Targeted RNA interference of phosphatidylinositol 3-kinase p110-beta induces apoptosis and proliferation arrest in endometrial carcinoma cells.

Phosphatidylinositol 3-kinase (PI3K) signalling plays a pivotal role in intracellular signal transduction pathways involved in cell growth, cellular transformation, and tumourigenesis. PI3K is overexpressed in many human cancers, including endometrial carcinomas, one of the most common female genital tract malignancies. Here, we used small interfering RNA (siRNA) targeted to PI3K p110-beta to determine whether inhibition of the beta isoform could be a potential therapeutic target for endometrial carcinoma. In this study, treatment of HEC-1B endometrial cancer cells with PI3K p110-beta-specific siRNA resulted in increased apoptosis and decreased tumour cell proliferation. Depletion of PI3K p110-beta decreased the protein levels of AKT1, AKT2, pAKT, and mTOR-downstream targets of PI3K. Knock-down of PI3K p110-beta by siRNA also induced decreased expression of cyclin E and Bcl-2, suggesting that PI3K p110-beta stimulates tumour growth, at least in part by regulating cyclin E and Bcl-2. Thus, our results indicate that siRNA-mediated gene silencing of PI3K p110-beta may be a useful therapeutic strategy for endometrial cancers overexpressing PI3K p110-beta.

Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients.

4-1BB is expressed on activated CD4(+) and CD8(+) T cells; its ligand, 4-1BB ligand is expressed on APCs. Despite expression on both T cell subpopulations, 4-1BB has been reported to predominantly affect CD8(+) T cell responses. By quantifying graft-vs-host disease alloresponses in vivo, we demonstrate that both CD4(+) and CD8(+) T cell-mediated alloresponses are regulated by 4-1BB/4-1BB ligand interactions to approximately the same extent. 4-1BB receptor-facilitated CD4(+) T cell-mediated alloresponses were partly CD28 independent. In two distinct marrow graft rejection systems, host CD8(+) and CD4(+) T cells each separately contributed to host anti-donor T cell-mediated allograft rejection. alpha 4-1BB mAb increased the graft-vs-leukemia effect of a suboptimal number of donor splenocytes given later post bone marrow transplantation by bolstering allogeneic responses resulting in leukemia elimination. In summary, 4-1BB ligation is a potent regulator of CD4(+) and CD8(+) T cell-mediated allogeneic responses in vivo. Modifying the ligation of 4-1BB represents a new approach to altering the graft-vs-host disease and graft-vs-leukemia effects of allogeneic T cells post bone marrow transplantation.

Cell-penetrating inhibitors of calpain block both membrane fusion and filamin cleavage in chick embryonic myoblasts.

Benzyloxycarbonyl(Z)-Leu-nLeu-H (calpeptin) and Z-Leu-Met-H, cell-penetrating inhibitors of calpain, were found to block myoblast fusion without any effect on cell proliferation and alignment along their bipolar axis. They also inhibited the accumulation of creatine kinase during myogenesis. These effects were dose-dependent, and could be reversed upon removal of the drug from the culture medium. Furthermore, treatment of the inhibitors prevented the hydrolysis of filamin, which is sensitive to cleavage by calpain in vitro and interferes with actin-myosin filament formation by cross-linking F-actin molecules. On the other hand, leupeptin, which can also inhibit calpain in vitro but can not penetrate into cells, showed little or no effect on both myoblast fusion and filamin clevage. These results suggest that calpain may play an important role in cytoskeletal reorganization that is requisite for myoblast fusion. The role of calpain on the expression of muscle-specific proteins remains unknown.

Increase in the level of m-calpain correlates with the elevated cleavage of filamin during myogenic differentiation of embryonic muscle cells.

