Insulin-like Growth Factor II mRNA Expression Research Articles

Alteration of Metabolic Conditions Impacts the Regulation of IGF-II/H19 Imprinting Status in Prostate Cancer.

Simple SummaryInsulin-like growth factor II (IGF-II) is a potent growth factor implicated in several cancer types. The IGF-II gene shares its locus with the long non-coding RNA, H19. IGF-II/H19 is an imprinted gene—a phenomenon where only the maternal copy is expressed. The silencing of the paternal copy is lost in many cancer types—including prostate. Our in vitro findings show it is possible to disrupt imprinting through the alteration of metabolic conditions, with changes occurring at the molecular level only. Comparing with prostate tissue samples we additionally found a positive correlation to exist between IGF-II and H19 mRNA expression, which was further confirmed by in silico data from the Cancer Genome Atlas.Prostate cancer is the second major cause of male cancer deaths. Obesity, type 2 diabetes, and cancer risk are linked. Insulin-like growth factor II (IGF-II) is involved in numerous cellular events, including proliferation and survival. The IGF-II gene shares its locus with the lncRNA, H19. IGF-II/H19 was the first gene to be identified as being “imprinted”—where the paternal copy is not transcribed—a silencing phenomenon lost in many cancer types. We disrupted imprinting behaviour in vitro by altering metabolic conditions and quantified it using RFLP, qPCR and pyrosequencing; changes to peptide were measured using RIA. Prostate tissue samples were analysed using ddPCR, pyrosequencing and IHC. We compared with in silico data, provided by TGCA on the cBIO Portal. We observed disruption of imprinting behaviour, in vitro, with a significant increase in IGF-II and a reciprocal decrease in H19 mRNA; the increased mRNA was not translated into peptides. In vivo, most specimens retained imprinting status apart from a small subset which showed reduced imprinting. A positive correlation was seen between IGF-II and H19 mRNA expression, which concurred with findings of larger Cancer Genome Atlas (TGCA) cohorts. This positive correlation did not affect IGF-II peptide. Our findings show that type 2 diabetes and/or obesity, can directly affect regulation growth factors involved in carcinogenesis, indirectly suggesting a modification of lifestyle habits may reduce cancer risk.

Biochemical Characterization of Individual Human Glycosylated pro-Insulin-like Growth Factor (IGF)-II and big-IGF-II Isoforms Associated with Cancer

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed "pro" and "big" IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling.

Correlation between epigenetic alterations in the insulin growth factor-II gene and hepatocellular carcinoma

To investigate whether epigenetic alterations in the insulin-like growth factor-II (IGF-II) gene that cause differential transcription or expression are correlated with onset and severity of hepatocellular carcinoma (HCC). Patient-matched specimens of HCC, paracancerous, and non-cancerous tissues were collected from 40 primary liver cancer patients. Epigenetic alterations in the promoter (P3) sequence of the IGF-II gene were analyzed by methylation-specific PCR (MSP) and IGF-II transcription was measured by RT-PCR. IGF-II protein expression and clinicopathological features were assessed by immunohistochemistry and microscopic observation. The rate of IGF-II P3 methylation was significantly lower in HCC tissues (0%) than in paracancerous tissues (vs. 47.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 100%; x2 = 80.000, P less than 0.001). IGF-II mRNA expression was significantly higher in HCC tissues (100%) than in paracancerous tissues (vs. 52.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 80.000, P less than 0.001). IGF-II protein expression was significantly higher in HCC tissues (82.5%) than in paracancerous tissues (vs. 45.0%; x2 = 12.170, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 56.170, P less than 0.001). IGF-II overexpression in HCC was significantly associated with degree of differentiation, extent of infiltrated serosa, size of tumor, and HBV-positive infection status. Epigenetic alterations in the IGF-II gene regulate its transcription and expression and are closely associated with HCC development and progression.

