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Frank Traganos

Publications and source records attributed to Frank Traganos.

35 records · Page 2Linked to original sources
PubMed2004

NO-donating nonsteroidal antiinflammatory drugs (NSAIDs) inhibit colon cancer cell growth more potently than traditional NSAIDs: a general pharmacological property?

The novel nitric oxide-donating nonsteroidal antiinflammatory drugs (NO-NSAIDs), consisting of a traditional NSAID to which a NO releasing moiety is covalently attached, may have an important role in colon cancer prevention and/or treatment. Preclinical studies have shown that NO-aspirin (NO-ASA) is more potent than traditional ASA in preventing colon cancer. Preclinical and clinical studies have also documented its superior safety, compared to traditional ASA. To evaluate the role of this structural modification on the cancer cell growth inhibitory effect of NSAIDs, we studied seven pairs of traditional NSAIDs (ASA, salicylic acid, indomethacin, sulindac, ibuprofen, flurbiprofen, piroxicam) and their corresponding NO-NSAIDs. All NO-NSAIDs (except NO-piroxicam which is a salt and not a true NO-NSAID) have greater potency in inhibiting HT-29 and HCT-15 colon cancer cell growth compared to their NSAID counterparts: the IC(50)s of the NO-NSAIDs were enhanced between 7- and 689-fold in HT-29 cells and 1.7- to 1083-fold in HCT-15 cells over those of the corresponding NSAIDs. Their growth inhibitory effect is due to a profound cell kinetic effect consisting of reduced cell proliferation and enhanced cell death. Since HT-29 cells express cyclooxygenases but HCT-15 do not, this effect appears independent of cyclooxygenase in the colon cancer cells. Thus the structural modification of these traditional NSAIDs leading to NO-NSAIDs enhances their potency in inhibiting colon cancer cell growth. Our findings suggest that the enhanced potency imparted on NSAIDs by this structural modification represents a pharmacological property that may be a general one for this class of compounds.

Analysis of Variance↗
PubMed2004

Angiotensin II stimulates nitric oxide production in pulmonary artery endothelium via the type 2 receptor.

We previously reported that angiotensin II stimulates an increase in nitric oxide production in pulmonary artery endothelial cells. The aims of this study were to determine which receptor subtype mediates the angiotensin II-dependent increase in nitric oxide production and to investigate the roles of the angiotensin type 1 and type 2 receptors in modulating angiotensin II-dependent vasoconstriction in pulmonary arteries. Pulmonary artery endothelial cells express both angiotensin II type 1 and type 2 receptors as assessed by RT-PCR, Western blot analysis, and flow cytometry. Treatment of the endothelial cells with PD-123319, a type 2 receptor antagonist, prevented the angiotensin II-dependent increase in nitric oxide synthase mRNA, protein levels, and nitric oxide production. In contrast, the type 1 receptor antagonist losartan enhanced nitric oxide synthase mRNA levels, protein expression, and nitric oxide production. Pretreatment of the endothelial cells with either PD-123319 or an anti-angiotensin II antibody prevented this losartan enhancement of nitric oxide production. Angiotensin II-dependent enhanced hypoxic contractions in pulmonary arteries were blocked by the type 1 receptor antagonist candesartan; however, PD-123319 enhanced hypoxic contractions in angiotensin II-treated endothelium-intact vessels. These data demonstrate that angiotensin II stimulates an increase in nitric oxide synthase mRNA, protein expression, and nitric oxide production via the type 2 receptor, whereas signaling via the type 1 receptor negatively regulates nitric oxide production in the pulmonary endothelium. This endothelial, type 2 receptor-dependent increase in nitric oxide may serve to counterbalance the angiotensin II-dependent vasoconstriction in smooth muscle cells, ultimately regulating pulmonary vascular tone.

Angiotensin I↗
PubMed2004

Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin.

