Caffeine inhibits the checkpoint kinase ATM.

The basis of many anti-cancer therapies is the use of genotoxic agents that damage DNA and thus kill dividing cells. Agents that cause cells to override the DNA-damage checkpoint are predicted to sensitize cells to killing by genotoxic agents. They have therefore been sought as adjuncts in radiation therapy and chemotherapy. One such compound, caffeine, uncouples cell-cycle progression from the replication and repair of DNA [1] [2]. Caffeine therefore servers as a model compound in establishing the principle that agents that override DNA-damage checkpoints can be used to sensitize cells to the killing effects of genotoxic drugs [3]. But despite more than 20 years of use, the molecular mechanisms by which caffeine affects the cell cycle and checkpoint responses have not been identified. We investigated the effects of caffeine on the G2/M DNA-damage checkpoint in human cells. We report that the radiation-induced activation of the kinase Cds1 [4] (also known as Chk2 [5]) is inhibited by caffeine in vivo and that ATM kinase activity is directly inhibited by caffeine in vitro. Inhibition of ATM provides a molecular explanation of the attenuation of DNA-damage checkpoint responses and for the increased radiosensitivity of caffeine-treated cells [6] [7] [8].

Ca2+ is essential for multistep activation of the heat shock factor in permeabilized cells.

We have developed a novel permeabilized-cell system to study transcription mechanisms. In permeabilized cells, heat-induced activation of the heat shock factor and transcription of the hsp70 gene require Ca2+. Activation involves at least two steps: Ca(2+)- and heat-dependent activation of heat shock factor binding and a second step, prior to transcription of hsp70, that requires ATP and is sensitive to genistein, a protein kinase inhibitor.

Reproducible and inexpensive probe preparation for oligonucleotide arrays.

We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.

DNA damage increases the levels of MDM2 messenger RNA in wtp53 human cells.

Damage to chromosomal DNA increases the levels of the transcriptional regulatory protein p53. We have investigated how the MDM2 protein, which binds to p53 and inactivates its transcriptional activity, may be controlled following DNA damage. Irradiation of human GM2149 fibroblast cells causes an increase in MDM2 mRNA levels within 1 h, and levels remain elevated for at least 8 h. The induction of MDM2 mRNA following irradiation is not blocked by inhibitors of protein synthesis and can be detected after doses of 2-5 Gy. In ataxia telangiectasia cells or cells where p53 is mutated/deleted, MDM2 mRNA levels are not increased after DNA damage. This suggests that p53 is required for transcription of the MDM2 gene following DNA damage.

The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes murine fibroblasts and human tumor cells to radiation and blocks induction of p53 following DNA damage.

AT cells are extremely sensitive to ionizing radiation. Since the AT gene has homology to phosphatidylinositol 3 kinases (PI 3-kinases), wortmannin, a specific inhibitor of PI 3-kinase, was used to determine if PI 3-kinase activity regulates radiation sensitivity. Human and murine cells exposed to wortmannin alone did not display significant cytotoxicity. Wortmannin in combination with radiation was an effective radiosensitizer of murine NIH-3T3 fibroblasts, with a sensitizer enhancement ratio of 1.8 at 10% survival, and had a similar effect on the human tumor cell lines HeLa, SW480, and MCF-7. Wortmannin inhibited the induction of p53 DNA-binding activity by actinomycin D and radiation and blocked the transcriptional activation of a p53 CAT reporter gene by actinomycin D. Wortmannin radiosensitized both wild-type (NIH-3T3 and MCF-7) and mutant (SW480 and HeLa) p53 cells, indicating that p53 induction was not required for radiosensitization by wortmannin. The results suggest that a wortmannin-sensitive pathway, possibly involving PI 3-kinase activity, may regulate the response of the cells to DNA damage.

Gadd45 and Gadd153 messenger RNA levels are increased during hypoxia and after exposure of cells to agents which elevate the levels of the glucose-regulated proteins.

