Hamster pancreatic beta cell lines with altered sensitivity towards apoptotic signalling by phosphatase inhibitors.
Specific inhibitors of serine/threonine phosphatases like okadaic acid can induce apoptotic cell death in the pancreatic beta cell line HIT. Cultivation in stepwise increased concentrations of okadaic acid enabled the isolation of HIT100R cells which proliferate at 100 nM okadaic acid (8 - 10 times the initially lethal concentration). These two cell lines were used to characterize the events triggered by okadaic acid that led to apoptosis. Biochemical markers, e.g. cytochrome c release from mitochondria and increase of caspase-3-like activity, revealed that induction of apoptosis by 100 nM okadaic acid in parental HIT cells started with the release of cytochrome c. In HIT100R cells 500 nM okadaic acid were necessary to induce alterations comparable to those observed with 100 nM okadaic acid in non-resistant HIT cells. In contrast to okadaic acid, the potency of the structurally different phosphatase inhibitor cantharidic acid to induce cytochrome c release, increase of caspase-3-like activity and DNA fragmentation was comparable in HIT and HIT100R cells. Thus, no cross-resistance between these phosphatase inhibitors seemed to exist. Phosphatase activity in extracts from HIT and HIT100R cells did not differ in its total amount or in its sensitivity for okadaic acid. Since higher concentrations of okadaic acid were needed to induce apoptosis in HIT100R cells, a compromised intracellular accumulation of the toxin appeared likely. Functional and structural analysis revealed that this was achieved by the development of the multidrug resistance phenotype in HIT100R cells. The underlying mechanism appeared to be the enhanced expression of the pgp1 but not the pgp2 gene.
Krautheim
A
A
Institute of Pharmacology and Toxicology, University of Gottingen, D-37075 Gottingen, Germany.
Brechlin
P
P
Becker
K
K
Winkler
M
M
Steinfelder
H J
HJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002193:Cantharidin / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D018432:Drug Resistance, Multiple; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology; D008387:Marine Toxins; D019319:Okadaic Acid / Q000494:pharmacology*; D010080:Oxazoles / Q000494:pharmacology*; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D019319:Okadaic Acid; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D002193:Cantharidin