Richard Grosse

Cloning and characterization of the mouse gene encoding mammary-derived growth inhibitor/heart-fatty acid-binding protein

From a mouse genomic library we isolated and characterized a gene, Fabph1, encoding mammary-derived growth inhibitor (MDGI)/heart fatty-acid-binding protein (H-FABP). Exon sequences were identical with a MDGI-encoding cDNA isolated previously from the mammary gland of pregnant mice. The product of this gene has also been detected in heart, where it had been termed H-FABP. It has an intron/exon structure similar to other FABP-encoding genes. In addition to this expressed gene, we isolated a related intronless pseudogene, Fabph-ps, with an open reading frame which was highly conserved when compared with Fabph1. Fabph1 was positioned on chromosome (Chr) 4 using interrelated sequence locus, Fabph-rs1, to Chr 8. A Mus spretus-specific related sequence, Fabph-rs2, was identified on Chr 17 by analysis of interspecies crosses. The 5-flanking region of Fabph1 contains putative transcription factor-binding elements which could account for its constitutive expression in muscle tissue, as well as for its developmental stage-dependent expression in mammary epithelium.

Purification of a growth inhibitor for Ehrlich ascites mammary carcinoma cells from bovine mammary gland

A growth inhibitor for Ehrlich ascites mammary carcinoma cells in vitro has been purified from bovine mammary gland. The purification procedure involving homogenization and differential centrifugation under hypotonic conditions, ammonium sulfate precipitation, ultrafiltration, gel chromatography and preparative polyacrylamide gel electrophoresis (PAGE) yielded an inhibitor showing half-maximal inhibition of cell proliferation in concentrations of 1-3 ng protein per ml. Upon 125I labelling and analysis by SDS gel electrophoresis, most purified preparations revealed a single band of 12-14 kD, likely to be representative for the inhibitory protein. The inhibitor was shown to affect resumption of proliferation of stationary cells; however, it was inactive towards cells stimulated by incubation with medium before adding the inhibitor. The inhibitor is heat-labile, does not act by exhausting essential components of culture medium, and its action is antagonized by insulin.

Specific neutralizing antiserum against a polypeptide growth inhibitor for mammary cells purified from bovine mammary gland

A recently published method for purification of a new inhibitor of growth of mammary cells in vitro from bovine mammary gland has been modified to yield several hundred micrograms of inhibitor per kg of glandular tissue. The inhibitory effect exerted by this preparation to Ehrlich ascites mammary carcinoma cells fulfilled all biological criteria of specificity established earlier for preparations obtained by other means, the most important being that the inhibitory effect is abolished by the epidermal growth factor and insulin. The preparation is shown to consist mainly of a protein of 13 kDa which appears to be not glycosylated. An antiserum raised in mice against the inhibitor is demonstrated to be specific for the 13 kDa bovine mammary gland protein. Neutralization of the inhibitory activity by the specific antiserum strongly supports the view that the 13 kDa protein is indeed the carrier of inhibitory activity. First data on tissue distribution obtained with an enzyme-linked immunosorbent assay revealed a high concentration of the anti-inhibitor-antiserum-reactive antigen in bovine lactating but not in non-lactating mammary gland tissue and in milk fat globule membranes. Some reactivity was also found in bovine lung. These data are interpreted with respect to a possible physiological significance of the growth inhibitor.

Detection of BRCA1 and BRCA2 mutations in breast cancer families by a comprehensive two‐stage screening procedure

