Biomedical subjects

J S Lazo

Publications and source records attributed to J S Lazo.

At least 55 records · Page 3Linked to original sources

PubMed1998

Paraquat-induced phosphatidylserine oxidation and apoptosis are independent of activation of PLA2.

Paraquat is a pneumotoxin that causes lung injury by enhancing oxidative stress; however, the cellular responses to these redox events are undefined. We previously showed that paraquat produced selective peroxidation of phosphatidylserine that preceded apoptosis in 32D cells. We now report that the phospholipase A2 (PLA2) inhibitor quinacrine can attenuate phosphatidylserine oxidation and also block paraquat-induced apoptosis. Therefore, we investigated the potential for PLA2 to mediate apoptosis after paraquat. We found that, in contrast to quinacrine, the PLA2 inhibitors manoalide, aristolochic acid, and arachidonyl trifluoromethylketone failed to prevent paraquat-induced apoptosis. Moreover, no evidence of PLA2 activation was observed within 7 h after paraquat exposure. Finally, quinacrine failed to inhibit basal and 4-bromo-A-23187-induced release of [3H]arachidonic acid at concentrations that protected paraquat-induced apoptosis. We conclude that paraquat-induced phosphatidylserine oxidation and apoptosis occurred in the absence of PLA2 activation and that quinacrine protected phosphatidylserine and cell viability after paraquat in a PLA2-independent manner.

Animals↗

PubMed1998

Differential transactivation of human metallothionein-IIa in cisplatin-resistant and -sensitive cells.

Cells with acquired anticancer drug resistance frequently exhibited broad cross-resistance to other anticancer agents. Increased human metallothionein (hMT) IIa transcription has been found in some cells with acquired resistance to cisplatin (CP). A panel of 5'hMT-IIa promoter deletions linked to the chloramphenicol acetyl transferase ((5'-hMT-IIaCAT) were used to demonstrate that certain cis-elements are important for the increased hMT-IIa transcription in CP-resistant cells (SCC25/CP) compared to CP-sensitive cells (SCC25). We further identified trans-acting factor differences between SCC25 and SCC25/CP cells using gel mobility shift assays. Significant increases in binding of specific factors to the -286 to -160 and -96 to -51 region were seen in CP-resistant SCC25/CP cells compared to sensitive SCC25 cells. Using cross-competition and super gel shift analyses, we identified enhanced Spl and AP-2 binding activity in SCC25/CP cells. By Western blot analysis and immunoprecipitation, we demonstrated that the level of Spl was unchanged between the two cells types whereas AP-2 was elevated twofold in SCC25/CP cells. Our results indicated that the selection of CP-resistant phenotype in SCC25/CP was accompanied by increased Spl and AP-2 DNA binding activities, which are likely not only to enhance hMT-IIa transcription but could also alter expression of other genes responsible for a broader anticancer drug cross-resistance. Thus, altered trans-acting factors could account for the complex cross-resistant phenotype found in some anticancer drug-resistant cells.

Antineoplastic Agents↗

PubMed1998

Disruption of insulin-like growth factor-1 signaling and down-regulation of cdc2 by SC-alphaalphadelta9, a novel small molecule antisignaling agent identified in a targeted array library.

