Recent advances in the development of anticancer agents targeting cell death inhibitors in the Bcl-2 protein family.

Hematopoietic malignancies frequently are characterized by defects in apoptosis signaling. This renders the malignant cells resistant to endogenous apoptotic stimuli, as well as exogenous stimuli, such as chemotherapy drugs and radiation. The defective apoptosis seen in human cancers often results from overexpression of antiapoptotic proteins in the Bcl-2 protein family, particularly Bcl-2 and Bcl-X(L). A great deal of effort is currently aimed at developing novel agents to inhibit the expression or function of these proteins. Antisense agents directed against Bcl-2 mRNA are showing considerable promise in clinical trials. In addition, detailed knowledge of the structures of Bcl-2 and Bcl-X(L), coupled with high-throughput and computer-assisted screening of chemical libraries, has led to the identification of a number of short peptides and small organic molecules capable of inhibiting Bcl-2 and Bcl-X(L) function. These newly described agents hold considerable promise for enhancing the chemo- and radiation sensitivities of Bcl-2- and Bcl-X(L)-overexpressing cancers. This review will highlight recent advances in the development and testing of agents targeting cell death inhibitors in the Bcl-2 protein family.

Purification and properties of four monocot lectins from the family Araceae.

Four new monocot lectins from the tubers of araceous plants, namely, Arisaema consanguineum Schott (ACA), A. curvatum Kunth (ACmA) and Sauromatum guttatum Schott (SGA) from the tribe Areae, and Gonatanthus pumilus D. Don (GPA) from the tribe Colocasieae have been purified by affinity chromatography on asialofetuin-linked amino activated silica beads. These lectins possess similar physicochemical and biological properties. All the lectins gave a single peak on HPLC size exclusion and cation exchange columns, and a single band on PAGE, (pH 4.5). In SDS-PAGE, all the lectins gave a single band corresponding to a subunit of M(r) 1,3000. All the lectins yielded multiple peaks on anion-exchange column, multiple bands on non-denatured PAGE (pH 8.3) and a family of bands on isoelectric focusing. The lectins agglutinate rabbit, rat and sheep red blood cells (RBCs) but are inactive towards human ABO erythrocytes. The haemagglutination activity of these lectins is inhibited by asialofetuin only, while simple sugars/derivatives including chitin, porcine mucin and fetuin did not react. In serological studies against rabbit anti-SGA serum, all four lectins produced immunoprecipitin lines. The lectins within each tribe were identical but the lectins belonging to the tribe Areae were only partially identical to the lectins from the tribe Colocasieae.

Amaranthus hypochondriacus and A. tricolor lectins: isolation and characterization.

Asialofetuin-linked amino activated silica was used for the affinity purification of lectins from Amaranthus hypochondriacus Linn (AHL) and A. tricolor Linn (ATL). Like a few other Amaranthus lectins, these lectins were also inhibited by N-acetyl-D-galactosamine, fetuin and asialofetuin; they agglutinated human and different animal erythrocytes. The purified lectins yielded a single band on PAGE pH 8.3, pH 4.5 and SDS-PAGE, pH 8.3. These also gave a single peak in gel exclusion on Biogel P-200, HPLC 300 SW and cation exchange columns. However, both lectins gave multiple peaks in anion exchange column and multiple bands in isoelectric focusing. AHL and ATL are dimeric proteins in which the subunits having M(r) 29,000 and 39,000, respectively, are not held together by disulphide linkages. The pure lectins are glycoproteins and do not require Ca2+, Mn2+ and Mg2+ for their agglutination activity.

New lymphocyte stimulating monocot lectins from family Araceae. II.

Two lectins purified from the tubers of Arisaema consanguineum Schott (ACA) and A. curvatum Kunth (ACmA) belonging to the monocot family Araceae were mitogenic for human peripheral blood mononuclear cells (PBMC) in the [3H]-thymidine uptake assay. ACA and ACmA had an optimum stimulatory concentration of 10-25 micrograms/ml and 50-100 micrograms/ml, respectively, as observed in PBMC from five different individuals. The mitogenic response of PBMC was inhibitable in a dose-dependent manner by asialofetuin. The lectins were T-cell specific, and stimulation kinetic studies using ACA and ACmA showed that they induce maximum thymidine uptake in PBMC at day 4 and 3, respectively.

New lymphocyte stimulating monocot lectins from family Araceae.

Three monocot lectins from underground tubers of plants belonging to the family Araceae were investigated for their mitogenic potential towards human peripheral blood lymphocytes. All the three lectins turned out to be potent mitogens in the [3H]-thymidine uptake assay. Gonatanthus pumilus lectin was mitogenic at an optimum concentration of 25 micrograms/ml while Alocasia indica and Sauromatum guttatum lectins were most effective at a concentration of 50 micrograms/ml. [3H]-thymidine incorporation studies further revealed that the lectins were T-cell mitogens and did not induce any appreciable DNA synthesis in B-enriched lymphocytes. The proliferation kinetic studies detected maximum incorporation on day 3 and the mitogenic response was shown to be inhibited by asialofetuin in a concentration-dependent manner.

Ionizing radiation exposure results in up-regulation of Ku70 via a p53/ataxia-telangiectasia-mutated protein-dependent mechanism.

Genome damaging events, such as gamma-irradiation exposure, result in the induction of pathways that activate DNA repair mechanisms, halt cell cycle progression, and/or trigger apoptosis. We have investigated the effects of gamma-irradiation on cellular levels of the Ku autoantigens. Ku70 and Ku80 have been shown to form a heterodimeric complex that can bind tightly to free DNA ends and activate the protein kinase DNA-PKcs. We have found that irradiation results in an up-regulation of cellular levels of Ku70, but not Ku80, and that this enhanced level of Ku70 accumulates within the nucleus. Further, we uncovered that the postirradiation up-regulation of Ku70 utilizes a mechanism that is dependent on both p53 and damage response protein kinase ATM (ataxia-telangiectasia-mutated); however, the activation of DNA-PK does not require Ku70 up-regulation. These findings suggest that Ku70 up-regulation provides the cell with a means of assuring either proper DNA repair or an appropriate response to DNA damage independent of DNA-PKcs activation.

Regulation of DNA-dependent protein kinase activity by ionizing radiation-activated abl kinase is an ATM-dependent process.

Ionizing radiation (IR) treatment results in activation of the nonreceptor tyrosine kinase c-Abl because of phosphorylation by ATM. In vitro evidence indicates that DNA-dependent protein kinase (DNA-PK) can also phosphorylate and thus potentially activate Abl kinase activity in response to IR exposure. To unravel the role of ATM and DNA-PK in the activation of Abl, we assayed Abl, ATM, and DNA-PK activity in ATM- and DNA-PKcs-deficient cells after irradiation. Our results show that despite the presence of higher than normal levels of DNA-PK kinase activity, c-Abl fails to become activated after IR exposure in ATM-deficient cells. Conversely, normal activation of both ATM and c-Abl occurs in DNA-PKcs-deficient cells, indicating that ATM but not DNA-PK is required for activation of Abl in response to IR treatment. Moreover, activation of Abl kinase activity by IR correlates well with activation of ATM activity in all phases of the cell cycle. These results indicate that ATM is primarily responsible for activation of Abl in response to IR exposure in a cell cycle-independent fashion. Examination of DNA-PK activity in response to IR treatment in Abl-deficient cells expressing mutant forms of Abl or in normal cells exposed to an inhibitor of Abl suggests an in vivo role for Abl in the down-regulation of DNA-PK activity. Collectively, these results suggest a convergence of the ATM and DNA-PK pathways in the cellular response to IR through c-Abl kinase.