Real-time molecular and cellular analysis: the new frontier of drug discovery.

The pharmaceutical industry is currently facing the challenge of maintaining increased efficiency and productivity while contending with a deluge of genomic and high-throughput screening data. To ease the bottlenecks at target validation and lead optimization, the industry must look to the living cell, the ultimate target of all drugs, as a source of new biological knowledge. This new 'cell-centric' perspective must integrate reagents that report on the state of molecular processes within the cell, automated detection and analysis of these processes, and cellular knowledge, building into a single platform.

Energy-dependent nuclear binding dictates metallothionein localization.

Metallothioneins (MTs) are low molecular weight, stress-activated proteins that protect cells against heavy metals, oxidants, and some electrophilic drugs. Both nuclear and cytoplasmic MT phenotypes have been observed in cells even though MTs (6 kDa) are well below the size exclusion limit for diffusion through the nuclear envelope. To study the factors controlling MT subcellular partitioning, we covalently linked MTII to a fluorescent label and examined its subcellular distribution in response both to pharmacologic and physical perturbations. Fluorescent MTII localized to the nucleus of digitonin-permeabilized human SCC25 carcinoma cells, consistent with its endogenous distribution in these cells. Nuclear sequestration of the fluorescent MTII was inhibited by a 100-fold molar excess of unlabeled MTII and by wheat germ agglutinin, indicating a saturable binding mechanism and the involvement of one or more glycoproteins, respectively. Depletion of adenosine triphosphate (ATP) inhibited MTII nuclear localization, implying energy-dependent nuclear translocation or retention of MT. Neither chilling nor the absence of cytosolic extracts inhibited nuclear sequestration of MTII, supporting diffusion-based entry mechanism. In situ biochemical extractions of the nuclear MTII revealed at least two distinct binding activities. Collectively, these data indicate that MTII diffuses into the nucleus of SCC25 cells, where it is selectively and actively retained by nuclear binding factors, imparting its localization phenotype.

Cell cycle dependent subcellular distribution of Cdc25B subtypes.

The dual specificity phosphatase and oncogene Cdc25B has been implicated in the G2/M cell cycle checkpoint, but the mode by which it is regulated remains poorly understood. Regional subcellular redistribution of proteins represents a unique potential regulatory mechanism. Thus, we examined in live cells the subcellular localization characteristics of Cdc25B2 and Cdc25B3 fused to green fluorescent protein. Cdc25B2 partitioned primarily in the cytoplasm during G1 and progressively migrated to the nucleus as cells transited from S to G2/M phase. In contrast, Cdc25B3 maintained a homogeneously staining diffuse phenotype irrespective of cell cycle phase. Treatment of the Cdc25B2-green fluorescent protein stable transfectants with vanadate inhibited the cell cycle dependency of intracellular distribution, while okadaic acid had little effect except in G1, suggesting regulation by at least one phosphorylation-dependent pathway. The DNA topoisomerase II poison and DNA damaging agent, etoposide, inhibited nuclear localization of Cdc25B2 in S phase, possibly by invoking a sequestration cascade. Thus, differences in the spatial distribution of Cdc25B subtypes exist within cells and the 41 amino acid insert in the N-terminus of the Cdc25B3 splice variant encodes an important inhibitory determinant for such regulation. The subcellular redistribution of Cdc25B2 could be functionally important for G2/M checkpoint regulation.

The protein thiol metallothionein as an antioxidant and protectant against antineoplastic drugs.

Metallothioneins (MTs) are major zinc-binding protein thiols that are readily inducible and whose functions remain unclear. Recent evidence supports a role for MT as an antioxidant. Mechanisms underlying this function may include direct interception of free radicals, complexation of redox sensitive transition metals, altered zinc homeostasis or interaction with glutathione (GSH). MT overexpression after direct gene transfer in cultured cells, decreases cytotoxicity, to partially reduce reactive oxygen and nitrogen species and markedly attenuates intracellular oxidation of reporter molecules including dichlorofluorescein and cis-parinaric acid. Conversely, enhanced intracellular oxidation is seen in cells derived from mice lacking both functional MTI and MTII genes. GSH levels are unaffected in MT null cells relative to wildtype, suggesting the antioxidant function of MT is independent of GSH. In tumor cells there is at least a 400-fold range in MT levels and a 10-fold difference in the ratio of nuclear to cytoplasmic distribution. No correlation exists between MT levels and GSH levels demonstrating the autonomous regulation of intracellular thiol pools. This may be important for cancer chemotherapies since MT overexpression is seen in human tumor cells with acquired drug resistance. The authors found no evidence for altered MT isoform profiles in drug resistant cells that overexpress MT. Recent evidence suggests MT subcellular location may dictate functionality and MT may help determine the threshold for apoptosis. Thus, MT is a stress-inducible protein with antioxidant attributes that may participate independently or in conjunction with GSH to protect cells against injurious agents.

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.

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.

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.

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.

Metallothionein and HSP-72 are induced in the liver by hemorrhagic shock and resuscitation but not by shock alone.

BACKGROUND: Previous reports have indicated that HSP-72 and metallothionein mRNA undergo induction in the liver after resuscitated hemorrhagic shock. In this study we investigated whether unresuscitated shock triggers induction and whether protein induction also occurs. METHODS: Rats were subjected to resuscitated and unresuscitated shock protocols of varying severity; livers were isolated and processed for Northern, Western, and immunohistochemical analysis. Cadmium binding assay was used to measure metallothionein protein. RESULTS: Unresuscitated shock led to no induction of HSP-72 or metallothionein. Severe resuscitated shock led to prompt induction of HSP-72 mRNA and protein in hepatocytes, up to 20-fold over sham group; metallothionein mRNA induction appeared later than HSP-72 and did not lead to elevated protein levels. Mild resuscitated shock had little effect. CONCLUSIONS: These findings indicate resuscitated severe shock, not shock alone, leads to induction of HSP-72 and metallothionein in the liver. Metallothionein expression lags behind HSP-72 expression.

