Okadaic acid-mediated induction of the c-fos gene in estrogen receptor-negative human breast carcinoma cells utilized, in part, posttranscriptional mechanisms involving adenosine-uridine-rich elements.

Signal transduction via modulation of phosphorylation after selective inhibition of protein phosphatase (PP) 1 and/or PP2A appears to play a role in okadaic acid (OA)-mediated effects. Treatment of several estrogen receptor-negative human breast carcinoma (HBC) cells with 100 nM OA resulted in induction of c-fos, c-myc, and cyclin-dependent kinase inhibitor p21WAF1/CIP1 genes. Transfections of various luciferase reporter constructs in HBC cells revealed involvement of activator protein-1-dependent as well as -independent pathways in induction of the c-fos gene by OA. MDA-MB-468 HBC cells were stably transfected with plasmids expressing luciferase, chimeric luciferase- c-fos 3' untranslated region (3'UTR), or chimeric luciferase-p21WAF1/CIP 3'UTR mRNAs. Expression of chimeric luciferase-c-fos and luciferase-p21WAF1/CIP1 mRNAs was elevated by OA in several independent sublines. Actinomycin D chase experiments revealed an enhanced rate of decay of luciferase-c-fos mRNA, whereas treatment with OA caused approximately 3.5-fold enhanced stability of the chimeric luciferase-c-fos mRNA only. By transfecting different plasmids containing deletions of c-fos 3'UTR, OA-responsive sequences were mapped to an 86-nucleotide, AU-rich region. UV cross-linking experiments using HBC cell cytosolic proteins showed multiple complexes with the AU-rich region subfragments of c-fos, as well as c-myc and p21WAF1/CIP1 mRNAs. OA enhanced binding of a novel Mr approximately 75,000 protein present in the cytosolic extracts of HBC cells to the AU-rich RNA probes of all of the above three genes. Taken together, OA regulation of HBC cell gene expression involves the activator protein-1 pathway, as well as enhanced binding of a novel Mr approximately 75,000 protein to an AU-rich region of the 3'UTRs of the target genes.     

Regulation of insulin-like growth factor-II expression and its role in autocrine growth of human neuroblastoma cells

Insulin-like growth factor-II (IGF-II) is highly expressed in fetal tissues and may act as an autocrine growth factor during early embryogenesis. The SH-SY5Y human neuroblastoma cell line also expresses IGF-II and its receptors and responds to exogenous IGF-II with increased DNA synthesis, cell division, and neuritic outgrowth. For this study, we tested the hypothesis that IGF-II mediates autocrine growth of SH-SY5Y cells in serum-free media. SH-SY5Y cells plated at high densities proliferated in serum-free media, whereas sparsely plated cells did not. IGF-II mRNA levels increased within 24 hours of serum deprivation and were associated with increased immunoreactive IGF-II protein. Exogenous addition of IGF-II increased ³H-TdR incorporation and cell number in a dose- and time-dependent fashion. By nuclear labelling experiments using 5-Bromo-2′ deoxyuridine (BrdU), we detected a twofold higher percentage of S phase nuclei after a 24-hour incubation in IGF-II. Treatment of SH-SY5Y cells with anti-IGF-II antibodies in serum-free media inhibited cell proliferation, and this inhibition was partially overcome by the addition of increasing concentrations of IGF-II. Collectively, our results indicate that IGF-II mediates an autocrine growth mechanism in SH-SY5Y cells that is associated with increased IGF-II expression. © 1993 Wiley-Liss, Inc.     

Identification of the insulin-like growth factor binding proteins 5 and 6 (IGFBP-5 and 6) in human breast cancer cells.

Four estrogen receptor-positive (ER+) [MCF-7, T47D, ZR75 and BT474] and 3 ER- [Hs578T, MDA-MB-468 and MDA-MB-231] human breast cancer cell lines were examined for expression of the IGFBP-5 and IGFBP-6 genes. Northern blot analysis revealed that all cell lines, except MDA-MB-231, expressed IGFBP-5 mRNA. IGFBP-6 mRNA, however, was expressed only by the ER- cell lines. Western immunoblotting indicated that the previously unidentified 31-kDa and 32-kDa IGF binding species secreted by these cell lines are IGFBP-5. The levels of IGFBP-4 and IGFBP-5 were increased in MCF-7 cells by estradiol and IGF-I, respectively, indicating that these BPs may contribute to the growth stimulatory response to these mitogens.     

Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells

The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the non-dividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the -701 to -921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.     

Regulation of activities of NK cells and CD4 expression in T cells by human HNP-1, -2, and -3

Human neutrophil defensin alpha (HNP) is a group of cationic peptides of diverse physiological roles. Recent studies revealed the nature of HNPs as the dominant HLA-DR binding peptides on malignant cancer cells, which may block the major histocompatibility complex for antigen presentation. Here we show that HNPs may inhibit T cells by downregulating CD4 expression, a molecule of critical importance for T cell's interaction with the target cell. HNPs also inhibited tumor-cell-lysis activities of NK cells by downregulating CD16-CD56 expression. More importantly, HNPs were markedly elevated in 14 cancer tissues out of 15 self-paired human colorectal cancers and their adjacent noncancerous tissues. The subset compositions of HNPs extracted from cancer tissues and neutrophils were identical. Immunohistochemical studies indicated that HNPs mainly distributed in the infiltrated neutrophils in the interstitium. The elevated HNPs in cancer tissues may create a microenvironment unfavorable for adaptive immune reaction, implicating the cancer evasion.     