We have developed a 2‐stage protocol for BRCA1 and BRCA2 mutation screening from blood spot paper. Stage 1 screening was aimed to analyze patients at highest risk for the most common disease‐associated sequence variants listed in the BIC database. Accordingly, stage1 testing implied detection of 18 disease‐ associated BRCA1 and 9 BRCA2 mutations by adapting the 5’ nuclease assay to heterozygote screening. For stage 2 screening, we applied the conformation sensitive gel electrophoresis (CSGE) method by adapting this technique to automated heteroduplex analysis of BRCA1 and BRCA2 using fragment scanning on an ABI 377 sequencing device. Of the 120 patients with a family history of breast and ovarian cancer who took part in this study so far, 45 entered stage 1 testing. Disease‐associated mutations were detected in 6 patients by stage 1 testing (13%). For these patients, the final result was available within 10 days. Mutation 300T→G was found in 2 patients. One patient with mutation 3036delACAA in BRCA2 reported only 1 sister with a multifocal bilateral breast cancer. New disease‐associated mutations were detected in 2 of the 114 patients who entered the stage 2 test (1.7%). Of particular interest was 1 patient who was diagnosed with a medullary breast carcinoma at age 39 and who had no family history of breast cancer. We conclude that pre‐screening by 5’ nuclease assay for the mutations most frequently seen in a given population represents a relatively effective first line of analysis. Subsequent detailed analysis by fluorescence conformation sensitive gel electrophoresis (F‐CSGE) and fragment sequencing is a sensitive alternative to full nucleotide sequencing. Int. J. Cancer 85:474–481, 2000.

EGF binding is quantitatively related to growth in node-positive breast cancer.

SummaryNumber of mitoses and EGF binding were determined in parallel in biopsies of 27 lymph-node positive and of 23 lymph-node negative breast cancer patients. For node-positive patients the parameters for cell growth and EGF binding were quantitatively correlated by the equation y = P3 + P1(1 − exp( − P2x)). For node-negative cases neither the non-linear model nor the linear approximation described the data unambiguously. The results strongly suggest that in node-positive patients, growth of breast cancer is related to an EGF-dependent acceleration of cell division.

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.

Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells

Ca2+-ATPase was isolated from plasma membranes of Ehrlich ascites mammary carcinoma cells by means of calmodulin affinity chromatography. The purification procedure included removal of endogenous calmodulin from a Triton X-100 solubilizate of the membranes by DEAE ion-exchange chromatography as an essential step. With respect to its molecular mass, activation by calmodulin, Ca2+-dependent phosphorylation and highly sensitive inhibition by orthovanadate, the purified enzyme resembles the Ca2+-ATPase of erythrocyte membranes. In contrast to the strong calmodulin dependence of the isolated enzyme the Ca2+-ATPase in native Ehrlich ascites carcinoma cell membranes cannot be remarkably stimulated by added calmodulin. It is suggested that the membrane-bound Ca2+-ATPase in the presence of Ca2+ is activated by interaction with endogenously bound calmodulin.

Local Signals for Growth Cessation and Differentiation in the Mammary Gland

The mammary gland provides a unique model to study postnatal processes of growth and differentiation. Development of the mouse mammary gland is a complex multistage process, which begins in the embryo with the mammary anlagen giving rise to primary and secondary sprouts. Sparsely branching ducts invade the stroma at puberty, followed by the development of lobuloalveolar structures, and functional differentiation during pregnancy (Topper and Freeman, 1980). By use of endocrine ablation, organ culture systems and various cell culture models, it has been demonstrated that several steroid hormones, prolactin and growth hormone regulate this process (for reviews, see Banerjee and Antoniou, 1985; Streuli and Bissell, 1991). The combined action of aldosterone, prolactin, insulin and cortisol is sufficient to promote lobuloalveolar development and functional differentiation in organ cultures of mammary glands from sexually immature mice, pretreated with estradiol and progesterone (Banerjee et al., 1973). Although the systemic importance of ovarian and pituitary hormones has been well documented, these hormones are virtually incapable of stimulating proliferation or inhibiting growth of mammary epithelial cells (MEC) in vitro.

Demonstration of an epidermal growth factor-dependent 58 kDa phosphoprotein secreted by rat kidney fibroblasts

Epidermal growth factor and 12‐O‐tetradecanoylphorbol‐13‐acetate increased the amount of 32Pi found as phosphoserine in a major, hitherto not described 58 kDa phosphoprotein (pp58) secreted by normal rat kidney fibroblasts. Platelet‐derived growth factor, insulin, nerve growth factor and fibroblast growth factor did not affect pp58 while transforming growth factor β decreased the accumulation of radioactivity into pp58. Cycloheximide, actinomycin D and ammonium chloride suppressed the labelling of pp58.