Cancer Letters

Cancer Letters

Volume 234, Issue 2, 28 March 2006, Pages 158-167
Cancer Letters

Evidence for CEA release from human colon cancer cells by an endogenous GPI-PLD enzyme

https://doi.org/10.1016/j.canlet.2005.03.028Get rights and content

Abstract

Elevated carcinoembryonic antigen (CEA) blood levels are found in a wide variety of epithelial neoplasms. The precise mechanism of the spontaneous CEA release from normal and cancer cells has not been established yet. In this study we investigated ‘in vitro’ the role of an endogenous glycosylphosphatidyl inositol phospholipase D (GPI-PLD) in spontaneous CEA release from human colon carcinoma cells. We detected GPI-PLD-specific transcript expression in four human colorectal tumor cell lines, LS180, HT29, HT29/219, and SW742 by RT-PCR. Furthermore, CEA release could be activated and inhibited by incubation of LS180 cells with suramin and 1,10-phenanthroline, compounds known to activate and inhibit GPI-PLD activity, respectively. The results suggest a mechanism for the involvement of an endogenous GPI-PLD in spontaneous CEA release from human colon cancer cells.

Introduction

Carcinoembryonic antigen (CEA) is one of the most extensively used clinical tumor markers. Serum CEA may be elevated in neoplastic diseases of the colon, breast, lung, prostate, bladder and stomach as well as in various gynecological malignancies. The blood CEA levels are highly variable from patient to patient. Some with cancer have normal levels despite the presence of CEA in their tumors [1], [2], [3].

The structure of CEA protein includes an N-terminal V-like Ig domain, followed by 6 I-like Ig domains, and a hydrophobic C-terminal domain. The C-terminal domain, consisting of 26 amino acids, is processed so that CEA binds to the plasma membrane through a glycophosphatidyl inositol (GPI) anchor [4].

CEA normal function(s) and its relevance to malignant transformation are not clear. The secretion of CEA by many colorectal tumors is associated with a worse prognosis and a greater likelihood of metastasis [5]. A number of functions that are ascribed to CEA include homophilic and heterophilic intercellular adhesion [6] and a possible instrumental role in carcinogenesis [7]. CEA has also been implicated in the process of human colon cancer liver metastasis [8], [9]. The latter function has been suggested to be facilitated by soluble CEA through induction of cytokine production by Kupffer cells [8], [10].

Although CEA has been for a long time known to be secreted or shed from the cell surface spontaneously, the exact mechanisms by which it is released into serum has not been demonstrated. Triton X-114 partitioning experiments showed that, in contrast to the membrane bound form, circulating CEA is mostly hydrophilic and partitions into the aqueous phase, indicating that it lacks the hydrophobic portion of the GPI moiety [11]. Khan and Hammarstrom demonstrated that CEA on isolated membranes and in cell lysates, but not on intact cells, could be cleaved by fresh human serum or purified GPI-PLD [12]. Studies have also indicated that CEA in normal colonic epithelial cells and colon cancer cells is released by a non-proteolytic cleavage, probably through the action of the endogenous phospholipases [13].

The endogenous GPI-PLD that is responsible for cleavage and release of several GPI-proteins from their anchors at the cell surface has been identified [14], [15], [16]. A GPI-specific phospholipase D (GPI-PLD) cDNA has also been cloned [17] and its expression has been shown in a number of human tissues including pancreatic β-cells, macrophages, keratinocytes, and liver [18]. The recent findings that cellular GPI-PLD appears to be involved in hydrolysis and spontaneous release of several GPI-proteins from various cells, including tumor cells [16], [17], [18], led us to examine the role of this enzyme in CEA release from colorectal carcinoma cells.

In this report, we investigated the mechanism of spontaneous CEA release in human colon carcinoma cells. We verified GPI-PLD enzyme expression in several human colon carcinoma cells. Using an activator and an inhibitor of this enzyme, we demonstrated that CEA release in LS180 colon cancer cells was readily stimulated or inhibited, respectively, by adding these chemicals to the culture medium. An understanding of the mechanism of CEA release might lead to improved methods of detection and therefore management of cancer patients.

Section snippets

Materials

Guanidinium thiocyanate, sodium citrate, phenol, 1,10-phenanthroline (PNT) were purchased from Fluka chemical company (Switzerland); l-glutamine, bovine serum albumin (BSA), suramin, Dulbecco's Modified Eagle's Medium (DMEM) were from (Sigma Co., St Louis, USA). Random primers, agarose MP and dNTPs were from Roche (Roche Diagnostics, Mannheim, Germany). Penicillin, streptomycin, ribonuclease inhibitor, M-MuLV reverse transcriptase, Taq DNA polymerase, and DNA markers were supplied and

Time course of CEA release from colon carcinoma cells

We first compared CEA release from various colorectal carcinoma cell lines into the medium as a function of time. The CEA release was studied in four human colon cancer cell lines LS180, HT29, HT29/219 and SW742 cells in the exponential phase and stationary phase (confluent cells in serum free medium). Fig. 1 compares the time course of CEA release from these cell lines. The CEA release from the cells peaked at confluence and declined in serum free medium. A great variation in CEA release was

Acknowledgements

We thank Dr Clifford P. Stanners of McGill University for a careful reading of the manuscript and Pooneh Mokarram for the technical assistance. This work was supported by the office of the Vice Chancellor for Research, Shiraz University of Medical Sciences, grant No. 81-1551.

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