Anne M. Galea

Controlling the size of lipid droplets: lipid and protein factors

Recent advances have transformed our understanding of lipid droplets (LDs). Once regarded as inert lipid storage granules, LDs are now recognized as multi-functional organelles that affect many aspects of cell biology and metabolism. However, fundamental questions concerning the biogenesis and growth of LDs remain unanswered. Recent studies have uncovered novel modes of LD growth (including rapid/homotypic as well as slow/atypical LD fusion), and identified key proteins (e.g. Fsp27, seipin, FITM2 and perilipin 1) and lipids (e.g. phosphatidylcholine and phosphatidic acid) that regulate the size of LDs. Phospholipids appear to have an evolutionarily conserved role in LD growth. Protein factors may regulate LD expansion directly and/or indirectly through modulating the level and composition of phospholipids on LD surface.

Special relationship between sterols and oxygen: were sterols an adaptation to aerobic life?

A fascinating link between sterols and molecular oxygen (O(2)) has been a common thread running through the fundamental work of Konrad Bloch, who elucidated the biosynthetic pathway for cholesterol, to recent work supporting a role of sterols in the sensing of O(2). Synthesis of sterols by eukaryotes is an O(2)-intensive process. In this review, we argue that increased levels of O(2) in the atmosphere not only made the evolution of sterols possible, but that these sterols may in turn have provided the eukaryote with an early defence mechanism against O(2). The idea that nature crafted sterols as a feedback loop to adapt to, or help protect against, the hazards of O(2) is novel and enticing. We marshal several lines of evidence to support this thesis: (1) coincidence of atmospheric O(2) and sterol evolution; (2) sterols regulate O(2) entry into eukaryotic cells and organelles; (3) sterols act as O(2) sensors across eukaryotic life; (4) sterols serve as a primitive cellular defence against O(2) (including reactive oxygen species). Therefore, sterols may have evolved in eukaryotes partially as an adaptive response to the rise of terrestrial O(2), rather than merely as a consequence of it.

The interaction of cisplatin and analogues with DNA in reconstituted chromatin

The influence of chromatin structure on cis-diamminedichloroplatinum(II) (cisplatin) DNA damage was investigated in a reconstituted nucleosome system. Nucleosomes were reconstituted on the somatic 5S rRNA gene from Xenopus borealis using the octamer transfer method of reconstitution. Footprinting techniques, utilising bleomycin and DNase I as the damaging agents, were employed to establish the precise location of positioned nucleosomes with respect to the DNA sequence. Reconstituted nucleosomal DNA was treated with cisplatin and drug-induced DNA adduct formation was quantitatively analysed with a polymerase stop assay using Taq DNA polymerase. A densitometric comparison of the relative damage band intensities between purified and reconstituted DNA revealed regions of relative protection corresponding to the sites of the positioned nucleosome cores. This indicated that the preferred site of cisplatin DNA binding was in the linker region of the nucleosome. Statistical analysis showed significant protection from cisplatin DNA damage in the core region of the nucleosome. Three cisplatin analogues were also investigated in this reconstituted nucleosome system. These analogues, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin), cis-dichlorobis(cyclohexylamine)platinum(II) (cis-[PtCl(2)(C(6)H(11)NH(2))(2)]) and dichloro(N-[3-[(2-aminoethyl)-amino]propyl]acridine-4-carboxamide)platinum(II) (ac-PtenCl(2)(n3)), were also found to target the linker region of the nucleosome. The latter DNA-targeted acridine-platinum complex gave rise to the most predominant footprints of all the Pt compounds tested.

CHOLESTEROL AS AN EVOLUTIONARY RESPONSE TO LIVING WITH OXYGEN

Although often considered in a negative light, cholesterol is an essential molecule with unusually diverse functions. Cholesterol and related sterols (ergosterol in yeast, phytosterols in plants) is considered a hallmark of eukaryotes, and may even have triggered the evolution of multicellular organisms. Synthesis of cholesterol is an extremely oxygen‐intensive process and requires sufficient terrestrial oxygen to proceed. In turn, several lines of evidence support the argument that cholesterol evolved at least in part as an adaptation to the hazards of oxygen. This evolutionary perspective usefully informs medical research on cholesterol to address health‐related issues, as illustrated by examples drawn from three prominent human diseases: cataracts, heart disease, and cancer.

The influence of chromatin structure on DNA damage induced by nitrogen mustard and cisplatin analogues.