We have investigated overlapping activation pathways for two families of stress genes that are expressed in cells exposed to hypoxia. The growth arrest and DNA damage (gadd) genes are induced by DNA damage and irradiation, and their expression is associated with growth arrest. The glucose-regulated proteins (GRPs) are induced by chemical agents that disrupt protein trafficking in the endoplasmic reticulum such as tunicamycin and A23187 and by hypoxia. Here, we demonstrate that the treatment of NIH-3T3 cells with chemical inducers of GRPs results in increased levels of gadd45 and gadd153 mRNA as well as GRP78 mRNA. In addition, hypoxia was also able to increase gadd45, gadd153, and GRP78 mRNA. Therefore the GRP and gadd genes can be activated by similar stimuli (e.g., hypoxia and chemical inducers). However, the mechanisms leading to increased levels of GRP78 and gadd gene mRNA are different and may involve distinct protein kinases. Increased expression of GRPs after treatment with chemical inducers is sensitive to cycloheximide and the protein kinase inhibitors genistein, 2-aminopurine, and H7, whereas the increase in gadd gene mRNA could be blocked by the protein kinase inhibitors H7 and 2-aminopurine but not by genistein or cycloheximide. GRP78 induction occurs by a pathway that requires protein synthesis and is sensitive to genistein, H7, and 2-aminopurine, whereas gadd gene induction is independent of protein synthesis and is inhibited by H7 and 2-aminopurine only.

Oxidative injury rapidly activates the heat shock transcription factor but fails to increase levels of heat shock proteins.

When cells are exposed to heat shock, heavy metals, amino acid analogues, and other stresses, the heat shock transcription factor (HSF) is activated. The HSF then binds to the promoter of the heat shock genes, stimulating transcription of the heat shock proteins. Here, we demonstrate that exposure of NIH-3T3 cells to oxidants (H2O2 or menadione) also causes activation of the HSF. This activation is not blocked by inhibitors of protein synthesis (cycloheximide) or by inhibitors of protein kinases (2-aminopurine or genistein). In addition, the oxidant activated HSF is located in the nucleus of the cells. However, oxidant activation of the HSF does not result in the accumulation of hsp70 mRNA or of heat shock proteins. This is in contrast to the accumulation of heat shock proteins seen after heat shock activation of the HSF. This suggests that oxidant induced activation of HSF binding may have a function different from that of heat induced activation of HSF binding.

Microtubule-active drugs taxol, vinblastine, and nocodazole increase the levels of transcriptionally active p53.

A range of DNA-damaging agents has been shown to increase cellular levels of the nuclear phosphoprotein p53 and to induce p53-dependent processes. We examined the ability of three microtubule-active agents, taxol, vinblastine, and nocodazole, to increase p53 levels and activate p53-dependent processes. When tested using a p53 DNA-binding assay, all three agents induced p53 in a dose-dependent manner. To varying degrees, these agents also induced p21WAF1/CIP1 mRNA and transcription in a chloramphenicol acetyl transferase reporter system. These data suggest there is an additional pathway for activating p53 and subsequent p53-dependent processes.

Increases in sequence specific DNA binding by p53 following treatment with chemotherapeutic and DNA damaging agents.

We have investigated the effect of chemotherapeutic and DNA damaging agents on binding of the tumor suppressor phosphoprotein p53 to its consensus DNA sequence. Activation of p53-DNA binding was seen for treatment with radiation, hydrogen peroxide, actinomycin D, Adriamycin, etoposide, camptothecin, 5-fluorouracil, mitomycin C, and cisplatin. These results showed that DNA strand breaks were sufficient to lead to increased levels of p53. The protein synthesis inhibitor cycloheximide blocks the increase in p53 following DNA damage. The increase in p53 activation in camptothecin treated cells may result, at least in part, from an increased half-life of the protein and consequent increases in intracellular protein concentration.

Highly selective isolation of unknown mutations in diverse DNA fragments: toward new multiplex screening in cancer.

Cancer research would greatly benefit from technologies that allow simultaneous screening of several unknown gene mutations. Lack of such methods currently hampers the large-scale detection of genetic alterations in complex DNA samples. We present a novel mismatch-capture methodology for the highly efficient isolation and amplification of mutation-containing DNA from diverse nucleic acid fragments of unknown sequence. To demonstrate the potential of this method, heteroduplexes with a single A/G mismatch are formed via cross-hybridization of mutant (T-->G) and wild-type DNA-fragment populations. Aldehydes are uniquely introduced at the position of mismatched adenines via the Escherichia coli glycosylase, MutY. Subsequent treatment with a biotinylated hydroxylamine results in highly specific and covalent biotinylation of the site of mismatch. For PCR amplification, synthetic linkers are then ligated to the DNA fragments. Biotinylated DNA is then isolated and PCR amplified. Mutation-containing DNA fragments can subsequently be sequenced to identify type and position of mutation. This method correctly detects a single T-->G transversion introduced into a 7-kb plasmid containing full-length cDNA from the p53 gene. In the presence of a high excess wild-type DNA (1:1000 mutant:normal plasmids) or in the presence of diverse DNA fragment sizes, the DNA fragments containing the mutation are readily detectable and can be isolated and amplified. The present Aldehyde-Linker-Based Ultrasensitive Mismatch Scanning has a current limit of detection of one base substitution in 7 Mb of DNA and increases the limit for unknown mutation scanning by two to three orders of magnitude. Homozygous and heterozygous p53 regions (G-->T, exon 4) from genomic DNA are also examined, and correct identification of mutations is demonstrated. This method should allow large-scale detection of genetic alterations in cancer samples without any assumption as to the genes of interest.

