Increased expression of the N-myc gene during normal and neoplastic rat liver growth

Experimental

Increased

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Expression of the N-myc Gene during Neoplastic Rat Liver Growth

CHRISTIANE Institut

Cell Research

Normal

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MARISOL CORRAL, BRIGITTE PARIS, GUGUEN-GUILLOUZO,* DANIEL CORCOS, JACQUES KRUH,’ and NICOLE DEFER

de Pathologie Molkulaire, Unite Associke CNRS, Facultt de Mtdecine Cochin-Port-Royal,24 rue du Faubourg-Saint-Jacques, 754014 Paris, France,and*Unitk INSERM de Recherches H&patologiques, H6pital Pontchaillou, 34011 Rennes Cedex, France

The activation of N-myc and c-myc genes has been implicated in the genesis of a variety of cancers. In order to determine whether this activation is releated to the process of cell growth, we examined N-myc and c-myc expression in rat hepatocytes when growth was stimulated by partial hepatectomy and in rat liver during neoplastic growth induced by diethylnitrosamine. The levels of N-myc and c-myc mRNAs, which are very low in hepatocytes from normal rats, were increased at least 20-fold within 3 h after partial hepatectomy and decreased rapidly by 6 h. A second transient peak of c-myc and N-myc mRNAs occurred around 9 and 48 h, respectively. N-myc and c-myc expressions were also induced in carcinoma nodules by diethylnitrosamine. A high level of N-myc transcript was observed in hepatocytes as early as 1 month after the carcinogen adiminstration, whereas c-myc transcript was detected at a high level only several months later in carcinoma nodules. Our results suggest that the transient expression of N-myc and c-myc oncogenes during the prereplicative stage of liver regeneration may be associated with the entry of hepatocytes into the cell cycle, and that N-myc expression is not limited to tumors of neural characteristics as has been previously shown but is also observed in cancer from epithelial origin. 6$ 1988 Academic Press, Inc.

Oncogenes represent a group of genes which play an important role in the development of organisms, in cell differentiation 11, 21, and in the control of cell proliferation [3, 51. Alteration of these genes or activation of their expression has been implicated in carcinogenesis. The N-myc gene was first identified by its homology to the second exon of the c-myc gene [6]. It was often found amplified and expressed at a high level in tumors of neural origin [7] and is able, as previously shown for c-myc, to contribute to neoplastic transformation of cultured mammalian cells [8,9]. N-myc mRNA has also been detected in tissues from newborn animals [lo]. In order to determine whether N-myc could play a role in the regulation of epithelial cell growth we measured the N-myc mRNA level during rat liver regeneration following a two-third hepatectomy, a model of regulated growth process. Liver regeneration involves a hypertrophy stage which lasts for 12 to 16 h, followed by a replicative stage during which almost all the hepatocytes undergo DNA replication (peak at 20-24 h) followed by mitosis (peak at 30 h), in a ’ To whom reprint requests should be addressed. OU:

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Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/88 $03.00

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synchronized manner [I I]. In a previous work we have shown that the c-fos gene expression is strongly increased in regenerating hepatocytes 30 h after partial hepatectomy [5]. Here we report that N-myc expression is induced transiently 3 h after partial hepatectomy and again 45 h later. This pattern of induction is partly different from that observed by us and by others [12, 131 for c-myc induction in the same cells. As a model of tumoral cell growth, we chose liver carcinoma induced by diethylnitrosamine (DENA) in which we have previously observed an increased expression of c-ras [ 141, c-fos [5], and other genes associated with liver transformation [15]. In this paper we show (a) that N-myc expression is induced early and transiently during the prereplicative stage of liver regeneration as is c-myc, which suggests that these two genes could be involved in normal cell growth control, and (b) that the N-myc mRNA level is increased in hepatocytes as early as 1 month after DENA administration whereas c-myc was detected only several months later in carcinoma nodules, indicating that N-myc as c-myc expression could be activited in various transformed cells. MATERIALS Hepatocytes

