Transcriptome analysis revealed the dynamic oil accumulation in Symplocos paniculata fruit.

BACKGROUND: Symplocos paniculata, asiatic sweetleaf or sapphire berry, is a widespread shrub or small tree from Symplocaceae with high oil content and excellent fatty acid composition in fruit. It has been used as feedstocks for biodiesel and cooking oil production in China. Little transcriptome information is available on the regulatory molecular mechanism of oil accumulation at different fruit development stages. RESULTS: The transcriptome at four different stages of fruit development (10, 80,140, and 170 days after flowering) of S. paniculata were analyzed. Approximately 28 million high quality clean reads were generated. These reads were trimmed and assembled into 182,904 non-redundant putative transcripts with a mean length of 592.91 bp and N50 length of 785 bp, respectively. Based on the functional annotation through Basic Local Alignment Search Tool (BLAST) with public protein database, the key enzymes involved in lipid metabolism were identified, and a schematic diagram of the pathway and temporal expression patterns of lipid metabolism was established. About 13,939 differentially expressed unigenes (DEGs) were screened out using differentially expressed sequencing (DESeq) method. The transcriptional regulatory patterns of the identified enzymes were highly related to the dynamic oil accumulation along with the fruit development of S. paniculata. In addition, quantitative real-time PCR (qRT-PCR) of six vital genes was significantly correlated with DESeq data. CONCLUSIONS: The transcriptome sequences obtained and deposited in NCBI would enrich the public database and provide an unprecedented resource for the discovery of the genes associated with lipid metabolism pathway in S. paniculata. Results in this study will lay the foundation for exploring transcriptional regulatory profiles, elucidating molecular regulatory mechanisms, and accelerating genetic engineering process to improve the yield and quality of seed oil of S. paniculata.

Advantages of Binomial Likelihood Maximization for Analyzing and Modeling Cell Survival Curves.

Mathematical analysis of cell survival allows parameter estimation for radiobiological models and selection of an appropriate model. To our knowledge, no rigorous comparisons on the accuracy of various methods used for such analysis have been performed. In this study we compared three methods: 1. maximization of binomial log-likelihood (BLL); 2. minimization of sum of squares (SS); and 3. method 2 using log-transformed data (SSlog). Analysis of Monte Carlo simulated data (A) generated from the linear-quadratic (LQ) model showed that model parameter estimates from the BLL method were more accurate and less affected by "noise" than those from other methods. Analysis of actual breast cancer cell data showed substantial differences among LQ parameters estimated by the three methods (B). To select among radiobiological models, we used: 1. Sample size-corrected Akaike information criterion (AICc), calculated from BLL method-generated log-likelihood values; and 2. Adjusted coefficient of determination (R(2)), calculated from SS/SSlog method-generated SS values. Analysis of data simulated from the repair-misrepair (RMR) formalism (C) showed that the first approach outperformed the second approach at identifying the true data-generating model. Examples of how the first approach discriminates between several models were explored using actual mouse (H2AX-proficient and -deficient) and human [DNA-dependent protein kinase (DNA-PK)-proficient and -deficient] cell data (D). Based on this work, we concluded that BLL maximization combined with AICc-based model selection constitutes an effective method for analyzing cell survival data.

Regulation of DNA Damage Response by Estrogen Receptor ß-Mediated Inhibition of Breast Cancer Associated Gene 2.

Accumulating evidence suggests that ubiquitin E3 ligases are involved in cancer development as their mutations correlate with genomic instability and genetic susceptibility to cancer. Despite significant findings of cancer-driving mutations in the BRCA1 gene, estrogen receptor (ER)-positive breast cancers progress upon treatment with DNA damaging-cytotoxic therapies. In order to understand the underlying mechanism by which ER-positive breast cancer cells develop resistance to DNA damaging agents, we employed an estrogen receptor agonist, Erb-041, to increase the activity of ERß and negatively regulate the expression and function of the estrogen receptor α (ERα) in MCF-7 breast cancer cells. Upon Erb-041-mediated ERα down-regulation, the transcription of an ERα downstream effector, BCA2 (Breast Cancer Associated gene 2), correspondingly decreased. The ubiquitination of chromatin-bound BCA2 was induced by ultraviolet C (UVC) irradiation but suppressed by Erb-041 pretreatment, resulting in a blunted DNA damage response. Upon BCA2 silencing, DNA double-stranded breaks increased with Rad51 up-regulation and ataxia telangiectasia mutated (ATM) activation. Mechanistically, UV-induced BCA2 ubiquitination and chromatin binding were found to promote DNA damage response and repair via the interaction of BCA2 with ATM, γH2AX and Rad51. Taken together, this study suggests that Erb-041 potentiates BCA2 dissociation from chromatin and co-localization with Rad51, resulting in inhibition of homologous recombination repair.

