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Biomedical subjects

John S Lazo

Publications and source records attributed to John S Lazo.

At least 37 records · Page 2Linked to original sources
PubMed2004

Induction of Cdc25B expression by epidermal growth factor and transforming growth factor-alpha.

The dual specificity protein phosphatase Cdc25B regulates of the mitotic cell cycle checkpoint and is over expressed in human tumors. Given the importance of growth factors in initiating and sustaining cell proliferation, we examined their effects on Cdc25B protein expression in human cancer cells. Within 1h after epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) treatment, Cdc25B protein levels increased in growth factor responsive A549 and SCC25 cells, but not in non-responsive MDA-MB-231 cells. A functional consequence of elevated Cdc25B was implied by the concomitant decrease in phosphorylated cyclin dependent kinase, a known Cdc25B substrate, after growth factor treatment of A549 and SCC25 cells. The EGF-mediated induction of Cdc25B required a functional EGF receptor (ErbB1), as mouse embryonic fibroblasts lacking ErbB1 did not have increased Cdc25B levels after EGF treatment. Moreover, the EGFR receptor-selective tyrosine kinase inhibitor AG1478 and mitogen activated kinase kinase inhibitor U0126 blocked growth factor-mediated Cdc25B induction. Thus, EGF and TGF-alpha appear to induce cellular Cdc25B through the mitogen-activated protein kinase pathway.

Butadienes↗
PubMed2004

The liver X receptor ligand T0901317 decreases amyloid beta production in vitro and in a mouse model of Alzheimer's disease.

Recent studies indicate that oxysterols, which are ligands for the nuclear hormone liver X receptors (LXR), decrease amyloid beta (Abeta) secretion in vitro. The effect was attributed primarily to the ATP-binding cassette transporter A1 (ABCA1) transcriptionally up-regulated by ligand-activated LXRs. We now examined the effect of the synthetic LXR ligand T0901317, which can be used in vivo, on Abeta production in vitro and in APP23 transgenic mice. T0901317 applied to a variety of in vitro models, including immortalized fibroblasts from Tangier patients, and primary embryonic mouse neurons caused a concentration-dependent decrease in Abeta secretion, and this effect was increased by the addition of apolipoprotein A-I. The inhibition of Abeta production by T0901317 was cell-type specific, being more prominent in primary neurons than in non-neuronal cells. Tangier fibroblasts lacking a functional ABCA1 secreted more Abeta than control fibroblasts, thus demonstrating the role of ABCA1 in amyloid precursor protein (APP) processing and Abeta generation. T0901317 treatment of 11-week-old APP23 mice for 6 days showed a significant increase in ABCA1 expression and a decrease in the ratio of soluble APP (sAPP)beta- to sAPPalpha-cleavage products. Most importantly, the treatment caused a statistically significant reduction in the levels of soluble Abeta40 and of Abeta42 in the brain these mice. Our experiments demonstrate that T0901317 decreases amyloidogenic processing of APP in vitro and in vivo, thus supporting the search for potent and specific LXR ligands with properties allowing therapeutic application.

Alzheimer Disease↗
PubMed2004

Discovery and characterization of novel small molecule inhibitors of human Cdc25B dual specificity phosphatase.

Cdc25A and Cdc25B dual-specificity phosphatases are key regulators of cell cycle transition and proliferation. They have oncogenic properties and are overexpressed in many human tumors. Because selective Cdc25 phosphatase inhibitors would be valuable biological tools and possible therapeutic agents, we have assayed a small molecule library for in vitro inhibition of Cdc25. We now report the identification of two new structurally distinct classes of Cdc25 inhibitors with cellular activity. The cyclopentaquinoline 3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-4,8-dicarboxylic acid (5661118) and the naphthofurandione 3-benzoyl-naphtho[1,2-b]furan-4,5-dione (5169131) had in vitro IC50 values of 2.5 to 11 microM against recombinant Cdc25 and were less potent inhibitors of other phosphatases. Unlike 5661118, 5169131 caused reversible inhibition of Cdc25B and displayed competitive inhibitor kinetics. No growth inhibitory activity was seen with 5661118, whereas 10 to 30 microM 5169131 caused G1/S and G2/M arrest. We also found that 5169131 inhibited human PC-3 prostate and MDA-MB-435 breast cancer cell proliferation. Concentration-dependent Tyr15 hyperphosphorylation was seen on cyclin-dependent kinase with a 1-h 5169131 treatment, consistent with Cdc25 inhibition. Cells resistant to DNA toposiomerase II inhibitors were as sensitive to 5169131 as parental cells, indicating that this quinone compound does not inhibit topoisomerase II in vivo. Molecular modeling was used to predict a potential interaction site between the inhibitor and Cdc25B and to provide insights as to the molecular origins of the experimental observations. Based on its kinetic profile and cellular activity, we suggest that 5169131 could be an excellent tool for further studies on the cellular roles of Cdc25.

