High UV damage and low repair, but not cytosine deamination, stimulate mutation hotspots at ETS binding sites in melanoma.

Noncoding mutation hotspots have been identified in melanoma and many of them occur at the binding sites of E26 transformation-specific (ETS) proteins; however, their formation mechanism and functional impacts are not fully understood. Here, we used UV (Ultraviolet) damage sequencing data and analyzed cyclobutane pyrimidine dimer (CPD) formation, DNA repair, and CPD deamination in human cells at single-nucleotide resolution. Our data show prominent CPD hotspots immediately after UV irradiation at ETS binding sites, particularly at sites with a conserved TTCCGG motif, which correlate with mutation hotspots identified in cutaneous melanoma. Additionally, CPDs are repaired slower at ETS binding sites than in flanking DNA. Cytosine deamination in CPDs to uracil is suggested as an important step for UV mutagenesis. However, we found that CPD deamination is significantly suppressed at ETS binding sites, particularly for the CPD hotspot on the 5' side of the ETS motif, arguing against a role for CPD deamination in promoting ETS-associated UV mutations. Finally, we analyzed a subset of frequently mutated promoters, including the ribosomal protein genes RPL13A and RPS20, and found that mutations in the ETS motif can significantly reduce the promoter activity. Thus, our data identify high UV damage and low repair, but not CPD deamination, as the main mechanism for ETS-associated mutations in melanoma and uncover important roles of often-overlooked mutation hotspots in perturbing gene transcription.

Who gets a license: DNA synthesis in quiescent cells re-entering the cell cycle.

The precise regulation of the entry into S phase is critical for preventing genome instability. The first step in the initiation of eukaryotic DNA synthesis occurs in G1 phase cells and involves the loading of the conserved MCM helicase onto multiple origins of replication in a process known as origin licensing. In proliferating metazoan cells, an origin-licensing checkpoint delays initiation until high levels of MCM loading occur, with excess origins being licensed. One function of this checkpoint is to ensure that S phase can be completed in the face of replication stress by activation of dormant MCM bound origins. However, when both metazoan and yeast cells enter S phase from quiescence or G0 phase, a non-growing but reversible cell cycle state, origins are significantly under-licensed. In metazoan cells, under-licensing is the result of a compromised origin-licensing checkpoint. In budding yeast, our study has revealed that under-licensing can be attributed to the chromatin structure at a class of origins that is inhibitory to the binding of MCM. Thus, defects in multiple pathways may contribute to the failure to fully license origins in quiescent cells re-entering the cell cycle, thereby promoting a higher risk of genome instability.

Chromatin structure restricts origin utilization when quiescent cells re-enter the cell cycle.

Quiescent cells reside in G0 phase, which is characterized by the absence of cell growth and proliferation. These cells remain viable and re-enter the cell cycle when prompted by appropriate signals. Using a budding yeast model of cellular quiescence, we investigated the program that initiated DNA replication when these G0 cells resumed growth. Quiescent cells contained very low levels of replication initiation factors, and their entry into S phase was delayed until these factors were re-synthesized. A longer S phase in these cells correlated with the activation of fewer origins of replication compared to G1 cells. The chromatin structure around inactive origins in G0 cells showed increased H3 occupancy and decreased nucleosome positioning compared to the same origins in G1 cells, inhibiting the origin binding of the Mcm4 subunit of the MCM licensing factor. Thus, quiescent yeast cells are under-licensed during their re-entry into S phase.

Regulation of UV damage repair in quiescent yeast cells.

Non-growing quiescent cells face special challenges when repairing lesions produced by exogenous DNA damaging agents. These challenges include the global repression of transcription and translation and a compacted chromatin structure. We investigated how quiescent yeast cells regulated the repair of DNA lesions produced by UV irradiation. We found that UV lesions were excised and repaired in quiescent cells before their re-entry into S phase, and that lesion repair was correlated with high levels of Rad7, a recognition factor in the global genome repair sub-pathway of nucleotide excision repair (GGR-NER). UV exposure led to an increased frequency of mutations that included C->T transitions and T > A transversions. Mutagenesis was dependent on the error-prone translesion synthesis (TLS) DNA polymerase, Pol zeta, which was the only DNA polymerase present in detectable levels in quiescent cells. Across the genome of quiescent cells, UV-induced mutations showed an association with exons that contained H3K36 or H3K79 trimethylation but not with those bound by RNA polymerase II. Together, the data suggest that the distinct physiological state and chromatin structure of quiescent cells contribute to its regulation of UV damage repair.

Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena.

The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress.

Developmental regulation of the Tetrahymena thermophila origin recognition complex.

The Tetrahymena thermophila DNA replication machinery faces unique demands due to the compartmentalization of two functionally distinct nuclei within a single cytoplasm, and complex developmental program. Here we present evidence for programmed changes in ORC and MCM abundance that are not consistent with conventional models for DNA replication. As a starting point, we show that ORC dosage is critical during the vegetative cell cycle and development. A moderate reduction in Orc1p induces genome instability in the diploid micronucleus, aberrant division of the polyploid macronucleus, and failure to generate a robust intra-S phase checkpoint response. In contrast to yeast ORC2 mutants, replication initiation is unaffected; instead, replication forks elongation is perturbed, as Mcm6p levels decline in parallel with Orc1p. Experimentally induced down-regulation of ORC and MCMs also impairs endoreplication and gene amplification, consistent with essential roles during development. Unexpectedly Orc1p and Mcm6p levels fluctuate dramatically in developing wild type conjugants, increasing for early cycles of conventional micronuclear DNA replication and macronuclear anlagen replication (endoreplication phase I, rDNA gene amplification). This increase does not reflect the DNA replication load, as much less DNA is synthesized during this developmental window compared to vegetative S phase. Furthermore, although Orc1p levels transiently increase prior to endoreplication phase II, Orc1p and Mcm6p levels decline when the replication load increases and unconventional DNA replication intermediates are produced. We propose that replication initiation is re-programmed to meet different requirements or challenges during the successive stages of Tetrahymena development.

Induction of necrosis and apoptosis to KB cancer cells by sanguinarine is associated with reactive oxygen species production and mitochondrial membrane depolarization.

Sanguinarine is a benzopheanthridine alkaloid present in the root of Sanguinaria canadensis L. and Chellidonium majus L. In this study, sanguinarine (2 and 3 microM) exhibited cytotoxicity to KB cancer cells by decreasing MTT reduction to 83% and 52% of control after 24-h of exposure. Sanguinarine also inhibited the colony forming capacity (>52-58%) and growth of KB cancer cells at concentrations higher than 0.5-1 microM. Short-term exposure to sanguinarine (>0.5 microM) effectively suppressed the adhesion of KB cells to collagen and fibronectin (FN). Sanguinarine (2 and 3 microM) induced evident apoptosis as indicated by an increase in sub-G0/G1 populations, which was detected after 6-h of exposure. Only a slight increase in cells arresting in S-phase and G2/M was noted. Induction of KB cell apoptosis and necrosis by sanguinarine (2 and 3 microM) was further confirmed by Annexin V-PI dual staining flow cytometry and the presence of DNA fragmentation. The cytotoxicity by sanguinarine was accompanied by an increase in production of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential as indicated by single cell flow cytometric analysis of DCF and rhodamine fluorescence. NAC (1 and 3 mM) and catalase (2000 U/ml) prevented the sanguinarine-induced ROS production and cytotoxicity, whereas dimethylthiourea (DMT) showed no marked preventive effect. These results suggest that sanguinarine has anticarcinogenic properties with induction of ROS production and mitochondrial membrane depolarization, which mediate cancer cell death.

Antiplatelet effect of sanguinarine is correlated to calcium mobilization, thromboxane and cAMP production.

