeLa cell line and their relation with gene NDRG1].

OBJECTIVE: To study the effect of arsenic trioxide (As2O3) and all-trans retinoic acid (ATRA) on human cervical carcinoma HeLa cell line. METHODS: HeLa cells were treated with As2O3 and ATRA. The cell proliferation was evaluated by MTT assay. The expressions of NDRG-1 protein and mRNA were determined by Western blot and RT-PCR analysis. RESULTS: MTT assay showed that As2O3 and ATRA inhibited the growth of human cervical carcinoma HeLa cells in vitro in a dose- and time-dependent manner. Western blot and RT-PCR techniques showed that As2O3 and ATRA down-regulated the expressions of NDRG-1 protein and mRNA (P < 0.05). CONCLUSION: As2O3 and ATRA can significantly inhibit the growth and proliferation of HeLa cells. The reason of these changes may be related with the down-regulation of expression of NDRG-1.

[Survivin antisense RNA induces apoptosis and sensitizes ovarian cancer cell line SKOV3 to docetaxel].

BACKGROUND & OBJECTIVE: Survivin gene overexpresses in a variety of human tumors, and plays an important role in cell apoptosis and drug resistance of tumors. This study was designed to establish Survivin antisense RNA, and explore its effects on apoptosis of ovarian cancer cell line SKOV3 and sensitivity to docetaxel. METHODS: Survivin antisense eukaryotic expression vector anti-pcDNA3-svv was established, and transfected into SKOV3 cells by electroperforation. Positive clones (SKOV3-SVVanti) were screened out. Survivin mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR); Survivin protein was detected by Western blot. The effect of Survivin antisense RNA on apoptosis of SKOV3 cells was measured by flow cytometry and observed under electron microscope; its effect on sensitivity of SKOV3 cells to docetaxel was examined by MTT assay. RESULTS: The mRNA and protein levels of Survivin were obviously lower in SKOV3-SVVanti cells than in control cells. Terminal apoptosis changes were observed under electron microscope after transfection. The apoptosis rate was 19%. The 50% inhibitory concentration (IC50) of docetaxel was significantly lower for SKOV3-SVVanti cells than for control cells [(13.3+/-2.2) ng/ml vs. (53.2+/-2.4) ng/ml, P<0.05]. CONCLUSION: Surivivin antisense RNA can induce apoptosis of SKOV3 cells, and sensitize SKOV3 cells to docetaxel.

Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria.

BLUF (a sensor of Blue-Light Using FAD) is a novel putative photoreceptor domain that is found in many bacteria and some eukaryotic algae. As found on genome analysis, certain cyanobacteria have BLUF proteins with a short C-terminal extension. As typical examples, Tll0078 from thermophilic Thermosynechococcus elongatus BP-1 and Slr1694 from mesophilic Synechocystis sp. PCC 6803 were comparatively studied. FAD of both proteins was hardly reduced by exogenous reductants or mediators except methylviologen but showed a typical spectral shift to a longer wavelength upon excitation with blue light. In particular, freshly prepared Tll0078 protein showed slow but reversible aggregation, indicative of light-induced conformational changes in the protein structure. Tll0078 is far more stable as to heat treatment than Slr1694, as judged from flavin fluorescence. The slr1694-disruptant showed phototactic motility away from the light source (negative phototaxis), while the wild type Synechocystis showed positive phototaxis toward the source. Yeast two-hybrid screening with slr1694 showed self-interaction of Slr1694 (PixD) with itself and interaction with a novel PatA-like response regulator, Slr1693 (PixE). These results were discussed in relation to the signaling mechanism of the "short" BLUF proteins in the regulation of cyanobacterial phototaxis.

Cyanobacterial phytochrome-like PixJ1 holoprotein shows novel reversible photoconversion between blue- and green-absorbing forms.

