Insulin-like Growth factor-II Gene Expression Research Articles

Rapamycin Suppresses the Expansion and Differentiation of Porcine Neonatal Pancreas Cell Clusters

The role of rapamycin in pancreas stem cells remains to be clearly elucidated. Herein, we evaluated the effects of rapamycin on porcine neonatal pancreas cell clusters (NPCCs), which primarily comprised pancreatic precursors, and attempted to find an intracellular mechanism about the harmful effects of rapamycin. Porcine NPCCs were treated with rapamycin in a monolayer, and the apoptosis and proliferation were determined via caspase-3 assay and H-thymidine uptake analysis. The expression of transcription factors was assessed via reverse-transcriptase polymerase chain reaction and Western blotting. For the in vivo study, the porcine NPCCs were transplanted into the kidney subcapsules of normal nude mice and treated with rapamycin. Rapamycin treatment significantly reduced the number of β cells, glucose-stimulated insulin secretion, and the insulin contents in the monolayer-cultured porcine NPCCs. Furthermore, rapamycin treatment increased the apoptosis and inhibited the proliferation of β cells in the culture dishes. The expressions of the insulin, pancreatic and duodenal homeobox-1, and NeuroD/Beta2 genes were down-regulated via rapamycin treatment. The expression of insulin-like growth factor-II was significantly down-regulated, but the expression of Foxo1 was simultaneously inversely increased, and the translocation of Foxo1 from the cytoplasm to the nucleus was induced by rapamycin treatment. Moreover, rapamycin treatment induced a marked reduction in the relative volume and absolute mass of β cells in the porcine NPCCs grafts at 8 weeks after transplantation in the normal nude mice. Here, we demonstrate that rapamycin treatment suppresses the expansion and differentiation of porcine NPCCs, and the alteration of Foxo1 and insulin-like growth factor-II gene expression might be the crucial factors.

CCAAT/Enhancer Binding Protein β2 Is Involved in Growth Hormone-Regulated Insulin-Like Growth Factor-II Gene Expression in the Liver of Rainbow Trout (Oncorhynchus mykiss)

Previously, we showed that levels of different CCAAT/enhancer binding protein (C/EBP) mRNAs in the liver of rainbow trout were modulated by GH and suggested that C/EBPs might be involved in GH-induced IGF-II gene expression. As a step toward further investigation, we have developed monospecific polyclonal antibodies to detect rainbow trout C/EBPalpha, -beta1, -beta2, and -delta2 isoform proteins. Injection of GH into adult rainbow trout resulted in a significant increase of C/EBPbeta1, C/EBPbeta2, and C/EBPdelta2 proteins in the liver. Chromatin immunoprecipitation analysis revealed that C/EBPbeta2 binds to multiple sites at the 5' promoter/regulatory region, introns, and the 3' untranslated region of the IGF-II gene. GH treatment reduced C/EBPbeta2 binding to several of these regions at 6 h after injection. The decreased occupancy of C/EBPbeta2 coincided well with an increase of histone H4 acetylation at the proximal promoter and elevation of the IGF-II mRNA level. Immunoblotting analysis showed that C/EBPbeta2 existed predominately as a truncated form in the liver, and cotransfection analysis further showed that the truncated C/EBPbeta2 acted as a negative regulator on IGF-II proximal promoter. GH treatment caused deacetylation of C/EBPbeta2 in the liver. In addition, we observed a GH-dependent interaction of C/EBPbeta2 with a complex involving histone H1. All together, these results suggest that C/EBPbeta2 was regulated at multiple levels by GH, and C/EBPbeta2 may play a suppressive role in mediating GH-induced IGF-II expression in the liver of rainbow trout.

