Inhibition of NF-κB-mediated signaling by the cyclin-dependent kinase inhibitor CR8 overcomes prosurvival stimuli to induce apoptosis in chronic lymphocytic leukemia cells.

PURPOSE: Chronic lymphocytic leukemia (CLL) is currently incurable with standard chemotherapeutic agents, highlighting the need for novel therapies. Overcoming proliferative and cytoprotective signals generated within the microenvironment of lymphoid organs is essential for limiting CLL progression and ultimately developing a cure. EXPERIMENTAL DESIGN: We assessed the potency of cyclin-dependent kinase (CDK) inhibitor CR8, a roscovitine analog, to induce apoptosis in primary CLL from distinct prognostic subsets using flow cytometry-based assays. CLL cells were cultured in in vitro prosurvival and proproliferative conditions to mimic microenvironmental signals in the lymphoid organs, to elucidate the mechanism of action of CR8 in quiescent and proliferating CLL cells using flow cytometry, Western blotting, and quantitative real-time PCR. RESULTS: CR8 was 100-fold more potent at inducing apoptosis in primary CLL cells than roscovitine, both in isolated culture and stromal-coculture conditions. Importantly, CR8 induced apoptosis in CD40-ligated CLL cells and preferentially targeted actively proliferating cells within these cultures. CR8 treatment induced downregulation of the antiapoptotic proteins Mcl-1 and XIAP, through inhibition of RNA polymerase II, and inhibition of NF-κB signaling at the transcriptional level and through inhibition of the inhibitor of IκB kinase (IKK) complex, resulting in stabilization of IκBα expression. CONCLUSIONS: CR8 is a potent CDK inhibitor that subverts pivotal prosurvival and proproliferative signals present in the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of selective CDK inhibitors as novel therapies for CLL.

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1.

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser(2). This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.

Identification of kinase inhibitors that target transcription initiation by RNA polymerase II.

Our current understanding of eukaryotic transcription has greatly benefited from use of small molecule inhibitors that have delineated multiple regulatory steps in site-specific initiation and elongation of RNA synthesis by multiple forms of RNA polymerase (RNAP). This class of "transcription" drugs is also of therapeutic interest and under evaluation in clinical trials. However, to date very few small molecules that directly abolish transcription have been identified, particularly those that act at the level of RNAP II initiation. Using a biochemical assay that measures transcription from recombinant, natural p53-responsive promoters and an artificial "super" promoter, we have identified three distinct small molecules that inhibit mRNA synthesis in vitro. Unexpectedly, these are kinase inhibitors, Hypericin, Rottlerin, and SP600125, with known substrates, which we find also strongly impair transcriptional initiation (IC50s = µM range) by targeting specific components of the RNAP II pre-initiation complex. When measured before and during transcription in vitro, one common target of inhibition by all three compounds is modification of the TATA Binding Protein (TBP) within the RNAP II holocomplex as it converts to an active transcribing enzyme. On this basis, by blocking the critical step of TBP modification, transcriptional initiation is effectively abolished even on structurally distinct core promoters.

Tumor therapy with Amanita phalloides (death cap): stabilization of B-cell chronic lymphatic leukemia.

BACKGROUND: Molecular events that cause tumor formation upregulate a number of HOX genes, called switch genes, coding for RNA polymerase II transcription factors. Thus, in tumor cells, RNA polymerase II is more active than in other somatic cells. Amanita phalloides contains amanitin, inhibiting RNA polymerase II. Partial inhibition with amanitin influences tumor cell--but not normal cell--activity. OBJECTIVES: To widen the treatment spectrum, homeopathic dilutions of Amanita phalloides, containing amanitin, were given to a patient with leukemia. Monitoring the leukemic cell count, different doses of amanitin were given. RESULTS: The former duplication time of leukemic cells was 21 months. Within a period of 21 months, the cell count is stabilized to around 10(5)/µL. No leukemia-associated symptoms, liver damage, or continuous erythrocyte deprivation occur. CONCLUSIONS: This new principle of tumor therapy shows high potential to provide a gentle medical treatment.

In vitro high-throughput screening assay for modulators of transcription.

We developed a 96-well microtiter-plate high-throughput screening (HTS) assay for the detection of modulators of transcription. This HTS assay consists of three steps: (1) the in vitro transcription reaction; (2) modification and hybridization of RNA products; and (3) washing and quantification. During the first step, a DNA template containing the promoter of interest upstream of a cassette lacking guanosine residues in one of its strands (G-less cassette) is incubated with nuclear extract and the necessary cofactors/activators and substrates. During the second step, the in vitro synthesized transcripts are digested with RNase T1 and hybridized to two DNA oligonucleotides. One oligonucleotide is biotinylated for trapping of the RNA products to a streptavidin-coated plate, and the other is europium-labeled for detection by time-resolved fluorescence. We show that this assay is highly reproducible and robust, yielding results comparable to those obtained by standard methodologies employing radioactive nucleotide incorporation and gel electrophoresis while offering a very significant advantage in terms of throughput (>2000 assay points per operator per day). We demonstrate the usefulness of the assay for the discovery of small molecule inhibitors of transcription, and applications of this approach for the high-throughput discovery of transcriptional modulators are discussed.

Quercetin, an inhibitor of heat shock protein synthesis, inhibits the acquisition of thermotolerance in a human colon carcinoma cell line.

Here, we describe the effects of quercetin on the induction of thermotolerance as examined by colony forming assay in a cell line derived from human colon carcinoma (COLO320 DM). Cells became resistant to heat treatment at 45 degrees C when they were preheated at 42 degrees C for 1.5 h or at 45 degrees C for 10 min. This induction of thermotolerance was almost completely inhibited by continuous treatment with 100 microM quercetin during the first and second heating sessions, and the interval between. This effect of quercetin was demonstrated to be dose-dependent over a concentration range of 50-200 microM. Quercetin did not increase the thermosensitivity of non-tolerant cells. The presence of quercetin during the first conditioning heating was more effective in inhibiting thermotolerance than its presence during the second heating. Quercetin was also found to inhibit the acquisition of thermotolerance induced by sodium arsenite. Cycloheximide, a nonspecific inhibitor of protein synthesis, did not affect the acquisition of thermotolerance by the same cell line. Quercetin specifically inhibits the synthesis of all heat shock proteins so far reported previously, and this leads to inhibition of the induction of thermotolerance. Such inhibition of thermotolerance by quercetin may improve the efficacy of clinical fractionated hyperthermia.

Selenotrisulfide inhibits initiation by RNA polymerase II but not elongation.

We previously reported that RNA polymerase II (purified from wheat germ) is inhibited by selenotrisulfides, the products of the reaction of selenite with sulfhydryl compounds [Frenkel, Walcott, and Middleton, Molecular Pharmacology 31, 112 (1987)]. We have now found that the initiation stage of the reaction is inhibited by selenotrisulfide but the elongation stage of the reaction is not. The actual start of the RNA chain is not inhibited by the selenotrisulfide, but rather the formation of the enzyme-DNA binary complex. Selenotrisulfide has a similar differential effect on initiation and elongation by RNA polymerase II from HeLa cells; in contrast, with E. coli RNA polymerase, it inhibits elongation as well.