The gene, pixJ1 (formerly pisJ1), is predicted to encode a phytochrome-like photoreceptor that is essential for positive phototaxis in the unicellular cyanobacterium Synechocystis sp. PCC 6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The PixJ1 protein was overexpressed as a fusion with a poly-histidine tag (His-PixJ1) and isolated from Synechocystis cells. A zinc-fluorescence assay suggested that a linear tetrapyrrole was covalently attached to the His-PixJ1 protein as a chromophore. His-PixJ1 showed novel photoreversible conversion between a blue light-absorbing form (Pb, lambdaAmax=425-435 nm) and a green light-absorbing form (Pg, lambdaAmax=535 nm). Dark incubation led Pg to revert to Pb, indicative of stability of the Pb form in darkness. Red or far-red light irradiation, which is effective for photochemical conversion of the known phytochromes, produced no change in the spectra of Pb and Pg forms. Site-directed mutagenesis revealed that a Cys-His motif in the second GAF domain of PixJ1 is responsible for binding of the chromophore. Possible chromophore species are discussed with regard to the novel photoconversion spectrum.

Biochemical and functional characterization of a eukaryotic-type protein kinase, SpkB, in the cyanobacterium, Synechocystis sp. PCC 6803.

On the basis of the genome sequence, the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for eukaryotic-type protein kinase belonging to Pkn2 subfamily ( spkA approximately spkG). Previously, SpkA was shown to have protein kinase activity and to be required for cell motility. Here, the role of the spkB was examined. The spkB gene was expressed in Escherichia coli as a fusion protein with His-tag, and the protein was purified by Ni(2+) affinity chromatography. The eukaryotic-type protein kinase activity of the expressed SpkB was demonstrated as autophosphorylation to itself and phosphorylation of the general substrate proteins. SpkB showed autophosphorylation activity in the presence of both Mg(2+) and Mn(2+), but not in Ca(2+). Phenotype analysis of spkB disruptant of Synechocystis revealed that spkB is required for cell motility, but not for phototaxis. These results suggest that SpkB is the eukaryotic-type protein kinase, which regulates cellular motility via protein phosphorylation like SpkA.

Biochemical examination of the potential eukaryotic-type protein kinase genes in the complete genome of the unicellular Cyanobacterium synechocystis sp. PCC 6803.

The complete genome of the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for a subfamily Pkn2 of the eukaryotic-type (or "Hanks-type") protein kinase. Previously, SpkA and SpkB were shown to have protein kinase activity and to be required for cell motility. Here, the other five genes were examined. These genes, except for spkG (slr0152), were successfully expressed in Escherichia coli. Eukaryotic-type protein kinase activity of the expressed SpkC (Slr0599), SpkD (S110776) and SpkF (Slr1225) was demonstrated as autophosphorylation and phosphorylation of the general substrate proteins. SpkE (Slr1443) did not show any activity, a finding consistent with its lack of several key amino acid residues in its kinase motif. Gene-disrupted mutants showed no discernible defect in phenotype except that spkD was apparently essential for survival.

pilG Gene cluster and split pilL genes involved in pilus biogenesis, motility and genetic transformation in the cyanobacterium Synechocystis sp. PCC 6803.

The unicellular motile cyanobacterium Synechocystis sp. PCC 6803 exhibits phototactic motility that depends on the type IV-like thick pilus structure. By gene disruption analysis, we showed that a gene cluster of slr1041, slr1042, slr1043 and slr1044, whose predicted products are homologous to PatA, CheY, CheW and MCP, respectively, was more or less required for pilus assembly, motility and natural transformation competency with extraneous DNA. By sequence homology, the missing cheA-like gene in this cluster was identified as novel split genes, slr0073 and slr0322, at separate loci on the genome. This was confirmed by non-motile phenotype of their disruptants. Unique hyperpiliation was observed in the slr1042 and slr0073 disruptants, suggestive of their specific interaction with pilT1. The genes, thus identified as pil genes in this study, were designated pilG (slr1041), pilH (slr1042), pilI (slr1043), pilJ (slr1044), pilL-N (slr0073) and pilL-C (slr0322).