Cell cycle pathway dysregulation in human keratinocytes during chronic exposure to low arsenite

Abstract

Background: Arsenic is naturally prevalent in the earth's crust and widely distributed in air and water. Chronic low arsenic exposure is associated with several cancers in vivo, including skin cancer, and with transformation in vitro of cell lines including immortalized human keratinocytes (HaCaT). Arsenic also is associated with cell cycle dysregulation at different exposure levels in multiple cell lines. In this work, we analyzed gene expression in HaCaT cells to gain an understanding of gene expression changes contributing to transformation at an early time point.

Methods: HaCaT cells were exposed to 0 or 100nM NaAsO₂ for 7weeks. Total RNA was purified and analyzed by microarray hybridization. Differential expression with fold change≥|1.5| and p-value≤0.05 was determined using Partek Genomic Suite™ and pathway and network analyses using MetaCore™ software (FDR≤0.05). Cell cycle analysis was performed using flow cytometry.

Results: 644 mRNAs were differentially expressed. Cell cycle/cell cycle regulation pathways predominated in the list of dysregulated pathways. Genes involved in replication origin licensing were enriched in the network. Cell cycle assay analysis showed an increase in G2/M compartment in arsenite-exposed cells.

Conclusions: Arsenite exposure induced differential gene expression indicating dysregulation of cell cycle control, which was confirmed by cell cycle analysis. The results suggest that cell cycle dysregulation is an early event in transformation manifested in cells unable to transit G2/M efficiently. Further study at later time points will reveal additional changes in gene expression related to transformation processes.

Keywords: Arsenic-induced skin cancer; Carcinogenesis; Cell cycle; Chronic exposure; Transformation; mRNA.

Figure 1

HaCaT cells were exposed to 0 or 100 nM NaAsO2 for 7 weeks. RNA was purified and expression determined on Affymetrix microarrays and analyzed using Metacore software. Cell cycle analyses were performed by flow cytometry.

Figure 2

Differential mRNA expression with arsenite exposure. A. Hierarchical clustering of mRNAs differentially expressed in HaCaT cells exposed to 0/100 nM arsenite for 7 weeks. Microarray hybridization data from 4 exposed and 4 unexposed cultures was analyzed using Parteck Genomic Suite to generate the heat map. Yellow indicates mRNAs induced by exposure, blue indicates suppression. B. Network of genes populating multiple dysregulated pathways at 7 weeks. Network of only direct interactions between genes populating multiple pathways at 7 weeks was built by Metacore^™ software.

Figure 3

HaCaT cells exposed to arsenite are increased in G2/M compartment. Four cultures each (exposed (+As)/unexposed (−As)) were analyzed by flow cytometry for cell cycle distribution. Fraction in each compartment (G1, S, G2/M) were determined and means ± SD are plotted. Student T-Test was used for statistical analyses. *p-value ≤ 0.05.