Differential effect of vinorelbine versus paclitaxel on ERK2 kinase activity during apoptosis in MCF-7 cells

The effects of vinorelbine and paclitaxel on the activity of extracellular signal-regulated protein kinase2 (ERK2), a member of MAP kinase, and its role in the induction of bcl-2 phosphorylation and apoptosis were evaluated in MCF-7 cells. We demonstrated that ERK2 was activated rapidly by vinorelbine, and was inhibited by either paclitaxel or estramustine. A 3-fold increase of ERK2 kinase activity was observed within 30 min when MCF-7 cells were treated with 0.1 μM vinorelbine. In contrast, the same treatment with paclitaxel resulted in a significant decrease of ERK2 kinase activity. We also demonstrated that elevated bcl-2 phosphorylation induced by vinorelbine is paralleled by decrease of a complex formation between bcl-2 and bax, cleavage of poly (ADP) ribose polymerase (PARP) protein, activation of caspase-7, and apoptosis. The levels of bcl-2 phosphorylation, bax, and PARP were not significantly affected by 2′-amino-3′-methoxyflavone (PD 98059), an ERK kinase specific inhibitor. Thus, our data suggest that the apoptosis induced by vinorelbine in MCF-7 cells is mediated through the bcl-2 phosphorylation/bax/caspases pathways, and that activation of ERK2 by vinorelbine does not directly lead to the drug-mediated apoptosis. Since decrease of PARP occurred quickly following the treatment of MCF-7 cells with either 0.1 μM of vinorelbine or paclitaxel, this protein may serve as an early indicator of apoptosis induced not only by DNA damaging agents, but also by antimicrotubule drugs.   http://www.bjcancer.com © 2001 Cancer Research Campaig

Cloning and expression of PTP-PEST. A novel, human, nontransmembrane protein tyrosine phosphatase

The polymerase chain reaction was used to amplify protein tyrosine phosphatase (PTPase)-related cDNA from a template of total RNA isolated from human skeletal muscle. A novel PTPase, which we term PTP-PEST, was detected by this method. The polymerase chain reaction fragment was used to screen two different HeLa cell libraries to obtain full length cDNA clones. The cDNA predicts a protein of 510 amino acids, approximately 60 kDa, that does not contain an obvious signal sequence or transmembrane segment suggesting it is a nonreceptor type enzyme. The PTPase domain is located in the N-terminal portion of the molecule and displays approximately 35% identity to other members of this family of enzymes. The C-terminal segment is rich in Pro, Glu, Asp, Ser, and Thr residues, possessing features of PEST motifs which have previously been identified in proteins with very short intracellular half-lives. The protein was expressed in Escherichia coli as a fusion product with glutathione S-transferase. Intrinsic activity was demonstrated in vitro against a variety of phosphotyrosine-containing substrates including BIRK, the autophosphorylated cytoplasmic kinase domain of the insulin receptor beta subunit. It did not dephosphorylate phosphoseryl-phosphorylase a. PTP-PEST mRNA is broadly distributed in a variety of cell lines. Stimulation of human rhabdomyosarcoma A204 cells, a transformed muscle line, with insulin led to an approximately 4-fold induction of PTP-PEST mRNA within 36 h