Abstract Background Cowden Syndrome (CS) patients with germ line point mutations in the PTEN gene are at high risk for developing breast cancer. It is believed that cells harboring these mutant PTEN alleles are predisposed to malignant conversion. This article will characterize the biochemical and biological properties of a mutant PTEN protein found in a commonly used metastatic breast cancer cell line. Methods The expression of PTEN in human breast carcinoma cell lines was evaluated by Western blotting analysis. Cell line MDA-MB-453 was selected for further analysis. Mutation analysis of the PTEN gene was carried out using DNA isolated from MDA-MB-453. Site-directed mutagenesis was used to generate a PTEN E307K mutant cDNA and ectopic expressed in PC3, U87MG, MCF7 and Pten -/- mouse embryo fibroblasts (MEFS). Histidine (His)-tagged PTEN fusion protein was generated in Sf9 baculovirus expression system. Lipid phosphatase and ubiquitination assays were carried out to characterize the biochemical properties of PTEN E307K mutant. The intracellular localization of PTEN E307K was determined by subcellular fractionation experiments. The ability of PTEN E307K to alter cell growth, migration and apoptosis was analyzed in multiple PTEN-null cell lines. Results We found a mutation in the PTEN gene at codon 307 in MDA-MB-453 cell line. The glutamate (E) to lysine (K) substitution rendered the mutant protein to migrate with a faster mobility on SDS-PAGE gels. Biochemically, the PTEN E307K mutant displayed similar lipid phosphatase and growth suppressing activities when compared to wild-type (WT) protein. However, the PTEN E307K mutant was present at higher levels in the membrane fraction and suppressed Akt activation to a greater extent than the WT protein. Additionally, the PTEN E307K mutant was polyubiquitinated to a greater extent by NEDD4-1 and displayed reduced nuclear localization. Finally, the PTEN E307K mutant failed to confer chemosensitivity to cisplatinum when re-expressed in Pten -/- MEFS. Conclusions Mutation at codon 307 in PTEN C2 loop alters its subcellular distribution with greater membrane localization while being excluded from the cell nucleus. This mutation may predispose breast epithelial cells to malignant transformation. Also, tumor cells harboring this mutation may be less susceptible to the cytotoxic effects of chemotherapeutics.
The trafficking of T-lymphocytes to peripheral draining lymph nodes is crucial for mounting an adaptive immune response. The role of chemokines in the activation of integrins via Ras-related small GTPases has been well established. R-Ras is a member of the Ras-subfamily of small guanosine-5'-triphosphate-binding proteins and its role in T cell trafficking has been investigated in R-Ras null mice (Rras−/−). An examination of the lymphoid organs of Rras−/− mice revealed a 40% reduction in the cellularity of the peripheral lymph nodes. Morphologically, the high endothelial venules of Rras−/− mice were more disorganized and less mature than those of wild-type mice. Furthermore, CD4+ and CD8+ T cells from Rras−/− mice had approximately 42% lower surface expression of L-selectin/CD62L. These aberrant peripheral lymph node phenotypes were associated with proliferative and trafficking defects in Rras−/− T cells. Furthermore, R-Ras could be activated by the chemokine, CCL21. Indeed, Rras−/− T cells had approximately 14.5% attenuation in binding to intercellular adhesion molecule 1 upon CCL21 stimulation. Finally, in a graft-versus host disease model, recipient mice that were transfused with Rras−/− T cells showed a significant reduction in disease severity when compared with mice transplanted with wild-type T cells. These findings implicate a role for R-Ras in T cell trafficking in the high endothelial venules during an effective immune response.
This Teaching Resource provides lecture notes and slides for a class covering three kinase-mediated signaling pathways and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The focus is three major signaling cascades that are implicated in cell proliferation, survival, and stress response. They are, respectively, the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and Jun N-terminal kinase (JNK) cascades. The aim of this lecture is to review the major players of these intracellular signaling cascades in mammalian cells. In addition, emphasis is placed on understanding the dynamic, rather than linear, nature of signal transduction in determining cellular responses to external stimuli.
Cultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes. Exemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1−/− cells and attenuated cytokine-induced cell death in FAT10−/− clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones. Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.
Abstract R-Ras belongs the RAS superfamily of small GTP-binding proteins implicated in cell adhesion signaling. Unlike classical Ras proteins, R-Ras is not activated by growth factor receptors. Rather, its GTPase activity is modulated by molecules implicated in cell-cell adhesion including semaphorins and notch. Using an R-Ras knockout mouse strain, we have previously reported that R-Ras is required for murine Dendritic cell functions. Further characterization of this mouse strain revealed reduced cellularity of peripheral lymph nodes (PLNs) by 40% with T cell regions in the paracortex in which high endothelial venules (HEV) resided were less prominent when compared with wild-type (WT) mice. Consistently, immunohistological analysis for MECA-79, a marker of mature HEV, showed a 3- to 4-fold decrease in staining intensity. This is correlated with a 30-40% reduction in the number of CD62L+ CD4 and CD8 T cells in PLNs. Furthermore, R-Ras null T cells had reduced proliferative and homing capacity when compared with WT. More importantly, we demonstrated for the first time that the chemokine CCL21 was able to stimulate the GTP-loading of R-Ras within 15 seconds of addition. Indeed, R-Ras null T-cells displayed reduced binding to soluble ICAM-1 as well as to 2H-11 endothelial cells. To explore the role of R-Ras in anti-tumor immunity, B16/F10 melanoma cells were injected subcutaneously to WT and R-Ras null mice. In addition, a mouse breast tumor model was generated ineither WT or R-Ras-null genetic background. Tumor development and the number of infiltrating immune cells were analyzed to examine if R-Ras was involved in either anti- or pro-tumor immune responses. This work was supported by NIH CA78509 (AMC), MH59771 (AMC). MY was supported by Hong Kong PhD Fellowship Scheme (PF12-13584) and AMC was supported by the Lo Kwee-Seong Seed Fund, and a General Research Fund Grant, 14120915, from Hong Kong University Grants Committee. Note: This abstract was not presented at the meeting. Citation Format: Andrew M. Chan, Xiaocai Yan, Wai Nam Liu, Mingfei Yan. R-Ras is required for T cell trafficking in high endothelial venues and implicated in anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2017-3711
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