Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro

BackgroundCancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation [1]. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process.MethodsIn order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point.ResultsMicroarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action.The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform.ConclusionNAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action.

[1]  D. Bernard,et al.  Mechanisms of inhibin signal transduction. , 2001, Recent progress in hormone research.

[2]  Mauro Piacentini,et al.  Transglutaminase 2: an enigmatic enzyme with diverse functions. , 2002, Trends in biochemical sciences.

[3]  Y. Chen,et al.  HEC, a novel nuclear protein rich in leucine heptad repeats specifically involved in mitosis , 1997, Molecular and cellular biology.

[4]  J. Ricort,et al.  Insulin-like Growth Factor-binding Protein-3 Activates a Phosphotyrosine Phosphatase , 2002, The Journal of Biological Chemistry.

[5]  W. Stetler-Stevenson,et al.  The role of matrix metalloproteases and their inhibitors in tumour invasion, metastasis and angiogenesis. , 1994, The European respiratory journal.

[6]  H. Shibai,et al.  Erythroid differentiation factor is encoded by the same mRNA as that of the inhibin beta A chain. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[7]  W. Dinjens,et al.  A novel gene which is up-regulated during colon epithelial cell differentiation and down-regulated in colorectal neoplasms. , 1997, Laboratory investigation; a journal of technical methods and pathology.

[8]  E. Pérez-Nadales,et al.  Essential function for ErbB3 in breast cancer proliferation , 2004, Breast Cancer Research.

[9]  R H Hruban,et al.  Gene expression profiles in normal and cancer cells. , 1997, Science.

[10]  A. Yang,et al.  REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species. , 2002, Molecular cell.

[11]  J. Morrow,et al.  Antioxidants reduce cyclooxygenase-2 expression, prostaglandin production, and proliferation in colorectal cancer cells. , 1998, Cancer research.

[12]  D. Yansura,et al.  Purification, receptor binding analysis, and biological characterization of human melanoma growth stimulating activity (MGSA). Evidence for a novel MGSA receptor. , 1993, The Journal of biological chemistry.

[13]  G. Silverman,et al.  Suppression of a squamous cell carcinoma (SCC)-related serpin, SCC antigen, inhibits tumor growth with increased intratumor infiltration of natural killer cells. , 2001, Cancer research.

[14]  Makoto M Taketo,et al.  Colonic polyposis caused by mTOR-mediated chromosomal instability in Apc+/Δ716 Cdx2+/− compound mutant mice , 2003, Nature Genetics.

[15]  J. Mandel,et al.  N-acetylcysteine suppression of the proliferative index in the colon of patients with previous adenomatous colonic polyps. , 1999, Cancer letters.

[16]  P. Agre,et al.  Aquaporin water channels: unanswered questions and unresolved controversies , 1995, Current Opinion in Cell Biology.

[17]  M. Noda,et al.  Identification of a Novel Suppressive Vitamin D Response Sequence in the 5′-Flanking Region of the Murine Id1 Gene* , 1997, The Journal of Biological Chemistry.

[18]  B. Craig,et al.  Gene expression profiling of Caco-2 BBe cells suggests a role for specific signaling pathways during intestinal differentiation. , 2003, Physiological genomics.

[19]  J. Campisi,et al.  Id-related genes encoding helix-loop-helix proteins are required for G1 progression and are repressed in senescent human fibroblasts. , 1994, The Journal of biological chemistry.

[20]  J. Murley,et al.  Effects of thiols on topoisomerase‐IIα activity and cell cycle progression , 1998 .

[21]  J. Seidelin,et al.  Transcriptome changes during intestinal cell differentiation. , 2002, Biochimica et biophysica acta.

[22]  Gregory J. Hannon,et al.  pl5INK4B is a potentia| effector of TGF-β-induced cell cycle arrest , 1994, Nature.

[23]  J. Nevins,et al.  Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  K. Sonoyama,et al.  Gene expression of activin, activin receptors, and follistatin in intestinal epithelial cells. , 2000, American journal of physiology. Gastrointestinal and liver physiology.

[25]  Sam W. Lee.  H–cadherin, a novel cadherin with growth inhibitory functions and diminished expression in human breast cancer , 1996, Nature Medicine.

