Altered transferrin gene expression in preneoplastic ... - Springer Link

Virchows Archiv B Cell Pathol (1992) 62:251-257

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Altered transferrin gene expression in preneoplastic and neoplastic liver lesions induced in rats with N-nitrosomorpholine K. Schiebel, H. Stumpf, H. Zerban, E. Pekel, and P. Bannasch Abteilung fiir Cytopathologie,DeutschesKrebsforschungszentrum,Im NeuenheimerFeld 280, W-6900 Heidelberg, Federal Republicof Germany ReceivedFebruary 26 / AcceptedJune 9, 1992 Summary. The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3' region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell loci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis. Key words: Transferrin - Gene expression - Hepatocarcinogenesis - Preneoplastic lesions - In situ hybridization

Introduction During carcinogenesis dramatic changes in the cellular enzyme pattern have been observed in early preneoplastic lesions in various tissues (Bannasch et al. 1991). Metabolic aberrations appearing in the hepatic parenchyma during hepatocarcinogenesis have been studied extensively and in particular, changes in carbohydrate metabolism have been investigated in detail (Mayer et al. 1989). The altered expression of several enzymes has been shown to be an early event in hepatocarcinogenesis and Correspondence to: P. Bannasch

is increasingly used for the detection of preneoplastic hepatic lesions (Bannasch 1990 a, b; Pitot 1990). We have screened a cDNA library of the rat liver RNA (Frain et al. 1989) for hitherto unknown genes differentially expressed in hepatocellular tumors and in lesions occurring during chemical hepatocarcinogenesis. The screening revealed, among other genes, the full length cDNA of the transferrin gene (Schiebel et al. in preparation). Transferrin is a major glycoprotein of vertebrate serum whose function is the transport of ferric iron from the intestine, reticuloendothelial system and liver parenchyma to erythroid precursors and proliferating cells in the body (for reviews see Kahn et al. 1987; Bowman et al. 1988; de Jong et al. 1990). Because of the involvement of iron in the synthesis of DNA and RNA, transferrin has been regarded as a growth factor required for the proliferation of normal and malignant cells (Barnes and Sato 1980), but a secondary growth effect unrelated to the iron metabolism has also been described (James and Bradshow 1984). The transferrin gene is mainly expressed in the liver, but also in the central nervous system, testis, lymphocytes and thymus. A lower level of synthesis varying during fetal development was found in rat heart, spleen, kidney and muscle (Aldred et al. 1987). The regulation of transferrin gene expression is not yet understood, but a few cis-acting elements of the human transferrin gene have been characterized (Adrian et al. 1986; Brunel et al. 1988). Positive and negative control by transacting agents, including metallic, hormonal, autocrine and mitotic factors has been proposed (Yang et al. 1990). In the present communication we report studies of the expression level of the rat transferrin gene during chemically induced hepatocarcinogenesis by Northern blotting and in situ hybridization.

Materials and methods Preneoplastic and neoplastic hepatic lesions were induced in male Sprague-Dawleyrats (purchased from the Zentralinstitut ffir Ver-

252 suchstierzucht, Hannover, FRG) by limited (7 weeks, stop model, Bannasch 1968) oral administration of N-nitrosomorpholine (120 or 240 mg/1 drinking water). Hepatocellular carcinomas developed between 30 and 80 weeks after withdrawal of the carcinogen. The animals were sacrificed between 9 and 10 a.m.; the livers were removed immediately and slices 5-10 mm thick were snap-frozen in isopentane precooled with liquid nitrogen at - 150~ C. The tissue was stored at - 8 0 ~ C until used.

Isolation of RNA and Northern blotting. DNA and RNA were isolated simultaneously from frozen tissue by the method of Chirgwin et al. (1979) in guanidine isothiocyanate. Samples of 20 pg total RNA were separated on 1.2% formaldehyde agarose gels and blotted by standard methods (Sambrock et al. 1989; Ausubel et al.

Fig. 1. Localization of the transferrin cDNA probes used for hybridization within the 2.3 kbp full length cDNA determined by nucleotide sequence comparison with human transferrin gene (Yang etal. 1984). B Barn HI1; E: Eco RI; K, Kpn I; P: PstI; Pv: Pvu I l l ; 1) sites determined by sequencing, additional sites within the gene not yet localized

1987). The amount of RNA was determined photometrically and corrected after ethidium bromide staining of the gel. The hybridization with a 150 bp partial eDNA fragment (see Fig. 1) was carried out in 5 x SSC, 5 x Denhardts, 0.5% SDS, and 20 lag/ml salmon sperm DNA at 68~ C, washes were started with 2 x SSC, 0.5% SDS at room temperature and finished with 0.1 x SSC, 0.5% SDS at 68~ C.

