Glutathione-S-transferase pi as a model protein for the characterisation of chemically reactive metabolites

Chemically reactive metabolites (CRMs) are thought to be responsible for a number of adverse drug reactions through modification of critical proteins. Methods that defined the chemistry of protein modification at an early stage would provide invaluable tools for drug safety assessment. Here, human GST pi (GSTP) was exploited as a model target protein to determine the chemical, biochemical and functional consequences of exposure to the hepatotoxic CRM of paracetamol (APAP), N-acetyl-p-benzoquinoneimine (NAPQI). Site-specific, dose-dependent modification of Cys47 in native and His-tagged GSTP was revealed by MS, and correlated with inhibition of glutathione (GSH) conjugating activity. In addition, the adaptation of iTRAQ labelling technology to define precisely the quantitative relationship between covalent modification and protein function is described. Multiple reaction monitoring (MRM)-MS of GSTP allowed high sensitivity detection of modified peptides at physiological levels of exposure. Finally, a bioengineered mutant cytochrome P450 with a broad spectrum of substrate specificities was used in an in vitro reaction system to bioactivate APAP: in this model, GSTP trapped the CRM and exhibited both reduced enzyme activity and site-specific modification of the protein. These studies provide the foundation for the development of novel test systems to predict the toxicological potential of CRMs produced by new therapeutic agents.     

Generation of a predictive melphalan resistance index by drug screen of B-cell cancer cell lines

Background

Recent reports indicate that in vitro drug screens combined with gene expression profiles (GEP) of cancer cell lines may generate informative signatures predicting the clinical outcome of chemotherapy. In multiple myeloma (MM) a range of new drugs have been introduced and now challenge conventional therapy including high dose melphalan. Consequently, the generation of predictive signatures for response to melphalan may have a clinical impact. The hypothesis is that melphalan screens and GEPs of B-cell cancer cell lines combined with multivariate statistics may provide predictive clinical information.

Materials and Methods

Microarray based GEPs and a melphalan growth inhibition screen of 59 cancer cell lines were downloaded from the National Cancer Institute database. Equivalent data were generated for 18 B-cell cancer cell lines. Linear discriminant analyses (LDA), sparse partial least squares (SPLS) and pairwise comparisons of cell line data were used to build resistance signatures from both cell line panels. A melphalan resistance index was defined and estimated for each MM patient in a publicly available clinical data set and evaluated retrospectively by Cox proportional hazards and Kaplan-Meier survival analysis.

Principal Findings

Both cell line panels performed well with respect to internal validation of the SPLS approach but only the B-cell panel was able to predict a significantly higher risk of relapse and death with increasing resistance index in the clinical data sets. The most sensitive and resistant cell lines, MOLP-2 and RPMI-8226 LR5, respectively, had high leverage, which suggests their differentially expressed genes to possess important predictive value.

Conclusion

The present study presents a melphalan resistance index generated by analysis of a B-cell panel of cancer cell lines. However, the resistance index needs to be functionally validated and correlated to known MM biomarkers in independent data sets in order to better understand the mechanism underlying the preparedness to melphalan resistance.     

A new determinant of glucocorticoid sensitivity in lymphoid cell lines

The SAK cell line, derived from a spontaneous thymic lymphoma in an AKR mouse, is resistant to lysis by glucocorticoids in spite of the presence of functional glucocorticoid receptor. Receptor function was determined by hormone binding analyses, as well as characterization of hormonal effects on cell growth and on the accumulation of murine leukemia virus and metallothionein mRNAs. SAK cells were fused with a receptor-defective (and therefore resistant) variant of a well- characterized murine thymoma line, W7. The resulting hybrids are glucocorticoid sensitive, demonstrating complementation of the receptor defect in W7 cells by the functional glucocorticoid receptor of SAK. This fusion shows that SAK cells are resistant to the hormone due to the absence of another function designated "I" for lysis. SAK cells were also fused with glucocorticoid-sensitive W7 cells (containing wild- type receptor), generating glucocorticoid-sensitive hybrids, which demonstrate that the dexamethasone-resistant phenotype of the SAK cells is recessive. Resistant derivatives of this hybrid were found which still contain the full amount of receptor. Chromosome analysis revealed that, on the average, the resistant derivatives had lost two chromosomes, suggesting segregation of chromosomes carrying genetic material necessary for the "lysis" function. The drug 5-azacytidine (a known inhibitor of DNA methylation) has been shown to cause heritable changes in gene expression. Treatment of SAK cells with 5-azacytidine generated glucocorticoid-sensitive clones at high frequency, suggesting that the gene(s) involved in the "lysis" function are intact and have been inactivated through a process such as differentiation.     

