Cripto Research Articles

FGF9 - Nodal signaling negatively control meiotic entry of postnatal male germ cells

Fibroblast growth factor 9 (FGF9) produced by the somatic cells of the testis acts directly on germ cells to inhibit meiosis by upregulating levels of the RNA binding protein Nanos2, both in fetal and postnatal gonad [1]. Several studies have recently demonstrated that the anti-meiotic effect of FGF9 in the fetal testis is mediated by Nodal, a member of the TGF-Beta morphogen family. In fetal male germ cells FGF9 upregulates Cripto, which is a Nodal co-receptor, making these cells unable to enter meiosis [2]. However it is currently unknown how FGF9 acts postnatally to inhibit entry into meiosis of male mitotic germ cells. We first investigated which was the signal transduction pathway activated by FGF9 on postnatal mouse spermatogonia. We found that FGF9 signaling is dependent on ERK but not on AKT activation, which is instead triggered by Kit ligand (an inducer of meiotic entry that cooperates with retinoic acid). By qRT-PCR we found that Nodal and Cripto are expressed in postnatal spermatogonia and that FGF9 (but not Kit ligand) treatment promotes their upregulation. By western blot we found that phospho-Smad2, an indicator of active Nodal signaling, is increased in spermatogonia upon FGF9 treatment, suggesting that Nodal mediates FGF9 action also in postnatal male germ cells.

EGF-related antisense oligonucleotides inhibit the proliferation of human ovarian carcinoma cells

The epidermal growth factor (EGF)-like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor alpha (TGFalpha) are expressed in human ovarian carcinomas. The expression of AR, CR and TGFalpha in ovarian carcinoma cell lines was assessed by immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). The antiproliferative effects of antisense phosphorothioate oligodeoxynucleotides (AS S-Oligos) directed against either AR, CR or TGFalpha was evaluated by using a clonogenic assay. A majority of the ovarian carcinoma cell lines was found to express TGFalpha, AR and CR mRNAs and proteins. AS S-Oligos directed against either AR, CR or TGFalpha were able to inhibit the anchorage-independent growth of NIH:OVCAR3 and NIH:OVCAR8 cells in a dose dependent manner. A 30%-50% growth inhibition was observed at a 2 microM concentration of the AS S-Oligos. Treatment of these cells with combinations of EGF-related AS S-Oligos resulted in a more significant growth inhibition when compared to treatment with a single AS S-oligo. A 60%-75% growth inhibition was observed using combinations of AR, CR and TGFalpha AS S-oligos at a total concentration of 2 microM. An additive growth-inhibitory effect occurred when ovarian carcinoma cells were exposed to the AS S-Oligos after treatment with either paclitaxel or cis-platinum. These data suggest that EGF-related peptides function as autocrine growth factors in ovarian carcinoma cells, and that they might represent targets for experimental therapy of ovarian carcinoma.

EGF-related peptides are involved in the proliferation and survival of MDA-MB-468 human breast carcinoma cells.

A majority of human breast carcinomas co-express the epidermal growth factor (EGF)-like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor alpha (TGF-alpha). MDA-MB-468 breast carcinoma cells express CR, AR and TGFalpha, while SK-BR-3 cells express CR and TGF-alpha. Anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against either CR or TGF-alpha inhibit the proliferation of both cell lines. A 40-50% growth inhibition was observed at a 2-microM concentration of each AS S-oligo. Treatment with the AR AS S-oligo also resulted in a significant inhibition of MDA-MB-468 anchorage dependent growth (ADG). No significant growth inhibition was observed when MDA-MB-468 or SK-BR-3 cells were treated with a mis-sense S-oligo. The AS S-oligos inhibited the expression of AR, CR or TGF-alpha proteins and mRNAs, as assessed by immuno-cytochemistry and semi-quantitative RT-PCR. An additive growth-inhibitory effect was observed when MDA-MB-468 cells were treated with a combination of EGF-related AS S-oligos. Indeed, treatment of MDA-MB-468 cells with a combination of AR, CR and TGF-alpha AS S-oligos resulted in about 70% growth inhibition at a concentration of 0.7 microM each. Finally, treatment of MDA-MB-468 cells with a combination either of the 3 AS S-oligos or of an EGF receptor-blocking antibody (MAb 225) and either CR, AR or TGFalpha AS S-oligos resulted in a significant increase in DNA fragmentation. Our data suggest that the EGF-related peptides are involved in the proliferation and survival of breast carcinoma cells.

