agar and Colorectal-Neoplasms

Control of E2F1 activity is restricted via its interactions with RB1 and HDAC1. However, the detailed regulatory mechanisms underlying the E2F1/HDAC1 complex remain elusive. Here, we report that Nemo-like kinase (NLK) boosts cell cycle progression, which facilitates tumor development by releasing the E2F1 protein from HDAC1. Deletion of NLK largely blocks colorectal tumor proliferation and development. Moreover, RNA-seq shows that cell cycle is arrested at the G1/S phase in NLK-deficient cells and that the expression of E2F complex-targeted genes are affected, whereas overexpression of NLK but not an NLK mutant restores the wild-type phenotype. Mechanistically, we show that NLK interacts with the E2F1 complex, leading to disassembly of the E2F1/HDAC1 complex and thus diminishing the ability of E2F1 to bind to target gene promoters. Our results indicate that NLK boosts cell proliferation and E2F1 activity and controls the cell cycle switch by releasing HDAC1 from the E2F1 complex.

Topics: Agar; Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Progression; E2F1 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; Histone Deacetylase 1; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Protein Serine-Threonine Kinases; RNA Interference; Transcriptional Activation

The generation of knock-out mice for E2F4 gene expression has suggested a role for this transcription factor in establishing and/or maintaining the intestinal crypt compartment. Having previously demonstrated that E2F4 is cytoplasmic in quiescent-differentiated cells but nuclear in growth factor-stimulated proliferative cells, the present study was aimed at determining the role of E2F4 in the control of human intestinal epithelial proliferation. Results herein demonstrate that lentiviral infection of an shRNA which specifically knocked-down E2F4 expression slowed down G1/S phase transition and the proliferation rate of normal human intestinal epithelial cells (HIEC) and of colon cancer cells. Protein expression of Cdk2, cyclins D1 and A, Cdc25A and c-myc was markedly down-regulated in shE2F4-expressing cells; by contrast, expression of the cell cycle inhibitors p21(Cip/Waf) and p27(Kip1) was increased. In addition, the expression of many genes involved in DNA synthesis was down-regulated in shE2F4-expressing cells, whereas no modulation in E2F1 expression was observed. A decrease in E2F4 in colon cancer cell lines also resulted in a reduction in soft-agar growth capacity. Immunofluorescence experiments in human fetal intestine revealed that cells expressing high nuclear levels of E2F4 also expressed cyclin A protein. Lastly, E2F4 and its target cyclin A were up-regulated and mostly nuclear in human colorectal tumor cells in comparison to the corresponding benign epithelium. These results indicate that nuclear E2F4 may be determinant in the promotion of proliferation of human intestinal epithelial crypt cells and colorectal cancer cells.

Topics: Agar; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Colorectal Neoplasms; Cyclin A; DNA; Down-Regulation; E2F4 Transcription Factor; Epithelial Cells; G1 Phase; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Intestinal Mucosa; Intestines; Protein Transport; S Phase

Wnt signaling is essential during development while deregulation of this pathway frequently leads to the formation of various tumors including colorectal carcinomas. A key component of the pathway is beta-catenin that, in association with TCF-4, directly regulates the expression of Wnt-responsive genes. To identify novel binding partners of beta-catenin that may control its transcriptional activity, we performed a mammalian two-hybrid screen and isolated the Tax-interacting protein (TIP-1). The in vivo complex formation between beta-catenin and TIP-1 was verified by coimmunoprecipitation, and a direct physical association was revealed by glutathione S-transferase pull-down experiments in vitro. By using a panel of deletion mutants of both proteins, we demonstrate that the interaction is mediated by the PDZ (PSD-95/DLG/ZO-1 homology) domain of TIP-1 and requires primarily the last four amino acids of beta-catenin. TIP-1 overexpression resulted in a dose-dependent decrease in the transcriptional activity of beta-catenin when tested on the TOP/FOPFLASH reporter system. Conversely, siRNA-mediated knock-down of endogenous TIP-1 slightly increased endogenous beta-catenin transactivation function. Moreover, we show that overexpression of TIP-1 reduced the proliferation and anchorage-independent growth of colorectal cancer cells. These data suggest that TIP-1 may represent a novel regulatory element in the Wnt/beta-catenin signaling pathway.

Topics: Agar; Animals; beta Catenin; Blotting, Western; Carrier Proteins; Cell Division; Cell Line; CHO Cells; Colorectal Neoplasms; Cricetinae; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Genes, Reporter; Glutaminase; Glutathione Transferase; Humans; Intracellular Signaling Peptides and Proteins; Luciferases; Mice; Microscopy, Fluorescence; Models, Genetic; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Trans-Activators; Transcription, Genetic; Transcriptional Activation; Transfection; Two-Hybrid System Techniques

It has been shown previously in ulcerative colitis tissue that E-cadherin can occasionally be mutated in the extracellular domain early in neoplastic progression. E-cadherin is known to maintain differentiation and inhibits invasion in vivo.. To assess the mechanisms by which such dysfunction occurs.. Four human colorectal cancer cell lines, HCA-7 colonies 1, 3, 6, and 30, derived from a single heterogeneous colorectal cancer were studied. The HCA-7 cell line has p53 mutations and a random errors of replication "positive" phenotype, as is seen in early colitis associated cancers or hereditary nonpolyposis coli cancer (HNPCC).. Cell lines 6 and 30 expressed E-cadherin abundantly and this correlated positively with their degree of differentiation and organisation; however, both cell lines had loss of heterozygosity of E-cadherin. Interestingly, E-cadherin production was downregulated in the poorly differentiated cell line 1, and this was associated with major chromosomal rearrangements of 16q. This cell line also had a mutation in the homophilic binding domain of exon 4, which was associated with disaggregation by low titres of a function blocking antibody, and an invasive phenotype.. These multiple biological alterations further characterise the complex association that E-cadherin has with tumour heterogeneity and suggest that this series of cell lines may be a useful model of colitis associated or HNPCC associated tumorigenesis.

Topics: Agar; Cadherins; Colitis, Ulcerative; Collagen; Colorectal Neoplasms; Culture Media; Humans; Karyotyping; Models, Biological; Mutation; Neoplasm Proteins; Polymorphism, Single-Stranded Conformational; RNA, Messenger; Tumor Cells, Cultured

In the present paper is studied the concentration in peripheral blood of immunoglobulins IgG, IgA, IgM and factors C3 and C4 of the complement system in a group of 120 patients with colorectal cancer operated on with radical intention. Preoperative values are compared to postoperative results and the values obtained on diagnosis of tumoral recurrence with the object of describing possible immunologic alterations existent in the patient with colorectal carcinoma. It is concluded that there are no significant relations between the biochemical parameters studied and age, sex, tumoral localization and Duke's clinical stages. Patients with pre and postoperative IgA values above normal present a poor prognosis, with an associated recurrence of 60% (p = 0.001) and 80% (p = 0.000), respectively; postoperative C3 levels above normal also represent a factor of poor prognosis, recurrence appearing in 80% (p = 0.001) and metastases in 85% (p = 0.000).

Topics: Adult; Agar; Aged; Aged, 80 and over; Colorectal Neoplasms; Complement C3; Complement C4; Complement System Proteins; Female; Humans; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Male; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Prospective Studies; Time Factors