BACKGROUND: DNA double-strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis-associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser-139; the phosphorylated H2AX is denoted gammaH2AX. The intensity of immunofluorescence (IF) of gammaH2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the drug-induced (DI) IF of gammaH2AX, and in particular to distinguish it from AA gammaH2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation. METHODS: HL-60 cells in cultures were treated with topotecan (TPT), mitoxantrone (MTX), or with DNA cross-linking drug cisplatin (CP); using multiparameter flow and laser-scanning cytometry, induction of gammaH2AX was correlated with: 1) caspase-3 activation; 2) chromatin condensation, 3) cell cycle phase, and 4) AA DNA fragmentation. The intensity of gammaH2AX IF was compensated for by an increase in histone/DNA content, which doubles during the cell cycle, and for the "programmed" H2AX phosphorylation, which occurs in untreated cells. RESULTS: In cells treated with TPT or MTX, the increase in DI-gammaH2AX IF peaked at 1.5 or 2 h, and was maximal in S- or G(1)-phase cells, respectively, for each drug. In cells treated with CP, compared with TPT, the gammaH2AX IF was less intense, peaked later (3 h) and showed no cell cycle-phase specificity. In the presence of phosphatase inhibitor calyculin A, a continuous increase in the TPT-induced gammaH2AX IF was still seen past 1.5 h, and after 3 h gammaH2AX IF was 2.7- to 3.4-fold higher than in the absence of the inhibitor. The AA gammaH2AX IF was distinguished from the DI-gammaH2AX IF by: 1) its greater intensity; 2) its prevention by caspase inhibitor zVAD-FMK; and 3) the concurrent activation of caspase-3 in the same cells. A decrease in AA gammaH2AX IF coinciding with AA chromatin condensation was seen in the late stages of apoptosis. CONCLUSIONS: Multiparameter analysis of gammaH2AX IF, caspase-3 activation, cellular DNA content, and chromatin condensation allowed us to distinguish the DI from AA H2AX phosphorylation and relate them to the cell cycle phase and stage of apoptosis. With a comparable degree of ds DNA breaks, the cells arrested at the G1 or G2/M checkpoint were less prone to undergo apoptosis than the cells replicating DNA. H2AX phosphorylation seen in CP-treated cells may be associated with DNA repair that involves nucleotide excision repair (NER) and nonhomologous end joining (NHEJ). When the primary drug-induced lesions do not involve ds DNA breaks, but ds DNA breaks are formed during DNA repair, as in the case of CP, analysis of H2AX phosphorylation may reflect extent of the repair process.

Antineoplastic Agents↗
PubMed2004

Histone H2AX phosphorylation induced by selective photolysis of BrdU-labeled DNA with UV light: relation to cell cycle phase.

BACKGROUND: The induction of DNA double-strand breaks (DSBs) in chromatin triggers histone H2AX phosphorylation (on Ser-139) by ATM-, ATR-, or DNA-dependent protein kinases (DNA-PK). Phosphorylated H2AX, denoted as gammaH2AX, can be detected immunocytochemically using an antibody that is specific to the Ser-139-phosphorylated epitope. We previously reported that the induction DSBs by DNA topoisomerase I or II inhibitors can be monitored in individual cells by measuring gammaH2AX immunofluorescence (IF) by cytometry. The present study explored whether the detection of gammaH2AX IF can serve as a marker of the presence of the DNA precursor bromodeoxyuridine (BrdU) that is incorporated into DNA. METHODS: HeLa cells growing on microscope slides were incubated with BrdU for 1 h, rinsed free of the precursor, and incubated for different periods for up to 12 h. The cells were then briefly incubated with Hoechst 33342 (to sensitize BrdU-labeled DNA to ultraviolet [UV] light), irradiated with 300 nm UV light to photolyze BrdU-labeled DNA, transferred back into culture for an additional hour, and fixed. Cells were concurrently immunostained for gammaH2AX (Alexa Fluor 633) and cyclin A (fluorescein isothiocyanate); their DNA was counterstained with 4,6-diamidino-2-phenylindole. The intensities of cellular far red (gammaH2AX), green (cyclin A), and blue (DNA) fluorescences were measured by laser scanning cytometry. RESULTS: After a 1-h pulse of BrdU followed by exposure to UV, nearly all cells with S-phase DNA content had many-fold higher gammaH2AX IF than G(1) or G(2)/M cells. The nonirradiated cells had minimal ("programmed") expression of gammaH2AX, whereas the irradiated cells incubated without BrdU had uniformly elevated levels of gammaH2AX IF independent of the cell cycle phase. Pulse-chase experiments showed that the cohort of BrdU-labeled (gammaH2AX-positive) cells progressed through G(2)/M and into G(1) phase after 8 and 12 h of growth in BrdU-free medium, respectively. Bivariate analysis of gammaH2AX versus cyclin A expression for the gated S-phase cells showed a correlation between these variables, suggesting that the rate of BrdU incorporation (DNA replication) correlates with expression of cyclin A. CONCLUSIONS: Photolysis of BrdU-labeled DNA induces DSBs and leads to H2AX phosphorylation. Detection of gammaH2AX IF indicates the presence of the incorporated BrdU, is compatible with concurrent detection of other intracellular antigens, and can be used to demonstrate cell cycle kinetics.

