H19 Transcription Research Articles

Prenatal Exposure to Maternal Hyperhomocysteinemia and Allele‐Specific Methylation and Expression of H19/Igf2

Epigenetic processes may play a role in the metabolic programming associated with prenatal exposure to maternal dietary factors. The goal of this study was to determine the effect of prenatal exposure to maternal hyperhomocysteinemia (HHcy) on allele‐specific methylation and expression of genomically imprinted H19 and Igf2. The H19 gene is located in close proximity to Igf2 with expression of H19 and Igf2 regulated by the methylation status of a differentially methylated domain (DMD). Female C57BL/6J mice with (Cbs +/−) and without (Cbs +/+) heterozygous targeted disruption of the gene for cystathionine‐β‐synthase were mated with male Cast/EiJ mice and fed a control or high methionine/low folate diet to induce HHcy (HH) during pregnancy. The F1 hybrid offspring mice were then challenged with the HH diet or control diet from weaning for 1 month. Mice exposed in utero to maternal HHcy and challenged with the HH diet post weaning had higher plasma total homocysteine than mice not exposed in utero to maternal HHcy but challenged with the HH diet post weaning (12.66±2.7 μM vs 6.99±0.9). This was accompanied by lower (p<0.05) methylation of liver H19 DMD on both the maternal and paternal alleles and higher H19 and lower Igf2 transcript levels in liver. These findings show that prenatal exposure to maternal HHcy results in tissue‐specific changes in H19/Igf2 methylation and expression.

The expression profile of the H19 gene in cattle

The expression of the H19 gene has been well studied in fetal human and mouse tissues but not in cattle. It is generally believed that H19 is abundantly expressed in the early stages of embryogenesis and is repressed postnatally. We report the expression pattern of this gene in cattle in a total of 120 fetal-organ combinations and in a total of 108 adult-organ combinations using quantitative real time PCR. In fetal tissues, H19 was abundantly expressed in amnion, chorion, and allantois. Fetal liver, lung, heart, spleen, eye, rib, mammary gland, and cotyledon showed moderate expression, while intestine and brain showed lower expression levels. For some organs examined in this study, the expression pattern in cattle fetal organs was similar to that of human, mouse, and sheep. For adult organs, H19 was highly expressed in muscle and moderately expressed in liver, lung, heart, kidney, pancreas, and ovary. Low expression levels were observed for adult spleen, caruncle, and endometrium. Of considerable interest was the observation that H19 transcripts have not been detected in the mouse and human kidney or in the mouse spleen. In contrast, we observed significant expression levels in adult kidney and low expression levels in adult spleen. In a comparison of adults and fetuses, significant differences in H19 expression levels were found for liver, lung, heart, and spleen. The expression pattern in adults implies that, in addition to possible roles in embryogenesis, the H19 gene may have other functions.

CTCF binding sites promote transcription initiation and prevent DNA methylation on the maternal allele at the imprinted H19/Igf2 locus

Imprinting at the H19/Igf2 locus depends on a differentially methylated domain (DMD) acting as a maternal-specific, methylation-sensitive insulator and a paternal-specific locus of hypermethylation. Four repeats in the DMD bind CTCF on the maternal allele and have been proposed to recruit methylation on the paternal allele. We deleted the four repeats and assayed the effects of the mutation at the endogenous locus. The H19DMD-DeltaR allele can successfully acquire methylation during spermatogenesis and silence paternal H19, indicating that these paternal-specific functions are independent of the CTCF binding sites. Maternal inheritance of the mutations leads to biallelic Igf2 expression, consistent with the loss of a functional insulator. Additionally, we uncovered two previously undescribed roles for the CTCF binding sites. On the mutant allele, H19 RNA is barely detectable in 6.5 d.p.c. embryos and 9.5 d.p.c. placenta, for the first time identifying the repeats as the elements responsible for initiating H19 transcription. Furthermore, methylation is abruptly acquired on the mutant maternal allele after implantation, a time when the embryo is undergoing genome-wide de novo methylation. Together, these experiments show that in addition to being essential for a functional insulator, the CTCF repeats facilitate initiation of H19 expression in the early embryo and are required to maintain the hypomethylated state of the entire DMD.

