Cloning of differentially expressed sequence tags from nickel-transformed human embryonic lung cells
Introduction
Epidemiological investigations have indicated that some nickel compounds, such as water-insoluble nickel compounds, are strongly related to the incidence of lung and nasal carcinomas [1], [2]. Some tumor models have been established in the rat, mouse and other animals after inhalation [3] or injection [4] of various nickel compounds. Nickel has been found to initiate lipid peroxidation [5] and reactive oxygen species (ROS) formation [6], and to induce chromosomal aberrations [7] and DNA–DNA, DNA–protein crosslinks [8], [9], which probably lead to abnormal gene expression. Some oncogenes, such as c-ras [10] and c-myc [4], and tumor suppressor genes, such as p53 [11] and the senescence gene [12], have been found to be mutated, overexpressed or reduced in nickel-induced tumors. Recently, some specifically expressed genes were separated from nickel-treated [13], [14] or transformed [15] cells. Cap43, a novel gene, specifically induced by nickel compounds, was cloned from Ni-treated A549 cells in a time- and concentration-dependent way [13]. Also, vimentin (hSNF2H homologue) and H ferritin were found to be up- or down-regulated in nickel-treated Chinese hamster ovary cells [14]. In nickel-transformed C3H10T1/2 mouse embryo fibroblasts, Ramnath et al. [15] identified four differentially expressed genes named R1-1, R2-3, R3-2 and R3-3. These genes may play important roles in nickel carcinogenesis. Epidemiological studies suggest that inhalation is the main route for human exposure to the nickel compound and nickel is primarily responsible for lung and other respiratory cancers [1], [2]. Accordingly, it is of great importance and interest to study nickel carcinogenesis in human lung cells, which may provide important information on nickel carcinogenesis in humans.
We selected water-insoluble nickel compounds (nickel oxide, NiO; and nickel subsulfide, Ni3S2) to induce transformation in the human embryonic lung (HEL) cells, MRC-9 and IMR-90 [16]. In the present study, differential display polymerase chain reaction (DD-PCR) [17] was applied to probe the differentially expressed sequence tags (ESTs) between the transformed cells and their control counterparts (cells without nickel treatment). Two genes, named MS515 and IC82, were found to be differentially expressed in nickel-transformed cells compared with their controls.
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Cell and cell culture
Normal HEL cells, MRC-9 (P7) and IMR-90 (P10), were obtained from the Japan Health Science Research Resources Bank (JHSRRB, Osaka, Japan). Cells were exposed to NiO and Ni3S2 for 3–5 months to achieve transformation [16]. MRC-9, IMR-90, and their transformants were cultured in DMEM (Nissui, Tokyo, Japan). SBC-2, a small cell lung cancer (SCLC) cell line, and EBC-1, a non-small cell lung cancer (NSCLC) cell line, were passaged in RMPI 1640 media (Nissui).
Isolation and purification of total RNA
The total RNA was extracted using an
Differential display of mRNA species in nickel-transformed HEL cells
Nickel can activate or suppress some genes by several means, such as inducing point mutation, DNA breaks and methylation [18], chromosomal aberrations and chromatin condensation, and DNA–protein crosslinks [8]. Based on the identification of Cap43, which is induced in A549 by nickel, but not other metals, such as zinc, cobalt, copper, lead, cadmium, arsenic or mercury, it is reasonable to speculate that some genes may be expressed specifically in response to nickel transformation. To test this,
Acknowledgements
This study is sponsored by the Fund of Doctoral Program, Ministry of Education, People's Republic of China, and the Association of International Education Japan (AIEJ). The authors gratefully acknowledge the contributions of Dr Eiji Sugiyama, Dr Toshiyasu Sasaoka, Dr Jun Araya, Dr Tadashi Fujita (The First Department of Internal Medicine), especially the kind help of Dr Yoshimi Takata in DNA sequencing (Department of Biochemistry), Toyama Medical and Pharmaceutical University. The authors also
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