Cloning and functional study of a novel human metallothionein-I isoform induced by paraquat

Abstract

Human metallothionein (hMT) is highly overexpressed in the resistant AML-2 cell line selected by paraquat, an intracellular superoxide generator. The total RNA obtained from the paraquat-resistant AML-2 cell subline was purified and reverse-transcribed into cDNA using an oligo(dT) primer. A PCR fragment for hMT was generated and cloned. Nucleotide sequence analysis revealed that the hMT transcript was a novel hMT-I isoform, which was designated hMT-Ip and showed homology with hMT-Ia (91.8%), -Ie (98.4%), -If (91.8%), -Ig (91.8%), -Ih (91.8%), -Il (86.9%), -Ir (96.7%), -Ix (86.9%), -Iy (85.2%), -IIa (91.8%), -III (83.8%), and -VI (62.9%). Polypeptide translation indicated that the hMt-Ip protein differs from the hMT-Ie protein by only one amino acid. hMT-Ip was also expressed in the peripheral lymphocytes of humans. The gastric cancer cell line SNU-601 was transfected by an expression vector harboring the hMT-Ip isoform. These stable transfected cells showed not only an inhibitory effect on dichlorofluorescin oxidation, a fluorometric probe, by hydrogen peroxide and paraquat, but also a high level of resistance to anthracyclines such as doxorubicin and pirarubicin. These results show that the novel MT-Ip isoform is closely associated with the protection against oxidative stress. Therefore, it can be utilized for preventing anthracycline-induced cardiac toxicity.