Localization of c-myc in HL-60 cells, neoplastic and normal tissues: An immunohistochemical and in situ hybridization study.

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  • AN IMMUNOHISTOCHEMICAL AND <i>IN SITU</i> HYBRIDIZATION STUDY

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Our preliminary immunohistochemical examination revealed that c-myc protein was localized in the cytoplasm of both normal and neoplastic cells. This finding did not correlate with the reported nuclear localization of c-myc protein by other analyses. To clarify this discrepancy, we investigated whether the cytoplasmic localization of c-myc protein in our preliminary immunohistochemical studies reflects true physiological habitat of the protein or the results of an artifact which occurred during tissue processing. For the immunohistochemical localization of c-myc protein, rabbit antisera against synthetic peptides which correspond to a portion of c-myc were utilized. Fixation conditions were examined by varying the temperature, the salt concentration, as well as the duration of fixation. With conventional fixation conditions used for the routine immunohistochemical localization of proteins, such as fixation with a solution of 4% formaldehyde with low salt concentration at low temperature, the c-myc protein was found in the cytoplasm of HL-60 cells, some gastric carcinoma cells, intestinal crypt epithelial cells and spermatogonia in seminiferous tubules from human and regenerating hepatocytes from rat. Whereas, when these cells and tissues were fixed at 42°C with a fixative containing 4% formaldehyde and a high concentration of NaCl, the c-myc protein was found in the nuclei of these cells. These results suggest that during fixation the c-myc protein translocates from nucleus to cytoplasm. To prove that these c-myc proteins were the product of the cells, in situ hybridization of c-myc mRNA was carried out using dinitrophenyl labeled c-myc cDNA. The gastric carcinoma cells which contained c-myc protein in their nuclei were morphologically similar to those contained the c-myc mRNA. The co-existance of c-myc protein and c-myc mRNA suggests that the c-myc protein found in the nuclei was the product of the cell.

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