Isolation of the transferrin eDNA. A cDNA library from Sprague Dawley rat liver mRNA (Frain et al. 1989, a generous gift from M. Frain and R. Cortese, EMBL, Heidelberg, FRG) in 2 gt 11 was comparatively screened with oligo dT-primed first-strand cDNA probes obtained by reverse transcription with 32p-labeled nucleotides of RNA isolated from a hepatocellular carcinoma and from a liver of an untreated rat, respectively. Each of these probes were hybridized directly with a replica filter of the cDNA library. The autoradiographs of filters hybridized with first-strand eDNA from untreated and treated rats were compared and clones exhibiting signals of different strength were used for two rescreens and tested on Northern blots. The differentially expressed transferrin clone was subcloned in bluescript II vector (Stratagene, Heidelberg, FRG) by standard methods as described by Maniatis et al. (1982) and Sambrock et al. (1989). Sequencing by the dideoxy chain termination method (Sanger et al. 1977) and a database search in the GenBank and EMBL-database were used to identify the clone. In situ hybridization. The in situ hybridization methods of Cox et al. (1984), Angerer et al. (1985) and Angerer and Angerer (1989) were slightly modified. Cryostat sections (6 lam thick) of the tissue were treated with 0.5 lag/ml proteinase K for 15 min and post-fixed in 4% paraformaldehyde. To avoid nonspecific sticking of the probe the sections were acetylated with 0.25% acetic acid anhydride (v/v) in 0.1 M triethanolamine. A 236 bp Kpn I-fragment

Fig. 2. Northern blot of RNAs isolated from ten hepatocellular carcinomas (3; 4; 31; 42; 247; 249; 251; 748; 780; 935) induced with N-nitrosomorpholine and the total RNA of a liver of an untreated rat (NL). Samples of 20 lag of RNA were separated on 1.2% formaldehyde-agarose gel, stained with ethidium bromide, blotted and hybridized by standard methods. A 150 bp partial eDNA probe depicted in Fig. 1 was used for hybridization, a Autoradiograph of the hybridized filter, b Agarose gel after staining with ethidium bromide

253

Fig. 3 a-f. Serial cryostat sections through a hepatocellular carcinoma and surrounding normal tissue (Border between tissue of normal appearance and carcinoma indicated by arrows), a Partial reduction in glycogen in neoplastic tissue demonstrated by the PASreaction and counterstaining with toluidine blue ( x 78). b Demonstration of the basophilic cell components by toluidine blue ( • 78). e and e in situ hybridization of transferrin mRNA with 35S_labeled

antisense RNA as detected by autoradiography and staining with hematoxylin and eosin (c • 78, e • 250). tl and f in situ hybridization of transferrin mRNA with 3SS-labeled sense RNA (control) shown after autoradiography and staining with hematoxylin and eosin (tl • 78, e x 250). e and f higher magnification of a detail of e and d, respectively

of the 3'coding region of the transferrin gene (Fig. 1) subcloned in bluescript II vector was used as template for the transcription by the T3/T7 polymerase system. To avoid transcription of the vector DNA the plasmids used as templates were linearized prior to the transcription. The 35S-labeled anti-sense and sense RNA (specific activity 5-10 • 107 cpm/~tg), served as hybridization probe and control, respectively. After prehybridization (1 h, 50% formamide, 0.3 M NaC1, 10 mM Tris pH 8, 1 mM EDTA, 10% dextransulfate, 100 mM DDT, 1 • Denhardts, 50 ~tg/ml tRNA, 50~ C) with unlabeled in vitro transcribed bluescript polylinker RNA, the slices

were hybridized overnight with labeled sense or antisense RNA (0.2 Ixg/ml/kb), respectively, (50% formamide, 0.3 M NaC1, 10 mM Tris pH 8, 1 mM EDTA, 10% dextransulfate, 100 mM DDT, 1 • Denhardts, 50 ~tg/ml tRNA, 50~ C). Two washing procedures with 2 • SSC, 0.1% 2-mercaptoethanol (15 min, 50~ C) were followed by a RNase A-digestion (20 ktg/ml, 37~ C) and finished by washing twice in 0.1 • SSC, 0.1% mercaptoethanol, 68 ~ C. For autoradiography the slices were dipped in Kodak NTB-2 emulsion and exposed for 1-3 days. The tissue was stained with hematoxylin and eosin.

