Cadmium induced cell apoptosis, DNA damage, decreased DNA repair capacity, and genomic instability during malignant transformation of human bronchial epithelial cells

Abstract

Cadmium and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood. Our study was designed to elucidate the mechanisms of DNA damage in cadmium-induced malignant transformation of human bronchial epithelial cells. We analyzed cell cycle, apoptosis, DNA damage, gene expression, genomic instability, and the sequence of exons in DNA repair genes in several kinds of cells. These cells consisted of untreated control cells, cells in the fifth, 15th, and 35th passage of cadmium-treated cells, and tumorigenic cells from nude mice using flow cytometry, Hoechst 33258 staining, comet assay, quantitative real-time polymerase chain reaction (PCR), Western blot analysis, random amplified polymorphic DNA (RAPD)-PCR, and sequence analysis. We observed a progressive increase in cell population of the G0/G1 phase of the cell cycle and the rate of apoptosis, DNA damage, and cadmium-induced apoptotic morphological changes in cerebral cortical neurons during malignant transformation. Gene expression analysis revealed increased expression of cell proliferation (PCNA), cell cycle (CyclinD1), pro-apoptotic activity (Bax), and DNA damage of the checkpoint genes ATM, ATR, Chk1, Chk2, Cdc25A. Decreased expression of the anti-apoptotic gene Bcl-2 and the DNA repair genes hMSH2, hMLH1, ERCC1, ERCC2, and hOGG1 was observed. RAPD-PCR revealed genomic instability in cadmium-exposed cells, and sequence analysis showed mutation of exons in hMSH2, ERCC1, XRCC1, and hOGG1 in tumorigenic cells. This study suggests that Cadmium can increase cell apoptosis and DNA damage, decrease DNA repair capacity, and cause mutations, and genomic instability leading to malignant transformation. This process could be a viable mechanism for cadmium-induced cancers.

Keywords: Cadmium chloride; DNA damage; DNA repair genes; genomic instability..

Figure 1

Effect of cadmium on cell cycle phase during cadmium-induced malignant transformation of human bronchial epithelial cells. Cell cycle phase was determined in untreated control cells, 5th, 15th, and 35th passage of cadmium-treated cells, and tumorigenic cells from nude mice through flow cytometric analysis. All values are mean±SD (n=5). Statistical (one-way ANOVA and Dunnett's multiple comparison test) differences between control and cadmium-treated cells (*P<0.05) are marked.

Figure 2

Apoptosis induced by cadmium during malignant transformation of human bronchial epithelial cells. Apoptosis was measured in untreated control cells, 5th, 15th, and 35th passage of cadmium-treated cells, and tumorigenic cells from nude mice through flow cytometry. Results are expressed as percentages of apoptosis with regard to the total cells. Data are mean±SD of three separate experiments, with each performed in triplicate. *P<0.05 compared with control using one-way ANOVA and Dunnett's multiple comparison test.

Figure 3

Effects of cadmium on apoptotic morphological changes during malignant transformation of human bronchial epithelial cells. Cells were incubated with cadmium (5th, 15th, and 35th passage) and tumorigenic cells. Nuclear chromatin changes (apoptosis) were assessed with Hoechst 33258 staining. Arrows identify apoptotic nuclei. Among the groups, (A) control; (B) 15th passage-transformed cells treated with cadmium; (C) 35th passage-transformed cells treated with cadmium; (D) tumorigenic cells induced by cadmium.

Figure 4

DNA damage to 16HBE cells treated with cadmium. (A) DNA damage to untransformed 16HBE cells with the comet assay; (B) DNA damage of 15th passage-transformed cells by comet assay; (C) DNA damage of 35th passage-transformed cells by comet assay; and (D) DNA damage of tumorigenic cells by comet assay.

Figure 5

Cadmium-induced changes in gene mRNA expression during cadmium-induced malignant transformation of 16HBE cells. The mRNA expression of selected cell cycle-related genes. (A) PCNA and CyclinD1, apoptosis-related genes; (B) Bal-2 and Bax, DNA damage checkpoint genes; (C) ATM, ATR, Chk1m, Chk2, and Cdc25A were determined in untransformed controls; 5th, 15th, and 35th passage-transformed cells; and tumorigenic cells using quantitative real-time PCR. The threshold cycle number (Ct value) for each gene obtained with real-time PCR was normalized to Ct value of β-actin from same sample, and fold changes in expression for each gene were obtained with delta-delta Ct method. The graph shows the mean±SD of triplicate values. *Statistically significant differences relative to the control group (P<0.05) analyzed using one-way ANOVA.

Figure 6

mRNA and protein expression of hMSH2, ERCC1, XRCC1, and hOGG1 during cadmium-induced malignant transformation of 16HBE cells. (A) mRNA and (B) protein expression of hMSH2, ERCC1, XRCC1, and hOGG1 was determined in untransformed controls; 5th, 15th, and 35th passage-transformed cells; and tumorigenic cells from nude mice with real-time PCR and Western blot analysis, respectively. mRNA and protein expression levels were calculated relative to β-actin.

Figure 7

Representative RAPD-PCR fingerprints generated from genomic DNA during cadmium-induced malignant transformation of 16HBE cells. (A) Fingerprints generated by primer OPC07(50-ATTCTGGTTT-30) showing affected genomic regions in cadmium-induced malignant transformation of 16HBE cells. Arrows indicate changes in RAPD-PCR-amplified loci in cadmium-induced cells compared with passage-matched control cells. (B) Fingerprints for passage-matched controls for cadmium-induced cells tested generated by primer OPC12 (50-TGTCATCCCC-30) showing unaffected genomic regions.

Figure 8

Sequence analysis of exons in hMSH2, ERCC1, XRCC1, and hOGG1. Sequence of exons in hMSH2, ERCC1, XRCC1, and hOGG1. Sequence analysis of exons in hMSH2, ERCC1, XRCC1, and hOGG1. Sequence of exons in hMSH2, ERCC1, XRCC1, and hOGG1 were detected by sequencing amplified PCR and primers in untransformed control and tumorigenic cells induced by cadmium chloride. (A) Sequence of exon8 in hMSH2; (B) Sequence of exon9 in hMSH2; (C) Sequence of exon12 in hMSH2; (D) Sequence of exon14 in ERCC1; (E) Sequence of exon17 in hOGG1. The mutation “↓” are marked.