Benzimidazoles↗
PubMed2004

Noncatalytic requirement for cyclin A-cdk2 in p27 turnover.

Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27(kip1) is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G(1) cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCF(skp2) complex.

CDC2-CDC28 Kinases↗
PubMed2004

Cycling without cyclins.

Complex oscillations in the activation and inactivation of cyclin-dependent kinase complexes propel mammalian cells through the cycle. A recent spate of studies seems to indicate that many, if not most, of these seemingly essential molecules may, in some senses, be dispensable. If true, that would mean the essential components of the mammalian cell cycle are not much different from those found in prokaryotes. That, in turn, would imply a fair amount redundancy exists in the system which may call into question current cancer treatment strategies designed to target some of these "dispensable" cell cycle control molecules.

Animals↗
PubMed2003

Cytometry of cell cycle regulatory proteins.

Immunocytochemical detection of cyclins, inhibitors of cyclin-dependent kinases and other cell cycle regulatory proteins, combined with analysis of cell cycle position (DNA content) by multiparameter cytometry offers unique analytical possibilities. This review focuses on applications of the methodology that yield information on the abundance, modification or interactions of these proteins in relation to cell cycle progression that cannot be obtained by other methods. Particular attention is given to the use of multiparameter cytometry in analysis of the mechanism by which antitumor drugs affect cell cycle progression and induce cell cycle phase-specific apoptosis.

Animals↗
PubMed2002

Differential effect of heme oxygenase-1 in endothelial and smooth muscle cell cycle progression.

Arterial remodeling in response to pathological insult is a complex process that depends in part on the balance between vascular cell apoptosis and proliferation. Studies in experimental models suggest that HO-1 mediates neointimal formation while limiting lumen stenosing, indicating a differential effect on vascular endothelial (EC) and smooth muscle cells (SMC). We investigated the effect of HO-1 expression on cell cycle progression in EC and SMC. The addition of SnMP (10 microM), an inhibitor of HO activity, to EC or SMC for 24h, resulted in significant abnormalities in DNA distribution and cell cycle progression compared to cells treated with the HO-1 inducers, heme (10 microM) or SnCl(2) (10 microM). SnMP increased G(1) phase and decreased S and G(2)/M phases in EC while heme or SnCl(2) decreased G(1) phase, but increased S and G(2)/M phases (p<0.05). Opposite effects were obtained in SMC. SnMP decreased G(1) phase and increased S and G(2)/M phases while heme or SnCl(2) increased G(1) phase but decreased S and G(2)/M phases (p<0.05). Our data demonstrate that HO-1 regulates the cell cycle in a cell-specific manner; it increases EC but decreases SMC cycle progression. The mechanisms underlying the HO-1 cell-specific effect on cell cycle progression within the vascular wall are yet to be explored. Nevertheless, these findings suggest that cell-specific targeting of HO-1 expression may provide a novel therapeutic strategy for the treatment of cardiovascular diseases.

