Susan C. Wright

Nicotine inhibition of apoptosis suggests a role in tumor promotion.

Recent evidence indicates that cell death through apoptosis may be an important mechanism to prevent tumor development. Therefore, agents that inhibit apoptosis may function as tumor promotors. The purpose of this study was to determine the effects of nicotine on the process of apoptosis. The results demonstrate that nicotine inhibits apoptosis induced by diverse stimuli including tumor necrosis factor (TNF), UV light, chemotherapeutic drugs, and calcium ionophore. This phenomenon was observed in normal and transformed cells derived from a variety of species and tissues, including tumor cell types related to tobacco use. The major nicotine metabolite, cotinine, also inhibited apoptosis, whereas N‐nitrosodiethylamine, a carcinogen found in tobacco, was without effect. Therefore, nicotine‐mediated inhibition of apoptosis may contribute to the pathogenesis of tobacco‐related cancer as well as decrease the efficacy of cancer therapies.—Wright, S. C., Zhong, J., Zheng, H., Larrick, J. W. Nicotine inhibition of apoptosis suggests a role in tumor production, FASEB J. 7: 1045‐1051; 1993.

Calmodulin-dependent protein kinase II mediates signal transduction in apoptosis.

The present studies describe a new function for calmodulin‐dependent protein kinase II (CaM‐KII) in signal transduction leading to apoptosis. Both tumor necrosis factor α (TNF) and UV light rapidly stimulated Ca2+‐independent activity of CaM‐KII in the monocytic leukemia, U937. Two mechanistically different inhibitors of CaM‐KII blocked activation of CaM‐KII and prevented DNA fragmentation and death. Activation of CaM‐KII during apoptosis and inhibition of DNA fragmentation by the two CaM‐KII inhibitors were reproduced in several other lines including KG1a, HL‐60, and YAC‐1. However, K562, which is relatively resistant to apoptosis induced by either TNF or UV light, did not activate CaM‐KII in response to these stimuli. A variant derived from U937 that is resistant to TNF‐ or UV light‐induced apoptosis also lacked a CaM‐KII response. Activation of Cam‐KII was blocked by two protease inhibitors, VAD‐fmk and TPCK, but not by other inhibitors of serine proteases. Both inhibitors of CaM‐KII and the protease inhibitors blocked activation of AP24, a serine protease originally isolated from apoptotic cells that induces DNA fragmentation in nuclei. Our evidence supports a model in which proteolytic activity functions upstream of CaM‐KII. This kinase then leads to activation of AP24, which transmits signals to the nucleus to initiate DNA fragmentation.—Wright, S. C., Schellenberger, U., Ji, L., Wang, H., Larrick, J. W. Calmodulin‐dependent protein kinase II mediates signal transduction in apoptosis. FASEB J. 11, 843–849 (1997)

Anti-microbial activity of human CAP18 peptides

BACKGROUND CAP18 derived from rabbit leukocytes is a 142-amino acid protein recently demonstrated to have Lipopolysaccharide (LPS) binding and anti-microbial activity. The C-terminal 37 amino acids of rabbit CAP18 (CAP18(106-142) comprise the LPS-binding and anti-microbial domain. The homologous domain of human CAP18 (huCAP18(104-140) was identified from the recently cloned human CAP18 cDNA. OBJECTIVES To evaluate the antimicrobial activity of C-terminal peptides derived from human CAP18. STUDY DESIGN Prepare synthetic human CAP18(104-140) and study anti-microbial activity versus various gram-negative and gram-positive bacteria. RESULTS Synthetic human CAP18(104-140) has broad anti-microbial activity versus both gram-positive (IC50 = 2.5 micrograms/ml) and gram-negative bacteria (IC50 = 0.5-5 micrograms/ml). Susceptible strains include Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium. A 32-amino acid peptide lacking five amino acids from the C-terminus of CAP18(104-140) has higher activity. Unlike previously characterized anti-microbial peptides derived from granulocyte proteins, CAP18(104-140) is active in serum. CONCLUSIONS Human CAP18(104-140) or a derivative peptide may have therapeutic potential for bacterial sepsis.

