The anti-cancer effects of bryostatin-1, a potent diacylglycerol analogue, have traditionally

The anti-cancer effects of bryostatin-1, a potent diacylglycerol analogue, have traditionally been attributed to its action on protein kinase C. mantle cell lymphoma cell lines underwent pico-induced apoptosis, as did BL2. In 107007-99-8 some cases, hallmarks of apoptosis were substantially diminished in the presence of mitogen-activated protein kinase kinase inhibitors. Pico treatment generally led to increased expression of proapoptotic Bik, although the absolute levels of Bik varied considerably between cell lines. A pico-resistant variant of Z138 exhibited decreased Bik induction compared to parental Z138 cells. Pico also generally decreased expression of anti-apoptotic Bcl-XL and Mcl1. Although, these changes in Bcl-2 family members seem unlikely to fully account for the Mouse monoclonal antibody to JMJD6. This gene encodes a nuclear protein with a JmjC domain. JmjC domain-containing proteins arepredicted to function as protein hydroxylases or histone demethylases. This protein was firstidentified as a putative phosphatidylserine receptor involved in phagocytosis of apoptotic cells;however, subsequent studies have indicated that it does not directly function in the clearance ofapoptotic cells, and questioned whether it is a true phosphatidylserine receptor. Multipletranscript variants encoding different isoforms have been found for this gene differential behavior of the cell lines, our demonstration of a potent apoptotic process in most cell lines derived from mantle cell lymphoma encourages a re-examination of diacylglycerol analogues in the treatment of this subset of B non-Hodgkin lymphoma cases. Bryostatin-1 is a potent diacylglycerol (DAG) analogue that exhibits a range of promising therapeutic activities. Unlike DAG analogues such as phorbol 12-myristate, 13-acetate, bryostatin-1 is not a tumor promoter [1]. Based on promising preclinical data, bryostatin-1 has been evaluated as 107007-99-8 both a single agent and an adjuvant to conventional anticancer therapies, including those aimed at leukemia and lymphoma [2]. Although bryostatin-1 is well tolerated, its efficacy has been documented on a sporadic basis only. Traditionally, the activities of both natural DAG and DAG analogues have been interpreted in terms of their binding to, and activation of, protein kinase C (PKC) family members. Conventional and novel species of PKC possess C1 domains that bind DAG and DAG 107007-99-8 analogues, contributing to membrane recruitment and activation of these enzymes. Because prolonged activation of PKCs can lead to proteolysis of these enzymes, DAG analogue effects have also been interpreted in terms of PKC downregulation [2]. Besides PKCs, several other classes of proteins with DAG and DAG-analogueCbinding C1 domains have been described [3,4]. We have shown that some members of the Ras guanyl-releasing protein (RasGRP) family of Ras activators bind DAG and DAG analogues such as phorbol esters and bryostatin-1 with low nanomolar affinity [5,6]. RasGRPs are functionally analogous to Sos proteins, but they are preferentially expressed in lymphocytes, where they link immune receptors and phospholipase C-generated DAG to Ras signaling. Recruitment of RasGRPs to cellular membranes by DAG and DAG analogues brings them into proximity with their substrate, membrane-bound Ras. DAG and DAG analogues additionally activate RasGRPs by PKC-mediated regulatory phosphorylation [7C9]. The interaction of activated RasGRPs with Ras at the cell membrane results in conversion of inactive Ras-GDPto active Ras-GTP, triggering signaling through the Raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) kinase cascade [10]. Activation of this pathway is a key process during immune receptor signaling and contributes to both immune cell development and effector function [10]. We have shown that bryostatin-1 and a synthetically accessible and tunable bryostatin-like compound called pico (or picolog) are indistinguishable in several RasGRP-based assays, including those based on primary human lymphocytes [11]. Additionally, we recently reported that exposure to either bryostatin-1 or pico induces extensive apoptosis in the cell line Toledo, which was derived from a germinal type B non-Hodgkin lymphoma (B-NHL) [12]. RasGRPs are almost certainly drug targets in this system because apoptosis was blocked effectively in the presence of MEK inhibitors or PKC inhibitors that block regulatory phosphorylation of RasGRPs. We also presented evidence that apoptosis arose from proapoptotic phosphorylation of the pro-death Bcl-2 family member Bim. Bim is a key regulator of lymphocyte apoptosis [13]. Bim has properties consistent with a tumor suppressor; Bim coding sequences are frequently mutated or epigenetically silenced in several types of B-NHL, including mantle cell lymphoma (MCL) and Burkitt lymphoma (BL) [14,15]. In light of our proposal that Bim 107007-99-8 played a key role in DAG analogue-induced apoptosis in Toledo B-NHL cells, we were interested in examining the response of cells lacking Bim to the bryostatin-1 analogue pico. Unexpectedly, pico induced apoptosis in most MCL cell lines, regardless of Bim status. The Bim-deficient BL cell line BL2 was also sensitive. Our results suggest that bryostatin-1 and related compounds might have efficacy in some cases of MCL and BL. Materials and methods Reagents and antibodies Pico was synthesized as described previously [16], dissolved in dimethyl sulfoxide (DMSO) at 1.0 mM and stored at ?20C. Except for the dose-response studies, pico was used.