Cancer Letters

Cancer Letters

Volume 139, Issue 2, 24 May 1999, Pages 137-143
Cancer Letters

Differential responses of staurosporine on protein kinase C activity and proliferation in two murine neuroblastoma cell lines

https://doi.org/10.1016/S0304-3835(99)00021-XGet rights and content

Abstract

We studied the effect of staurosporine (SSP), a broad-spectrum protein kinase inhibitor, on the levels of protein kinase C (PKC) activity and proliferation in two murine neuroblastoma cell lines, Neuro2a and its clone NB41A3. The PKC activity was examined in whole cell lysate, cytosolic and particulate fractions. A differential response to SSP treatment in the enzyme activity in whole cell lysate and particulate fractions was demonstrated in the two cell lines. The data on proliferation indicated that Neuro2a cells were more sensitive to the SSP treatment with significant inhibition in DNA synthesis in a time course study. Our findings suggest that the data on basal levels of PKC activity in tumors will be of significance in studies using PKC inhibitors as an approach for therapeutic intervention.

Introduction

Protein kinase C (PKC) is a family of serine/threonine kinases, that are directly involved in a variety of diverse processes including cell proliferation and differentiation [1], [3], [14], [27]. Staurosporine (SSP), an alkaloid isolated from Streptomyces species, is a broad-spectrum PKC inhibitor [4], [23]. It has been shown to inhibit adhesion, invasion and tumor growth in different carcinoma models [16], [27].

Neuroblastoma cell lines are an excellent paradigm to study the regulation of neural crest differentiation at the cellular and molecular levels [19]. The cell lines, Neuro2a and NB41A3, provide a large population of primary neuroblastoma (NB) cells to understand the mechanism of differentiation under controlled conditions [17]. Various agents are known to induce in vitro differentiation in murine neuroblastomas [19].

In the present study we compared the basal levels of PKC activity and the effect of SSP on the activity in murine NB cell lines Neuro2a and its clone NB41A3 and examined its significance on the proliferation of these two cell lines. The differential response of the two cell lines in regard to the PKC activity and inhibition of proliferation is discussed.

Section snippets

Cell cultures and reagents

The murine (mouse) neuroblastoma cell lines Neuro2a and NB41A3, ATCC CCL 131 and ATCC CCL 147, respectively, were obtained from the American Type Culture Collection, USA. Neuro2a cells were grown in Minimum Essential Medium with Earl's salt and NB41A3 cells were grown in Ham F-10 tissue culture medium (Gibco-BRL, Gaithensburg, MD), supplemented with 10% fetal calf serum and 15% horse serum plus 2.5% FCS, respectively, and a penicillin (200 units/ml)– streptomycin (0.2 μg/ml) mixture. Cells

Proliferation assay

There was dose- and time-dependent inhibition of proliferation upon treatment with SSP in both Neuro2a and NB41A3 cell lines. At 24 h, Neuro2a cells demonstrated significantly higher inhibition compared to NB41A3, at all the concentrations used. At 48 h and 72 h time points the difference was significant up to 25 nM but not at 50 nM concentration of SSP (Fig. 1). The trends in the results clearly demonstrate the differences in response to SSP treatment in regard to growth and DNA synthesis in

Discussion

PKC is regarded as a marker enzyme for tumorigenesis [7], [8], [16], [25]. PKC activity has been linked to metastasis through studies of PKC blockers that inhibited cell adhesion, growth in tumor cells and growth factor phosphorylation [5], [12], [22]. These findings suggest that PKC may be a potent target for therapeutic intervention. Staurosporine, a protein kinase inhibitor has been used in the present study to examine its effect on two NB cell lines as regards PKC activity and its influence

Acknowledgements

We thank Prof. S.P. Mahadik, Dept. of Psychiatry, University of Georgia for helpful suggestions. We are grateful to the director Dr. G.C. Mishra and former director Dr. U.V. Wagh of the National Centre for Cell Science for providing the facilities.

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