Induction chemotherapy with docetaxel, cisplatin and fluorouracil followed by surgery and concurrent chemoradiotherapy improves outcome of recurrent advanced head and neck squamous cell carcinoma.

BACKGROUND: Locally recurrent rate of advanced head and neck squamous cell carcinoma (HNSCC) still remains high and the treatment is controversial. PATIENTS AND METHODS: We retrospectively analyzed ninety-three patients with recurrent advanced oral cancer from 2009 to 2013. Sixty-four patients are in the docetaxel with cisplatin and 5'-fluorouracil (TPF) group and the remaining twenty-nine patients are in the cisplatin and 5'-fluorouracil (PF) group. RESULTS: The overall response rate was better in the TPF group (p=0.005) than the PF group. Patients who received induction chemotherapy, TPF, followed by surgery and concurrent chemoradiotherapy (CRT) had better overall survival (OS) (p=0.012) and progression-free survival (PFS) (p=0.038), while patients with prior intra-arterial-infusion-chemotherapy had an adverse impact on OS (p=0.039). CONCLUSION: We showed that induction chemotherapy with TPF, followed by surgery and consolidation CRT, is the ideal choice for recurrent advanced HNSCC with improving response rates and survival. However, prior intra-arterial-infusion-chemotherapy showed an adverse impact.

Ursolic Acid-Regulated Energy Metabolism-Reliever or Propeller of Ultraviolet-Induced Oxidative Stress and DNA Damage?

Ultraviolet (UV) light is a leading cause of diseases, such as skin cancers and cataracts. A main process mediating UV-induced pathogenesis is the production of reactive oxygen species (ROS). Excessive ROS levels induce the formation of DNA adducts (e.g., pyrimidine dimers) and result in stalled DNA replication forks. In addition, ROS promotes phosphorylation of tyrosine kinase-coupled hormone receptors and alters downstream energy metabolism. With respect to the risk of UV-induced photocarcinogenesis and photodamage, the antitumoral and antioxidant functions of natural compounds become important for reducing UV-induced adverse effects. One important question in the field is what determines the differential sensitivity of various types of cells to UV light and how exogenous molecules, such as phytochemicals, protect normal cells from UV-inflicted damage while potentiating tumor cell death, presumably via interaction with intracellular target molecules and signaling pathways. Several endogenous molecules have emerged as possible players mediating UV-triggered DNA damage responses. Specifically, UV activates the PIKK (phosphatidylinositol 3-kinase-related kinase) family members, which include DNA-PKcs, ATM (ataxia telangiectasia mutated) and mTOR (mammalian target of rapamycin), whose signaling can be affected by energy metabolism; however, it remains unclear to what extent the activation of hormone receptors regulates PIKKs and whether this crosstalk occurs in all types of cells in response to UV. This review focuses on proteomic descriptions of the relationships between cellular photosensitivity and the phenotypic expression of the insulin/insulin-like growth receptor. It covers the cAMP-dependent pathways, which have recently been shown to regulate the DNA repair machinery through interactions with the PIKK family members. Finally, this review provides a strategic illustration of how UV-induced mitogenic activity is modulated by the insulin sensitizer, ursolic acid (UA), which results in the metabolic adaptation of normal cells against UV-induced ROS, and the metabolic switch of tumor cells subject to UV-induced damage. The multifaceted natural compound, UA, specifically inhibits photo-oxidative DNA damage in retinal pigment epithelial cells while enhancing that in skin melanoma. Considering the UA-mediated differential effects on cell bioenergetics, this article reviews the disparities in glucose metabolism between tumor and normal cells, along with (peroxisome proliferator-activated receptor-γ coactivator 1α)-dependent mitochondrial metabolism and redox (reduction-oxidation) control to demonstrate UA-induced synthetic lethality in tumor cells.