Insulin-Like Growth Factor-II Is Increased in Systemic Sclerosis-Associated Pulmonary Fibrosis and Contributes to the Fibrotic Process via Jun N-Terminal Kinase- and Phosphatidylinositol-3 Kinase-Dependent Pathways

Systemic sclerosis (SSc)-related pulmonary fibrosis, for which there are few effective therapies, is the most common cause of SSc-related mortality. We examined insulin-like growth factor (IGF)-II expression in explanted lung tissues from control and SSc patients to determine its role in the pathogenesis of fibrosis. IGF-II levels in vivo were detected using immunohistochemistry. Primary lung fibroblasts were cultured from lung tissues, and IGF-II mRNA was measured using reverse transcriptase-polymerase chain reaction. Western blot analysis measured extracellular matrix (ECM) production and phosphorylated signaling molecules. Immunostaining revealed increased IGF-II expression in fibroblastic foci of SSc lungs. Furthermore, primary SSc lung fibroblasts had a fourfold increase in IGF-II mRNA and a twofold increase in IGF-II protein compared with normal lung fibroblasts. IGF-II mRNA in SSc lung fibroblasts was expressed primarily from the P3 promoter of the IGF-II gene, and IGF-II induced both a dose- and time-dependent increase in collagen type I and fibronectin production. IGF-II triggered the activation of both phosphatidylinositol-3 kinase and Jun N-terminal kinase signaling cascades, the inhibition of which diminished IGF-II-induced ECM production. Our study demonstrates increased local IGF-II expression in SSc-associated pulmonary fibrosis both in vitro and in vivo as well as IGF-II-induced ECM production through both phosphatidylinositol-3 kinase- and Jun N-terminal kinase-dependent pathways. Our results provide novel insights into the role of IGF-II in the pathogenesis of SSc-associated pulmonary fibrosis.

The RNA-binding Protein IMP-3 Is a Translational Activator of Insulin-like Growth Factor II Leader-3 mRNA during Proliferation of Human K562 Leukemia Cells

IMP-3, a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein (IMP) family, is expressed mainly during embryonic development and in some tumors. Thus, IMP-3 is considered to be an oncofetal protein. The functional significance of IMP-3 is not clear. To identify the functions of IMP-3 in target gene expression and cell proliferation, RNA interference was employed to knock down IMP-3 expression. Using human K562 leukemia cells as a model, we show that IMP-3 protein associates with IGF-II leader-3 and leader-4 mRNAs and H19 RNA but not c-myc and beta-actin mRNAs in vivo by messenger ribonucleoprotein immunoprecipitation analyses. IMP-3 knock down significantly decreased levels of intracellular and secreted IGF-II without affecting IGF-II leader-3, leader-4, c-myc, or beta-actin mRNA levels and H19 RNA levels compared with the negative control siRNA treatment. Moreover, IMP-3 knock down specifically suppressed translation of chimeric IGF-II leader-3/luciferase mRNA without altering reporter mRNA levels. Together, these results suggest that IMP-3 knock down reduced IGF-II expression by inhibiting translation of IGF-II mRNA. IMP-3 knock down also markedly inhibited cell proliferation. The addition of recombinant human IGF-II peptide to these cells restored cell proliferation rates to normal. IMP-3 and IMP-1, two members of the IMP family with significant structural similarity, appear to have some distinct RNA targets and functions in K562 cells. Thus, we have identified IMP-3 as a translational activator of IGF-II leader-3 mRNA. IMP-3 plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells.

Targeted Knockdown of the RNA-binding Protein CRD-BP Promotes Cell Proliferation via an Insulin-like Growth Factor II-dependent Pathway in Human K562 Leukemia Cells

