Effective administration of flavopiridol in advanced-stage chronic lymphocytic leukemia (CLL) is often associated with early biochemical evidence of tumor cell lysis. Previous work using other cell types showed that flavopiridol impacts mitochondria, and in CLL cells flavopiridol down-regulates the mitochondrial protein Mcl-1. We therefore investigated mitochondrial structure and function in flavopiridol-treated CLL patient cells and in the lymphoblastic cell line 697 using concentrations and times at which tumor lysis is observed in treated patients. Mitochondrial membrane depolarization was detected in flavopiridol-treated CLL cells by 6 hours, well before the onset of cell death. Flavopiridol-induced mitochondrial depolarization was not blocked by caspase inhibitors or by the calcium chelator EGTA, but was reduced by Bcl-2 overexpression. Intracellular calcium mobilization was noted at early time points using fluorescence microscopy. Furthermore, electron paramagnetic resonance oximetry showed a gradual but significant reduction in cellular oxygen consumption rate by 6 hours, corresponding with ultrastructural mitochondrial damage detected by electron microscopy. IntroductionFlavopiridol is a semisynthetic flavone (N-methylpiperidinyl chlorophenyl flavone) that is considered to act broadly as a cyclindependent kinase (CDK) inhibitor. 1 However, the in vivo mechanism of action of flavopiridol is not well understood, and may involve actions other than or inclusive of CDK inhibition. Our group recently demonstrated significant clinical efficacy of flavopiridol in patients with refractory chronic lymphocytic leukemia (CLL) using a novel schedule of administration. 2 Approximately 50% of CLL patients who receive flavopiridol using this schedule exhibit biochemical signs of tumor lysis (elevated potassium and phosphate levels, reduced calcium levels) occurring as early as 4.5 hours after treatment initiation. In limited cases with highly elevated peripheral white blood cell counts, this tumor lysis can be severe enough to require dialysis. This observation suggests that flavopiridol, when effectively administered, induces very rapid cell death that is atypical of classical apoptosis. However, more work is needed to understand this process and to better predict which patients may experience severe tumor lysis.Multiple previous studies in different cell types have incriminated mitochondrial mechanisms in flavopiridol-induced apoptosis, although conditions and time points under which this is noted vary widely and are often reported in combination with other agents. [3][4][5][6][7][8][9][10][11] Several reports showed that flavopiridol induces mitochondrial membrane disruption and release of cytochrome c in the U937 human monoblastic leukemia cell line and that these effects were potentiated by phorbol myristate acetate (PMA). 4,9,10 This process in U937 cells was noted to be partially caspase independent, as a general caspase inhibitor blocked flavopiridol-induced loss of mitochondrial membrane potential (⌬⌿ m ) but ...

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Slug (Snail2) is a member of the Snail family of zinc-finger transcription factors with regulatory functions in development, tissue morphogenesis, and tumor progression. Little is known about Slug in normal adult tissue; however, a role for Slug in the skin was suggested by our previous observations of Slug expression in normal murine keratinocytes and Slug induction at wound margins. To study the impact of Slug in the skin, we compared patterns of gene expression in epidermis from Slug-null and wild-type mice. A total of 139 genes had significantly increased, and 109 genes had significantly decreased expression in Slug knockout epidermis. Altered expression of selected genes in Slug knockout epidermis was validated by real-time PCR and immunohistochemistry. Previously reported Slug targets were identified, in addition to novel genes, including cytokeratins, adhesion molecules, and extracellular matrix components. Functional classification of altered gene expression was consistent with a role for Slug in keratinocyte development and differentiation, proliferation, apoptosis, adhesion, motility, as well as angiogenesis and response to environmental stimuli. These results highlight the utility of genetic models to study the in vivo impact of regulatory factors in unperturbed skin and suggest that Slug has significant activities in the adult epidermis.

The current classification of sporadic parathyroid neoplasia, specifically the distinction of adenoma from multiple gland neoplasia (double adenoma and nonfamilial primary hyperplasia) is problematic and results in a relatively high rate of clinical error. Oligonucleotide microarrays (Affymetrix U133A) were used to evaluate parathyroid samples from 61 patients; 35 adenomas, 10 nonfamilial multiple gland neoplasia, 3 familial primary hyperplasia, 8 renalinduced hyperplasia, and 5 from patients without parathyroid disease (normals). A multiclass comparison using supervised clustering identified distinct gene signatures for each class of parathyroid samples. We developed a predictor model that correctly identified 34 of 35 cases of adenoma, 9 of 10 cases of nonfamilial multiple gland neoplasia, and identified a minimum set of 11 genes for the distinction of adenoma versus multiple gland neoplasia. All methods of unsupervised clustering showed two related but different types of parathyroid adenomas that we have arbitrarily designated as type 1 and type 2 adenomas. Multiple gland parathyroid neoplasia, which represents either synchronous or asynchronous autonomous growth in two, three, or all four parathyroid glands, is a distinct molecular entity and does not represent the molecular pathogenesis of adenoma occurring in multiple glands.

The present study compared directly the ability of precipitating and nonprecipitating antibodies to form and sustain glomerular immune deposits. The antigen phenylated gelatin (DNP-GL) was injected i.v. into rats. DNP-GL is cleared from the circulation rapidly and becomes localized in glomeruli. Two hours later, rats received either precipitating or nonprecipitating mouse monoclonal anti-DNP antibodies. These antibodies were comparable with respect to size, affinity, number of antigen combining sites and isoelectric point. However, in vitro, nonprecipitating antibodies demonstrated a faster dissociation rate from antigen than precipitating antibodies. Control rats received DNP-GL alone or antibody alone. Antibody deposition in glomeruli was quantitated by Computerized Image Analysis (CIA) of immunoperoxidase stained tissue sections and by glomerular radioactive counts in experiments using 125I labelled antibodies. We demonstrated that glomerular uptake of anti-DNP antibody was similar two hours after injection of precipitating and nonprecipitating antibodies. However, six or more hours after injection, significantly less antibody was present in the glomeruli of rats injected with nonprecipitating antibodies. These differences could not be explained by a greater rate of antigen removal from kidney in rats injected with nonprecipitating antibodies. To assess whether nonprecipitating antibodies modify the glomerular binding and retention of precipitating antibody, in a separate series of experiments rats were injected with equal amounts of precipitating and nonprecipitating antibodies. Both types of antibody bound to glomeruli. However, with time glomerular antibody levels paralleled those found in rats injected with nonprecipitating antibody alone. We conclude that the precipitating characteristics of antibodies do not affect their ability to deposit in kidney.(ABSTRACT TRUNCATED AT 250 WORDS)