Cell Cycle↗
PubMed2002

Proteasome-dependent downregulation of p21(Waf1/Cip1) induced by reactive oxygen species.

After hydrogen peroxide (H(2)O(2)) treatment, the p21 (p21(Waf1/Cip1)) protein level in GM00637 fibroblast cells was rapidly decreased, reaching its nadir around 3 h. However, it rebounded within 5 hours to a level higher than that before treatment. Fluorescence microscopic analyses revealed that nuclear p21 was downregulated during the initial oxidative stress. H(2)O(2)-induced downregulation of p21 protein was accompanied by a gradual increase in p21 mRNA levels. Other inducers of genotoxic stress, such as treatment with adriamycin, a DNA damage compound, did not cause a significant decrease in p21 protein levels. Pretreatment of GM00637 cells with the proteasome inhibitors, lactacystin or MG132, completely blocked H(2)O(2)-induced p21 downregulation, suggesting that H(2)O(2) treatment accelerated p21 degradation. Conversely, cotreatment of cells with a protein synthesis inhibitor, cycloheximide, and H(2)O(2) drastically shortened the half-life of p21. Moreover, p21 mRNA levels were not downregulated by treatment with proteasome or protein synthesis inhibitors. Taken together, our studies indicate that oxidative stress induces rapid, but reversible, downregulation of functional p21 by accelerating its protein turnover.

Acetylcysteine↗
PubMed2002

Nitric oxide-donating nonsteroidal anti-inflammatory drugs inhibit the growth of various cultured human cancer cells: evidence of a tissue type-independent effect.

The novel nitric oxide (NO)-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs), which are safer than their NSAID counterparts, inhibit the growth of colon cancer cells with far greater potency than traditional NSAIDs. We examined whether NO-NSAIDs inhibit the growth of cancer cells arising from other human tissues. Human pancreatic, colon, prostate, lung, and tongue cancer cell lines were treated with NO-aspirin, -sulindac, -ibuprofen, and -indomethacin or their traditional counterparts. We determined IC(50) values, cell proliferation, apoptosis, cell cycle, cyclooxygenase (COX) protein levels, and morphological changes (light and electron microscopy). All NO-NSAIDs inhibited the growth of all cancer cell lines studied. The potency of NO-NSAIDs was 11- to 6000-fold greater than that of their counterparts (except for the effect of sulindac on lung cancer cells). NO-aspirin was consistently the most potent NO-NSAID in all cell lines tested (except for the lung cancer cell line), sometimes in excess of 100-fold over the other three NO-NSAIDs. NO-NSAIDs inhibited cell proliferation, induced apoptosis, and altered cell cycle phase distribution (G2/M to G0/G1 block). All altered cellular morphology, whereas NO-aspirin induced nuclear disintegration ("atypical" cells) established by electron microscopy. NO-aspirin showed similar effects on two pancreatic cancer cell lines, BxPC-3 (expresses COX) and MIA PaCa-2 (no COX expression), suggesting a COX-independent effect. NO-NSAIDs showed a tissue-type-independent effect. Their pleiotropic effects involve cell renewal, cell death, and cell cycle phase transitions. These results raise the possibility that NO-NSAIDs possess chemopreventive and/or chemotherapeutic activity against a wide variety of human cancers.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2002

Cell cycle arrest and apoptosis induced by human Polo-like kinase 3 is mediated through perturbation of microtubule integrity.