Complementary roles of genes regulated by two paternally methylated imprinted regions on chromosomes 7 and 12 in mouse placentation

Imprinted genes have prominent effects on placentation; however, there is limited knowledge about the manner in which the genes controlled by two paternally methylated regions on chromosomes 7 and 12 contribute to placentation. In order to clarify the functions of these genes in mouse placentation, we examined transcription levels of the paternally methylated genes, tissue differentiation and development and the circulatory system in placentae derived from three types of bi-maternal conceptuses that contained genomes of non-growing (ng) and fully grown (fg) oocytes. The genetic backgrounds of the ng oocytes were as follows: one was derived from the wild-type (ngWT) and another from mutant mice carrying a 13 kb deletion in the H19 transcription unit including the germline-derived differentially methylated region (H19-DMR) on chromosome 7 (ngDeltach7). Another set of oocytes was derived from mutant mice carrying a 4.15 kb deletion in the intergenic germline-derived DMR (IG-DMR) on chromosome 12 (ngDeltach12). Although placental mass was lower in the ngWT/fg placentae compared with that in the WT placentae, it was recovered in the ngDeltach7/fg placentae, but not in the ngDeltach12/fg placentae. The ngDeltach7/fg placental growth improvement was associated with severe dysplasia such as an expanded spongiotrophoblast layer and a malformed labyrinthine zone. In contrast, the ngDeltach12/fg placentae retained the layer structures with expanded giant cells, but their total masses were smaller with a normal circulatory system in order. Our findings demonstrate that the genes controlled by the two paternally methylated regions, H19-DMR and IG-DMR, complementarily organize placentation.

979: DNA Based Therapy Using the H19 Transcription Regulatory Sequence in Bladder Cancer Models

979: DNA Based Therapy Using the H19 Transcription Regulatory Sequence in Bladder Cancer Models

Role of H19 3′ sequences in controlling H19 and Igf2 imprinting and expression

The regulation of H19 and Igf2 imprinting and expression depends on common elements. Using comparative analysis between human and mouse, we identified conserved regions 3′ of the H19 transcription unit, including the H19/ Igf2 endodermal enhancers and elements within a 4.2-kb domain between the H19 transcription unit and the enhancers. Transgene experiments implicate these elements in imprinting regulation. To establish whether they are required at the endogenous locus, first we replaced the endodermal enhancers with the α -fetoprotein endodermal enhancers ( H19 Afp ). Second, we deleted the 4.2-kb region ( H19 Δ 4.2 ). Our analysis revealed that H19 and Igf2 imprinting and tissue-specific expression were maintained for both mutations, except for a slight reduction in paternal Igf2 expression from the H19 Afp allele in liver. These results demonstrate that the H19 insulator can interact with heterologous enhancers to imprint Igf2. Furthermore, for H19, chromatin context or additional sequences possibly compensate for loss of conserved 3′ elements.

Overexpression of an ectopic H19 gene enhances the tumorigenic properties of breast cancer cells.

The maternally expressed H19 gene is transcribed as an untranslated RNA that serves as a riboregulator. We have previously reported that this transcript accumulates in epithelial cells in approximately 10% of breast cancers. To gain further insight on how the overexpression of the H19 gene affects the phenotype of human breast epithelial cells, we investigated the oncogenic potential of RNA that was abundantly expressed from MDA-MB-231 breast cancer cells stably transfected with the genomic sequence of the human H19 gene. The amount of H19 RNA did not affect cell proliferation capacity, timing of cell cycle phases or anchorage-dependent ability of H19-transfected clones in vitro. But in anchorage-independent growth assays the H19-recombined cells formed more and larger colonies in soft-agar versus control cells. To explore this phenotypic change, we analysed tumour development after subcutaneous injection of H19-recombined cells into scid mice. Results showed that H19 overexpression promotes tumour progression. These data support the hypothesis that an overload of H19 transcript is associated with cells exhibiting higher tumorigenic phenotypes and therefore we conclude that the H19 gene has oncogenic properties in breast epithelial cells.

