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The Expression of the Imprinted Genes H19 and IGF-2 in Choriocarcinoma Cell Lines

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The Expression of the Imprinted Genes H19 and IGF-2 in Choriocarcinoma Cell Lines Oncogene (1997) 15, 169 ± 177 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 The expression of the imprinted genes H19 and IGF-2 in choriocarcinoma cell lines. Is H19 a tumor suppressor gene? O Lustig-Yariv1, E Schulze2, D Komitowski3, V Erdmann4, T Schneider4, N de Groot1 and A Hochberg1 1The Department of Biological Chemistry, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; 2The Zoologisches Institut-Entwicklungs biologie, Gottingen University, Gottingen, Germany; 3The Department of Histodiagnostics & Pathomorphology, DKFZ, Heidelberg, Germany; 4The Department of Chemistry, Institute for Biochemistry, Freie Universitat, Berlin, Germany H19 is a paternally imprinted gene with unknown nearly all precursors of mRNAs). It is bound in 28S function. It is located in close proximity to the cytoplasmic RNPs and not to ribosomes (Brannan et maternally imprinted IGF-2 gene on chromosome al., 1990). Its function is enigmatic. Genetic analysis of 11p15.5. In this study no consistent relationship between the 11p15.5 region revealed a frequent correlation the expression of these two genes in clones derived from between the loss of the maternally inherited 11p15.5 JEG-3 and JAr cell lines could be detected. Nor could a region and many cases of Wilm's tumor (Junien, 1992) consistent relationship be detected between the expres- and some embryonal rhabdomyosarcomas (Scrable et sion levels of these two genes and between certain al., 1990). These correlations and other experimental characteristic tumorigenic properties of these clones. We ®ndings can be explained by assuming the existence of included in this study clones, expressing low H19 levels, a paternally imprinted (maternally expressed) tumor which after transfection with an H19 expression suppressor gene in this chromosomal area. In order to construct highly expressed the H19 gene. In tumors, provide evidence for the proposal that H19 functions formed by the injection of cells of JAr or JEG-3 clones as a tumor suppressor gene, Hao et al. (1993) into nude mice, the H19 expression was high and introduced a construct expressing the H19 gene under irrelevant to the expression level in the cells before the the control of the metallothionein promoter into cells injection. The same phenomenon was found for IGF-2 derived from a kidney tumor. Under conditions of high expression during tumorigenesis caused by cells of H19 expression, the cells, which showed prominent dierent JEG-3 clones and in some but not all JAr morphological changes, grew at a slower rate, had a derived clones. Both H19 and IGF-2 are biallelicly much lower anchorage independent growth rate in soft expressed in all the JAr and JEG-3 clones. In summary, agar, and did not develop tumors when injected into our observations point to the conclusion that H19 is not nude mice as did cells prior to their transfection with a tumor suppressor gene. However, its high expression in the H19 expression construct. However, recently, all the tumors formed after injection of cells of the JAr ®ndings have been reported, which are contradictory and JEG-3 clones, leaves its role, if any, in choriocarci- to the proposed tumor suppressor function for the H19 nogenesis an open question. gene. H19, which is not expressed in normal adult tissues, is expressed in several human cancers such as Keywords: H19; IGF2; choriocarcinoma cell lines choriocarcinoma and placental site trophoblastic tumor (Ariel et al., 1994), bladder carcinoma (Cooper et al., 1996; Elkin et al., 1995), lung cancer (Kondo et al., 1995), breast adeno-carcinoma (Dugimont et al., 1995). Introduction Cervical carcinoma (Douc-Rasy et al., 1996) and esophageal cancer (Hibi et al., 1996). Moreover, in H19 and IGF-2 belong to the family of imprinted tumors formed by the injection of cells of a genes whose expression depends upon their parental choriocarcinoma derived cell line, JEG-3, into nude origin. The two genes are located in close physical mice, the level of H19 RNA was 3 ± 5 times higher than proximity both in human (11p15.5) and mice (distal in the cells before injection (Rachmilewitz et al., 1995). chromosome 7) (Zemel et al., 1992) and are Also, cells of human bladder carcinoma derived cell reciprocally imprinted. The H19 gene is expressed lines which do not express H19 (or express it at a very from the maternally inherited allele and the IGF-2 low level) form tumors in nude mice in which H19 from the paternally derived allele (Rainier et al., 1993). RNA can easily be detected (Elkin et al., 1995). The H19 gene is highly expressed in many embryonic However, all these observations leaves the question tissues but not expressed in neural