Trp53 regulates Notch 4 signaling through Mdm2.

Notch receptors and their ligands have crucial roles in development and tumorigenesis. We present evidence demonstrating the existence of an antagonistic relationship between Notch 4 and Trp53, which is controlled by the Mdm2-dependent ubiquitylation and degradation of the Notch receptor. We show that this signal-controlling mechanism is mediated by physical interactions between Mdm2 and Notch 4 and suggest the existence of a trimeric complex between Trp53, Notch 4 and Mdm2, which ultimately regulates Notch activity. Functional studies indicate that Trp53 can suppress NICD4-induced anchorage-independent growth in mammary epithelial cells and present evidence showing that Trp53 has a pivotal role in the suppression of Notch-associated tumorigenesis in the mammary gland.

Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1.

Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor-like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum-Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the posttranslational maturation of Notch receptors.

Smad2 functions as a co-activator of canonical Wnt/beta-catenin signaling pathway independent of Smad4 through histone acetyltransferase activity of p300.

Both canonical Wnt/beta-catenin and TGFbeta/Smad signaling pathways coordinately regulate pattern formation during embryogenesis as well as tumor progression. Evidence of cross-talk between these two pathways has been reported. Here we demonstrated that the Activin-like kinase 4 (Alk4)/Smad2 pathway facilitates the transcriptional activity of the oncogenic Wnt/beta-catenin/Tcf4 pathway through a novel Smad4-independent mechanism. Upon activation, Smad2 physically interacted with Tcf4, beta-catenin and the co-activator p300 to enhance transcriptional activity of beta-catenin/Tcf4 through the histone acetyltransferase activity of p300. Transactivation by Smad2 was independent of a Smad-binding element (SBE) and Smad4. Indeed, the enhancement of beta-catenin/Tcf4 transcriptional activity by activated Smad2 was negatively regulated by the presence of Smad4. Moreover, a tumor-derived missense mutant of Smad2, lacking the ability to bind to Smad4 was still able to enhance the Tcf4 transcriptional reporter in the presence of beta-catenin and Tcf4. Our findings suggest that Smad2 may function as an activator of canonical Wnt/beta-catenin/Tcf4 signaling through a SBE/Smad4-independent pathway.

Netrin-1 regulates invasion and migration of mouse mammary epithelial cells overexpressing Cripto-1 in vitro and in vivo.

The neuronal guidance molecule, Netrin-1, has been suggested to play a role in the adhesion and migration of the mammary gland epithelium. Human and mouse Cripto-1 induce proliferation, migration, invasion and colony formation by epithelial cells in 3D matrices. Here we investigate whether Netrin-1 affects these Cripto-1-dependent activities in mouse mammary epithelial cells. Overexpression of Cripto-1 in EpH4 and HC-11 cells (EpH4/Cripto-1 or HC-11/Cripto-1) was associated with low expression of Netrin-1 and increased expression of its receptor Neogenin compared to that of wild-type cells. No change was observed in the expression of the other Netrin-1 receptor, UNC5H1. Treating EpH4/Cripto-1 or HC-11/Cripto-1 mammary cells with exogenous soluble Netrin-1 resulted in increased expression of E-cadherin and UNC5H1, decreased expression of vimentin and decreased activation of Akt as determined by western blotting. Colony formation by Eph4/Cripto-1 cells in 3D gels was significantly reduced in proximity to a Netrin-1 source, and mammary glands of transgenic mice overexpressing human Cripto-1 showed altered ductal growth in proximity to implanted Netrin-1-releasing pellets. Terminal end buds in the treated transgenic mice mammary glands also showed increased expression of E-cadherin and UNC5H1 and decreased expression of active Akt determined by immunohistochemistry. Together, these results suggest that regulation of Netrin-1 expression is important in regulating Cripto-1-dependent invasion and migration of mammary epithelial cells.

Modulation of TGF-beta signaling by EGF-CFC proteins.