Mouse Parthenogenetic Embryos with Monoallelic H19 Expression Can Develop to Day 17.5 of Gestation

In mammals, both maternal and paternal genomes are required for a fetus to develop normally to term. This requirement is due to the epigenetic modification of genomes during gametogenesis, which leads to an unequivalent expression of imprinted genes between parental alleles. Parthenogenetic mouse embryos that contain genomes from nongrowing (ng) and fully grown (fg) oocytes can develop into 13.5-day-old fetuses, in which paternally and maternally expressed imprinted genes are expressed and repressed, respectively, from the ng oocyte allele. The H19 gene, however, is biallelically expressed with the silent status Igf2 in such parthenotes. In this study, we examined whether the regulation of H19 monoallelic expression enhances the survival of parthenogenetic embryos. The results clearly show that the ngH19-KO/fgwt parthenogenetic embryos carrying the ng-oocyte genome that had been deleted by the H19 transcription unit successfully developed as live fetuses for 17.5 gestation days. Control experiments revealed that this unique phenomenon occurs irrespective of the genetic background effect. Quantitative gene expression analysis showed that day 12.5 ngH19-KO/fgwt parthenogenetic fetuses expressed Igf2 and H19 genes at <2 and 82% of the levels in the controls. Histological analysis demonstrated that the placenta of ngH19-KO/fgwt parthenotes was afflicted with atrophia with severe necrosis and other anomalies. The present results suggest that the cessation of H19 gene expression from the ng-allele causes extended development of the fetus and that functional defects in the placenta could be fatal for the ontogeny.

Elevated IGF-II mRNA and phosphorylation of 4E-BP1 and p70(S6k) in muscle showing clenbuterol-induced anabolism.

Muscle wasting affects large numbers of people, but few therapeutic approaches exist to treat and/or reverse this condition. The beta(2)-adrenoceptor agonist clenbuterol produces a muscle-specific protein anabolism in both normal and catabolic muscle and has been used to limit muscle wasting in humans. Because clenbuterol appears to interact with or mimic innervation, its effect on the expression of the neurotrophic agents insulin-like growth factor (IGF)-II and H19 and their putative pathways was examined in normal rat plantaris muscle. The results showed that the well-documented early effects of clenbuterol on protein metabolism were preceded by elevated levels of IGF-II and H19 transcripts together with increased phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1) and p70(S6k). By 3 days, transcript levels for IGF-II and H19 and 4E-BP1 and p70(S6k) phosphorylation had returned to control values. These novel findings indicate that clenbuterol-induced muscle anabolism is potentially mediated, at least in part, by an IGF-II-induced activation of 4E-BP1 and p70(S6k).

Evidence for evolutionarily conserved secondary structure in the H19 tumor suppressor RNA.

