Ultrastructural localization of mRNA encoding for the EGF receptor in human breast cell cancer line BT20 by in situ hybridization.
The EGF receptor (EGF-R), a 170 KD transmembrane glycoprotein, is found at a high level in the BT20 human mammary carcinoma cell line (1 +/- 0.4 x 10(6) sites per cell). In this study, we examined the expression of the EGF-R gene in BT20 cell line by in situ hybridization at the light and electron microscopic level using a human cDNA, corresponding to EGF-R transmembrane and protein kinase domains, labeled with [3H]-, [35S]-, or [32P]-d-ATP. Two treatments were tested to embed cells in Lowicryl resin: the first used fixation and dehydration by progressive lowering of temperature, the second quick freezing and cryosubstitution. The best ultrastructural preservation was obtained with the second procedure without modification of the hybridization signal. EGF-R mRNA was observed principally at the cytoplasmic level, on organelles involved in the protein synthesis process. Labeling was also located on the microvilli which extend into the intercellular space, suggesting that some mRNA would be located in sites where EGF-R is utilized. Some mRNA was observed in the nucleus. This study demonstrates that post-embedding in situ hybridization, after quick freezing and cryosubstitution, is a powerful EM in situ hybridization procedure to study the expression of the EGF-R gene.
Le Guellec
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Laboratoire d'Histologie Expérimentale, UPR CNRS 412 (DLG), Université Claude Bernard, Villeurbanne, France.
Frappart
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Desprez
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D001943:Breast Neoplasms / Q000648:ultrastructure*; D003593:Cytoplasm / Q000737:chemistry; D015342:DNA Probes; D004721:Endoplasmic Reticulum / Q000737:chemistry; D066246:ErbB Receptors / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008854:Microscopy, Electron; D008871:Microvilli / Q000737:chemistry; D009685:Nuclear Envelope / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis*; D012259:Ribonuclease, Pancreatic; D013462:Sulfur Radioisotopes; D014316:Tritium; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D014316:Tritium; D066246:ErbB Receptors; D012259:Ribonuclease, Pancreatic