Cloning and functional study of a novel human metallothionein-I isoform induced by paraquat.
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.
Choi
Cheol-Hee
CH
Department of Pharmacology, Chosun University Medical School, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, Republic of Korea.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000633:toxicity; D005452:Fluoresceins / Q000737:chemistry; D005822:Genetic Vectors; D006801:Humans; D008668:Metallothionein / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010269:Paraquat / Q000494:pharmacology*; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D017382:Reactive Oxygen Species / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D005452:Fluoresceins; D020033:Protein Isoforms; D017382:Reactive Oxygen Species; C037631:2',7'-dichlorofluorescein; D004317:Doxorubicin; D008668:Metallothionein; C027260:pirarubicin; D010269:Paraquat