The molecular basis for function of the mammalian H19 as a tumor suppressor is poorly understood. Large, conserved open reading frames (ORFs) are absent from both the human and mouse cDNAs, suggesting that it may act as an RNA. Contradicting earlier reports, however, recent studies have shown that the H19 transcript exists in polysomal form and is likely translated. To distinguish between possible functional roles for the gene product, we have characterized the sequence requirements for H19-mediated in vitro suppression of tumor cell clonogenicity and analyzed the sequence of the gene cloned from a range of mammals. A cDNA version of the human gene, lacking the unusually short introns characteristic of imprinted genes, is as effective as a genomic copy in blocking anchorage-independent growth by G401 cells. The first 710 nucleotides of the gene can be deleted with no effect on in vitro activity. Further truncations from either the 5'- or 3'-end, however, cause a loss of suppression of clonogenicity. Using conserved sequences within the H19 gene as PCR primers, genomic DNA fragments were amplified from a range of mammalian species that span the functional domain defined by deletion analysis. Sequences from cat, lynx, elephant, gopher and orangutan complement the previous database of sequences from human, mouse, rat and rabbit. Hypothetical translation of the resulting sequences shows an absence of conserved ORFs of any size. Free energy and covariational analysis of the RNA sequences was used to identify potential helical pairings within the H19 transcript. A set of 16 helices are supported by covariation (i.e. conservation of base pairing potential in the absence of primary sequence conservation). The predicted RNA pairings consist largely of local hairpins but also include several long range interactions that bridge the 5'- and 3'-ends of the functional domain. Given the evolutionary conservation of structure at the RNA level and the absence of conservation at the protein level, we presume that the functional product of the H19 gene is a structured RNA.

Symmetric and Asymmetric DNA Methylation in the Human IGF2–H19 Imprinted Region

The two contiguous IGF2 (human insulin-like growth factor II) and H19 genes are reciprocally imprinted in both human and mouse. In most tissues, IGF2 is transcribed only from the paternal chromosome while H19 is transcribed only from the maternal allele. The presence of a differential methylation region (DMR) on the two parental alleles at the 5′ flanking region of H19 has been proposed to constitute the gametic imprint, which controls the reciprocal allelic expression of the two genes. Using bisulfite genomic sequencing, we have assessed the methylation status of cytosine (including 154 CpG sites) in six CpG-rich regions of the human IGF2–H19 genes. In a CpG island near promoter P3 of the IGF2 gene, more than 99.8% of all cytosines were converted to thymidine by sodium bisulfite mutagenesis, indicating that none of the CpGs was methylated. In the IGF2 exon 8–9 region, mosaic methylation of 56 CpG sites was observed in fetal tissues and in adult blood DNA. In contrast to the mosaic methylation of IGF2, the allelic methylation of the human H19 DMR was uniform. In the CpG region located 2 kb upstream (−2362 to −1911) of the H19 transcription site, all 25 CpG sites were completely methylated on only one parental allele. Uniform allele-specific methylation was also observed in the CpG island proximal to the H19 promoter (−711 to −290) with complete methylation of all 25 CpG sites in one parental allele. In contrast, the CpG region in the H19 promoter (−292 to +15) was mosaically methylated in all tissues. In addition, cytosine was methylated at three CpNpG and GpNpC sites on the top DNA strand and one CpNpG site on the bottom DNA strand from the fetal brain. The cytosines at CpG sites were methylated on both DNA strands (symmetric methylation) while cytosines at the CpNpG and GpNpC sites were methylated on only one DNA strand (asymmetric methylation). The asymmetric methylation was associated with tissue-specific disruption of H19 genomic imprinting in fetal brain.

Biallelic transcription of Igf2 and H19 in individual cells suggests a post-transcriptional contribution to genomic imprinting

The H19 and insulin-like growth factor 2 (Igf2) genes in the mouse are models for genomic imprinting during development. The genes are located only 90 kb apart in the same transcriptional orientation [1], but are reciprocally imprinted: Igf2 is paternally expressed while H19 is maternally expressed. It has been suggested that expression of H19 and repression of Igf2 (or the converse) on a given chromosome are mechanistically linked and that the parental imprint operates at the level of transcription [2]. Although expression of Igf2 and H19 is thought to be monoallelic, the data have so far been obtained exclusively by looking at steady-state RNA levels using techniques that reflect the average activity of the genes in a cell population [3,4]. Here, we have adapted a fluorescent in situ hybridisation (FISH) method to detect nascent RNA molecules of Igf2 and H19 at the initial transcription sites in the nuclei of wild-type mouse embryonic liver cells. Nine different transcription patterns were observed, reflecting a high heterogeneity of transcription at the single-cell level. Our observations suggest that regulation of Igf2 and H19 by parental imprinting is much more complex than previously proposed and acts at both transcriptional and post-transcriptional levels.