Glutathione-S-transferase in human renal cell carcinoma

Homogenates of human renal cell carcinomas were tested for glutathione-S-transferase, an enzyme of normal proximal tubule cells. All tumors were positive; mean tumor fraction enzyme activity was 0.040 +/- 0.02 mumol/min/microgram protein. Glutathione-S-transferase activity in homogenates from normal kidney was 0.022 and 0.054 mumol/min/microgram protein. Finding similar levels of a major cytosolic enzyme in tumor and renal cortex confirms the origin of renal cell carcinoma in the proximal nephron. Glutathione-S-transferase, which binds carcinogens and steroids, may play a role in carcinogenesis and serve as a marker for this tumor.     

Human cell lines as models for multidrug resistance in solid tumours

In spite of our expanding knowledge on the molecular biology of cancer, relatively little progress has been made in improving therapy for the solid tumours which are major killers, e.g., lung, colon, breast. Significant advances over the past 10–15 years in chemotherapy of some tumours such as testicular cancer and some leukaemias indicates that, in spite of the undesirable side-effects, chemotherapy has the potential to effect cure in the majority of patients with certain types of cancer. Multidrug resistance, inherent or acquired, is one important limiting factor in extending this success to most solid tumours.In vitro studies described in this review are now uncovering a diversity of possible mechanisms of cross-resistance to different types of drug. Sensitive methods such as immunocytochemistry, RT-PCR orin situ RNA hybridisation may be necessary to identify corresponding changes in clinical material. Only by classifying individual tumours according to their specific resistance mechanisms will it be possible to define the multidrug resistance problem properly. Such rigorous definition is a prerequisite to design (and choice on an individual basis) of specific therapies suited to individual patients. Since a much larger proportion of cancer biopsies should be susceptible to accurate analysis by the immunochemical and molecular biological techniques described above than to direct assessment of drug response, it seems reasonable to hope that this approach will succeed in improving results for cancer chemotherapy of solid tumours where other approaches such as individualisedin vitro chemosensitivity testing have essentially failed. Results from clinical trials using cyclosporin A or verapamil are encouraging, but these agents are far from ideal, and reverse resistance in only a subset of resistant tumours. Proper definition of the other mechanisms of MDR, and how to antagonize them, is an urgent research priority.Abbreviations MDR multiple drug resistance - P-170=pgp P-glycoprotein=product ofmdr-1 gene     

Drug resistance to cis-diamminedichloroplatinum (II) in Chinese hamster ovary cell lines transfected with glutathione S-transferase pi gene

Establishment of Chinese hamster ovary (CHO) cell lines expressing human glutathione S-transferase-pi (GST-pi) was performed after cotransfection of pSV2-neo and human GST-pi cDNA-carrying plasmid p beta actGPi-2. About 30 G418-resistant clones were tested for their expression of GST-pi by Northern blot analysis. Two clones, beta 2-3 and beta 2-5, expressed a significant amount of GST-pi mRNA; and one clone, beta 1-1, that did not was also used for further study. Western blot analysis with anti-GST-pi antibody showed significant increases of GST-pi in beta 2-3 and beta 2-5, but not in beta 1-1. Northern blot analysis with the human GST-pi cDNA probe showed that the increase in the expression of GST-pi-mRNA in beta 2-3 and beta 2-5 was respectively 2- and 4-fold higher than that in beta 1-1. Southern blotting analysis showed that beta 1-1, beta 2-3 and beta 2-5 contained about one copy of the human GST-pi cDNA sequence. beta 2-3 and beta 2-5 were resistant to 1.4- and 3.0-fold higher doses of CDDP than CHO, respectively, but beta 1-1 was not. Increased expression of GST-pi might be associated with CDDP-resistance in CHO cells.     

NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-alpha and -beta, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.     

NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-α and -β, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.     

Expression of a drug resistance gene in human neuroblastoma cell lines: modulation by retinoic acid-induced differentiation.

Expression of a multidrug resistance gene (mdr1) and its protein product, P-glycoprotein (Pgp), has been correlated with the onset of multidrug resistance in vitro in human cell lines selected for resistance to chemotherapeutic agents derived from natural products. Expression of this gene has also been observed in normal tissues and human tumors, including neuroblastoma. We therefore examined total RNA prepared from human neuroblastoma cell lines before and after differentiation with retinoic acid or sodium butyrate. An increase in the level of mdr1 mRNA was observed after retinoic acid treatment of four neuroblastoma cell lines, including the SK-N-SH cell line. Western blot (immunoblot) analysis demonstrated concomitant increases in Pgp. However, studies of 3H-vinblastine uptake failed to show a concomitant Pgp-mediated decrease in cytotoxic drug accumulation. To provide evidence that Pgp was localized on the cell surface, an immunotoxin conjugate directed against Pgp was added to cells before and after treatment with retinoic acid. Incorporation of [3H]leucine was decreased by the immunotoxin in the retinoic acid-treated cells compared with the undifferentiated cells. These results demonstrate that whereas expression of the mdr1 gene can be modulated by differentiating agents, increased levels of expression are not necessarily associated with increased cytotoxic drug accumulation.     

Mitochondrial quality control as a key determinant of cell survival

Mitochondria are the energy producing dynamic double-membraned organelles essential for cellular and organismal survival. A multitude of intra- and extra-cellular signals involved in the regulation of energy metabolism and cell fate determination converge on mitochondria to promote or prevent cell survival by modulating mitochondrial function and structure. Mitochondrial fitness is maintained by mitophagy, a pathway of selective degradation of dysfunctional organelles. Mitophagy impairment and altered clearance results in increased levels of dysfunctional and structurally aberrant mitochondria, changes in energy production, loss of responsiveness to intra- and extra-cellular signals and ultimately cell death. The decline of mitochondrial function and homeostasis with age is reported to be central to age-related pathologies. Here we discuss the molecular mechanisms controlling mitochondrial dynamics, mitophagy and cell death signalling and how their perturbation may contribute to ageing and age-related illness.     

Comparing solid tumors with cell lines: implications for identifying drug resistance genes in cancer

Gene expression arrays allow researchers to profile the differences between cell lines or tissues and they may identify genetic markers of development, organ maturation, or tumor progression. Although a primary tumor that grows in a host and a tumor-cell-line derived from that primary tumor and grown in vitro share similar gene expression profiles, there are, not unexpectedly, some important differences. In fact, Stein and colleagues have found that genes that are differentially expressed in primary tumors as compared to the specific genes expressed in their cell-line derivatives are more reliably predictive of tumor tractability. Thus, sensitivity in vitro might not reflect sensitivity in vivo. Because anti-tumor compounds are largely evaluated in cell culture assays, these compounds' therapeutic utility must be judged in light of genes described by Stein et al. that better predict tractability.     

Conditionally immortalized cell lines as model systems for high-throughput biology in drug discovery

There is an increasing emphasis on the need for high-quality biological data much earlier in the drug-discovery process. This has led to the development of high-throughput approaches to biology, many of which rely on the use of cell-culture models. Unfortunately, available cell-culture models often reflect poorly the characteristics of the tissue they are supposed to represent. However, the conditional-immortalization approach as applied by Xcellsyz offers the possibility of producing human cell lines on demand, which are truly representative of the tissue from which they derive.     