Fig. 4a-h. Cryostat sections through different types of foci of altered hepatocytes as demonstrated by the PAS-reaction and counterstaining with toluidine blue (a, c, e, g) or by in situ hybridization of transferrin m R N A with 35S-labeled antisense RNA in autoradiographs of the respective serial sections (b, d, f, h) (border between tissue of normal appearance and carcinoma indicated by arrows), a and b Glycogen storage focus exhibiting increased trans-

ferrin mRNA (x 78). e and d pronounced glycogen storage focus showing unchanged to slightly reduced transferrin mRNA level (x 62.5). e and f Mixed cell focus containing many glycogenotic cells in addition to glycogen-poor cells and exhibiting a reduced transferrin mRNA level ( x 62.5). g and h Mixed cell focus poor in glycogen exhibiting a reduced transferrin m R N A level ( x 78)

255 The high expression level of the transferrin gene in liver tissue made a quantitation of silver grains neither possible nor necessary, since there were obvious differences seen by low magnification microscopy and overview photographs. For the identification of preneoplastic lesions, serial sections of the tissues were used. Sections were treated by the periodic acid-Schiff reaction (PAS) and counterstained with toluidine blue or stained with toluidine blue only. The lesions were classified according to Bannasch and Zerban (1990).

Results

In order to identify genes that change their expression level during chemically induced hepatocarcinogenesis, a cDNA library from rat liver mRNA was screened for differentially expressed genes. Using RNA isolated from a hepatocellular carcinoma of a rat treated with N-nitrosomorpholine compared with the RNA of a liver of an untreated rat, 17 different clones which were expressed at different levels (data not shown) were isolated. One clone identified as the full length cDNA of the transferrin gene by sequence analysis was used for further investigations. The hybridization of the 32p-nick translated 150 bp partial cDNA fragment of the transferrin gene with the Northern blot of the RNA of ten different hepatocellular carcinomas compared with the RNA of an untreated control rat is shown in Fig. 2. Four well differentiated HCCs (see Fig. 2, tumors: 3; 247; 249; 251) showed no or only slight reduction in transferrin gene expression compared with the normal liver parenchyma. Five HCCs showing a lower differentiation (see Fig. 2, tumors: 4; 31; 748; 780; 935) expressed transferrin at a reduced level. However, one poorly differentiated HCC (see Fig. 2, tumor 42) revealed normal expression of the transferrin gene, which might be the consequence of an admixture with less altered tissue. To avoid misinterpretation due to the admixture of different stages of neoplastic transformation or differences in the amounts of RNA loaded for Northern blotting, in situ hybridization was used for the direct correlation of preneoplastic and neoplastic lesions with the expression levels. In situ hybridization of the antisense RNA probe with sections of two additional different hepatocellular carcinomas revealed a marked decrease in the expression level of the transferrin gene in carcinoma cells compared with those of the surrounding tissue showing a normal phenotype (Fig. 3). Analysis of 50 preneoplastic lesions revealed remarkable differences in the expression of the transferrin gene in early glycogenotic and tigroid cell lesions on the one hand and in mixed cell lesions on the other hand. The early preneoplastic lesions characterized by a high content of glycogen expressed increased levels of transferrin RNA in 9 out of 16 lesions, compared with the surrounding tissue. Five lesions had an unchanged expression level and only two lesions with decreased transferrin expression were detected (Fig. 4 and Table 1). Among 29 mixed cell loci appearing at later time points, only two foci showed higher expression levels than the sur-

Table 1. Summary of alterations of the transferrin expression in different types of preneoplastic lesions. A total of 50 preneoplastic lesions of different types were analysed for their transferrin expression level in autoradiographs of in situ hybridized sections Preneoplastic

N

Transferrin expression

lesion Increased Glycogen storage foci Mixed cell foci Tigroid cell foci

Unchanged

Decreased

16

9

5

2

29 5

2 4

11 0

16 1

rounding tissue, whereas 16 foci exhibited a reduced transferrin expression, and 11 lesions were not distinguishable by their transferrin mRNA content from the surrounding tissue (Fig. 4 and Table 1). Four of the five tigroid cell foci observed had increased transferrin expression and one such focus showed a reduced expression level. A basophilic adenoma in which the tigroid cell pattern predominated was characterized by a homogeneously high expression level of the transferrin gene like the majority of the tigroid cell foci.