Antimicrobial activity of rabbit CAP18-derived peptides.

A cationic antimicrobial protein of 18 kDa (CAP18) was originally isolated from rabbit granulocytes by using as an assay the agglutination of Re-lipopolysaccharide-coated erythrocytes. The C-terminal 37 amino acids of CAP18 (CAP18(106-142)) make up the lipopolysaccharide-binding domain. Synthetic CAP18(106-142) has broad antimicrobial activity against both gram-positive (50% inhibitory concentration, 130 to 200 nM) and gram-negative (50% inhibitory concentration, 20 to 100 nM) bacteria. Susceptible strains include Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium. Antimicrobial activity is highly dependent on peptide structure. Although a 32-amino-acid peptide resulting from the truncation of 5 amino acids from the C terminus of CAP18(106-142) is highly active, other fragments of CAP18(106-142), including CAP18(106-142) with a truncated N terminus, do not exhibit antimicrobial activity. Unlike previously characterized antimicrobial peptides derived from granulocyte proteins, CAP18(106-142) is active in serum. CAP18(106-142) or a derivative peptide may have therapeutic potential for bacterial sepsis. Images

Tumor cell resistance to apoptosis due to a defect in the activation of sphingomyelinase and the 24 kDa apoptotic protease (AP24).

Signal transduction pathways involved in apoptotic cell death are poorly understood, although recent studies have implicated sphingomyelin hydrolysis and generation of the second messenger, ceramide. Previous work in this laboratory demonstrated that a serine protease termed AP24 was activated by TNF or UV light and induced DNA fragmentation in isolated nuclei. This study extended these findings to examine the role of these enzymes in apoptosis of the U937 cell line and the mechanism of resistance of its variant, U9‐TR. Although this subclone was selected by growth in TNF, it was unexpectedly found to resist apoptosis induced by UV light, but was still sensitive to anti‐Fas‐induced DNA fragmentation. Here we show that in contrast to normal U937 cells, UV light and TNF both failed to activate neutral or acidic sphingomyelinase or AP24 in the U9‐TR variant. However, anti‐Fas activated both neutral and acidic sphingomyelinase in the variant comparable to that seen in parental U937. The U9‐TR variant could be sensitized to TNF or UV light activation of both sphingomyelinase and DNA fragmentation by the protein phosphatase inhibitors okadaic acid and caly‐culin A. Furthermore, exogenous bacterial‐derived sphingomyelinase caused U9‐TR activation of AP24 and DNA fragmentation comparable to that in the parental U937. Exposure of permeabilized U937 cells to ceramide caused internucleosomal DNA cleavage that was blocked by an inhibitor of AP24. Taken altogether, these findings demonstrate that TNF or UV light activate sphingomyelinase that leads to generation of ceramide resulting in activation of AP24 and DNA fragmentation in sensitive cells. A selective defect in signals leading to sphingomyelinase activation can confer resistance to apoptosis even though the variant is still sensitive to downstream apoptotic signals such as nuclear DNA fragmentation by activated exogenous AP24.—Wright, S. C., Zheng, H., Zhong, J. Tumor cell resistance to apoptosis due to a defect in the activation of sphingomyelinase and the 24 kDa apoptotic protease (AP24). FASEB J. 10, 325‐332 (1996)

Structural, functional analysis and localization of the human CAP18 gene.

CAP18 is an antimicrobial protein found in specific granules of PMNs. The human CAP18 (HCAP18) gene was cloned from a human genomic phage library. Sequence analysis revealed the HCAP18 gene to have 4 exons spanning 3 kb, including 700 bp of upstream DNA. Using 3′ RACE no homologs of human HCAP18 were found in human bone marrow or leukocyte populations. By PCR analysis of a somatic cell mapping panel and fluorescence in situ hybridization of a genomic clone to metaphase chromosomes the gene was mapped to chromosome band 3p21.3. Like several other genes expressed late in PMN development the CAP18 gene did not contain typical TATA box or CCAAT sequences. Expression in Cos 7 cells permitted limited mapping of the promoter function in upstream fragments of the HCAP18 gene. Western blot, Northern blot and RT‐PCR analysis show HCAP18 to be produced specifically in granulocytes. This work forms the groundwork for future analysis of the genetic regulation of this antimicrobial protein during PMN differentiation.

