Genetic control of cell proliferation and cancer susceptibility in zebrafish.

摘要

Zebrafish is an ideal system for cancer gene discovery owing to its combination of forward genetics and use as a carcinogenesis model. Zebrafish exposed to carcinogens develop a wide variety of tumors in virtually all organs with a histology closely resembling that of human tumors. In order to explore the genetic pathways that control cellular proliferation and cancer in the zebrafish, we performed a haploid screen using whole-mount anti-phosphohistone H3 antibody staining to detect mutations causing alterations in cell proliferation. Eight homozygous lethal mutants with distinct cell cycle abnormalities were identified: crash&burn, cease&desist, standstill, self-destruct, sleepyhead, dillydally, lollygag and shortstop.; One mutant, crash&burn (crb), has defects in mitotic progression and exhibits genomic instability associated with mitotic spindle defects. Positional cloning demonstrated that crb represents a loss of function mutation in bmyb, a transcriptional regulator and member of a putative proto-oncogene family. Our work suggests that regulation of cyclin B levels by bmyb is the mechanism of mitotic accumulation in crb. Carcinogenesis studies revealed increased cancer susceptibility in adult crb heterozygotes that seems to occur via haplo-insufficiency of bmyb . Through a novel chemical suppressor screening approach, a chemical, persynthamide (psy), was discovered that suppressed bmyb-dependent prometaphase accumulation, polyploidy and cell death, and restored cyclin B1 levels. Our studies show that the mechanism of crb suppression by psy involves activation of the intra-S-phase checkpoint and subsequent upregulation of cyclin B1 transcription.; Another mutant, cease&desist (cds), exhibits a high percentage of polyploid and aneuploid cells. Positional cloning revealed that a mutation in separase, a gene involved in regulating sister chromatid cohesion, is responsible for the cds phenotype. cds embryos also exhibit multiple mitotic spindle abnormalities. Carcinogenesis studies have shown that cds adult heterozygotes have a 2.5 fold increase in tumor formation with a specific increase in epithelial tumors, indicating that the separase hemizygous state triggers formation of these tumors.; Another genetic screening approach was undertaken to identify zebrafish embryos with defects in cell cycle checkpoints. Haploid embryos from ENU-derived F1 females were screened for the ability to bypass a radiation-induced G2 checkpoint. Three mutants were identified in this screen and they exhibit both general and tissue-specific defects in checkpoint regulation.; This thesis demonstrates that zebrafish screens can be used to uncover genes that underlie cell cycle and cancer pathways. Both crb and cds represent genes that had not previously been shown to increase cancer susceptibility through loss of function. This work provides new avenues for cancer research and treatment and demonstrates that zebrafish is a powerful tool for dissecting cell proliferation and cancer pathways.