Expression of insulin-like growth factor-II, matrix metalloproteinases, and their tissue inhibitors as predictive markers in the peripheral blood of HCC patients

Background/aim: Elevated relative expression of insulin-like growth factor-II (IGF-II) was observed in hepatocellular carcinoma (HCC) liver tissues with a role in neovascularization and associated with poor prognosis. IGF-II is influenced by the proteolytic cleavage of IGF-binding protein 3 and by matrix metalloproteinases (MMP), which are further regulated by their tissue inhibitors tissue inhibitor of metalloprotienase-1 (TIMP-1). Our aim is to study new molecular markers for HCC.Patients/methods: RNA was extracted from the peripheral blood for evaluating the relative expression of IGF-II, MMP-9, and TIMP-1 in correlation with clinical staging of 39 HCC patients and 15 healthy controls using TaqMan real-time PCR.Results: The relative expression of IGF-II and MMP-9 mRNA were significantly elevated in HCC patients compared with healthy controls; P-value <0.0001 for both. There was a significant correlation between MMP-9 and different HCC stages. On the other hand, TIMP-1 was significantly down-regulated in HCC patients; P = 0.0003 with the elevation of the IGF-II/TIMP-1 ratio. Significant correlation between TIMP-1 and HCC Stage III and Stage IV was found; P-value = 0.0138.Conclusion: These results highlight the importance of profiling the expression of IGF-II, MMP-9, and TIMP-1 in the peripheral blood as prognostic molecular biomarkers in HCC.

IGF-II promoter specific methylation and expression in epithelial ovarian cancer and their associations with disease characteristics

High expression of insulin-like growth factor-II (IGF-II) in epithelial ovarian cancer is associated with aggressive disease and poor prognosis. IGF-II transcription is initiated from multiple promoters. Promoter-specific expression is regulated by DNA methylation, which is often dysregulated in cancer. Here, the effects of promoter-specific methylation on IGF-II expression are investigated in ovarian cancer. Fresh tumor samples were collected from 211 patients for analyses of IGF-II promoter methylation using methylation-specific PCR, and of promoter-specific expression of IGF-II mRNA with qRT-PCR, as well as tissue levels of IGF-II peptide with an ELISA. Cox regression analysis was performed to assess IGF-II methylation and expression in association with progression-free and overall survival. DNA methylation was high in IGF-II promoters 2 (P2, 64.2%) and 3 (P3, 52.1%) and low in promoter 4 (P4, 9.8%). High methylation was associated with low mRNA expression in a promoter-specific manner. P3 methylation and expression appeared to be critical in ovarian cancer compared to other promoters. While methylation in an individual promoter was not associated with the disease, a methylation pattern involving P2 and P3 was significantly different among patients with distinct tumor grade, debulking results, residual tumor size and treatment response. The methylation pattern was also associated with disease progression. The study suggests that DNA methylation regulates IGF-II promoter-specific expression in ovarian cancer and the regulation may play a role in disease progression. Assessing methylation patterns in IGF-II promoters may have clinical implications.

Igf-Ii Mrna Expression in Breast Cancer: Predictive Value and Relationship to Other Prognostic Factors

Insulin-like growth factor-II (IGF-II) is an important regulator of tumor growth in breast cancer. In this study we have examined the prognostic value of IGF-II mRNA expression in breast cancer and its relationship to other predictive parameters. Sixty-eight women with infiltrating ductal carcinoma were given the same treatments including mastectomy and antitumoral therapies and followed up for 5 years. The overall 5-year survival rate was 73.5% (55/68). IGF-II mRNA was expressed in 33/64 patients (51.6%) and had no significant impact on survival. The expression of estrogen receptor (ER) and progesterone receptor (PgR) did not significantly affect the 5-year survival, but in the presence of an IGF-II mRNA signal, the survival of ER- and PgR-negative patients (n=9) was lower than that of ER- and PgR-positive patients (n=15), although the difference was not significant. The 5-year survival was not significantly different between Ki-67-positive and negative patients, but in the IGF-II positive group Ki-67-positive patients (n=7) had a significantly poorer prognosis than Ki-67-negative patients (n=26). The expression of p53 protein was associated with a poorer prognosis: 6/11 (54.5%) p53-positive patients died in the first 26 months of follow-up and 5 of these 6 patients (83.3%) also had positive IGF-II mRNA expression. IGF-II mRNA expression per se is not an independent predictive factor in breast cancer but may be a marker of poor prognosis when associated with other prognostic factors such as Ki-67 index and p53 expression.

