Original article
Fetal colon cell line FHC exhibits tumorigenic phenotype, complex karyotype, and TP53 gene mutation

https://doi.org/10.1016/j.cancergencyto.2009.11.009Get rights and content

Abstract

Stable cell lines obtained by spontaneous immortalization might represent early stages of malignant transformation and be useful experimental models for studies of mechanisms of cancer development. The FHC (fetal human cells) cell line has been established from normal fetal colonic mucosa. Detailed characterization of this cell line and mechanism of spontaneously acquired immortality have not been described yet. Therefore, we characterized the FHC cell line in terms of its tumorigenicity, cytogenetics, and TP53 gene mutation analysis. FHC cells displayed capability for anchorage-independent growth in semisolid media in vitro and formed solid tumors after transplantation into SCID (severe combined immunodeficiency) mice. This tumorigenic phenotype was associated with hypotriploidy and chromosome number ranging from 66 to 69. Results of comparative genetic hybridization arrays showed that most chromosomes included regions of copy number gains or losses. Region 8q23∼8q24.3 (containing, e.g., MYC proto-oncogene) was present in more than 20 copies per nucleus. Moreover, we identified mutation of TP53 gene in codon 273; triplet CGT coding Arg was changed to CAG coding His. Expression of Pro codon 72 polymorphic variant of p53 was also detected. Mutation of TP53 gene was associated with abolished induction of p21Waf1/Cip1 and MDM-2 proteins and resistance to apoptosis after genotoxic treatment. Because of their origin from normal fetal colon and their relative resistance to the induction of apoptosis, FHC cells can be considered a valuable experimental model for various studies.

Introduction

Isolation of human immortalized cells and establishment of permanent cell lines from nontransformed cell populations are limited. However, it has been described in human prostate epithelial cells [1], human breast epithelial cells [2], and colon-derived fibroblasts [3]. Deregulation of several particular signaling pathways has been shown to be responsible for induction of senescence/immortalization [4]. It often includes defects associated with genome stability, epigenetic deregulation of gene expression, reactivation of telomerase activity, inactivation of cell cycle regulators, overexpression of oncogenes such as MYC, or viral oncogenes [4]. Mechanisms of spontaneous immortalization are not usually known. Events associated with spontaneous immortalization in breast epithelial cell line MCF10A [5], [6], [7] have been described in relative detail. This cell line shows several genomic alternations that include, for example, gain of MYC and ERBB2. Moreover, it shows increased telomerase activity and interestingly expresses wild-type p53 [7]. Cell lines established by spontaneous immortalization might represent early stages of malignant transformation and could be useful experimental models to study mechanisms of cancer development [8].

Colorectal cancer is a frequent disease in Western countries. It can be categorized according to type of genomic instability as chromosomally unstable (CIN+), characterized by aneuploid/polyploid karyotype, or as microsatellite unstable (MSI+), characterized by near-diploid karyotype and defective mismatch repair [9]. Approximately 40% of colorectal tumors contain point mutations in the TP53 gene (http://www-p53.iarc.fr/Graph.asp). These point mutations are clustered in exons 4–9 with five hot spots: 273, 248, 175, 245, and 282 (http://www-p53.iarc.fr/Graph.asp). TP53 gene product, the p53 tumor suppressor protein, is a transcription factor activated in response to stress signals, in particular to genotoxic stress. It is a crucial barrier against cancer development. Generally, detailed cell line characterization is considered necessary for validity of experimental studies. The FHC (fetal human cells) cell line has been established by Siddiqui and Chopra [10] from normal fetal colonic mucosa. This cell line has been used in various studies focusing on mechanisms of cancer progression, regulation of differentiation, and apoptosis [11], [12], [13], [14], [15], [16]. However, to our knowledge, detailed characterization of this cell line has not yet been published. In this study, we present cytogenetic, array comparative genetic hybridization (CGH), and TP53 gene characterization of this cell line.

Section snippets

Cell lines, cultivation, and treatment

FHC was obtained from the American Type Culture Collection (CRL-1831, LGC Standards, Lomianki, Poland). Cells were cultured in 1:1 mixture of Ham F-12 medium and Dulbecco modified Eagle medium (Gibco, Invitrogen) supplemented with 25 mmol/L HEPES (N-[2-hydroxyethyl] piperazine-N′[2-ethanesulfonic acid]), 10 ng/mL cholera toxin (Calbiochem, Merck, Darmstadt, Germany), 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 100 ng/mL hydrocortisone (all from Sigma-Aldrich, St. Louis, MO), and 10% fetal bovine

Cytogenetics and array CGH analysis

Karyotype analysis of FHC cell line was made by conventional staining with Giemsa (GTG-banding) at passage 13 and 40; a representative karyotype is shown in Figure 1A. FHC is a hypotriploid cell line with a chromosome number ranging from 66 to 69. With regard to the complex rearrangements present in all tested metaphase spreads, karyotyping was supplemented with array comparative genomic hybridization analysis (Fig. 1B). With the exception of few chromosomes (1, 2, 10, and 16), all the other

Discussion

We analyzed tumorigenic characteristics and molecular cytogenetic features of spontaneously immortalized cell line FHC isolated from normal fetal colonic mucosa. FHC cells exhibit epithelial morphology and were originally established by Siddiqui and Chopra [10]. Basic characteristics of these cells have been published [10]; however, other detailed molecular cytogenetic characteristics are missing. Through their origin from normal colonic mucosa and capability to differentiate after various

Acknowledgments

The authors would like to thank B. Vogelstein for providing HCT-116 p53−/− and p53+/+ clones, Iva Lišková, Jaromíra Netíková, and Petra Jelínková for technical assistance, Jiřina Holá for help with γ irradiation, and John P. Smith for English-language help. This work was supported by grants 204/07/0834, 501100041, and 204/08/1560 of the Czech Science Foundation; grants ME-919 and MSM6198959216 of the Ministry of Education, Youth and Sport; grant 9600-4/2008 of the Ministry of Health; and grants

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