[26]  M. Fishman,et al.  Impaired Defense of Intestinal Mucosa in Mice Lacking Intestinal Trefoil Factor , 1996, Science.

[27]  D. Arango,et al.  A gene expression profile that defines colon cell maturation in vitro. , 2002, Cancer research.

[28]  David E. Misek,et al.  The Hepatocyte Nuclear Factor 3 α Gene, HNF3α (FOXA1), on Chromosome Band 14q13 Is Amplified and Overexpressed in Esophageal and Lung Adenocarcinomas , 2002 .

[29]  K. Takata,et al.  Water Channel Protein AQP3 Is Present in Epithelia Exposed to the Environment of Possible Water Loss , 1999, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[30]  E. Hammond,et al.  Cell cycle blockade and differentiation of ovarian cancer cells by the histone deacetylase inhibitor trichostatin A are associated with changes in p21, Rb, and Id proteins. , 2002, Molecular cancer therapeutics.

[31]  J. Lundeberg,et al.  Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms , 2005, Cell Death and Differentiation.

[32]  C. Delporte,et al.  Aquaporin 1 expression during the cell cycle in A5 cells. , 1996, Biochemical and biophysical research communications.

[33]  W. Rubas,et al.  A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model. , 1997, Critical reviews in therapeutic drug carrier systems.

[34]  Douglas A. Hosack,et al.  Identifying biological themes within lists of genes with EASE , 2003, Genome Biology.

[35]  Joel I. Pritchard,et al.  BMP-2 mediates retinoid-induced apoptosis in medulloblastoma cells through a paracrine effect , 2003, Nature Medicine.

[36]  M. Buendia,et al.  Identification of the LIM Protein FHL2 as a Coactivator of β-Catenin* , 2003, The Journal of Biological Chemistry.

[37]  Andreas Scorilas,et al.  Expression analysis of the human kallikrein 7 (KLK7) in breast tumors: a new potential biomarker for prognosis of breast carcinoma , 2003, Thrombosis and Haemostasis.

[38]  Joanna Groden,et al.  The APC tumor suppressor controls entry into S-phase through its ability to regulate the cyclin D/RB pathway. , 2002, Gastroenterology.

[39]  D. Goeddel,et al.  Identification of Heregulin, a Specific Activator of p185erbB2 , 1992, Science.

[40]  M. Ffrench,et al.  BTG1, a member of a new family of antiproliferative genes. , 1992, The EMBO journal.

[41]  J. Kellerth,et al.  Sensory neuroprotection, mitochondrial preservation, and therapeutic potential of n-acetyl-cysteine after nerve injury , 2004, Neuroscience.

[42]  G. Hannon,et al.  p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest. , 1994, Nature.

[43]  R. DuBois,et al.  Cyclooxygenase Regulates Angiogenesis Induced by Colon Cancer Cells , 1998, Cell.

[44]  M. Buendia,et al.  Identification of the LIM protein FHL2 as a coactivator of beta-catenin. , 2003, The Journal of biological chemistry.

[45]  H. Shih,et al.  HBP1: a HMG box transcriptional repressor that is targeted by the retinoblastoma family. , 1997, Genes & development.

[46]  G. Peters,et al.  Opposing effects of Ets and Id proteins on p16INK4a expression during cellular senescence , 2001, Nature.

[47]  Tony Kouzarides,et al.  The HMG-box transcription factor HBP1 is targeted by the pocket proteins and E1A , 1997, Oncogene.

[48]  S. Calvano,et al.  The mature bone morphogenetic protein-2 is aberrantly expressed in non-small cell lung carcinomas and stimulates tumor growth of A549 cells. , 2003, Carcinogenesis.

[49]  J. Massagué,et al.  Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. , 1995, Genes & development.

[50]  L. Coussens,et al.  Matrix metalloproteinases and the development of cancer. , 1996, Chemistry & biology.

[51]  M. Nachtigal,et al.  Identification of a putative autocrine bone morphogenetic protein-signaling pathway in human ovarian surface epithelium and ovarian cancer cells. , 2003, Endocrinology.

[52]  Suresh Mishra,et al.  Tissue Transglutaminase Has Intrinsic Kinase Activity , 2004, Journal of Biological Chemistry.

[53]  A. Wickrema,et al.  Role of JunB in Erythroid Differentiation* , 2002, The Journal of Biological Chemistry.