Members of the transforming growth factor-beta (TGF-beta) family of ligands exhibit potent growth-suppressive and/or apoptosis-inducing effects on different types of cells. They perform essential roles in the elimination of damaged or abnormal cells from healthy tissues. On the other hand, TGF-betas have also been shown to act as tumor-promoting cytokines in a number of malignancies that are capable of stimulating extracellular matrix production, cell migration, invasion, angiogenesis, and immune suppression. Dissecting the complex, multifaceted roles of different TGF-beta-related peptides especially during the development of pathological conditions and in carcinogenesis is an area of continuous research and development. The characterization of EGF-CFC proteins as essential co-receptors that contribute to the modulation of the physiological activities of some of the TGF-beta ligands will be beneficial for future medical research and the adaptation and possible readjustment of currently applied therapeutic regimes.

Overexpression of human Cripto-1 in transgenic mice delays mammary gland development and differentiation and induces mammary tumorigenesis.

Overexpression of Cripto-1 has been reported in several types of human cancers including breast cancer. To investigate the role of human Cripto-1 (CR-1) in mammary gland development and tumorigenesis, we developed transgenic mice that express the human CR-1 transgene under the regulation of the whey acidic protein (WAP) promoter in the FVB/N mouse background. The CR-1 transgene was detected in the mammary gland of 15-week-old virgin WAP-CR-1 female mice that eventually developed hyperplastic lesions. From mid-pregnancy to early lactation, mammary lobulo-alveolar structures in WAP-CR-1 mice were less differentiated and delayed in their development due to decreased cell proliferation as compared to FVB/N mice. Early involution, due to increased apoptosis, was observed in the mammary glands of WAP-CR-1 mice. Higher levels of phosphorylated AKT and MAPK were detected in mammary glands of multiparous WAP-CR-1 mice as compared to multiparous FVB/N mice suggesting increased cell proliferation and survival of the transgenic mammary gland. In addition, more than half (15 of 29) of the WAP-CR-1 multiparous female mice developed multifocal mammary tumors of mixed histological subtypes. These results demonstrate that overexpression of CR-1 during pregnancy and lactation can lead to alterations in mammary gland development and to production of mammary tumors in multiparous mice.

Notch4 intracellular domain binding to Smad3 and inhibition of the TGF-beta signaling.

We present evidence that Notch4ICD attenuates TGF-beta signaling. Cells expressing the activated form of the Notch4 receptor (ICD4) were resistant to the growth-inhibitory effects of TGF-beta. Notch4ICD was found to bind to Smad2, Smad3 and Smad4 but with higher affinity to Smad3. Deletion analysis showed that binding of Smad3 to ICD4 was mediated by its MH2 domain and was not dependent on the presence of the RAM23 region in ICD4. Using two TGF-beta/Activin reporter luciferase assays, RT-PCR and Western blot analysis, we demonstrate that ICD4 and ICD4 deltaRAM23 inhibit Smad-binding element and 3TP luciferase reporter activity and PAI-1 gene expression. MCF-7 human breast cancer cells express Notch4ICD (ICD4) and are resistant to the growth-inhibitory effects of TGF-beta. Blockage of Notch4 processing to ICD4 by gamma-secretase inhibitor renders MCF-7 cells sensitive to growth inhibition by TGF-beta. The interplay between these two signaling pathways may be a significant determinant during mammary tumorigenesis.

Human Cripto-1 overexpression in the mouse mammary gland results in the development of hyperplasia and adenocarcinoma.

Human Cripto-1 (CR-1) is overexpressed in approximately 80% of human breast, colon and lung carcinomas. Mouse Cr-1 upregulation is also observed in a number of transgenic (Tg) mouse mammary tumors. To determine whether CR-1 can alter mammary gland development and/or may contribute to tumorigenesis in vivo, we have generated Tg mouse lines that express human CR-1 under the transcriptional control of the mouse mammary tumor virus (MMTV). Stable Tg MMTV/CR-1 FVB/N lines expressing different levels of CR-1 were analysed. Virgin female MMTV/CR-1 Tg mice exhibited enhanced ductal branching, dilated ducts, intraductal hyperplasia, hyperplastic alveolar nodules and condensation of the mammary stroma. Virgin aged MMTV/CR-1 Tg mice also possessed persistent end buds. In aged multiparous MMTV/CR-1 mice, the hyperplastic phenotype was most pronounced with multifocal hyperplasias. In the highest CR-1-expressing subline, G4, 38% (12/31) of the multiparous animals aged 12-20 months developed hyperplasias and approximately 33% (11/31) developed papillary adenocarcinomas. The long latency period suggests that additional genetic alterations are required to facilitate mammary tumor formation in conjunction with CR-1. This is the first in vivo study that shows hyperplasia and tumor growth in CR-1-overexpressing animals.

