Therapeutic implications of Src independent calcium mobilization in diffuse large B-cell lymphoma.

We report that 38% of primary large B-cell lymphoma (DLBCL) tested expressed active Src family kinases, which are targeted by dasatinib. The expression of active Src family of kinases (SFK) in primary DLBCL tumors correlated with unfavorable prognostic markers such as Ki67 and Mum1. Using four DLBCL cell lines we found that: (1) sensitivity to dasatinib (but not imatinib) varied 400-fold; (2) dasatinib resistance was associated with distinct signaling profiles downstream of BCR activation. In particular, although Src family kinase phosphorylation was inhibited by 100-150 nM dasatinib in all cell lines, this failed to inhibit BCR-mediated Blnk phosphorylation, calcium signaling and proliferation in a dasatinib resistant cell line.

Photophysics of dopamine-modified quantum dots and effects on biological systems.

Semiconductor quantum dots (QDs) have been widely used for fluorescent labelling. However, their ability to transfer electrons and holes to biomolecules leads to spectral changes and effects on living systems that have yet to be exploited. Here we report the first cell-based biosensor based on electron transfer between a small molecule (the neurotransmitter dopamine) and CdSe/ZnS QDs. QD-dopamine conjugates label living cells in a redox-sensitive pattern: under reducing conditions, fluorescence is only seen in the cell periphery and lysosomes. As the cell becomes more oxidizing, QD labelling appears in the perinuclear region, including in or on mitochondria. With the most-oxidizing cellular conditions, QD labelling throughout the cell is seen. Phototoxicity results from the creation of singlet oxygen, and can be reduced with antioxidants. This work suggests methods for the creation of phototoxic drugs and for redox-specific fluorescent labelling that are generalizable to any QD conjugated to an electron donor.

Constitutive activation of extracellular signal-regulated kinase predisposes diffuse large B-cell lymphoma cell lines to CD40-mediated cell death.

CD40 promotes survival, proliferation, and differentiation of normal B cells but can cause activation-induced cell death in malignant B lymphocytes. CD40 ligand and anti-CD40 antibodies have been used successfully to induce apoptosis in lymphoma lines both in vitro and in xenograft tumor models. Although this makes CD40 an attractive target for antitumor therapies, the response of malignant B cells to CD40 signaling is variable, and CD40 stimulation can enhance proliferation and can increase chemoresistance in some cell lines. It would therefore be useful to identify markers that predict whether a specific cell line or tumor will undergo apoptosis when stimulated with CD40 and to identify targets downstream of CD40 that affect only the apoptotic arm of CD40 signaling. We have analyzed gene expression patterns in CD40-sensitive and CD40-resistant diffuse large B-cell lymphoma (DLBCL) cell lines to identify signaling pathways that are involved in CD40-mediated apoptosis. CD40-resistant lines expressed pre-B-cell markers, including RAG and VPREB, whereas CD40-sensitive cells resembled mature B cells and expressed higher levels of transcripts encoding several members of the CD40 signaling pathway, including LCK and VAV. In addition, CD40-sensitive DLBCL cell lines also displayed constitutive activation of extracellular signal-regulated kinase (ERK) and failed to undergo apoptosis when ERK phosphorylation was inhibited. In contrast, CD40-resistant lines showed no constitutive activation of ERK and no increase in ERK activity in response to CD40 stimulation. Our results suggest that constitutive activation of ERK may be required for death signaling by CD40.