Sanguinarine is a plant alkaloid present in the root of Sanguinaria canadensis and Poppy fumaria species. Sanguinarine has been used as an antiseptic mouth rinse and a toothpaste additive to reduce dental plaque and gingival inflammation. In this study, we investigated the antiplatelet effects of sanguinarine, aiming to extend its potential pharmacological applications. Sanguinarine inhibited platelet aggregation induced by arachidonic acid (AA), collagen, U46619 and sub-threshold concentration of thrombin (0.05 U/ml) with IC(50) concentrations of 8.3, 7.7, 8.6 and 4.4 microM, respectively. Sanguinarine (5-10 microM) inhibited 10-31% of platelet TXB(2) production, but not platelet aggregation induced by higher concentration of thrombin (0.1 U/ml). SQ29548, a thromboxane receptor antagonist, inhibited the AA-induced platelet aggregation but not TXB(2) production. Sanguinarine suppressed cyclooxygenase-1 (COX-1) activity (IC(50)=28 microM), whereas its effect on COX-2 activity was minimal. Sanguinarine (8, 10 microM) further inhibited the AA-induced Ca(2+) mobilization by 27-62%. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the inhibitory effect of sanguinarine toward AA-induced platelet Ca(2+) mobilization and aggregation. These results suggest that sanguinarine is a potent antiplatelet agent, which activates adenylate cyclase, inhibits platelet Ca(2+) mobilization, TXB(2) production as well as suppresses COX-1 enzyme activity. Sanguinarine may have therapeutic potential for treatment of cardiovascular diseases related to platelet aggregation.

Toxic and metabolic effect of sodium butyrate on SAS tongue cancer cells: role of cell cycle deregulation and redox changes.

Butyrate is a metabolite produced by oral and colonic microorganism. Butyrate has been shown to reduce colon cancer, whereas its role in oral carcinogenesis is not clear. Butyrate concentration in dental plaque and saliva ranged from 0.2 to 16 mM. In this study, we found that sodium butyrate inhibited the growth of SAS tongue cancer cells by 32% and 53% at concentrations of 1 and 2mM, respectively. Low concentrations of sodium butyrate (1-8mM) induced G0/G1 cell cycle arrest of SAS cells, whereas concentrations of 4-16 mM elicited G2/M arrest and a slight increase in apoptotic cell populations. These events were concomitant with induction of intracellular reactive oxygen species (ROS) production. An elevation in p21 mRNA and protein level was noted in SAS cells by sodium butyrate. On the contrary, a decline of cyclin Bl, cdc2 and cdc25C mRNA and protein expression in SAS cells was found after exposure to sodium butyrate. In addition, no evident increase in cdc2 inhibitory phosphorylation was found in sodium butyrate-treated SAS cancer cells. Inclusion of N-acetyl-l-cysteine (NAC) (3mM), catalase (1000 U/ml) and dimethylthiourea (DMT, 5mM), and also SOD (500 U/ml) attenuated the sodium butyrate-induced ROS production in SAS cells. However, they were not able to prevent the cell cycle arrest, apoptosis and growth inhibition in SAS cells induced by 1, 2 and 16 mM of sodium butyrate. These results indicate that sodium butyrate is toxic and inhibits the tongue cancer cell growth via induction of cell cycle arrest and apoptosis. Sodium butyrate mediates these events by mechanisms additional to ROS production.

Prolonged exposure to arecoline arrested human KB epithelial cell growth: regulatory mechanisms of cell cycle and apoptosis.

Arecoline, the main areca alkaloid in betel quid (BQ), is reported to have cytotoxic, genotoxic, and mutagenic effects in various cells. It shows strong correlation to the incidence of oral submucous fibrosis, leukoplakia, and oral cancer. To clarify the role of arecoline in BQ-induced carcinogenesis, primary human gingival keratinocyes (GK) and human KB epithelial cells were used for studying the molecular mechanisms of arecoline-mediated cell cycle deregulation for comparison. After 24 h of exposure, arecoline (0.2-0.8 mM) inhibited KB cell growth in a dose- and time-dependent manner with a reduction in cell number by 27-37 and 37-58%, respectively, as determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and sulforhodamine B (SRB) assays. Incubation of KB cells with arecoline (0.1-0.4 mM) caused late-S and G2/M phases' cell cycle arrest. Western blot analysis revealed that arecoline induced cyclin Bl, Wee 1, and phosphorylated cdc2 protein levels whereas it declined p21 protein expression in KB cancer cells. Nevertheless, arecoline induced p21, but decreased cdc2 and cyclin B1 protein levels in GK. We demonstrated that higher concentrations of arecoline (0.2-1.2 mM) induced both cell necrosis and apoptosis as detected by DNA fragmentation and Annexin V-PI staining after long-term (48 h) treatment. Our results suggest that differential regulation of S and/or G2/M cell cycle-related proteins in the GK and KB cells play a crucial role in different stages of BQ-mediated carcinogenesis.

