Identification of Genes Responsive to Intracellular Zinc Depletion in the Human Colon Adenocarcinoma Cell Line HT-29

Abstract

Zinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. To identify genes with altered mRNA expression level after zinc depletion, we employed oligonucleotide arrays with approximately 10,000 targets and used the human colon adenocarcinoma epithelial cell line HT-29 as a model. A low intracellular zinc concentration caused alterations in the steady-state mRNA levels of 309 genes at a threshold factor of 2.0. Northern blot analysis and/or real-time RT-PCR confirmed the array results for 12 of 14 selected targets. Genes identified as regulated based on microarray data encode mainly proteins involved in central pathways of intermediary metabolism (79 genes) including protein metabolism (21). We also identified five groups of genes important for basic cellular functions such as signaling (30), cell cycle control and growth (15), vesicular trafficking (15), cell-cell interaction (13), cytoskeleton (10) and transcription control (19). The latter group comprises several zinc finger-containing transcription factors of which the Kruppel-like factor 4 showed the most pronounced changes. Western blot analysis confirmed the increased expression level of this protein in cells grown under low zinc conditions. Our findings in a homogeneous cell population demonstrate that the molecular mechanisms by which cellular functions are altered at a low zinc status, occur via pleiotropic effects on gene expression. In conclusion, the pattern of zinc-affected genes may represent a reference for further studies to define the zinc regulon in mammalian cells.

Krüppel-like factor 4 regulates adaptive expression of the zinc transporterZip4in mouse small intestine

“…By contrast, we have found that excess dietary zinc has minimal effects on Zip4 mRNA (7). It has been demonstrated that elevated zinc in cells leads to posttranslational processing, endocytosis of plasma membrane-localized Zip4, and degradation or ubiquitination (14). Those findings agree with our results on ZIP4 protein induction and that such degradative pathways are negligible during zinc-limited conditions.…”

“…Particularly relevant to our present findings are the experiments reported by Kindermann et al (13,14) that demonstrated the marked upregulation of Klf4 by zinc chelation with TPEN in a human colon carcinoma cell line (HT-29). They suggested that the metal-responsive transcription factor MTF-1 was involved in regulation of Klf4 expression, since the human KLF4 promoter has MRE sequences upstream in the 65 Zn (57,000 cpm/ml) without DTPA and incubated for up to 60 min at 37°C.…”

A novel small molecule with potent anticancer activity inhibits cell growth by modulating intracellular labile zinc homeostasis

“…Although labile zinc has been shown to be involved in several cellular pathways related to the regulation of cell fate, these mechanisms are not well characterized (3). Reduction of intracellular labile zinc has been associated with the induction of apoptosis, decreased cell proliferation, and altered cell cycle progression in a number of cancer cell types, including mammary adenocarcinoma (4), melanoma (5), colon adenocarcinoma (6), and lymphocytic leukemia (7)(8)(9)(10).…”

“…Studies of zinc-responsive gene regulation induced by intracellular labile zinc depletion in colon carcinoma HT-29 cells identified Krüppel-like factor 4 (KLF4, also known as GKLF) as one of the genes whose expression is most significantly changed (up-regulated) among >10,000 target genes tested (6). KLFs are members of the SP/XKLF family of transcription factors defined by an amino acid binding domain at the C termini that comprises three C2H2-type zinc fingers with similarity to the developmental gene Krüppel of Drosophila melanogaster (11).…”

Increased Uptake of Zinc in Malignant Cells is Associated with Enhanced Activation of MAPK Signalling and P53-Dependent Cell Injury

“…concentrations Free intracellular zinc in Hep-2 cells treated with zinc as described above was monitored using microfluorometry of the zinc-specific dye Newport Green diacetate (6). Briefly, cells grown in black-bottom 96-well plates were incubated with Newport Green diacetate (5 μmol in PBS, dark, 30 min at 37 °C) and fluorescence intensity was determined by a multiplate reader TECAN SpectraFluor Plus (TECAN Austria GmbH, Grödig, Austria) at 485 and 535 nm, respectively with stable integration time 1000 ms and gain 150.…”