Abstract 834: IGF-II promoter specific methylation patterns are associated with its expression and disease progression in epithelial ovarian cancer

Abstract Purpose: High insulin-like growth factor-II (IGF-II) expression in epithelial ovarian cancer is associated with aggressive disease and poor prognosis. IGF-II expression is regulated by promoter methylation; aberrant methylation is common in cancer. The effect of promoter methylation on IGF-II expression and the role of methylation in ovarian cancer are investigated in a clinical study. Patients and Methods: The study enrolled 211 epithelial ovarian cancer patients who were followed after surgery for disease outcomes. Fresh tumor samples were collected from the patients and were analyzed for IGF-II promoter methylation using methylation-specific PCR, as well as for IGF-II mRNA expression with qRT-PCR. IGF-II peptide concentrations were measured with an ELISA kit in tumor tissues. Results: The study found different levels of methylation in 3 IGF-II promoters (P2-P4) which are located in a large CpG island, high methylation in P2 (64.2%) and P3 (52.1%) and low in P4 (9.8%). Promoter-specific mRNA expression was lower in methylated than in unmethylated tumors. P3 methylation and expression appeared to play a major role for IGF-II in ovarian cancer. While methylation in individual promoter was not related to the disease, a methylation pattern between P2 and P3 was significantly associated with clinical and pathological features of ovarian cancer, including tumor grade, debulking results, residual tumor size and treatment response. The methylation pattern was also linked to disease progression, but not overall survival. Conclusions: Our findings suggest that IGF-II expression affects tumor progression and the expression is regulated by methylation which may have clinical implications in disease management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 834.

Abstract A9: Expression of insulin-like growth factor II and of the type 1 receptor gene in esophageal adenocarcinoma

Abstract A9 Aim To explore potential biological mechanisms underlying the association between obesity and esophageal malignancy, we studied the expression of insulin-like growth factor-II (IGF-II) and of the type 1 receptor (IGF-IR) gene in a well characterized series of patients with primary esophageal adenocarcinoma (EADC) and matched histologically normal esophageal epithelia (NE). Patients and Methods Banked esophageal tissues (EADC and matched NE, obtained with informed consent) were available from a consecutive series of 52 patients who underwent esophageal resection without preoperative chemoradiation therapy. Following extraction of RNA, expression of IGF-II and IGF-IR mRNA was determined using reverse transcription and real time polymerase chain reaction (RT-PCR). The 2-ΔΔCT (Livak) method was used to quantitate tissue expression of IGF-II and IGF-IR mRNA, which was correlated with measured body mass index (BMI), clinicopathological findings, and outcome. Results As expected, significant correlations between tumor stage, differentiation and survival were found for the 49 male and 3 female patients in this series, who ranged in age from 36 to 85 years (median: 60.5 years). A significant association was seen between BMI and IGF-II mRNA expression in NE tissues (rs=0.26, p=0.075). Significantly higher IGF-II mRNA expression was seen in EADC tumors (median 6.45, 95%CI 7.00-13.46) compared to matched NE tissues (median 1.32, 95%CI 1.37-4.31, p&amp;lt;0.01), particularly in poorly differentiated tumors. On multivariate analysis, IGF-II expression was an independent predictor of disease-free survival (p=0.02) and overall survival (p=0.03). Expression levels of IGF-II mRNA correlated significantly with IGF-IR in both EADC (rs=0.47, p&amp;lt;0.01) and NE (rs=0.46, p&amp;lt;0.01). Furthermore, IGF-IR mRNA expression was found to be significantly higher in EADC (median 1.57; 95%CI 1.47-3.86) compared with matched NE (median 0.57; 95%CI 0.50-0.85) (p&amp;lt;0.01), but only in patients with the A variant of the common IGF-IR G3174A polymorphism, which has recently been shown to modulate the obesity risk for EADC. Conclusions These studies further implicate perturbations of the IGF axis in the molecular pathogenesis of human EADC, and suggest a plausible mechanistic basis to explain the association between obesity and risk for malignancy. The significant association between IGF-II expression and survival suggests that IGF-II may be a clinically relevant biomarker for prognosis, or molecular target for novel anti-cancer therapies. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A9.

