p21 functions in a post-mitotic block checkpoint in the apoptotic response to vinblastine.

We have shown previously that in KB-3 (HeLa) cells vinblastine causes downregulation of the CDK inhibitor p21 through a c-Jun regulated pathway. To test the hypothesis that p21 downregulation is necessary to alleviate a protective function, we transfected p21 in KB-3 cells and examined the apoptotic response to vinblastine. The results showed that cells overexpressing p21 were apoptosis-resistant, not through an ability of p21 to cause cell cycle arrest prior to mitotic arrest, but through altering the fate of mitotically arrested cells after drug treatment.

Induction of apoptosis by vinblastine via c-Jun autoamplification and p53-independent down-regulation of p21WAF1/CIP1.

Vinblastine treatment in all cell lines examined causes a robust increase in c-Jun protein expression and phosphorylation and a corresponding increase in activator protein-1 (AP-1) transcriptional activity. We show in KB-3 carcinoma cells that this is due to a strong autoamplification loop involving the proximal AP-1 site in the c-Jun promoter, resulting in highly increased c-Jun mRNA and c-Jun protein. Inhibitors of RNA transcription and protein translation blocked both vinblastine-induced c-Jun expression and apoptotic cell death, suggesting that apoptosis is dependent, at least in part, on transcription/translation.

Subcellular localization as a limiting factor for utilization of decoy oligonucleotides.

Transfection of cells with short double-stranded synthetic DNA molecules that contain a transcription factor binding site, known as decoy oligodeoxynucleotides (ODNs), has been proposed as a novel approach in vitro and in vivo for the study of gene regulation and for gene therapy. Once delivered into cells, decoy ODNs are predicted to bind to nuclear transcription factors, preventing their binding to consensus sequences in target genes. Using a fluorescein-labeled decoy ODN containing a consensus sequence for the AP-1 transcription factor, we show that lipid-complexed decoys were readily transfectable into cells, but were consistently detectable in the cytoplasm and not in the nucleus.

Glycosyl modification facilitates homo- and hetero-oligomerization of the serotonin transporter. A specific role for sialic acid residues.

The serotonin transporter (SERT) is an oligomeric glycoprotein with two sialic acid residues on each of two complex oligosaccharide molecules. In this study, we investigated the contribution of N-glycosyl modification to the structure and function of SERT in two model systems: wild-type SERT expressed in sialic acid-defective Lec4 Chinese hamster ovary (CHO) cells and a mutant form (after site-directed mutagenesis of Asn-208 and Asn-217 to Gln) of SERT, QQ, expressed in parental CHO cells. In both systems, SERT monomers required modification with both complex oligosaccharide residues to associate with each other and to function in homo-oligomeric forms.