We previously reported the generation of a library of hydrophobic oxazole-based small molecules designed as inhibitors of phosphatases involved in cellular signaling and cell cycle control. One member of the targeted array library, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alphaalphadelta9), inhibited cell growth in the G0/G1 phase of the cell cycle. To investigate potential mechanisms for SC-alphaalphadelta9 antiproliferative activity, we have used mouse embryonic fibroblasts transformed with simian virus 40 large T antigen mouse embryonic fibroblasts as a model system for a malignant phenotype that depends on overexpression of cell cycle regulators and autocrine stimulation by insulin-like growth factor-1. Structure-activity relationship studies with SC-alphaalphadelta9 and four library congeners demonstrated that antiproliferative activity was not a result of overall hydrophobicity. Rather, SC-alphaalphadelta9 decreased insulin-like growth factor-1 receptor tyrosine phosphorylation, receptor expression, mitogen-activated protein kinase activation and levels of the cyclin-dependent kinase Cdc2. Less toxic congeners only partially affected receptor expression, receptor tyrosine phosphorylation and Cdc2 levels. Thus SC-alphaalphadelta9, which is structurally distinct from other known small molecules that decrease intracellular Cdc2 levels, has profound effects on intracellular signaling. Furthermore, SC-alphaalphadelta9, but not vanadate or okadaic acid, selectively inhibited the growth of simian virus 40 large T antigen mouse embryonic fibroblasts compared to the parental cells. These results suggest that overexpression of Cdc2 and increased dependence on insulin-like growth factor-1 autocrine stimulation are responsible for the increased sensitivity of simian virus 40 large T antigen mouse embryonic fibroblasts to SC-alphaalphadelta9. The SC-alphaalphadelta9 pharmacophore could be a useful platform for the development of novel antisignaling agents.

Animals↗

PubMed1997

A targeted library of small-molecule, tyrosine, and dual-specificity phosphatase inhibitors derived from a rational core design and random side chain variation.

Tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosines while dual-specificity phosphatases (DSPases) dephosphorylate contiguous and semicontiguous phosphothreonine and phosphotyrosine on cyclin dependent kinases and mitogen-activated protein kinases. Consequently, PTPases and DSPases have a central role controlling signal transduction and cell cycle progression. Currently, there are few readily available potent inhibitors of PTPases or DSPases other than vanadate. Using a pharmacophore modeled on natural product inhibitors of phosphothreonine phosphatases, we generated a refined library of novel, phosphate-free, small-molecule compounds synthesized by a parallel, solid-phase combinatorial-based approach. Among the initial 18 members of this targeted diversity library, we identified several inhibitors of DSPases: Cdc25A, -B, and -C and the PTPase PTP1B. These compounds at 100 microM did not significantly inhibit the protein serine/threonine phosphatases PP1 and PP2A. Kinetic studies with two members of this library indicated competitive inhibition for Cdc25 DSPases and noncompetitive inhibition for PTP1B. Compound AC-alphaalpha69 had a Ki of approximately 10 microM for recombinant human Cdc25A, -B, and -C, and a Ki of 0.85 microM for the PTP1B. The marked differences in Cdc25 inhibition as compared to PTP1B inhibition seen with relatively modest chemical modifications in the modular side chains demonstrate the structurally demanding nature of the DSPase catalytic site distinct from the PTPase catalytic site. These results represent the first fundamental advance toward a readily modifiable pharmacophore for synthetic PTPase and DSPase inhibitors and illustrate the significant potential of a combinatorial-based strategy that supplements the rational design of a core structure by a randomized variation of peripheral substituents.

Binding, Competitive↗

PubMed1997

Genomic structure and genetic mapping of the human neutral cysteine protease bleomycin hydrolase.

Bleomycin hydrolase (BH) is the only known eukaryotic enzyme that inactivates the widely used antineoplastic agent bleomycin (BLM) and is a primary candidate gene for protection against lethal BLM-induced pulmonary fibrosis and for BLM resistance in tumors. Human BH was found to exist as a single gene that was mapped to chromosome 17 using National Institute of General Medical Sciences human/rodent hybrid mapping panels and localized to 17q11.1-11.2 by linkage analysis using the Centre d'Etude du Polymorphisme Humain reference database. The human BH gene consisted of 11 exons ranging in size from 69-198 bp separated by introns of approximately 1 kb, reflecting the archetypal genomic structure of the cysteine protease family. A polymorphic site was identified in the eleventh exon at bp 1450 encoding either valine or isoleucine. These findings provide essential tools required to define the role of BH in BLM-induced pulmonary fibrosis and BLM resistance in tumors.

Animals↗

PubMed1997

Nucleocytoplasmic functionality of metallothionein.