HCG increases trophoblast migration in vitro via the insulin-like growth factor-II/mannose-6 phosphate receptor

We have previously shown that both HCG and insulin-like growth factor-II (IGF-II) stimulate trophoblastic invasion. Furthermore, the invasion-promoting function of IGF-II resulted from IGF-II mannose 6-phosphate receptor (IGF-II/M6PR) activation. Since HCG and IGF-II did not have an additive effect on cell migration of extravillous trophoblast (EVT) cell line, HTR-8 SVneo, we hypothesized that HCG actions are mediated via alterations in the expression and/or function of IGF-II axis. HCG treatment (50-50,000 mU/ml) of the HTR-8/SVneo cells did not alter the expression of either insulin-like growth factor-I or IGF-II mRNA or peptide synthesis, but caused (i) an increase in the (125)I-IGF-II binding to EVT cells, and (ii) an increase in the externalization rate of the IGF-II binding sites without affecting their internalization. This effect was due to the increase in the number of IGF-II binding sites in the plasma membrane without any change in the IGF-II binding affinity. Although HCG did not influence the abundance of IGF-II/M6PR mRNA or protein, anti-IGF-II/M6PR antibody decreased HCG-induced migration of EVT, supporting the hypothesis that HCG might stimulate EVT migration by increasing IGF-II binding to the plasma membrane and subsequently by increasing the IGF-II effect probably mediated via the IGF-II/M6PR.

Induction of insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells by cells of trophoblast origin.

To determine the influence of the conceptus on the induction of decidualization in endometrial stromal cells from the baboon. For in vivo studies, implantation sites from day 22 of pregnancy in the baboon were analyzed for insulin-like growth factor-II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) mRNAs using in situ hybridization. For in vitro studies, Jeg-3 cells or primary cytotrophoblasts isolated from term placenta were cocultured with a monolayer of stromal cells from the baboon endometrium. Cytotrophoblasts were placed either directly on top of the stromal cell monolayer or in transwell cell culture inserts and cultured for 7 days. No exogenous hormones were added. Stromal cells were analyzed for IGFBP-1 mRNA and protein by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Examination of early implantation sites in the baboon revealed a high expression of IGF-II mRNA in the invading cytotrophoblasts. Conversely, the stromal cells of the endometrium directly adjacent to the cytotrophoblasts expressed IGFBP-1 mRNA. Endometrial stromal cells cocultured with Jeg-3 cells or primary cytotrophoblasts for 7 days expressed IGFBP-1 mRNA and protein. This expression occurred in both the direct coculture system and coculture using the cell culture inserts. Cytotrophoblasts also induced the expression of prolactin in stromal fibroblasts following coculture. The conceptus is capable of inducing decidualization of endometrial stromal cells. This is shown in both the in vivo and in vitro systems.

Expression of insulin-like growth factor-II and insulin-like growth factor binding protein-1 in the placental basal plate from pre-eclamptic pregnancies

Objectives: To determine whether expressions of insulin-like growth factor-II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) are altered in pre-eclamptic placenta and to elucidate the possible relationship between their expressions and a mechanism for inadequate trophoblast invasion in pre-eclampsia. Methods: Placental tissues were obtained at cesarean delivery from five normotensive, nine mild pre-eclamptic and five severe pre-eclamptic women at 33–39 completed weeks of gestation. After total ribonucleic acid was extracted, reverse transcriptase-polymerase chain reaction was performed to determine IGF-II and IGFBP-1 mRNA expression. Product bands were quantitated by scanning densitometry and results were expressed as ratio of cytokines/β-actin. Western blot analysis was also done to determine IGF-II and IGFBP-1 protein expression. Statistical analysis was determined by Kruskal–Wallis analysis of variance with the Scheffe multiple post-hoc test. Results: The IGF-II mRNA levels of mild and severe pre-eclamptic placenta were significantly lower than that of uncomplicated placenta (P<0.005, P<0.001, respectively), with the level of severe pre-eclamptic placenta being significantly lower than that of mild pre-eclamptic placenta (P<0.05). As for the IGF-II protein expression, a significant decrease was found among the three groups (P<0.001), correlating with the IGF-II mRNA results. However, the mean IGFBP-1 mRNA levels of mild and severe pre-eclamptic placenta were significantly higher than that of uncomplicated placenta (P<0.05, P<0.005, respectively), with the level of severe pre-eclamptic placenta being significantly raised compared with that of mild pre-eclamptic placenta (P<0.05). Finally, a significant increase of IGFBP-1 protein expression was noted among the three groups (P<0.001), correlating with the IGFBP-1 mRNA results. Conclusions: This study suggests that IGF-II and IGFBP-1 might be associated with the impaired trophoblastic invasion that may lead to pathogenesis of pre-eclampsia.

