FHA-ATG12-ATG5) complex (A) as well as that of the endogenous MCL1 (endog. with other autophagy mediators, such as ATG5. We found that the ability of ATG12 to kill oncogenic RAS-carrying malignant cells does not require covalent binding of ATG12 to other proteins. In summary, we have identified a novel mechanism by which oncogenic RAS promotes survival of malignant intestinal epithelial cells. This mechanism is usually driven by RAS-dependent loss of ATG12 in these cells. allele and their mutant knockout derivatives DKO-3 and DKS-8 were assayed for ATG12 expression by western blot. (C) Human colon cancer cells HT29 (left) and CaCo2 (right) carrying the wild-type and human Mouse monoclonal to EGF colon cancer cells LoVo, LS180 and SKCO1 carrying a mutant GSK2838232A allele (left and right) were assayed for ATG12 expression by western blot. (D) Stable cell lines CaCo2-cont and CaCo2-ras generated by contamination of human colon cancer cells CaCo2 with either a control retrovirus (CaCo2-cont) or HA-tagged an oncogenic KRAS mutant-encoding retrovirus (CaCo2-ras) were assayed for KRAS (left) or ATG12 (right) expression by western blot. CDC25 (A, left), CDK4 (A, right, C, and D) and MAPK14/p38 MAP kinase (B) served as loading controls. Positions of unconjugated ATG12 (ATG12), the ATG12-ATG5 (ATG12-ATG5) conjugate and that of HA-tagged KRAS around the blots are indicated. GSK2838232A Covalent complexes between ATG12 and ATG531 and possibly between ATG12 and ATG337,38 GSK2838232A promote autophagy. ATG12 can also cause autophagy-independent apoptosis.32 Apoptosis is mediated by the release of CYCS/cytochrome c from the mitochondria to the cytoplasm where it triggers activation of caspases,39 proteases that cleave vital cellular targets.40 CYCS release is caused by the pro-apoptotic BCL2-family proteins using a Bcl-2 homology 3 domain name to bind and neutralize the anti-apoptotic BCL2 family members (which block CYCS release).41 ATG12 contains such domain and kills cells by the same mechanisms.32 GSK2838232A This effect of ATG12 does not require the ability of ATG12 to covalently bind other autophagy mediators.32 The effect of RAS on ATG12 was not unique to rat cells as human colon cancer cells DLD142 carrying a mutant allele showed lower free ATG12 levels than their variants DKO3 and DKS8, in which this allele was ablated by homologous recombination (Fig.?1B).42 GSK2838232A Furthermore, mutant (Fig.?1C). Finally, we observed that introduction of the mutant gene in mutant KRAS-negative cells CaCo2 resulted in a noticeable downregulation of free ATG12 (Fig.?1D). Thus, oncogenic RAS reduces free ATG12 levels in malignant intestinal epithelial cells. RAS-induced ATG12 downregulation is critical for clonogenic survival of malignant intestinal epithelial cells To test the role of ATG12 in cancer cell growth we infected ras-4 cells with a control murine stem cell virus (MSCV) or MCSV encoding ATG12. Contamination efficiency was close to 100% as puromycin (resistance to which was encoded by MSCV) killed essentially all uninfected cells but essentially all cells were clonogenic in the presence of puromycin after being infected with a control MSCV (not shown). We found that ras-4 cells infected with ATG12-encoding viruses produced free ATG12 at levels that were significantly higher than those in the cells infected with a control virus and comparable to those in the parental IEC-18 cells (Fig.?2A). We also observed a band recognized by the anti-ATG12 and anti-ATG5 antibodies around the respective western blots that displayed a reduced mobility compared with the ATG12-ATG5 complex, most likely, due to the conjugation of ectopic ATG12 with endogenous ATG5 (Fig.?2A and ?andB).B). We found that exogenous ATG12 strongly blocked clonogenicity of these cells (Fig.?2C). This observation was not unique to ras-4 cells as ectopic ATG12 also noticeably blocked clonogenicity of human mutant KRAS-positive cells LS180 (Fig.?2D and ?andE).E). Unlike the case with mutant RAS-carrying cells, exogenous ATG12 did not have a significant effect on clonogenicity of nonmalignant IEC-18 cells (Fig.?2F and ?andGG). Open in a separate.