The activity of Ca(2+)-activated proteinase requiring millimolar Ca2+ (m-calpain) was found to increase dramatically in cultured chick embryonic myoblasts during the early period of myogenic differentiation. Furthermore, the protein level of m-calpain also markedly increased in parallel with the rise in its activity, and both remained elevated thereafter. On the other hand, the activity level of calpastatin, an endogenous inhibitor of the proteinase, remained similar during the entire period of the culture. In addition, the activity of Ca(2+)-activated proteinase requiring micromolar Ca2+ (mu-calpain) was not detected in either proliferating or differentiated myoblasts. Thus, the overall capacity of Ca(2+)-dependent proteolysis is likely to increase in differentiating myoblasts and should be contributed by m-calpain. Filamin (250 kDa), that is known to facilitate actin microfilament assembly and interfere with actin-myosin filament formation, was found to be cleaved in cultured myoblasts to 240 kDa products. This filamin-cleavage occurred in a manner similar to the in vitro cleavage of the cytoskeletal protein by the purified m-calpain. Moreover, the filamin-cleavage was most evident at the period of the cell fusion. Thus, it seems likely that the in vivo cleavage of filamin is mediated by m-calpain. These results suggest that m-calpain may play an important role in cytoskeletal reorganization that is requisite for myoblast fusion.

Developmental regulation of proteolytic activities and subunit pattern of 20 S proteasome in chick embryonic muscle.

The proteolytic activities of the 20 S proteasome were found to change in their levels during the development of chick embryonic muscle. The peptide-cleaving activities against N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-beta-naphthylamide gradually decreased with the time of development. On the other hand, the casein-degrading activity in the presence of poly-L-lysine markedly increased from embryonic day 11 and reached a maximal level by day 17. These changes appeared to be tissue-specific because little or no change in any of the proteolytic activities was observed with developing embryonic brain, while dramatic alterations occurred in the extents of the peptide hydrolyses in liver. Furthermore, a number, but not all, of the proteasome subunits in embryonic muscle were changed in their amounts during the development. These results suggest that the alterations in the proteasome activities and subunit pattern are developmentally regulated and may be correlated.

Preferential degradation of the KMnO4-oxidized or N-ethylmaleimide-modified form of sarcoplasmic reticulum ATPase by calpain from chick skeletal muscle.

KMnO4 and N-ethylmaleimide at low concentrations (i.e., below 0.2 and 1.5 mM, respectively) are known to interact specifically with four to five sulfhydryl residues per Ca2+/Mg2(+)-ATPase molecule in sarcoplasmic reticulum. Purified calpain preferentially hydrolyzes the ATPase that was treated with either agent but not the native form of the enzyme. Exposure to each agent with increasing concentrations results in a greater loss of the ATPase activity and renders the enzyme more susceptible to calpain. In addition, beta,r-methylene-ATP, when added during the treatment of KMnO4 or N-ethylmaleimide, can partially protect the ATPase against the degradation. These results suggest that the covalent modification at the specific sulfhydryl residues in sarcoplasmic reticulum ATPase may mark the enzyme for degradation by intracellular proteinases, such as calpain.

Purothionin from wheat endosperm reversibly blocks myogenic differentiation of chick embryonic muscle cells in culture.

Purothionin from wheat endosperm is a cysteine-rich, basic polypeptide of about 5000 Da, which modifies membrane permeability of cultured mammalian cells. This peptide was found to block fusion of chick embryonic muscle cells in culture but allows proliferation and alignment. A purothionin concentration of 6 micrograms/ml (1.2 microM) was necessary for the complete prevention of myotube formation. Under similar conditions, incorporation of [35S]methionine occurred normally but the synthesis of muscle-specific proteins including creatine kinase and acetylcholine receptor was strongly inhibited. In addition, purothionin blocked the uptake of 86Rb+, immediately after its addition to the cultured myoblasts. No such effects were found with the purothionin chemically modified with acetic or succinic anhydride. Thus, the basic residues in purothionin appear to be associated with the inhibition of myogenic differentiation. These results suggest that purothionin exerts its regulatory effect on the transition from proliferative to differentiative myoblasts by interfering with membrane permeability or intercellular contact and recognition, which are necessary for the initiation of muscle differentiation.