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  • ICANCERRÃœSI-ARCHÕS,3i5-3622.Augustis. i

    a-Fetoprotein (AFP) Expression in Clones of McA-RH 7777 Rat Hepatoma:

    Correlation with the Occurrence of Homogeneously Staining Regions on Chromosome 14 '

    Leila Khamzina,2 Tatjana Eraiser, and Pierre Borgeat

    Centre Je Recherche en Rhumatologie el Immunologie. Centre tie Recherche Ju Centre Hospitalier de l'UniversitéLaval, UniversitéLaval, Québec,Canada, HIV 4G2

    ABSTRACT

    a-fetoprotein (AFP) is a well-established cell differentiation and tumor marker. We showed previously that McA-RH 7777 hepatoma cells are

    heterogeneous in terms of their AFP cellular expression. In the present study, we developed stable and unstable 7777 hepatoma clones in terms of their AFP phenotype: AFP-producing (AFP+) or AFP-nonproducing

    (AI-T ) clones, and investigated in these clones (a) AFP phenotype related

    to protein and mRNA levels; (/»cellular morphology; and in expression of several liver-specific markers. Our results demonstrated that a-albu- min expression paralleled that of AFP, from the absence of a-albumin message in All' clones to high expression in Ml'1 clones, suggesting

    that common mechanisms control the expression of both proteins in this hepatoma cell population. In addition, the karyotypes of the McA-RH

    7777 hepatoma cell line and its 15 generated clones were analyzed and correlated to their AFP phenotypes. Only the stable AFP+ clones showed

    homogeneously staining regions on the chromosome carrying the Ml'

    gene. These results strongly suggest that amplification of either structural or regulatory sequences of the .1/7' gene is involved in maintaining its

    high expression.

    INTRODUCTION

    AFP3 is a well-known oncodevelopmental protein. Actively tran

    scribed by fetal and postnatal hepatocytes, AFP is almost undetectable in adult liver parenchymal cells; however, its production can resume in the liver under pathological conditions such as primary hepatocel- lular carcinoma (1-5). Therefore, this protein is considered a pathol

    ogy marker and is used in clinical diagnosis (6). AFP is a member of a multigene family (albumin family) that includes ALB, DBP, and the recently discovered a-ALB (7-9). All four of these proteins are

    synthesized in mammalian liver parenchymal cells and share a similar secondary folding structure with three related domains (10, 11). Their genes remain in a single copy per haploid genome and have been mapped to chromosome 14 in rats (12).

    The disappearance of AFP postnatally and its reappearance in malignancy reflect sequential gene activation and repression. The hepatoma-derived cell lines, therefore, provide a very interesting

    model for studying AFP gene regulation during normal liver devel opment, differentiation, and oncogenesis. The McA-RH 7777 cell line (13) is a well-established, chemically induced rat hepatoma, which we

    adopted for our studies. Using several clonal approaches, we have shown (14, 15) that individual AFP-producing (AFP+) and

    Received 4/3/95; accepled 6/19/95. The costs of publication of this article were defrayed in part by the payment of page

    charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    1This work was supported by Medical Research Council of Canada grants. This work

    is the first in the series: a-Fetoprotein (AFP) Expression in Clones of McA-RH 7777 Rat

    Hepatoma. 2 To whom requests for reprints should be addressed, at Centre dc Recherche en

    Rhumatologie et Immunologie, Local 9800, CHUL, 2705 boul. Laurier, Ste-Foy, Quebec, Canada, G1V 4G2.

    ' The abbreviations used are: AFP. a-fetoprotein: AFP ', a-fetoprotein-producing phenotype: AFP , a-fetoprotein-nonproducing phenotype; AFP', phenotype with mixed

    a-fetoprotein production: ALB, albumin; a-ALB, a-albumin; DBP. vitamin D-binding

    protein; TAT, tyrosine aminotransferase; TO, tryptophan oxygcnase; dsr. differential staining regions; hsr. homogeneous staining regions.

    AFP-nonproducing (AFP ) cells give rise to AFP+ and AFP clones,

    respectively. However, clones of alternative AFP phenotypes were sometimes generated with a frequency that far exceeded that of gene mutation. In addition, these studies established a method of stabilizing cloning (7-step selection) that allows development of both stable and

    unstable clones by selective pressure in function of the AFP phenotype (AFP f or AFP ).

    The present study introduces a unique model system of 7777 hepatoma clones. We describe the isolation and characterization of eight new clones (AFP+ and AFP ) derived from the McA-RH 7777

    cell line, their phenotype analysis, and the regulatory mechanisms that lead to their generation. We also analyzed the expression of AFP and other liver-specific markers. Our results demonstrated an absence of a-ALB gene expression in AFP" clones and significant high expres

    sion in AFP+ clones that reached maximal levels in stable clones.

