The gene, encompassing an active common fragile site, FRA3B, is frequently silenced in preneoplasia and cancer, through gene rearrangement or methylation of regulatory sequences. has slowed general acceptance of a role for Fhit in tumor suppression, despite strong evidence of Fhit association with multiple cancer\associated functions. This skepticism has hindered consideration of Fhit\associated therapeutic targets for the many Fhit\deficient human cancers. For example, the accumulation of genome mutations due to Fhit loss and the ability to stop the accumulation of genome damage by thymidine supplementation13 hint at possible preneoplasia prevention strategies. In addition, Fhit loss\induced DNA damage creates optimal single\stranded DNA substrates for the APOBEC3B enzyme (a cytidine deaminase that converts cytosines to uracils in single\stranded DNA), illustrating a key role for Fhit loss15 in hypermutation genotypes observed in most common cancers, a major source of cancer\associated genetic heterogeneity.16 The APOBEC3B enzyme, which causes hypermutations selectively in Fhit\deficient cells, is likely a critical diagnostic and therapeutic target.16 The purpose of the current study was to show that Fhit deficiency supports neoplastic progression. We followed expression changes from establishment, through proliferation in the face of selective pressures, to transformation and nascent neoplastic changes, in epithelial cells from Fhit knockout and wild\type 76095-16-4 mice. 76095-16-4 We have observed that Fhit loss is followed by genomic and functional changes in response to selective pressures that allow survival of clonally expanded populations, supporting the conclusion that Fhit loss\induced genome instability enables selection for transformation and neoplastic progression. Materials and Methods Ethics statement Mice were maintained and animal experiments carried out in accord with institutional guidelines established by the Animal Care and Use Committee at Ohio State University (Columbus, OH, USA). Cell lines and reagents Mouse kidney cell lines were established by culturing minced mouse kidney tissue from three C57Bl6 (B6 +/+ kd cell lines 1, 2, 3) and three (B6x129SvJ backcross, >99% B6 at genomic level)17 5\week\old mice (?/? kd cell lines 2, 3, 4). After emergence of epithelial cells from minced kidney fragments, cells could be subcultured; these epithelial kidney cell lines did not show an obvious TNFRSF10B crisis phase but rather grew steadily from first subculturing. Early passage +/+ and ?/? kidney lines did not show obvious morphological or proliferation differences (Figs S1,S2). However, late passage ?/? kidney lines grew more rapidly than +/+ (Fig. S2). RNA, DNA, and protein were isolated at alternate passages. To establish 7,12\dimethylbenz[a]anthracene (DMBA) survivor (DS) cell lines, late passage (p40) cells were treated with two sequential 24\h, 20\M DMBA doses, followed by plating and culturing of surviving colonies 8 days post\treatment; +/+ cells did not survive DMBA treatment. To establish nutritionally stressed (NS) cell lines, early passage cells were maintained without replenishing medium for several months, followed by fresh medium and culture of surviving colonies; +/+ cell lines did not survive nutritional stress. The NS cell lines exhibited new morphological features as they transitioned from epithelial to mesenchymal phenotype (Fig. S1). Nutritionally stressed cells also grew to a higher density than 76095-16-4 +/+ cells (Fig. S2). Some DS and NS cell lines formed colonies in soft agar. Colonies were isolated and replated to establish colony\forming cell lines (Table 1 summarizes cell line characteristics). The mouse cell lines were cultured in MEM with 5% FBS and 100 g/mL gentamicin. H1299, a human non\small\cell lung carcinoma cell line, was cultured in MEM with 10% FBS and 76095-16-4 100 g/mL gentamicin. Table 1 Derivation of mouse kidney cell lines Immunoblots, soft agar growth, and invasion assays Immunoblots were carried out as described.13 Antisera18, 19 used and working dilutions are available in Table S1. Soft agar20.