Although the biochemical mechanism of action of phorbol ester tumour promoters is not fully understood, it is known that phorbol ester binding to the cell surface causes rapid changes in calcium flux and phospholipid metabolism. A protein kinase activity has recently been described which is dependent on calcium and acidic phospholipids and is further enhanced by diacylglycerol. Previously, we have observed that phorbol ester treatment of EL4 mouse thymoma cells causes a rapid decrease in cytosolic calcium, phospholipid-dependent protein kinase (Ca, PL-PK) activity, which is mediated through the specific phorbol ester cell-surface receptors identified on EL4 cells. We now show that treatment of parietal yolk sacs (PYS-2) cells with biologically active 12-O-tetradecanoyl phorbol-13-acetate (TPA) provokes a rapid decrease in cytosolic Ca, PL-PK activity that is accompanied by a significant increase in the amount of Ca, PL-PK activity associated with the plasma membrane fraction. These results suggest that the rapid and tight association of Ca, PL-PK activity with the plasma membrane may be an early event in mediating some of the effects of phorbol esters.

Phorbol ester tumor promoters, such as phorbol 12-myristate 13-acetate (PMA), are potent activators of extracellular signal-regulated kinase 2 (ERK2), stress-activated protein kinase (SAPK), and p38 mitogenactivated protein kinase (MAPK) in U937 human leukemic cells. These kinases are regulated by the reversible dual phosphorylation of conserved threonine and tyrosine residues. The dual specificity protein phosphatase MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylate and inactivate ERK2, SAPK, and p38 MAPK in transient transfection studies. Here we demonstrate that PMA treatment induces MKP-1 protein expression in U937 cells, which is detectable within 30 min with maximal levels attained after 4 h. This time course coincides with the rapid inactivation of PMAinduced SAPK activity, but not ERK2 phosphorylation, which remains elevated for up to 6 h. To examine directly the role of MKP-1 in the regulation of these protein kinases in vivo, we established a U937 cell line that conditionally expresses MKP-1 from the human metallothionein IIa promoter. Conditional expression of MKP-1 inhibited PMA-induced ERK2, SAPK, and p38 MAPK activity. By titrating the levels of MKP-1 expression from the human metallothionein IIa promoter, however, it was found that p38 MAPK and SAPK were much more sensitive to inhibition by MKP-1 than ERK2. This differential substrate specificity of MKP-1 can be functionally extended to nuclear transcriptional events in that PMAinduced c-Jun transcriptional activity was more sensitive to inhibition by MKP-1 than either Elk-1 or c-Myc. Conditional expression of MKP-1 also abolished the induction of endogenous MKP-1 protein expression in response to PMA treatment. This negative feedback regulatory mechanism is likely due to MKP-1-mediated inhibition of ERK2, as studies utilizing the MEK1/2 inhibitor PD98059 suggest that ERK2 activation is required for PMA-induced MKP-1 expression. These findings suggest that ERK2-mediated induction of MKP-1 may play an important role in preferentially attenuating signaling through the p38 MAPK and SAPK signal transduction pathways. Mitogen-activated protein (MAP)1 kinases play a key role in transducing various extracellular signals to the nucleus (1). The MAP kinases (MAPKs) consist of three major subgroups that include the ERK, SAPK/JNK, and p38 MAPK families (2). The ERKs, SAPKs, and p38 MAPK are activated by the reversible dual threonine and tyrosine phosphorylation of a conserved TEY, TPY, or TGY motif, respectively (1-5). Although distinct and selective activators of the MAPKs have been cloned and characterized (6 -9), less in known about the negative regulation of these kinases. The reversible nature of MAPK phosphorylation suggests that protein phosphatases play an important role in regulating MAPK activity. An expanding subfamily of dual specificity protein tyrosine phosphatases has been identified which is capable of dephosphorylating and inactivating various members of the MAPK family. This class of phosphatases is characterized by MKP-1 (also k...

An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21 WAF1/Cip1 gene. We show that the gene encoding the gut-enriched Krü ppel-like factor (GKLF, KLF4) is concurrently induced with p21 WAF1/Cip1during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21 WAF1/Cip1 due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21, suggesting that GKLF may be involved in the induction of p21 Indeed, GKLF activates p21WAF1/Cip1 through a specific Sp1-like cis-element in the p21 WAF1/Cip1 proximal promoter. The same element is also required by p53 to activate the p21 WAF1/Cip1 promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21 WAF1/Cip1 promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21 WAF1/Cip1 promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21 WAF1/Cip1 is demonstrated by the ability of antisense Gklf oligonucleotides to block the production of p21 WAF1/Cip1 in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21 WAF1/Cip1 and may be part of a novel pathway by which cellular responses to stress are modulated.

PURPOSE Since activity of sorafenib was observed in sarcoma patients in a phase I study, we performed a multicenter phase II study of daily oral sorafenib in patients with recurrent or metastatic sarcoma. PATIENTS AND METHODS We employed a multiarm study design, each representing a sarcoma subtype with its own Simon optimal two-stage design. In each arm, 12 patients who received 0 to 1 prior lines of therapy were treated (0 to 3 for angiosarcoma and malignant peripheral-nerve sheath tumor). If at least one Response Evaluation Criteria in Solid Tumors (RECIST) was observed, 25 further patients with that sarcoma subtype were accrued. Results Between October 2005 and November 2007, 145 patients were treated; 144 were eligible for toxicity and 122 for response. Median age was 55 years; female-male ratio was 1.8:1. The median number of cycles was 3. Five of 37 patients with angiosarcoma had a partial response (response rate, 14%). This was the only arm to meet the RECIST response rate primary end point. Median progression-free survival was 3.2 months; median overall survival was 14.3 months. Adverse events (typically dermatological) necessitated dose reduction for 61% of patients. Statistical modeling in this limited patient cohort indicated sorafenib toxicity was correlated inversely to patient height. There was no correlation between phosphorylated extracellular signal regulated kinase expression and response in six patients with angiosarcoma with paired pre- and post-therapy biopsies. CONCLUSION As a single agent, sorafenib has activity against angiosarcoma and minimal activity against other sarcomas. Further evaluation of sorafenib in these and possibly other sarcoma subtypes appears warranted, presumably in combination with cytotoxic or kinase-specific agents.