Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro.
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. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/beta-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.


CONCLUSIONS
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.
Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden. annag@kth.se
Anna C
AC
Gustafsson
Ilya
I
Kupershmidt
Esther
E
Edlundh-Rose
Giulia
G
Greco
Annalucia
A
Serafino
Eva K
EK
Krasnowska
Thomas
T
Lundeberg
Luisa
L
Bracci-Laudiero
Maria-Concetta
MC
Romano
Tiziana
T
Parasassi
Joakim
J
Lundeberg
D000111:Acetylcysteine / Q000737:chemistry*; D000975:Antioxidants / Q000737:chemistry; D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D045744:Cell Line, Tumor; D019070:Cell Lineage; D049109:Cell Proliferation; D003599:Cytoskeleton / Q000378:metabolism; D015536:Down-Regulation; D004848:Epithelium / Q000378:metabolism / Q000473:pathology*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D015894:Genome, Human; D006801:Humans; D015603:Keratinocytes / Q000166:cytology; D007700:Kinetics; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000378:metabolism / Q000473:pathology*; D013997:Time Factors; D014158:Transcription, Genetic; D015854:Up-Regulation
D000975:Antioxidants; D015820:Cadherins; D000111:Acetylcysteine