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Acquired Cadmium Resistance in Metallothionein-I/II(–/–) Knockout Cells: Role of the T-Type Calcium Channel Cacn_1G in Cadmium Uptake

Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute and National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (E.M.L., J.L., M.P.W.); and Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas (C.D.K.)

Metallothioneins (MTs) are cytoplasmic proteins that sequester certain divalent cations and are considered a primary cellular defense against the toxic transition metal cadmium (Cd²⁺). MT-I/II(–/–) knockout [MT(–/–)] cells are available and serve as an excellent tool to study non–MT-related mechanisms in metal tolerance. In the current study, Cd²⁺-resistant MT(–/–) (CdR) and CdR revertant (CdR-rev) cell lines were developed and characterized to investigate non–MT-mediated cellular protection mechanisms. Resistance to Cd²⁺ was approximately 70-fold higher in CdR than the parental MT(–/–) cell line (IC₅₀ = 20 versus 0.3 µM, respectively) and was stable in the absence of Cd²⁺ for 35 days. Accumulation of Cd²⁺ by the CdR cell line was reduced by approximately 95% compared with parental cells, primarily because of a decreased Cd²⁺ uptake. Cd²⁺ uptake by the MT(–/–) parental cell line was independent of sodium, energy, and electrogenic potential. Uptake was saturable (K_m = 65 nM; V_max = 4.9 pmol/mg/min) and pH-dependent (maximal at pH 6.5–7). Potent inhibitors of Cd²⁺ uptake included Zn²⁺ (IC₅₀ = 7 µM), Mn²⁺ (IC₅₀ = 0.4 µM), and the T-type Ca²⁺ channel antagonist mibefradil (IC₅₀ = 5 µM), whereas other metals (including Fe²⁺) and L-type Ca²⁺ channel antagonists had little effect. Immunoblot and real-time reverse transcription-polymerase chain reaction analysis indicated that the Cacn_1G T-type Ca²⁺ channel was expressed at a reduced level in CdR compared with the parental MT(–/–) cell line, suggesting it is important for Cd²⁺ uptake. The CdR1-rev cell line was found to have a Cd²⁺ uptake and sensitivity level in between that of the CdR1 and MT(–/–) cell lines. Consistent with this was an intermediate expression of Cacn_1G in the CdR-rev cell line. These data suggest that decreased expression of Cacn_1G protects cells from Cd²⁺ exposure by limiting Cd²⁺ uptake.

Address correspondence to: Dr. Michael P. Waalkes, Inorganic Carcinogenesis Section, National Cancer Institute, National Institute of Environmental Health Sciences, P.O. Box 12233, Mail Drop F0-09, 111 Alexander Dr., Research Triangle Park, NC 27709. E-mail: waalkes{at}niehs.nih.gov