Glucose-dependent de novo lipogenesis in B lymphocytes: a requirement for atp-citrate lyase in lipopolysaccharide-induced differentiation.

Bacterially derived lipopolysaccharide (LPS) stimulates naive B lymphocytes to differentiate into immunoglobulin (Ig)-secreting plasma cells. Differentiation of B lymphocytes is characterized by a proliferative phase followed by expansion of the intracellular membrane secretory network to support Ig production. A key question in lymphocyte biology is how naive B cells reprogram metabolism to support de novo lipogenesis necessary for proliferation and expansion of the endomembrane network in response to LPS. We report that extracellularly acquired glucose is metabolized, in part, to support de novo lipogenesis in response to LPS stimulation of splenic B lymphocytes. LPS stimulation leads to increased levels of endogenous ATP-citrate lyase (ACLY), and this is accompanied by increased ACLY enzymatic activity. ACLY produces cytosolic acetyl-CoA from mitochondrially derived citrate. Inhibition of ACLY activity in LPS-stimulated B cells with the selective inhibitor 2-hydroxy-N-arylbenzenesulfonamide (compound-9; C-9) blocks glucose incorporation into de novo lipid biosynthesis, including cholesterol, free fatty acids, and neutral and acidic phospholipids. Moreover, inhibition of ACLY activity in splenic B cells results in inhibition of proliferation and defective endomembrane expansion and reduced expression of CD138 and Blimp-1, markers for plasma-like B cell differentiation. ACLY activity is also required for LPS-induced IgM production in CH12 B lymphoma cells. These data demonstrate that ACLY mediates glucose-dependent de novo lipogenesis in response to LPS signaling and identify a role for ACLY in several phenotypic changes that define plasma cell differentiation.

Cyclin D3 is dispensable for human diffuse large B-cell lymphoma survival and growth: evidence for redundancy with cyclin E.

Genomic changes disrupting the expression of cyclin D3 are associated with aberrant growth of several human B-lymphoid malignancies. We demonstrate that the human diffuse large B-cell lymphoma (DLBCL), OCI-LY18 (LY18) expresses cyclin D3 but not cyclins D1 and D2. RNA interference was used to deplete cyclin D3 from LY18 cells. Surprisingly, knockdown of cyclin D3 did not inhibit pRb phosphorylation on cdk4/6- and cdk2-specific residues or measurably affect viability and proliferation. These results suggest that cyclin D3 is dispensable in LY18 cell proliferation and survival. Similar results were obtained following depletion of cyclin E. By contrast, combined knockdown of cyclins D3 and E had substantial consequences leading to G(1)-phase arrest and inhibition of proliferation. Whereas cell cycle distribution was not affected following individual depletion of cdk4, cdk6 or cdk2, the combined knockdown of cdk4 and cdk6 led to accumulation of LY18 cells in G(1)-phase of the cell cycle and inhibition of proliferation. Likewise treatment of LY18 cells with 2-Bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, a selective inhibitor of cdk4/6, led to inhibition of proliferation. Taken together, these results uncover a built-in redundancy with cyclins D3 and E for G(1)-S progression. Moreover these findings highlight the rationale for simultaneous disruption of cdk4/6 as a potential therapeutic cancer strategy.

Interaction between carbon nanotubes and mammalian cells: characterization by flow cytometry and application.

We show herein that CNT-cell complexes are formed in the presence of a magnetic field. The complexes were analyzed by flow cytometry as a quantitative method for monitoring the physical interactions between CNTs and cells. We observed an increase in side scattering signals, where the amplitude was proportional to the amount of CNTs that are associated with cells. Even after the formation of CNT-cell complexes, cell viability was not significantly decreased. The association between CNTs and cells was strong enough to be used for manipulating the complexes and thereby conducting cell separation with magnetic force. In addition, the CNT-cell complexes were also utilized to facilitate electroporation. We observed a time constant from CNT-cell complexes but not from cells alone, indicating a high level of pore formation in cell membranes. Experimentally, we achieved the expression of enhanced green fluorescence protein by using a low electroporation voltage after the formation of CNT-cell complexes. These results suggest that higher transfection efficiency, lower electroporation voltage, and miniaturized setup dimension of electroporation may be accomplished through the CNT strategy outlined herein.

Cutting edge: IL-4-mediated protection of primary B lymphocytes from apoptosis via Stat6-dependent regulation of glycolytic metabolism.

IL-4 prevents the death of naive B lymphocytes through the up-regulation of antiapoptotic proteins such as Bcl-x(L). Despite studies implicating glucose utilization in growth factor-dependent survival of hemopoietic cells, the role of glucose energy metabolism in maintaining B cell viability by IL-4 is unknown. We show that IL-4 triggers glucose uptake, Glut1 expression, and glycolysis in splenic B cells; this is accompanied by increased cellular ATP. Glycolysis inhibition results in apoptosis, even in the presence of IL-4. IL-4-induced glycolysis occurs normally in B cells deficient in insulin receptor substrate-2 or the p85alpha subunit of PI3K and is not affected by pretreatment with PI3K or MAPK pathway inhibitors. Stat6-deficient B cells exhibit impaired IL-4-induced glycolysis. Cell-permeable, constitutively active Stat6 is effective in restoring IL-4-induced glycolysis in Stat6-deficient B cells. Therefore, besides controlling antiapoptotic proteins, IL-4 mediates B cell survival by regulating glucose energy metabolism via a Stat6-dependent pathway.

Disruption of cyclin D3 blocks proliferation of normal B-1a cells, but loss of cyclin D3 is compensated by cyclin D2 in cyclin D3-deficient mice.

Peritoneal B-1a cells differ from splenic B-2 cells in the molecular mechanisms that control G(0)-S progression. In contrast to B-2 cells, cyclin D2 is up-regulated in a rapid and transient manner in phorbol ester (PMA)-stimulated B-1a cells, whereas cyclin D3 does not accumulate until late G(1) phase. This nonoverlapping expression of cyclins D2 and D3 suggests distinct functions for these proteins in B-1a cells. To investigate the contribution of cyclin D3 in the proliferation of B-1a cells, we transduced p16(INK4a) peptidyl mimetics (TAT-p16) into B-1a cells before cyclin D3 induction to specifically block cyclin D3-cyclin-dependent kinase 4/6 assembly. TAT-p16 inhibited DNA synthesis in B-1a cells stimulated by PMA, CD40L, or LPS as well as endogenous pRb phosphorylation by cyclin D-cyclin-dependent kinase 4/6. Unexpectedly, however, cyclin D3-deficient B-1a cells proliferated in a manner similar to wild-type B-1a cells following PMA or LPS stimulation. This was due, at least in part, to the compensatory sustained accumulation of cyclin D2 throughout G(0)-S progression. Taken together, experiments in which cyclin D3 was inhibited in real time demonstrate the key role this cyclin plays in normal B-1a cell mitogenesis, whereas experiments with cyclin D3-deficient B-1a cells show that cyclin D2 can compensate for cyclin D3 loss in mutant mice.