title: Inhibition of prolidase activity by nickel causes decreased growth of proline auxotrophic CHO cells.
abstract: Occupational exposure to nickel has been epidemiologically linked to increased cancer risk in the respiratory tract. Nickel-induced cell transformation is associated with both genotoxic and epigenetic mechanisms that are poorly understood. Prolidase [E.C.3.4.13.9] is a cytosolic Mn(II)-activated metalloproteinase that specifically hydrolyzes imidodipeptides with C-terminal proline or hydroxyproline and plays an important role in the recycling of proline for protein synthesis and cell growth. Prolidase also provides free proline as substrate for proline oxidase, whose gene is activated by p53 during apoptosis. The inhibition of prolidase activity by nickel has not yet been studied. We first showed that Ni(II) chloride specifically inhibited prolidase activity in CHO-K1 cells in situ. This interpretation was possible because CHO-K1 cells are proline auxotrophs requiring added free proline or proline released from added Gly-Pro by prolidase. In a dose-dependent fashion, Ni(II) inhibited growth on Gly-Pro but did not inhibit growth on proline, thereby showing inhibition of prolidase in situ in the absence of nonspecific toxicity. Studies using cell-free extracts showed that Ni(II) inhibited prolidase activity when present during prolidase activation with Mn(II) or during incubation with Gly-Pro. In kinetic studies, we found that Ni(II) inhibition of prolidase varied with respect to Mn(II) concentration. Analysis of these data suggested that increasing concentrations of Mn(II) stabilized the enzyme protein against Ni(II) inhibition. Because prolidase is an important enzyme in collagen metabolism, inhibition of the enzyme activity by nickel could alter the metabolism of collagen and other matrix proteins, and thereby alter cell-matrix and cell-cell interactions involved in gene expression, genomic stability, cellular differentiation, and cell proliferation.
authors.affiliation: Metabolism and Cancer Susceptibility Section, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Miltyk
authors.forename: Arkadiusz
authors.initials: A
authors.lastname: Surazynski
authors.forename: Kazimierz S
authors.initials: KS
authors.lastname: Kasprzak
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Fivash
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Buzard
authors.forename: James M
authors.initials: JM
authors.lastname: Phang
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004150:Dipeptidases / Q000037:antagonists & inhibitors*; D008345:Manganese / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology*
chemical_list: D008345:Manganese; D009532:Nickel; D004150:Dipeptidases; C022449:proline dipeptidase