Pharmacological inhibitors of NF-kappaB accelerate apoptosis in chronic lymphocytic leukaemia cells.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a critical role in the inappropriate survival of various types of malignant cells. Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy in the Western world. Although overexpression and regulation of NF-kappaB has been described in CLL, its function remains unclear. Exposure of CLL cells to BAY117082 or Kamebakaurin, potent pharmacological inhibitors of the NF-kappaB pathway, accelerated apoptosis in approximately 70% of cases. Sensitivity to NF-kappaB pathway inhibitors was not related to the prognostic markers VH status, CD38 or Zap70 expression, or to the levels of nuclear NF-kappaB. Normal peripheral B cells were resistant to the apoptosis-inducing effects of these compounds. Cell death induced by the inhibitors was associated with activation of caspase-9 and -3, and loss of mitochondrial membrane polarization, but did not involve changes in the expression of Bcl-2 or Mcl-1. Inhibitors caused an increase in c-jun NH2-terminal kinase activity in CLL, but this did not appear to be important for apoptosis. Microarray analysis identified some potential novel NF-kappaB target genes, including interleukin-16- and the Bcl-2- related survival protein Bcl-w. These results demonstrate that a substantial proportion of CLL are dependent on NF-kappaB for enhanced survival and suggest that inhibition of NF-kappaB may have therapeutic potential.

Distinct promoters mediate constitutive and inducible Bcl-XL expression in malignant lymphocytes.

Bcl-X(L) is a Bcl-2-related survival protein that is essential for normal development. Bcl-X(L) expression is rapidly induced by a wide range of survival signals and many cancer cells constitutively express high levels. The Bcl-X gene has a complex organization with multiple promoters giving rise to RNAs with alternate 5' non-coding exons. Here we have investigated the mechanisms that control basal and induced expression of Bcl-X(L) in B-lymphoma cells. Antisense experiments demonstrated that Bcl-X(L) was essential for survival of Akata6 B-lymphoma cells. The levels of RNAs containing the IB Bcl-X non-coding exon, derived from the distal 1B promoter, correlated with basal expression of Bcl-X(L) in primary malignant B cells and this promoter was highly active in B-cell lines. The activity of this promoter was largely dependent on a single Ets binding site and Ets family proteins were bound at this promoter in intact cells. CD40 ligand (CD40L)-induced cell survival was associated with increased Bcl-X(L) expression and accumulation of exon IA-containing RNAs, derived from the proximal 1A promoter. Nuclear factor-kappaB (NF-kappaB) inhibition prevented induction of Bcl-X(L) protein and exon IA-containing RNAs by CD40L. Therefore, the distal Bcl-X 1B promoter plays a critical role in driving constitutive expression-mediated via Ets family proteins in malignant B cells, whereas NF-kappaB plays a central role in the induction of Bcl-X(L) in response to CD40 signalling via the proximal 1A promoter.

Novel sulfasalazine analogues with enhanced NF-kB inhibitory and apoptosis promoting activity.

The NF-kB transcription factor plays a key role in the regulation of apoptosis by modulating expression of a wide range of cell death control molecules. NF-kB also plays an important role in human diseases by promoting inappropriate cell survival. Small molecule inhibitors of NF-kB are therefore likely to provide novel therapeutic opportunities. Sulfasalazine (SFZ) is a synthetic anti-inflammatory comprising an aminosalicylate, 5-amino salicylic acid (5-ASA), linked to an antibiotic, sulfapyridine (SPY). SFZ, but not 5-ASA or SPY, inhibits activation of NF-kB. We synthesised a small number of SFZ analogues and determined their ability to inhibit NF-kB activity and promote apoptosis in chronic lymphocytic leukaemia and hepatic stellate cells, where NF-kB plays an important role in cell survival. Remarkably, 3 of the 6 analogues synthesised were significantly more effective (up to 8-fold) inhibitors of NF-kB dependent transcription and this increased activity was associated with enhanced apoptosis. Therefore, it is possible to readily improve the NF-kB inhibiting activity of SFZ and analogues of SFZ may be attractive therapeutic agents for malignancies and chronic liver disease where NF-kB is thought to play a significant role.

Characteristics of the pulse waveform during altered nitric oxide synthesis in the rabbit.

Nitrovasodilators produce characteristic changes in the shape of the peripheral pulse wave. Similar changes might also be caused by alteration of endogenous NO activity, which would allow such activity to be assessed in vivo. We investigated whether manipulation of the NO pathway influences the pulse waveform, and the mechanisms involved. The pulse wave in the ear of normal rabbits was examined by reflectance photoplethysmography before and during infusion of vasoactive agents. Pulse wave velocity was assessed by using an additional sensor on the rear foot. A diastolic peak was observed in the ear pulse; its timing was consistent with it being a reflection of the systolic peak from the lower body. The height of the dicrotic notch marking the start of this diastolic wave was decreased by acetylcholine or an NO donor, and further decreased by a phosphodiesterase type V inhibitor. The acetylcholine-induced decreases were blocked by inhibiting NO synthesis with N(G)-nitro-L-arginine methyl ester (L-NAME) but were unaffected by the inactive enantiomer D-NAME. These data demonstrate that NO influences the height of the notch in the pulse wave. Heart rate and blood pressure were altered during acetylcholine or L-NAME infusion, but there were no changes in pulse wave amplitude or velocity, or in the timing of the diastolic peak or dicrotic notch. The slope of the pulse wave between the systolic peak and notch changed substantially. These effects are most convincingly explained by changes in wave reflection, not only from the lower body but also from more proximal sites.

Contraction-induced muscle fiber damage is increased in soleus muscle of streptozotocin-diabetic rats and is associated with elevated expression of brain-derived neurotrophic factor mRNA in muscle fibers and activated satellite cells.

The expression of brain-derived neurotrophic factor (BDNF) is elevated in the soleus muscle of streptozotocin-diabetic rats. To determine whether this diabetes-induced elevation was associated with or enhanced by muscle activity we have induced high-intensity muscle contraction by electrically stimulating the sciatic nerve. In 6-week diabetic rats, intense contraction of the soleus muscle resulted in a two- to four-fold elevation of BDNF mRNA and increased plasma levels of creatine kinase that were associated with severe focal muscle fiber damage and concomitant satellite cell activation. Focal muscle fiber damage and concomitant satellite cell activation were also observed in the soleus muscle of nonstimulated diabetic rats, but to a much lesser extent. No effects of muscle contraction, i.e., experimentally induced or during normal daily activity, on muscle fiber structure or BDNF mRNA expression were seen in diabetic extensor digitorum longus (EDL) muscle. Using a nonradioactive in situ hybridization technique for electron microscopy, the elevated expression of BDNF mRNA in the diabetic soleus muscle was localized within muscle fibers as well as activated satellite cells. This study shows that diabetic soleus muscle, in contrast to diabetic EDL and to soleus and EDL muscle of normal animals, is highly susceptible to contraction-induced damage. Intense contraction and the associated muscle fiber damage in the diabetic soleus muscle result in an upregulation of BDNF mRNA in muscle fibers and activated satellite cells, which may be involved in the restoration and/or maintenance of nerve/muscle integrity.