The c-myc mRNA coding region determinant-binding protein (CRD-BP) was first identified as a masking protein that stabilizes c-myc mRNA in a cell-free mRNA degradation system. Thus, CRD-BP is thought to promote cell proliferation by maintaining c-Myc at critical levels. CRD-BP also appears to be an oncofetal protein, based upon its expression during mammalian development and in some tumors. By using K562 leukemia cells as a model, we show that CRD-BP gene silencing by RNA interference significantly promoted proliferation, indicating an inhibitory effect of CRD-BP on proliferation. Unexpectedly, CRD-BP knockdown had no discernible effect on c-myc mRNA levels. CRD-BP is also known as insulin-like growth factor II (IGF-II) mRNA-binding protein-1. It has been reported to repress translation of a luciferase reporter mRNA containing an IGF-II 5'-untranslated region known as leader 3 but not one containing IGF-II leader 4. CRD-BP knockdown markedly increased IGF-II mRNA and protein levels but did not alter translation of luciferase reporter mRNAs containing 5'-untranslated regions consisting of either IGF-II leader 3 or leader 4. Addition of antibody against IGF-II to cell cultures inhibited the proliferative effect of CRD-BP knockdown, suggesting that regulation of IGF-II gene expression, rather than c-myc mRNA levels, mediates the proliferative effect of CRD-BP knockdown. Thus, we have identified a dominant function for CRD-BP in cell proliferation of human K562 cells, involving a possible IGF-II-dependent mechanism that appears independent of its ability to serve as a c-myc mRNA masking protein.

Loss of imprinting of IGF-II gene in children with acute lymphoblastic leukemia

Insulin-like growth factor-II (IGF-II) is known to be involved in the regulation of growth, differentiation and cell death in normal human tissues. In a variety of human tumors, the IGF-II gene is overexpressed and considered to be a stimulator for tumor growth through autocrine and paracrine mechanisms. The IGF-II gene is normally parental imprinted, only the paternal allele being expressed in most tissues. Several reports about biallelic expression (loss of imprinting (LOI)) of the IGF-II gene in different tumors suggest a role of dysregulation of IGF-II imprinting in tumorigenesis. However, biallelic expression of IGF-II gene has also been reported in different tissues of a significant number of normal controls, indicating either a normal phenomenon or an elevated cancer risk in this group of persons. Although LOI of IGF-II presumably promotes tumorigenesis by increasing IGF-II expression, elevated IGF-II levels in those patients have not been reported. We studied IGF-II gene expression in malignant lymphoblasts of 124 children suffering from acute lymphoblastic leukemia, 196 cord blood samples from healthy newborns and mononuclear cells (MNC) from 50 healthy age matched children. The ApaI polymorphism in exon 9 of the IGF-II gene and allele-specific exon-connection RT-PCR was used for determination of the imprinting status. From 44 informative ALL-patients, 24 (54%) showed LOI of the IGF-II gene. Twenty percent of the informative cord blood samples ( N=56) and 14% of the informative MNC samples from healthy controls ( N=22) showed biallelic expression of IGF-II. In the ALL-patients, no statistical significant correlation between LOI patients and relapse rate, surviving rate and risk groups could be detected. We conclude that LOI of IGF-II occurs in malignant lymphoblasts of children suffering from acute lymphoblastic leukemia in more than 50% of the patients. In MNC from cord blood and peripheral MNC from healthy controls, biallelic expression could be detected in up to 20% of all cases. The importance of LOI in ALL-patients needs to be further evaluated to determine its impact in leukemogenesis.

Relaxin causes proliferation of human amniotic epithelium by stimulation of insulin-like growth factor-II

Objective: The study was conducted to determine whether relaxin has a proliferative effect on amniotic epithelial cells and to show that this effect is caused by its stimulation of the insulin-like growth factor-II (IGF-II) gene. Study Design: Immunolocalization and Northern analysis were used to confirm the expression of IGF-II by the fetal cells in the membranes. Human amniotic epithelial (WISH) cells were treated with doses of IGF-II or human relaxin and their proliferative effects measured. The mechanism of the effect of relaxin on cellular proliferation was studied with the use of an IGF-II-blocking antibody and Northern analysis for IGF-II gene expression after treatment with relaxin. An in vivo correlate was sought by quantitation of relaxin gene expression in 10 fetal membranes from women with normally grown and large for gestational age infants. Results: The amniotic epithelial and cytotrophoblast cells of the fetal membranes expressed IGF-II, as did the amniotic epithelial-like (WISH) cell line. Treatment of WISH cells with IGF-II or relaxin caused a significant (P < .03) and dose-related increase in WISH cell proliferation over 5 days. The concurrent treatment with a blocking antibody to IGF-II significantly decreased the proliferative response to IGF-II (P < .002) and relaxin (P < .002). Treatment with relaxin caused a significant increase (P < .003) in the transcription of IGF-II in 24 hours. In fetal membranes, the levels of relaxin gene expression correlated with fetal membrane surface area (r = 0.76) and was significantly greater (P < .008) in the membranes from macrosomic infants (4020-4729 g) compared with those normally grown (2855-3830 g). Conclusion: IGF-II and relaxin both caused the proliferation of WISH cells. Concurrent treatment with an IGF-II-blocking antibody abrogated the proliferative effects of both hormones. Relaxin increased the transcription of IGF-II, and its expression levels in the fetal membranes correlated with the membrane surface area as well as neonatal birth weight. These data suggest that relaxin is a growth factor for the fetal membranes. Am J Obstet Gynecol 2003;188:234-41.

