Complement factor H-derived short consensus repeat 18-20 enhanced complement-dependent cytotoxicity of ofatumumab on chronic lymphocytic leukemia cells.

The antitumor activity of monoclonal antibodies in the treatment of chronic lymphocytic leukemia is mediated mainly by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Unfortunately, the efficacy of complement-dependent cytotoxicity is strongly restricted due to the expression and acquisition of regulators of complement activation by lymphocytic leukemia cells. Whereas the role of membrane regulators of complement activation, such as CD55 and CD59, has been investigated in detail in chronic lymphocytic leukemia, the involvement of soluble regulators of complement activation, such as complement factor H, has not yet been reported. Propidium iodide staining was performed to investigate the efficacy of ofatumumab and factor H-derived short-consensus-repeat 18-20 in the induction of complement-dependent cytotoxicity on primary chronic lymphocytic leukemia cells from 20 patients. Deposition of complement C3 fragments was monitored by western blot analysis. Expression of CD20, CD55 or CD59 was determined by FACS analysis. Replacement of factor H with short consensus repeat 18-20 significantly increased the susceptibility of primary chronic lymphocytic leukemia cells to ofatumumab-induced complement-dependent cytotoxicity. More importantly, addition of short-consensus-repeat 18-20 was able to overcome complement- resistance occurring during treatment with ofatumumab alone. Use of short consensus repeat 18-20 is likely to prolong the turnover time of active C3b fragments generated on the target cells following ofatumumab-induced complement activation, thereby improving specific killing of chronic lymphocytic leukemia cells by complement-dependent cytotoxicity. The relative contribution of factor H to the protection of chronic lymphocytic leukemia cells against complement-dependent cytotoxicity was comparable to that of CD55. Our data suggest that, by abrogating factor H function, short consensus repeat 18-20 may provide a novel approach that improves the complement-dependent efficacy of therapeutic monoclonal antibodies.

ENaC proteolytic regulation by channel-activating protease 2.

Epithelial sodium channels (ENaCs) perform diverse physiological roles by mediating Na(+) absorption across epithelial surfaces throughout the body. Excessive Na(+) absorption in kidney and colon elevates blood pressure and in the airways disrupts mucociliary clearance. Potential therapies for disorders of Na(+) absorption require better understanding of ENaC regulation. Recent work has established partial and selective proteolysis of ENaCs as an important means of channel activation. In particular, channel-activating transmembrane serine proteases (CAPs) and cognate inhibitors may be important in tissue-specific regulation of ENaCs. Although CAP2 (TMPRSS4) requires catalytic activity to activate ENaCs, there is not yet evidence of ENaC fragments produced by this serine protease and/or identification of the site(s) where CAP2 cleaves ENaCs. Here, we report that CAP2 cleaves at multiple sites in all three ENaC subunits, including cleavage at a conserved basic residue located in the vicinity of the degenerin site (alpha-K561, beta-R503, and gamma-R515). Sites in alpha-ENaC at K149/R164/K169/R177 and furin-consensus sites in alpha-ENaC (R205/R231) and gamma-ENaC (R138) are responsible for ENaC fragments observed in oocytes coexpressing CAP2. However, the only one of these demonstrated cleavage events that is relevant for the channel activation by CAP2 takes place in gamma-ENaC at position R138, the previously identified furin-consensus cleavage site. Replacement of arginine by alanine or glutamine (alpha,beta,gammaR138A/Q) completely abolished both the Na(+) current (I(Na)) and a 75-kD gamma-ENaC fragment at the cell surface stimulated by CAP2. Replacement of gamma-ENaC R138 with a conserved basic residue, lysine, preserved both the CAP2-induced I(Na) and the 75-kD gamma-ENaC fragment. These data strongly support a model where CAP2 activates ENaCs by cleaving at R138 in gamma-ENaC.

Signaling protein inhibitors via the combinatorial modification of peptide scaffolds.

