Project name: Dictyostelium discoideum

Description: Dictyostelium discoideum is a useful model for studying mechanisms of cisplatin drug sensitivity. Our previous findings, that mutations in sphingolipid-metabolism genes confer cisplatin resistance in D. discoideum and in human cells, raised interest in the resistance mechanisms and their implications for cisplatin chemotherapy. Here we used expression microarrays to monitor physiological changes and to identify pathways that are affected by cisplatin treatment of D. discoideum. We found over 400 genes whose regulation was altered by cisplatin treatment of wild type cells, including groups of genes that participate in cell proliferation and in nucleotide and protein metabolism. These findings show that the cisplatin response is orderly and multifaceted. Transcriptional profiling of two isogenic cisplatin-resistant mutants, impaired in different sphingolipid metabolism steps, showed that the effect of cisplatin treatment was greater than the effect of the mutations, indicating that cisplatin-resistance in the mutants is due to specific abilities to overcome the drug effects rather than to general drug insensitivity. Nevertheless, the mutants exhibited significantly different responses to cisplatin compared to the parent and over 200 genes accounted for that difference. We mutated some of the cisplatin-response genes and found that the mutants had altered drug sensitivity. These findings reveal the power of this system to identify pathways and genes that are affected by drugs and mutations. Our data illustrate how modeling complex cellular responses to drugs in genetically stable and tractable systems can uncover new targets with the potential for improving chemotherapy.Keywords: Dictyostelium discoideum, cisplatin treatment, transcriptional profiles, physiological changesOverall design: Exponentially growing cells (2x10E6 cells/ml) were divided into twelve 50 ml aliquots in 500 ml flasks. The cells were shaken for 30 min, 300 µM cisplatin was added to 6 cultures (PT buffer was added to the 6 controls), and the cells were shaken for 3 hr at 22C in the dark. The cells were washed with phosphate buffered saline (PBS) and resuspended in 1.0 ml TRIzol reagent (Life Technologies, Gaithersburg, MD) and RNA was extracted. For technical variation, RNA samples from each experiment were hybridized to at least 5 arrays (technical replication). For biological variation, each treatment (with or without cisplatin) was repeated twice for each mutant and 6 times for the wild type (biological replication). We analyzed both wild type and 2 mutant strains that have a higher resistance to cisplatin than wild type.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: ModelOrganism

Release date: 2007-08-31

Last updated: 2007-06-25