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Gene expression analysis of human epidermal keratinocytes after N-acetyl L-cysteine treatment demonstrates cell cycle arrest and increased differentiation
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0002-4657-8532
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2005 (English)In: Pathobiology (Basel), ISSN 1015-2008, E-ISSN 1423-0291, Vol. 72, no 4, 203-212 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: Several cancer prevention programmes have previously been executed using treatment of antioxidant compounds. The antioxidant N-acetyl L-cysteine (NAC), a membrane-permeable aminothiol, is a sulfhydryl reductant reducing oxidised glutathione, as well as being a precursor of intracellular cysteine and glutathione. A previous report based on the cellular response to NAC treatment showed that NAC induced a 10-fold more rapid differentiation in normal primary keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation. In order to investigate molecular events underlying the changes in proliferation and differentiation induced by NAC treatment, we performed global gene expression analysis of normal human epidermal keratinocytes in a time series. Methods: Treated samples were compared to untreated samples through a reference design using a spotted cDNA array comprising approximately 30,000 features. B statistics was used to identify differentially expressed genes, and RT-PCR of a selected set of genes was performed to verify differential expression. Results: The number of differentially expressed genes increased over time, starting with 0 at 30 min, 73 at 3 h and increasing to 952 genes at 48 h. Results of the expression analysis showed arrest of the cell cycle and an upregulation of cytoskeletal reorganisation, implicating increased differentiation. A comparison to gene ontology groups indicated downregulation of a large number of genes involved in cell proliferation and regulation of the cell cycle. Conclusions: A significant fraction of the differentially expressed genes could be classified according to their role in the differentiation process, demonstrating that NAC regulates the conversion from proliferation to differentiation at a transcriptional level.

Place, publisher, year, edition, pages
2005. Vol. 72, no 4, 203-212 p.
Keyword [en]
Cell cycle, Gene expression, Keratinocytes, Microarray; N-acetyl L-cysteine, NHEK, Real-time, RT-PCR
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-5716DOI: 10.1159/000086790ISI: 000231700000005Scopus ID: 2-s2.0-24044509171OAI: oai:DiVA.org:kth-5716DiVA: diva2:10171
Note
QC 20100915Available 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
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Note
QC 20110121Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2011-01-21Bibliographically approved

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Edlundh-Rose, EstherKupershmidt, IlyaGustafsson, AnnaNilsson, Peter M.Lundeberg, Joakim
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