Cell Cycle↗
PubMed2004

NAD(P)H:quinone oxidoreductase-1-dependent and -independent cytotoxicity of potent quinone Cdc25 phosphatase inhibitors.

Cdc25 dual-specificity phosphatases coordinate cell cycle progression and cellular signaling. Consequently, Cdc25 inhibitors represent potential anticancer agents. We evaluated >10,000 compounds for inhibition of human Cdc25 phosphatases and identified many potent and selective inhibitors, which all contained a quinone. Bioreductive enzymes frequently detoxify or activate quinones. Therefore, we evaluated the effect of NAD(P)H:quinone oxidoreductase-1 (NQO1) and reductase-rich microsomes on the activity of three quinone-containing Cdc25 inhibitors: 2-(2-hydroxyethylsulfanyl)-3-methyl-1,4-naphthoquinone (Cpd 5, compound 5; NSC 672121), 2,3-bis-(2-hydroxyethylsulfanyl)-1,4-naphthoquinone (NSC 95397), and 6-chloro-7-(2-morpholin-4-yl-ethylamino)quinoline-5,8-dione (NSC 663284). Each inhibitor was reduced by human NQO1 (K(m) of 0.3-0.5 microM) but none by microsomes. Compounds were evaluated with six cancer cell lines containing different amounts of NQO1: HT-29 (1056 nmol/mg/min), HCT116 (660 nmol/mg/min), sublines HCT116-R30A (28 nmol/mg/min) and HCT-116R30A/NQ5 (934 nmol/mg/min), MDA-MB-231/Q2 (null NQO1), and subline MDA-MB-231/Q6 (124 nmol/mg/min) but containing similar amounts of microsomal cytochrome P450 reductase and cytochrome b(5) reductase. Growth inhibition and G2/M arrest by Cpd 5 was proportional to NQO1 levels, requiring 4- to 5-fold more Cpd 5 to inhibit HCT-116 or HCT-116R30A/NQ5 compared with HCT-116R30A. In contrast, in all tested cell lines irrespective of NQO1 level, growth inhibition and G2/M arrest by NSC 95375 and NSC 663284 were similar (average IC(50) of 1.3 +/- 0.3 and 2.6 +/- 0.4 microM, respectively). NSC 95375 and NSC 663284 also caused similar Cdk1 hyperphosphorylation, indicating similar Cdc25 inhibition. However, lower Cpd 5 concentrations were needed to produce Cdk1 hyperphosphorylation in sublines with minimal NQO1. Thus, NQO1 detoxified Cpd 5, probably by reducing it to a less active hydroquinone, whereas NSC 95397- and NSC 663284-generated cytotoxicity was unaffected by NQO1.

CDC2 Protein Kinase↗
PubMed2004

Activation of the Raf-1/MEK/Erk kinase pathway by a novel Cdc25 inhibitor in human prostate cancer cells.

BACKGROUND: The serine/threonine kinase Raf-1 is a major regulator of the mitogen activated protein kinase (MAPK) pathway, which has been associated with the progression of prostate cancer to the more advanced and androgen-independent disease. Cdc25A phosphatase has been implicated in the regulation of Raf-1 and the MAPK pathway. METHODS: We used a novel and potent Cdc25A inhibitor, 2,3-bis-[2-hydroxyethylsulfonyl]-[1,4] naphthoquinone (NSC 95397), and its congener (2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone (NSC 672121) to study the role of Cdc25A on the MAPK pathway in human prostate cancer cells. RESULTS: We found Raf-1 physically interacted with Cdc25A in PC-3 and LNCap cells and inhibitors of Cdc25A induced both extracellular signal-regulated kinase (Erk) activation and Raf-1 tyrosine phosphorylation. NSC 95397 attenuated Cdc25A and Raf-1 interactions due to accelerated degradation of Cdc25A, which was mediated by proteasome degradation. The MAPK kinase (MEK) inhibitor U0126 completely inhibited Erk activation by NSC 95397 and NSC 672121. CONCLUSIONS: These results indicate Cdc25A phosphatase regulates Raf-1/MEK/Erk kinase activation in human prostate cancer cells.