Signaling mechanism of thrombin-induced gingival fibroblast-populated collagen gel contraction.

1.--Thrombin is activated during gingival tissue injury and inflammation. Thrombin (platelet)-rich plasma has been used for periodontal regeneration with success. Thrombin and other bacterial proteases also affect the functions of adjacent periodontal cells via stimulation of protease-activated receptors (PARs). 2.--We noted that thrombin (0.1-2 U ml(-1)), human, and frog PAR-1 agonist peptide (20-240 microM) induced the gingival fibroblast (GF)-populated collagen gel contraction within 2 h of exposure. However, PAR-2, PAR-3, and PAR-4 agonist peptide (20-240 microM) showed little effect on collagen gel contraction. U73122 (phospholipase C inhibitor) and 2-APB (IP3 antagonist) were effective in inhibition of GF contraction. 3.--Thrombin-induced GF contraction was inhibited by 5 mM EGTA (an extracellular calcium chelator) and verapamil (an L-type calcium channel blocker). In addition, W7 (10 and 25 microM, a calcium/calmodulin (CaM) inhibitor), ML-7 (50 microM, myosin light chain kinase (MLCK) inhibitor), and HA1077 (100 microM, Rho kinase inhibitor) completely inhibited the thrombin-induced collagen gel contraction. Thrombin also induced the phosphorylation of ERK1/ERK2 and elevated the Rho-GTP levels in GF. 4.--However, U0126 only partially inhibited the thrombin-induced GF contraction. Similarly, wortmannin (100 nM), LY294002 (20 microM) (two PI3K inhibitor) and genistein also showed partial inhibition. Moreover, NAC was not able to suppress the GF contraction, as supported by the slight decrease in reactive oxygen species production in GF by thrombin. 5.--Thrombin also stimulated metalloproteinase-2 (MMP-2) and MMP-3 production in GF. But addition of GM6001 or 1,10-phenanthroline, two MMP inhibitors, could not inhibit the thrombin-induced GF contraction. 6.--These results indicate that thrombin is crucial in the periodontal inflammation and wound healing by promoting GF contraction. This event is mainly mediated via PAR-1 activation, PLC activation, extracellular calcium influx via L-type calcium channel, and the calcium/CaM-MLCK and Rho kinase activation pathway.

The induction of prostaglandin E2 production, interleukin-6 production, cell cycle arrest, and cytotoxicity in primary oral keratinocytes and KB cancer cells by areca nut ingredients is differentially regulated by MEK/ERK activation.

There are about 200-600 million betel quid (BQ) chewers in the world. BQ chewing is one of the major risk factor of hepatocarcinoma, oropharyngeal, and esophagus cancers in Taiwan, India, and Southeast Asian countries. Thus, the precise molecular mechanisms deserve investigation. We used cultured primary keratinocytes and KB cells, RT-PCR, flow cytometry, Western blotting, and ELISA to evaluate whether alterations in early gene expression is crucial in the carcinogenic processes of BQ. We observed the induction of c-Fos mRNA expression in human gingival keratinocyte (GK) and KB carcinoma cells by areca nut (AN) extract and arecoline. A maximal increment in c-fos gene expression was shown at about 30 min after challenge. AN extract (100-800 microg/ml) and arecoline (0.1-0.8 mM) also stimulated ERK1/ERK2 phosphorylation with a maximal stimulation at 5-10 min of exposure. Pretreatment by U0126 (30 microM), a MEK inhibitor, markedly inhibited the c-Fos, cyclooxygenase-2 (COX-2), and IL-6 mRNA expression of the KB epithelial cells. In addition, U0126 and PD98059 (50 microM) also decreased AN extract- and arecoline-associated PGE2 and IL-6 production in GK and KB cells. However, U0126 by itself arrested the cells in G0/G1 phase, but was not able to prevent AN- and arecoline-induced cell death or apoptosis. In contrast, U0126 enhanced the AN-induced apoptosis of KB cells. AN ingredients thus play a significant role in the pathogenesis of oropharyngeal cancer by activation of MEK1/ERK/c-Fos pathway, which promotes keratinocyte inflammation, cell survival, and affects cell cycle progression.