Role of human cripto-1 in tumor angiogenesis.

BACKGROUND: Human cripto-1 (CR-1) promotes cell transformation and increases migration and invasion of various mouse and human epithelial cell lines. We investigated whether CR-1 also stimulates angiogenesis. METHODS: We used human umbilical vein endothelial cells (HUVECs) to measure in vitro migration with fibronectin-coated Boyden chambers, invasion with Matrigel-coated Boyden chambers, proliferation with a tetrazolium salt, and differentiation with an in vitro Matrigel assay. We investigated new blood vessel formation in vivo by use of Matrigel-filled silicone cylinders implanted under the skin of nude mice and by use of a breast cancer xenograft model with CR-1-transfected or control Neo-transfected MCF-7 human breast cancer cells. We also used a blocking anti-CR-1 monoclonal antibody to investigate the role of CR-1 in angiogenesis in vivo and in vitro. All statistical tests were two-sided. RESULTS: CR-1 stimulated HUVEC proliferation, migration, and invasion and induced HUVEC differentiation into vascular-like structures on Matrigel. In vivo, recombinant CR-1 protein induced microvessel formation in Matrigel-filled silicone cylinders, and microvessel formation was statistically significantly inhibited with a blocking anti-CR-1 monoclonal antibody (CR-1 and antibody = 127% of microvessel formation compared with that in untreated control cylinders and CR-1 alone = 259%; difference = 132%, 95% confidence interval [CI] = 123% to 140%; P<.001). Tumors formed by CR-1-transfected MCF-7 cells in the cleared mammary fat pad of nude mice had higher microvessel density than tumors formed by control Neo-transfected MCF-7 cells (CR-1-transfected cells = 4.66 vessels per field and Neo-transfected cells = 2.33 vessels per field; difference = 2.33 vessels per field, 95% CI = 1.2 to 2.8; P = .004). CONCLUSION: CR-1 appears to have an important role in the multistep process of angiogenesis.

Epithelial mesenchymal transition is a characteristic of hyperplasias and tumors in mammary gland from MMTV-Cripto-1 transgenic mice.

Epithelial-mesenchymal transition (EMT) facilitates migration and invasion of epithelial tumor cells. Cripto-1 (CR-1), a member of the epidermal growth factor-CFC protein family increases migration of cells in vitro. Here the expression of molecular markers and signaling molecules characteristic of EMT were assessed in mammary gland hyperplasias and tumors from mice expressing the human CR-1 transgene by the MMTV promoter (MMTV-CR-1) and in mouse mammary epithelial cell line HC-11 overexpressing CR-1 (HC-11/CR-1). Western blot analysis showed decreased expression of E-cadherin in MMTV-CR-1 tumors and in HC-11/CR-1 cells. The expression of N-cadherin, vimentin, cyclin-D1, and of the zinc-finger transcription factor, snail, was increased in MMTV-CR-1 tumors. Increased snail mRNA was also found in HC-11/CR-1 cells. Expression of phosphorylated (P)-c-Src, P-focal adhesion kinase (FAK), P-Akt, P-glycogen synthease kinase 3beta (GSK-3beta), dephosphorylated (DP)-beta-catenin, and various integrins such as, alpha 3, alpha v, beta 1, beta 3, and beta 4 was also increased in MMTV-CR-1 tumors. Immunohistochemistry showed positive staining for vimentin, N-cadherin, cyclin-D1, smooth muscle actin, fibronectin, snail, and beta-catenin in MMTV-CR-1 tumor sections. HC-11/CR-1 cells treated with the c-Src inhibitor PP2 reduced the expression of P-c-Src and of P-FAK, P-Akt, P-GSK-3beta, DP-beta-catenin all known to be activated by c-Src. Migration of HC-11/CR-1 cells was also reduced by PP2 treatment. These results suggest that CR-1 may play a significant role in promoting the increased expression of markers and signaling molecules associated with EMT.

