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Here, we report that the calcium ionophore ionomycin induces a massive Ca2+-dependent apoptosis in wildtype DT-40 chicken B lymphoma cells, as well as in BTK-deficient, PLCgamma2-deficient and IP3 receptor-deficient DT-40 cells, but not in LYN- or SYK-deficient DT-40 cells. Notably, the deficiency of CSK, a negative regulator of Src-family PTK, promoted ionomycin-induced apoptosis of DT-40 cells. Reconstitution of SYK-deficient cells with wild-type SYK restored the apoptotic response of the cells to ionomycin, but the expression of FYN or LCK in LYN-deficient cells did not restore the apoptotic response of LYN-deficient cells. Taken together, our data suggests that both LYN and SYK, but not BTK, FYN or LCK, are crucial mediators of BCR-independent Ca2+-induced apoptosis in DT-40 lymphoma B cells.

We examined the effects of cathepsin inhibitor 1 (CATI-1), a selective inhibitor of cysteine cathepsins, on human leukemia and lymphoma cells. CATI-1 induced apoptosis in all 12 cell lines tested. Apoptosis of CATI-1-treated leukemia/lymphoma cells was caspase-independent, p53-independent, BAX-independent as well as MAP kinase-independent. Our findings provide unprecedented experimental evidence that cathepsins play a pivotal role for the survival of human leukemia/lymphoma cells. Therefore, cathepsin inhibitors may provide the basis for new treatment programs against leukemia and lymphoma.

Calphostin C is a potent inhibitor of protein kinase C and can induce Ca2+-dependent apoptosis in human ALL cells. Further development of calphostin C will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high-performance liquid chromatography (HPLC)-based quantitative detection method for the measurement of calphostin C levels in plasma. Extraction of calphostin C from plasma was performed by precipitation of plasma protein using acetonitrile and an aliquot of extracted supernatant was injected onto a Hewlett-Packard HPLC system constituting a 250x4 mm LiChrospher 100, RP-18 (5 microm) in conjunction with a 4x4 mm LiChrospher 100, RP-18 guard column (5 microm). The eluted compounds were detected by diode array detection set at a wavelength of 479 nm. Acetonitrile-water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (70:30, v/v) was used as the mobile phase. The average extraction recovery from plasma was 97.3%. Good linearity (r0.999) was observed throughout the concentration range of 0.05-40 microM for calphostin C in 50 microl of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 microl plasma was 0.02 microM at a signal-to-noise ratio of approximately 3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo.

Purpose. To examine the pharmacokinetic features and metabolism of calphostin C, a naturally occurring perylenequinone with potent antileukemic activity.

The surface adsorption and micelle formation of aqueous solutions of C12H25(OC2H4)jSO4NaC10H21SOCH3 systems (j=0, 1, 3) have been investigated by the surface tension method. It has been shown that there are strong molecular interactions between the two surfactant components in the surface layers and the mixed micelles. The interactions become weaker as the length of the oxyethylene (EO) chain increases. This is illustrated by the fact that there exist a maximum surface adsorption (ΓT.cmc) and a minimum c.m.c. in the C12H25SO4NaC10H21SOCH3 system (I), and a minimum c.m.c. in the C12H25OC2H4SO4NaC10H21SOCH3 system (II) (no ΓT.cmc maximum), but neither the ΓT.cmc maximum nor the c.m.c. minimum occurs in the C12H25(OC2H4)3SO4NaC10H21SOCH3 system (III). Furthermore, the absolute values of molecular interaction parameters in the micelles (βm) and in the surface layers (βs) of the three systems are in the following order: (I) ⪢ (II) ⪢ (III). Explanations for these findings are given.

