ABSTRACT
Septin9 (Sept9) is a member of the filament-forming septin family of structural proteins and is associated with a variety of cancers and with hereditary neuralgic amyotrophy. We have generated mice with constitutive and conditional Sept9 knockout alleles. Homozygous deletion of Sept9 results in embryonic lethality around day 10 of gestation whereas mice homozygous for the conditional allele develop normally. Here we report the consequences of homozygous loss of Sept9 in immortalized murine embryonic fibroblasts. Proliferation rate was not changed but cells without Sept9 had an altered morphology compared to normal cells, particularly under low serum stress. Abnormal, fragmented, and multiple nuclei were more frequent in cells without Sept9. Cell migration, as measured by gap-filling and filter-invasion assays, was impaired, but individual cells did not move less than wild-type cells. Sept9 knockout cells showed a reduced resistance to hypo-osmotic stress. Stress fiber and vinculin staining at focal adhesion points was less prominent. Long septin filaments stained for Sept7 disappeared. Instead, staining was found in short, often curved filaments and rings. Furthermore, Sept7 was no longer localized to the mitotic spindle. Together, these data reveal the importance of Sept9 for septin filament formation and general cell stability.
Subject(s)
Septins/metabolism , Animals , Blotting, Western , Cell Movement/genetics , Cell Movement/physiology , Cell Proliferation , Cells, Cultured , Cytoskeleton/genetics , Cytoskeleton/metabolism , Fluorescent Antibody Technique , Mice , Mice, Knockout , Polymerase Chain Reaction , Septins/geneticsABSTRACT
The aim of the study was to investigate the expression of angio- and lymphangiogenic molecules (vascular endothelial growth factors VEGF and VEGF-C and their receptors Flt-1, KDR, and Flt-4) in non-Hodgkin lymphomas (NHL) treated in the pre-rituximab era. Pre-therapeutic lymph-node biopsies from 155 patients with NHL (64 follicular lymphomas (FLs), 47 de novo diffuse large B-cell lymphomas (DLBCL) and 44 peripheral T-cell lymphomas (PTCL)) were stained by in situ hybridization and immunohistochemistry. Tumor cell expression of VEGF, VEGF-C and their receptors was detected in most of the analyzed biopsies. In FL, diffuse intratumoral VEGF staining correlated with shorter overall survival (OS) (p = 0.008) and diffuse KDR staining was associated with a higher risk of histologic transformation (p = 0.05). In DLBCL, high KDR expression predicted poor treatment response (p = 0.03) and had a significant adverse impact on OS (p < 0.001). In PTCL, diffuse tissue distribution of VEGF mRNA correlated with an unfavorable 5-year OS (p = 0.004).
Subject(s)
Lymphoma, Follicular/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, T-Cell, Peripheral/genetics , Neoplasm Proteins/analysis , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor C/analysis , Vascular Endothelial Growth Factor Receptor-1/analysis , Vascular Endothelial Growth Factor Receptor-2/analysis , Aged , Cell Nucleus/chemistry , Cytoplasm/chemistry , Female , Gene Expression Profiling , Humans , Lymph Nodes/chemistry , Lymph Nodes/pathology , Lymphoma, Follicular/mortality , Lymphoma, Follicular/pathology , Lymphoma, Large B-Cell, Diffuse/mortality , Lymphoma, Large B-Cell, Diffuse/pathology , Lymphoma, T-Cell, Peripheral/mortality , Lymphoma, T-Cell, Peripheral/pathology , Male , Middle Aged , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neovascularization, Pathologic/etiology , Neovascularization, Pathologic/genetics , Prognosis , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/analysis , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Retrospective Studies , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor C/biosynthesis , Vascular Endothelial Growth Factor C/genetics , Vascular Endothelial Growth Factor Receptor-1/biosynthesis , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-2/biosynthesis , Vascular Endothelial Growth Factor Receptor-2/geneticsABSTRACT
The CCAAT enhancer binding protein alpha (C/EBPalpha) is an important myeloid tumor suppressor that is frequently mutated in human acute myeloid leukemia (AML). We have previously shown that mice homozygous for the E2F repression-deficient Cebpa(BRM2) allele develop nonfatal AML with long latency and incomplete penetrance, suggesting that accumulation of secondary mutations is necessary for disease progression. Here, we use SRS19-6-driven retroviral insertional mutagenesis to compare the phenotypes of leukemias arising in Cebpa(+/+), Cebpa(+/BRM2), and Cebpa(BRM2/BRM2) mice, with respect to disease type, latency of tumor development, and identity of the retroviral insertion sites (RISs). Both Cebpa(+/BRM2) and Cebpa(BRM2/BRM2) mice preferentially develop myeloid leukemias, but with differing latencies, thereby demonstrating the importance of gene dosage. Determination of RISs led to the identification of several novel candidate oncogenes, some of which may collaborate specifically with the E2F repression-deficient allele of Cebpa. Finally, we used an in silico pathway analysis approach to extract additional information from single RISs, leading to the identification of signaling pathways which were preferentially deregulated in a disease- and/or genotype-specific manner.