The interaction of anti‐tumour drugs with reconstituted chromatin has been investigated using defined nucleosomal complexes. This allowed the effect of nucleosome cores on drug‐induced DNA damage to be assessed for four nitrogen mustard analogues, dimethylsulphate and three cisplatin analogues. A defined nucleosomal complex was employed that contained two precisely positioned nucleosome cores. The construct was then subjected to drug treatment, and the resulting DNA damage was quantitatively analysed using a Taq DNA polymerase stop assay. At the sites of damage, densitometric comparisons between purified and reconstituted DNA were used to evaluate the influence of nucleosomal core proteins on specific drug–DNA interactions. Results were combined with previous data obtained for other DNA‐damaging drugs investigated using the same nucleosomal construct. For most of the DNA‐damaging agents studied, this method revealed protection at the positioned nucleosome cores and indicated that the preferred site of DNA binding for these compounds was in the linker region of the construct. Statistical analyses confirmed the significant level of damage protection conferred by the nucleosome cores and revealed differences between the examined compounds. Larger compounds generally displayed a greater tendency to target the linker region of the nucleosomal DNA and were impeded from damaging nucleosomal core DNA. In contrast, smaller molecules had greater access to nucleosomal core DNA.

Characterising the atypical 5′-CG DNA sequence specificity of 9-aminoacridine carboxamide Pt complexes

In this study, the DNA sequence specificity of four DNA-targeted 9-aminoacridine carboxamide Pt complexes was compared with cisplatin, using two specially constructed plasmid templates. One plasmid contained 5′-CG and 5′-GA insert sequences while the other plasmid contained a G-rich transferrin receptor gene promoter insert sequence. The damage profiles of each compound on the different DNA templates were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. With the plasmid that contained 5′-CG and 5′-GA dinucleotides, the four 9-aminoacridine carboxamide Pt complexes produced distinctly different damage profiles as compared with cisplatin. These 9-aminoacridine complexes had greatly increased levels of DNA damage at CG and GA dinucleotides as compared with cisplatin. It was shown that the presence of a CG or GA dinucleotide was sufficient to reveal the altered DNA sequence selectivity of the 9-aminoacridine carboxamide Pt analogues. The DNA sequence specificity of the Pt complexes was also found to be similarly altered utilising the transferrin receptor DNA sequence.

The Anti-tumour Agent, Cisplatin, and its Clinically Ineffective Isomer, Transplatin, Produce Unique Gene Expression Profiles in Human Cells

Cisplatin is a DNA-damaging anti-cancer agent that is widely used to treat a range of tumour types. Despite its clinical success, cisplatin treatment is still associated with a number of dose-limiting toxic side effects. The purpose of this study was to clarify the molecular events that are important in the anti-tumour activity of cisplatin, using gene expression profiling techniques. Currently, our incomplete understanding of this drugs mechanism of action hinders the development of more efficient and less harmful cisplatin-based chemotherapeutics. In this study the effect of cisplatin on gene expression in human foreskin fibroblasts has been investigated using human 19K oligonucleotide microarrays. In addition its clinically inactive isomer, transplatin, was also tested. Dual-fluor microarray experiments comparing treated and untreated cells were performed in quadruplicate. Cisplatin treatment was shown to significantly up- or down-regulate a consistent subset of genes. Many of these genes responded similarly to treatment with transplatin, the therapeutically inactive isomer of cisplatin. However, a smaller proportion of these transcripts underwent differential expression changes in response to the two isomers. Some of these genes may constitute part of the DNA damage response induced by cisplatin that is critical for its anti-tumour activity. Ultimately, the identification of gene expression responses unique to clinically active compounds, like cisplatin, could thus greatly benefit the design and development of improved chemotherapeutics.

Gene Expression Profiling of the cancer chemotherapeutic agent, Cisplatin, compared with its ineffective Isomer, Transplatin

