Riitta Koistinen

Primary structure of human insulin-like growth factor-binding protein/placental protein 12 and tissue-specific expression of its mRNA.

The low‐molecular‐mass insulin‐like growth factor‐binding protein (IGF‐BP) and placental protein 12 (PP12) are identical proteins that are present in human serum, amniotic fluid, secretory endometrium and decidua. IGF‐BP/PP12 is believed to act as an autocrine or paracrine regulator of cell growth. A cDNA clone encompassing the entire protein coding region of this protein was isolated from a human decidual cDNA library. The authenticity of the cDNA was verified by in vitro transcription/translation experiments and by the identity of the 10 N‐terminal amino acids deduced for the mature peptide with those obtained by direct protein sequencing. The amino acid sequence indicates that pre‐IGF‐BP/PP12 consists of 259 amino acid residues. The putative signal peptide is 25 residues long, and the mature protein thus contains 234 amino acids and has a molecular mass of 25 293 Da. The sequence is very cysteine‐rich at the N‐terminus after which there are regions of clustered Pro, Glu, Ser and Thr residues (so‐called PEST regions), which exist in proteins with short half‐lives. The amino acid sequence also includes an Arg‐Gly‐Asp tripeptide that may function as a cell recognition signal. The IGF‐BP/PP12 gene encodes a single 1.6 kb mRNA species that is expressed in decidua, secretory endometrium, liver and a human hepatoma cell line (HepG2). Southern blot analysis suggests that there is a single IGF‐BP/PP12 gene in the human genome.

Uterine endocrinology and paracrinology: insulin-like growth factor binding protein-1 and placental protein 14 revisited

A large number of proteins and peptides have been identified in the endometrium where they are likely to exert local biological effects. These substances may be enzymes, their inhibitors, proteinases, proteinase inhibitors, hormones, or bioactive peptides with diverse functions. Endometrial function and embryo-endometrial interactions require synchronized actions between endocrine and local factors. As examples of local factors recent studies on the insulin-like growth factor (IGF) system and placental protein 14 (PP14) are reviewed. IGF binding protein-1 (IGFBP-1) and PP14 are products of the secretory phase endometrium: IGFBP-1 is produced by decidualized stromal cells and PP14 by glandular epithelial cells. IGFBP-1 may inhibit the action of IGFs at the endometrial-trophoblastic interphase, and it may also have a role in stromal-epithelial interaction. PP14 has immunosuppressive properties, and recent findings indicate that it may play a part in the fertilization process by inhibiting binding of spermatozoa to the zona.

HUMAN GRANULOSA CELLS CONTAIN INSULIN‐LIKE GROWTH FACTOR‐BINDING PROTEIN (IGF BP‐1) mRNA

Previous studies have demonstrated expression of insulin‐like growth factor‐binding protein (IGF BP‐1) in secretory and decidualized endometrium, in adult and fetal liver, and in HepG2 liver cancer cells. We have studied the expression of IGF BP‐1 in various types of ovarian neoplasias, normal ovary, and granulosa cells from hyperstimulated human ovarian follicles by RNA blot hybridization. A single 1.6 kb mRNA species, similar to that present in human decidua, was identified in poly(A)RNA‐containing preparations of granulosa cells and of a borderline malignant ovarian cystadenoma. This finding verifies the postulated production of IGF BP‐1 by the human ovary.

The gene encoding human low-molecular weight insulin-like growth-factor binding protein (IGF-BP25): regional localization to 7p12-p13 and description of a DNA polymorphism

SummaryThe low-molecular weight insulin-like growth-factor binding protein (IGF-BP25) is synthesized by human liver, secretory endometrium and decidua, and is also present in human serum. It binds insulin-like growth factors IGF-I and IGF-II with high affinity, and is proposed to act as a paracrine regulator of cell growth. In situ hybridization studies with a cDNA encompassing the entire protein coding region of IGF-BP25 localized the gene to bands p12–p13 on chromosome 7. Southern blot analysis with the enzyme BglII revealed a common restriction fragment length polymorphism: the presence of the polymorphic BglII site results in the formation of two fragments 4.6 kb and 1.6 kb in size whereas its absence produces a single 6.2 kb fragment. The frequencies of the two alleles were 0.73 and 0.27, respectively. IGF-BP25 constitutes a useful genetic marker for the proximal short arm of chromosome 7.

