Cancer Letters

Cancer Letters

Volume 211, Issue 2, 10 August 2004, Pages 209-218
Cancer Letters

Expression of AIE-75 PDZ-domain protein induces G2/M cell cycle arrest in human colorectal adenocarcinoma SW480 cells

https://doi.org/10.1016/j.canlet.2004.02.005Get rights and content

Abstract

AIE-75 has been known as a 75-kDa autoantigen detected in the serum of autoimmune enteropathy (AIE) and as a colon cancer-related antigen, and now designated as a gene causative of Usher syndrome type 1C hereditary syndromic hearing loss. It binds to a novel putative tumor suppressor MCC2 that is homologous to MCC (mutated in colon cancer) through a PSD-95/Dlg/ZO-1 (PDZ) domain. To clarify the functional role in colon cancer cells, we transfected AIE-75 gene into SW480 colon cancer cells which do not express AIE-75. Expression of AIE-75 suppressed growth of SW480 cells in vitro in correlation with the expression levels. It was due mainly to G2/M phase cell cycle arrest associated with mitotic slippage, resulting in emergence of hyperploid giant-nucleated or multi-nucleated cells. Screening of proteins that bound to PDZ domains of AIE-75 by a yeast two hybrid system showed that three serine/threonine phosphatase catalytic subunits (PP2AC-α, PP2AC-β, and PPP6C) could bind to AIE-75. Since PP2AC is known to regulate G2/M checkpoint, we suggest that AIE-75 interacts with PP2AC and prevent cells to transit mitotic phase.

Introduction

AIE-75/harmonin is a protein of 75-kDa identified as an autoantigen specific to X-linked autoimmune enteropathy (AIE) [1], and as a colon cancer-related antigen [2]. AIE-75/harmonin germline mutation has been found to be a cause of Usher syndrome type 1C, an autosomal recessive disorder characterized by congenital sensorineural deafness and retinitis pigmentosa [3], [4]. Alternatively spliced transcripts of AIE-75/harmonin [4] predict at least 10 protein isoforms which can be grouped into three classes: harmonin a, b, and c [5]. All the three isoform classes of AIE-75/harmonin have three PSD-95/Dlg/ZO-1 (PDZ) domains [1], [5], which have been known to interact with the specific carboxyl terminus sequence motif X-S/T/F/Y-X-Φ(Φ: hydrophobic amino acid residue) of target proteins [6], [7]. Several PDZ domain-containing proteins have been known to interact with tumor suppressor proteins, for example, binding of discs large-1 (dlg) tumor suppressor (hDLG) to APC [8], [9], membrane-associated guanylate kinases (MAGI) to PTEN [10], [11], and Na+/H+ exchanger regulatory factor-1 to merlin [12]. We have recently identified an interaction of AIE-75 with a novel protein MCC2 which has high homology to the tumor suppressor MCC (mutated in colon cancer) [13]. AIE-75/harmonin binds to cadherin 23 and myosin VIIa, via its PDZ domains [5], [14]. In addition, the longest isoform b of AIE-75/harmonin is also known to interact with F-actin through the second coiled-coil domain [5].

AIE-75 is physiologically expressed in epithelial cells with microvilli such as those of small intestine, colon, kidney, pancreas, and inner ear [1], [4], [13], [15]. It is present at the apical membranes of ileal villous cells and renal proximal tubular cells, while it is present diffusely in the cytoplasm of colonic mucosa cells. In colon cancer cells, loss of AIE-75 expression is a rather rare phenomenon, whereas it is frequent in renal cancer cells [13]. Although the above pieces of evidence suggest a unique functional role of AIE-75 in colonic mucosa cells and colon cancer cells, it remains totally unknown. In order to find out the potential function of AIE-75, we transfected AIE-75 gene into SW480 human colon adenocarcinoma cells. Here, we report that expression of AIE-75 induces G2/M cell cycle arrest. We also identified a serine/threonine phosphatase PP2A as a candidate mediator of the checkpoint induction.

Section snippets

Cells

Human colon cancer cell line SW480 [16] was provided by Institute of Development, Aging and Cancer, Tohoku University. They were maintained in the mixture of DMEM and Ham's F12 (NISSUI, Tokyo) with 10% fetal bovine serum (FBS; Bio Whittaker, Walkersville, MD) at 37 °C in a humidified 5% CO2 atmosphere.

AIE-75 transfection and cell cloning

AIE-75 expression vector pcDNA3.1/HisC-AIE-75 was made by subcloning an AIE-75 (harmonin a; GenBank AB006955) coding region into a vector, pcDNA3.1/HisC-Neo (Invitrogen, Carlsbad, CA). The

Expression of AIE-75

Expression of AIE-75 mRNA in each transfectant clone was assessed by a semi-quantitative RT-PCR assay. Expression level of AIE-75 was high in Clone 1-2 and Clone 1-4, and relatively low in Clone 1-5 and Clone 1-6. In contrast, neither parent cells nor the cells transfected with vector only (Neo1, Neo2) showed expression of AIE-75(Fig. 1).

Cell growth and morphological changes

Using AIE-75 transfectants, we evaluated the effect of AIE-75 expression on cell growth (Fig. 2). Compared to Neo1 and Neo2 control vector transfectants, all

Discussion

AIE-75 is a protein of 75 kDa recently identified as the autoantigen that reacts to the autoantibody in the sera of patients with AIE [1]. This protein was also identified by another group as the antigen reactive to autoantibody in the sera of colon cancer patients [2], [15]. We have recently demonstrated that AIE-75 can bind to a novel candidate tumor suppressor MCC2 that is homologous to the tumor suppressor MCC (mutated in colon cancer) [13]. In this context, we were interested in the

Acknowledgements

We thank Ms Narumi Furuuchi for technical help and Ms Masako Yanome for help in preparing manuscript.

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