The erbB2 gene is required for the development of terminally differentiated spinal cord oligodendrocytes.

Development of oligodendrocytes and the generation of myelin internodes within the spinal cord depends on regional signals derived from the notochord and axonally derived signals. Neuregulin 1 (NRG)-1, localized in the floor plate as well as in motor and sensory neurons, is necessary for normal oligodendrocyte development. Oligodendrocytes respond to NRGs by activating members of the erbB receptor tyrosine kinase family. Here, we show that erbB2 is not necessary for the early stages of oligodendrocyte precursor development, but is essential for proligodendroblasts to differentiate into galactosylcerebroside-positive (GalC+) oligodendrocytes. In the presence of erbB2, oligodendrocyte development is normal. In the absence of erbB2 (erbB2-/-), however, oligodendrocyte development is halted at the proligodendroblast stage with a >10-fold reduction in the number of GalC+ oligodendrocytes. ErbB2 appears to function in the transition of proligodendroblast to oligodendrocyte by transducing a terminal differentiation signal, since there is no evidence of increased oligodendrocyte death in the absence of erbB2. Furthermore, known survival signals for oligodendrocytes increase oligodendrocyte numbers in the presence of erbB2, but fail to do so in the absence of erbB2. Of the erbB2-/- oligodendrocytes that do differentiate, all fail to ensheath neurites. These data suggest that erbB2 is required for the terminal differentiation of oligodendrocytes and for development of myelin.

Neu differentiation factor (NDF), a dominant oncogene, causes apoptosis in vitro and in vivo.

Neu differentiation factor (NDF, also called neuregulin) is a potent inducer of epithelial cell proliferation and has been shown to induce mammary carcinomas in transgenic mice. Notwithstanding this proliferative effect, we have shown that a novel isoform of NDF can induce apoptosis when overexpressed. Here we report that this property also extends to other NDF isoforms and that the cytoplasmic portion of NDF is largely responsible for the apoptotic effect, whereas the proliferative activity is likely to depend upon the secreted version of NDF. In accordance with these contradictory properties, we find that tumors induced by NDF display extensive apoptosis in vivo. NDF is therefore an oncogene whose deregulation can induce transformation as well as apoptosis.

NDF/heregulin induces persistence of terminal end buds and adenocarcinomas in the mammary glands of transgenic mice.

Neu differentiation factor (NDF), a member of the neuregulin family of ligands of erbB receptors, induces both differentiative and mitogenic effects on cultured human mammary epithelial cells. Since members of the epidermal growth factor receptor family, including Neu/erbB2, have been implicated in mammary carcinoma, we wished to know whether a potential ligand of this family, NDF, could induce such effects in the mammary gland in vivo. We therefore targeted expression of NDF to the mammary gland of transgenic mice using the mouse mammary tumor virus (MMTV) promoter in a fusion construct. There was a clear, but subtle effect on development of the adult virgin gland of female transgenic animals. Terminal end bud structures (TEBs), which normally disappear from the mammary gland at the age of approximately 8 weeks in wild type mice, persist in glands of virgin MMTV-NDF transgenic females, suggesting that NDF inhibits signals that normally lead to the terminal differentiation of these structures. Further, female mice, bred continuously to maximize expression of the transgene in the mammary gland, develop mammary adenocarcinomas at a median age of 12 months. Since these tumors arise in a solitary fashion, we infer that NDF is necessary, but not sufficient for their formation. In order to explore the signal transduction pathways potentially activated by NDF, we examined expression of the receptors erbB2, erbB3 and erbB4 in mammary epithelial cells established from an NDF-induced tumor. All three receptors were expressed, though only the erbB3 receptor was phosphorylated, suggesting that overexpression of NDF might operate through this receptor. Additionally, about 50% of MMTV-NDF transgenic mice developed Harderian (lachrymal) gland hyperplasia, a benign tumor that does not progress to frank malignancy.

Thyroid hormone decreases the stability and the poly(A) tract length of rat thyrotropin beta-subunit messenger RNA.

Thyroid hormone (T3 and T4) down-regulation of TSH subunit steady state mRNA levels and subunit gene transcription in vitro and in vivo has been well studied. We present evidence here that T3 can also regulate the turnover of TSH subunit mRNA. The apparent half-life of the TSH beta-subunit mRNA was determined by adding actinomycin-D (2 microM) to dispersed rat pituitary cultures in hypothyroid medium or medium containing 10(-7) M T3 and analyzing the decline in subunit mRNA levels with time. The half-life of the TSH beta mRNA from those cultures treated with T3 was shorter than that of the control cultures (9 vs. greater than 24 h, respectively). A possible mechanism by which TSH beta-subunit mRNA stability is altered is through a change in the size of each mRNA's poly(A) tail. Northern blot analysis of total RNA from the above cultures revealed that T3 treatment reduces the size of the TSH beta-subunit mRNA. To determine if this alteration of mRNA size was due to a loss of a portion of the poly(A) tract and not to alternative splicing of the transcript or use of a secondary transcriptional start site, pooled RNAs were hybridized with oligo(dT) and subsequently digested with RNAse-H to remove the poly(A) tract. RNA blot analysis of these RNAs showed that T3 treatment results in the loss of most of the TSH beta poly(A) tail.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of cDNAs encoding the human bombesin-like peptide neuromedin B. Chromosomal localization and comparison to cDNAs encoding its amphibian homolog ranatensin.

