Elsevier

Biochemical Pharmacology

Volume 62, Issue 11, December 2001, Pages 1469-1480
Biochemical Pharmacology

Accompanying protein alterations in malignant cells with a microtubule-polymerizing drug-resistance phenotype and a primary resistance mechanism1

https://doi.org/10.1016/S0006-2952(01)00804-8Get rights and content

Abstract

Microtubules (MTs) are cytoskeletal components whose structural integrity is mandatory for the execution of many basic cell functions. Utilizing parental and drug-resistant ovarian carcinoma cell lines that have acquired point mutations in β-tubulin and p53, we studied the level of expression and modification of proteins involved in apoptosis and MT integrity. Extending previous results, we demonstrated phosphorylation of pro-survival Bcl-xL in an epothilone-A resistant cell line, correlating it with drug sensitivity to tubulin-active compounds. Furthermore, Mcl-1 protein turned over more rapidly following exposure to tubulin-modifying agents, the stability of Mcl-1 protein paralleling the drug sensitivity profile of the paclitaxel or epothilone-A resistant cell lines. The observed decreases in Mcl-1 were not a consequence of G2M arrest, as determined by flow cytometry analysis, which showed prominent levels of Mcl-1 in the absence of any drug treatment in populations enriched in mitotic cells. We also observed that a paclitaxel-resistant cell line expressed Bax at a much lower level than the sensitive parental line [A2780(1A9)], consistent with its mutant p53 status. MT-associated protein-4 (MAP4), whose phosphorylation during specific phases of the cell cycle reduces its MT-polymerizing and -stabilizing capabilities, was phosphorylated in response to drug challenge without a change in expression. Phosphorylation of MAP4 correlated with sensitivity to tubulin-binding drugs and with a dissociation from MTs. We propose that the tubulin mutations, which result in a compromised paclitaxel:tubulin or epothilone:tubulin interaction and paclitaxel or epothilone resistance, indirectly inhibit downstream events that lead to cell death, and this, in turn, may contribute to the drug-resistance phenotype

Introduction

The regulatory mechanisms that control normal cell proliferation and cell death remain balanced through orchestrated cascades of multiple interacting signaling pathways. Members of such networks exercise triggers and constraints, resulting in normal cell metabolism, motility, division, growth arrest, and proliferation. When this rhythmic scheme is perturbed, either permanently as might be envisioned in a transformed cell [1], [2], [3], or transiently as after drug challenge, the effects could provide survival advantages to the cell.

One of several obstacles to the effective treatment of cancer has been the resistance of cells to chemotherapeutic agents [2]. The ability to circumvent such barriers would be improved by the identification of the factors that contribute to the ability of the cell to survive and evade the toxic effects of a drug. With regard to apoptosis, it is well appreciated that the Bcl-2 family of proteins that mediate this process is expanding in number, and encompasses proteins facilitating cell death (e.g. Bax and Bad) and those promoting survival (e.g. Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and viral protein E1B-19K) [4], [5]. When the pendulum swings towards cell death, events culminate in activation of caspases [5], leading ultimately to cell demise [5], [6]. We, as well as others, have shown that several of the pro-survival Bcl-2 family members, such as Bcl-2 and Bcl-xL, become phosphorylated following treatment of cells with tubulin-targeting drugs [7], [8], [9]. This modification requires an uncompromised drug:tubulin interaction [7], is accompanied by cell cycle arrest, and is often followed by apoptosis. Since several family members interact to form dimers and heterodimers, the levels of each protein and their regulation have been acknowledged as bridling the “cell-death rheostat” [4], [5], [10]. Therefore, we studied the responses of sensitive and resistant ovarian carcinoma cells to tubulin-targeting drugs, and examined the expression of two additional members of the Bcl-2 family with opposing functions, Bax and Mcl-1, while considering the drug-resistance profile of each cell. The pro-survival family member MCL-1 gene [4] encodes a 46-kDa protein that has sequence similarity to Bcl-2 at its carboxyl terminus [11], an overlapping intracellular localization pattern [12], and is well studied in ovarian cell differentiation and follicle atresia [11]. To assess the possible contribution of Mcl-1 protein to support a previously determined drug-resistance phenotype and promote survival, we determined its basal level of expression in each ovarian cell line and compared it with that observed after drug treatment.

