Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis

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Highlights

  • Short-term exposure of BEAS-2B cells to arsenic or Cr(VI) activates p53 and p21.

  • Chronic exposure of BEAS-2B cells to arsenic or Cr(VI) causes cell transformation and tumorigenesis.

  • Arsenic-transformed cells exhibit reduced activities of p53 and p21.

  • Cr(VI)-transformed cells exhibit increased activities of p53 and p21.

Abstract

Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG  TCG) at codon 47 and the codon 72 polymorphism (CGC  CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer.

Introduction

Heavy metals, such as arsenic and Cr(VI) are confirmed human lung carcinogens by International Agency for Research on Cancer (IARC) (International Agency for Research on Cancer (IARC), 2012a, International Agency for Research on Cancer (IARC), 2012b, Martinez et al., 2011, Morales et al., 2000, Machle and Gregorius, 1948, Sorahan et al., 1998). While short-term study of those metals' toxicity is an initial step and necessary, chronic exposure at a low dose which simulates human environmental exposure is mostly adopted by researchers. Therefore, instead of primary cells, immortalized cell lines from normal human bronchial epithelium are essential for those studies of long-term exposure to metals. Among all available immortalized human bronchial/lung cell lines, BEAS-2B cells and human bronchial epithelial cells (HBECs) are most frequently used. BEAS-2B cells, were originally generated by infection with Ad12-SV40 virus (Lechner et al., 1982, Lechner et al., 1984). Wild type p53 was found in BEAS-2B cells with missense mutation (CCG  TCG) at codon 47 which causes a Pro to Ser substitution and the codon 72 polymorphism (CGC  CCC) which alters an Arg residue to a Pro residue (Matlashewski et al., 1987). p53 in BEAS-2B cells binds to SV40 T antigen protein but does not bind to the heat shock 70 protein (Reddel et al., 1988). Transfection of p53 plasmid with codon 47 mutation into human lung carcinoma cell line inhibited cell proliferation, indicating that p53 in BEAS-2B cells has normal wild type properties (Lehman et al., 1993). It has been reported that BEAS-2B cells are non-malignant as evidences by in vitro cell transformation assay and in vivo xenograft tumor model (Ramirez et al., 2004). The cell line provider ATCC has described that BEAS-2B cells are p53 wild type (http://www.atcc.org/Products/All/CRL-9609.aspx#documentation). However, the missense mutation at codon 47 of p53 remains to be a concern for the scientists who utilize BEAS-2B cells as an in vitro model in the various research areas including respiratory cytotoxicity of chemicals/agents and malignant cell transformation of carcinogens and related cancer prevention.

The p53 signaling pathway is activated in response to a variety of stress signals, allowing p53 to coordinate transcription programs that ultimately contribute to tumor suppression (Vousden and Prives, 2009). Loss of p53 function, through mutations in p53 itself or perturbations in pathways signaling to p53, is a common feature in the majority of human cancers. More than 75% of the mutations result in the expression of a p53 protein that has in most cases lost wild-type functions and may exert a dominant-negative regulation over any remaining wild-type p53 (Petitjean et al., 2007). To evaluate whether exposure of BEAS-2B cells to the heavy metals is able to activate p53, in the present study BEAS-2B cells were exposed to arsenic or Cr(VI) in a short term, activities and expressions at mRNA and protein level of p53 were examined. p21, downstream target protein of p53, its activity and expressions were also evaluated. To best simulate the human environmental exposure, BEAS-2B cells were also chronically exposed to low dose of arsenic or Cr(VI) to generate malignant transformed cells. Expressions of p53 and p21 were examined in arsenic- or Cr(VI)-transformed cells. Tumorigenicity of arsenic- or Cr(VI)-transformed cells was also conducted in vivo.

Section snippets

Chemicals and reagents

Sodium arsenite (NaAsO2) and sodium dichromate dehydrate (Na2Cr2O7) were from Sigma (St Louis, MO). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), gentamicin, l-glutamine, Oligo (dT)20, AccuPrime Taq DNA polymerase high fiedelity were from Invitrogen (Carlsbad, CA). RNeasy mini kit and plasmid prep kit were from Qiagen (Valencia, CA). M-MLV reverse transcriptase was from Promega (Madison, WI). Luciferase assay system was from Promega (Fitchburg, WI). Antibodies against p53

Activation of p53 upon short-term exposure of BEAS-2B cells to arsenic or Cr(VI) exposure

To investigate whether short term exposure of BEAS-2B cells to arsenic or Cr(VI) is able to activate p53, promoter activity, phosphorylation, and expression of p53 at both transcription and translation levels were measured. The results show that both arsenic and Cr(VI) treatment for 24 h increased p53 activity in a dose-dependent manner (Figs. 1A and 3A). Similarly, mRNA level of p53 was also elevated upon arsenic or Cr(VI) treatment (Figs. 1B and 3B). To confirm that exposure of these two

Discussion

The results from present study show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) increased promoter activity and mRNA expression of p53. While Cr(VI) exposure increased phosphorylation of p53 at Ser15, but not at Ser392, arsenic exposure increased phosphorylation of p53 at Ser392. Total p53 protein level remained the same upon Cr(VI) or arsenic treatment compared to control without treatment. p53, a well-known tumor suppressor, functions as a node for organizing whether the

Conflict of interest

The authors declare that there is no conflict interest.

Transparency document

Transparency document.

Acknowledgments

This research was supported by the National Institutes of Health/National Institutes of Environmental Health Sciences (R01ES018883 and R01ES021771).

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