Appropriate nucleocytoplasmic partitioning of proteins can direct diverse cellular processes. Metallothioneins (MTs) are thiol-rich, stress-inducible proteins that can afford protection against oxidants, mutagens, and anticancer drugs. MTs display discrete nucleocytoplasmic sequestration patterns despite their small size (Mr 6,000). We demonstrate subcellular location-specific functionality of MT using a regulated expression system that restricts MT expression to the nucleus or the cytoplasm in MT-null fibroblasts. Specifically, we found that cytoplasmic but not nuclear expression of MT decreases the level of intracellular reactive oxygen species and is more cytoprotective against the prototypic oxidizing agent tert-butyl hydroperoxide. Cytoplasmic MT expression also protects against the cytotoxicity of the heavy metal CdCl2, whereas nuclear expression protects against the cytotoxicity of the mutagenic agent N-methyl-N'-nitro-N-nitrosoguanidine. These data support the hypothesis that essential cytotoxic targets of both oxidants and heavy metals reside in the cytoplasm and establish the importance of nucleocytoplasmic partitioning for the function of small protective proteins such as MTs.

Animals↗

PubMed1997

Chemical synthesis and biological activity of a novel fluorescent etoposide derivative.

The antineoplastic activity of etoposide resides in its ability to poison the nuclear enzyme DNA topoisomerase II (topo II). The factors that control the cellular entry and subcellular distribution of etoposide remain poorly understood. Therefore, we have synthesized a novel fluorescence-labeled etoposide (Bodipyetoposide) by coupling 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionylethylenediamine (Bodipy) to 4'-benzyloxycarbonyl-4'-demethylepipodophyllotoxin beta-D-glucopyranoside, a precursor of etoposide. Bodipy-etoposide retained the ability to stabilize topo II-DNA covalent complexes in isolated nuclei, although it was significantly less potent and efficacious than etoposide. The growth inhibitory activity of Bodipy-etoposide was also approximately 200-fold less than that of etoposide in human leukemia K562 and DU-145 prostatic carcinoma cells. Nonetheless, etoposide-resistant K/VP.5 and K/VP.5-1 leukemia cells were cross-resistant to Bodipy-etoposide compared with parental K562 cells. Analysis by flow cytometry revealed a concentration-dependent Bodipy-etoposide cell association with no significant difference in drug association in the etoposide-resistant cell lines relative to the parental K562 cells. Using confocal laser scanning microscopy, we found significant cytoplasmic perinuclear localization of Bodipy-etoposide. Thus, Bodipy-etoposide displays promise as a tool to probe the factors controlling entry and subcellular distribution of etoposide-like compounds in live cells.

Antineoplastic Agents↗

PubMed1997

Diversity of metallothionein content and subcellular localization in the National Cancer Institute tumor panel.

UNLABELLED: Metallothioneins (MTs) are major thiol-containing intracellular proteins that bind metals, are induced by stress, and have been implicated in resistance to drugs and heavy metals. PURPOSE: To examine the hypothesis that the protective functionality of MT may be dictated by its subcellular localization. METHODS: We analyzed the basal MT content in 53 adherent cell lines of the National Cancer Institute (NCI) tumor panel and quantified the nuclear/cytoplasmic distribution of MT using confocal laser scanning microscopy and a recently described immunofluorescence-based algorithm. RESULTS: Among these cell types we found a 400-fold range in the basal MT levels and a tenfold range in the ratio of the nuclear to cytoplasmic MT immunostaining that was independent of basal MT content. Total MT levels and nuclear/cytoplasmic distribution were independent of total glutathione content, suggesting autonomous regulation of these protective protein and nonprotein thiol pools. Approximately 50% (29/53) of the cell lines had a greater nuclear than cytoplasmic MT density and were defined as having a karyophilic phenotype. Tissue specificity of MT localization was seen with breast cancer cell lines, which were cytoplasmophilic, whereas prostate-derived cells were karyophilic. Among the 25000 unrestricted compounds in the NCI database, we detected a correlation between total basal MT levels and resistance to CdCl2, four Pt- and two Cu-containing compounds. High nuclear/cytoplasmic MT values correlated with resistance to six Cu-, six Pb-, and one Zn-containing compounds. CONCLUSIONS: These results demonstrated significant diversity in MT content and subcellular localization in human tumor cells. Moreover, both basal MT levels and subcellular distribution appeared to be determinants of cellular responsiveness to metal-containing compounds.

