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1.  Conditional drug screening shows that mitotic inhibitors induce AKT/PKB-insensitive apoptosis 
Journal of Chemical Biology  2009;2(2):81-87.
The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is frequently upregulated in human cancer. Activation of this pathway has been reported to be associated with resistance to various chemotherapeutical agents. We here used a chemical biology/chemical informatic approach to identify apoptotic mechanisms that are insensitive to activation of the PI3K/AKT pathway. The National Cancer Institute (NCI) Mechanistic Set drug library was screened for agents that induce apoptosis in colon carcinoma cells expressing a constitutively active form of AKT1. The cytotoxicity screening data available as self-organized maps at the Developmental Therapeutics Program (DTP) of the NCI was then used to classify the identified compounds according to mechanism of action. The results showed that drugs that interfere with the mitotic process induce apoptosis which is comparatively insensitive to constitutive AKT1 activity. The conditional screening approach described here is expected to be useful for identifying relationships between the state of activation of signaling pathways and sensitivity to anticancer agents.
PMCID: PMC2701489  PMID: 19568785
Chemical biology; Apoptosis; AKT; Microtubuli-interacting agents
2.  Charting calcium-regulated apoptosis pathways using chemical biology: role of calmodulin kinase II 
Intracellular free calcium ([Ca2+]i) is a key element in apoptotic signaling and a number of calcium-dependent apoptosis pathways have been described. We here used a chemical biology strategy to elucidate the relative importance of such different pathways.
A set of 40 agents ("bioprobes") that induce apoptosis was first identified by screening of a chemical library. Using p53, AP-1, NFAT and NF-κB reporter cell lines, these bioprobes were verified to induce different patterns of signaling. Experiments using the calcium chelator BAPTA-AM showed that Ca2+ was involved in induction of apoptosis by the majority of the bioprobes and that Ca2+ was in general required several hours into the apoptosis process. Further studies showed that the calmodulin pathway was an important mediator of the apoptotic response. Inhibition of calmodulin kinase II (CaMKII) resulted in more effective inhibition of apoptosis compared to inhibition of calpain, calcineurin/PP2B or DAP kinase. We used one of the bioprobes, the plant alkaloid helenalin, to study the role of CaMKII in apoptosis. Helenalin induced CaMKII, ASK1 and Jun-N-terminal kinase (JNK) activity, and inhibition of these kinases inhibited apoptosis.
Our study shows that calcium signaling is generally not an early event during the apoptosis process and suggests that a CaMKII/ASK1 signaling mechanism is important for sustained JNK activation and apoptosis by some types of stimuli.
PMCID: PMC2518916  PMID: 18673549
3.  Is translational research compatible with preclinical publication strategies? 
The term "translational research" is used to describe the transfer of basic biological knowledge into practical medicine, a process necessary for motivation of public spending. In the area of cancer therapeutics, it is becoming increasingly evident that results obtained in vitro and in animal models are difficult to translate into clinical medicine. We here argue that a number of factors contribute to making the translation process inefficient. These factors include the use of sensitive cell lines and fast growing experimental tumors as targets for novel therapies, and the use of unrealistic drug concentrations and radiation doses. We also argue that aggressive interpretation of data, successful in hypothesis-building biological research, does not form a solid base for development of clinically useful treatment modalities. We question whether "clean" results obtained in simplified models, expected for publication in high-impact journals, represent solid foundations for improved treatment of patients. Open-access journals such as Radiation Oncology have a large mission to fulfill by publishing relevant data to be used for making actual progress in translational cancer research.
PMCID: PMC1459183  PMID: 16722592
4.  Doxorubicin Requires the Sequential Activation of Caspase-2, Protein Kinase Cδ, and c-Jun NH2-terminal Kinase to Induce ApoptosisD⃞ 
Molecular Biology of the Cell  2005;16(8):3821-3831.
