Nancy E. Hynes

Epidermal growth factor receptor, but not c-erbB-2, activation prevents lactogenic hormone induction of the beta-casein gene in mouse mammary epithelial cells.

The HC11 cell line was isolated from mammary gland cells of pregnant mice. The cells displayed a normal phenotype and retained some characteristics of mammary epithelial cell differentiation. After treatment with the lactogenic hormones prolactin and glucocorticoids, the HC11 cells expressed the milk protein beta-casein. Various oncogenes were transfected and expressed in HC11 cells. The oncogenes were tested for their transformation ability and for their effects upon the differentiation of the HC11 cells. All of the oncogenes tested, including activated human Ha-ras, human transforming growth factor-alpha, activated rat neuT, and human c-erbB-2 activated by a point mutation in the transmembrane domain, caused transformation of the HC11 cells, as shown by tumor formation in nude mice. HC11 cells expressing the neuT and activated c-erbB-2 genes synthesized beta-casein in response to lactogenic hormones, whereas those expressing the Ha-ras or transforming growth factor-alpha oncogenes were no longer able to respond to the lactogenic hormones. This inhibition of beta-casein production occurs at the transcriptional level and in the transforming growth factor-alpha-transformed cells is due to an autocrine mechanism involving the activation of the epidermal growth factor receptor. This suggests that, although the c-erbB-2 and epidermal growth factor receptors are structurally quite similar, their activation has different effects upon mammary epithelial cell differentiation.

Oncogenic activation of the human trk proto-oncogene by recombination with the ribosomal large subunit protein L7a.

The trk‐2h oncogene, isolated from the human breast carcinoma cell line MDA‐MB 231 by genomic DNA‐transfection into NIH3T3 cells, consists of the trk proto‐oncogene receptor kinase domain fused to a N‐terminal 41 amino acid activating sequence (Kozma, S.C., Redmond, S.M.S., Xiao‐Chang, F., Saurer, S.M., Groner, B. and Hynes, N.E. (1988) EMBO J., 7, 147‐154). Antibodies raised against a bacterially produced beta gal‐trk receptor kinase fusion protein recognized a 44 kd phosphoprotein phosphorylated on serine, threonine and tyrosine in extracts of trk‐2h transformed NIH3T3 cells. In vitro, in the presence of Mn2+/gamma ATP, this protein became phosphorylated extensively on tyrosine. Cells transformed by trk‐2h did not, however, show an elevation in total phosphotyrosine. We have cloned and sequenced the cDNA encoding the amino terminal activating sequences of trk‐2h (Kozma et al., 1988). The encoded protein has a high basic amino acid content and the gene is expressed as an abundant 1.2 kb mRNA in human, rat and mouse cells. Antipeptide antibodies raised against a C‐terminal peptide recognized specifically a 30 kd protein on Western blots of human, rat and mouse cell extracts. Immunofluorescence revealed, in addition to granular cytoplasmic fluorescence, intense nucleolar staining. The high basic amino acid content and nucleolar staining prompted us to investigate whether the 30 kd protein could be a ribosomal protein. Western immunoblotting analysis of 2D‐electrophoretically resolved ribosomal proteins indicated that the 30 kd protein is the ribosomal large subunit protein L7a.(ABSTRACT TRUNCATED AT 250 WORDS)

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.

Activation of the receptor kinase domain of the trk oncogene by recombination with two different cellular sequences.

A new chimeric oncogene, trk‐2h, has been generated by recombination of two segments of MDA‐MB231 human breast carcinoma cell line DNA after transfection in NIH/3T3 cells. The rearranged DNA segments form a fused transcriptional unit. Sequences at the 3′ end are homologous to the tyrosine kinase receptor moiety found in the trk oncogene which resembles a truncated growth factor receptor lacking part of its extracellular domain (Martin‐Zanca et al., 1986). The 5′ sequence of the trk‐2h oncogene is contributed by a gene which is expressed in all human cells tested, and is not related to any known gene. Transfection of the receptor kinase domain DNA fragment into NIH/3T3 cells generated another oncogene, trk‐3mh, which contains a mouse‐specific sequence fused 5′ to the receptor kinase. All three trk recombinants have the receptor kinase moiety fused to an activating amino terminus at the same nucleotide in their transcriptional product.