Kinetics and regulation of site-specific endonucleolytic cleavage of human IGF-II mRNAs.

Human insulin-like growth factor II (IGF-II) mRNA can be cleaved at a specific site in its 4 kb long 3'-UTR. This yields a stable 3' cleavage product of 1.8 kb consisting of a 3'-UTR and a poly(A) tail and an unstable 5' cleavage product containing the IGF-II coding region. After cleavage, the 5' cleavage product is targeted to rapid degradation and consequently is no longer involved in IGF-II protein synthesis. Cleavage is therefore thought to provide an additional way to control IGF-II gene expression. In this paper the kinetics and the efficiency of cleavage of IGF-II mRNAs are examined. The cleavage efficiency of IGF-II mRNAs carrying four different leaders (L1-L4) is enhanced in the highly structured leaders L1 and L3. Additionally, under standard cell culture conditions cleavage is a slow process that only plays a limited role in destabilisation and translation of the IGF-II mRNAs. However, in human Hep3B cells and CaCo2 cells which express IGF-II endogenously, cleavage is upregulated 3-5-fold at high cell densities. Regulated endonucleolytic cleavage of IGF-II mRNAs is restricted to cells in which IGF-II expression is related to specific cell processes.

Hepatitis C virus core protein transactivates insulin-like growth factor II gene transcription through acting concurrently on Egr1 and Sp1 sites.

The possibility that hepatitis C virus core gene product (HCV-core) acts as a transactivator in insulin-like growth factor II (IGF-II) gene transcription was tested. HCV-core protein increases endogenous IGF-II expression from promoter 4 (P4) of the IGF-II gene through two cis-acting elements: Sp1 and Egr1 binding sites. Sp1 and Egr1 both bind to IGF-II P4 and functionally cooperate in mediating the maximal activity of IGF-II P4. HCV-core protein induced the binding of Sp1 and Egr1 on its binding sites on IGF-II P4. In addition, Sp1 and Egr1 were stimulated to phosphorylate by HCV-core, and its DNA binding activity was up-regulated upon HCV-core transfection. Transfection with HCV-core in HepG2 cells stimulated the membrane translocation of protein kinase C (PKC) and the treatment of HCV-core transfected cells with calphostin C, a PKC inhibitor, blocked induction of Sp1 and Egr1 DNA binding activity, and eventually transcriptional transactivations of the IGF-II gene. Increasing the DNA binding activity of the phosphorylated form of Sp1 and Egr1 might be an important mechanism for regulating IGF-II gene expression and for promoting cell division during hepatic carcinogenesis. These results indicate that HCV-core functions as a positive regulator of IGF-II transcription through the PKC pathway and that Sp1 and Egr1 are direct targets of the transcriptional regulation of the IGF-II gene which plays an important role in hepatitis C virus pathogenesis during the formation of hepatocellular carcinoma (HCC).