Cytoplasm↗

PubMed1997

Combinatorial synthesis and biological evaluation of library of small-molecule Ser/Thr-protein phosphatase inhibitors.

In eukaryotes, phosphorylation of serine, threonine, and tyrosine residues on proteins is a fundamental posttranslational regulatory process for such functions as signal transduction, gene transcription, RNA splicing, cellular adhesion, apoptosis, and cell cycle control. Based on functional groups present in natural product serine/threonine protein phosphatase (PSTPase) inhibitors, we have designed pharmacophore model 1 and demonstrated the feasibility of a combinatorial chemistry approach for the preparation of functional analogues of 1. Preliminary biological testing of 18 structural variants of 1 has identified two compounds with growth inhibitory activity against cultured human breast cancer cells. In vitro inhibition of the PSTPase PP2A was demonstrated with compound 1d. Using flow cytometry we observed that compound f1 caused prominent inhibition in the G1 phase of the cell cycle. Thus, the combinatorial modifications of the minimal pharmacophore 1 can generate biologically interesting antiproliterative agents.

Breast Neoplasms↗

PubMed1997

Enhanced apoptosis in metallothionein null cells.

Metallothioneins (MTs) are major intracellular, zinc-binding proteins with antioxidant properties. Mouse embryonic cells null for MT due to loss of functional MT I and II genes (MT-/-) were more susceptible to apoptotic death after exposure to tert-butyl hydroperoxide or the anti-cancer agents cytosine arabinoside, bleomycin, melphalan, and cis-dichlorodiammineplatinum(II) compared with wild-type mouse embryonic cells (MT+/+). We measured basal levels of the tumor suppressor protein p53 and the death effector protein Bax and found the basal levels of both proteins were higher in MT null cells compared with MT+/+ cells. After treatment with the DNA-damaging agent cis-dichlorodiammineplatinum(II), p53 protein levels were induced in both MT+/+ and MT-/- cells with MT null cells always maintaining the highest p53 levels. The elevated sensitivity to apoptosis was not restricted to embryonic cells. Primary pulmonary fibroblasts were isolated from distinct litters of MT null, heterozygous, and wild-type mice, and all had undetectable basal MT levels. Zinc exposure increased MT levels in the wild-type and heterozygous fibroblasts but not in the MT null fibroblasts. Consistent with the induced MT levels, we found MT+/+ and MT+/- embryonic cells were less sensitive to cis-dichlorodiammineplatinum(II)-induced apoptosis compared with MT-/- cells. Our results implicate MT as a stress-responsive factor that can regulate apoptotic engagement.

Animals↗

PubMed1997

Bcl-2 inhibits selective oxidation and externalization of phosphatidylserine during paraquat-induced apoptosis.

Oxidative stress is a potential component of the final common pathway leading to apoptosis following many diverse stimuli. Here, we document that the oxidant paraquat caused apoptosis in mouse 32D cells. We examined early paraquat-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid (cis-PA) into phospholipids and high-performance liquid chromatography separation of specific phospholipid classes. Paraquat induced peroxidation of cis-PA primarily in phosphatidylserine (PS) and to a lesser extent in phosphatidylinositol (PI) within 2 h. The selective oxidation of PS occurred before signs of cytotoxicity and preceded the externalization of PS as assessed by annexin V binding. Overexpression of Bcl-2 afforded significant protection against paraquat-induced apoptosis, early PS and PI oxidation, and PS externalization but not the ultimate formation of high-molecular-weight DNA fragments. Therefore, both selective phospholipid peroxidation and DNA damage occurred after paraquat exposure, but only the former was specifically associated with apoptosis. We suggest Bcl-2 may inhibit oxidant-induced apoptosis by preventing the peroxidation of specific membrane phospholipids.

Animals↗

PubMed1997

Integrin activation protects pulmonary endothelial cells from the genotoxic effects of bleomycin.

