Irene Rappold

Expression of MUC-1 Epitopes on Normal Bone Marrow: Implications for the Detection of Micrometastatic Tumor Cells

PURPOSE The expression of the carcinoma-associated mucin MUC-1 is thought to be restricted to epithelial cells and is used for micrometastatic tumor cell detection in patients with solid tumors, including those with breast cancer. Little is known, however, about the expression of MUC-1 epitopes in normal hematopoietic cells. MATERIALS AND METHODS MUC-1 expression was analyzed by flow cytometry and immunocytology on bone marrow (BM) mononuclear cells and purified CD34+ cells from healthy volunteers, using different anti-MUC-1-specific monoclonal antibodies. In addition, Western blotting of MUC-1 proteins was performed. RESULTS Surprisingly, 2% to 10% of normal human BM mononuclear cells expressed MUC-1, as defined by the anti-MUC-1 antibodies BM-2 (2E11), BM-7, 12H12, MAM-6, and HMFG-1. In contrast, two antibodies recognizing the BM-8 and the HMFG-2 epitopes of MUC-1 were not detected. MUC-1+ cells from normal BM consisted primarily of erythroblasts and normoblasts. In agreement with this, normal CD34+ cells cultured in vitro to differentiate into the erythroid lineage showed a strong MUC-1 expression on day 7 proerythroblasts. Western blotting of these cells confirmed that the reactive species is the known high molecular weight MUC-1 protein. CONCLUSION Our data demonstrate that some MUC-1 epitopes are expressed on normal BM cells and particularly on cells of the erythroid lineage. Hence the application of anti-MUC-1 antibodies for disseminated tumor cell detection in BM or peripheral blood progenitor cells may provide false-positive results, and only carefully evaluated anti-MUC-1 antibodies (eg, HMFG-2) might be selected. Furthermore, MUC-1-targeted immunotherapy in cancer patients might be hampered by the suppression of erythropoiesis.

Expression and Signal Transduction of the FLT3 Tyrosine Kinase Receptor

FLT3 is a receptor tyrosine kinase of 130-55 kDa expressed on normal bone marrow stem and early progenitor cells and on leukemic blasts from patients with acute leukemias. The FLT3 ligand, FL, is a new cytokine which acts on hematopoietic progenitors in synergy with other cytokines. FLT3 transduces FL-mediated signal through interaction with a number of cytoplasmic substrates.

Analysis of Tie receptor tyrosine kinase in haemopoietic progenitor and leukaemia cells

We generated a panel of monoclonal antibodies against the extracellular domain of the Tie receptor tyrosine kinase and studied its expression in human haemopoietic and tumour cell lines and in samples from leukaemia patients. Most of the erythroblastic/megakaryoblastic (6/8), 2/7 myeloid and 3/6 B‐lymphoblastic leukaemia cell lines were Tie‐positive. The erythroblastic/megakaryoblastic leukaemia cell lines also expressed the related Tie‐2/Tek gene and, surprisingly, its recently cloned ligand gene angiopoietin‐1, which was located in chromosome 8q23.1. In addition, 16% of freshly isolated leukaemia samples were Tie positive. Peripheral blood mononuclear cells were Tie negative, but a few Tie positive cells were found in immunoperoxidase staining of mobilized peripheral blood stem cells. Long‐term culture of isolated umbilical cord blood CD34+Tie+ and CD34+Tie−cells indicated that the Tie+ fraction contained a slightly higher frequency of cobblestone area forming cells (CAFC). Thus, Tie is expressed on haemopoietic progenitor cells and some leukaemic blasts. The coexpression of Tie‐2 and angiopoietin‐1 in megakaryoblastic leukaemia cell lines suggests the existence of an autocrine ligand/receptor signalling loop in these cells.

CD164 Monoclonal Antibodies That Block Hemopoietic Progenitor Cell Adhesion and Proliferation Interact with the First Mucin Domain of the CD164 Receptor

The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by the mAbs, 103B2/9E10 and 105A5. We report here the precise epitope mapping of these mAbs together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined CD164 splice variants and a set of soluble recombinant chimeric proteins encoded by exons 1–6 of the CD164 gene, we demonstrate that the 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function.

Re‐entrainment, phase‐response and range of entrainment of circadian rhythms in Owl Monkeys (Aotus lemurinus g.) of different age

Abstract To evaluate whether there are age‐related differences in the characteristics of the circadian system in owl monkeys (Aotus lemurinus griseimembra) experiments analysing the resynchronization behaviour, the phase response to 3 h‐light pulses (LP), and the range of entrainment were carried out with 4 young (2–6 years), 5 middle‐aged (10–20 years), and 3 old (>20 years) animals. Following an 8 h‐delay shift of the entraining light‐dark cycle (LD 12:12; 100/230 lx : 0.2 lx) the monkeys’ locomotor activity rhythm re‐entrained significantly faster than after an 8 h‐advance shift. While no age‐associated differences in time needed for re‐entrainment (tr) could be detected following the delay‐shift, a difference appeared after the advance‐shift with faster re‐entrainment in the older animals. In a second experiment, the phase‐shifting effects of 3 h‐light pulses of 100 lx and 230 lx, respectively, applied at various phases of the circadian activity rhythm free‐running in constant dim light (LL) of 0.2 lx...