Here, we identified caspase-2, protein kinase C (PKC)δ, and c-Jun NH2-terminal kinase (JNK) as key components of the doxorubicin-induced apoptotic cascade. Using cells stably transfected with an antisense construct for caspase-2 (AS2) as well as a chemical caspase-2 inhibitor, we demonstrate that caspase-2 is required in doxorubicin-induced apoptosis. We also identified PKCδ as a novel caspase-2 substrate. PKCδ was cleaved/activated in a caspase-2–dependent manner after doxorubicin treatment both in cells and in vitro. PKCδ is furthermore required for efficient doxorubicin-induced apoptosis because its chemical inhibition as well as adenoviral expression of a kinase dead (KD) mutant of PKCδ severely attenuated doxorubicin-induced apoptosis. Furthermore, PKCδ and JNK inhibition show that PKCδ lies upstream of JNK in doxorubicin-induced death. Jnk-deficient mouse embryo fibroblasts (MEFs) were highly resistant to doxorubicin compared with wild type (WT), as were WT Jurkat cells treated with SP600125, further supporting the importance of JNK in doxorubicin-induced apoptosis. Chemical inhibitors for PKCδ and JNK do not synergize and do not function in doxorubicin-treated AS2 cells. Caspase-2, PKCδ, and JNK were furthermore implicated in doxorubicin-induced apoptosis of primary acute lymphoblastic leukemia blasts. The data thus support a sequential model involving caspase-2, PKCδ, and JNK signaling in response to doxorubicin, leading to the activation of Bak and execution of apoptosis.
PMCID: PMC1182319  PMID: 15917298
5.  Calpain-Mediated Bid Cleavage and Calpain-Independent Bak Modulation: Two Separate Pathways in Cisplatin-Induced Apoptosis 
Molecular and Cellular Biology  2002;22(9):3003-3013.
Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.
PMCID: PMC133754  PMID: 11940658
6.  Cisplatin Induces the Proapoptotic Conformation of Bak in a ΔMEKK1-Dependent Manner 
Molecular and Cellular Biology  2001;21(11):3684-3691.
In a panel of four human melanoma cell lines, equitoxic doses of cisplatin induced the proapoptotic conformation of the Bcl-2 family protein Bak prior to the execution phase of apoptosis. Because cisplatin-induced modulation of the related Bax protein was seen in only one cell line, a degree of specificity in the signal to Bak is indicated. Little is known about upstream regulation of Bak activity. In this study, we examined whether the apoptosis-specific pathway mediated by a kinase fragment of MEKK1 (ΔMEKK1) is involved in the observed Bak modulation. We report that expression of a kinase-inactive fragment of MEKK1 (dominant negative MEKK [dnMEKK]) efficiently blocked cisplatin-induced modulation of Bak and cytochrome c release and consequently also reduced DEVDase activation and nuclear fragmentation. Accordingly, expression of a kinase-active MEKK1 fragment (dominant positive MEKK) was sufficient to induce modulation of Bak in three cell lines and to induce apoptosis in two of these. dnMEKK did not block cisplatin-induced c-Jun N-terminal kinase (JNK) activation, in agreement with a specifically proapoptotic role for the ΔMEKK1 pathway. Finally, we show that reduction of Bak expression by antisense Bak reduced cisplatin-induced loss of mitochondrial integrity and caspase cleavage activity in breast cancer cell lines. In summary, we have identified Bak as a cisplatin-regulated component downstream in a proapoptotic, JNK-independent ΔMEKK1 pathway.
PMCID: PMC86999  PMID: 11340162
7.  Two Novel Adenovirus Vector Systems Permitting Regulated Protein Expression in Gene Transfer Experiments 
Journal of Virology  1998;72(10):8358-8361.
Two new adenovirus vector systems based on the tetracycline-regulated Tet-ON- (Gossen, M., et al., Science 268:1766–1769, 1995) and the RU 486-regulated progesterone antagonist (Wang, Y., et al., Proc. Natl. Acad. Sci. USA 91:8180–8184, 1994)-induced gene expression systems are described. We show that both systems permit a tight control of chloramphenicol acetyltransferase reporter gene expression in a variety of cell types, with induction levels of approximately 1,800-fold (Tet-ON system) and 600-fold (RU 486-regulated system), respectively. A significant advantage of our vector systems is that reporter protein expression can be adjusted over a wide range by varying the amount of inducer. The Tet-ON system is also shown to permit an efficient control of reporter gene expression in mice.
PMCID: PMC110212  PMID: 9733884

Results 1-7 (7)