Academic journal article Genetics

Synthetic Lethality of Retinoblastoma Mutant Cells in the Drosophila Eye by Mutation of a Novel Peptidyl Prolyl Isomerase Gene

Academic journal article Genetics

Synthetic Lethality of Retinoblastoma Mutant Cells in the Drosophila Eye by Mutation of a Novel Peptidyl Prolyl Isomerase Gene

Article excerpt

ABSTRACT

Mutations that inactivate the retinoblastoma (Rb) pathway are common in human tumors. Such mutations promote tumor growth by deregulating the G1 cell cycle checkpoint. However, uncontrolled cell cycle progression can also produce new liabilities for cell survival. To uncover such liabilities in Rb mutant cells, we performed a clonal screen in the Drosophila eye to identify second-site mutations that eliminate Rbf^sup -^ cells, but allow Rbf^sup +^ cells to survive. Here we report the identification of a mutation in a novel highly conserved peptidyl prolyl isomerase (PPIase) that selectively eliminates Rbf^sup -^ cells from the Drosophila eye.

AN important goal of novel cancer therapy is to elicit the death of mutant tumor cells in the patient, while allowing normal cells to survive. The identification of gene products required for tumor cell survival can provide highly validated drug targets for the development of therapeutic inhibitors. Ideally, targets could be identified that would kill cancer cells while sparing normal cells. A synthetic lethal screen is one method of identifying such targets. In this type of screen, cells are genetically altered to model tumor cells and one then screens for mutations that eliminate the model tumor cells but have little or no effect on wild-type cells.

One way to model tumor cells is to functionally inactivate the RBl gene. In addition to being mutated in retinoblastomas, where it was initially discovered, RBl is mutated in many other cancers including prostate (KUBOTA et al 1995), bladder (MiYAMOTO et al 1995), parathyroid (CRYNS et al 1994), and 90% of small cell lung cancers (SCLCs) (MiNNA et al 2002). RBl is also functionally inactivated in tumors that do not harbor mutations in the RBl locus itself, but do carry mutations that target the pathway through the loss of cyclin-dependentkinase (Cdk) inhibitors or overexpression of Cyclin Dl or Cdk4 (reviewed in SHERR and McCoRMiCK 2002). Additionally, the transforming activities of DNA tumor virus oncoproteins are mediated via their interaction with RBl (HELT and GALLOWAY 2003).

The RBl protein acts as a critical regulator of Gl/S phase progression by binding to members of the E2F family of transcription factors (DvSON 1998; NEVINS 2001). E2F-RB1 complexes prevent entry into S phase by actively repressing transcription through the recruitment of histone deacetylases and other chromatin modifiers to E2F-responsive promoters (HARBOUR and DEAN 2000; OGAWA et al 2002). Progression from Gl through S phase occurs when RBl is inactivated through phosphorylation by the Cdk complexes Cyclin D/Cdk4 or Cyclin D/Cdk6 and Cyclin E/Cdk2 (LUNDBERG and WEINBERG 1998). Phosphorylation relieves transcriptional repression and allows E2F-dependent transcription of target genes required for S phase progression, such as Cyclin E (MORRIS et al. 2000) as well as enzymes required for DNA synthesis and metabolism ( STEVAUX and DYSON 2002). In addition to its effects on cell proliferation, loss of RBl predisposes cells to apoptosis through the actions of E2F on p53 (reviewed in CHAU and WANG 2003), thereby creating a selective pressure for tumors to accumulate mutations in p53.

Components of the RBl pathway are being investigated as potential anticancer targets. These include the upstream kinases, Cdk2, Cdk4, and Cdk6, and the downstream effector of retinoblastoma (Rb), E2F (McLAUGHLIN et al. 2003; VERMEULEN et al. 2003). These targeted approaches could lead to therapies with an improved profile of efficacy vs. toxicity compared to conventional treatment. It would also be of interest to identify novel targets involved in RBl biology, especially those necessary for the viability of cells mutant for RBl. We therefore carried out a synthetic lethal screen in Drosophila to look for RB1-interacting genes.

Like its mammalian counterpart, Drosophila Rbf (CG7413) binds to E2F1 and regulates E2F target gene expression (Du et al. …

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