Research using the fruit fly Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and the genetics of early embryonic development. These breakthroughs led to additional discoveries, including the mechanisms of body patterning, organogenesis and control of proliferation. Although great progress has been made using the whole fly as a model for development and disease, in vitro experiments have also contributed to our understanding of biological processes.
Cell lines are important tools for biologists primarily because millions of homogeneous cells can be cultured under defined conditions and analyzed. This is particularly important for biochemical analysis. More recently the use of RNAi in cell lines, a mechanism for gene silencing, has allowed the systematic examination of the entire genome in vitro. These RNAi screens have resulted in the discovery of functions for uncharacterized genes. These assays depend critically on the existence of appropriate cell lines and only a few relatively uncharacterized Drosophila cell lines exist. This is in part because the mechanisms that lead to the establishment of cell lines are not defined. Currently, cell lines are established by homogenization of embryos or tissues to produce primary cultures, and subsequently subculturing these cells to produce lines. During this time, cultures presumably acquire mutations, leading to the establishment of cells that can survive indefinitely in vitro. Work done on mammalian cells have revealed that genetic modifications can alter cellular activity in vitro. Activation of telomerase, and genetic alteration of oncogenes and tumor suppressors profoundly alters the behavior of cells in culture and leads these cells to acquire characteristics that allow cells to thrive in vitro. In order to identify genetic elements with similar roles in the establishment of Drosophila cell lines, I have used two approaches.
The first approach was an in vitro screen of Drosophila tumor suppressors. Tumor suppressors regulate the proliferation of cells through control of the cell cycle or apoptosis, and the loss of these genes usually results in overgrowth of tissues and the formation of tumors. Homologs of mammalian tumor suppressors have been identified in the fruit fly and the loss of function of these genes often results in increased cell proliferation and/or decreased apoptosis, and consequently the growth of tumors. Because of the importance of tumor suppressors in the regulation of cell cycle and/or control of apoptosis, and because it has been observed that loss of tumor suppressors can cause tumor formation in vivo in flies, I examined the effects of loss of various tumor suppressors in vitro. I hypothesize that the loss of tumor suppressor activity in Drosophila cells in vitro results in increased cell proliferation and may help establish immortal cell cultures. I observed a significant decrease in time to confluency in primary cultures with loss of function tumor suppressor genes. Additionally, I observed a very dramatic decrease in time to confluency in primary cultures with the Pten loss of function mutation. The results of loss of Pten on cells in vitro were examined further through the use of western blotting and differential expression analysis of microarrays on a loss of Pten cell line. Pten cell lines show increased activation of Akt and upregulation of genes involved in metabolism of carbohydrates, lipids and amino acids.
The second approach I used was to identify genes involved in the establishment of cell lines, and was designed to take advantage of the wealth of information generated from microarray experiments. Comparison of the transcriptional profiles of various cell types was used to identify differentially regulated genes. We identified 52 genes with differential expression between cell lines and embryos/imaginal discs. Among these 52 genes we identified 18 genes, which are differentially expressed as part of the adaptation to the new tissue culture environment. Of the 34 remaining genes 6 genes were identified as being differentially expressed exclusively in cell lines. Additional experiments are underway to determine the significance of the differential expression of these genes and identify genes underlie the immortalization and/or transformation of cell line cells.