AND

METHODS

and Tumor Cells

Hepatocytes were obtained by collagenase perfusion of rat livers [16]. A homogeneous suspension of single cells was obtained. Isolated hepatocytes were easily purified from the nonparenchymal cells, including inflammatory cells, by three differential centrifugations. For the experiments with regenerating livers, rats were partially hepatectomized by the method of Higgins and Anderson [17] under oxygenxther anaesthesia. The rats were killed from 3 to 168 h after the operation and the livers were isolated immediately. Several rats were submitted to anesthesia and sham-operated. They were killed 3, 6, and 9 h after the operation. Liver carcinogenesis was induced as follows: female Sprague-Dawley rats weighing 1X1-175 g were given three doses of DENA (25 mg/kg) through stomach tubes 24, 48, and 72 h after a two-third hepatectomy. For rats sacrificed from 1 to 7 months after the carcinogen administration, total hepatocytes were isolated. For rats sacrificed from 12 to 18 months after DENA administration, each liver was perfused via the portal vein with a collagenase solution. A mixture of isolated cells and packs of undissociated cells resulted from the perfusion. The undissociated cells, corresponding to malignant nodules, were collected by filtration of the suspension through a 150~urn nylon mesh.

Nucleic Acid Preparation RNA isolation. Frozen tumors and hepatocytes were lysed in 5 M guanidium thiocyanate, 0.1 M sodium acetate (pH 5.5), 1 mM EDTA, 5 % (v/v) 2-mercaptoethanol, and 2% (v/v) N-lauroyl sarkosyl. Total cellular RNAs were prepared by the method of Chirgwin er al. [18] as modified by Raymondjean et al. [193. Poly(A)+ RNAs were isolated by oligodeoxythymidylate-cellulose chromatography as described by Aviv and Leder [20]. DNA isolation. High molecular weight DNA was prepared according to the method of GrossBellard er al. [21] from normal livers and from four carcinoma nodules isolated from rats killed 18 months after the DENA administration. Hybridization probes. The probe we used for the N-myc gene was the I-kb EcoR-BumHI fragment excized from the plasmid pNb1 corresponding to the human N-myc gene [63. The c-myc probe corresponds to the third exon of the human c-myc gene isolated after the excision of the plasmid with EcoRI and CluI enzymes. The albumin probe was the cDNA clone pRSA 8 kindly provided by Dr. J. Sala-Trepat.

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Norrhern blot hybridizations. Samples of poly(A)+ RNAs or total RNAs were separated by electrophoresis on 1% agarose gels containing 2.2 M formaldehyde. RNAs were transferred onto GeneScreen Plus filters (NEN, Boston, MA). The filters were then baked and hybridized with the 32Plabeled probes. In order to visualize the amounts of RNA the gels were stained, before transfer, with 0.5 ug/ml of ethidium bromide in 0.3 M ammonium acetate. Prehybridizations of Northern blots were performed at 42°C for 16 h in a solution containing 50% formamide, 5% dextran sulfate, 1% sodium dodecyl sulfate, 10 ug/ml of poly(A), 50 &ml of herring sperm DNA, 50 ug/ml of heparin, and 0.75 M NaCl. Hybridizations were performed in the same buffer in the presence of the specific fragments labeled by nick-translation (3-5 x 10’ cpm/ug DNA) for 24 to 48 h at 42°C. After hybridization the filters were washed for 1 h at 56°C in O.~XSSC, 0.2% sodium dodecyl sulfate. Under these conditions of stringency no cross-hybridization between the N-myc and the cmyc probes was observed, whereas some nonspecific labeling was detected with the pNb1 probe at the level of the 28 S and 18 S rRNAs when total RNAs were used for Northern blot experiments. After washing, the bands hybridizing with the labeled probes were detected by radioautography after l-4 days of exposure and quantified by densitometry. For sequential hybridization of the same filters with various probes, dehybridizations were performed by incubation at 68°C in a solution of 0.1% sodium dodecyl sulfate. Before each subsequent hybridization, radioautographies of the filters were made to ensure the complete elimination of the radioactivity. Southern blot and dot blot assays. High molecular weight DNAs from normal tissues and from tumors were digested to completion with EcoRI and Hind111 restriction endonucleases. DNA fragments were separated by electrophoresis in 0.8% agarose horizontal gels and transferred to nylon filters (Amersham International, Amersham, UK). The Southern blots were prehybridized and hybridized as described for the RNA assays. For the dot blot assays 5 and 1 ug of DNA digested with the EcoRI enzyme and denatured by heating at 90°C in 0.5 N NaOH were spotted on a nylon sheet before prehybridization and hybridization.