Enhancement of insulin-like growth factor signaling in human breast cancer: estrogen regulation of insulin receptor substrate-1 expression in vitro and in vivo.

Cross-talk between insulin-like growth factor (IGF)- and estrogen receptor (ER)-signaling pathways results in synergistic growth. We show here that estrogen enhances IGF signaling by inducing expression of three key IGF-regulatory molecules, the type 1 IGF receptor (IGFR1) and its downstream signaling molecules, insulin receptor substrate (IRS)-1 and IRS-2. Estrogen induction of IGFR1 and IRS expression resulted in enhanced tyrosine phosphorylation of IRS-1 after IGF-I stimulation, followed by enhanced mitogen-activated protein kinase activation. To examine whether these pathways were similarly activated in vivo, we examined MCF-7 cells grown as xenografts in athymic mice. IRS-1 was expressed at high levels in estrogen-dependent growth of MCF-7 xenografts, but withdrawal of estrogen, which decreased tumor growth, resulted in a dramatic decrease in IRS-1 expression. Finally, we have shown that high IRS-1 expression is an indicator of early disease recurrence in ER-positive human primary breast tumors. Taken together, these data not only reinforce the concept of cross-talk between IGF- and ER-signaling pathways, but indicate that IGF molecules may be critical regulators of estrogen-mediated growth and breast cancer pathogenesis.     

Regulation of transforming growth factor-beta secretion by human peritoneal mesothelial and ovarian carcinoma cells

This study was conducted to compare the secretion of TGF-beta isoforms by human ovarian carcinoma (OVCA) cell lines (n=12) and human peritoneal mesothelial cells (HPMC;n=6) and to examine the regulation of their production by inflammatory cytokines. TGF-beta isoforms were furthermore analysed in OVCA-associated ascitic fluids. HPMC constitutively produced considerable amounts of TGF-beta1 (median 42 pg/10(5)cells; range 7-98) but only minimal amounts of TGF-beta2 (median 0.8 pg/10(5)cells; range 0-1.5). Treatment of HPMC with IL-1beta (10 ng/ml) resulted in a significant elevation of the secretion of both TGF-beta1 (median 187 pg/10(5)cells; range 71-264;P<0.001) and TGF-beta2 (median 1.8 pg/10(5)cells; range 0-13;P<0.01). In OVCA TGF-beta1 and TGF-beta2 were detected in 7/12 and 11/12 of the cell lines, respectively. The levels detected varied widely for TGF-beta1 (median 25 pg/10(5)cells; range 0-410) as well as for TGF-beta2 (median 14 pg/10(5)cells; range 0-419) and there was no correlation between the two isoforms. In contrast to HPMC, TGF-beta secretion by OVCA was not affected by any of the inflammatory cytokines tested. TGF-beta3 could not be detected in supernatants, neither in OVCA nor in HPMC. In ascitic fluids the median level of TGF-beta1 (median 5443 pg/ml; range 737-14687) was 10-fold higher than the level of TGF-beta2 (median 545 pg/ml; range 172-3537). The present data provide a model for the analysis of the molecular mechanisms of aberrant TGF-beta production by OVCA and support the hypothesis that HPMC are an important source of ascitic TGF-beta.     

Regulation of transforming growth factor gene expression in human endometrial adenocarcinoma cells

We have examined the effects of medroxyprogesterone acetate (MPA) and 4-hydroxytamoxifen (OH-TAM) on the cell proliferation and the expression of TGF- and TGF-β genes in Ishikawa cells and HEC-50 human endometrial adenocarcinoma cells. The effects of exogenous TGF-, TGF-β and anti-EGF receptor monoclonal antibody on cell proliferation were also determined. Antisense oligonucleotides were used to determine the effects of endogenous expression of TGF- and TGF-β. In both cell lines, MPA resulted in a time and dose-dependent inhibition of cell proliferation whereas OH-TAM had no effect on HEC-50 cell proliferation. The relative abundance of TGF- mRNA was significantly reduced by MPA in Ishikawa cells but not in HEC-50 cells. In Ishikawa cells, a reduction in TGF- mRNA abundance was observed with OH-TAM under conditions where both inhibition and stimulation of cell proliferation were demonstrated. Anti-EGF receptor monoclonal antibody inhibited Ishikawa cell growth but had little effect on HEC-50 cell proliferation. Exogenous TGF- stimulated proliferation of both cell lines whereas exogenous TGF-β inhibited proliferation of Ishikawa cells but stimulated proliferation of HEC-50 cells. Antisense oligonucleotides to TGF-β inhibited proliferation of HEC-50 cells. From these data we conclude that the antiproliferative effects of progestins and OH-TAM on endometrial cancer cells appear to be mediated by different mechanisms.