Anti-sense oligonucleotides directed against EGF-related growth factors enhance anti-proliferative effect of conventional anti-tumor drugs in human colon-cancer cells.

We have demonstrated that anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against the EGF-like growth factors CRIPTO (CR), amphiregulin (AR) or transforming-growth-factor-alpha(TGFalpha) mRNA, are equipotent in their ability to inhibit the growth of human colon-carcinoma GEO cells. In this study, we evaluated the effect of combinations of these AS S-oligos and conventional anti tumor drugs, such as 5-fluorouracil (5-FU), adriamycin (ADR), mitomycin C (MIT) and cis-platinum (CDDP), on GEO cell growth. Dose-dependent growth inhibition was observed by treatment either with AS S-oligos or with anti-tumor drugs, using a clonogenic assay. Furthermore, an additive growth inhibitory effect occurred when GEO cells were exposed to the AS S-oligos after treatment with different concentrations of either 5-FU, MIT, ADR or CDDP. For example, treatment of GEO cells with a combination of low concentrations of 5-FU and any of the 3 AS S-oligos resulted in up to 70% growth inhibition. However, treatment of GEO cells with AS S-oligos before exposure to 5-FU or CDDP resulted in reduced efficacy of both drugs. Flow-cytometric analysis of DNA content demonstrated that treatment with the AS S-oligos caused a slight reduction of the percentage of cells in the S-phase of the cell cycle. These data suggest that combinations of AS S-oligos directed against EGF-related growth factors and of conventional anti-tumor drugs may result in efficient inhibition of colon-carcinoma cell growth.

Anti‐sense oligonucleotides directed against EGF‐related growth factors enhance anti‐proliferative effect of conventional anti‐tumor drugs in human colon‐cancer cells

We have demonstrated that anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against the EGF-like growth factors CRIPTO (CR), amphiregulin (AR) or transforming-growth-factor-α(TGFα) mRNA, are equipotent in their ability to inhibit the growth of human colon-carcinoma GEO cells. In this study, we evaluated the effect of combinations of these AS S-oligos and conventional anti-tumor drugs, such as 5-fluorouracil (5-FU), adriamycin (ADR), mitomycin C (MIT) and cis-platinum (CDDP), on GEO cell growth. Dose-dependent growth inhibition was observed by treatment either with AS S-oligos or with anti-tumor drugs, using a clonogenic assay. Furthermore, an additive growth-inhibitory effect occurred when GEO cells were exposed to the AS S-oligos after treatment with different concentrations of either 5-FU, MIT, ADR or CDDP. For example, treatment of GEO cells with a combination of low concentrations of 5-FU and any of the 3 AS S-oligos resulted in up to 70% growth inhibition. However, treatment of GEO cells with AS S-oligos before exposure to 5-FU or CDDP resulted in reduced efficacy of both drugs. Flow-cytometric analysis of DNA content demonstrated that treatment with the AS S-oligos caused a slight reduction of the percentage of cells in the S-phase of the cell cycle. These data suggest that combinations of AS S-oligos directed against EGF-related growth factors and of conventional anti-tumor drugs may result in efficient inhibition of colon-carcinoma cell growth. Int. J. Cancer 73:277–282, 1997. © 1997 Wiley-Liss, Inc.

Expression of epidermal growth factor (EGF) family and expression of epidermal growth factor receptor (EGFR) in human chorionic villi

