Gene expression analysis of human epidermal keratinocytes after N-acetyl L-cysteine treatment demonstrates cell cycle arrest and increased differentiation.
OBJECTIVE
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.
Edlundh-Rose
Esther
E
Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, and Rehabilitation Medicine, Karolinska University Hospital, Stockholm, Sweden.
Kupershmidt
Ilya
I
Gustafsson
Anna C
AC
Parasassi
Tiziana
T
Serafino
Annalucia
A
Bracci-Laudiero
Luisa
L
Greco
Giulia
G
Krasnowska
Ewa K
EK
Romano
Maria-Concetta
MC
Lundeberg
Thomas
T
Nilsson
Peter
P
Lundeberg
Joakim
J
D000111:Acetylcysteine / Q000008:administration & dosage / Q000494:pharmacology*; D000975:Antioxidants / Q000008:administration & dosage / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000166:cytology; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors
D000975:Antioxidants; D018076:DNA, Complementary; D013936:Thymidine; D000111:Acetylcysteine