Cyclin D1 is an important regulator of cell cycle progression and can function as a transcriptionl co-regulator. The overexpression of cyclin D1 has been linked to the development and progression of cancer. Deregulated cyclin D1 degradation appears to be responsible for the increased levels of cyclin D1 in several cancers. Recent findings have identified novel mechanisms involved in the regulation of cyclin D1 stability. A number of therapeutic agents have been shown to induce cyclin D1 degradation. The therapeutic ablation of cyclin D1 may be useful for the prevention and treatment of cancer. In this review, current knowledge on the regulation of cyclin D1 degradation is discussed. Novel insights into cyclin D1 degradation are also discussed in the context of ablative therapy. A number of unresolved questions regarding the regulation of cellular cyclin D1 levels are also addressed.

Conclusions: Tamoxifen has been shown to inhibit ER␣-mediated cyclin D1 transcription, and acquired resistance to tamoxifen is associated with a shift to ER␣-independent cyclin D1 up-regulation. Taken together, our data show that TSA effectively induces cyclin D1 down-regulation through both ER␣-dependent and ER␣-independent mechanisms, providing an important new strategy for combating resistance to antiestrogens.

ABSTRACT:Trichostatin A is a potent and specific histone deacetylase inhibitor with promising antitumor activity in preclinical models. Plasma pharmacokinetics of trichostatin A were studied following singledose intraperitoneal administration of 80 mg/kg (high dose) or 0.5 mg/kg (low dose) to female BALB/c mice. Plasma trichostatin A concentrations were quantified by high performance liquid chromatography (HPLC)-UV assay (high dose) or by HPLC-multiple reaction monitoring assay (low dose). Trichostatin A was rapidly absorbed from the peritoneum and detectable in plasma within 2 min. C max of 40 g/ml and 8 ng/ml occurred within 5 min, followed by rapid exponential decay in plasma trichostatin A concentration with t 1/2 of 6.3 min and 9.6 min (high and low doses, respectively). Phase I metabolites at the high dose were identified by simultaneous UV and positive ion electrospray mass spectrometry. Trichostatin A underwent extensive metabolism: primary metabolic pathways were Ndemethylation, reduction of the hydroxamic acid to the corresponding trichostatin A amide, and oxidative deamination to trichostatic acid. N-Monomethyl trichostatin A amide was the major plasma metabolite. No didemethylated compounds were identified. Trichostatic acid underwent further biotransformation: reduction and ␤-oxidation of the carboxylic acid, with or without N-demethylation, resulted in formation of dihydro trichostatic acid and dinor dihydro trichostatic acids. HPLC fractions corresponding to trichostatin A and Ndemethylated trichostatin A exhibited histone deacetylase-inhibitory activity; no other fractions were biologically active. We conclude that trichostatin A is rapidly and extensively metabolized in vivo following intraperitoneal administration to mice, and N-demethylation does not compromise histone deacetylase-inhibitory activity.

Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both S chizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S . pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S . pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25.

A series of 3-substituted-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines have been designed, synthesized, and evaluated as RET protein kinase inhibitors. On the basis of docking results, a small library of pyrazolopyrimidine compounds with an extended hydrophobic side arm was synthesized. The most promising of the compounds (7a) displayed efficient inhibition in vitro and good selectivity when tested on a panel of kinases. Furthermore, 7a inhibited GDNF-induced RET phosphorylation of ERK1/2 in MCF-7 breast cancer cells at concentrations as low as 100 nM.

Histone deacetylase inhibitors (HDACI) are emerging as growth inhibitory compounds that modulate gene expression and inhibit tumor cell proliferation. We assessed whether 3 ¶-deoxy-3 ¶-[18 F]fluorothymidine-positron emission tomography ([ 18 F]FLT-PET) could be used to noninvasively measure the biological activity of a novel HDACI LAQ824 in vivo. We initially showed that thymidine kinase 1 (TK1; EC2.7.1.21), the enzyme responsible for [18 F]FLT retention in cells, was regulated by LAQ824 in a drug concentration-dependent manner in vitro. In HCT116 colon carcinoma xenograftbearing mice, LAQ824 significantly decreased tumor [18 F]FLT uptake in a dose-dependent manner. At day 4 of treatment, [ 18 F]FLT tumor-to-heart ratios at 60 minutes (NUV60) were 2.16 F 0.15, 1.86 F 0.13, and 1.45 F 0.20 in vehicle, and 5 and 25 mg/kg LAQ824 treatment groups, respectively (P V 0.05). LAQ825 at 5 mg/kg also significantly reduced both TK1 levels and [18 F]FLT uptake at day 10 but not at day 2 (P V 0.05). [ 18 F]FLT NUV60 correlated significantly with cellular proliferation (r = 0.68; P = 0.0019) and was associated with druginduced histone H4 hyperacetylation. Of interest to [18 F]FLT-PET imaging, both TK1 mRNA copy numbers and protein levels decreased in the order vehicle >5 mg/kg LAQ824 > 25 mg/kg LAQ824, providing a rationale for the use of [ 18 F]FLT-PET in this setting. We also observed increases in Rb hypophosphorylation and p21 levels, factors that could have contributed to the alteration in TK1 transcription in vivo. In conclusion, we have shown the utility of [18 F]FLT-PET for monitoring the biological activity of the HDACI, LAQ824. Drug-induced changes in tumor [18 F]FLT uptake were due, at least in part, to reductions in TK1 transcription and translation. (Cancer Res 2006; 66(15): 7621-9)