Placental protein 5 is related to blood coagulation and fibrinolytic systems

Previous studies have shown that placental protein 5 (PP5) forms complexes with heparin. In order to further elucidate the biological role of PP5 we studied the effect of plasmin and thrombin on the immunoreactivity of PP5, and the possible functional antiplasmin and antithrombin effects of purified PP5. Varying concentrations of plasmin and thrombin were added to pregnancy plasma, and the PP5 levels, measured by radioimmunoassay, were found to be elevated by 558% (plasmin and 48-87% (thrombin). Incubation of radiolabeled PP5 with plasmin resulted in the formation of radioactive fragments with smaller molecular weights. Functional studies using a chromogenic substrate confirmed that purified PP5 has an antiplasmin activity. An average increase of 15% was observed in the antiplasmin activity when 200 ng purified PP5 was added to 150 microliters of pregnancy serum. Thus, there are certain similarities between PP5 and antihrombin III. Both form complexes with heparin and have antiplasmin properties, and both were found to be heat labile. But, functional studies utilizing a chromogenic substrate failed to demonstrate any antithrombin III-like activity in the purified PP5 preparation that had antiplasmin activity. Our results show that the function of PP5 is related to the blood coagulation and fibrinolytic systems, at least through its inhibitory action on plasmin.

The effect of estrogen level on glucose-induced changes in serum insulin-like growth factor binding protein-1 concentration *

Objective To investigate the regulation of insulin-like growth factor binding protein-1 (IGFBP-1) concentration during ovarian stimulation. Design A prospective study of patients undergoing in vitro fertilization treatment. Setting Infertility unit at the University Central Hospital of Oulu, a tertiary referral center. Patients Sixteen healthy, regularly menstruating lean tubal infertility patients. Interventions Oral glucose tolerance test was performed first in a hypoestrogenic state after suppression by long-term gonadotropin-releasing hormone (GnRH) agonist and, second, in a hyperestrogenic state after stimulation by human menopausal gonadotropins. Main Outcome Measures Serum concentrations of IGFBP-1, insulin-like growth factor I (IGF-I), insulin and sex hormone-binding globulin were measured before and 2hours after glucose administration. Results Before and after glucose administration, the serum IGFBP-1 concentrations were significantly higher in the hyperestrogenic state (estradiol [E 2 ] level 3.5±0.57nmol/L) after ovarian stimulation than in the GnRH-analogue-induced hypoestrogenic state before the gonadotropin treatment (E 2 level 0.10±0.02nmol/L). On both occasions glucose-induced hyperinsulinemia caused a significant decrease in the circulating IGFBP-1 levels, whereas the IGF-I levels remained unchanged. There was a significant correlation between E 2 and the insulin-suppressed IGFBP-1 level. The sum of follicular diameters correlated positively with the serum IGFBP-1 concentration. Conclusions Gonadotropin-induced hyperestrogenism is related to elevated serum IGFBP-1 levels, either via estrogen-stimulated synthesis or via increased contribution from multiple follicles. Glucose-induced hyperinsulinemia suppresses serum IBFBP-1 concentration equally both in the hypoestrogenic and hyperestrogenic states. Because of similar IGF-I levels, it is likely that the biological activity of IGF-I is different before and after gonadotropin stimulations.

Mutations in two short noncoding mononucleotide repeats in most microsatellite-unstable colorectal cancers.

DNA mismatch repair (MMR)-deficient cells typically accumulate mutations in short repetitive DNA tracts. This microsatellite instability (MSI) facilitates malignant transformation when affecting genes with growth-related and caretaker functions. To date, several putative MSI target genes have been proposed mainly based on high mutation frequency within their coding regions. However, some intronic repeat mutations have also been suggested to associate with MSI tumorigenesis, indicating the need for additional analyses on noncoding repeats. Here we have analyzed an intronic T9 repeat of semenogelin I (SEMG1) and report mutation frequencies of 51% (75 of 146) and 62% (8 of 13) in MMR-deficient primary colorectal cancers and cell lines, respectively. The putative effect of the SEMG1 mutations was assessed by RNA and protein level analyses, but no differences were detected between colorectal cancer cell lines with different SEMG1 status. Subsequently, the general background mutation frequency of MSI colorectal cancers was assessed by screening for intergenic T9 repeat alterations. One of 10 examined repeats was mutated in 70% (102 of 145) of the colorectal cancers evaluated. The frequencies observed here are notably higher than previously published in noncoding repeats shorter than 10 bp in MMR-deficient primary tumors. Our results indicate that high mutation frequencies, similar or higher than those observed in proposed and approved target genes, can be detected in repeat tracts of MSI tumors without any apparent selection pressure. These data call for urgent and thorough large-scale evaluation of mutation frequencies in neutral short repetitive sequences in MMR-deficient tumors.

Use of a rapid radioimmunoassay for pregnancy-specific beta-1 glycoprotein in gynecologic emergencies: Comparison with routine urinary pregnancy test and a rapid radioimmunoassay for human chorionic gonadotropin

The use of a rapid test for pregnancy-specific beta-1 glycoprotein (SP1) in emergency patients is described and the results of using this test on 141 women with gynecologic emergencies are presented. Serum samples were obtained from 141 women of reproductive age with lower abdominal pain or vaginal bleeding. The radioimmunoassay for SP1 used only 1 standard consisting of pregnancy serum diluted with normal female serum to give an SP1 concentration of 20 mcgm/liter. Serum (undiluted) was incubated with anti-SP1 antiserum diluted to yield 30% binding in zero standard and iodinated SP1 at 37 degrees centigrade for 3 hours. Antibody-bound material was precipitated and the result was judged positive if radioactivity in the precipitate was less than that in the reference tube. Results were compared with routine urinary pregnancy tests and rapid serum human chorionic gonadotropin beta subunit radioimmunoassay. Clinical/histological evidence of complicated intrauterine or ectopic pregnancy was found in 38 patients (27%). Rapid SP1 radioimmunoassay was positive in 18% of all patients rapid chorionic radioimmunoassay was positive in 24% and urinary tests were positive in 7%. 17 patients had verified ectopic pregnancy and among these cases 59% were positive by SP1 23% by urinary test and all 17 patients were positive by chorionic gonadotropin.

Characterisation, Biological Action and Clinical Studies of Endometrial Proteins

Placental proteins 12 and 14 (PP12 and PP14) were originally isolated from the placenta (Bohn and Kraus 1980; Bohn et al. 1982) but were later shown to derive predominantly from decidua and endometrium (Rutanen et al. 1986; Julkunen et al. 1986a—c). Both are abundant in amniotic fluid (Rutanen et al. 1982; Julkunen et al. 1985a, b). We have characterised human placental/decidual PP12 and PP14 and purified these proteins from human mid-trimester amniotic fluid. In this chapter we shall review these studies and our recent research on the biological action and clinical significance of PP12 and PP14.

Abstract 4066: Glycodelin abolishes PMA-induced migration of MCF-7 breast cancer cells

Glycodelin is a lipocalin protein mainly expressed in well-differentiated epithelial cells in reproductive tissues. Previously, glycodelin has been shown to induce cell differentiation in endometrial and breast cancer cells. We have used glycodelin-transfected MCF-7 breast cancer cells to study the glycodelin-induced differentiation. Glycodelin expression changed the cellular morphology towards normal, less malignant direction. In addition, glycodelin-producing cells formed significantly smaller tumors in xenograft mice. We have found that the effects of glycodelin are at least partly mediated by repressed PKCδ activity, rendering the cells resistant to activation of phorbol 12-myristate 13-acetate (PMA), a known activator of PKCδ. Here we continued these previous studies by addressing the effect of glycodelin on the invasion and migration of MCF-7 breast cancer cells, and studying the affected signaling route in more detail. Activation of signaling proteins was detected with an antibody array of 43 phospho kinases. Cell migration was studied using wound healing test. The cells were grown on chamber slides until they formed a monolayer and then a scratch was made using a pipet tip. After addition of PMA or DMSO control, the migration of the cells was monitored under microscope. In some of the control cells, PKCδ was down-regulated with RNAi prior the migration assay. For invasion through Matrigel basement membrane preparation, a Boyden chamber assay was used. The cells were grown with and without PMA and the amount of invasive cells was quantified. Phospho-kinase array did not reveal any widespread changes in the amount of phosphorylated signaling molecules between the glycodelin-producing and control cells irrespective of the PMA treatment. However, in addition to PKCδ some changes were found in the levels of phosphoproteins such as p38α and p27. In wound healing test PMA caused a two-fold increase in the migration of the control cells, but had no effect on the glycodelin-producing cells. When the expression of PKCδ was blocked in the control cells using siRNA, the addition of PMA did not increase the migration of the cells. PMA also increased the invasiveness of the cells, but that was detected in both cells clones regardless of glycodelin expression. Without PMA treatment both glycodelin-expressing and control cells behaved similarly in wound healing and invasion tests. In conclusion, glycodelin abolished PMA-induced migration in MCF-7 breast cancer cells, while it did not have any effect on the invasion of the cells. Our results suggest that the effect of glycodelin in migration is mediated by reduced activation of PKCδ. Citation Format: Laura C. Hautala, Riitta Koistinen, Hannu Koistinen. Glycodelin abolishes PMA-induced migration of MCF-7 breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4066. doi:10.1158/1538-7445.AM2014-4066