Effect of refeeding on IGFI, IGFII, IGF receptors, FGF2, FGF6, and myostatin mRNA expression in rainbow trout myotomal muscle

Fish endure long periods of fasting and demonstrate an extensive capacity for rapid and complete recovery after refeeding. The underlying mechanisms through which nutrient intake activates an increase in somatic growth and especially in muscle growth is poorly understood. In this study we examined the expression profile of major muscle growth regulators in trout white muscle 4, 12, and 34 days after refeeding, using real-time quantitative RT-PCR. Mean insulin-like growth factor I (IGFI) mRNA level in muscle increased dramatically 8- and 15-fold, 4 and 12 days, respectively, after refeeding compared to fasted trout. This declined thereafter. Conversely, only a weak but gradual increase in mean insulin-like growth factor II (IGFII) mRNA level was observed during refeeding. Inversely to IGFI, mean IGF receptor Ia (IGFRIa) mRNA level declined after ingestion of food. In contrast, IGF receptor Ib (IGFRIb) mRNA level was not affected by refeeding. Mean fibroblast growth factor 2 (FGF2) mRNA level increased by 2.5-fold both 4 and 12 days after refeeding, whereas fibroblast growth factor 6 (FGF6) and myostatin mRNA levels were unchanged. Subsequent to IGFI and FGF2 gene activation, an increase in myogenin mRNA accumulation was observed at 12 days post-refeeding suggesting that an active differentiation of myogenic cells succeeds their proliferation. In conclusion, among the potential growth factors we examined in this study, IGFI and FGF2 were identified as candidate genes whose expression may contribute to muscle compensatory growth induced by refeeding.

Endometrial insulin-like growth factor II and type I receptor in unexplained infertility and its steroid regulation

OBJECTIVE: To investigate the endometrial expression of insulin-like growth factor II(IGF-II) and type I receptor(IGF-1R) in unexplained infertility. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect and quantify endometrial IGF-II and IGF-I R mRNA experession in the mid-luteal phase. Serum concentrations of estradiol(E(2)) and progesterone (P) were measured by radioimmunoassays. Thirty-four women with unexplained infertility were included in the study group. Twenty-one women with normal fertility were selected as the control group. RESULTS: Endometrial IGF-II and IGF-I R mRNA experession during the midluteal phase were significantly lower in the study group than in the control group (0.662 +/- 0.371 compared with 0.961+/- 0.389, P< 0.05; 0.582 +/- 0.257 compared with 0.829 +/-0.341, P< 0.05 respectively). The average serum P level in the study group was significantly lower than in the control group (23.782 +/-15.459 compared with 43.142 +/- 16.549nmol/L, P< 0.005). IGF-II mRNA expression correlated positively with IGF-I R mRNA expression in the two groups. Serum P level correlated positively with IGF-II and IGF-I R mRNA expression in the two groups. CONCLUSION: IGF-II and IGF-I R may play an important role in the implantation process. Their decreased expression relating to decreased P level may be important in unexplained infertility.

Differential Expression of Insulin-like Growth Factor I and II mRNAs during Embryogenesis and Early Larval Development in Rabbitfish, Siganus guttatus