Integrin activation promotes the survival of endothelial cells undergoing diverse forms of stress. Here we determined the ability of integrins to inhibit DNA strand breakage by bleomycin (BLM), a DNA-cleaving antitumor antibiotic that causes acute endothelial injury and subsequent pulmonary fibrosis. We found that BLM produced DNA breakage in cultured murine lung endothelial cells (MLEC) within 45 min of treatment as measured by DNA sedimentation and in situ labeling of 3'-OH by nick translation (ISNT). Two hours after the removal of BLM, we found a marked but incomplete reduction in DNA strand breakage as measured by ISNT, indicating that the damage was reversible. DNA sedimentation and ISNT demonstrated that strand breakage due to BLM was inhibited in MLEC cultured on fibronectin, and no evidence of breakage was found 2 h after removal of the drug in ISNT experiments. Gelatin, type IV collagen, laminin, and the integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro, but not the inactive Gly-Arg-Ala-Asp-Ser-Pro peptide, also inhibited DNA strand breakage. Activation of integrins, either by coating surfaces with antibodies to alpha 5-, beta 1-, or beta 3-integrin subunits or by receptor clustering with the soluble antibodies, inhibited BLM-induced DNA breakage. Inhibition of BLM-induced DNA strand breakage by soluble beta 1-integrin antibody increased with increasing antibody concentration and duration of receptor clustering before BLM treatment. Thus integrin activation protects pulmonary endothelial cells from the genotoxic effects of BLM.

Animals↗

PubMed1997

Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury.

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein with extensive metal binding capacity and potential nonenzymatic antioxidant activity. Despite the sensitivity of vascular endothelium to either heavy metal toxicity or oxidative stress, little is known regarding the role of MT in endothelial cells. Accordingly, we determined the sensitivity of cultured sheep pulmonary artery endothelial cells (SPAEC) that overexpressed MT to tert-butyl hydroperoxide (t-BOOH), hyperoxia, or 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN; peroxyl radical generator). Nontoxic doses of 10 microM Cd increased MT levels from 0.21 +/- 0.03 to 2.07 +/- 0.24 microg/mg and resulted in resistance to t-BOOH and hyperoxia as determined by reduction of Alamar blue or [3H]serotonin transport, respectively. SPAEC stably transfected with plasmids containing either mouse or human cDNA for MT were resistant to both t-BOOH and hyperoxia. In addition, we examined transition metal-independent, noncytotoxic AMVN-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid into phospholipids and high-performance liquid chromatography separation. SPAEC that overexpressed MT after gene transfer completely inhibited peroxyl oxidation of phosphatidylserine, phosphatidylcholine, and sphingomyelin (but not phosphatidylethanolamine) noted in wild-type SPAEC. These data show for the first time that MT can 1) protect pulmonary artery endothelium against a diverse array of prooxidant stimuli and 2) directly intercept peroxyl radicals in a metal-independent fashion, thereby preventing lipid peroxidation in intact cells.

Animals↗

PubMed1997

Bleomycin.

Explore the source record for details and available documents.

Animals↗

PubMed1996

Integrin activation suppresses etoposide-induced DNA strand breakage in cultured murine tumor-derived endothelial cells.

Tumor endothelium is critical for solid tumor growth and is a potential site for anticancer drug action. Within 2 h, etoposide caused marked DNA strand breakage in xenograft tumor-derived endothelial cells (TDECs). Etoposide-induced DNA breakage was inhibited by culturing TDECs on gelatin, type IV collagen, laminin, fibronectin, and the integrin ligand hexapeptide, GRGDSP, but not the inactive peptide, GRADSP. It was also inhibited when TDECs were on surfaces coated with antibodies to alpha 5, beta 1, or beta 3 integrin subunits and by clustering integrins with soluble antibodies. After 8 h with etoposide, TDECs detached from the monolayer, and 50-kb DNA fragments were seen. Fibronectin inhibited both processes. Thus, integrins are survival factors for TDEC that inhibit the genotoxicity of etoposide and may influence the sensitivity of tumors to drugs.

Animals↗

PubMed1996

Nucleophilic distribution of metallothionein in human tumor cells.