Presently, an intense interest exists in the cytoskeleton as a chemotherapeutic target, since it is well recognized that its components provide a framework where signal transduction pathways can interact [13], [14], [15] and form crucial structural scaffolds that dismantle and then reform during mitosis and cytokinesis [16], [17]. The latter events require the functional integrity of the MT network and the coordinated reorganization of MTs into spindles to achieve accurate chromosome segregation. Many compounds have been identified that interfere with MTs and spindle formation or maintenance, either by depolymerizing MTs (e.g. VCR, COL, nocodazole) or by polymerizing tubulin (e.g. PTX, docetaxel, epothilones, discodermolide, sarcodyctin, eleutherobin) [18]. The fidelity of MT dynamics and function throughout the cell cycle is influenced by many components, one being the action of MAPs like tau and MAP4 [19], [20], [21]. MAPs promote MT integrity, and the phosphorylation of MAPs results in their inability to stabilize MTs, altering their dynamics [19], [21], [22], [23], [24], [25], [26]. Given the close physical and functional relationship between MTs and MAP4, and the ubiquitous nature of the latter, we studied the level of MAP4 protein expression and its modification after treating cells with tubulin-binding drugs.

In the present study, we examined representative opposing apoptotic regulatory proteins to determine if their modification and expression might contribute to the drug-resistance phenotype. Our observations made on pro-survival proteins Mcl-1 and Bcl-xL, pro-death Bax, and the regulation and modification of MAP4 parallel the drug sensitivity profile for each cell line. Our results suggest that although the mutations in tubulin confer drug resistance due to impaired drug:tubulin interactions, proteins mediating the apoptotic pathway or MT structure are affected indirectly, and these secondary effects might support the drug-resistance phenotype.

Section snippets

Cells and culture

A2780(1A9) is a single cell clone of the human ovarian carcinoma cell line A2780. Two PTX-resistant sublines, PTX10 and PTX22, have been described previously and are maintained in 15 ng/mL of PTX and 5 μg/mL of verapamil [3]. An epothilone-A resistant cell line, EPOA-R, was selected in EPOA and maintained in 30 nM EPOA as described [27], [28]. Cells were removed from the drug 5–7 days prior to an experiment. SKOV3 is a human ovarian carcinoma line. All cells were cultured in RPMI 1640

Effect of tubulin-targeted drugs on phosphorylation of Bcl-xL and expression of Mcl-1

Cell death following exposure to an MT-active agent could occur as a result of the drug:tubulin interaction and the ensuing sequelae, and/or as a consequence of events independent of drug binding to tubulin. Development of drug resistance could then involve one or several mechanisms. The present studies were designed to examine additional changes that occur as a consequence of drug:tubulin interaction, and how these might contribute to, or be affected during, the development of drug resistance.

Discussion

Chemotherapeutic strategies against many cancers utilize drugs such as the vinca alkaloids (VCR, vinorelbine), COL, epothilones, PTX, and others, which, by targeting cytoskeletal elements like tubulin, impede cell division, proliferation, signaling, motility, and transport, culminating in cell death. The ability to bypass the resistance of a malignant cell to chemotherapeutic agents would be a significant accomplishment in the clinical setting and requires elucidating the factors contributing

Acknowledgements

The authors thank Zhirong Zhan for her technical assistance.

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    Abbreviations: MTs, microtubules; MAPs, microtubule-associated proteins; MAP4, microtubule-associated protein-4; PTX, paclitaxel; EPOA, epothilone A; EPOB, epothilone B; EPOA-R, epothilone A-resistant; COL, colchicine; VCR, vincristine; and VBL, vinblastine.

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