The chum salmon insulin-like growth factor II promoter requires Sp1 for its activation by C/EBPβ

The chum salmon insulin-like growth factor II (IGF-II) gene is highly expressed in liver tissue. In this study we demonstrate that two transcription factors, Sp1 and C/EBPβ, are involved in the enhanced expression of the salmon IGF-II gene. The presence of the fish homolog for C/EBPβ in salmon liver RNA was confirmed by Northern blotting. The sIGF-II promoter was activated up to 20-fold by co-transfection with C/EBPβ. The functional importance of four out of the five putative C/EBPβ binding sites was demonstrated with mutational analysis in transient transfection assays. The transcription factor Sp1 binds to two sites within the salmon IGF-II promoter. Interestingly, mutation of the Sp1 binding sites decreases not only the basal IGF-II promoter activity but also the C/EBPβ-induced transactivation. These results demonstrate that liver-enriched C/EBPβ and ubiquitously expressed Sp1 each activate the sIGF-II promoter and that Sp1 is required for full transactivation of the sIGF-II gene by C/EBPβ. This suggests that C/EBPβ and Sp1 act in synergy.

Cellular distribution of insulin-like growth factor II (IGF-II) mRNA and hormonal regulation of IGF-I and IGF-II mRNA expression in rainbow trout testis (Oncorhynchus mykiss)

In this study, Northern blot analysis of RNA from trout testis revealed a single transcript of insulin-like growth factor II (IGF-II) around 4.7 kb. The cellular distribution of IGF-II mRNA was studied and quantified in different testicular cells enriched populations by RT-PCR. IGF-II mRNA appears to be expressed in all cellular types tested: spermatogonia A and B, primary spermatocytes, spermatids and secondary spermatocytes and Sertoli cells. A significantly higher expression of IGF-II was found in premeiotic germ cells. The levels of IGF-II mRNA appear to be higher than those of IGF-I in immature trout testis, as judged from the semi-quantitative RT-PCR results. These data suggest that in addition to IGF-I, IGF-II may play a role in testicular physiology in fish. The hormonal regulation of IGF-I and IGF-II gene expression was investigated both in vitro and in vivo using RT-PCR approach. Gonadotropin (GtH) added to testicular explants increased IGF-II mRNA levels but had no effect on IGF-I. No statistically significant effect was observed with androgens. In vivo, GH and pituitary extracts resulted in an 8 fold and 2-3 fold increase in both IGF-I and IGF-II mRNA levels, respectively. Taken together, our study suggests that IGF-I and IGF-II may act as local mediators of GH and GtHs in fish testis. Moreover, our results imply that in fish testicular cells, IGFs are potential paracrine/autocrine regulators inside the spermatogenic compartment and can act directly on germ cells to stimulate their proliferation.

The human hepatitis B virus transactivator X gene product regulates Sp1 mediated transcription of an insulin-like growth factor II promoter 4.

Human hepatitis B virus (HBV) is one of the causative agents of hepatocellular carcinoma (HCC). The virus encodes a 17 kDa protein, X, which is known to be a causative agent in the formation of HCC. An insulin-like growth factor-II (IGF-II) is expressed during the formation of HCC. Among the four promoters of the IGF-II gene, promoters 2, 3 and 4 become activated during the formation of HCC. The high frequency of detection of hepatitis B virus X (HBV-X) antigen in liver cells from patients with chronic hepatitis, cirrhosis, and liver cancer suggested that the expressions of HBV-X and IGF-II are associated. Studies were carried out to test the relationship between the HBV-X gene product and the activation of IGF-II promoter 4. We demonstrated that the HBV-X protein increases the endogenous IGF-II expression from promoter 3 and 4 of IGF-II gene. Analysis of the fourth promoter of IGF-II gene showed that the HBV-X gene product positively regulates transcription. Two copies of a motif are responsible for conferring HBV-X regulation on the fourth promoter of IGF-II. These motifs have been identified as Sp1 binding sites. Sp1 binding to IGF-II P4 promoter was identified by gel mobility shift assay using purified Sp1. By using a GAL4-Sp1 fusion protein it was demonstrated that HBV-X positively regulates the Spl mediated transcriptional activity of IGF-II in vivo. A protein-affinity chromatography experiment showed that HBV-X protein does not bind directly to Sp1, but HBV-X does augment the DNA binding activity of the phosphorylated form of Sp1 in HepG2 cells. Sp1 was phosphorylated by HBV-X and its DNA-binding activity was up-regulated upon HBV-X transfections. Various HBV-X mutant expression vectors were used for the demonstration of specific interactions between Sp1 and HBV-X. These results indicate that HBV-X functions as a positive regulator of transcription, and that Sp1 is a direct target for the transcriptional regulation of IGF-II. Increasing the DNA binding ability of the phosphorylated form of Sp1 by HBV-X might be an important mechanism for regulating the IGF-II gene expression and possibly promoting cell division during hepatic carcinogenesis. Our experimental results suggest that expression of HBV-X might induce the expression of IGF-II and the IGF-II might play a role in hepatitis B virus pathogenesis during the formation of HCC.