Increased sequence-specific p53-DNA binding activity after DNA damage is attenuated by phorbol esters.

Damage to cellular DNA greatly increases the levels of the tumor-suppressor gene p53 and induces cell cycle arrest in G1. A critical function of wild-type p53 is its ability to bind to specific DNA sequences. The effect of DNA damage on the sequence-specific DNA-binding properties of cellular p53 was investigated using DNA gel mobility-shift assays with nuclear extracts from NIH-3T3 cells. DNA damage (initiated by radiation) induced a rapid, cycloheximide-sensitive increase in the levels of nuclear p53-DNA binding activity and an increase in the half-life of the p53 protein. Increased p53-DNA binding activity could be detected at low (0.2 Gy), non-lethal doses of radiation. The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) attenuated the DNA damage-induced increase in p53-DNA binding activity by decreasing the half-life of the p53 protein. The tumor promoter properties of TPA may therefore be mediated by interfering with the cellular p53 response to DNA damage. The increased levels of p53 bound to specific DNA sequences following DNA damage may induce cell cycle arrest. p53-mediated growth arrest could occur by inhibition of DNA replication and/or alterations in transcription of cell cycle genes.

Cdk2 kinase phosphorylates serine 315 of human p53 in vitro.

DNA damage increases p53 protein levels and activates transcription of the p21 gene. The p21 protein binds to and inhibits cdk2 kinase, causing G1 arrest. Here, we have investigated if a p53 fusion protein is a substrate for cdk2 kinase in vitro. Cdk2 kinase was immunoprecipitated from NIH3T3 cells and allowed to phosphorylate a human p53-GST (glutathione-s-transferase) fusion protein. Cdk2 and cyclin E-cdk2 efficiently phosphorylated both wild-type (wt) and mutant p53-GST. Cdk2 immunoprecipitated from cells in Go and early G1 exhibited minimal p53 kinase activity, whereas cells in S-phase displayed high levels of p53 kinase activity. If NIH3T3 cells were X-ray irradiated to induce DNA damage, cdk2 p53 kinase activity was rapidly inhibited within 1 h, but had recovered by 4 h post irradiation. Mutation of serine 315 of p53 to alanine (p53-S315A) abolished phosphorylation by cdk2 kinase. However, wtp53 and p53-S315A were equally effective at activating transcription when cotransfected with a p53 reporter construct. The results demonstrate that ser 315 of p53 is phosphorylated by cdk2 in vitro. However, ser 315 of wtp53 is not required for transcriptional activity in vivo, suggesting that cdk2 phosphorylation of p53 may be involved in regulating other cellular functions of wtp53.

Activation of p53 transcriptional activity requires ATM's kinase domain and multiple N-terminal serine residues of p53.

The ATM protein kinase regulates the cell's response to DNA damage by regulating cell cycle checkpoints and DNA repair. ATM phosphorylates several proteins involved in the DNA-damage response, including p53. We have examined the mechanism by which ATM regulates p53's transcriptional activity. Here, we demonstrate that reintroduction of ATM into AT cells restores the activation of p53 by the radio-mimetic agent bleomycin. Further, p53 activation is lost when a kinase inactive ATM is used, or if the N-terminal of ATM is deleted. In addition, AT cells stably expressing ATM showed decreased sensitivity to Ionizing Radiation-induced cell killing, whereas cells expressing kinase inactive ATM or N-terminally deleted ATM were indistinguishable from AT cells. Finally, single point-mutations of serines 15, 20, 33 or 37 did not individually block the ATM-dependent activation of p53 transcriptional activity by bleomycin. However, double mutations of either serines 15 and 20 or serines 33 and 37 blocked the ability of ATM to activate p53. Our results indicate that the N-terminal of ATM and ATM's kinase activity are required for activation of p53's transcriptional activity and restoration of normal sensitivity to DNA damage. In addition, activation of p53 by ATM requires multiple serine residues in p53's transactivation domain.

Constitutive activation of IkappaB kinase alpha and NF-kappaB in prostate cancer cells is inhibited by ibuprofen.