RESULTS Expression of N-myc and c-myc in Proliferating Partial Hepatectomy

Hepatocytes after

In order to measure the levels of N-myc and c-myc mRNAs during liver regeneration we prepared total RNAs from hepatocytes from rats sacrificed from 3 to 168 h after the operation. Fifteen micrograms of each RNA was separated by electrophoresis under denaturing conditions, transferred to nitrocellulose filters, and hybridized sequentially to N-myc, c-myc and albumin probes (Fig. 1A). The level of the 3.1-kb N-myc mRNA was verly low but detectable in hepatocytes from normal rats. It was strongly increased 3 h after partial hepatectomy, the earliest time examined. The N-myc mRNA level was increased approximately 20fold but declined at 6 h and remained low for at least 30 h. A second peak of Nmyc mRNA was observed 48 h after the hepatectomy. At this time, the N-myc mRNA level was approximately the same as that at the third hour, whereas 168 h after the operation it had returned to the baseline value. The 2.4-kb c-myc mRNA increased transiently 3 h after partial hepatectomy, and a second peak of c-myc expression was observed 6 h later, in agreement with the recent result of Thompson et al. [22]. The c-myc mRNA level then decreased slowly and was still two to three times higher than that in quiescent hepatocytes 31 h after the partial hepatectomy. Densitometric analysis of the c-myc band indicated that the levels of c-myc mRNA at 3 and 9 h after partial hepatectomy were approximately 25 to 30-fold higher than those in normal hepatocytes. This observation was confirmed in three separate experiments using different rats to

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ALB

C-MYC 2.4

kbe

Fig. I. Steady-state levels of N-myc and c-myc in RNAs from normal and regerating livers and from livers of sham-operated rats. Fifteen micrograms of each total RNA preparation was separated by electrophoresis on formaldehyde-agarose gel, transferred to a Gene Screen Plus filter, and hybridized with the “P-labeled I-kb BamHI-EcoRI fragment of the human N-myc PNbl plasmid or with the 32Plabeled human c-myc third exon probe. The filters were then rehybridized with the 32P-labeled rat serum albumin probe as control. (A) Northern blot analysis of total RNAs from hepatocytes from nonhepatectomized rats (0) and from regenerating hepatocytes at various times after partial hepatectomy. (B) Northern blots of total RNAs from hepatocytes from normal rats (0), rats submitted to anesthesia and sham-operation and sacrificed 3, 6, and 9 h later (S), and rats submitted to partial hepatectomy (R) and sacrificed 3, 6, and 9 h later. Hybridizations were performed with the same probes and the radioautographies were submitted to the same exposure time.

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Fig. 2. Variation of N-myc and c-myc expression during liver regeneration after partial hepatectomy. The level of N-myc and c-myc mRNAs has been measured at various times after partial hepatectomy by densitometric scanning of the bands revealed by the specific probes in Northern blots. The lowest curve shows the level of [‘Hlthymidine incorporation after hepatectomy.

obtain RNA for each measure, in contrast to the fivefold increased expression found by Thompson et al. [22], but close to the observation reported by Makino et al. [13].