Transcriptional Regulation of Insulin-like Growth Factor-II Gene Expression by Cortisol in Fetal Sheep during Late Gestation

The objective of this study was to determine the mechanisms by which cortisol down-regulates hepatic insulin-like growth factor-II (IGF-II) gene expression in late gestation. Leader exons 6 and 7 of the ovine IGF-II gene, with their 5'-flanking regions, were first isolated. Characterization of transcription start sites revealed a unique site for exon 6 and three dispersed sites for exon 7. Nuclear run-on assays showed a 5-fold higher transcription rate of the IGF-II gene in liver of adrenalectomized fetuses compared with control animals, suggesting that regulation of IGF-II gene expression by cortisol is at the transcriptional level. RNase protection assays demonstrated hepatic leader exon 7 expression in adrenalectomized fetuses to be more than 2-fold higher than in controls, whereas it was reduced by 50% in cortisol-infused fetuses compared with controls. There was no effect on the expression of other leader exons. Functions of the upstream regulatory region of leader exon 7 (i.e. promoter P4) were investigated by luciferase transient expression. A region of -172 bases downstream relative to the first transcription site of leader exon 7 was shown to retain basal promoter activity and respond to cortisol. These results suggest that cortisol may induce the prenatal decline in ovine hepatic IGF-II expression by suppressing promoter P4 of the IGF-II gene.

P53 regulates human insulin-like growth factor II gene expression through active P4 promoter in rhabdomyosarcoma cells.

The developmentally regulated human insulin-like growth factor II (IGFII) gene is expressed at high levels in many types of tumors and promotes the proliferation of tumor cells with a high incidence of p53 gene defects. We have previously shown that p53 inhibits IGFII P3 promoter activity and decreases endogenous IGFII gene expression derived from the P3 promoter in rhabdomyosarcomas by interfering with TBP binding to the TATA element of the IGFII P3 promoter. In this report, we demonstrate that wild-type p53 expression in rhabdomyosarcoma cell lines containing mutant p53 leads to a decrease in the activity of another active IGFII promoter, P4, and a 5-fold reduction of IGFII mRNA derived from the P4 promoter. This inhibition of P4 activity is associated with direct binding of p53 to the P4 proximal promoter element despite the lack of a p53 consensus binding site. Our results suggest that p53 inhibits IGFII P4 promoter activity by a mechanism different than its effect on the P3 promoter. These data also supply further evidence of cross-talk between the IGF and p53 signaling pathways.

Different protein-binding patterns in the P3 promoter region of the human insulin-like growth factor II gene in the human liver cirrhosis and hepatocellular carcinoma tissues.