Compounds that selectively interfere with protein-protein interactions are not only invaluable as biological reagents, but may ultimately serve as therapeutically useful drugs for the treatment of a wide variety of disease states. However, unlike active site directed inhibitors that bind to a relatively small, well-defined, hydrophobic pocket, reagents that disrupt protein-protein interactions must contend with a protein surface that is comparatively large, ill defined, and solvent exposed. We have developed a straightforward method for the acquisition of protein-protein interaction inhibitors. The library-based strategy starts with low affinity consensus sequence peptides, which are then transformed in a stepwise fashion into high affinity inhibitors. The approach has been used to create potent ligands for SH2 and SH3 domains, as well as powerful and highly selective inhibitors for protein kinases and phosphatases. The protocol is easily automated and therefore has the potential to be routinely applied, in a high throughput fashion.

Alteration of nuclear factor-kappaB (NF-kappaB) expression in bone marrow stromal cells treated with etoposide.

Bone marrow stromal cells are an essential regulatory component in the hematopoietic microenvironment. Regulation of hematopoietic cell development is mediated, in part, through interaction of progenitor cells with stromal cell vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 expression has been shown to be driven primarily by binding of nuclear factor-kappaB (NF-kappaB) to two consensus binding sites in the promoter region. In this study, we show that down-regulation of VCAM-1 by the chemotherapeutic agent etoposide (VP-16) is associated with altered cellular localization of NF-kappaB. We demonstrated that VCAM-1 was diminished at the transcriptional level following treatment of stromal cells with VP-16, without alteration of VCAM-1 stability. Culture of bone marrow stromal cells in VP-16 resulted in reduced nuclear RelA (p65), a modest increase in nuclear NF-kappaB1 (p50), and reduced NF-kappaB binding to its DNA consensus sequence. Total levels of the NF-kappaB inhibitor Ikappa-Balpha were reduced during exposure to VP-16. Following removal of VP-16 from the culture, p65 and p50 nuclear profiles approximated those of untreated stromal cells, and VCAM-1 protein expression was restored. The current study indicates that NF-kappaB is a target molecule that is responsive to VP-16-induced damage in bone marrow stromal cells. As the primary transcription factor that promotes VCAM-1 expression, the observed changes in p65 and p50 cellular localization during treatment have a direct consequence for stromal cell function. The myriad of genes regulated by NF-kappaB, including both adhesion molecules and cytokines that contribute to stromal cell function, make chemotherapy-induced disruption of NF-kappaB biologically significant. Alterations in NF-kappaB activity may provide one measure by which the effects of aggressive treatment strategies on the bone marrow microenvironment can be evaluated.

Mechanisms of organoselenium compounds in chemoprevention: effects on transcription factor-DNA binding.

Data obtained on the effects of selenium compounds on regulatory transcription factor-DNA binding by other laboratories are briefly reviewed, and some of our own results in this area are also presented. We assessed the in vitro and in vivo effects of the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate (p-XSC) on the binding activities of the transcription factors nuclear factor-kappa B (NF-kappa B), activator protein-1 (AP-1), Sp1, and Sp3 using the HCT-116 (human colorectal adenocarcinoma) cell line as a model system. Using nuclear extracts, electrophoretic mobility shift assays were carried out to determine the extent of binding of the transcription factors to their respective consensus recognition sites on radiolabeled oligonucleotides. p-XSC and sodium selenite reduced the consensus site binding activity of NF-kappa B in a concentration-dependent manner when nuclear extracts from cells stimulated with tumor necrosis factor-alpha were incubated with either compound ("in vitro"). However, only p-XSC inhibited NF-kappa B consensus recognition site binding when the cells were pretreated with either compound and were then stimulated with tumor necrosis factor-alpha ("in vivo"). In contrast, the consensus site binding activity of AP-1 was inhibited only with sodium selenite, but not with p-XSC in vitro or in vivo. p-XSC or sodium selenite reduced the consensus site binding of transcription factors Sp1 and Sp3 in concentration- and time-dependent manners when nuclear extracts from cells treated with either compound in vivo were assayed by electrophoretic mobility shift assay. 1,4-Phenylenebis(methylene)thiocyanate, the sulfur analog of p-XSC, which is inactive in chemoprevention, had no effect on the oligonucleotide binding of Sp1 and Sp3. Our observations could provide further clues as to the mechanisms involved in the chemoprevention of cancer by p-XSC.

