Rapid dimerization of quercetin through an oxidative mechanism in the presence of serum albumin decreases its ability to induce cytotoxicity in MDA-MB-231 cells.

Quercetin is a member of the flavonoid family and has been previously shown to have a variety of anti-cancer activities. We and others have reported anti-proliferation, cell cycle arrest, and induction of apoptosis of cancer cells after treatment with quercetin. Quercetin has also been shown to undergo oxidation. However, it is unclear if quercetin or one of its oxidized forms is responsible for cell death. Here we report that quercetin rapidly oxidized in cell culture media to form a dimer. The quercetin dimer is identical to a dimer that is naturally produced by onions. The quercetin dimer and quercetin-3-O-glucopyranoside are unable to cross the cell membrane and do not kill MDA-MB-231 cells. Finally, supplementing the media with ascorbic acid increases quercetin's ability to induce cell death probably by reduction oxidative dimerization. Our results suggest that an unmodified quercetin is the compound that elicits cell death.

Mastermind-Like 1 Is Ubiquitinated: Functional Consequences for Notch Signaling.

Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1). MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.

Characterization of 3',5' cyclic nucleotide phosphodiesterase activity in Y79 retinoblastoma cells: absence of functional PDE6.

PURPOSE: Previous studies identified rod photoreceptor cyclic GMP phosphodiesterase (PDE6) transcripts in the human Y79 retinoblastoma cell line. To assess the potential to utilize this cell line for structure/function studies of PDE6, we analyzed 3',5' cyclic nucleotide phosphodiesterase activity focusing on expression of PDE6. METHODS: DEAE-chromatography was used to fractionate PDE activity from Y79 cell homogenates. PCR was performed on cDNA generated from Y79 cells and retina with PDE isoform specific primers. Western blots were performed with antibodies to PDE1, PDE4, or rod PDE6. DNA sequencing and protein truncation tests were performed with plasmids containing the entire coding region of Y79 rod PDE6 transcripts. Proteasome mediated degradation of PDE6 subunits was analyzed with a pathway specific inhibitor. Polysome isolation was performed by fractionation on sucrose gradients followed by RT-PCR for the PDE6 transcripts. RESULTS: Of three peaks of PDE activity, peaks 1 and 2 were activated by Ca2+/calmodulin, inhibited by dipyridamole and zaprinast, and were reactive with a PDE1 antibody. Peak 3 hydrolyzed only cAMP and was rolipram sensitive, indicative of PDE4. Transcripts for rod and cone PDE6 isoforms were detected in Y79 total RNA, however PDE6 antibodies recognized only a single 99 kDa polypeptide from immunoprecipitated 35S labeled Y79 extracts. DNA sequencing of PDE6 alpha, beta, gamma, and PDE6 associated delta-subunit cDNA revealed some polymorphism, but no apparent mutations. Each of the PDE6 transcripts could be translated into protein of the correct length. The concentration of cGMP in the cells was greatly reduced in comparison to that reported in the photoreceptor cell. Addition of cyclic nucleotide analogues, zinc, or butyrate did not enhance the expression of PDE6. Transduction into Y79 cells of adenovirus expressing PDE6 subunits failed to produce functional enzyme CONCLUSIONS: PDE1 and PDE4 enzyme activities predominate in Y79 cells. Despite the presence of PDE6 transcripts and the ability to translate each into protein in vitro, a functional PDE6 enzyme could not be detected. Attempts to enhance expression with cell culture or with introduction of virus expressing PDE6 were not successful. The results indicate that expression of a fully active stable PDE6 enzyme requires other post-transcriptional events that do not occur or are inhibited in Y79 cells.

Profiling flavonoid cytotoxicity in human breast cancer cell lines: determination of structure-function relationships.

