bromochloroacetic-acid and Leukemia--Promyelocytic--Acute

Topics: Avian Myeloblastosis Virus; Gene Rearrangement; Humans; Keratins; Leukemia, Promyelocytic, Acute; Moloney murine leukemia virus; Oncogene Proteins, Fusion; Polymerase Chain Reaction; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA-Directed DNA Polymerase; Sensitivity and Specificity

A novel technique in multiparameter flow cytometry (FCM) using dual laser excitation of three fluorescent dyes has been developed to differentiate breast epithelial cells from stromal components. This technique has been applied to determine the expression of the oncoprotein c-erbB-2 in breast epithelial tumors. SK-BR-3, a breast cancer cell line with c-erbB-2 overexpression, can be identified by FCM from a mixed cell suspension using a monoclonal anti-human cytokeratin antibody conjugated with fluorescein isothiocyanate. Using the mouse monoclonal anti-c-erbB-2 antibody, TA-1 (4.8 +/- 1.0 x isotype control), the c-erbB-2 oncoprotein overexpression in breast epithelial cells can be detected as an increase in indirect blue fluorescence by aminomethyl coumarin. MCF-7, a breast cancer cell line with normal c-erbB-2 expression, has baseline blue fluorescence (1.0 +/- 0.5 x isotope control). Twenty-one fresh breast specimens have been examined by FCM. Overexpression of c-erbB-2, defined by blue fluorescence ratio of TA-1/isotype control > or = 1.5 (> 3 SD from baseline), is detected in 0 of 3 patients with Stage I cancer, 5 of 14 patients with Stage II and III cancer, and 3 of 4 patients with proliferative disease. Patients with elevation of oncoprotein detected by FCM have corresponding RNA overexpression detected by Northern blot hybridization and increased gene amplification detected by Southern blot hybridization. FCM allows for the simultaneous identification of breast epithelial cells and the selective examination of these cells for the expression of c-erbB-2 oncoprotein, thus minimizing stromal contamination. This represents a novel application of FCM with potential for wide clinical applicability.

Topics: Biomarkers, Tumor; Blotting, Northern; Blotting, Southern; Breast Diseases; Breast Neoplasms; DNA; DNA, Neoplasm; Female; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression; Humans; Keratins; Leukemia, Promyelocytic, Acute; Neoplasm Staging; Proto-Oncogene Proteins; Proto-Oncogenes; Receptor, ErbB-2; RNA; RNA, Neoplasm; Tumor Cells, Cultured

We examined the feasibility of performing non-radioactive in situ hybridization (ISH) in flow cytometrically sorted (tumor) cells with a chromosome #1 specific centromere probe. The study was performed in a model system of HL60 cells mixed with different quantities of HeLa cells. These latter cells were sorted directly onto poly-l-lysine coated glass slides on the basis of their keratin content, a cytoskeletal component not present in HL60 cells. Overall morphology of the separated HeLa cells was excellent and, after the ISH procedure, the appropriate number of ISH spots was observed in more than 85% of the sorted cells. This percentage did not differ significantly in cell mixtures with different percentages of HeLa cells (down to 1%). Sorting of HeLa cells in different phases of the cell cycle, and subsequent ISH, revealed the same spot number for chromosome #1 in all cell cycle stages, including mitosis. In the latter phase of the cell cycle we did not find a duplication of the chromosome #1 centromere, not even after sorting of the mitotic cells on the basis of specific labeling with an antibody to mitotin. The early G2 mitotin negative fraction, however, showed a significant percentage of cells with a duplicate spot number, most likely representing a tetraploid cell fraction in this HeLa cell culture. The protocol that evolved from these model studies was applied to cell suspensions of malignant body cavity effusions as well as solid bladder carcinomas. In several of these cases numerical chromosome aberrations could be detected by ISH more evidently after sorting on the basis of keratin labeling.

Topics: Antibodies, Monoclonal; Cell Cycle; Cell Cycle Proteins; Cell Separation; Centromere; Chromosomes, Human; Chromosomes, Human, Pair 1; DNA Probes; Flow Cytometry; HeLa Cells; Humans; Interphase; Keratins; Leukemia, Promyelocytic, Acute; Minichromosome Maintenance Complex Component 2; Neoplasms; Nuclear Proteins; Nucleic Acid Hybridization; Pleural Effusion; Tumor Cells, Cultured; Urinary Bladder Neoplasms