Down-regulation of growth factor-stimulated MAP kinase signaling in cytotoxic drug-resistant human neuroblastoma cells

doi:10.1016/S0898-6568(01)00173-5

Cellular Signalling

Volume 13, Issue 7, July 2001, Pages 499-505

https://doi.org/10.1016/S0898-6568(01)00173-5 Get rights and content

Abstract

The mitogen-activated protein kinase (MAPk) signaling pathway, which plays a critical role in the proliferation of mammalian cells, is frequently up-regulated in human tumors and may contribute to the transformed phenotype. Since a major limitation of current cancer chemotherapy is prevalent resistance to cytotoxic drugs, this study determined whether alterations in growth factor signaling through MAPk may contribute to this phenomenon in human neuroblastoma cell lines. Drug-resistant SKNSH cell lines were established by long-term incubation with increasing concentrations to 10⁻⁶ M doxorubicin (SKNSH rDOX6) or MDL 28842 (SKNSH rMDL6). The expression of epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF)-induced EGFR tyrosine phosphorylation were lower in drug-resistant SKNSH cells than their wild-type counterparts. In SKNSH rDOX6 cells, decreased activation and reduced nuclear translocation of MAPk in response to EGF, or lysophosphatidic acid (LPA), or phorbol 12-myristate 13-acetate (PMA), were observed. In SKNSH rMDL6 cells, although MAPk could be activated to wild-type levels by ligand stimulation, the translocation of active MAPk to the nucleus was also reduced. These results suggest that resistance to cytotoxic drugs in human neuroblastoma cell lines is associated with a decrease in growth factor signaling through the MAPk pathway.

Introduction

A major pathway for regulation of cellular growth and differentiation is through the ubiquitous mitogen-activated protein kinase (MAPk) cascade that transmits growth factor signals from the cell membrane to the nucleus [1]. This evolutionarily conserved signaling pathway is thought to be the principal mechanism through which signals from both polypeptide growth factor receptors with intrinsic tyrosine kinase activity and receptors coupled to heterotrimeric G proteins stimulate cell cycle progression and proliferation [2]. Oncogenic Ras proteins [3], overexpressed growth factor receptors [4], and enhanced autocrine growth factor loops [5] are commonly found in human tumors, and can induce constitutive and inappropriate activation of MAPk [6]. Thus pharmacological intervention in the MAPk cascade has been identified as a promising new approach to cancer therapy [7].

A major limitation in response to current chemotherapy of human cancers is the widespread occurrence of inherent or acquired resistance to cytotoxic drugs [8]. In certain cases, including neuroblastoma [9], [10], altered growth factor signaling has been suggested to provide chemoprotection [11], [12]. Modulation of growth factor receptors in neuroblastoma tumor cells has been shown to occur under a variety of clinical [13], [14], [15] and experimental [16], [17] conditions. In murine neuroblastoma models, homologous and heterologous down-regulation of growth factor receptors occurs following overexpression of nerve growth factor (NGF). Specifically, retroviral infection with the NGF gene induces down-regulation of the NGF receptor in C-1300 neuroblastoma cells and of the epidermal growth factor (EGF) receptor in Neuro-2A neuroblastoma cells [12]. In both cases there is a marked decrease in cell replication and proliferation rate following transfer of the NGF gene. Neuroblastoma cells that become drug-resistant also manifest a decrease in proliferation rate, suggesting that changes in the expression or activation of components of growth factor signaling pathways may be associated with failure to respond to cytotoxic agents [12], [18], [19].

The current study has determined the relationship that exists between acquired drug resistance in human neuroblastoma cells to doxorubicin, a topoisomerase-2 inhibitor [20], and MDL-28842, a mechanism-based inhibitor of S-adenosylhomocysteine hydrolase [21], [22], [23], the expression of epidermal growth factor receptor (EGFR), and the activation and nuclear translocation of MAPk.

Section snippets

Cell culture

SKNSH cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. The generation of resistant lines was performed by incubating cells with stepwise increments of drugs from concentrations of 10⁻⁹ to 10⁻⁶ M. Cytotoxicity was assessed by determining cell survival with 3-(4,5-dimethyl-2-thiazoyl)2,5-diphenyl tetrazolium bromide.

Western blots

Western blots for the EGFR and phosphotyrosine were performed by standard procedures using cell lysates that had been prepared in a

EGFR expression and tyrosine kinase activity in drug-resistant SKNSH neuroblastoma cells

Drug-resistant SKNSH neuroblastoma cell lines were generated by sustained, incremental incubation up to 1 μM doxorubicin (SKNSH rDOX6) or MDL 28842 (SKNSH rMDL6). The lines exhibited alterations in cellular morphology, substrate attachment, and reduced cellular proliferation rates that persisted even after subculturing in medium that lacked the drugs. The doubling time of wild-type cells was 49 h, whereas that of SKNSH rMDL6 and SKNSH rDOX6 lines were 63 and 92 h, respectively. Corollary

Discussion

The p44 MAPk and p42 MAPk (also termed ERK-1 and -2) are serine/threonine protein kinases that are activated by dual phosphorylation on particular threonine and tyrosine residues [28] by the enzyme termed MAPk/ERK kinase 1 (MEK1) [29] or MAPk kinase [30]. MEK1 is itself activated upon phosphorylation by upstream kinases such as Raf [31] or Mos [32]. The kinase Raf provides a link to this cascade from agonists that can induce activation of the small GTPase Ras, as Ras is known to complex with

Acknowledgements

We thank Sandra H. Clark and Irina Laer for technical assistance, Eric N. March for data quantification, and Roberta Gerard for preparation of the manuscript. This work was supported by the Anderson Foundation and the Medical Research Institute Council (to B.L.M.) and a Research Starter Grant and a Faculty Development Award from the PhRMA Foundation and Grant No. DAMD17-00-1-0544 from the Department of the Army (to R.R.M.). Confocal facilities were supported in part by the EHS Center in

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Down-regulation of growth factor-stimulated MAP kinase signaling in cytotoxic drug-resistant human neuroblastoma cells

Abstract

Introduction

Section snippets

Cell culture

Western blots

EGFR expression and tyrosine kinase activity in drug-resistant SKNSH neuroblastoma cells

Discussion

Acknowledgements

Cell Signal

J Biol Chem

Cell

J Biol Chem

Curr Opin Cell Biol

J Biol Chem

J Biol Chem

Curr Biol

Cell Signal

FASEB J

Nature

Cancer Surv

J Cell Physiol, Suppl

Cancer Res

Nat Med

Curr Opin Oncol

Cancer Res

Cell Biol Int

J Neurosci Res

J Histochem Cytochem

Cancer Res

Cell Growth Differ

J Cell Biochem

Cancer Res