The molecular mechanism and thermodynamics of the interactions between plasmid DNA and cationic surfactants were investigated by isothermal titration calorimetry (ITC), dynamic light scattering, surface tension measurements, and UV spectroscopy. The cationic surfactants studied include benzyldimethyldodecylammonium chloride, benzyldimethyltetradecylammonium chloride, cetylpyridinium chloride, and cetyltrimethylammonium chloride. The results indicate a critical aggregation concentration (cac) of a surfactant: above the cac the surfactant forms aggregates with plasmid DNA; below the cac, however, there is no detectable interaction between DNA and surfactant. Surfactants with longer hydrocarbon chains have smaller cac, indicating that hydrophobic interaction plays a key role in DNA-surfactant complexation. Moreover, an increase in ionic strength (I) increases the cac but decreases the critical micellization concentration (cmc). These opposite effects lead to a critical ionic strength (I(c)) at which cac = cmc; when II(c), caccmc; when II(c), DNA does not form complexes with surfactant micelles. In the interaction DNA exhibits a pseudophase property as the cac is a constant over a wide range of DNA concentrations. ITC data showed that the reaction is solely driven by entropy because both deltaH(o) (approximately 2-6 kJ mol(-1)) and deltaS(o) (approximately 70-110 J K(-1) mol(-1)) have positive values. In the complex, the molar ratio of DNA phosphate to surfactant is in the range of 0.63-1.05. The reaction forms sub-micrometer-sized primary particles; those aggregate at high surfactant concentrations. Taken together, the results led to an inference that there is no interaction between surfactant monomers and DNA molecules and demonstrated that DNA-cationic surfactant interactions are mediated by the hydrophobic interactions of surfactant molecules and counterion binding of DNA phosphates to the cationic surfactant aggregates.

We have evaluated the stability profiles of adenovirus type-5 (Ad5)-based vaccine formulations to identify liquid formulations that are stable during long-term storage at 4 degrees C. By identifying the major physiochemical inactivation pathway(s) during storage, formulations of Ad5 were designed with specific pharmaceutical excipients leading to greatly enhanced stability. For example, results indicate that Ad5 is stabilized by non-ionic surfactants and cryoprotectants as well as excipients known to inhibit free-radical oxidation. A non-ionic surfactant is necessary to prevent adsorption of adenovirus to glass surfaces during storage, and a cryoprotectant is needed to prevent freeze-thaw-induced virus inactivation. In a base formulation (A105) containing sucrose as the cryoprotectant and polysorbate-80 as the non-ionic surfactant, metal-ion catalyzed free-radical oxidation is an important mechanism of Ad5 inactivation. The free-radical oxidation inhibitors ethanol and histidine, combined with the metal-ion chelator ethylenediaminetetraacetic acid (EDTA), were determined to be effective stabilizers of Ad5. Arrhenius plots of stability data are consistent with a first-order inactivation mechanism with apparent activation energies for virus inactivation of 26.5 +/- 0.9 and 28.7 +/- 0.6 kcal/mol in the absence and presence of free-radical oxidation inhibitors, respectively. Optimization of formulation pH, as well as the EDTA and ethanol concentrations, allowed for the identification of formulations that further enhanced long-term storage stability. For example, Ad5 in an optimized liquid formulation (A195) lost

We describe the physiochemical characterization and immunological evaluation of plasmid DNA vaccine formulations containing a nonionic triblock copolymer adjuvant (CRL1005) in the presence and absence of a cationic surfactant, benzalkonium chloride (BAK). CRL1005 forms particles of 1-10 microns upon warming above its phase-transition temperature (approximately 6-8 degrees C) and the physical properties of the particles are altered by BAK. DNA/CRL1005 vaccines formulated with and without BAK were evaluated in rhesus macaques to determine the effect of CRL1005 and BAK on the ability of plasmid DNA to induce a cellular immune response. Immunogenicity results indicate that the addition of CRL1005 to human immunodeficiency virus-1 gag plasmid DNA formulated in phosphate-buffered saline leads to an enhancement in the gag-specific cellular immune response. Moreover, the addition of BAK to human immunodeficiency virus-1 gag plasmid DNA/CRL1005 formulations produces an additional enhancement in gag-specific cellular immunity. In vitro characterization studies of DNA/CRL1005 formulations indicate no detectable binding of DNA to CRL1005 particles in the absence of BAK, suggesting that the enhancement of cellular immunity induced by DNA/CRL1005 formulations is not due to enhanced DNA delivery. In the presence of BAK, however, results indicate that BAK binds to CRL1005 particles, producing cationic microparticles that bind DNA through electrostatic interactions. If BAK is present at the phase-transition temperature, it reduces the particle size from approximately 2 microns to approximately 300 nm, presumably by binding to hydrophobic surfaces during particle formation. Zeta potential measurements indicate that the surface charge of CRL1005-BAK particles changes from positive to negative upon DNA binding, and DNA bound to the surface of CRL1005-BAK particles was visualized by fluorescence microscopy. These results indicate that the addition of BAK to DNA/CRL1005 formulations leads to the formation of approximately 300 nm CRL1005-BAK-DNA particles that enhance the cellular immune response in rhesus monkeys.

Cellular immune responses, particularly those associated with CD3 + CD8 + cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5- gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4 + and CD8 + T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5- gag -induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5- gag . The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5- gag . These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

Cellular signaling mediated by inositol (1,4,5)trisphosphate (Ins(1, 4,5)P(3)) results in oscillatory intracellular calcium (Ca(2+)) release. Because the amplitude of the Ca(2+) spikes is relatively invariant, the extent of the agonist-mediated effects must reside in their ability to regulate the oscillating frequency. Using electroporation techniques, we show that Ins(1,4,5)P(3), Ins(1,3,4, 5)P(4), and Ins(1,3,4,6)P(4) cause a rapid intracellular Ca(2+) release in resting HeLa cells and a transient increase in the frequency of ongoing Ca(2+) oscillations stimulated by histamine. Two poorly metabolizable analogs of Ins(1,4,5)P(3), Ins(2,4,5)P(3), and 2,3-dideoxy-Ins(1,4,5)P(3), gave a single Ca(2+) spike and failed to alter the frequency of ongoing oscillations. Complete inhibition of Ins(1,4,5)P(3) 3-kinase (IP3K) by either adriamycin or its specific antibody blocked Ca(2+) oscillations. Partial inhibition of IP3K causes a significant reduction in frequency. Taken together, our results indicate that Ins(1,3,4,5)P(4) is the frequency regulator in vivo, and IP3K, which phosphorylates Ins(1,4, 5)P(3) to Ins(1,3,4,5)P(4), plays a major regulatory role in intracellular Ca(2+) oscillations.

To investigate the mechanism by which the tryptophan synthase beta subunit accelerates the cleavage of the indole-3-glycerol phosphate catalyzed by the alpha subunit (alpha reaction), kinetic experiments were carried out with wild-type and mutant forms of the alpha 2 beta 2 complex. Previous studies indicate that this activation can be attributed to the conformational changes associated with the formation of a Schiff base between aminoacrylate and pyridoxal phosphate at the beta site. To test this hypothesis, we investigated a mutant form of the alpha 2 beta 2 complex having the lysine-87 of its beta subunits replaced by threonine. The mutant alpha 2 beta 2 complex (K87T) exhibits normal activity for the alpha reaction but fails to catalyze formation of L-tryptophan from L-serine and indole (beta reaction). However, the mutant enzyme can form a Schiff base intermediate with L-serine at the beta site. Using a "chemical rescue" method, we converted K87T L-serine intermediate to an aminoacrylate intermediate. Steady-state kinetic studies reveal that the aminoacrylate derivative exhibits a 7-fold enhancement in kcat/Km for the alpha reaction relative to that of the L-serine derivative of the mutant or the wild-type enzyme in the absence of L-serine. Rapid kinetic data show that the aminoacrylate derivative of the mutant enzyme exhibits a 6-fold increase in the rate constant for the indole-3-glycerol phosphate cleavage reaction. In addition, rate constants for the reverse reaction and product release steps are also altered. Together, these changes lead to a decrease in Km and an increase in kcat.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and its incidence continues to increase. Although there are approved therapies for HCC, the prognosis remains poor. The 5-year survival rate is < 5%. Nucleic acid-based therapeutics such as RNAi may improve patient outcome. Nevertheless, target identification/validation for HCC and delivery of siRNA are major challenges facing the development of HCC-specific RNAi based therapeutics. To enable target validation in vivo using RNAi, we have developed nanolipid siRNA formulations for the in vivo delivery of siRNA to orthotopic tumors. PK/PD studies in model systems demonstrate that up to 80% knockdown of target protein is achievable within tumors and that significant knockdown persists for at least 10 days following a single dose of formulated siRNA. Efficacy studies using similar formulations of siRNAs against potential HCC tumor targets, including mTOR, will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1646. doi:10.1158/1538-7445.AM2011-1646