Metallothionein (MT), a major zinc-binding intracellular protein thiol, has been associated with cytoprotection from heavy metals, antineoplastic drugs, mutagens, and cellular oxidants. Despite its small mass (7 kDa), nuclear partitioning of MT has been observed in both normal and malignant tissues. The factors controlling MT sequestration are unknown. Thus, we examined the regulation of MT subcellular distribution in human cancer cell lines that exhibit prominent nuclear MT. The nuclear disposition of MT was unaltered during cell cycle passage in synchronized cells. MT redistributed to the cytoplasm when cells were exposed to reduced temperature. Cytoplasmic redistribution was also seen in DU-145 and HPC36M prostatic cancer cells after ATP depletion, but not in PC3-MA2 and SCC25/CP cells. Pretreatment with 10 microM CdCl2 did not significantly alter MT distribution but did render all cells sensitive to cytoplasmic redistribution after either reduced temperature or ATP depletion. Thus, nuclear retention of MT is energy requiring and this ability of MT to accumulate in subcellular compartments against its concentration gradient may be important in the capacity of MT to supply Zn or other metals to target sites within the cell.

Adenosine Triphosphate↗

PubMed1996

Downregulation of protein kinase C suppresses induction of apoptosis in human prostatic carcinoma cells.

Protein kinase C (PKC) has been implicated in propagating signals for apoptosis. We have investigated the effect of pharmacological modulation of PKC activity in DU-145 human androgen-independent prostatic carcinoma cells. The apoptotic death of these cells is characterized by the acquisition of classical apoptotic morphology and generation of > or = 1-mbp and 450- to 600-kbp DNA fragments in attached preapoptotic cell populations prior to cellular detachment and accrual of 30- to 50-kbp DNA fragments. We found that induction of apoptosis was arrested by downregulation of PKC activity and not by transient activation or inhibition of the enzyme. Concentrations and durations of exposure to phorbol esters that downregulated PKC activity correlated with inhibition of VP-16 or melphalan-induced morphological apoptosis and generation of the 30-to-50-kbp DNA fragments. Chronic exposure to phorbol-12,13-dibutyrate (PDBu) did not, however, suppress production of the > or = 1-mbp and 450- to 600-kbp DNA fragments found in preapoptotic cell populations, suggesting that PKC downregulation may interfere with the transition between a preapoptotic cell and an apoptotic cell. PKC isozyme analysis revealed that chronic PDBu treatment caused downregulation of PKC-alpha and -epsilon in DU-145 cells. Using concentrations of the PKC inhibitor UCN-01 that were consistent with PKC-alpha inhibition (but not PKC-epsilon inhibition), however, did not mimic the effects of chronic PDBu treatment, implying that downregulation of PKC-epsilon may be of particular importance. Together, these findings suggest that phorbol esters may act as tumor promoters by suppressing apoptosis.

Antineoplastic Agents↗

PubMed1996

Synthesis of chemoreversible prodrugs of ara-C with variable time-release profiles. Biological evaluation of their apoptotic activity.

N4-Dipeptidyl slow-release forms of the anticancer drug ara-C were prepared by acylation of the lithiated nucleotide with 4,4-dialkyloxazolinones. An azapeptide prodrug of ara-C was obtained by condensation of an amino acid hydrazide with an activated nucleotide urea. The use of unnatural amino acid residues at N4 prevented nonspecific proteolytic cleavage in biological medium. Ara-C prodrugs 10, 15, 17, and 19 released active drug with half-lives from a few minutes to several days, respectively. Activation via intramolecular N4-deacylation did not require enzymatic intervention but was strictly dependent on the structure of the peptide chain. The prodrugs 10, 15, and 17 produced similar growth inhibition as ara-C in cultured murine leukemia cells while the azapeptide prodrug 19 was less potent reflecting the slow release of active drug with this compound. All four prodrugs retained the ability to induce apoptosis in human HL-60 leukemia cells with kinetics dictated by the rate of intramolecular N4-deacylation. This the first demonstration for the control of apoptotic cell death by the modulation of drug release from prodrugs.

Animals↗