Caffeine stabilises fission yeast Wee1 in a Rad24-dependent manner but attenuates its expression in response to DNA damage identifying a putative role for TORC1 in mediating its effects on cell cycle progression

The widely consumed neuroactive compound caffeine has generated much interest due to its ability to override the DNA damage and replication checkpoints. Previously Rad3 and its homologues was thought to be the target of caffeine’s inhibitory activity. Later findings indicate that the Target of Rapamycin Complex 1 (TORC1) is the preferred target of caffeine. Effective Cdc2 inhibition requires both the activation of the Wee1 kinase and inhibition of the Cdc25 phosphatase. The TORC1, DNA damage, and environmental stress response pathways all converge on Cdc25 and Wee1. We previously demonstrated that caffeine overrides DNA damage checkpoints by modulating Cdc25 stability. The effect of caffeine on cell cycle progression resembles that of TORC1 inhibition. Furthermore, caffeine activates the Sty1 regulated environmental stress response. Caffeine may thus modulate multiple signalling pathways that regulate Cdc25 and Wee1 levels, localisation and activity. Here we show that the activity of caffeine stabilises both Cdc25 and Wee1. The stabilising effect of caffeine and genotoxic agents on Wee1 was dependent on the Rad24 chaperone. Interestingly, caffeine inhibited the accumulation of Wee1 in response to DNA damage. Caffeine therefore modulates cell cycle progression contextually through increased Cdc25 activity and Wee1 repression following DNA damage via TORC1 inhibition

Caffeine Stabilises Fission Yeast Wee1 in a Rad24-Dependent Manner but Attenuates Its Expression in Response to DNA Damage

The widely consumed neuroactive compound caffeine has generated much interest due to its ability to override the DNA damage and replication checkpoints. Previously Rad3 and its homologues was thought to be the target of caffeine’s inhibitory activity. Later findings indicate that the Target of Rapamycin Complex 1 (TORC1) is the preferred target of caffeine. Effective Cdc2 inhibition requires both the activation of the Wee1 kinase and inhibition of the Cdc25 phosphatase. The TORC1, DNA damage, and environmental stress response pathways all converge on Cdc25 and Wee1. We previously demonstrated that caffeine overrides DNA damage checkpoints by modulating Cdc25 stability. The effect of caffeine on cell cycle progression resembles that of TORC1 inhibition. Furthermore, caffeine activates the Sty1 regulated environmental stress response. Caffeine may thus modulate multiple signalling pathways that regulate Cdc25 and Wee1 levels, localisation and activity. Here we show that the activity of caffeine stabilises both Cdc25 and Wee1. The stabilising effect of caffeine and genotoxic agents on Wee1 was dependent on the Rad24 chaperone. Interestingly, caffeine inhibited the accumulation of Wee1 in response to DNA damage. Caffeine may modulate cell cycle progression through increased Cdc25 activity and Wee1 repression following DNA damage via TORC1 inhibition, as TORC1 inhibition increased DNA damage sensitivity

The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation

The ataxia telangiectasia mutated (ATM) and the ATM- related (ATR) kinases play a central role in facilitating the resistance of cancer cells to genotoxic treatment regimens. The components of the ATM and ATR regulated signaling pathways thus provide attractive pharmacological targets, since their inhibition enhances cellular sensitivity to chemo- and radiotherapy. Caffeine as well as more specific inhibitors of ATM (KU55933) or ATM and ATR (CGK733) have recently been shown to induce cell death in drug-induced senescent tumor cells. Addition of these agents to cancer cells previously rendered senescent by exposure to genotoxins suppressed the ATM mediated p21 expression required for the survival of these cells. The precise molecular pharmacology of these agents however, is not well characterized. Herein, we report that caffeine, CGK733, and to a lesser extent KU55933, inhibit the proliferation of otherwise untreated human cancer and non-transformed mouse fibroblast cell lines. Exposure of human cancer cell lines to caffeine and CGK733 was associated with a rapid decline in cyclin D1 protein levels and a reduction in the levels of both phosphorylated and total retinoblastoma protein (RB). Our studies suggest that observations based on the effects of these compounds on cell proliferation and survival must be interpreted with caution. The differential effects of caffeine/CGK733 and KU55933 on cyclin D1 protein levels suggest that these agents will exhibit dissimilar molecular pharmacological profiles

T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-α. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein-protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexe

Suppression of Sensitivity to Drugs and Antibiotics by High External Cation Concentrations in Fission Yeast

