06-864 Sigma-AldrichAnti-FLIP Antibody, CT

Keratin 8 and 18 (K8/K18) are major intermediate filament proteins of liver hepatocytes. They provide mechanical and nonmechanical stability, thereby protecting cells from stress. Hence, K8-null mice are highly sensitive to Fas-mediated liver cell apoptosis. However, the role of c-Flip protein in K8-null related susceptibility to liver injury is controversial. Here we analyzed c-Flip protein expression in various K8 or K18 null/mutant transgenic livers and show that they are similar in all analyzed transgenic livers and that previously reported c-Flip protein changes are due to antibody cross-reaction with mouse K18. Furthermore, analysis of various apoptosis- or cell survival-related proteins demonstrated that inhibition of phosphorylation of NF-κB and various stress activated protein kinases (SAPKs), such as p38 MAPK, p44/42 MAPK and JNK1/2, is related to the higher sensitivity of K8-null hepatocytes whose nuclear NF-κB is rapidly depleted through Fas-mediated apoptosis. Notably, we found that NF-κB and the studied protein kinases are associated with the K8/K18 complex and are released upon phosphorylation. Therefore, interaction of keratins with cell survival-related protein kinases and transcription factors is another important factor for hepatocyte survival.

  Personalized cancer treatment strategies depend on comprehensive and detailed characterization of individual human malignancies. Clinical pathology, particularly immunohistochemical evaluation of biomarkers in tissues, is considered to be the approved standard for diagnostic and therapeutic decisions, having a direct influence on patient management and therapy. Although antibody-based approaches are established and integrated successfully into both clinical and research applications, for personalized treatment regimens new demands have been placed on the quality, reproducibility and accuracy of antibody-based assays. To ensure the accuracy of specific antigen detection in immunohistochemistry, we introduce a novel approach for antibody validation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces selective apoptotic death of human cancer cells while sparing normal human cells. Although TRAIL holds great promise as a potential anticancer agent, some tumors develop resistance to TRAIL. Previously, we have shown that the activator protein 1 (AP-1) family member, c-Fos, is an important modulator of apoptosis. Although F- box protein 10 (FBXL10) has been implicated to regulate an AP-1 family protein, c-Jun, its role in mediating apoptotic pathways has not been previously investigated. Here, we report that FBXL10 is a transcriptional repressor of c-Fos and a target gene of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-p65 in human cancers. We demonstrate that FBXL10 is an important anti-apoptotic molecule, which directly binds and represses c-Fos promoter in order for cancer cells to resist TRAIL-induced apoptosis. FBXL10 indirectly regulates c-FLIP(L) levels via c-Fos-dependent pathways. Silencing of FBXL10 sensitizes resistant cells to TRAIL, while, overexpression of FBXL10 represses TRAIL-induced apoptosis. Moreover, our results indicate that expression of FBXL10 functions via an NF-κB-dependent pathway, and TRAIL or proteasome inhibitors downregulate FBXL10 via inhibiting NF-κB signaling. Taken together, we find a novel functional role for FBXL10 as an anti-apoptotic molecule, and describe a new apoptotic-related pathway that involves NF-κB/FBXL10/c-Fos/c-FLIP. Therefore, silencing FBXL10 can help overcome resistant cancer cells for pro-apoptotic therapies.

M1 myeloid leukemic cells were used to dissect the molecular mechanisms of myeloid cell survival and apoptosis. A salient feature of M1 cells is that they respond to the physiological survival factor interleukin-6 (IL-6), yet lack the tumor suppressor gene p53. Functional wild-type activation of temperature-sensitive p53 protein (p53 val) at permissive temperature in M1-t-p53 cells results in rapid apoptosis, which is blocked by IL-6. How p53 induces M1 apoptosis and how IL-6 protects against p53-induced apoptosis are not fully understood. Here it is shown that p53-mediated apoptosis of M1 cells involves rapid activation of the proapoptotic Fas/CD95 death pathway, which activates caspases 8 and 10. Functional p53 also targets the mitochondria, causing upregulation of proapoptotic Bax, downregulation of prosurvival Bcl-2 and activation of caspase 9. IL-6 was found to protect against p53-induced apoptosis via activation of the PI3K/Akt survival pathway, which in turn counters both the Fas/CD95 and mitochondrial apoptotic pathways and activates the prosurvival transcription factor nuclear factor-kappaB (NF-kappaB). Taken together, this work supports a novel model for leukemic progression where cells that acquire the ability to produce an autocrine survival factor, such as IL-6, can bypass normal p53 surveillance function by targeting Akt, which in turn can exert effects on the regulators of apoptosis, such as the Fas/CD95 pathway, the mitochondria and NF-kappaB.

The widely expressed protein Fas is a member of the tumour necrosis factor receptor family which can trigger apoptosis. However, Fas surface expression does not necessarily render cells susceptible to Fas ligand-induced death signals, indicating that inhibitors of the apoptosis-signalling pathway must exist. Here we report the characterization of an inhibitor of apoptosis, designated FLIP (for FLICE-inhibitory protein), which is predominantly expressed in muscle and lymphoid tissues. The short form, FLIPs, contains two death effector domains and is structurally related to the viral FLIP inhibitors of apoptosis, whereas the long form, FLIP(L), contains in addition a caspase-like domain in which the active-centre cysteine residue is substituted by a tyrosine residue. FLIPs and FLIP(L) interact with the adaptor protein FADD and the protease FLICE, and potently inhibit apoptosis induced by all known human death receptors. FLIP(L) is expressed during the early stage of T-cell activation, but disappears when T cells become susceptible to Fas ligand-mediated apoptosis. High levels of FLIP(L) protein are also detectable in melanoma cell lines and malignant melanoma tumours. Thus FLIP may be implicated in tissue homeostasis as an important regulator of apoptosis.