Role of the Stress-activated/c-Jun NH2-terminal Protein Kinase Pathway in the Cellular Response to Adriamycin and Other Chemotherapeutic Drugs

Maudie T Osborn,Timothy C Chambers

doi:10.1074/jbc.271.48.30950

Abstract

c-Jun NH2-terminal protein kinase (JNK), a member of the mitogen-activated protein kinase family, is activated in response to many stressful stimuli including heat shock, UV irradiation, protein synthesis inhibitors, and inflammatory cytokines. In this study, we investigated whether JNK plays a role in the cellular response to different drugs commonly used in cancer chemotherapy. Treatment of human KB-3 carcinoma cells with Adriamycin resulted in a time- and dose-dependent activation of JNK of up to 40-fold. Treatment with vinblastine or etoposide (VP-16) also activated JNK, with maximum increases of 6.5- and 4.3-fold, respectively. Consistent with these findings, increased c-Jun phosphorylation was observed after drug treatment of cells. In contrast, none of the drugs significantly activated the extracellular response kinase/mitogen-activated protein kinase pathway. Since these drugs are transport substrates for the MDR1 gene product, P-glycoprotein, JNK was assayed in two multidrug-resistant (MDR) KB cell lines, KB-A1 and KB-V1, selected for resistance to Adriamycin and vinblastine, respectively. Relative to KB-3 cells, basal JNK activity was increased 7-fold in KB-A1 cells and 4-fold in KB-V1 cells, with no change in JNK protein expression, indicating that JNK is present in a more highly activated form in the MDR cell lines. Under conditions optimal for JNK activation, Adriamycin, vinblastine, and VP-16 all induced MDR1 mRNA expression in KB-3 cells. Our findings suggest that JNK activation is an important component of the cellular response to several structurally and functionally distinct anticancer drugs and may also play a role in the MDR phenotype.

Highlights

Like ERK and Jun NH2-terminal protein kinase (JNK), p38 is activated by dual phosphorylation on a tyrosine and a threonine residue, and this is catalyzed by a MEK family member, MKK3 [11]. p38 is activated by inflammatory cytokines and environmental stress including osmotic shock and UV irradiation [12]
Activation of JNK by Adriamycin—To validate the JNK assay in KB-3 cell extracts and to examine possible effects on activity of Adriamycin treatment, lysates were prepared from cells exposed to 500 ng/ml Adriamycin for either 1 or 16 h
We have shown that three structurally and mechanistically distinct anticancer drugs activate the stressactivated kinase, JNK, in human carcinoma cells

Summary

Chemotherapeutic Drugs*

The classical MAPK module consists of Raf kinase, MEK, and ERK and is activated in response to a variety of mitogenic signals operating through different mechanisms. One consists of MEKK, MKK4 (or SEK1), and c-Jun NH2-terminal kinase (JNK), which, like Raf/MEK/ERK, operate in a phosphorylation cascade [3, 4]. Unlike the classical MAPK pathway, the MEKK/SEK1/JNK module is only modestly activated by growth factors and phorbol esters and is instead strongly activated by cellular stress including heat shock, UV irradiation, protein synthesis inhibitors, and inflammatory cytokines [5]. An important physiological substrate of p38 is MAPK-activated protein kinase-2, which phosphorylates heat shock protein hsp as part of the cellular response to stress [9].

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION