Paul H. Steenbergh

A second human calcitonin/CGRP gene

The calcitonin (CT) gene is alternatively expressed in a tissue‐specific fashion producing either the calcium regulatory hormone CT in the thyroid or the neuropeptide calcitonin gene related peptide (CGRP) in the brain. In medullary carcinoma of the thyroid both peptides are produced. We present here evidence for the existence in the human genome of a second CT gene, which is also expressed in human medullary thyroid carcinoma. This gene encodes a second human CGRP, differing from the known human CGRP in 3 of the 37 amino acids.

Mitogenic Signaling of Insulin-like Growth Factor I in MCF-7 Human Breast Cancer Cells Requires Phosphatidylinositol 3-Kinase and Is Independent of Mitogen-activated Protein Kinase

Addition of insulin-like growth factor I (IGF-I) to quiescent breast tumor-derived MCF-7 cells causes stimulation of cyclin D1 synthesis, hyperphosphorylation of the retinoblastoma protein pRb, DNA synthesis, and cell division. All of these effects are independent of the mitogen-activated protein kinase (MAPK) pathway since none of them is blocked by PD098059, the specific inhibitor of the MAPK activating kinase MEK1. This observation is consistent with the finding that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong inducer of MAPK activity in MCF-7 cells, effectively inhibits proliferation. The anti-proliferative effect of TPA in these cells may be accounted for, at least in part, by the MAPK-dependent stimulation of the synthesis of p21WAF1/CIP1, an inhibitor of cyclin/cyclin-dependent kinase complexes. In contrast, all of the observed stimulatory effects of IGF-I on cell cycle progression, cyclin D1 synthesis, and pRb hyperphosphorylation were blocked by the specific phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that phosphatidylinositol 3-kinase activity but not MAPK activity is required for transduction of the mitogenic IGF-I signal in MCF-7 cells.

The role of MAP kinase in TPA-mediated cell cycle arrest of human breast cancer cells

In MCF7 breast cancer cells, mitogen-activated protein (MAP) kinase (i.e. Erk-1/2) is activated by the mitogen insulin, but also by the growth inhibiting agent TPA, though with very different kinetics. Insulin induces a relatively transient activation of Erk2 (<15 min), whereas TPA is able to induce a prolonged activation of Erk2 (>6 h). Expression of immediate-early genes of the c-fos and c-jun families, whose transcription and activation are regulated by MAP kinases, is differentially induced by insulin and TPA. Whereas insulin stimulates prolonged induction of c-jun, but not of junB mRNA, resulting in c-jun expression during the entire G1 period, the growth inhibitor TPA induces junB much longer than c-jun. Inhibition of the Erk2 pathway by PD98059, specific for the upstream MAP kinase kinase (MEK1), abolishes TPA-stimulated junB but not insulin-induced c-jun. In agreement with this, insulin readily stimulates Jun kinase (JNK), whereas TPA does not. Furthermore, insulin-induced pRB hyperphosphorylation at the G1-S transition and S-phase entry is insensitive to MAP kinase inhibition by PD98059. On the other hand, PD98059 reverts the inhibitory effect of TPA on cell cycle entry as well as on pRB hyperphosphorylation, indicating that Erk effectors function as inhibitors of proliferation in MCF7 cells.

Localization of the polymorphic human calcitonin gene on chromosome 11

SummaryA molecular probe containing a 584 base pairs sequence corresponding to part of the human calcitonin mRNA was used for the chromosomal assignment of the calcitonin gene. Restriction endonuclease analysis of DNA from human-Chinese hamster and human-mouse somatic cell hybrids, including some containing a translocation of human chromosomes, placed the calcitonin gene in the p14→qter region of chromosome 11.Analysis of human DNA showed that the calcitonin gene has a polymorphic site for restriction endonuclease TaqI.

The human IGF-I gene contains two cell type-specifically regulated promoters

The human insulin-like growth factor-I (IGF-I) gene contains two alternative leader exons: exons 1 and 2. We have identified, by transient transfection experiments, the putative promoters P1 and P2 upstream of these leader exons. The promoter regions were cloned in front of the luciferase reporter gene and their promoter activities were measured in transfected SK-N-MC (human neuroepithelioma) and OVCAR-3 (human ovarian carcinoma) cells. Both of these cell lines express the IGF-I gene endogenously, resulting in normally sized IGF-I mRNAs of 7.6, 1.3 and 1.1 kb. In SK-N-MC cells, in which P1 is the most active IGF-I promoter, P2 displayed a three times lower promoter activity than P1. However, in OVCAR-3 cells, P2 is four times more active than P1, resulting in an overall 12-fold difference in the relative promoter activities of the two IGF-I gene promoters in these two cell types. This indicates that the IGF-I promoters show a cell type-specific expression pattern.