Hybridization with a rat albumin cDNA probe is shown in Fig. 1A. Little modification of the 2.1-kb mRNA albumin level was observed during liver regeneration. Thus the biphasic pattern of increase observed for N-myc and cmyc transcripts in regenerating hepatocytes represents a true modification in the abundance of these oncogene messenger RNAs. Total RNAs have been prepared from hepatocytes from sham-operated rats 3, 6, and 9 h after the operation. Figure 1B shows a minor increase of N-myc and c-myc mRNAs. However this increase is negligable as compared to those observed in RNAs from regenerating liver. Figure 2 shows that the expression of N-myc and c-myc occurs a long time before the onset of DNA synthesis ([23, 241 and C. Guguen-Guillouzo, unpublished results) whereas the second peak of N-myc mRNA at 48 h could be related to a second round of hepatocyte replication. Expression of N-myc and c-myc Genes during Neoplastic Growth of Hepatocytes

In order to determine whether N-myc and c-myc expressions are also increased in hepatocytes during neoplastic growth we induced liver carcinogenesis by administration of three doses of DENA to adult rats after partial hepatectomy. In this system no cytotoxic effect of this carcinogen was observed and hyperplastic foci of hepatocyte origin developed in the livers as early as 2 months after DENA administration; the majority of them regressed and only a small number became a-888331

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Fig. 3. Analysis of N-myc and c-myc transcripts in carcinoma nodules. Five micrograms of poly(A)+ RNA preparations was separated by electrophoresis on a formaldehyde-agarose gel and transferred to a GeneScreen Plus filter. RNA blots were first hybridized with “P-labeled N-myc probe (3-5 10’ cpm/ug of DNA). After complete dehydbridization of the filter, hybridizations were performed with the “P-labeled third human c-myc exon (3-5 10’ cpm/ug of DNA). As control the same filters were hybridized with the albumin cDNA probe. No difference in the concentration of poly(A)+ RNA was observed in all the samples (data not shown). Lane 1, RNA from hepatocytes isolated from normal livers; lanes 2, 3, 4, 5, RNAs from DENA-induced carcinoma nodules.

malignant nodules [25, 261. Total hepatocytes were purified from rats sacrificed from 1 to 7 months after DENA administration. In rats sacrificed from 12 to 18 months after the treatment the carcinoma nodules were separated from the nonnodular hepatocytes. Northern blot analyses of poly(A)+ RNAs were performed using the human N-myc clone pNbl and the the third human c-myc exon as probes (Fig. 3). We observed a strong increase in the level of the 3.1-kb N-myc mRNA and of the 2.4-kb c-myc mRNA in carcinoma nodules prepared from rats 18 months after the DENA treatment (Fig. 3, lanes 2 to 5) as compared to the levels found in hepatocytes from nontreated rats (lane 1). Figure 4 shows that N-myc mRNA was increased as early as 1 month after the DENA treatment and a high level was found at 4 months. At this time only hyperplastic foci and nodules were present in the liver. Some minor variations occurred in the intensity of the band corresponding to the N-myc mRNA from several normal rats. In all the cases these variations were small and could not have biological significance. A high level of the 2.4-kb c-myc mRNA was only reached 7-12 months after DENA administration, a time at which most of the nodules present in the liver are very likely to be malignant. These observations were confirmed by two series of similar experiments (data not shown). Hybridizations with a rat albumin cDNA probe indicates that the RNA samples obtained at various times after DENA administration contained similar amounts of albumin messenger RNA. The genomic DNAs from the tumors studied in Fig. 3 and those from normal liver were analyzed by Southern blots and dot blots in order to determine

