ALBERT

Description: Vascular endothelial cell growth factor (VEGF) is a multifunctional cytokine involved in tumor formation. In chronic lymphocytic leukemia (CLL), it is known that the malignant cells secrete VEGF and possess VEGF receptors. This suggests that an autocrine loop might be important in the pathogenesis of CLL. Here we show that, in patients with lymphadenopathy, autocrine VEGF and α4β1 integrin are involved in the chemokine-dependent motility of CLL cells on and through endothelium—processes important for the invasion of lymphoreticular tissues, a major determinant of disease outcome. In contrast, normal lymphocytes were not dependent on autocrine VEGF or α4β1 for either type of cell movement. Moreover, in contrast to normal B lymphocytes, CLL cells failed to cluster and activate αLβ2 in response to chemokines, unless VEGF receptor(s) and α4β1 were also engaged by their respective ligands. This is the first demonstration that autocrine VEGF is involved in CLL-cell motility, and that the αLβ2 on the malignant cells is functionally altered compared with that of normal B cells in not undergoing activation in response to chemokine alone. Given the importance of cell motility for tissue invasion, the present results provide a rationale for a trial of VEGF and α4 blockade in patients with CLL who have tissue disease. (Blood. 2005;105:4813-4819)

Description: Antisense oligodeoxynucleotides targeted to bcr-abl are potential ex vivo purging agents for use with autologous bone marrow transplantation in the treatment of chronic myeloid leukemia (CML). We investigated, in a cell-free system, the activity and nuclease resistance of phosphodiester, phosphorothioate, chimeric methylphosphonate/phosphodiester, and chimeric methylphosphonate/phosphorothioate antisense octadecamers directed against either b2a2 or b3a2 bcr-abl breakpoint RNAs. Certain chimeric compounds were shown to possess targeted activity broadly equal to the parent phosphodiester or phosphorothioate forms and greater resistance to the nucleases present in cell extracts. Selected chimeric structures were compared with phosphodiester and phosphorothioate analogues for antisense activity in human CML cells containing either b2a2 or b3a2 bcr-abl breakpoint mRNAs. We present results showing that all four structures can suppress bcr-abl mRNA level in vivo. The rank of in vivo activity is chimeric methylphosphonate/phosphodiester 〉 or = phosphodiester 〉 phosphorothioate 〉 methylphosphonate/phosphorothioate. We show that b2a2 breakpoint RNAs can be more effectively targeted than b3a2 sequence RNAs both in vitro and in vivo and suggest that RNA secondary structure may be a possible explanation for this phenomenon.

Description: Mcl-1 is a member of the Bcl-2 protein family, which has been shown to delay apoptosis in transfection and/or overexpression experiments. As yet no gene knockout mice have been engineered, and so there is little evidence to show that loss of Mcl-1 expression is sufficient to trigger apoptosis. U937 cells constitutively express the antiapoptotic protein Bcl-2; but during differentiation, in response to the phorbol ester PMA (phorbol 12 β-myristate 13 α-acetate), Mcl-1 is transiently induced. The purpose of this investigation was to determine the functional role played by Mcl-1 in this differentiation program. Mcl-1 expression was specifically disrupted by chimeric methylphosphonate/phosphodiester antisense oligodeoxynucleotides to just 5% of control levels. The depletion of Mcl-1 messenger RNA (mRNA) and protein was both rapid and specific, as indicated by the use of control oligodeoxynucleotides and analysis of the expression of otherBCL2 family members and PMA-induced tumor necrosis factor–α (TNF-α). Specific depletion of Mcl-1 mRNA and protein, in the absence of changes in cellular levels of Bcl-2, results in a rapid entry into apoptosis. Levels of the proapoptotic protein Bax remained unchanged during differentiation, while Bak expression doubled within 24 hours. Apoptosis was detected within 4 hours of Mcl-1 antisense treatment by a variety of parameters including a novel live cell imaging technique allowing correlation of antisense treatment and apoptosis in individual cells. The induction of Mcl-1 is required to prevent apoptosis during differentiation of U937 cells, and the constitutive expression of Bcl-2 is unable to compensate for the loss of Mcl-1.

Description: Key PointsClot contraction has 3 phases differentially affected by platelet and fibrin mechanics, RBC compaction, and various blood components. A new dynamic quantitative clot contraction assay can reveal novel aspects of formation and evolution of hemostatic clots and thrombi.

Description: The hybrid gene BCR-ABL that typifies chronic myeloid leukemia (CML) represents an attractive target for therapy with antisense oligodeoxyribonucleotides (ODN). A central obstacle in the therapeutic application of ODN is their poor cellular uptake. Adding various lipophilic conjugates to the ODN backbone has been reported to improve uptake, and electroporation of target cells has also been shown to enhance intracellular ODN delivery. We have shown that (1)BCR-ABL–directed ODN will specifically decrease the level ofBCR-ABL mRNA, provided that cells are first permeabilized with Streptolysin-O (SL-O), and (2) chimeric methylphosphonodiester:phosphodiester ODN directed against 9 bases either side of the BCR-ABL junction are more efficient ODN effectors than structures composed solely of phosphodiester or phosphorothioate linkages. In this study, we compared the efficacy of lipophilic conjugation, SL-O permeabilization and electroporation on the intracellular delivery and molecular effect ofBCR-ABL–directed ODN. b2a2- and b3a2-directed chimeric ODN were synthesized either unmodified or with one of the following groups at the 5′ end: cholesterol, vitamin E, polyethylene glycol of average molecular weight 2,000 or 5,000, N-octyl-oligo-oxyethylene, or dodecanol. ODN associated with Lipofectin was also studied. Comparison was made in untreated, electroporated, and SL-O permeabilized KYO1 cells. Uptake was examined by fluorescence microscopy and flow cytometry, using ODN structures that were 3′ labeled with fluorescein. The effect on target BCR-ABL mRNA expression was analyzed by Northern blotting. Several conjugated structures associated avidly with the cell membrane without achieving significant intracellular uptake or molecular effect. Similarly, ODN:Lipofectin complexes moderately increased cell association, without enhancing intracellular levels of ODN or inducing detectable molecular effect. In SL-O permeabilized or electroporated cells, uptake was approximately 1 to 2 logs greater than in untreated cells, and rapid nuclear localization was seen, especially with unmodified chimeric ODN. In SL-O permeabilized cells treated with ODN directed to the b2a2 and b3a2 junctions respectively, b2a2 BCR-ABL mRNA levels at 4 hours were reduced to 2.6% ± 2.1% and 38.4% ± 1.3% of control values. In cells permeabilized by electroporation, BCR-ABL mRNA levels were decreased to 4.0% ± 1.4% of control levels by b2a2 directed ODN, although very little nontargeted suppression was seen with b3a2-targeted ODN (93.4% ± 4.2% of control). Greater cell to cell variation in ODN uptake was seen for SL-O permeabilized cells when compared with electroporated cells, suggesting that, after SL-O permeabilization, relatively unpermeabilized and overpermeabilized populations may coexist. No structure had any effect on the level of irrelevant (p53, MYC, and GADPH) mRNA levels. We conclude that the conjugation of chimeric ODN with one of the above-mentioned lipophilic groups or the complexing of ODN with Liopfectin does not improve either intracellular delivery of ODN or the molecular effect. In contrast, both electroporation and SL-O permeabilization (1) considerably enhanced uptake of chimeric ODN (even for structures without a conjugate group) and (2) achieved significant suppression of target mRNA levels.

Description: VEGF is a multifunctional cytokine involved in tumourigenesis. In chronic lymphocytic leukaemia (CLL), it is known that the malignant cells secrete VEGF and possess VEGF receptors. This suggests that an autocrine loop might be important in the pathogenesis of CLL. Here we show that, in patients with lymphadenopathy, autocrine VEGF and α4β1-integrin are involved in the chemokine-dependent motility of CLL cells on and through endothelium - processes important for the invasion of lymphoreticular tissues. In contrast, normal lymphocytes were not dependent on autocrine VEGF or α4β1 for either type of cell movement. Moreover, in contrast to normal B lymphocytes, CLL cells failed to cluster and activate αLβ2 in response to chemokines, unless VEGF receptor(s) and α4β1 were also engaged by their respective ligands. This is the first demonstration that autocrine VEGF is involved in CLL-cell motility and that the αLβ2 on the malignant cells is functionally altered in the disease. Taken together, the results suggest that VEGF and α4 blockade may be therapeutically useful in CLL patients with tissue disease.

Description: The hybrid gene BCR-ABL that typifies chronic myeloid leukemia (CML) represents an attractive target for therapy with antisense oligodeoxyribonucleotides (ODN). A central obstacle in the therapeutic application of ODN is their poor cellular uptake. Adding various lipophilic conjugates to the ODN backbone has been reported to improve uptake, and electroporation of target cells has also been shown to enhance intracellular ODN delivery. We have shown that (1)BCR-ABL–directed ODN will specifically decrease the level ofBCR-ABL mRNA, provided that cells are first permeabilized with Streptolysin-O (SL-O), and (2) chimeric methylphosphonodiester:phosphodiester ODN directed against 9 bases either side of the BCR-ABL junction are more efficient ODN effectors than structures composed solely of phosphodiester or phosphorothioate linkages. In this study, we compared the efficacy of lipophilic conjugation, SL-O permeabilization and electroporation on the intracellular delivery and molecular effect ofBCR-ABL–directed ODN. b2a2- and b3a2-directed chimeric ODN were synthesized either unmodified or with one of the following groups at the 5′ end: cholesterol, vitamin E, polyethylene glycol of average molecular weight 2,000 or 5,000, N-octyl-oligo-oxyethylene, or dodecanol. ODN associated with Lipofectin was also studied. Comparison was made in untreated, electroporated, and SL-O permeabilized KYO1 cells. Uptake was examined by fluorescence microscopy and flow cytometry, using ODN structures that were 3′ labeled with fluorescein. The effect on target BCR-ABL mRNA expression was analyzed by Northern blotting. Several conjugated structures associated avidly with the cell membrane without achieving significant intracellular uptake or molecular effect. Similarly, ODN:Lipofectin complexes moderately increased cell association, without enhancing intracellular levels of ODN or inducing detectable molecular effect. In SL-O permeabilized or electroporated cells, uptake was approximately 1 to 2 logs greater than in untreated cells, and rapid nuclear localization was seen, especially with unmodified chimeric ODN. In SL-O permeabilized cells treated with ODN directed to the b2a2 and b3a2 junctions respectively, b2a2 BCR-ABL mRNA levels at 4 hours were reduced to 2.6% ± 2.1% and 38.4% ± 1.3% of control values. In cells permeabilized by electroporation, BCR-ABL mRNA levels were decreased to 4.0% ± 1.4% of control levels by b2a2 directed ODN, although very little nontargeted suppression was seen with b3a2-targeted ODN (93.4% ± 4.2% of control). Greater cell to cell variation in ODN uptake was seen for SL-O permeabilized cells when compared with electroporated cells, suggesting that, after SL-O permeabilization, relatively unpermeabilized and overpermeabilized populations may coexist. No structure had any effect on the level of irrelevant (p53, MYC, and GADPH) mRNA levels. We conclude that the conjugation of chimeric ODN with one of the above-mentioned lipophilic groups or the complexing of ODN with Liopfectin does not improve either intracellular delivery of ODN or the molecular effect. In contrast, both electroporation and SL-O permeabilization (1) considerably enhanced uptake of chimeric ODN (even for structures without a conjugate group) and (2) achieved significant suppression of target mRNA levels.

Description: Mcl-1 is a member of the Bcl-2 protein family, which has been shown to delay apoptosis in transfection and/or overexpression experiments. As yet no gene knockout mice have been engineered, and so there is little evidence to show that loss of Mcl-1 expression is sufficient to trigger apoptosis. U937 cells constitutively express the antiapoptotic protein Bcl-2; but during differentiation, in response to the phorbol ester PMA (phorbol 12 β-myristate 13 α-acetate), Mcl-1 is transiently induced. The purpose of this investigation was to determine the functional role played by Mcl-1 in this differentiation program. Mcl-1 expression was specifically disrupted by chimeric methylphosphonate/phosphodiester antisense oligodeoxynucleotides to just 5% of control levels. The depletion of Mcl-1 messenger RNA (mRNA) and protein was both rapid and specific, as indicated by the use of control oligodeoxynucleotides and analysis of the expression of otherBCL2 family members and PMA-induced tumor necrosis factor–α (TNF-α). Specific depletion of Mcl-1 mRNA and protein, in the absence of changes in cellular levels of Bcl-2, results in a rapid entry into apoptosis. Levels of the proapoptotic protein Bax remained unchanged during differentiation, while Bak expression doubled within 24 hours. Apoptosis was detected within 4 hours of Mcl-1 antisense treatment by a variety of parameters including a novel live cell imaging technique allowing correlation of antisense treatment and apoptosis in individual cells. The induction of Mcl-1 is required to prevent apoptosis during differentiation of U937 cells, and the constitutive expression of Bcl-2 is unable to compensate for the loss of Mcl-1.