In rodents, the expression of insulin-like growth factor II (IGF-II) is higher than that of insulin-like growth factor I (IGF-I) during fetal life while the reverse is true after birth. We wanted to examine whether this is also true in fish and whether IGF-I and IGF-II are differentially regulated during different stages of embryogenesis and early larval development in rabbitfish. We first cloned the cDNAs of rabbitfish IGF-I and IGF-II from the liver. Rabbitfish IGF-I has an open reading frame of 558 bp that codes for a signal peptide of 44 amino acids (aa), a mature protein of 68 aa, and a single form of E domain of 74 aa. Rabbitfish IGF-II, on the other hand, has an open reading frame of 645 bp that codes for a signal peptide of 47 aa, a mature protein of 70 aa, and an E domain of 98 aa. On the amino acid level, rabbitfish IGF-I shares 68% similarity with IGF-II. We then examined the relative expression of the two IGFs in unfertilized eggs, during different stages of embryogenesis, and in early larval stages of rabbitfish by a semiquantitative reverse transcription-polymerase chain reaction. Primers that amplify the mature peptide region of both IGFs were used and PCR for both peptides was done simultaneously, with identical PCR conditions for both. The identity of the PCR products was confirmed by direct sequencing. Contrary to published reports for seabream and rainbow trout, IGF-I mRNA was not detected in rabbitfish unfertilized eggs; it was first expressed in larvae soon after hatching. IGF-II mRNA, however, was expressed in unfertilized eggs, albeit weakly, and was already strongly expressed during the cleavage stage. mRNAs for both peptides were strongly expressed in the larvae, although IGF-II mRNA expression was higher than IGF-I expression.

ONTOGENY OF THE H19 GENE IN SHEEP AND EFFECT OF MATERNAL FASTING ON ITS EXPRESSION IN THE FETUS

Mapped on the same chromosome as the Insulin-like Growth Factor II (IGF-II), an important factor regulating fetal growth, the H19 gene, is believed to play a role during embryogenesis and to share similar regulatory elements with IGF-II possibly by an enhancer competition system. This study was designed to characterize the ontogeny of H19 in sheep and the effect of maternal fasting on the expression of fetal IGF-II and H19 mRNA. A partial cDNA clone for the ovine H19 gene was isolated and used as a probe for RNase protection analysis. The ontogeny of H19 in liver, skeletal muscle and heart of ovine fetuses at 62, 100 and 130 days, lambs at 1 month and adult sheep revealed high tissue levels of H19 mRNA during fetal life that decreased significantly after birth. Maternal fasting significantly decreased fetal liver H19 mRNA expression but did not alter fetal IGF-II mRNA expression. These results suggest that H19, like IGF-II, may play an important role in the regulation of fetal growth and define an environmental condition whereby these two genes are regulated independently.

Developmental expression of and effect of betamethasone on the messenger ribonucleic acid levels for peptide growth factors in the baboon fetal adrenal gland.

In the present study, we determined whether expression of the messenger ribonucleic acids (mRNAs) for insulin-like growth factor-II (IGF-II), and its principal IGF type-1 receptor and IGF-binding protein-2 (IGFBP-2), as well as basic fibroblast growth factor (bFGF), was developmentally regulated in the baboon fetal adrenal gland. In the second phase of this study, fetal pituitary ACTH was suppressed by the administration of betamethasone to determine the possible effect on the mRNA levels for those factors, i.e. IGF-II and IGFBP-2, shown to be expressed at high levels in the adrenal late in fetal development. Adrenals were obtained from fetuses delivered via Cesarean section on days 60 (early), 100 (mid), and 165 (late) of gestation (term=184 days) from untreated baboons and on day 165 from baboons in which betamethasone was administered to the fetus, or to fetus and mother, every other day between days 150 and 164 of gestation. Although the mRNA levels of IGF-II in the fetal adrenal were similar at early, mid and late gestation, IGF type-1 receptor mRNA levels were approximately 2- to 3-fold greater (P<0.01) at mid than at early or late gestation. In contrast, there was an increase (P<0.001) in fetal adrenal IGFBP-2 and bFGF mRNA levels in late gestation. Although fetal adrenal weights and width of the zone of definitive/transitional cells exhibiting immunocytochemical staining for Delta(5)-3beta-hydroxysteroid dehydrogenase (3beta-HSD) were markedly suppressed (P<0.01) by the administration of betamethasone, IGF-II and IGFBP-2 mRNA expression was not decreased. In summary, very different patterns of mRNA levels for IGF-II, IGF type-1 receptor, IGFBP-2 and bFGF were exhibited in the developing baboon fetal adrenal gland, which may reflect functionally important differences in their respective cellular localization within the cortex, as well as a divergence in the functional development of the fetal, transitional and definitive zones of the baboon fetal adrenal cortex.