Apoptotic pathways controlled by the Rel/NF-kappaB family of transcription factors may regulate the response of cells to DNA damage. Here, we have examined the NF-kappaB status of several prostate tumor cell lines. In the androgen-independent prostate tumor cells PC-3 and DU-145, the DNA-binding activity of NF-kappaB was constitutively activated and IkappaB-alpha levels were decreased. In contrast, the androgen-sensitive prostate tumor cell line LNCaP had low levels of NF-kappaB which were upregulated following exposure to cytokines or DNA damage. The activity of the IkappaB-alpha kinase, IKKalpha, which mediates NF-kappaB activation, was also measured. In PC-3 cells, IKKalpha activity was constitutively active, whereas LNCaP cells had minimal IKKalpha activity that was activated by cytokines. The anti-inflammatory agent ibuprofen inhibited the constitutive activation of NF-kappaB and IKKalpha in PC-3 and DU-145 cells, and blocked stimulated activation of NF-kappaB in LNCaP cells. However, ibuprofen did not directly inhibit IkappaB-alpha kinase. The results demonstrate that NF-kappaB is constitutively activated in the hormone-insensitive prostate tumor cell lines PC-3 and DU-145, but not in the hormone responsive LNCaP cell line. The constitutive activation of NF-kappaB in prostate tumor cells may increase expression of anti-apoptotic proteins, thereby decreasing the effectiveness of anti-tumor therapy and contributing to the development of the malignant phenotype.

The isolation and characterization of a Drosophila gene encoding a putative NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.

Mammalian NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase is a bifunctional mitochondrial enzyme expressed in most established cell lines but only in developing normal tissues. We report the cloning and molecular characterization of a Drosophila gene (DNMDMC) that encodes a protein with 56% identity to the mammalian bifunctional protein. Like the mammalian bifunctional proteins, the Drosophila protein contains a putative mitochondrial targeting sequence and its transcripts are expressed in developing tissues. Unlike its mammalian homologs, DNMDMC is expressed at high levels in adult tissues. DNMDMC maps to polytene chromosome band 85C, is encoded in three exons, and is closely flanked by two additional genes.

Signalling across the endoplasmic reticulum membrane: potential mechanisms.

The endoplasmic reticulum (ER) is a membrane-bound organelle responsible for the synthesis, assembly and post-translational modification of proteins destined for the lysosomes, Golgi and for secretion. The processes which occur in the lumen of the ER are vital to the correct functioning of the cell, and mechanisms must exist to enable the cell to monitor events within the lumen of the ER. How the cell is able to do this is not known, but it would apparently require the passage of signals from the lumen of the ER to the cytosol, from where signals can be sent to, for example, the nucleus to effect changes in transcription. Here, it is suggested that the membrane of the ER may contain the components (i.e. receptors, kinases, etc.) required for transmembrane signalling in much the same way as the plasma membrane does. This hypothesis will be discussed in relation to known ER proteins which might act as signalling proteins.

Radiosensitization of human tumor cells by the phosphatidylinositol3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay.

Members of the phosphatidylinositol (PI) 3-kinase gene family, including the ataxia telangiectasia gene and the DNA-dependent protein kinase (DNA-PK), are involved in regulating cellular radiosensitivity. We have investigated two structurally unrelated PI 3-kinase inhibitors, wortmannin and LY294002, to determine whether they inhibit DNA-PK and increase cellular radiosensitivity. The PI 3-kinase inhibitors wortmannin and LY294002 were effective radiosensitizers of human tumor cells, with sensitizer enhancement ratios (at 10% survival) of 2.8 and 1.9, respectively, in SW480 cells. Wortmannin and LY294002 inhibited the kinase activity of purified DNA-PK and inactivated cellular DNA-PK kinase activity. Inhibition of cellular DNA-PK activity occurred at the same concentrations of wortmannin that caused radiosensitization, and this correlation was found in a range of tumor cell lines. However, cells deficient in either DNA-PK (scid cells) or the ataxia telangiectasia protein were also partly sensitized to radiation by wortmannin, indicating the involvement of more than one protein kinase in the mechanism of action of wortmannin. Wortmannin also affected the G2-M checkpoint. SW480 cells had a reversible G2-M delay of 20 h following irradiation. However, wortmannin-treated SW480 cells had a prolonged G2-M delay; more than 75% of cells were arrested in G2 at 50 h postirradiation. This suggests the accumulation of significant unrepaired DNA damage following inhibition of PI 3-kinase family members. Therefore, PI 3-kinase inhibitors may represent a new class of radiosensitizers that inhibit the repair of DNA damage.

Suramin increases p53 protein levels but does not activate the p53-dependent G1 checkpoint.

