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The HTLV-1 transactivator protein Tax is essential for malignant transformation of CD4 T cells, ultimately leading to adult T-cell leukemia/lymphoma (ATL). Malignant transformation may involve development of apoptosis resistance. In this study we investigated the molecular mechanisms by which HTLV-1 Tax confers resistance toward CD95-mediated apoptosis. We show that Tax-expressing T-cell lines derived from HTLV-1–infected patients express elevated levels of c-FLIPL and c-FLIPS. The levels of c-FLIP correlated with resistance toward CD95-mediated apoptosis. Using an inducible system we demonstrated that both resistance toward CD95-mediated apoptosis and induction of c-FLIP are dependent on Tax. In addition, analysis of early cleavage of the BH3-only Bcl-2 family member Bid, a direct caspase-8 substrate, revealed that apoptosis is inhibited at a CD95 death receptor proximal level in Tax-expressing cells. Finally, using siRNA we directly showed that c-FLIP confers Tax-mediated resistance toward CD95-mediated apoptosis. In conclusion, our data suggest an important mechanism by which expression of HTLV-1 Tax may lead to immune escape of infected T cells and, thus, to persistent infection and transformation.

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34(+) hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34(+) HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60(+), yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60(+)), with variable yield (6 to500 TRA-1-60(+) iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP-reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34(+) HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14(+) monocytes do not yield iPSC colonies under these reprogramming conditions.

Herbs have successfully been used in traditional Chinese medicine for centuries. However, their curative mechanisms remain largely unknown. In this study, we show that Wogonin, derived from the traditional Chinese medicine Huang-Qin (Scutellaria baicalensis Georgi), induces apoptosis in malignant T cells in vitro and suppresses growth of human T-cell leukemia xenografts in vivo. Importantly, Wogonin shows almost no toxicity on T lymphocytes from healthy donors. Wogonin induces prolonged activation of PLCgamma1 via H(2)O(2) signaling in malignant T cells, which leads to sustained elevation of cytosolic Ca(2+) in malignant but not normal T cells. Subsequently, a Ca(2+) overload leads to disruption of the mitochondrial membrane. The selective effect of Wogonin is due to its differential regulation of the redox status of malignant versus normal T cells. In addition, we show that the L-type voltage-dependent Ca(2+) channels are involved in the intracellular Ca(2+) mobilization in T cells. Furthermore, we show that malignant T cells possess elevated amounts of voltage-dependent Ca(2+) channels compared with normal T cells, which further enhance the cytotoxicity of Wogonin for malignant T cells. Taken together, our data show a therapeutic potential of Wogonin for the treatment of hematologic malignancies.

The existence of an ecto-sialyltransferase (ecto-ST) on B lymphocytes with increasing activity at late maturation stages is shown using a novel flow cytometric enzyme assay. This ecto-ST is effective in reconstituting different surface glycoconjugates on desialylated B cells in the presence of exogenous CMP-NeuAc. We found that this ecto- ST is distinct in its activity from soluble ST released into the culture supernatant. Surface sialylation was independent of the amount of ST secreted into the culture supernatant and followed different kinetics than sialylation of exogenous substrate by soluble ST. Four human B-cell lines representing different maturation stages were analyzed for secreted and ecto-ST activity. The myeloma cell line U266 and the lymphoblastoid cell line JOK-1 showed higher activity of both ST forms than the acute lymphoblastic leukemia B-cell line Nalm-6. ST activity in culture supernatants of U266, JOK-1, and Nalm-6 cells consisted predominantly of the alpha 2,6 ST type with specificity for N- linked oligosaccharides. As an exception, the myeloma cell line IM-9, deficient of alpha 2,6 ST activity, secreted only small amounts of ST and showed low activity of ecto-ST. Sialylation of surface-expressed glycoconjugates by ecto-ST was measured by incubating B-cell lines in the presence of fluorescent CMP-sialic acid. Surface structures labeled with fluorescent sialic acid under this condition were visualized by confocal laser scanning microscopy and fluorescent label was quantitatively assessed by flow cytometric analysis on live cells. Incubation of cells in acidified culture medium, to release possibly receptor-bound ST, did not alter the intensity of cell surface sialylation. Inhibition of internalization and membrane traffic by various approaches (reduced incubation temperature and chloroquine or brefeldin A treatment) did not block surface sialylation. Together, these observations point to cell surface sialylation in B lymphocytes mediated by a cell surface-expressed ecto-ST distinct from the secreted ST form. On desialylated JOK-1 cells, ecto-ST in the presence of exogenous CMP-NeuAc was able to resialylate the B-cell surface sialoglycans CDw75 and HB-6 and major surface glycoproteins of B cells, such as HLA class I and II antigens, transferrin receptor, and surface IgM. In contrast, cell surface glycans of coincubated desialylated erythrocytes were not sialylated by the B-cell ecto-ST. Ecto-alpha 2,6 ST of B cells may be involved in the sialylation of distinct differentiation glycan antigens.