Estrogen Receptor (ER)-α, but Not ER-β, Mediates Regulation of the Insulin-like Growth Factor I Gene by Antiestrogens

The importance of insulin-like growth factor I (IGF-I) on maintenance of skeletal integrity has been widely recognized. Although osteoblasts secrete some IGF-I, the liver is the primary endocrine source for IGF-I. We have studied the regulation of the human IGF-I promoter in the hepatocyte cell line Hep3B, and we have shown that the IGF-I promoter, when co-transfected in Hep3B cells together with an estrogen receptor (ER)-α expression vector, was transcriptionally regulated by raloxifene or raloxifene-like molecules but not by 17β-estradiol and 4(OH)-tamoxifen. The induction mediated by raloxifene is antagonized by 17β-estradiol and mediated selectively by ER-α, but not by ER-β. Transfer of IGF-I promoter sequences from −733 to −65 or from −375 to −65 to a minimal Fos promoter resulted in a comparable responsiveness to raloxifene. This region contains two CAAT/enhancer-binding protein sites and an activator protein 1 site, both of which have been shown to be involved in estrogen receptor-mediated transactivation. When the CAAT/enhancer-binding protein sites were mutated in a construct bearing the sequence from −375 to −65 in front of the minimal Fos promoter, raloxifene induction was reduced, whereas mutation of the other elements did not affect induction. In addition, using chimeric proteins, we delineated the domains of ER-α that confer to ER-α transactivation abilities on the IGF-I promoter that are not exhibited by ER-β. These data shed new light on the mechanism of action of antiestrogens and might help explain, at least in part, the bone-protective effects observed for some antiestrogens in ovariectomized animals.

THE HEPATOCYTE NUCLEAR FACTOR 3BETA STIMULATES THE TRANSCRIPTION OF THE HUMAN INSULIN-LIKE GROWTH FACTOR I GENE IN A DIRECT AND INDIRECT MANNER

Promoter 1 (P1) of the human insulin-like growth factor I (IGF-I) gene is most active in adult liver. In this study we show that HNF-3β, a member of the winged helix protein family of liver-enriched transcription factors, has a strong stimulatory effect on the activity of P1. Transient transfection experiments in combination with bandshift and DNase I footprinting analysis revealed the presence of two HNF-3 binding sites in the proximal promoter region of P1. Both binding sites, which are well conserved in evolution, are required for maximal transactivation. Studies employing HNF-3 mutant constructs indicated that IGF-I expression is also regulated indirectly by HNF-3β as a consequence of enhanced expression of HNF-1α. This liver-enriched transcription factor has previously been shown to transactivate P1. Thus, HNF-3β regulates the expression of the human IGF-I gene via two distinct mechanisms.

Growth hormone induces insulin‐like growth factor‐I gene transcription by a synergistic action of STAT5 and HNF‐1α

Salmon insulin‐like growth factor‐I (sIGF‐I) expression is, as in mammals, induced by growth hormone (GH). To elucidate the mechanism by which GH stimulates the transcription of the IGF‐I gene, we transiently transfected Hep3B cells expressing the rat GH receptor with a sIGF‐I promoter‐luciferase reporter construct. Activation of the construct by GH added to the medium of the transfected cells was observed when two specific transcription factors, STAT5 and HNF‐1α, were simultaneously overexpressed in these cells. This finding demonstrates for the first time a GH‐dependent activation of an IGF‐I promoter construct in an immortalized laboratory cell line.

17β-Estradiol responsiveness of MCF-7 laboratory strains is dependent on an autocrine signal activating the IGF type I receptor

BackgroundHuman MCF-7 cells have been studied extensively as a model for breast cancer cell growth. Many reports have established that serum-starved MCF-7 cells can be induced to proliferate upon the sole addition of 17β-estradiol (E2). However, the extent of the mitogenic response to E2 varies in different MCF-7 strains and may even be absent. In this study we compared the E2-sensitivity of three MCF-7 laboratory strains.ResultsThe MCF-7S line is non-responsive to E2, the MCF-7 ATCC has an intermediate response to E2, while the MCF-7 NKI is highly E2-sensitive, although the levels and activities of the estrogen receptor (ER) are not significantly different. Both suramin and IGF type I receptor blocking antibodies are able to inhibit the mitogenic response to E2-treatment in MCF-7 ATCC and MCF-7 NKI cells. From this we conclude that E2-induced proliferation is dependent on IGF type I receptor activation in all three MCF-7 strains.ConclusionsThe results presented in this article suggest that E2-responsiveness of MCF-7 cells is dependent on the secretion of an autocrine factor activating the IGF-IR. All three strains of MCF-7 breast cancer cells investigated do not respond to E2 if the IGF-RI-pathway is blocked. Generally, breast cancer therapy is targeted at inhibiting estrogen action. This study suggests that inhibition of IGF-action in combination with anti-estrogen-treatment may provide a more effective way in treatment or even prevention of breast cancer.

A third human CALC (pseudo)gene on chromosome 11

A genomic locus in man (CALC‐III) containing nucleotide sequences highly homologous to both exon 2 and exon 3 of the CALC‐I and ‐II genes, is described in this paper. The CALC‐I gene produces calcitonin (CT) (encoded by exon 4) or calcitonin gene‐related peptide (CGRP) (encoded by exon 5) in a tissue‐specific fashion. The CALC‐II gene produces a second human CGRP, but probably not a second CT. The CALC‐III gene does not seem to encode a CT‐ or CGRP‐related polypeptide hormone and is probably a pseudogene. Like the other two CALC genes, the CALC‐III gene is located on human chromosome 11.