Suramin is an antineoplastic agent which has a cytostatic effect on both normal and tumor-derived cells. We have investigated whether the induction of growth arrest by suramin requires the p53 protein, a tumor suppressor gene product involved in the initiation of growth arrest following DNA damage. Activation of the p53 protein by genotoxic agents causes increased p53 protein levels and p53-dependent transcription of the p21 gene. The p21 protein then inhibits cyclin-dependent kinases, initiating G1 arrest. Exposure of NIH-3T3 cells to suramin caused a rapid (1-2 h) increase in the level of p53-DNA-binding activity. Flow cytometric analysis indicated that suramin arrested NIH-3T3 cells in G0-G1. However, suramin did not increase the p53-dependent transcription of the p21 gene or inhibit cyclin-dependent kinase 2 kinase activity. If NIH-3T3 cells were exposed to radiation or suramin plus radiation, p21 mRNA levels were increased and cyclin-dependent kinase 2 kinase activity was inhibited, indicating that suramin does not block the cells' ability to increase p21 levels. To determine whether the G0-G1 arrest induced by suramin required p53, NIH-3T3 cells transfected with a dominant negative mutant p53 gene to eliminate wild-type p53 function (NMP cells) were exposed to suramin. NMP cells still exhibited G0-G1 arrest after suramin treatment. Suramin increases p53 protein levels, but fails to increase p21 mRNA levels or to activate the G1 checkpoint. These data suggest that suramin induces growth arrest in NIH-3T3 cells by a mechanism that is independent of cellular p53 status.

Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity.

BACKGROUND: The p53 protein is activated by genotoxic stress, oncogene expression and during senescence, p53 transcriptionally activates genes involved in growth arrest and apoptosis. p53 activation is regulated by post-translational modification, including phosphorylation of the N-terminal transactivation domain. Here, we have examined how Glycogen Synthase Kinase (GSK3), a protein kinase involved in tumorigenesis, differentiation and apoptosis, phosphorylates and regulates p53. RESULTS: The 2 isoforms of GSK3, GSK3alpha and GSK3beta, phosphorylate the sequence Ser-X-X-X-Ser(P) when the C-terminal serine residue is already phosphorylated. Several p53 kinases were examined for their ability to create GSK3 phosphorylation sites on the p53 protein. Our results demonstrate that phosphorylation of serine 37 of p53 by DNA-PK creates a site for GSK3beta phosphorylation at serine 33 in vitro. GSK3alpha did not phosphorylate p53 under any condition. GSK3beta increased the transcriptional activity of the p53 protein in vivo. Mutation of either serine 33 or serine 37 of p53 to alanine blocked the ability of GSK3beta to regulate p53 transcriptional activity. GSK3beta is therefore able to regulate p53 function in vivo. p53's transcriptional activity is commonly increased by DNA damage. However, GSK3beta kinase activity was inhibited in response to DNA damage, suggesting that GSK3beta regulation of p53 is not involved in the p53-DNA damage response. CONCLUSIONS: GSK3beta can regulate p53's transcriptional activity by phosphorylating serine 33. However, GSK3beta does not appear to be part of the p53-DNA damage response pathway. Instead, GSK3beta may provide the link between p53 and non-DNA damage mechanisms for p53 activation.

An essential role of NFkappaB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia.

We have recently demonstrated that myocardial adaptation to ischemia triggers a tyrosine kinase regulated signaling pathway leading to the translocation and activation of p38 MAP kinase and MAPKAP kinase 2. Since oxidative stress is developed during ischemic adaptation and since free radicals have recently been shown to function as an intracellular signaling agent leading to the activation of nuclear transcription factor, NFkappaB, we examined whether NFkappaB was involved in the ischemic adaptation process. Isolated perfused rat hearts were adapted to ischemic stress by repeated ischemia and reperfusion. Hearts were pretreated with genistein to block tyrosine kinase while SB 203580 was used to inhibit p38 MAP kinases. Ischemic adaptation was associated with the nuclear translocation and activation of NFkappaB which was significantly blocked by both genistein and SB 203580. The ischemically adapted hearts were more resistant to ischemic reperfusion injury as evidenced by better function recovery and less tissue injury during post-ischemic reperfusion. Ischemic adaptation developed oxidative stress which was reflected by increased malonaldehyde formation. A synthetic peptide containing a cell membrane-permeable motif and nuclear sequence, SN 50, which blocked nuclear translocation of NFkappaB during ischemic adaptation, significantly inhibited the beneficial effects of adaptation on functional recovery and tissue injury. In concert, SN 50 reduced the oxidative stress developed in the adapted myocardium. These results demonstrate that p38 MAP kinase might be upstream of NFkappaB which plays a role in ischemic preconditioning of heart.