The amidated decapeptide neuromedin B (NMB) is the mammalian homolog of the amphibian bombesin-like peptide ranatensin. cDNAs encoding human neuromedin B and amphibian ranatensin were isolated from human hypothalamic and Rana pipiens skin libraries, respectively. Sequence analysis revealed that NMB is encoded in a 76-amino acid precursor and ranatensin in an 82-amino acid precursor. In the NMB preprohormone, the sequence of the large form of NMB (NMB-22) immediately follows the signal peptide and is, in turn, followed by a dibasic cleavage site and a 17-amino acid carboxyl-terminal extension peptide. The structure for the ranatensin preprohormone is very similar. RNA blot analysis shows two NMB mRNA species, each approximately 800 bases, with wide distribution in brain and gastrointestinal tract. Genomic DNA blot analysis is consistent with a single human NMB gene. Analysis of mouse-human somatic cell hybrids indicates that this gene is localized on the long arm of human chromosome 15. Since the gene for human gastrin-releasing peptide is on chromosome 18, this analysis demonstrates that the bombesin-like peptide genes are not clustered.

Mullerian inhibiting substance reduction of colony growth of human gynecologic cancers in a stem cell assay.

Mullerian Inhibiting Substance (MIS), a fetal testicular product that causes regression of the Mullerian duct in the male mammalian embryo, was evaluated for its antitumor effect on the premise that a substance active against this genital precursor in the fetus might also be active against tumors derived from these tissues. Increasingly pure fractions of biologically active MIS, prepared from newborn calf testes, were tested in the soft agar colony inhibition assay against single cell suspensions of fresh tumors derived in ascitic or solid form from patients with gynecologic malignancies. Twenty-eight tumor specimens placed in soft agar culture have provided sufficient growth to assess an MIS effect. Twenty-five of these 28 tumors showed significant colony inhibition after incubation with MIS. Increased antitumor response correlated with increased purification of MIS when the same tumor was treated with preparations of different purity. Samples obtained from the same patient at different times, from both ascites and solid tumor sources, produced nearly identical responses to MIS. MIS preparations, previously shown to be active in microcytotoxicity and colony inhibition assays against established human ovarian and endometrial carcinoma lines demonstrate consistent antitumor activity against fresh human gynecologic cancers removed at surgery.

Subrenal capsule assay to test the viability of parenterally delivered müllerian inhibiting substance.

Production of bovine müllerian inhibiting substance (MIS) has been increased to allow generation of large quantities of biologically active purified material. The limited MIS previously available allowed only pretreatment of tumors prior to colony inhibition or implanting in nude mice. In preparation for posttransplantation tumor treatment, a subrenal capsule assay, which was first used against human tumors heterotransplanted into nude mice and subsequently against those heterotransplanted into immunocompetent mice, was adapted to determine (1) if MIS preparations could traverse the bloodstream without degradation and (2) the optimal dose required to produce a biologic effect. Urogenital ridges from female 14-day-old rat embryos were transferred atraumatically to small pouches beneath the renal capsule of the immunocompetent male CDF1 mice. The cranial-caudal orientation of the ridge with its müllerian duct was maintained. Over the next 72 hours, the mice were injected via the tail vein with 0.1 mL of an MIS-containing solution over a 100-fold concentration range. After three days, the kidneys were removed and shaved just below the ridge, which was then placed in soft agar for orientation and subsequent serial sectioning. After fixation, dehydration, and paraffin embedding, sections were stained and regression of the müllerian duct was graded and compared according to concentration and number of MIS doses administered. Regression diminished from almost complete (4+) at the highest dose, to minimal (1 to 2+) at 1/100 of that dose. Heat-inactivated and vehicle controls caused no regression of the müllerian ducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum levels of type I and III procollagen fragments in Paget's disease of bone.

Patients with Paget's disease of bone were found to have elevated serum levels of type I procollagen carboxyterminal peptide (pColl-I-C) which correlated with other measurements of disease activity. The elevated levels of pColl-I-C decreased within hours after the injection of salmon calcitonin and within weeks after oral dichloromethylene diphosphonate treatment. The decrease in serum pColl-I-C after a single injection of salmon calcitonin was associated with a decrease in urinary hydroxyproline excretion, both of which rose toward pretreatment values within 7 h. The pColl-I-C levels remained normal for months after dichloromethylene diphosphonate therapy was discontinued. Using a RIA for the type III procollagen amino-terminal peptide (pColl-III-N), it was found that serum levels were also elevated in patients with Paget's disease. The levels of pColl-III-N also decreased after the injection of salmon calcitonin, but not to the same extent as those of pColl-I-C. After chronic therapy with dichloromethylene diphosphonate, serum levels of pColl-III-N decreased, but not into the normal range. We postulate that whereas pColl-I-C is derived from synthesis of mineralized bone collagen, pColl-III-N is derived from the loose fibrous stroma replacing marrow in areas closely associated with active Pagetic bone disease.

Müllerian inhibiting substance inhibition of a human endometrial carcinoma cell line xenografted in nude mice.

Müllerian inhibiting substance (MIS) is a fetal testicular product which causes regression of the müllerian duct in the male mammalian embryo. This material has been partially purified from a neonatal bovine source and in cruder fractions has shown antitumor effects when tested against the HOC-21 ovarian carcinoma line in monolayer cytotoxicity, in soft agar colony inhibition assay, and in nude mouse xenografts. The glycoprotein used for the present studies was more highly purified by sequential ion exchange, carbohydrate affinity, and dye affinity chromatography. After a 1-hr exposure with 1.0 X 10(6) tumor cells prior to heterotransplantation, this more purified preparation with MIS biological activity as determined by organ culture assay of embryonic urogenital ridges delayed the appearance of palpable tumor nodules. That this response may be specific for tumors derived from the coelomic epithelium of the embryo is further supported by the absence of any antitumor effect when this substance was tested against the SW-48 colon carcinoma line. It is of interest that the antitumor activity followed the biological activity as the preparation was further purified to 30,000-fold.