T bacterial genus Streptomyces has long been appreciated for its ability to produce various kinds of medically important secondary metabolites such as antibiotic actinorhodin (ACT) in S. coelicolor and anti-tumor doxorubicin (DXR) in S. peucetius. Although traditional random mutation has been one of the most widely-practiced strategies for Streptomyces strain improvement, genome sequencing, targeted-gene disruption, and omics-guided reverse engineering approaches were successfully used to identify, analyze, and modify specific biosynthetic and regulatory genes involved in most of the secondary metabolites in Streptomyces species. Here, I present an example of rational polyketide pathway redesign strategies through Streptomyces genome engineering. Recursive comparative transcriptome analyses using S. coelicolor microarrays, followed by sequential targeted-gene disruptions of independently-functioning regulatory as well as precursor flux-controlling systems could be synergistically optimized for ACT and DXR productions in Streptomyces species.R progress in the field of metabolomics has created an opportunity to advance our understanding of physiological and pathological processes. It also created a number of bioinformatics challenges associated with data analysis and interpretation. Experience with genomic data has shown that automated annotations linking genes, transcripts and proteins to published biomedical literature is useful for a wide range of bioinformatics applications including pathway analysis and identification of disease genes. With this in mind we recently developed Metab2MeSH, a web-based tool that uses a statistical approach (Fisher’s exact test) to annotate compounds with Medical Subject Headings (MeSH) used by the National Library of Medicine to manually index articles for MEDLINE/PubMed. The resulting data set contains statistically significant associations (p-value<0.005) between compounds and MeSH terms linked to PubMed articles (http://metab2mesh.ncibi.org).C genomic and metabonomic approaches based on culture-independent (e.g., pyrosequencing, FISH, DGGE) and culture-dependent methods together with 1H-NMR and GC-MS/SPME analyses were used to determine the metabolic changes triggered by gut microbiota and dietary variation. This new “integrated” approach lead to understand the “collaboration” between human host and microorganisms in relation to phenotype, diet and diseases. The impact of the diet on the gut microbiota of children having cow’s milk protein allergy (CMPA) and celiac disease (CD) was determined. Fecal slurry and urine of two groups of children were analyzed: the first one is CMPA children before and after 2 months of the hydrolyzed and ultra-filtered whey protein formula (eHF) with lactose intake in the diet; and the second one, symptom-free CD children, who had been on a gluten free diet (GFD) for at least 2 years. Children without known food intolerance (healthy children; HC) were also studied. The addition of lactose to eHF formula is able to positively modulate the composition of microbiota by increasing the total fecal counts of Lactobacillus/Bifidobacteria and decreasing that of Bacteroides/Clostridia. The GFD lasting at least two years did not completely restore the microbiota of the CD children. The levels of Lactobacillus, Enterococcus and Bifidobacteria were significantly higher in HC than in CD children. On the contrary, Bacteroides, Staphylococcus, Salmonella, Shighella and Klebsiella were higher in CD compared to HC children. As showed by GC-MS/SPME and 1H-NMR, significant differences between molecules belonging to short chain fatty acids (SCFAs), esters, alcohols, aldehydes, ketones, monosaccharides and amino acids, before and after the lactose intake or GFD were found. Some molecules seems to be metabolic signatures of food allergy and intolerance.T fate of the last intermediate of glycolysis, phosphoenolpyruvate (PEP), controls much of cellular metabolism, e.g. the balance of glycolysis and gluconeogenesis. How are the key enzymes consuming PEP controlled? Here we examine this issue in the bacterium Escherichia coli and the budding yeast Saccharomyces cerevisiae. In both organisms, removal of glucose results in a paradoxical increased in PEP, which goes up the most of any canonical metabolite. What mechanisms lead this product of glucose to rise when glucose is removed? Enzyme activity can be regulated at the level of transcription, translation, degradation, covalent modification, and allostery.We show that allostery predominates in both organisms, with PEP consumption activated in an ultrasensitive (switch-like) manner by the upstream glycolytic intermediate fructose-1,6-bisphosphate. Mutations that eliminate this regulation do not impair growth on steady glucose, but they render the microbes defective in gluconeogenesis and ingrowth in an oscillating glucose environment. Thus, microbial central carbon metabolism is intrinsically programmed with ultrasensitive feed-forward regulation to enable rapid adaptation to changing environmental conditions.R 13C-labeled tracers have been incorporated into metabolomic studies to complement the conventional 1H NMRbased metabolomic studies that result in concentration profiles of metabolites. These Stable Isotope Resolved Metabolomics (SIRM) studies produce comprehensive metabolic data that unequivocally quantifies whether the metabolite concentrations changes discovered in conventional metabolomic studies are due to increased or decreased pathway production. Cell culture is an ideal biosystem to apply this relatively new methodology because all major substrates in the central compartment (i.e., media) and tissue can be quantified, and thus accounting for all input and output to the biosystem. Cultured hepatocytes prove especially useful in demonstrating the effectiveness of untargeted SIRM because their normal function is to create glucose under fasted conditions, and thus consumption rates for glucose are often ambiguous based on concentration data alone since the carbon source could be a multitude of compounds found in the media. The comparison of rat and human cultured hepatocytes will be discussed using several 13C-labeled substrates added to the media, in order to elucidate the metabolome of these two species’ liver cells in 2D culture (Winnike et al., 2011). A strategy for targeted metabolomics will be described using a human cell model for resistance chronic myelogenous leukemia, the Myl and Myl-R cell lines. In this approach, an initial conventional 1H NMR-based metabolomic analysis of Myl and Myl-R cells revealed that creatine is 7-fold higher in the resistant cell-line, MylR (Dewar et al., 2010). Glycine is a substrate for creatine, and therefore, 2-13C-glycine was used in a targeted SIRM study to elucidate the mechanism of increased creatine in the Myl-R, and propose a potential mechanism of chemoresistance.A parasites are responsible for high impact human diseases such as malaria, toxoplasmosis and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes and the membranes of vacuoles within which they reside. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized. However, the relative contribution of each of these biosynthetic pathways to total fatty acid composition of intracellular parasite stages remains poorly defined. Here we use a combine metabolomics with genetic and biochemical approaches to delineate the contribution of fatty acid biosynthetic pathways in Toxoplasma gondii. Metabolic labeling studies with 13C-glucose and 13C-acetate showed that intracellular tachyzoites synthesized a range of long and very long chain fatty acids (C14:0-26:1). Genetic disruption of type II fatty acid synthase (FASII) resulted in greatly reduced synthesis of saturated fatty acids up to eighteen carbons long, leading to reduced intracellular growth that was partially restored by addition long chain fatty acids. In contrast, synthesis of very long chain fatty acids was primarily dependent on a fatty acid elongation system comprising three elongases, two reductases and a dehydratase that were localized to the endoplasmic reticulum. The function of these enzymes was confirmed by metabolomics and heterologous expression in yeast. This elongase pathway appears to have a unique role in generating very long unsaturated fatty acids (C26:1) that cannot be salvaged from the host.T Clostridia are a diverse group of Gram-positive bacteria that include several pathogens and many terrestrial species that produce solvents and organic acids through fermentation of a variety of carbon sources. However, the knowledge about carbohydrate utilization pathways and their regulation in Clostridium spp. is rather limited. Accurate projection of known carbohydrate catabolic pathways across diverse bacteria with complete genomes is quite challenging due to frequent variations in components of these pathways.A 70% of newly diagnosed cases of invasive breast cancer in the U.S. will be estrogen receptor i positive (ER+). Endocrine therapy is the least toxic and most effective means to manage the hormone-dependent breast cancer in these patients, administered as an antiestrogen, e.g., Tamoxifen (TAM) or Faslodex (Fulvestrant; ICI 182,780) or an aromatase inhibitor (AI), e.g., Letrozole (LET). TAM produces a 26% proportional reduction in mortality; however, advanced ER+ breast cancer that has become resistant to endocrine therapy remains a significant clinical problem.We have shown that antiestrogen resistant breast cancer cells over-express X-Box Binding Protein 1 (XBP1) and glucose regulated protein-78 (GRP78;BiP), two integral signaling components of the unfolded protein response (UPR).XBP1 can regulate glucose homeostasis, and as glucose levels fall, GRP78 activates the UPR. Antiestrogen resistant breast cancer cells (MCF7/LCC9) utilize prosurvival UPR to maintain a higher level of basal autophagy compared to sensitive cells (MCF7/LCC1) that can provide raw materials to promote cell survival under stress from therapeutic insults. Abundance of metabolites from antiestrogen sensitive and resistant cells was compared and analyzed using UPLC-MS. Changes in selected metabolites were independently validated. Our findings indicate that resistant MCF7/LCC9 cells have an abundance of cAMP compared to MCF7/LCC1 cells. Glucose uptake in MCF/LCC9 control cells was 28-fold higherwhen compared to MCF7/LCC1 control cells, yet ATP levels in MCF7/LCC9 cells was 40% lower compared with MCF7/LCC1 control cells. cAMP has been recently identified as a potent inducer of autophagy. Our findings suggest that antiestrogen resistant breast cancer cells may have higher glucose (from the Warburg effect) and energy requirements, resulting in increased cAMPthat helps to maintain survival via autophagy under basal or treatment conditions. These metabolic adaptations are critical to the coordinated signaling from the UPR that both suppresses apoptosis and activates a prosurvival autophagy. Further studies will help to uncover the signaling mechanism involved in regulating the pathways that connect autophagy and metabolic pathways to maintain cell survival in resistance. The overall goal of this study is to provide more affordable diagnostic tools and to identify effective therapies and reliable biomarkers to predict accurately the response to antiestrogen therapy.R emergences of glycobiology, glycotechnology and glycomics have been clarifying enormous roles of carbohydrates in both physiological and pathological recognition systems. Glycan arrays have become important tools for the analysis of carbohydrate–biomacromolecule interactions such as the specificities of lectins, antibodies, cells, and viruses. However, the critical limitations of glycan array applications are restricted epitopes available from both synthesis and isolation and less mode of glycan presentation on the array surface. Conventional glycan arrays are based on two dimensional (2D) surface chemistries that result in low signal intensity and substantial non-specific binding of target proteins because of an insufficient number of binding sites and the presence of surface-protein interactions. We present here cytomimetic glycan microarray platforms based on glycopolymer immobilization and glycosylated liposome immobilization strategies. First, an oriented and density controlled glyco-marcroligand array formation was demonstrated by end-point immobilization of glycopolymer imprinted with boronic acid ligands in different sizes. Glycoarray and SPR results confirmed the same trend of density-dependent binding of specific lectins. Second, liposomes carrying ganglioside and lipid-triphenylphosphine as anchor lipid were printed onto azide-modified glass slide via Staudinger ligation. Specific lectin and toxin bindings onto liposomal glycan arrays, containing GM1 and GM3 in different densities, were confirmed by florescence scanning. The reported glycan array platforms present multivalent glycans in defined orientation and density configurations that are critical for glycan recognition. It is, thus, uniquely useful tool for probing the ligand specificities of glycan-binding molecules and for molecular and cellular proteomic applications.T public availability of high throughput datasets from a