Human Polo-like kinase 3 (Plk3, previously termed Prk or Fnk) is involved in regulation of cell cycle progression through the M phase (B. Ouyang, H. Pan, L. Lu, J. Li, P. Stambrook, B. Li, and W. Dai, J. Biol. Chem. 272:28646-28651, 1997). Here we report that in most interphase cells endogenous Plk3 was predominantly localized around the nuclear membrane. Double labeling with Plk3 and gamma-tubulin, the latter a major component of pericentriole materials, revealed that Plk3 was closely associated with centrosomes and that its localization to centrosomes was dependent on the integrity of microtubules. Throughout mitosis, Plk3 appeared to be localized to mitotic apparatus such as spindle poles and mitotic spindles. During telophase, a significant amount of Plk3 was also detected in the midbody. Ectopic expression of Plk3 mutants dramatically changed cell morphology primarily due to their effects on microtubule dynamics. Expression of a constitutively active Plk3 (Plk3-A) resulted in rapid cell shrinkage, which led to formation of cells with an elongated, unsevered, and taxol-sensitive midbody. In contrast, cells expressing a kinase-defective Plk3 (Plk3(K52R)) mutant exhibited extended, deformed cytoplasmic structures, the phenotype of which was somewhat refractory to taxol treatment. Expression of both Plk3-A and Plk3(K52R) induced apparent G(2)/M arrest followed by apoptosis, although the kinase-defective mutant was less effective. Taken together, our studies strongly suggest that Plk3 plays an important role in the regulation of microtubule dynamics and centrosomal function in the cell and that deregulated expression of Plk3 results in cell cycle arrest and apoptosis.

Apoptosis↗
PubMed2002

Histone deacetylase inhibitors all induce p21 but differentially cause tubulin acetylation, mitotic arrest, and cytotoxicity.

By preventing deacetylation of histones, histone deacetylase inhibitors (HDIs) transcriptionally induce p21. Here we show that the HDIs sodium butyrate (Bu), trichostatin A (TSA) and depsipeptide (FR901228) all induced p21, but only TSA and FR901228 caused mitotic arrest (in addition to arrest in G1 and G2). The ability to cause mitotic arrest correlated with the higher cytotoxicity of these compounds. Although causing mitotic arrest, TSA and FR901228 (unlike paclitaxel) did not affect tubulin polymerization. Unlike FR9012208, TSA caused acetylation of tubulin at lysine 40; both soluble tubulin and microtubules were acetylated. Whereas the induction of p21 reached a maximum by 8 h, tubulin was maximally acetylated after only 1 h of TSA treatment. Tubulin acetylation was detectable after treatment with 12-25 ng/ml TSA although acetylation plateaued at 50 ng/ml TSA, coinciding with G2-M arrest, appearance of cells with a sub-2N DNA content, poly(ADP-ribose) polymerase cleavage, and rapid cell death. We conclude that HDIs have differential effects on non-histone deacetylases and that rapid acetylation of tubulin caused by TSA is a marker of nontranscriptional effects of TSA.

Anesthetics, Intravenous↗
PubMedDate not supplied

Effects of PC-SPES on proliferation and expression of AR/PSA in androgen-responsive LNCaP cells are independent of estradiol.

Previous studies have suggested that the clinical efficacy of PC-SPES, a dietary supplement used frequently by men diagnosed with androgen-dependent (AD) or androgen-independent (AI) prostate cancer (CaP), is mechanistically attributed to estrogenic components present in the herbal mixture. To test this hypothesis, we compared estradiol (1 nM), potentially an active principle in PC-SPES, with PC-SPES (using an amount equivalent to 1 nM estradiol) on cell proliferation, induction of apoptosis, and regulation of prostate specific genes, PSA and AR, in androgen-responsive LNCaP cells. Cells cultured in steroid-proficient (FBS) or-deficient (CS-FBS) media to simulate hormonal status pre- and post-castration in vivo, were incubated with estradiol or PC-SPES. Proliferation was reduced in PC-SPES treated cells cultured in media supplemented with FBS or CS-FBS; in contrast, addition of estradiol had no effect on proliferation in FBS cultures, and elicited a 45% growth increase in CS-PBS-supplemented cultures. The differential proliferative response of LNCaP cells to PC-SPES vs. estradiol was also supported by changes in PCNA expression, cell viability, cell cycle phase distribution, and induction of apoptosis. Estradiol elicited time-dependent increases in secreted PSA, whereas PC-SPES suppressed PSA secretion, in both culture conditions. In FBS cultures, PC-SPES lowered intracellular AR and PSA by 61% and 17%, respectively, while estradiol increased intracellular PSA, in parallel with a 42% decrease in AR expression. In comparison with cells maintained with CS-FBS, estradiol induced substantial increases in both intracellular PSA and AR, whereas PC-SPES resulted in a smaller increase in intracellular PSA without affecting the expression of AR. These studies show that the antiproliferative and gene modulatory effects of PC-SPES in androgen-dependent human prostate cancer cells are mechanistically and functionally distinct from effects attributable to estradiol.