Hamsters chewing betel quid or areca nut directly show a decrease in body weight and survival rates with concomitant epithelial hyperplasia of cheek pouch.

Betel quid (BQ) chewing is strongly associated with the occurrence of oral leukoplakia, oral submucous fibrosis, and oral cancer. There are about 200-600 million BQ chewers in the world. Previous animal studies support the potential carcinogenicity of BQ in different test systems. However, little animal experiment has let hamsters or rats to chew BQ directly, similar to that in humans. In the present study, we established a hamster model of chewing BQ or areca nut (AN). A total of 81 2-week-old hamsters were randomly divided into three groups: 25 for control group, 28 for BQ-chewing group, and 28 for AN-chewing group. These animals were fed with powdered diet with/without BQ or AN for 18 months. Although the consumption of BQ or AN showed some variations, hamsters fed with powdered diet could chew and grind AN or BQ into small pieces of coarse fibers during the entire experimental period. The survival rate of AN-chewing hamsters decreased significantly after 6 months of exposure. The mean survival time was 15.6 +/- 0.9 months for control animals, 13.6 +/- 0.98 months for AN-chewing animals, and 15.7 +/- 0.55 months for BQ-chewing animals. The body weight of BQ- or AN-chewing animals also decreased after 4-13 months. Hamsters fed with AN for 18 months showed hyperkeratosis in 80% and acanthosis in 50% of cheek pouches. Animals fed with BQ for 18 months also showed hyperkeratosis in 93% and acanthosis in 14% of cheek pouches. These results indicate that AN and BQ components may induce alterations in proliferation and differentiation of oral epithelial cells. Animal model of chewing BQ or AN can be useful for future tumor initiation, promotion and chemoprevention experiments simulating the condition of BQ chewing in humans.

Roles of keratinocyte inflammation in oral cancer: regulating the prostaglandin E2, interleukin-6 and TNF-alpha production of oral epithelial cells by areca nut extract and arecoline.

Betel quid (BQ) chewing is an etiologic factor of oral cancer and submucus fibrosis (OSF). Keratinocyte inflammation is crucial for the pathogenesis of cancer and tissue fibrosis. We found that areca nut (AN) extract (100-400 micro g/ml) induced PGE2 production by KB cells by 2.34- to 23.1-fold and also TNF-alpha production by gingival keratinocytes (GK). Arecoline (0.2-1.2 mM) elevated PGE2 production by KB cells by 2.5- to 6.1-fold. AN extract (200-400 micro g/ml) also induced IL-6 production by GK (7.5- to 8.4-fold) and KB cells. In contrast, arecoline (0.1-1.2 mM) suppressed IL-6 production by GK and KB cells, with 42-81 and 41-63% inhibition, respectively. A 48 h exposure of GK to 800-1200 micro g/ml AN extract led to 37-69% cell death. Arecoline cytotoxicity to GK was noted at concentrations of 0.8-1.2 mM, which led to 28-38% cell death. AN extract (400-800 micro g/ml) induced Cox-2 and IL-6 mRNA expression and also COX-2 protein production by KB cells. IL-6 (5-100 ng/ml) suppressed GK growth by 20-33%, but enhanced oral fibroblast (OMF) and KB cell growth. PGE2 (0.05-5 micro g/ml) and anti-IL-6 antibody (ab) (50-1000 ng/ml) showed little effect on GK and KB cell growth. Incubation of GK and KB cells with aspirin, anti-IL-6 ab and anti-TNF-alpha ab showed little effect on arecoline- and AN-induced cytotoxicity, cell cycle arrest and apoptosis. Exposure to anti-TNF-alpha ab slightly affected arecoline- and AN-modulated PGE2 and IL-6 production by GK and KB cells. Arecoline- and AN-conditioned medium decreased phytohemagglutinin-mediated CD4+ and CD8+ T cell activation. These results indicate that BQ chewing contributes to the pathogenesis of cancer and OSF by impairing T cell activation and by induction of PGE2, TNF-alpha and IL-6 production, which affect oral mucosal inflammation and growth of OMF and oral epithelial cells.