Methylation Sequencing Analysis Refines the Region ofH19 Epimutation in Wilms Tumor

Differential DNA methylation of the parental alleles has been implicated in the establishment and maintenance of the monoallelic expression of imprinted genes. H19 and IGF2 are oppositely imprinted with only the maternal and the paternal alleles expressed, respectively. In Wilms tumor, a childhood renal neoplasm, loss of the H19/IGF2 imprinted expression pattern results in silencing of H19 and biallelic expression of IGF2. This was shown to be associated with biallelic methylation of the H19 promoter in the tumor and the adjacent kidney tissue suggesting that epigenetic H19 silencing is an early event in Wilms tumorigenesis. An imprinting mark region characterized by paternal allele-specific methylation has been suggested to reside in a GC-rich region of 400-base pair direct repeats starting at -2 kilobase pairs (kb) relative to the H19 transcription start and extending upstream. The upstream boundary of the potential paternal methylation imprint of the H19 gene has yet to be defined. We sought to define this upstream imprint boundary and investigate whether Wilms tumors with loss of imprinting are biallelically methylated in this imprinting mark region. The analysis of 6.6 kb of new upstream H19 sequence determined in this study identified a series of the direct 400-base pair repeats that extends to approximately -5.3 kb relative to the transcription start. DNA methylation analyses indicated that the upstream boundary of the potential imprint may coincide with the 5' end of the direct repeats. We found that Wilms tumors with loss of imprinting are biallelically methylated in the H19 upstream repeat region, and we suggest that pathological methylation in this region is the epigenetic error that initiates H19 silencing.

Genomic Imprinting of IGF-II and H19 in Adult Human Pancreatic Tissues

Background/Aim: Genomic imprinting is a chromosomal modification causing differential expression of maternal and paternal alleles. Loss of imprinting (LOI) of IGF-II and H19 has been suggested to be an early oncogenic event in cancerogenesis. Aim of the present study was to describe the status of IGF-II and H19 imprinting in adult human pancreatic tissues. Methods: Allele-specific gene expression was studied using RNA and DNA from human pancreatic cancer, chronic pancreatitis, and normal pancreas tissues heterozygous for ApaI (IGF-II) or RsaI (H19) restriction fragment length polymorphism. Reverse-transcriptase polymerase chain reaction products were digested with either ApaI or RsaI and analyzed on agarose gels to study the status of allelic expression. The expression level of H19 and IGF-II was studied on Northern blots or by polymerase chain reaction. Results: H19 was imprinted in normal pancreas and in chronic pancreatitis. H19 LOI was observed in 1 of 4 informative cancer tissues and was not associated with increased H19 transcript levels. Biallelic expression of IGF-II was found in 6 of 10 informative cancer tissues and in 6 of 9 informative normal tissues. In chronic pancreatitis, the IGF-II gene was imprinted in all informative samples. IGF-II mRNA was not overexpressed in the tissues showing LOI. Conclusion: Low frequencies of H19 LOI and the lack of correlation between biallelic expression and overexpression observed for both H19 and IGF-II suggest that LOI of H19 and IGF-II is not a relevant oncogenic factor during human exocrine pancreatic cancerogenesis.

Steroid hormones modulate H19 gene expression in both mammary gland and uterus.

