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Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0003-4313-1601
2005 (English)In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 5, 75- p.Article in journal (Refereed) Published
Abstract [en]

Background: Cancer 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. Methods: In 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. Results: Microarray 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. Conclusion: NAC 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.

Place, publisher, year, edition, pages
2005. Vol. 5, 75- p.
Keyword [en]
acetylcysteine, animal cell, article, cancer cell, cell differentiation, cell proliferation, cell strain CACO 2, cell type, computer program, controlled study, DNA microarray, down regulation, drug mechanism, epithelium, gene activation, gene control, gene expression, gene identification, gene repression, gene sequence, genetic transcription, growth inhibition, human, human cell, in vitro study, keratinocyte, nonhuman, nucleotide sequence, reverse transcription polymerase chain reaction, time series analysis, validation process
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-5717DOI: 10.1186/1471-2407-5-75ISI: 000230966200001Scopus ID: 2-s2.0-26844509194OAI: oai:DiVA.org:kth-5717DiVA: diva2:10172
Note
QC 20100916Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Molecular Signatures of Cancer
Open this publication in new window or tab >>Molecular Signatures of Cancer
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Cancer is an important public health concern in the western world, responsible for around 25% of all deaths. Although improvements have been made in the diagnosis of cancer, treatment of disseminated disease is inefficient, highlighting the need for new and improved methods of diagnosis and therapy. Tumours arise when the balance between proliferation and differentiation is perturbed and result from genetic and epigenetic alterations.

Due to the heterogeneity of cancer, analysis of the disease is difficult and a wide range of methods is required. In this thesis, a number of techniques are demonstrated for the analysis of genetic, epigenetic and transcriptional alterations involved in cancer, with the purpose of identifying a number of molecular signatures. Pyrosequencing proved to be a valuable tool for the analysis of both point mutations and CpG methylation. Using this method, we showed that oncogenes BRAF and NRAS, members of the Ras-Raf-MAPK pathway, were mutated in 82% of melanoma tumours and were mutually exclusive. Furthermore, tumours with BRAF mutations were more often associated with infiltrating lymphocytes, suggesting a possible target for immunotherapy. In addition, methylation of the promoter region of the DNA repair gene MGMT was studied to find a possible correlation to clinical response to chemotherapy. Results showed a higher frequency of promoter methylation in non-responders as compared to responders, providing a possible predictive role and a potential basis for individually tailored chemotherapy. Microarray technology was used for transcriptional analysis of epithelial cells, with the purpose of characterization of molecular pathways of anti-tumourigenic agents and to identify possible target genes. Normal keratinocytes and colon cancer cells were treated with the antioxidant N-acetyl L-cysteine (NAC) in a time series and gene expression profiling revealed that inhibition of proliferation and stimulation of differentiation was induced upon treatment. ID-1, a secreted protein, was proposed as a possible early mediator of NAC action. In a similar study, colon cancer cells were treated with the naturally occurring bile acid ursodeoxycholic acid (UDCA) in a time series and analysed by microarray and FACS analysis. Results suggest a chemopreventive role of UDCA by G1 arrest and inhibition of cell proliferation, possibly through the secreted protein GDF15.

These investigations give further evidence as to the diversity of cancer and its underlying mechanisms. Through the application of several molecular methods, we have found a number of potential targets for cancer therapy. Follow up studies are already in progress and may hopefully lead to novel methods of treatment.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 59 p.
Keyword
: BRAF, NRAS, MGMT, methylation, pyrosequencing, microarray technology, gene expression analysis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-3954 (URN)91-7178-348-2 (ISBN)
Public defence
2006-05-24, Sal FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20110121Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2011-01-21Bibliographically approved

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