variety of biological sources has prompted the creation of a multitude of databases that significantly facilitate biomedical research. However, although each individual experiment may successfully address a focused set of hypotheses, a more comprehensive understanding of biological processes may emerge by integrating results from multiple datasets into a coherent framework. Furthermore, knowledge about higher-order, more clinically relevant aspects of any complex disease may be only achieved by these kind of analyses. Unfortunately, such integrative approaches to complex diseases have not been yet realized in part due to the lack of a theoretical framework and appropriate analytical tools to implement them.M neoplasms (MPNs) are a kind of bone marrow diseases and Ten-Eleven Transcription 2 (TET2) mutations are found in MPN patients.Located on 4q24, TET2 can catalyze the conversion from 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. TET2 mutations,such as nonsense mutations, out-of-frame insertions, deletions, and splice site mutations, alter catalytic activity andlead to low level of 5hmC. However, it was unclear why some MPN patients with wild type TET2 also revealed both low level of 5hmC.To explore other factors affecting the 5hmc level, we investigated the microRNAs’ post-transcriptional regulation of TET2 and its associated genes using 11 bioinformatics resources in our previous study. It was found that TET2 is associatedeight genes and they are targets of miR-152 and miR-29b.Importantly, DNMT-1,an associated gene, is a DNA methyltransferase catalyze the methylation, in whicha methyl group is added to cytosine and 5mC is formed.Involvement of microRNAs may explain the low 5hmC level in MPN patients with wild type TET2.D liver injury (DILI) is the leading cause of drug failure. The prevalence of serious adverse effects is due in part to inefficient and inaccurate biomarkers of toxicity in preclinical studies. ALT and BILI are the commonly measured parameters to assess liver injury. However, ALT elevation can occur without signs of liver injury so it not specific enough. BILI only elevates upon severe liver damage. Therefore, there is a need for more specific biomarkers of liver injury and dysfunction. The omics methods have the potential to detect early biomarkers of toxicity in biofluid samples. In an effort to identify biomarkers of DILI, metabolomics and transcriptomics data were acquired on urine and serum samples in two separate studies. In the first study, Sprague Dawley rats were dosed with 0, 100 or 1250 mg acetaminophen (APAP)/kg body weight. Urine, serum, and tissue were collected 6 hr, 1 d, 3 d, and 7 d post-dosing. Metabolites in pathways involving oxidative stress, bile acids, and lipid ketones were altered. The transcriptomics data indicated genes within the same pathways were altered. In the second study, rats were dosed with 0, 50, or 2000 mg carbon tetrachloride (CCl4)/kg body weight and samples collected 6 hr, 1 d, and 3 d post-dosing. Similar to the results in the APAP study, metabolites and genes involved in oxidative stress, bile acids, and lipid ketones were altered. The arginine metabolism and glycolysis pathways were also affected following administration of CCl4. Omics technologies can provide potential new biomarkers and pathway information.C cell proliferation depends on increased supply of nutrients including carbon sources and molecular oxygen. However, solid tumors frequently outgrow the blood supply, resulting in insufficient supply of oxygen, glucose and glutamine. Particularly, carbon sources are critical for the generation of ATP and building blocks, and for the maintenance of intracellular redox. Two metabolic features of cancers are the Warburg effect and glutaminolysis, underlines the importance of carbon utilization. While hypoxic adaptation of cancer cells has been well studied, how cancer cells respond to lack of carbon sources remain elusive. Using microarray technology, we compared the gene expression profiles of Hep3B cells under a series of defined culture conditions. Data analysis reveals that depletion of glucose and depletion of glutamine have dramatically different effects on transcriptional reprogramming. This observation suggests that glucose and glutamine are two different types of carbon sources, each having some specific metabolic roles in cell proliferation. Analysis of differentially expressed genes and their functional networks reveals that lack of either glucose or glutamine may lead to inhibition of multiple anabolic pathways and cell growth. Considering metabolite homeostasis in cancer cells, we are trying to validate and interpret the data, expecting to eventually establish a systemic view of the sensing, signaling, transcription reprogramming and metabolic reprogramming of cancer cells in response to carbon source insufficiency. A better understanding of the molecular mechanisms that link the carbon source sensing, signaling, transcriptional reprogramming and adaptive metabolic reprogramming may pinpoint novel targets for drug development and cancer prevention.M methods hold promise as part of aplatform which is highly complementary to other systems biology tools such as proteomics, transcriptomics and genomics. The two main analytical platforms employed are nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). These two platforms have unique characteristics which suggest that acquiring data from both platforms would be advantageous. In this presentation, a systematic relationship between serum biomarkers from model systems studies in vivo will first be examined. The successful application of NMR and GC-MS to these model systems serve as a template for clinicalbiomarkers discovery using a combination of NMR and GC-MS in cancer studies. Data will be presented from cohorts of pancreatic, colon and brain tumors patients.Analysis of these biomarkers is performed using apattern-driven approach, and as such analytical techniques which are both quantitative and high-throughput are favoured. The outcome is information with respect to a ‘biopattern’ of disease without the requirement for comprehensively attempting to characterize the entire metabolome. Furthermore, the resulting multivariate data is characterized by concerted changes in multiple markers, in contrast with traditional biomarker-driven approaches that rely on single markers. Finally, several challenges in the evolution of the field will be discussed, including interpreting coherent biological meaning from a combination of both NMR and MS data, and reliable assessment of candidate markers using multivariate statistics.