Synthesis and evaluation of antibacterial activities of andrographolide analogues

Andrographolide (Andro), the main active component of the herb Andrographis paniculata, has been used for many years to treat a variety of diseases including bacterial and viral infections. Andro was recently reported to act by inhibiting the bacterial quorum sensing system. We have synthesized several Andro analogues and investigated their antibacterial activity and mechanism of action. The new compounds were found to be much more potent than the parent Andro in inhibiting bacterial growth and quorum sensing system. Compounds 5 and 7 significantly reduced virulence factor production. Compound 7 completely inhibited Pseudomonas aeruginosa (P. aeruginosa) biofilm formation, and exhibited synergistic activity with conventional antibiotics. These findings suggest that compound 7 may be the basis for future drug development to combat the unmet needs of virulence factor production, biofilm formation and antibiotic resistance.

Synthesis and preliminary cytotoxicity study of glucuronide derivatives of CC-1065 analogues.

Glucuronide derivatives of CBI-bearing CC-1065 analogues have been synthesized, and their cytotoxicities tested against U937 leukemia cells. The new compounds show potent antitumor activity in vitro. Compounds 1 and 2, and their corresponding glucuronides 3 and 4 have IC(50) values of 0.6, 0.1, 1.4 and 0.6 nM, respectively. Glucuronide 3 is approximately 2-fold less toxic than its hydroxyl counterpart 1, and glucuronide 4 is approximately 6-fold less toxic than its hydroxyl counterpart 2. Glucuronides 3 and 4 may have limited use in the ADEPT approach. However, they may be used as antitumor agents in a conventional way.

Synthesis and preliminary cytotoxicity study of a cephalosporin-CC-1065 analogue prodrug

Background Antibody-directed enzyme prodrug therapy (ADEPT) is a promising new approach to deliver anticancer drugs selectively to tumor cells. In this approach, an enzyme is conjugated to a tumor-specific antibody. The antibody selectively localizes the enzyme to the tumor cell surface. Subsequent administration of a prodrug substrate of the enzyme leads to the enzyme-catalyzed release of the free drug at the tumor site. The free drug will destroy the tumor cells selectively, thus, reducing side effects. Results A CC-1065 analogue was conjugated to a cephalosporin affording prodrug 2. The prodrug and its corresponding free drug, 1, have IC50 values of 0.9 and 0.09 nM, respectively, against U937 leukemia cells in vitro. Conclusions For the first time, a prodrug comprised of a cephalosporin and a CC-1065 analogue has been synthesized. The preliminary in vitro studies show that the prodrug was 10-fold less toxic than the free drug. Prodrug 2 has the potential to be useful in cancer treatment using the ADEPT approach.

Native cytokine antagonists

Cytokines orchestrate the complex homeostasis of cells and tissues by acting in both an autocrine and paracrine fashion. The processes responsible for regulation of cytokines is not well understood. This chapter has summarized what is known about antagonism and inhibition of the action of cytokines. Several concepts have emerged from work in this area. At least two cytokines (IL-1 alpha and IL-1 beta) have an endogenous receptor antagonist, the IL-1 receptor antagonist. This is the first example of one endogenous molecule directly blocking the binding of another molecule to its receptor: most forms of regulation occur through independent receptors. Several cytokines, including TNF, IFN-gamma, IL-2 and IL-4, are inhibited by soluble receptors. Several cytokines, including IL-10, TGF-beta and MDF, act to inhibit other cytokines. It is likely that these inhibitors will be found to have pleiotropic actions in vivo. Finally, we describe antibody inhibition of cytokines. Detailed studies will be required to understand the complex interplay of the aforementioned cytokine inhibitors and the processes they regulate.