Cancer Letters

Cancer Letters

Volume 235, Issue 1, 8 April 2006, Pages 84-92
Cancer Letters

Localization of the ABCG2 mitoxantrone resistance-associated protein in normal tissues

Presented in part at the United States and Canadian Academy of Pathology annual meeting, Washington, DC, March 2003, Abstract No. 1348
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Abstract

Reduced drug accumulation due to overexpression of individual members of the ATP binding cassette (ABC) superfamily of membrane transporters has been investigated as a cause of multidrug resistance and treatment failure in oncology. This study was designed to develop an immunohistochemical assay to determine the expression and localization of the 72 kDa ABC half-transporter ABCG2 in normal tissues. Formalin-fixed, paraffin embedded archival tissue from 31 distinct normal tissues with an average of eight separate tissue samples of each were immunostained with rabbit-anti-ABCG2 antibody 405 using a modified avidin–biotin procedure. As a negative control, each sample was also stained with antibody pre-adsorbed with peptide to assess background staining. As a means of verification, selected tissues were also stained with the commercially available monoclonal antibody 5D3.

ABCG2 positivity was consistently found in alveolar pneumocytes, sebaceous glands, transitional epithelium of bladder, interstitial cells of testes, prostate epithelium, endocervical cells of uterus, squamous epithelium of cervix, small and large intestinal mucosa/epithelial cells, islet and acinar cells of pancreas, zona reticularis layer of adrenal gland, kidney cortical tubules and hepatocytes. Placental syncytiotrophoblasts showed both cytoplasmic and surface staining.

Our results support a hypothesis concluding that ABCG2 plays a role in the protection of organs from cytotoxins. However, many of the cell types expressing ABCG2 have a significant secretory function. These data suggest a dual function for ABCG2 in some tissues: the excretion of toxins and xenobiotics including anti-cancer agents and a potential, as-yet undefined role in the secretion of endogenous substrates.

Introduction

A major cause of failure in cancer chemotherapy is the development of multidrug resistance. As classically defined, the multidrug resistance phenotype is characterized by a diminished intracellular drug concentration and overexpression of individual members of the ABC (ATP binding cassette) superfamily of membrane transporters [1], [2]. The ABC transporter superfamily consists of proteins that bind ATP and use the energy of hydrolysis to drive the transport of various molecules across the plasma membrane. These transporters are expressed in both normal and neoplastic cell types, and function to transport exogenously administered drugs as well as numerous endogenous substrates.

Cancer cells can become resistant to anti-cancer drugs by several mechanisms, including alterations in survival or apoptotic pathways, mutation or altered expression of the target, and altered uptake or efflux transporters. By this latter mechanism, drugs are expelled from cells by increasing the activity of efflux pumps, such as the ATP-dependent transporters. Thus, resistance results because increased drug efflux lowers intracellular drug concentrations.

The breast cancer resistance protein (BCRP) [3], also called mitoxantrone resistance protein (MXR) [4] or ABC transporter in placenta (ABC-P) [5], is a member of the ABCG subfamily of ABC transport proteins (gene symbol ABCG2). Overexpression of the ABCG2 gene product has been shown to confer resistance to mitoxantrone [3], [6], and various camptothecin analogs including topotecan and irinotecan (CPT-11) [7], [8], [9], likely acting as an efflux pump. Previous immunohistochemical studies using several drug resistant cell lines demonstrated that the ABCG2 protein was predominantly expressed in the plasma membrane [10], [11].

As a means of exploring the normal role of ABCG2 and understanding possible consequences of its inhibition, this study was designed to determine the expression and localization of this protein in normal tissues utilizing immunohistochemistry with a polyclonal ABCG2 antibody generated by our institution.

Section snippets

Immunohistochemistry

Archival formalin-fixed, paraffin embedded tissue blocks from 31 distinct normal sites with an average of eight separate tissue samples of each retrieved from either the autopsy and surgical files of the Laboratory of Pathology/National Cancer Institute or from the Cooperative Human Tissue Network were cut in 5 μm sections and mounted on charged microscope slides (Fisher Scientific, Pittsburgh, PA). Placental tissues were retrieved from the files of the Laboratory of Pathology/New York

Results

We previously reported results of immunoanalysis using a rabbit polyclonal antibody generated by immunization with a peptide encompassing amino acids 56–70 from ABCG2. This sequence is located just proximal to the N-terminal Walker A motif (56–70: RKPVEKEILSNINGI) and has 100% identity only with ABCG2. The antibody detected ABCG2 by immunoblot analysis, immunofluorescence, and immunohistochemistry. By immunofluorescence, cell surface expression was observed in selected drug resistant cell lines

Discussion

The ABC transporter superfamily consists of proteins that bind ATP and use the energy of hydrolysis to drive the transport of various molecules across the plasma membrane [15]. These transporters are expressed in both normal and neoplastic cell types and function as efflux pumps that transport exogenously administered drugs as well as numerous endogenous substrates [2]. ABC transporters expressed in normal brain, testes and placenta are thought to protect these organs from cytotoxins; in the

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