We used the estrogen-responsive MCF-7 breast cancer cell line as a relevant model to study the anti-proliferative effects of ICI182,780 and identified the negative cell cycle regulator p21Waf1 as a specific target of ICI182,780. Furthermore, silencing of the p21 Waf1 expression by small interfering RNA overcame the G 0 /G 1 cell cycle arrest induced by ICI182,780, suggesting that the induction of p21Waf1 expression has a direct role in mediating the ICI182,780-induced G 0 /G 1 arrest. We further demonstrated that the induction of p21Waf1 by ICI182,780 is mediated at transcriptional and gene promoter levels through the proximal Sp1 sites located near the transcription start site. Co-immunoprecipitation, DNA "pulldown," and chromatin immunoprecipitation experiments together showed that in cycling cells, estrogen receptor ␣ and histone deacetylase 1 (HDAC1) are recruited to the proximal Sp1 sites of the promoter to repress p21 Estradiol (E 2 ) 1 is the principle steroid mitogen for normal breast epithelial cells and has been shown to be important for the development and progression of de novo breast cancers. E 2 exerts its effect on target cells predominantly through binding to and activating the estrogen receptor ␣ (ER␣), which is a member of the steroid/thyroid hormone superfamily of liganddependent transcription factors. The ligand-bound receptor binds to promoter regions of estrogen-regulated genes, where it recruits co-activators or co-repressors as well as the transcriptional machinery necessary to modulate gene expression. The recruitment of ligand-bound ER to the promoter regions of target genes can occur directly through binding to specific DNA sequences in target genes known as estrogen response elements (EREs) or indirectly through interaction with other DNA-binding transcription factors, such as AP1 and Sp1 (1). In addition to estradiol, the transcription activity of ER can also be modulated by a group of ligands called selective estrogen receptor modulators (SERMs), which have agonist and/or antagonist functions depending on the target promoters and tissues. Some of these SERMs, including tamoxifen, raloxifene, and ICI182,780 (also clinically termed faslodex or fulvestrant), have been employed to antagonize estrogen actions in order to treat breast cancer and protect high risk individuals against breast cancer (2). However, SERMs, such as tamoxifen and raloxifene, also behave as agonists in certain tissues. In fact, the modes of action of SERMs are often promoter context-and cell type-specific. For example, whereas tamoxifen is an effective adjuvant treatment for breast cancer, it can also promote endometrial cancer. Recent gene array data showed that antiestrogens, such as tamoxifen, raloxifene, and ICI182,780, function by either partially or completely antagonizing the actions of estrogen on most genes in breast cancer cells (3). Although it was initially believed that anti-estrogens function merely by competing with endogenous estrogens for receptor binding, previous studies using mutated ERs and re...

The dichloromethane/methanol (1:1) extracts of the roots of Pentas longiflora and Pentas lanceolata showed low micromolar (IC(50) = 0.9-3 µg/mL) IN VITRO antiplasmodial activity against chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of PLASMODIUM FALCIPARUM. Chromatographic separation of the extract of PENTAS LONGIFLORA led to the isolation of the pyranonaphthoquinones pentalongin (1) and psychorubrin (2) with IC(50) values below 1 µg/mL and the naphthalene derivative mollugin (3), which showed marginal activity. Similar treatment of Pentas lanceolata led to the isolation of eight anthraquinones ( 4-11, IC(50) = 5-31 µg/mL) of which one is new (5,6-dihydroxydamnacanthol, 11), while three--nordamnacanthal (7), lucidin-ω-methyl ether (9), and damnacanthol (10)--are reported here for the first time from the genus Pentas. The compounds were identified by NMR and mass spectroscopic techniques.