Blotting, Western↗
PubMed2004

Marine sesquiterpenoids that inhibit the lyase activity of DNA polymerase beta.

Bioassay-directed fractionation of an extract of the marine species Spongia sp. led to the discovery of the new sesquiterpenoid derivative 17-O-isoprenyldictyoceratin-C (1), the known sesquiterpenoid derivative dictyoceratin-C (2), and the sesquiterpenoid quinone ilimaquinone (3), in addition to the nucleoside 2'-deoxyuridine. The structure of the new compound 1 was determined on the basis of spectroscopic methods and by conversion of dictyoceratin-C (2) to 1.

Animals↗
PubMed2004

A scalable high-content cytotoxicity assay insensitive to changes in mitochondrial metabolic activity.

Quantitative assessments of cell proliferation and cytotoxicity are key components of anticancer drug discovery. Advancements in genomics and combinatorial chemistry have increased the demand for cytotoxicity measurements, and almost all assays for cell proliferation that are compatible with high-throughput screening are single-parameter, homogeneous biochemical assays. Furthermore, the most popular methods currently in use are based on measurements of mitochondrial activity and therefore provide only an indirect measure of cell growth. Here we describe a high-content screening (HCS) methodology that permits the direct quantification of cell numbers as well as multiple measurements of drug activity in individual cells without the ambiguities of previously described homogenous metabolism-based assays. Using two human tumor cell lines, we compared our HCS method with a commonly used homogenous colorimetric assay that detects mitochondrial activity and found the colorimetric assay substantially underrepresented the cytotoxic effects of two mechanistically diverse, clinically used, anticancer drugs: a DNA-damaging agent, bleomycin, and a tubulin stabilizer, paclitaxel. Simultaneous evaluation of cell numbers, mitochondrial mass, and nuclear morphology by HCS provided an explanation for the differential toxicity. Furthermore, we expanded the scope of the HCS assay to include tubulin mass measurements in paclitaxel-treated cells as an example for a specific drug target. The data illustrate the utility of HCS as a powerful analysis tool compatible with the demands of high-throughput screening, and adaptable to include fluorescence-based markers of drug activity.

Antineoplastic Agents↗
PubMed2003

Low molecular weight inhibitors of Myc-Max interaction and function.

c-Myc is helix-loop-helix-leucine zipper (HLH-ZIP) oncoprotein that is frequently deregulated in human cancers. In order to bind DNA, regulate target gene expression, and function in a biological context, c-Myc must dimerize with another HLH-ZIP protein, Max. A large number of c-Myc target genes have been identified, and many of the encoded proteins are transforming. Such functional redundancy, however, complicates therapeutic strategies aimed at inhibiting any single target gene product. Given this consideration, we have instead attempted to identify ways by which c-Myc itself could be effectively disabled. We have used a yeast two-hybrid approach to identify low-molecular-weight compounds that inhibit c-Myc-Max association. All of the compounds prevented transactivation by c-Myc-Max heterodimers, inhibited cell cycle progression, and prevented the in vitro growth of fibroblasts in a c-Myc-dependent manner. Several of the compounds also inhibited tumor growth in vivo. These results show that the yeast two-hybrid screen is useful for identifying compounds that can be exploited in mammalian cells. More specifically, they provide a means by which structural analogs, based upon these first-generation Myc-Max inhibitors, can be developed to enhance antitumor efficacy.

Animals↗
PubMed2003

Binding and inhibition of Cdc25 phosphatases by vitamin K analogues.