    Moreover, because the malignant phenotype often arises as a result of consistent chromosome aberrations (16), we examined whether AFP production could be associated with specific changes at the chromo some level. The chromosome constitution of the 7777 hepatoma cell line and 15 generated clones were analyzed and correlated to the AFP phenotypes. High-level AFP stable expression correlated significantly

    with specific gene amplification characteristics.

    MATERIALS AND METHODS

    Cell Culture and Cloning. McA-RH 7777 rat hepatoma cells were ob

    tained from the American Type Culture Collection (ATCC, Rockville, MD) and grown as described elsewhere (13). The method of stabilizing cloning consisted of seven cycles and was performed according to Eraiser and Mi.-nii/iiu (14, 15). Each cycle spaned over al least 4 weeks and included the

    following steps: (n) mass hepatoma culture cell cloning; (/;) random selection of 10-20 clones on days 7-10 of culture; (

  • AFP EXPRESSION CORRELATES WITH HSR ON CHROMOSOME 14

    Cytogenetic Analysis. Colchicine (1 ngjmi) was added to the culture medium for 2 h, and then the cells were trypsinized, centrifuged, treated with hypotonie KG (75 mM), and fixed with methanol:acetic acid (v/v; 3:1). The slides were prepared by air-drying technique. Chromosomes were banded by a modification of the trypsin-Giemsa method of Seabright (23). For each cell

    population, 50 metaphases were counted, photographed, and analyzed. The standard karyotype for trypsin-Giemsa-banded rat chromosomes was used

    (24), and the nomenclature for the bands was derived from Levan (25).

    RESULTS AND DISCUSSION

    The stabilizing cloning was started from the mixed (AFP21) clone D7 and carried out in two contrasting phenotypic directions: AFP+ and

    AFP . The selected clones underwent a maximum of six culture passages before starting the next cloning cycle. Stability-gaining selection through the stabilizing cloning was quite efficient allowing us to obtain AFP+ and AFP" clones with different levels of AFP phenotype stability. From the

    panel of generated clones, eight were chosen for additional characteriza tion: mixed (AFP^ clone D7; the unstable AFP" clones, F4 and Hll; the unstable AFP+ clones, A3 and G6; the stable AFP" clone 7H10; and

    the stable AFP4 clones, 7G3 and 7G4. The filiation of these clones is

    schematically summarized in Fig. 1. Analysis of AFP Phenotype in McA-RH 7777 Hepatoma Clones.

    The AFP phenotype of each clone was characterized by two inde pendent and complementary methods: immunocytochemical staining, which localizes the intracellular protein and reflects the homogeneity of the population, and Northern blot analysis, which estimates the actual AFP biosynthesis. As shown in Fig. 2, the parental 7777 cells consisted of a mixture of stained and nonstained cells that yielded three types of clones: AFP+, AFP^ and AFP". Staining appeared as

    discrete brown granules within the cytoplasm, with stronger staining near the perinuclear space. For the AFP±phenotype, some variations

    in staining intensity were noted from one cell to another (Fig. 3A). AFP+ clones had staining profiles that were much more homogene

    ous, with hardly visible variations whether stable or unstable (Fig. 3, B and C). On the other hand, AFP" clones did not show any staining

    (Fig. 3, D and E). Growing our stable clones for more than 6 months showed that cells with alternative phenotypes appear within each passage with a frequency not exceeding 0.01-0.005% but did not

    form clones with selective advantages. This latter observation is in agreement with our previous results (14, 15).

    The results of our Northern blot analysis demonstrated that the absence of AFP production in AFP" clones correlated with the absence of the

    corresponding mRNA. As illustrated in Fig. 4, a single band of 2.2 kb was observed in the parental McA-RH 7777 cell line and its AFP+

    clones, which corresponds to the reported size of AFP mRNA in rat liver (26). Densitometric analysis of the bands showed that the level of AFP mRNA of the stable AFP+ clones was twice that of unstable clones and

    20 times that of the parental line. The reporter chloramphenicol acetyl- transferase gene driven by 7 kb of the 5 ' flanking region of the AFP gene, which was active only in the AFP+ clones (27), indicated a transcrip-

    tional control of AFP gene expression in our system. Clones of 7777 hepatoma vary not only in the function of their AFP

    phenotype but also morphologically (Fig. 2A). Some had fibroblast-

    like morphology with regular epitheli