Collagen processing in ras-transfected mouse mammary epithelial cells

A mouse mammary epithelial cell line (NMuMG), after transfection with the c-rasH oncogene, forms invasive tumors in nude mice. NMuMG and NMuMG/p-rasH cells produce similar amounts of collagen (mostly type IV) when grown on plastic. NMuMG cells respond to growth on collagen gels by increasing the rate of collagen synthesis and deposition by 100%, unlike NMuMG/p-rasH cells which synthesize similar amounts of collagen whether grown on plastic or collagen gels. These results suggest that ras transformation partially inhibits the interaction between epithelial cells and the surrounding stroma that is necessary for basement membrane deposition in vivo and consequently may facilitate the invasion of the stroma by transfected cells.

Epidermal Growth Factor, Glucocorticoid Hormones and Prolactin Act Sequentially in the Induction of Milk Protein Gene Expression

The status of growth and differentiation of individual cells is governed by multiple signals transmitted to the cell from its environment. Small molecules, hormones and growth factors act as extracellular signals which are recognised by cellular receptors, present on the cell surface or intracellularly. These signals are converted into enzymatic or regulatory activities which trigger specific signal transduction pathways. They result in the alteration of transcriptional activity and finally an adapted cellular phenotype. Considerable progress has been made over the past years in the molecular description of individual signal transduction pathways.

The v-raf and Ha-ras Oncogenes Inhibit Transcription from the Beta-Casein Gene Promoter by Suppression of a Mammary Gland Specific Transcription Factor

Under the influence of pregnancy, the epithelial cells of the mammary gland undergo a complex pattern of growth and differentiation. Multiple steroid and peptide hormones cooperate in this process that ultimately leads to the production of the milk proteins. In order to study these processes at the molecular level we have simplified the complexity of the interactions by developing an in vitro cultured cell system. The HC11 cell line was isolated from mid-pregnant mammary gland cells of Balb/c mice (1). The cells display a normal phenotype and have retained some characteristics of mammary epithelial cell differentiation. After treatment with the lactogenic hormones prolactin and glucocorticoids, the HC11 cells express the milk protein beta-casein. It has been shown that the hormones act in a synergistic fashion to regulate transcription from the beta-casein promoter (2).

Hormonal Control of Mouse Mammary Tumor Virus Transcription

Mouse mammary tumor virus (MMTV) is a RNA-containing tumor virus that causes mammary cancer in mice. MMTV has a life cycle similar to that of other characterized retroviruses, yet MMTV is not completely analogous to the other viruses (1). Some of its unique features have attracted considerable attention in the past few years. First, MMTV transcription is regulated by glucocorticoid hormones. The isolation of MMTV proviruses by gene cloning followed by their transfer into cultured cells has allowed major insights into the mechanism of steroid hormone action (2,3). Second, MMTV belongs to the group of retroviruses that do not carry an oncogene. Recent results suggest that the transformation of mammary gland cells is related to the integration site of the MMTV viral DNA in the host genome. Thus, MMTV most likely transforms cells via insertional mutagenesis (4,5). In this article we will limit our discussion to experiments concerning the hormonal control of MMTV transcription. We will show that glucocorticoid as well as gonadal steroid hormones can enhance MMTV transcription. The role which MMTV plays in mammary tumor formation will be described in other chapters in this book

Cooperation between mutant p53 and the ras, raf, erbb-2 and fgf-3 oncogenes for transformation of mammary epithelial-cells.

The murine mammary epithelial cells HC11 were used as a model to examine cooperation between mutated p53 and activated oncogenes for cell growth and transformation. These cells lack wild-type (wt) p53 and their proliferation in monolayer is inhibited by reitroduction of wt p53. Expression of the ras, raf, erbB-2 and fgf-3 (former int-2) oncogenes in HC11 cells leads to their growth in soft-agar, a parameter of cell transformation. Clonogenicity in soft-agar of the ras, raf and erbB-2 transformed cells was inhibited by a temperature-sensitive (ts) p53 at 32 degrees C, when the ts p53 protein is wt. Thus these oncogenes act synergistically with mutant p53 to induce anchorage-independent growth. Proliferation in monolayer of erbB-2, but not ras, raf, or fgf-3, transformed cells was retarded by ts p53 at 32 degrees C. Thus, ras, raf and fgf-3 oncogenes can partly or completely overcome the proliferation inhibitory function of wt p53, while erbB-2 cannot. These data indicate that specific oncogenes can distinctly cooperate with p53 for growth and transformation of mammary cells.