The unique gene expression profile of the anti-tumour agent, cisplatin, compared with its clinically ineffective isomer, transplatin

Cisplatin is one of the most widely used cancer chemotherapeutic agents and is utilised to treat testicular and ovarian carcinomas as well as certain sarcomas and lymphomas. However, despite its clinical success, cisplatin treatment is still associated with a number of dose-limiting toxic side effects. The purpose of this study was to clarify the molecular events that are important in the anti-tumour activity of cisplatin, using gene expression profiling techniques. Cisplatin acts by forming covalent adducts with DNA. As well as inhibiting DNA replication and cell division, cisplatin DNA adducts also affect the level of transcription of human genes. In our study we found hundreds of human genes that were down-regulated in response to cisplatin as well genes that were up-regulated. We utilised a particularly powerful analysis technique to reveal the genes that were important in the anti-tumour activity of cisplatin. This involved the cisplatin analogue, transplatin, that although it produces DNA adducts, it does not have any anti-tumour activity. Hence by comparing the gene expression profiles of cisplatin and transplatin, the genes that are important in the anti-tumour activity of cisplatin can be revealed. Using this technique, we identified 27 genes that were up-regulated and 12 genes that were down-regulated, in response to cisplatin (but not transplatin) treatment of human cells. Ultimately, the identification of gene expression responses unique to cisplatin, could greatly benefit the design and development of improved cancer chemotherapeutics.