A synthetic K vitamin analogue, 2-(2-mercaptothenol)-3-methyl-1,4-naphthoquinone or Cpd 5, was previously found to be a potent inhibitor of cell growth [Nishikawa et al., (1995) J. Biol. Chem. 270, 28304-28310]. The mechanisms of cell growth were hypothesized to include the inactivation of cellular protein tyrosine phosphatases, especially the Cdc25 family [Tamura et al. (2000) Cancer Res. 60, 1317-1325]. In this study, we synthesized PD 49, a new biotin containing Cpd 5 derivative, to search for evidence of direct interaction of these arylating analogues with Cdc25A, Cdc25B, and Cdc25C phosphatases. PD 49 was shown to directly bind to GST-Cdc25A, GST-Cdc25B, their catalytic fragments, and GST-Cdc25C. The binding could be competed with excess glutathione or Cpd 5, and a cysteine-to-serine mutation of the catalytic cysteine abolished binding. This was consistent with an involvement in binding of cysteine in the catalytic domain. This interaction between PD 49 and Cdc25 also occurred in lysates of treated cells. PD 49 also bound to protein phosphatases other than Cdc25. We found that the new analogue also inhibited Hep3B human hepatoma cell growth. This growth inhibition involved ERK1/2 phosphorylation and was inhibited by a MEK antagonist. The results demonstrate a direct interaction and binding between this growth-inhibiting K vitamin derivative with both purified as well as with cellular Cdc25A, Cdc25B, and Cdc25C.

Antifibrinolytic Agents↗
PubMed2003

Regulation of Cdc25A half-life in interphase by cyclin-dependent kinase 2 activity.

Cdc25A regulates cell cycle progression, has oncogenic and anti-apoptotic activity, and is over-expressed in many human tumors. Phosphorylation by Chk1 and Cds1/Chk2 down-regulates Cdc25A levels in response to genotoxic stresses. Nevertheless, it remains unclear whether Chk1 and Cds1/Chk2 are uniquely responsible for regulating Cdc25A stability during interphase or if other kinase activities contribute. Here we report that treatment of HeLa cells with the cyclin-dependent kinase inhibitor roscovitine caused a concentration- and time-dependent increase in Cdc25A protein levels. Transfection with dominant-negative Cdk mutants demonstrated that only a Cdk2 mutant increased Cdc25A protein levels; Cdk1 and Cdk3 mutants had no effect. The increased Cdc25A protein levels were the result of an increase in the half-life of the protein; no increase in Cdc25A mRNA levels was observed. These results demonstrate Cdk2 kinase activity contributes to the labile nature of Cdc25A during interphase and redefine the nature of the Cdc25A-Cdk2 autoamplification feedback loop.

CDC2-CDC28 Kinases↗
PubMed2003

A Cdc25A antagonizing K vitamin inhibits hepatocyte DNA synthesis in vitro and in vivo.

Thioalkyl containing K vitamin analogs have been shown to be potent inhibitors of hepatoma cell growth and antagonizers of protein tyrosine phosphatase activity. We now show that they inhibit the activity of specific protein tyrosine phosphatases (PTP) in cell-free conditions in vitro, particularly the dual specificity phosphatase Cdc25A. Using primary cultures of adult rat hepatocytes that are in G0/G1 phase until stimulated into DNA synthesis by epidermal growth factor, we found that 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5) inhibited hepatocyte DNA synthesis and PTP activity in cell culture and in vivo after a two-thirds partial hepatectomy. We found a selective inhibition of Cdc25A activity in vitro, using both synthetic substrates and authentic cellular substrate, immunoprecipitated phospho-Cdk4. Intact Cpd 5-treated cells had decreased cellular Cdc25A activity and increased tyrosine phosphorylation of Cdk4, resulting in decreased phosphorylation of retinoblastoma (Rb). Loss of Cdk4 activity was confirmed using Cdk4 immunoprecipitates from either Cpd 5-treated or untreated cells and measuring its kinase activity using GST-Rb as target. We found a similar order of activity for inhibition of growth and Cdc25A activity using several thiol-containing analogs. Cdc25A inhibitors may thus be useful for defining biochemical pathways involving protein tyrosine phosphorylation that mediate cell growth inhibition.

Animals↗
PubMed2003

The carcinogen (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene induces Cdc25B expression in human bronchial and lung cancer cells.

