A human neuroblastoma cell line with an altered ornithine decarboxylase
Abstract:
A human neuroblastoma cell line (Paju) was resistant to 10 mM difluoromethylornithine, a concentration at which the growth of all mammalian cells normally stops. Ornithine decarboxylase from Paju was very resistant to inhibition by difluoromethylornithine in vitro (Ki = 10 microM compared to 0.5 microM for mouse kidney ornithine decarboxylase). After purification, apparently homogeneous Paju ornithine decarboxylase was inactivated with [3H]difluoromethylornithine and analyzed by polyacrylamide gel electrophoresis. Under denaturing conditions it was found to have an altered molecular structure, i.e. two nonidentical subunits of Mr = 55,000 and 60,000. Another unusual feature of Paju ornithine decarboxylase was its long half-life in vivo (T 1/2 = 8 h compared with 36 min in human HL-60 promyelocytic leukemia cells). The disappearance of immunoreactive protein was only slightly slower than the loss of catalytic activity. The long half-life of Paju ornithine decarboxylase was not shared by adenosylmethionine decarboxylase. Despite the altered structure of Paju ornithine decarboxylase, it was recognized by a specific antisera raised in rabbit against mouse kidney ornithine decarboxylase. The Paju karyotype did not contain double minute chromosomes or any large homogeneously staining region such as that seen in a mouse lymphoma cell mutant that is resistant to difluoromethylornithine and overproduces ornithine decarboxylase (McConlogue, L., and Coffino, P. (1983) J. Biol. Chem. 258, 12083-12086).
Insights
A human neuroblastoma cell line exhibits resistance to difluoromethylornithine due to an altered ornithine decarboxylase enzyme with a unique structure and extended lifespan, offering insights into drug resistance mechanisms.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Mammalian cell growth is typically inhibited by difluoromethylornithine.
- Ornithine decarboxylase (ODC) is a key enzyme in polyamine synthesis and a target for ODC inhibitors.
Purpose of the Study:
- To investigate the molecular basis of difluoromethylornithine resistance in a human neuroblastoma cell line (Paju).
- To characterize the properties of ODC from the resistant cell line.
Main Methods:
- Enzyme inhibition assays using difluoromethylornithine.
- Purification and structural analysis of Paju ODC via SDS-PAGE.
- Determination of ODC half-life in vivo.
- Immunological characterization of Paju ODC.
Main Results:
- Paju cells showed resistance to difluoromethylornithine, with ODC exhibiting low sensitivity to inhibition (Ki = 10 microM).
- Purified Paju ODC displayed an altered molecular structure with two non-identical subunits (Mr = 55,000 and 60,000).
- Paju ODC had a significantly longer in vivo half-life (8 hours) compared to normal cells (36 minutes).
- Despite structural alterations, Paju ODC was recognized by antibodies against mouse ODC.
Conclusions:
- The Paju cell line's resistance to difluoromethylornithine is linked to a structurally altered ODC enzyme with reduced sensitivity to the inhibitor and an extended half-life.
- The findings suggest a novel mechanism of drug resistance independent of gene amplification typically seen in other resistant cell lines.