Inhibition of growth, production of insulin-like growth factor-II (IGF-II), and expression of IGF-II mRNA of human cancer cell lines by antagonistic analogs of growth hormone-releasing hormone in vitro.

Antagonistic analogs of growth hormone-releasing hormone (GHRH) suppress growth of various tumors in vivo. This effect is exerted in part through inhibition of the GHRH-GH-insulin-like growth factor (IGF)-I axis. Nevertheless, because autocrine/paracrine control of proliferation by IGF-II also is a major factor in many tumors, the interference with this growth-stimulating pathway would offer another approach to tumor control. We thus investigated whether GHRH antagonists MZ-4-71 and MZ-5-156 also act on the tumor cells directly by blocking the production of IGF-II. An increase in the IGF-II concentration in the media during culture was found in 13 of 26 human cancer cell lines tested. Reverse transcription-PCR studies on 8 of these cell lines showed that they also expressed IGF-II mRNA. Antagonists of GHRH significantly inhibited the rate of proliferation of mammary (MDA-MB-468 and ZR-75-1), prostatic (PC-3 and DU-145), and pancreatic (MiaPaCa-2, SW-1990, and Capan-2) cancer cell lines as shown by colorimetric and [3H]thymidine incorporation tests and reduced the expression of IGF-II mRNA in the cells and the concentration of IGF-II secreted into the culture medium. Growth and IGF-II production of lung (H-23 and H-69) and ovarian (OV-1063) cancer cells that express mRNA for IGF-II and excrete large quantities of IGF-II also was marginally suppressed by the antagonists. These findings suggest that antagonistic analogs of GHRH can inhibit growth of certain tumors not only by inhibiting the GHRH-GH-IGF-I axis, but also by reducing the IGF-II production and by interfering with the autocrine regulatory pathway.

Presence of GH-dependent IGF-II mRNA in the diffuse pancreatic tissue of a teleost

Previously, insulin-like growth factor II (IGF-II) mRNA steady state levels were found to be elevated in the pyloric caeca and surrounding diffuse pancreatic and adipose tissues of rainbow trout treated with bovine growth hormone (bGH) (Shamblott MJ, Cheng CM, Bolt D, Chen TT. Proc Natl Acad Sci USA 1995;A 92:6943-6946). We now report on the GH-dependent expression of IGFs in the pyloric caeca and the surrounding diffuse pancreatic tissue, using a non-radioactive in situ hybridization method employing IGF-I, IGF-II, and insulin specific digoxigenin-labeled antisense cRNA probes. IGF-II mRNA was localized to diffuse pancreatic tissue and to a lesser extent to epithelial cells of the pyloric caeca in rainbow trout treated with bGH. IGF-II mRNA was also detected in epithelial cells of the pyloric caeca in control trout. IGF-I mRNA was undetectable in the pyloric caeca or pancreatic tissue of bGH treated or control rainbow trout. Insulin specific cRNA probes were used in situ to confirm the specificity of IGF-II mRNA expression. We hypothesize that IGF-II may be acting in a local manner as a GH-dependent growth factor in the pancreatic tissue surrounding the pyloric caeca and within the pyloric caeca.

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.

Caprine testicular hypoplasia associated with sexual reversion decreases the expression of insulin-like growth factor II (IGF-II) mRNA in testes.

The genetic expression of insulin-like growth factor II (IGF-II) mRNA was studied in healthy adult testes and in hypoplastic testes of polled Murciano-Granadina goats by means of in situ hybridization. A positive reaction in spermatogonia, pachytene spermatocytes and a few peritubular myoid cells was observed using the ovine antisense oligonucleotide in healthy testes. The hypoplastic testes displayed a loss of germinal epithelium and a slight thickening of the basement membranes. A limited number of immature germinal cells displayed a lesser hybridization reaction, while the expression of IGF-II mRNA observed in the peritubular myoid cells was similar to that seen in healthy testes. In hypoplastic testes, IGF-II mRNA expression within germinal cells decreased with increasing hypoplasia within the seminiferous epithelium and there was no hybridization within the tubules in cases of severely disrupted spermatogenesis. These results suggest that testicular hypoplasia is associated with changes in the expression of IGF-II mRNA.