tissues (Goshen et of the role of H19 in tumorigenesis open. al., 1993; Lustig et al., 1994). H19 expression is down The IGF-2 gene is highly expressed in the embryo regulated postnatally in nearly all tissues. and in some fetal tissues. The tissue-speci®c expression No protein product is known to be produced by the pattern of IGF-2 is very similar to that of the H19 gene H19 gene, nevertheless its primary transcript undergoes (Rappolee et al., 1992; Goshen et al., 1993) and, its processing (capping, polyadenylation and splicing like expression is also postnatally depressed in many adult tissues (Rechler and Nissley, 1990). Prominent excep- Correspondence: N de Groot tions are the adult liver and the choroid plexus which Received 8 November 1996; revised 24 March 1997; accepted 24 continue to express IGF-2 at relatively high levels March 1997 postnatally. It has to be mentioned here that the IFG-2 H19 and IGF-2, in choriocarcinoma cell lines O Lustig-Yariv et al 170 gene can be transcribed from four dierent promoters, and IGF-2 expression, we followed IGF-2 expression three of which are active during fetal life and in cancer in the same JEG-3 and JAr clones. As reported before, tissues and are imprinted (P2, P3, P4). The fourth (P1)) no expression of IGF-2 was detected in cells of the is not imprinted, and is responsible for IGF-2 original JAr cell line (de Groot et al., 1994). Similarly expression in the adult liver and in the choroid plexus in cells of the JAr derived clones IGF-2 expression was (Holthuizen et al., 1993; Robertson, 1995; Ekstrom et not detected. The sole exception was clone JAr-C4 al., 1995). which showed a very low level of expression (data not The relationship between the regulation of IGF-2 shown). It is very interesting to note that no consistent and H19 is a complex one. It has been proposed that in mice H19 and IGF-2 expression are under the control of a common enhancer, which is *3 kb downstream a from the H19 coding region (Sasaki et al., 1992; Ferguson-Smith et al., 1993; Batholomei et al., 1993). JEG-3 C1 C2 C3 C4 C5 C6 This enhancer, or protein(s), bound to the enhancer region, can activate the H19 promoter on the maternally inherited gene or the IGF-2 promoters on the paternally inherited allele because paternally 28S — inherited H19 gene has a more fully methylated promoter which is unable to interact with the H19 downstream enhancer. However, this proposed me- 18S — ¨ H19 chanism can only partly explain the regulation of the H19 and IGF-2 expression. The presence of gene and tissue-speci®c transcription factors is decisive for the actual expression levels of the two genes, and therefore b loss of imprinting of either the H19 or the IGF-2 gene is not necessarily accompanied by an increase in the gene's expression. The relationship between H19 and IGF-2 expression in human diers from that in mouse 28S — (Adam et al., 1996). It has been claimed that in the human both genes can be expressed from the same chromosome (Eversole-Cire et al., 1995; Jinno et al., 18S — 1995). In order to establish more clearly the relationship between the level of H19 and IGF-2 expression and tumorigenicity, we purposely compared the tumori- c genic properties of choriocarcinoma cell line derived clones which express H19 and IGF-2 at dierent levels. However, since we were aware that those clones were still heterogeneous in many other properties, we 28S — introduced an H19 expressing construct into some of the very low H19-expressing clones and looked for phenotypic dierences related exclusively to the change 18S — in H19 expression. Figure 1 Expression of H19 and IGF-2 in JEG-3 derived clones. Results Autoradiogram of Northern blots containing RNA isolated from the original JEG-3 cells and their derived clones (C1 ± C6). The Individual clones were isolated from JAr and JEG-3 positions of 28S and 18S ribosomal RNAs are indicated. (a) original cell cultures. Special care was taken during the Expression of H19 (exposure time 3 h). (b) Expression of IGF-2. (exposure time 72 h). (c) Relative amounts of RNA loaded in cloning procedure in order to ensure that each clone is each lane. Visualized by methylene blue staining of the blot derived from one cell only. (Sambrook et al., 1989) In Figure 1a we show H19 expression in several of the JEG-3 clones. H19 RNA appears as 2.3 kb band corresponding to the sum of the ®ve exons of the H19 gene (Brannan et al., 1990). In the majority of the blots JAr done by us and others a second band of approximately C1 C2 C3 C4 C5 C6 C7 4 kb is visible. The clones C1, C3 and C6 express H19 approximately at the same level as the original culture 28S — from which they were derived, but other clones showed very dierent levels of H19 expression, such as clones ¨ H19 C2 and C5, in which the H19 level was de®nitely lower 18S — and clone C4 which expresses H19 at a signi®cantly higher level.
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