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-MYC

Fig. 4. Time course of N-myc and c-myc transcript appearance in hepatocytes after DENA administration. Total RNAs were prepared from hepatocytes isolated from 1 to 7 months after carcinogen administration and from carcinoma nodules isolated 12 months after DENA treatment. Fifteen micrograms of each RNA preparation was separated by electrophoresis as described under Materials and Methods. The hybridizations were successively performed with the N-myc, and the albumin probes.

whether the increase of N-myc and c-myc expression in liver tumors induced by DENA was the consequence of gene amplification and/or rearrangement, as it has been reported for other types of tumors [27, 281. We did not find any amplification or modification in the pattern of N-myc and c-myc hybridizations when genomic DNAs from normal and tumor cells were digested with restriction enzymes EcoRI and Hind111 (data not shown). DISCUSSION The N-myc oncogene has been implicated in the formation and the progression of tumors from neuroectodermal origin where it is frequently amplified and expressed at high level [6, 4, 281. A high expression of this gene has also been observed in teratocarcinoma cells, in mouse embryo [29], and in liver from newborn mouse [lo], which suggests a possible biological role in development and in cell differentiation. In this work we show for the first time that the N-myc expression could also be implicated in nontumoral cell growth. Indeed the N-myc transcript is strongly increased within the first hours following partial hepatectomy as observed by us and by others for the c-myc mRNA [12, 131. The increase of N-myc and c-myc transcripts in the first hours after partial hepatectomy might be associated with the entry of quiescent hepatocytes into the cell cycle as a response to growth or humoral factors. The concentration of some hormones such as insuline/glucagon and vasopressine or mitogens like EGF can

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stimulate DNA synthesis in primary culture hepatocytes effectively [3&321. They may increase rapidly in liver as a consequence of tissue loss and may contribute to the entry of the hepatocytes into the cell cycle. The subsequent expression of N-myc and c-myc genes in the stimulated hepatocytes could be implicated into the progression of these cells toward DNA synthesis and cell division. We also reported that the expression of N-myc and c-myc genes is strongly increased in carcinoma nodules of hepatocyte origin. This expression is not the consequence of gene amplification or large chromosome rearrangement. This is the first report of an induction of N-myc expression during hepatocarcinogenesis. N-myc mRNA has been found to be present at a high level in hepatocytes as early as l-2 months after the administration of DENA, a long time before the formation of carcinoma nodules. This period could correspond to the early phases of neoplastic transformation. Furthermore the high expression of the two myc genes in regenerating liver and at the first stages of liver transformation cannot be attributed to some macrophage infiltration, implicated in the inflammatory process; indeed in all our experiments all nonhepatocyte cells of the liver have been discarded. Concerning the mechanism leading to the increase of the N-myc and cmyc mRNA levels, we cannot exclude that it could result from a genetically controlled stabilization of the transcripts rather than an activation of gene expression, as has been shown for the transcripts of c-myc [33, 341 and c-fos 1351. Recently Nisen et al. have reported an enhanced N-myc expression in Wilms tumors, a neoplasm which derives from primitive kidney cells [36]. In this system they have shown that a high expression of N-myc gene was accompanied by a down-regulation of c-myc expression. In this paper we show that during normal and neoplastic liver growths, the kinetics of induction of N-myc and c-myc transcripts are different, suggesting that their expression levels are probably subject to different regulations. However, high amounts of the two gene products were observed sismultaneously 3 h after partial hepatectomy and in carcinoma nodules induced by DENA. This argues against the hypothesis that in rat liver one gene of the myc family could be capable of down-regulating the expression of another myc gene. In conclusion these observations suggest that the N-myc product, in addition to its role in cell differentiation and mammalian development [lo, 281, could play a regulatory role in cell proliferation in normal as well as in tumoral cells, as previously shown for c-fos, c-myb, and c-myc [37-391. This work was financially supported by the Institut National de la Sante et de la Recherche Medicale, the Centre National de la Recherche Scientifique, and the Comitt de Paris de la Ligue Nationale Francaise contre le Cancer. We thank Yu-Chun Lone for useful discussions.

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