Colchicine resistance in mammalian cell lines.

Colchicine-resistant variants derived from mouse and Syrian hamster lines are described. The resistant cells do not appear to be true mutants, since they appear at a high frequency, unaffected by treatment with ethyl methyl sulphonate, and are unstable in the absence of the drug. They are cross-resistant to other drugs, show a reduced rate of binding of colchicine in monolayer, and give extracts with colchicine-binding properties identical to those of the wild type. Thus the resistance is due to a permeability barrier. The naturally occurring resistance of the Syrian hamster line is specific for colchicine, and may be due to a specific permeability barrier. The Syrian hamster line is also shown to have an extra colchicine-binding pool.     

O-glycosylation as a sorting determinant for cell surface delivery in yeast

Little is known about the mechanisms that determine localization of proteins to the plasma membrane in Saccharomyces cerevisiae. The length of the transmembrane domains and association of proteins with lipid rafts have been proposed to play a role in sorting to the cell surface. Here, we report that Fus1p, an O-glycosylated integral membrane protein involved in cell fusion during yeast mating, requires O-glycosylation for cell surface delivery. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A chimeric protein lacking O-glycosylation motif was missorted to the vacuole and accumulated in late Golgi in wild-type cells. Exocytosis of this protein could be restored by addition of a 33-amino acid portion of an O-glycosylated sequence from Fus1p. Our data suggest that O-glycosylation functions as a sorting determinant for cell surface delivery of Fus1p.     

Increased T-type Ca2+ channel activity as a determinant of cellular toxicity in neuronal cell lines expressing polyglutamine-expanded human androgen receptors

We have analyzed Ca²⁺ currents in two neuroblastoma-motor neuron hybrid cell lines that expressed normal or glutamine-expanded human androgen receptors (polyGln-expanded AR) either transiently or stably. The cell lines express a unique, low-threshold, transient type of Ca²⁺ current that is not affected by L-type Ca²⁺ channel blocker (PN 200-110), N-type Ca²⁺ channel blocker (-conotoxin GVIA) or P-type Ca²⁺ channel blocker (Agatoxin IVA) but is blocked by either Cd²⁺ or Ni²⁺. This pharmacological profile most closely resembles that of T-type Ca²⁺ channels [1-3]. Exposure to androgen had no effect on control cell lines or cells transfected with normal AR but significantly changed the steady-state activation in cells transfected with expanded AR. The observed negative shift in steady-state activation results in a large increase in the T-type Ca²⁺ channel window current. We suggest that Ca²⁺ overload due to abnormal voltage-dependence of transient Ca²⁺ channel activation may contribute to motor neuron toxicity in spinobulbar muscular atrophy (SBMA). This hypothesis is supported by the additional finding that, at concentrations that selectively block T-type Ca²⁺ channel currents, Ni²⁺ significantly reduced cell death in cell lines transfected with polyGln-expanded AR.     

Resistance of proteinuric rats to furosemide: Urinary drug protein binding as a determinant of drug effect

The effect of drug binding to urinary proteins on the diuretic response to furosemide was assessed in normal and nephrotic rats. Nephrosis was induced by treating Sprague-Dawley rats with puromycin aminonucleoside. Binding of furosemide to urinary proteins was found to range from 60 to 95% depending on the concentration of urinary protein. The diuretic response to furosemide reaching the renal tubular lumen was inversely correlated with the degree of proteinuria, a finding that was independent of serum protein concentration of glomerular filtration rate. These data suggest that the binding of furosemide to urinary protein decreases the diuretic effect of furosemide and that drug-protein interactions of this type may also be important in modulating the activity of other lumenally-active drugs or endogenous substances exhibiting a high degree of protein binding. The binding of furosemide to urinary protein may explain the refractoriness of some patients with proteinuria to this agent.