H19 is an imprinted and developmentally regulated gene whose product remains apparently untranslated. In a previous study on breast adenocarcinomas, we reported that overexpression of the H19 gene was significantly correlated with the presence of steroid receptors, suggesting the putative role of hormones in H19 transcription. To determine the mode of steroid action, we have detected levels of H19 RNA synthesis during mammary gland development by in situ hybridization (ISH): two peaks of H19 transcription occur during puberty and pregnancy. Furthermore, we demonstrated by ISH that in the uterus H19 RNA synthesis is high during estrus and metestrus phases. To test steroid control of H19 transcription, ovariectomized and adrenalectomized mice were supplemented, 1 week after surgery, with 17-beta-estradiol (E2, 20 microg/kg/day), progesterone (P, 1 mg/kg/day) or corticosterone (B, 0.3 mg/ kg/day) for 2 weeks. According to ISH data, E2 and to a lesser extent B stimulated H19 transcription in the uterus, whereas P inhibited it. To confirm the in vivo results, in vitro experiments were performed using cultures of MCF-7 cells (a hormone-sensitive mammary cell line). E2 stimulated the endogenous H19 gene of this cell line and tamoxifen inhibited this effect. Furthermore, we performed transient cotransfections in MCF-7, in HBL-100 (another hormone-sensitive mammary cell line) and in BT-20 (a hormone-insensitive mammary cell line) with various constructs of ERalpha (WT or mutated) and PR-A, in presence or absence of steroid hormones. We demonstrated that ERalpha up-regulated the H19 promoter in MCF-7 and in HBL-100, whereas PR-A did not have any effect per se. Moreover, in MCF-7, PR-A antagonized clearly the ERalpha-mediated promoter enhancement, but in HBL-100 this counteracting effect on the ERalpha up-regulation was not found. Interestingly, the same experiments performed in BT-20 cell line provided very similar results as those obtained in MCF-7 cells, with a clear down-regulation mediated by PR-A on the H19 promoter. All these in vitro data are in agreement with in vivo results. In addition, data obtained with ERalpha mutants indicate that H19 promoter activation is both ligand-dependent and ligand-independent. We have thus demonstrated that H19 gene expression is controlled by steroid hormones; furthermore, this gene is highly expressed in hormone-sensitive organs when the hormonal stimulation is accompanied with a morphological repair.

The genotype and epigenotype synergize to diversify the spatial pattern of expression of the imprinted H19 gene

Little is known of how the genetic background effects the phenomenon of genomic imprinting. The H19 gene belongs to a cluster of imprinted genes on human chromosome 11. Here we show that the alternative splicing of a human H19 transcript is genotype-specific. Moreover, this variant transcript, which lacks exon 4, is either not found at all, is widely expressed or is confined to extra-villous cytotrophoblasts in first trimester placenta, depending on a combination of the genotype and the sex of the transmitting parent.

The H19 Transcript Is Associated with Polysomes and May Regulate IGF2 Expression in trans

The imprinted H19 gene produces a fully processed transcript that does not exhibit any conserved open reading frame between mouse and man. Although transcriptional control elements associated with the mouse H19 locus have been shown to control the neighboring Igf2 gene in cis, the prevailing view is that the cytoplasmic H19 transcript does not display any function. In contrast to earlier reports, we show here that the H19 transcript is associated with polysomes in a variety of cell types, in both mouse and man. A possible trans-function of the H19 gene is suggested by a reciprocal correlation in trans between cytoplasmic H19 and IGF2 mRNA levels, as well as IGF2 mRNA translatability. We discuss these results in terms of their challenge to the prevailing dogma on the function of the enigmatic H19 gene, as well as with respect to the ontogeny of the Beckwith-Wiedemann syndrome, and propose that the human H19 gene is an antagonist of IGF2 expressivity in trans.

H19 and Igf2 are expressed and differentially imprinted in neuroectoderm-derived cells in the mouse brain.