Flavonoids have been shown to be cytotoxic to cancer cells. However, the mechanism of cytotoxicity has not been clearly defined. It has previously been reported that HER2/ERBB2, the estrogen receptor, progesterone receptor, and p53 were required for flavonoid induced cytotoxicity in breast cancer cell lines. We have used a panel of breast cancer cell lines, known to contain as well as be deficient in these signaling pathways, to screen fourteen different flavonoids. Comparing the cytotoxicity for all flavonoids allows us to determine if a structure-functional relationship exists between cytotoxicity and flavonoid, and if a particular signaling pathway is required for cytotoxicity. We show that several flavonoids are cytotoxic to all cell lines including primary mammary epithelial cells tested. The cytotoxic flavonoids are also able to inhibit Mitochondrial Outer Membrane Permeability while at the same time stimulate ATP levels whereas the non-cytotoxic flavonoids are not able to do this. We also show that both cytotoxic and non-cytotoxic flavonoids can transverse the cell membrane to enter MDA-MB-231 cells at different levels. Finally, all flavonoids regardless of their cytotoxicity were able to induce some form of cell cycle arrest. We conclude that for flavonoids to be strongly cytotoxic, they must possess the 2,3-double bond in the C-ring and we believe the cytotoxicity occurs through mitochondrial poisoning in both cancer and normal cells.

Some natural flavonoids are competitive inhibitors of Caspase-1, -3 and -7 despite their cellular toxicity.

A common feature of both apoptosis and inflammation is the activation of caspases. Caspases are aspartate-directed cysteine proteases that have numerous cellular targets. It has been discovered that several flavonoids are inhibitors of caspases. Flavonoids are members of a family of polyphenolic compounds from plants that have many biological properties, one of which is the ability to induce cell death. Some flavonoids are selective inhibitors of particular caspases. Since some of the inhibitory flavonoids are nevertheless cytotoxic, these results suggest that flavonoid-induced cell death may be occurring through a non-classical apoptosis pathway that is not dependent on caspase activity.

Profiling flavonoid cytotoxicity in human breast cancer cell lines: determination of structure-function relationships.

Flavonoids have been shown to be cytotoxic to cancer cells. However, the mechanism of cytotoxicity has not been clearly defined. It has previously been reported that HER2/ERBB2, the estrogen receptor, progesterone receptor, and p53 were required for flavonoid induced cytotoxicity in breast cancer cell lines. We have used a panel of breast cancer cell lines, known to contain as well as be deficient in these signaling pathways, to screen fourteen different flavonoids. Comparing the cytotoxicity for all flavonoids allows us to determine if a structure-functional relationship exists between cytotoxicity and flavonoid, and if a particular signaling pathway is required for cytotoxicity. We show that several flavonoids are cytotoxic to all cell lines including primary mammary epithelial cells tested. The cytotoxic flavonoids are also able to inhibit Mitochondrial Outer Membrane Permeability while at the same time stimulate ATP levels whereas the non-cytotoxic flavonoids are not able to do this. We also show that both cytotoxic and non-cytotoxic flavonoids can transverse the cell membrane to enter MDA-MB-231 cells at different levels. Finally, all flavonoids regardless of their cytotoxicity were able to induce some form of cell cycle arrest. We conclude that for flavonoids to be strongly cytotoxic, they must possess the 2,3-double bond in the C-ring and we believe the cytotoxicity occurs through mitochondrial poisoning in both cancer and normal cells.

Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover.

Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. We previously reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. Here we show that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes.

Functional analysis of the rod photoreceptor cGMP phosphodiesterase alpha-subunit gene promoter: Nrl and Crx are required for full transcriptional activity.

To understand the factors controlling expression of the cGMP phosphodiesterase type 6 (PDE6) genes, we have characterized the promoter of the human PDE6A gene that encodes the catalytic alpha-subunit. In vivo DNase I hypersensitivity assays revealed two sites immediately upstream of the PDE6A core promoter region. Transient transfection assay in Y79 cells of constructs containing varying lengths of the promoter region showed a decrease in promoter activity with increasing length. The most active segment contained a 177-bp upstream sequence including apparent Crx and Nrl transcription factor binding sites. Both Crx and Nrl transactivated the PDE6A promoter in HEK293 cells and showed a >100-fold increase when coexpressed. Coexpression of a dominant negative inhibitor of Nrl abolished Nrl transactivation but had no effect on Crx. DNase I footprinting assays identified three potential Crx binding sites within a 55-bp segment beginning 29 bp upstream of the transcription start point. Mutation of two of these sites reduced reporter gene activity by as much as 69%. Gel shifts showed that all three Crx sites required a TAAT sequence for efficient binding. Consistent with a requirement for Crx and Nrl in Pde6a promoter activity, Pde6a mRNA is reduced by 87% in the retina of Crx(-/-) mice and is undetectable in Nrl(-/-) mice at postnatal day 10. These results establish that both Nrl and Crx are required for full transcriptional activity of the PDE6A gene.