Adenoviral vectors (Ad), known for its efficiency in gene delivery, are currently used for Merck's developmental HIV vaccine. A formulation has been developed to stabilize the adenovirus serotype 5 (Ad5) at 2-8|[deg]|C storage. In support of formulation development, we have used QPCR, in vitro antigen expression (IVAE), flow cytometry, microscopy, an enzymatic assay, and animal study to investigate the inactivation pathway of Ad5 vector containing HIV-1 gag gene (Ad5gag) induced by thermal stress (15-37|[deg]|C). The infectivity of Ad5gag in the permissive HEK-293T cell line was determined by QPCR, and antigen expression of Ad5gag in the nonpermissive A549 cell line was determined by ELISA. We found that infectivity loss of Ad5gag observed by QPCR is consistent with less antigen expression shown from IVAE, and the reduced immunogenicity demonstrated with the mice study; but the infectivity loss has no effect on CAR-mediated Ad5gag binding to the HEK-293T cells, internalization of the bound Ad5gag, and intercellular trafficking of Ad5gag to the membrane of cell nucleus. However, we found that the reduced Ad cysteine protease activity is highly correlated to Ad5gag infectivity loss in the HEK-293T cell line and to a decrease in HIV-1 gag antigen expression in A549 cells. These results suggest that the inactivation of Ad5gag cysteine protease is, at least partially, responsible for thermal stress-induced infectivity loss of Ad5gag. Therefore preserving Ad5 cysteine protease activity may be essential to enhance the stability of Ad5 viral vectors.

Janus kinase 3 (JAK3), a member of the Janus family protein-tyrosine kinases, is expressed in mast cells, and its enzymatic activity is enhanced by IgE receptor/FcepsilonRI cross-linking. Selective inhibition of JAK3 in mast cells with 4-(4'-hydroxylphenyl)-amino-6, 7-dimethoxyquinazoline) (WHI-P131) blocked the phospholipase C activation, calcium mobilization, and activation of microtubule-associated protein kinase after lgE receptor/FcepsilonRI cross-linking. Treatment of IgE-sensitized rodent as well as human mast cells with WHI-P131 effectively inhibited the activation-associated morphological changes, degranulation, and proinflammatory mediator release after specific antigen challenge without affecting the functional integrity of the distal secretory machinery. In vivo administration of the JAK3 inhibitor WHI-P131 prevented mast cell degranulation and development of cutaneous as well as systemic fatal anaphylaxis in mice at nontoxic dose levels. Thus, JAK3 plays a pivotal role in IgE receptor/FcepsilonRI-mediated mast cell responses, and targeting JAK3 with a specific inhibitor, such as WHI-P131, may provide the basis for new and effective treatment as well as prevention programs for mast cell-mediated allergic reactions.

The surface adsorption and micelle formation of the aqueous solutions of alkyl (C8 and C10) methylsulfoxide [A]-ionic surfactant (C7F15COONa [I], C10H21SO4Na [II] or C10H21N(CH3)3Br [III]) mixtures were investigated by the surface tension method. It was found that there is significant synergism in the surface activity of the A-I and II systems, but not in that of the A-III system. This is shown by the fact that we can obtain the γCMC and CMC minima in the A-I system and the CMC minimum in the A-II system, but neither of these in the A-III system; furthermore, the negative values of βσ and βm are much larger in the A-I and II systems than in the A-III system.