Antineoplastic Agents, Phytogenic↗
PubMedDate not supplied

Arsenic trioxide arrests cells early in mitosis leading to apoptosis.

Arsenic trioxide (As(2)O(3)) is highly effective in the treatment of acute promyelocytic leukemias that express the promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARalpha) fusion protein. However, evidence has accumulated that As(2)O(3) induces apoptosis regardless of PML-RARalpha status. Here we show that, at clinically relevant concentrations, As(2)O(3) causes S and G(2)M phase arrest of both PML-RARalpha-positive and -negative leukemia cell lines, thus inhibiting their growth. Apoptotic cells are generated predominately from the G(2)M fraction. Using several independent methods, we demonstrate that the cells accumulated in the G(2)M peak consist primarily of cells arrested in the early stages of mitosis, prophase, prometaphase and metaphase. In mitotic cells, there was significant activation of caspases, PARP cleavage, and morphological changes characteristic of apoptosis. Unlike microtubule-active drugs that arrest cells in metaphase, arsenic trioxide did not affect the architecture of microtubules. Our data suggest that the antileukemic activities of arsenic may be a result of mitotic arrest which culminates in apoptosis.

Antineoplastic Agents↗
PubMedDate not supplied

DNA damage induced by DNA topoisomerase I- and topoisomerase II-inhibitors detected by histone H2AX phosphorylation in relation to the cell cycle phase and apoptosis.

Histone H2AX is phosphorylated on Ser-139 by ATM kinase in response to damage that induces dsDNA breaks. Immunocytochemical detection of phosphorylated H2AX (gammaH2AX), thus, reveals the presence of dsDNA breaks in chromatin. Multiparameter cytometry was presently used to correlate the appearance of gammaH2AX with: a. cell cycle phase; b. caspase-3 activation; and c. apoptosis-associated DNA fragmentation in individual human leukemic HL-60 cells treated with the DNA topoisomerase I (topo1) inhibitors topotecan (TPT) and camptothecin (CPT) or with the topo2 inhibitor mitoxantrone (MTX). In response to TPT or CPT maximal increase of gammaH2AX immunofluorescence was seen in S-phase cells by 90 min. In contrast, following MTX treatment the maximal rise of gammaH2AX was detected at 2 h in G1 cells and the cell cycle phase specificity was much less apparent. A linear relationship between the drug concentration and increase of gammaH2AX immunofluorescence was seen only up to 200 nM TPT; a decline in gammaH2AX was apparent at a concentration range between 0.4 and 1.6 microM TPT. Thus, the intensity of gammaH2AX immunofluorescence, as a marker of cell survival following TPT treatment, can be used only within a limited range of drug concentration. Following treatment with TPT, CPT or MTX the peak of H2AX phosphorylation preceded caspase-3 activation and the appearance of apoptosis-associated DNA fragmentation, both selective to S-phase cells. Progression of apoptosis was paralleled by a decrease in gammaH2AX immunofluorescence. The data also indicate that regardless whether treated with inhibitors of topo1 or topo2, at comparable levels of dsDNA breaks, the cells replicating DNA have a higher proclivity to undergo apoptosis compared to G1 or G2/M cells.

Apoptosis↗