A Nodal- and ALK4-independent signaling pathway activated by Cripto-1 through Glypican-1 and c-Src.

Human Cripto-1 (CR-1) is a member of the epidermal growth factor-Cripto FRL1 Cryptic family that has been shown to function as a coreceptor with the type I Activin serine-threonine kinase receptor ALK4 for the transforming growth factor beta-related peptide Nodal. However, CR-1 can also activate the mitogen-activated protein kinase and Akt pathways independently of Nodal and ALK4 by an unknown mechanism. Here, we demonstrate that CR-1 specifically binds to Glypican-1, a membrane-associated heparan sulfate proteoglycan, and activates the tyrosine kinase c-Src, triggering the mitogen-activated protein kinase and Akt signaling pathways. Finally, an active Src kinase is necessary for CR-1 to induce in vitro transformation and migration in mouse mammary epithelial cells.

Cripto-1 activates nodal- and ALK4-dependent and -independent signaling pathways in mammary epithelial Cells.

Cripto-1 (CR-1), an epidermal growth factor-CFC (EGF-CFC) family member, has a demonstrated role in embryogenesis and mammary gland development and is overexpressed in several human tumors. Recently, EGF-CFC proteins were implicated as essential signaling cofactors for Nodal, a transforming growth factor beta family member whose expression has previously been defined as embryo specific. To identify a receptor for CR-1, a human brain cDNA phage display library was screened using CR-1 protein as bait. Phage inserts with identity to ALK4, a type I serine/threonine kinase receptor for Activin, were identified. CR-1 binds to cell surface ALK4 expressed on mammalian epithelial cells in fluorescence-activated cell sorter analysis, as well as by coimmunoprecipitation. Nodal is coexpressed with mouse Cr-1 in the mammary gland, and CR-1 can phosphorylate the transcription factor Smad-2 in EpH-4 mammary epithelial cells only in the presence of Nodal and ALK4. In contrast, CR-1 stimulation of mitogen-activated protein kinase and AKT in these cells is independent of Nodal and ALK4, suggesting that CR-1 may modulate different signaling pathways to mediate its different functional roles.

Detection and localization of Cripto-1 binding in mouse mammary epithelial cells and in the mouse mammary gland using an immunoglobulin-cripto-1 fusion protein.

Human Cripto-1 (CR-1), a member of the epidermal growth factor-CFC (EGF-CFC) family of peptides, is expressed in the developing mouse mammary gland and can modulate mammary epithelial cell migration, branching morphogenesis and milk protein expression in vitro. In order to screen for a CR-1 receptor and to identify potential CR-1 target tissues, we constructed a fusion protein comprising the EGF-like domain of CR-1 and the Fc domain of a human IgG1. The recombinant CR-1 fusion protein (CR-1-Fc) was biologically active as it was able to activate the ras/raf/mitogen activated protein kinase (MAPK) pathway and to inhibit transcription of the milk protein beta-casein in NMuMG and HC-11 mouse mammary epithelial cells. By using immunocytochemistry and by an in situ enzyme-linked immunosorbent assay (ELISA), CR-1-Fc was found to specifically bind to NMuMG and HC-11 cells. Finally, immunohistochemical analysis using CR-1-Fc showed a specific localization of CR-1 binding to tissue sections from mouse mammary gland. In particular, more than 60% of the epithelial cells were intensely stained with the CR-1-Fc fusion protein in the lactating mouse mammary gland, whereas approximately 25% of the mammary epithelial cells were stained in the gland from pregnant mouse. Since expression of mouse cripto-1 (Cr-1) in the pregnant and lactating mouse mammary gland as well as its presence in milk has been previously demonstrated, these data strongly suggest that an autocrine pathway involving Cr-1 and its putative receptor is operating in the mouse mammary gland during pregnancy and lactation.