Eva Spitzer

Histochemical localization of heart-type fatty-acid binding protein in human and murine tissues

Cellular fatty acid-binding proteins (FABP) are a highly conserved family of proteins consisting of several subtypes, among them the mammary-derived growth inhibitor (MDGI) which is quite homologous to or even identical with the heart-type FABP (H-FABP). The FABPs and MDGI have been suggested to be involved in intracellular fatty acid metabolism and trafficking. Recently, evidence for growth and differentiation regulating properties of MDGI and H-FABP was provided. Using four affinity-purified polyclonal antibodies against bovine and human antigen preparations, the cellular localization of MDGI/H-FABP in human and mouse tissues and organs was studied. The antibodies were weakly cross-reactive with adipose tissue extracts known to lack H-FABP, but failed to react by Western blot analysis with liver-type FABP (L-FABP) and intestinal-type FABP (I-FABP). MDGI/H-FABP protein was mainly detected in myocardium, skeletal and smooth muscle fibres, lipid and/or steroid synthesising cells (adrenals, Leydig cells, sebaceous glands, lactating mammary gland) and terminally differentiated epithelia of the respiratory, intestinal and urogenital tracts. The results provide evidence that expression of H-FABP is associated with an irreversibly postmitotic and terminally differentiated status of cells. Since all the antisera employed showed spatially identical and qualitatively equal immunostaining, it is suggested that human, bovine and mouse MDGI/H-FABP proteins share highly homologous epitopes.

Hormonal induction of functional differentiation and mammary-derived growth inhibitor expression in cultured mouse mammary gland explants

SummaryA method for the cultivation of organ explants from abdominal mammary glands of virgin mice has been established. In a serum-free medium containing aldosterone, prolactin, insulin, and cortisol (APIH medium) mammary gland development was documented by lobuloalveolar morphogenesis. The hormonal requirements for in vitro expression of beta-casein and of the mammary-derived growth inhibitor (MDGI) were tested. To this end, a full length cDNA coding for mouse MDGI was prepared displaying strong homologies to a mouse heart fatty acid binding protein, which is also expressed in the mammary gland. MDGI and beta-casein transcripts were found to be absent in the mammary tissue from primed virgin mice, and were induced upon culture of mammary explants in the APIH medium. An immunohistochemical analysis with specific antibodies against MDGI and casein revealed a different pattern of expression for the two proteins. In the APIH medium, MDGI was expressed mainly in differentiating alveolar cells of the lobuloalveolar structures, whereas beta-casein was present in both ductules and alveoli. The relationship between functional differentiation and MDGI expression was further studied in explants from glands of late-pregnant mice. At this stage of development, MDGI is found both in ducts and in alveoli. If explants were cultured with epidermal growth factor (EGF) and insulin, the lobuloalveolar structure was still present, whereas MDGI disappeared. Reinduction of MDGI expression was achieved by subsequent PIH treatment. Independent on developmental stage, EGF strongly inhibits MDGI mRNA expression. It is concluded that MDGI-expression is associated with functional differentiation in the normal gland.

C-erbB-2 overexpression in primary breast cancer: independent prognostic factor in patients at high risk

SummaryThe prognostic value of c-erbB-2 protein overexpression has been evaluated in 463 patients with operable breast cancer after a median follow-up of 66 months. Overexpression was observed in 99/463 (21%) of the breast tumors. It showed significant positive correlation to histological grade (p < 0.0001) and tumor size (p < 0.02). A relationship of borderline significance was observed between c-erbB-2 protein overexpression and negative or low estrogen receptor (ER) content. No significant correlation was found to lymph node involvement or proliferating tumor cell fraction as determined by the proliferating cell nuclear antigen (PCNA). After a median follow-up of 66 months (range 6 to 109 months), the overall survival of all patients amounted to 63%. Multivariate analysis revealed lymph node involvement, tumor size, histological grade, histological type, c-erbB-2 protein overexpression, progesterone receptor (PR) content, and oral contraceptive use as independent prognostic factors. In an univariate analysis, the overall survival amounted to 72% and 38% of tumor patients with negative and positive c-erbB-2 protein overexpression, respectively. The most significant finding is that c-erbB-2 overexpression has been recognized as an independent predictive factor in subsets of tumor patients who would be expected to have a generally poor prognosis, such as those indicating axillary lymph node involvement, large tumor size (> 2 cm), and PR negativity.

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.

Significance of immunohistochemical c-erbB-2 product localization pattern for prognosis of primary human breast cancer

Concerning immunohistochemistry of the c-erbB-2 receptor in human mammary carcinoma, membranous immunostaining of tumor cells has been generally considered as a potential risk factor for early recurrence, whereas cytoplasmic reactivity has been neglected. An archival study on 463 patients with primary breast cancers demonstrates that cytoplasmic localization of p185 is significantly correlated with high estrogen and progesterone receptor levels, low histological grade and a low proliferating tumor cell fraction. In accordance with these data, patients bearing mammary carcinomas with cytoplasmic localization of p185 reactivity have a significant better overall survival than those with membranous immunostaining.

Mammary-Derived Growth Inhibitor (MDGI)

In contrast to the steadily increasing number of growth factors capable of stimulating cells to synthesize DNA and to divide, very little is known about their potential antagonists, which are supposed to contribute to balanced growth and development by inhibiting cellular proliferation.

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.