In situ hybridization for the detection of low copy numbers of c-abl oncogene mRNA in lymphoma cells: technical approach and comparison with results with anti-oncoprotein antibodies.
We investigated the practical value of antisense RNA/mRNA in situ hybridization for the detection of low level expression of the c-abl oncogene in non-Hodgkin lymphomas. This is of clinical relevance, since we recently showed that low level expression of this proto-oncogene mainly occurs in advanced stage disease of non-Hodgkin lymphomas and in cases of chronic lymphocytic leukemia with progressive course of the disease (Greil R, Gattringer C, Fasching B, Cleveland J, Thaler J, Radaskiewicz T, Gastl G, Huber C, Rapp U, Huber H: Int J Cancer 42:529 1988). When numerous technical parameters were tested for the adaptation of the method, fixation with 4% paraformaldehyde, gelatin coating of the slides, the time concentration product of proteinase K, and the kind of labeling had the greatest impact on results and successful performance of the technique. When the optimized method was applied to the v-abl-transformed NIH 3T3-, the K 562 CML blast cell line and to nine cases of lowly malignant non-Hodgkin lymphomas it semiquantitatively discriminated the varying amounts of v-abl, bcr/c-abl and c-abl mRNA expressed within these cells. Parallel analysis with Northern blotting confirmed the specificity of the method and pointed to a very high sensitivity, including the capacity to detect only few c-abl mRNA molecules/cell. An essential advantage of in situ hybridization was the detection of inhomogeneous expression of the c-abl mRNA within subpopulations of the malignant clone. In addition, this technique might be of particular importance when a gene is only weakly expressed on a small fraction of cells which might easily escape the detection by Northern blotting. Immunocytochemical investigation suggested parallel expression of the oncoprotein in six of seven c-abl mRNA positive cases as well as high specificity and sensitivity for the polyclonal and to a lesser extent for one monoclonal antibody. However, because of the high potential of cross-reactivity of anti-oncoprotein antibodies, parallel investigations on the mRNA level should be performed particularly when new anti-oncoprotein antibodies are applied. Our results demonstrate that this can be performed using in situ hybridization, even when the number of mRNA targets is very low.
Greil
R
R
Department of Internal Medicine, University of Innsbruck, Austria.
Fasching
B
B
Huber
H
H
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D015152:Blotting, Northern; D002460:Cell Line; D006801:Humans; D007150:Immunohistochemistry; D008223:Lymphoma / Q000032:analysis* / Q000235:genetics / Q000473:pathology; D015335:Molecular Probes; D009693:Nucleic Acid Hybridization; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000276:immunology; D016315:Proto-Oncogene Proteins c-abl; D011519:Proto-Oncogenes; D012333:RNA, Messenger / Q000032:analysis*
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; C000715470:MAS1 protein, human; D015335:Molecular Probes; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016315:Proto-Oncogene Proteins c-abl