Summary Among epidermal growth factor (EGF) family growth factors, EGF, transforming growth factor-α (TGF-α) and amphiregulin (AR) are considered to exert their effects through binding to epidermal growth factor receptor (EGFR). Cripto (CR) is homologous to EGF in its structure, but it is postulated that it might not bind with EGFR. We examined localization of TGF-α, AR, CR and EGFR in human chorionic villi of various stages. Chorionic villi were collected from thirty-four cases. Immunohistochemistry was performed on 4% paraformaldehyde fixed and paraffin embedded specimens using an avidin biotin complex-perioxidase technique. TGF-α immunoreactivity was intense in the cytoplasm of the cytotrophoblast (CT), especially in first trimester. AR immunoreactivity was observed predominantly in the nuclei of the CT and some syncytiotrophoblast (ST) only in first trimester. EGFR immunoreactivity was intense in the ST, and weak in the CT and its frequency appeared to decrease as gestational period progressed. CR immunoreactivity was mainly observed in the ST, but marked heterogeneity was observed in its expression and its frequency appeared to increase as pregnancy progressed. Based on these findings, TGF-α is considered to act mainly in a paracrine manner in first trimester chorionic villi. AR is produced to modify the biological function of the trophoblast cells during the first trimester. In contrast, CR may modify ST function, especially during the third trimester.

Expression of epidermal growth factor family proteins and epidermal growth factor receptor in human endometrium

The biological significance of epidermal growth factor (EGF)—related proteins in the development and progression of endometrioid endometrial carcinoma was studied. Expression of EGF-related proteins including EGF, transforming growth factor-α (TGF-α), cripto (CR), amphiregulin (AR), and EGF receptor (EGFR) were immunohistochemically examined. Results were then correlated with clinicopathologic findings and steroid receptor status in 12 specimens with normal endometrium, 13 with endometrial hyperplasia, and 40 with endometrioid endometrial carcinoma. EGFR, EGF, TGF-α, and CR iinmunoreactivities were observed in 58.3%, 66.7%, 91.6%, and 66.7% of normal endometrial specimens; 100%, 15.4%, 100%, and 30.8% of endometrial hyperplasia specimens; and 67.5%, 32.5%, 65.0%, and 65.0% of endometrial carcinoma specimens, respectively. AR immunoreactivity was not observed in any of the normal, hyperplastic, or neoplastic endometrium. The presence or absence of EGFR or TGF-α in endometrial carcinoma correlated with surgical stage, depth of myometrial invasion, and findings from peritoneal washing cytology. EGF expression significantly correlated with the age of the patients and that of CR with surgical stage and peritoneal washing cytological findings. There was a significant correlation between EGFR and TGF-α expression, and between EGF and TGF-α. Coexpression of EGFR and TGF-α, EGFR and CR, and TGF-α and CR in carcinoma specimens significantly correlated with advanced surgical stage, deeper myometrial invasion, and positive peritoneal washing cytology. In normal as well as hyperplastic endometrium, endometrial glands immunohistochemically positive for TGF-α were generally positive for ER, but in poorly differentiated endometrial carcinoma, cells positive for TGF-α tended to be negative for ER. The results of the present study show that among EGF-related proteins, expression of TGF-α and CR seem to be associated with the progression of human endometrioid endometrial carcinoma. Additionally, expression of TGF-α became increasingly estrogen independent with increasing histological carcinoma grades.

Expression of amphiregulin and transforming growth factor alpha mRNA in gestational tissue at term

Among epidermal growth factor (EGF) family growth factors, EGF, transforming growth factor-α (TGF-α) and amphiregulin (AR) are considered to exert their effects through binding to epidermal growth factor receptor (EGFR). Cripto (CR) is homologous to EGF in its structure, but it is postulated that it might not bind with EGFR. We examined localization of TGF-α, AR, CR and EGFR in human chorionic villi of various stages. Chorionic villi were collected from thirty-four cases. Immunohistochemistry was performed on 4% paraformaldehyde fixed and paraffin embedded specimens using an avidin biotin complex-perioxidase technique. TGF-α immunoreactivity was intense in the cytoplasm of the cytotrophoblast (CT), especially in first trimester. AR immunoreactivity was observed predominantly in the nuclei of the CT and some syncytiotrophoblast (ST) only in first trimester. EGFR immunoreactivity was intense in the ST, and weak in the CT and its frequency appeared to decrease as gestational period progressed. CR immunoreactivity was mainly observed in the ST, but marked heterogeneity was observed in its expression and its frequency appeared to increase as pregnancy progressed. Based on these findings, TGF-α is considered to act mainly in a paracrine manner in first trimester chorionic villi. AR is produced to modify the biological function of the trophoblast cells during the first trimester. In contrast, CR may modify ST function, especially during the third trimester.