The P3 promoter of the human insulin-like growth factor II (IGF-II) is the major IGF-II promoter in fetal liver (FL) and hepatocellular carcinoma (HCC). However, little information is available on the transcriptional factors (TFs) controlling IGF-II gene expression in human liver cirrhosis (LC) and HCC tissues. To evaluate the protein-binding patterns in the P3 promoter region, we performed electromobility shift assay (EMSA) and DNase I footprinting assay using nuclear extracts from human FL, LC and HCC tissues. EMSA showed considerable differences in binding patterns of proteins to P3 promoter region according to different nuclear extracts used in this study. By footprinting assay, eight footprints were observed in extracts. In addition, LC extract showed two specific binding at L1 [-80:+30] and L2 [-126:-80] regions, and HCC showed two specific binding at H1 [-176:-120] and H2 [-210:-177] as well as two liver specific binding (L1 and L2). Footprinting after immunoprecipitation indicates that Egr1, Egr2 and Sp1 could bind to P3 promoter directly, while c-jun and c-fos could not bind to these region directly. Further study is required to determine the function of these proteins.

Expression and imprinting of the insulin‐like growth factor II gene in neonatal mouse cerebellum

Insulin-like growth factor II (IGF-II) plays significant roles in the growth and development of mammals through the regulation of mitogenesis and cell survival. Previously, IGF-II mRNA transcripts within the CNS were detected in the choroid plexus and leptomeninges (DeChiara et al., 1991). The objective of this study was to determine the expression pattern of IGF-II mRNA in different cell types of the cerebellum during development. We report here that the IGF-II gene is transcribed in granule and glial cells within the cerebellar parenchyma at various times during the early postnatal period in mice. IGF-II gene expression is further regulated by parent-specific imprinting such that only the paternal IGF-II allele is expressed in granule cells. In contrast, choroid plexus and leptomeninges express IGF-II mRNAs biallelically, indicating that cell type-specific regulation of genomic imprinting occurs within the mammalian CNS. J. Neurosci. Res. 50:958–966, 1997. © 1997 Wiley-Liss, Inc.

Expression and imprinting of the insulin-like growth factor II gene in neonatal mouse cerebellum.

Insulin-like growth factor II (IGF-II) plays significant roles in the growth and development of mammals through the regulation of mitogenesis and cell survival. Previously, IGF-II mRNA transcripts within the CNS were detected in the choroid plexus and leptomeninges (DeChiara et al., 1991). The objective of this study was to determine the expression pattern of IGF-II mRNA in different cell types of the cerebellum during development. We report here that the IGF-II gene is transcribed in granule and glial cells within the cerebellar parenchyma at various times during the early postnatal period in mice. IGF-II gene expression is further regulated by parent-specific imprinting such that only the paternal IGF-II allele is expressed in granule cells. In contrast, choroid plexus and leptomeninges express IGF-II mRNAs biallelically, indicating that cell type-specific regulation of genomic imprinting occurs within the mammalian CNS.

Coexpression of stromelysin-3 and insulin-like growth factor II in tumors of ectodermal, mesodermal, and endodermal origin: indicator of a fetal cell phenotype.

Stromelysin-3 (ST-3) is thought to play an important role in invasion and tumor progression. We have analyzed ST-3 expression in fibroblasts with defined topographical relations to breast cancers. We demonstrate that these fibroblasts exhibit the same distinctive pattern of messenger ribonucleic acid (mRNA) expression that we have previously shown for insulin-like growth factor II (IGF-II). Tumor-derived fibroblasts and skin fibroblasts produce abundant ST-3 mRNA. Fibroblasts from normal breast stroma distant from the malignant tumor in the same patient express considerably less ST-3 mRNA. When we analyzed ST-3 and IGF-II gene expression in sarcomas, we found a similar pattern of coexpression. Immunohistochemical analysis of IGF-II and ST-3 protein expression in sarcomas and breast tumors confirmed the mRNA data. ST-3 mRNA expression was also seen in most colon cancer cell lines, again matching reports of IGF-II gene expression. As the two proteins are known to play an important role during fetal growth and development, their coexpression in fibroblasts from malignant tumors of ectodermal (breast cancer) and mesodermal (sarcoma) origin and in epithelial cells of endodermal origin (colon cancer) implies a more primitive cellular phenotype. The regained ability to express such developmentally regulated proteins might, therefore, be a more general marker indicating a fetal-type phenotype of cells in a malignant tumor.