<div><p>Background</p><p>Potassium ion homeostasis plays an important role in regulating membrane potential and therefore resistance to cations, antibiotics and chemotherapeutic agents in <i>Schizosaccharomyces pombe</i> and other yeasts. However, the precise relationship between drug resistance in <i>S</i>. <i>pombe</i> and external potassium concentrations (particularly in its natural habitats) remains unclear. <i>S</i>. <i>pombe</i> can tolerate a wide range of external potassium concentrations which in turn affect plasma membrane polarization. We thus hypothesized that high external potassium concentrations suppress the sensitivity of this yeast to various drugs.</p><p>Methods</p><p>We have investigated the effect of external KCl concentrations on the sensitivity of <i>S</i>. <i>pombe</i> cells to a wide range of antibiotics, antimicrobial agents and chemotherapeutic drugs. We employed survival assays, immunoblotting and microscopy for these studies.</p><p>Results</p><p>We demonstrate that KCl, and to a lesser extent NaCl and RbCl can suppress the sensitivity of <i>S</i>. <i>pombe</i> to a wide range of antibiotics. Ammonium chloride and potassium hydrogen sulphate also suppressed drug sensitivity. This effect appears to depend in part on changes to membrane polarization and membrane transport proteins. Interestingly, we have found little relationship between the suppressive effect of KCl on sensitivity and the structure, polarity or solubility of the various compounds investigated.</p><p>Conclusions</p><p>High concentrations of external potassium and other cations suppress sensitivity to a wide range of drugs in <i>S</i>. <i>pombe</i>. Potassium-rich environments may thus provide <i>S</i>. <i>pombe</i> a competitive advantage in nature. Modulating potassium ion homeostasis may sensitize pathogenic fungi to antifungal agents.</p></div

<i>S</i>. <i>pombe</i> strains used in this study.

<p>YGRC, Yeast Genetic Resource Center, Osaka, Japan</p><p><i>S</i>. <i>pombe</i> strains used in this study.</p

Relative effect of external KCl on drug sensitivity in <i>S</i>. <i>pombe</i>.

<p>Relative effect of external KCl on drug sensitivity in <i>S</i>. <i>pombe</i>.</p

Suppression of drug sensitivity in <i>S</i>. <i>pombe</i> by alkali metal ions.

<p><b>A- C.</b> Wt <i>S</i>. <i>pombe</i> cells were incubated with various concentrations of phleomycin (phleo), G418 and hygromycin (hygro) ± 0.6 M KCl for 24 h at 30°C. Equal cell numbers were serially diluted and plated on YES agar. Plates were incubated at 30°C for 2–3 days. <b>D.</b> Wt <i>S</i>. <i>pombe</i> cells were incubated with 10 μg/ ml phleomycin ± 0.1 M CaCl₂ for 4 h at 30°C and treated as in A. <b>E.</b> Wt S. pombe cells were exposed to 10 μg/ ml phleomycin ± 0.6 M KCl or 1.2 M sorbitol for 6 h, fixed in 70% ethanol, stained with DAPI and examined by microscopy. <b>F.</b> Wt S. pombe cells were treated as in E. Equal cell numbers were serially diluted and plated on YES agar. Plates were incubated at 30°C for 2–3 days. <b>G.</b> Wt <i>S</i>. <i>pombe</i> cells were incubated with 10 μg/ ml phleomycin or in the presence of the indicated NaCl concentrations for 24 h at 30°C and treated as in A. <b>H.</b> Wt cells were incubated with 10 μg/ ml phleomycin ± 0.25 M K₂HPO for 4 h at 30°C and treated as in A. <b>I.</b> Wt cells were incubated with 10 μg/ ml phleomycin ± 0.25 M NH₄Cl for 24 h at 30°C and treated as in A. <b>H. I.</b><i>S</i>. <i>pombe</i> cells were incubated with 0.6 M RbCl ± 0.6 M KCl for 24 h at 30°C. <b>J.</b><i>S</i>. <i>pombe</i> cells were incubated with 40 μg/ ml doxorubicin ± 1 M NH₄Cl for 24 h at 30°C. <b>K.</b> Cells were treated as in I, fixed in 70% ethanol, stained with DAPI and examined by microscopy. <b>L.</b> Log phase wt cultures were serially diluted and plated on YES agar containing 100 μg/ ml hygromycin ± 0.6 M KCl for 72 h at 30°C.</p

List of drugs investigated in this study.

<p>^a Refers to the ability of high external KCl concentrations to suppress drug sensitivity.</p><p>^b 0.6 M KCl enhanced sensitivity to sub-lethal concentrations of valinomycin.</p><p>^c Sensitivity to amphotericin B was suppressed by 0.06 M KCl and increased by 0.6 M KCl.</p><p>^d 0.6 M KCl enhanced sensitivity to sub-lethal concentrations of nystatin.</p><p>List of drugs investigated in this study.</p