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.

Metallothionein null cells have increased sensitivity to anticancer drugs.

Overexpression of metallothioneins (MTs) protects some cells against heavy metals, mutagens, anticancer agents, and reactive oxygen species. We have examined the effect of the loss of MT expression on the cytotoxicity of anticancer agents and mutagens using embryonic fibroblast cells from transgenic mice with targeted disruptions of MT I and II genes (MT -/-). MT -/- cells expressed no detectable MT. Compared to wild type cells, MT -/- cells showed enhanced sensitivity to a 2-h exposure to cisplatin, melphalan, bleomycin, cytarabine, or N-methyl-N'-nitro-N-nitrosoguanidine but were equally sensitive to doxorubicin and neocarzinostatin. Basal expression of the DNA damage-response genes, gadd 45 and gadd 153, were elevated in MT -/- cells compared to MT +/+ cells. Anticancer drug treatment, however, did not produce a greater increase in gadd 45 or gadd 153 expression in MT null cells compared to MT +/+ cells. These results support the hypothesis that endogenous MT levels affect the sensitivity of mammalian cells to mutagens and clinically important anticancer drugs.

Human metallothionein isoform gene expression in cisplatin-sensitive and resistant cells.

Overexpression of metallothioneins (MTs) has been observed in some cis-diamminedichloroplatinum (CDDP)-resistant cells. We have developed oligonucleotide probes for each of the six non-neuronal human MT (hMT) isoforms and used them to assay hMT isoform expression in three pairs of CDDP-resistant and -sensitive human carcinoma cell lines, i.e., SCC25/CP versus SCC25 cells, H69/CP versus H69 cells, and SW2/CP versus SW2 cells. We found a 9-fold increase in basal hMT-IIa mRNA levels and a 5-fold increase in hMT-le mRNA levels in SCC25/CP cells, compared with SCC25 cells. Nuclear run-on studies also revealed a 3-fold increase in hMT-IIa transcription rate. Basal hMT-IIa steady state mRNA levels were 2-3.6-fold greater in H69/CP and SW2/CP cells, compared with their parental cells. No significant basal expression of hMT-Ia, -Ib, -If, or Ig was detected in any cells, suggesting that overexpression of these isoforms was not commonly associated with the CDDP-resistant phenotype. Levels of constitutively expressed hMT isoforms, as well as hMT-If, could be elevated by treatment of all cells with 100 microM zinc. The universal overexpression of hMT-IIa suggests a role of this particular isoform in CDDP resistance. Using our isoform-specific hMT-IIa probe and the demethylating agent 5'-azacytidine (AZC), we found that AZC pretreatment increased basal hMT-IIa mRNA levels in SCC25 but not SCC25/CP cells, suggesting that DNA hypomethylation was responsible for higher basal hMT-IIa mRNA levels in SCC25/CP cells. AZC had little or no effect on hMT-If or -Ig expression. Limited restriction analysis by methylation-sensitive enzymes, however, revealed no obvious differences in the methylation status of the hMT-IIa promoter in either SCC25 or SCC25/CP cells.

DNA polymerase arrest by adducted trivalent chromium.

Carcinogenic chromium (Cr6+) enters cells via the sulfate transport system and undergoes intracellular reduction to trivalent chromium, which strongly adducts to DNA. In this study, the effect of adducted trivalent chromium on in vitro DNA synthesis was analyzed with a polymerase-arrest assay in which prematurely terminated replication products were separated on a DNA sequencing gel. A synthetic DNA replication template was treated with increasing concentrations of chromium(III) chloride. The two lowest chromium doses used resulted in biologically relevant adduct levels (6 and 21 adducts per 1,000 DNA nucleotides) comparable with those measured in nuclear matrix DNA from cells treated with a 50% cytotoxic dose of sodium chromate in vivo. In vitro replication of the chromium-treated template DNA using the Sequenase version 2.0 T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) resulted in dose-dependent polymerase arrest beginning at the lowest adduct levels analyzed. The pattern of polymerase arrest remained consistent as chromium adduct levels increased, with the most intense arrest sites occurring 1 base upstream of guanine residues on the template strand. Replication by the DNA polymerase I large (Klenow) fragment as well as by unmodified T7 DNA polymerase also resulted in similar chromium-induced polymerase arrest. Interstrand cross-linking between complementary strands was detected in template DNA containing 62, 111, and 223 chromium adducts per 1,000 DNA nucleotides but not in template containing 6 or 21 adducts per 1,000 DNA nucleotides, in which arrest nevertheless did occur. Low-level, dose-dependent interstrand cross-linking between primer and template DNA, however, was detectable even at the lowest chromium dose analyzed. Since only 9% of chromium adducts resulted in polymerase arrest in this system, we hypothesized that arrest occurred when the enzyme encountered chromium-mediated interstrand DNA-DNA cross-links between either the template and a separate DNA molecule or the template and its complementary strand in the same molecule. These results suggest that the obstruction of DNA replication by chromium-mediated DNA-DNA cross-links is a potential mechanism of chromium-induced genotoxicity in vivo.