Igf2 and H19 are reciprocally imprinted genes that are closely linked and coexpressed in tissues of mesodermal and endodermal origin. Here we report that coexpression of these genes is also found in specific fetal tissues of neuroectodermal origin, that is in the ventral midline region of both the hindbrain and spinal cord. For cells of neuroectodermal origin, complete absence of Igf2 and H19 transcription was previously described. Analysis of allele-specific expression of both Igf2 and H19 in the ventral midline region of the hindbrain shows that H19 is expressed monoallelically, with the paternal allele being silent, whereas Igf2 is expressed biallelically. Furthermore, we observed a strong influence of the parental species background, in that the Mus musculus allele was always expressed at higher levels than the M. spretus allele. This was observed when the M. spretus allele was contributed by the mother or by the father. An analysis of Igf2 methylation by bisulphite genomic sequencing provided no clear answer as to whether Igf2 expression and methylation are linked in a tissue of neuroectodermal origin. Taken together, our results provide novel information on H19 and Igf2 expression and imprinting patterns in the fetal mouse brain. In addition, they indicate that some aspects of Igf2 regulation in cells of neuroectodermal origin do not follow the pattern that exists in mesoderm- and endoderm-derived tissues. Apart from the ventral midline region, H19 and Igf2 were found to be coexpressed in the ectodermally derived Rathke's pouch and in some circumventricular organs of the brain, such as the organum vasculosum of the lamina terminalis (OVLT) and the pineal gland.

The H19 TATA-less promoter is efficiently repressed by wild-type tumor suppressor gene product p53.

The developmentally regulated H19 gene displays several remarkable properties: expression of an apparently non-translated mRNA, genomic imprinting (maternal allele only expressed), relaxation of the imprinting and/or epigenetic lesions demonstrated in some tumors. Despite several observations after relaxation of imprinting status of the gene, data on trans and cis-acting factors required for the human H19 gene expression are still missing. As a first approach to address identification of factors involved in the regulation of the gene, we found that cells from a p53 antisense-transfected HeLa clone displayed increased amounts of H19 transcripts when compared to the non-transfected cells. Moreover, a HeLa clone stably transfected with a temperature sensitive (ts) 143 Ala p53 mutant exhibited temperature-dependent regulation of H19 expression. This preliminary indication of the repressing effect of the p53 protein on H19 expression has been confirmed by transient cotransfection experiments in HeLa cells, using luciferase surrogate constructs under the control of the 823 bp sequence immediately upstream of the transcription start point of the H19 gene, and different constructs containing sense, antisense or a ts 143 Ala mutant p53 cDNA. We observed an increase of H19 promoter-driven activity in transient cotransfections with the antisense p53 cDNA and the temperature sensitive mutant p53 at the non-permissive temperature, but a decrease with sense wild-type p53 cDNA. Furthermore, the cotransfection experiments were repeated in a cell line lacking endogenous p53. (Calu 6 cells) and the results provided additional evidence for a down regulation of the expression of the H19 gene by the p53 protein.

In situ detection of insulin-like growth factor II (IGF2) and H19 gene expression in hepatocellular carcinoma.

To assess the relationship between insulin-like growth factor II (IGF2) and H19 gene expression at the cellular level, we have examined the distribution of IGF2 and H19 mRNA by means of an situ hybridization in hepatic malignancies consisting of hepatocellular carcinoma (HCC), cholangiocellular carcinoma (CCC), and metastatic liver cancer (MLC). In HCC, 15 of 27 tumors (56%) and 11 of 27 tumors (41%) demonstrated increased IGF2 and H19 gene expression, respectively. Of 16 HCCs with increased expression of either IGF2 or H19, 10 tumors coexpressed both transcripts at comparable levels. Moreover, the spatiotemporal distribution and the cellular localization of the two gene transcripts were almost identical, suggesting the presence of a reciprocal relation between IGF2 and H19. In addition, 5 HCCs showed increased IGF2 expression without concomitant H19 expression, whereas 1 HCC showed increased H19 expression without IGF2 transcripts. However, 11 HCCs showed no IGF2 or H19 expression. On the other hand, neither IGF2 transcripts nor H19 transcripts were detected in 2 CCCs or 10 MLCs studied. The data suggest that IGF2 and/or H19 gene expression may be characteristic of some HCCs.