Cdc25B regulates cell cycle progression and genetic stability. Here, we report that exposure to the environmental carcinogen (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) causes a marked increase in the expression of Cdc25B mRNA and protein levels in terminal squamous differentiated human bronchial epithelial cells and in lung cancer cells, but not in undifferentiated bronchial cells. In addition, the growth rate of lung cancer cells was increased significantly in comparison with untreated cells after chronic exposure to 0.1 micro M anti-BPDE. Furthermore, increased Cdc25B expression and decreased Cdk1 phosphorylation were observed in anti-BPDE-treated cells. We postulate that the induction of Cdc25B expression in lung cancer cells by the ultimate carcinogen anti-BPDE accelerates the further development of lung carcinogenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2003

22R-hydroxycholesterol and 9-cis-retinoic acid induce ATP-binding cassette transporter A1 expression and cholesterol efflux in brain cells and decrease amyloid beta secretion.

The ATP-binding cassette transporter A1 (ABCA1) is a major regulator of peripheral cholesterol efflux and plasma high density lipoprotein metabolism. In adult rat brain we found high expression of ABCA1 in neurons in the hypothalamus, thalamus, amygdala, cholinergic basal forebrain, and hippocampus. Large neurons of the cholinergic nucleus basalis together with CA1 and CA3 pyramidal neurons were among the most abundantly immunolabeled neurons. Glia cells were largely negative. Because cholesterol homeostasis may have an essential role in central nervous system function and neurodegeneration, we examined ABCA1 expression and function in different brain cell types using cultures of primary neurons, astrocytes, and microglia isolated from embryonic rat brain. The basal ABCA1 mRNA and protein levels detected in these cell types were increased markedly after exposure to oxysterols and 9-cis-retinoic acid, which are ligands for the nuclear hormone liver X receptors and retinoic X receptors, respectively. Functionally, the increased ABCA1 expression caused by these ligands was followed by elevated apoA-I- and apoE-specific cholesterol efflux in neurons and glia. In non-neuronal and neuronal cells overexpressing a human Swedish variant of amyloid precursor protein, 22R-hydroxycholesterol and 9-cis-retinoic acid induced ABCA1 expression and increased apoA-I-mediated cholesterol efflux consequently decreasing cellular cholesterol content. More importantly, we demonstrated that these ligands alone or in combination with apoA-I caused a substantial reduction in the stability of amyloid precursor protein C-terminal fragments and decreased amyloid beta production. These effects of 22R-hydroxycholesterol may provide a novel strategy to decrease amyloid beta secretion and consequently reduce the amyloid burden in the brain.

ATP Binding Cassette Transporter 1↗
PubMed2003

Desperately seeking genotype-selective anticancer agents.

An approach has been described to identify new agents that selectively kill mammalian cells with multiple genetic abnormalities. Compounds of this type could lead to innovative cancer treatments based on tumor genotype rather than by histology or anatomical location.

Animals↗
PubMed2003

Cell-active dual specificity phosphatase inhibitors identified by high-content screening.

Phosphorylation of extracellular signal-regulated kinase (Erk) is tightly controlled by dual specificity phosphatases (DSPases), but few inhibitors of Erk dephosphorylation have been identified. Using a high-content, fluorescence-based cellular assay and the National Cancer Institute's 1990 agent Diversity Set, we identified ten compounds (0.5%) that significantly increased phospho-Erk cytonuclear differences in intact cells. Three of the ten positive compounds inhibited the mitogen-activated protein kinase phosphatase-3 (MKP-3/PYST-1) in vitro without affecting VHR or PTP1B phosphatases. The most potent inhibitor of MKP-3 had an IC(50) of <10 microM and inhibited MKP-3 in a novel, fluorescence-based multiparameter chemical complementation assay. These results suggest that the phospho-Erk nuclear accumulation assay may be a useful tool to discover DSPase inhibitors with biological activity.

Animals↗
PubMed2003

Small molecule regulation of phosphatase-dependent cell signaling pathways.

Small molecules provide exceptionally useful tools for probing signaling targets relevant for cancer and stem cell differentiation. In contrast to genetic approaches, the application of small molecules generally offers a graded and reversible disruption of a particular pathway. The vast array of theoretical chemical entities that exist in the chemical universe are now becoming available through the production and distribution of chemical libraries generated by both traditional and combinatorial methods, which are suitable for pharmacological use. Convenient and inexpensive cell-free and cell-based assays can be used to identify chemicals that exhibit desirable antisignaling properties. We illustrate a model of how agents targeted against signaling macromolecules involved in cancer, namely dual specificity protein phosphatases, can be identified.

Antineoplastic Agents↗