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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.

The flavonoid flavone contained in a variety of fruits and vegetables was identified as a very potent apoptosis inducer in human colonic cancer cells. In search of the molecular targets of flavone action in HT-29 cells we analyzed changes in mRNA and protein expression levels by proteomics and oligonucleotide array technologies. Proteome analysis identified several heat-shock proteins, annexins, and cytoskeletal caspase substrates as regulated by flavone and these protein classes are known to play a role in apoptosis induction and execution. Protein kinase C-beta, which serves as an ultimate marker for colon cancer development was no longer detectable in HT-29 cells exposed to flavone. Besides proteins involved in gene regulation or detoxification pathways, proteins involved in intermediary metabolism were altered by flavone exposure and this was associated with changes in the flux of energetic substrates. Oligonucleotide arrays, using chips with around 10 000 oligonucleotides spotted, revealed numerous changes in transcript levels of genes related to signaling, transcription, cancer development but also to metabolism. In conclusion, flavone has a surprisingly broad spectrum of effects on mRNA and protein expression in a human colonic cancer cell line with clusters of targets related to its apoptosis-inducing activity and to cellular metabolism.

Zinc is an essential trace element that serves as a structural constituent of a large number of transcription factors, which explains its pivotal role in the control of gene expression. Previous studies investigating the effect of zinc deficiency and zinc supplementation on gene expression in the human adenocarcinoma cell line HT-29 led to the identification of a considerable number of genes responding to alterations in cellular zinc status with changes in steady state mRNA levels. For 9 of 20 genes from these previous screenings that were studied in more detail, mRNA steady state levels responded to both high and low media zinc concentrations. As they are primarily zinc-dependent, we assessed whether these genes are controlled by the zinc-finger metal transcription factor MTF-1. To test this hypothesis we generated a doxycyline-inducible Tet-On HT-29 cell line overexpressing MTF-1. Using this conditional expression system, we present evidence that Kruppel-like factor 4 (klf4), hepatitis A virus cellular receptor 1 (hhav), and complement factor B (cfbp) are 3 potential new target genes of MTF-1. To support this, we used in silico analysis to screen for metal-responsive elements (MREs) within promotors of zinc-sensitive genes. We conclude that zinc responsiveness of klf4, hhav, and cfbp in HT-29 cells is mediated at least in part by MTF-1. Résumé :Le zinc est un oligo-élément essentiel qui agit comme constituant structural d'un grand nombre de facteurs de transcription, ce qui explique son rôle pivot dans le contrôle de l'expression génique. Des études précédentes des effets d'une déficience ou d'une supplémentation en zinc sur l'expression génique dans la lignée d'adénocarcinome humain HT-29 ont mené à l'identification d'un nombre considérable de gènes répondant aux altérations du statut cellulaire en zinc par des changements dans les niveaux d'ARNm. Parmi les 20 gènes étudiés plus en détail dans les criblages précédents, neuf ont répondu tant à des concentrations élevées que faibles de zinc dans le milieu, par des variations dans les niveaux d'ARNm. Puisqu'ils sont considérés comme dépendant du zinc, nous avons déterminé si ces gènes étaient contrôlés par le facteur de transcription à doigt de zinc MTF-1. Afin de tester cette hypothèse, nous avons généré une lignée HT-29 inductible par la Dox dans le système « Tet-on » qui surexprime MTF-1. À l'aide de ce système d'expression conditionnel, nous présentons des évidences que le facteur de type Kruppel (klf4), le récepteur cellulaire du virus de l'hépatite A (hhav) et le facteur du complément (cfbp) constituent trois gènes cibles potentiels de MTF-1. En parallèle, en utilisant une analyse in silico, nous avons recherché des éléments de réponse aux métaux (MREs) à l'intérieur de promoteurs de gènes sensibles au zinc. Nous concluons que la réponse au zinc de klf4, hhav et cfbp dans les cellules HT-29 dépend en partie du facteur MTF-1.Mots clés : gènes sensibles au zinc, gènes cibles, MTF-1, HT-29, élément de réponse aux métaux.[Traduit par la Rédaction] Kindermann et...

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