Both ATP sites of human P-glycoprotein are essential but not symmetric.

Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.

AML1A and AML1B can transactivate the human IL-3 promoter.

The AML1 gene encodes several transcription factors, including AML1A and AML1B, which bind to DNA via a TGT/cGGT consensus sequence that is found in several promoters, including the human IL-3 promoter. We performed cotransfection experiments in T cells, and demonstrated that although AML1A lacks a putative transactivation domain, it can transactivate the IL-3 promoter nearly as effectively as AML1B, a known activator. A consensus AML1 binding site (TGTGGT), located in the previously identified DNase I footprint region A of the human IL-3 promoter (extending from bp -165 to -128), and a sequence similar to the consensus binding site (TGTGGG), located in footprint region B (bp -55 to -42), specifically bind AML1 proteins in gel shift assays. The affinity for the TGTGGG sequence was much less than that for the TGTGGT sequence, and mutating the TGTGGG sequence did not alter the IL-3 promoter activity, whereas mutation of the consensus binding site decreased the basal promoter activity and nearly eliminated transactivation by AML1A and AML1B. The AML1/ETO fusion protein, generated by the t(8;21) translocation, repressed IL-3 promoter activity, although the AML1 portion of AML1/ETO (amino acids 1-177) lacked transcriptional regulatory activity and did not bind to DNA in vitro. The 60- to 177-amino acid portion of AML1 readily bound DNA, suggesting that the first 59 amino acids may function as an inhibitory domain for DNA binding. Demonstration of the transactivation by AML1A and localization of a putative inhibitory binding domain suggest additional complexity within this family of transcription factors.

beta-Cyclodextrin derivatives as carriers to enhance the antiviral activity of an antisense oligonucleotide directed toward a coronavirus intergenic consensus sequence.

The ability of cyclodextrins to enhance the antiviral activity of a phosphodiester oligodeoxynucleotide has been investigated. A 18-mer oligodeoxynucleotide complementary to the initiation region of the mRNA coding for the spike protein and containing the intergenic consensus sequence of an enteric coronavirus has been tested for antiviral action against virus growth in human adenocarcinoma cells. The phosphodiester oligodeoxynucleotide only showed a limited effect on virus growth rate (from 12 to 34% viral inhibition in cells treated with 7.5 to 25 microM oligodeoxynucleotide, respectively, at a multiplicity of infection of 0.1 infectious particle per cell). In the same conditions, the phosphorothioate analogue exhibited stronger antiviral activity, the inhibition increased from 56 to 90%. The inhibitory effect of this analogue was antisense and sequence-specific. Northern blot analysis showed that the sequence-dependent mechanism of action appears to be the inhibition of mRNA transcription. We conclude that the coronavirus intergenic consensus sequence is a good target for an antisense oligonucleotide antiviral action. The properties of the phosphodiester oligonucleotide was improved after its complexation with cyclodextrins. The most important increase of the antiviral activity (90% inhibition) was obtained with only 7.5 microM oligonucleotide complexed to a cyclodextrin derivative, 6-deoxy-6-S-beta-D-galactopyranosyl-6-thio-cyclomalto-heptaose+ ++ in a molar ratio of 1:100. These studies suggest that the use of cyclodextrin derivatives as carrier for phosphodiester oligonucleotides delivery may be an effective method for increasing the therapeutic potential of these compounds in viral infections.