Activation of fetal promoters of insulinlike growth factors II gene in hepatitis C virus-related chronic hepatitis, cirrhosis, and hepatocellular carcinoma

Increased prevalence of hepatitis C virus (HCV) infection has been found in patients with hepatocellular carcinoma (HCC). The expression of insulinlike growth factor II (IGF-II) has been linked to hepatocarcinogenesis in the experimental animal and in humans. Since reactivation of fetal IGF-II transcripts has been observed in human HCC, we have analyzed the levels of adult P1 and fetal P3 and P4 IGF-II promoter-derived transcripts in the liver of patients with HCV-related chronic active hepatitis (CAH), cirrhosis, and HCC by means of a semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) assay. Transcripts derived from adult P1 promoter were increasingly expressed from normals to patients with CAH and cirrhosis, but were undetectable in the tumorous area of 5 of 7 HCC patients and present at low levels in the nontumorous area of all HCC patients. Transcripts derived from fetal P3 promoter were not detectable in normal subjects, while they were expressed abundantly in most CAH and all cirrhotic patients. Transcripts from fetal P4 promoter were detected at high levels in 3 of 9 CAH patients and in the majority of cirrhotic patients. Increased expression of fetal promoter-derived transcripts was also found in the liver of HCC patients, although levels were lower than in cirrhosis. Also, the activity of fetal P3 and P4 promoters was higher in the nontumorous than in the tumorous area of the liver of HCC patients. The expression of IGF-II transcripts was correlated with the rate of cell mitotic activity by measuring the expression of the proliferating cell nuclear antigen (PCNA) gene. PCNA messenger RNA (mRNA) levels progressively increased from normals to CAH and to cirrhotic patients, and persisted at a high level in the tumorous and in the nontumorous area of HCC subjects, thus showing that the increase of IGF-II transcripts in CAH and cirrhosis is accompanied by an activation of cell mitosis in these samples. These data suggest that the activation of IGF-II gene expression from adult and fetal promoters may play a role in premalignant proliferation observed in HCV-related chronic liver disease. (Hepatology 1996 Jun;23(6):1304-12)

Activation of fetal promoters of insulinlike growth factors II gene in hepatitis C virus-related chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

Increased prevalence of hepatitis C virus (HCV) infection has been found in patients with hepatocellular carcinoma (HCC). The expression of insulinlike growth factor II (IGF-II) has been linked to hepatocarcinogenesis in the experimental animal and in humans. Since reactivation of fetal IGF-II transcripts has been observed in human HCC, we have analyzed the levels of adult P1 and fetal P3 and P4 IGF-II promoter-derived transcripts in the liver of patients with HCV-related chronic active hepatitis (CAH), cirrhosis, and HCC by means of a semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) assay. Transcripts derived from adult P1 promoter were increasingly expressed from normals to patients with CAH and cirrhosis, but were undetectable in the tumorous area of 5 of 7 HCC patients and present at low levels in the nontumorous area of all HCC patients. Transcripts derived from fetal P3 promoter were not detectable in normal subjects, while they were expressed abundantly in most CAH and all cirrhotic patients. Transcripts from fetal P4 promoter were detected at high levels in 3 of 9 CAH patients and in the majority of cirrhotic patients. Increased expression of fetal promoter-derived transcripts was also found in the liver of HCC patients, although levels were lower than in cirrhosis. Also, the activity of fetal P3 and P4 promoters was higher in the nontumorous than in the tumorous area of the liver of HCC patients. The expression of IGF-II transcripts was correlated with the rate of cell mitotic activity by measuring the expression of the proliferating cell nuclear antigen (PCNA) gene. PCNA messenger RNA (mRNA) levels progressively increased from normals to CAH and to cirrhotic patients, and persisted at a high level in the tumorous and in the nontumorous area of HCC subjects, thus showing that the increase of IGF-II transcripts in CAH and cirrhosis is accompanied by an activation of cell mitosis in these samples. These data suggest that the activation of IGF-II gene expression from adult and fetal promoters may play a role in premalignant proliferation observed in HCV-related chronic liver disease.