Discussion

Changes in the iron metabolism of preneoplastic and neoplastic hepatic lesions have been recognized for a number of years. Williams et al. (1976) described rats treated with N-2-fluorenylacetamide and fed with ferrous gluconate, in which focal preneoplastic lesions could be recognized by the absence of histochemically demonstrated iron. These authors stated that these ironfree lesions were also characterized by changes in the activity of various enzymes and an excessive storage of glycogen, as demonstrated previously by other workers in foci of altered hepatocytes. A reduced uptake of iron has also been observed in hepatoceUular nodules induced by the Solt-Farber-procedure, although the number of transferrin receptors was increased in these nodules (Eriksson et al. 1990). More recently, Stitzel et al. (1990) reported that basophilic hepatocellular loci, which correspond to the tigroid cell loci described by Bannasch et al. (1985) and frequently occur spontaneously in old female Fischer rats, do not exclude iron. The finding of a reduced transferrin expression level in some glycogenotic clear cell foci and in the majority of mixed cell foci appears to correlate with the iron deficiency of such lesions reported by Williams et al. (1976). However, in the majority of the early glycogenotic foci there was an elevated transferrin expression, and it remains to be clarified whether or not these lesions exclude iron. The tigroid cell foci showing an increased expression of the transferrin gene probably represent the same cell population which, according to Stitzel et al. (1990), does not exclude iron. The glycogenotic and mixed cell foci have been shown to constitute successive stages in the predominant sequence of cellular changes leading to hepatocellular car-

256 cinomas (Bannasch 1990a). During this process several phenotypic changes appear which suggest a progressive cellular dedifferentiation. The changes in the expression of the transferrin gene described in this communication seem to be associated with this dedifferentiation process. Thus, in the early glycogenotic foci, which are composed of highly differentiated hepatocytes, the expression level of the transferrin gene is normal or even increased compared with that of the surrounding parenchyma, whereas a reduction in gene expression becomes increasingly evident when less differentiated glycogen-poor cells emerge in mixed cell populations. The high expression level o f the transferrin gene in the tigroid cell foci appears to indicate that these lesions are also endowed with a relatively high cellular differentiation. Although this interpretation is consistent with the concept that the tigroid cell foci have only a low potential for progression to hepatocellular tumors (Bannasch and Zerban 1992), more detailed studies on larger number of these focal lesions are required before final conclusions are drawn. The relationship of transferrin gene expression to the differentiation of hepatocytes under cell culture conditions was studied in detail by Clayton and Darnell (1983). When hepatocytes were cultured in serum-supplemented standard tissue medium, both the normal phenotype of the hepatocytes and liver-specific gene expression were rapidly lost, indicating that dedifferentiation was closely associated with increased cellular proliferation. Thus the expression of the transferrin gene can be used as a marker of the maintenance of differentiation and the liver-specific expression pattern o f enzymes in hepatocytes in cell culture (Isom et al. 1987). Interestingly, the sequential changes in cellular phenotype during the transition of glycogenotic foci to mixed cell lesions and hepatic tumors are similarly related to increasing cell proliferation as demonstrated by the incorporation of 3H-thymidine (Zerban et al. 1989). Obviously, although transferrin can be regarded as a growth factor in the broader sense (Barnes and Sato 1980; James and Bradshow 1984), dedifferentiated and proliferating liver cells are able to grow without themselves expressing the transferrin gene. Until now, very little is known about the regulation of the transferrin gene. Studies investigating a correlation between iron uptake and transferrin gene expression have produced contradictory results. Tuil et al. (1985) observed that neither iron overload nor iron depletion resulted in changes in transferrin transcription and concluded that transferrin expression in rat liver is not influenced by iron. However, McKnight et al. (1983)found that the chicken transferrin m R N A transcription rate was increased after iron depletion. At the molecular level, transcription factors and transcription factor-binding sites for the rat transferrin gene have not yet been characterized. However, it is known that in dedifferentiated hepatocytes in culture and in dedifferentiated hepatoma cells the liver-specific transcription factors which may also be involved in the regulation of the transferrin expression are either decreased or occur in a variant form (Johnson 1990; Cereghini et al. 1988, 1990). These observations support the con-

cept that the decrease in transferrin gene expression in mixed cell lesions and HCCs mirrors the dedifferentiation process appearing during hepatocarcinogenesis.

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