[54]  P. Russell,et al.  Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15. , 1993, The EMBO journal.

[55]  J. Godleski,et al.  N-acetylcysteine prevents lung inflammation after short-term inhalation exposure to concentrated ambient particles. , 2004, Toxicological sciences : an official journal of the Society of Toxicology.

[56]  R. DuBois,et al.  Cyclooxygenase Regulates Angiogenesis Induced by Colon Cancer Cells , 1998, Cell.

[57]  Ruo-Pan Huang,et al.  The Transcription Factor EGR-1 Suppresses Transformation of Human Fibrosarcoma HT1080 Cells by Coordinated Induction of Transforming Growth Factor-β1, Fibronectin, and Plasminogen Activator Inhibitor-1* , 1999, The Journal of Biological Chemistry.

[58]  S. Sato,et al.  In situ expression of platelet-activating factor (PAF)-receptor gene in rat skin and effects of PAF on proliferation and differentiation of cultured human keratinocytes. , 1998, The Journal of investigative dermatology.

[59]  S. De Flora,et al.  Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points. , 2001, Carcinogenesis.

[60]  G. Novelli,et al.  Evidence for differential S100 gene over-expression in psoriatic patients from genetically heterogeneous pedigrees , 2002, Human Genetics.

[61]  J. Parvin,et al.  DNA topoisomerase IIα is required for RNA polymerase II transcription on chromatin templates , 2001, Nature.

[62]  G. Yousef,et al.  Tissue kallikreins: new players in normal and abnormal cell growth? , 2003, Thrombosis and Haemostasis.

[63]  Ann-Marie Wennberg,et al.  The helix-loop-helix transcription factor Id1 is highly expressed in psoriatic involved skin. , 2003, Acta dermato-venereologica.

[64]  David E. Misek,et al.  The hepatocyte nuclear factor 3 alpha gene, HNF3alpha (FOXA1), on chromosome band 14q13 is amplified and overexpressed in esophageal and lung adenocarcinomas. , 2002, Cancer research.

[65]  S. Chakrabarty,et al.  Platelet-activating factor activates mitogen-activated protein kinases, inhibits proliferation, induces differentiation and suppresses the malignant phenotype of human colon carcinoma cells , 2003, Oncogene.

[66]  Xiangjian Zheng,et al.  Aquaporin 3 colocates with phospholipase d2 in caveolin-rich membrane microdomains and is downregulated upon keratinocyte differentiation. , 2003, The Journal of investigative dermatology.

[67]  E. Langenfeld,et al.  Bone morphogenetic protein-2 stimulates angiogenesis in developing tumors. , 2004, Molecular cancer research : MCR.

[68]  Chris Albanese,et al.  Negative regulation of the Wnt–β‐catenin pathway by the transcriptional repressor HBP1 , 2001, The EMBO journal.

[69]  J. Beaulieu,et al.  Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line. , 1992, Gastroenterology.

[70]  S. De Flora,et al.  N-acetylcysteine inhibits endothelial cell invasion and angiogenesis. , 1999, Laboratory investigation; a journal of technical methods and pathology.

[71]  R. Benezra,et al.  Transcription of the dominant-negative helix-loop-helix protein Id1 is regulated by a protein complex containing the immediate-early response gene Egr-1 , 1996, Molecular and cellular biology.

[72]  K. Hatakeyama,et al.  Expression and localization of aquaporins in rat gastrointestinal tract. , 1999, American journal of physiology. Cell physiology.

[73]  R. DuBois,et al.  Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[74]  F. Marks,et al.  Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[75]  Xiao-Hong Sun.  Constitutive expression of the Id1 gene impairs mouse B cell development , 1994, Cell.

[76]  Y. Yuasa,et al.  BMP-2 modulates the proliferation and differentiation of normal and cancerous gastric cells. , 2004, Biochemical and biophysical research communications.

[77]  A. Tabilio,et al.  Apoptosis of human primary B lymphocytes is inhibited by N‐acetyl‐L‐cysteine , 2004, Journal of leukocyte biology.

[78]  E. Wagner,et al.  Chronic Myeloid Leukemia with Increased Granulocyte Progenitors in Mice Lacking JunB Expression in the Myeloid Lineage , 2001, Cell.

[79]  A. Mccarthy.  Development , 1996, Current Opinion in Neurobiology.