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Isolation of cDNA clones corresponding to genes differentially expressed in two colon‐carcinoma cell lines differing by their tumorigenicity
Author(s) -
Denis Marc G.,
Chadeneau Corinne,
Lustenberger Patrick,
Mevel Bernard Le,
Meflah Khaled
Publication year - 1992
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.2910500619
Subject(s) - complementary dna , biology , microbiology and biotechnology , messenger rna , northern blot , cdna library , gene , gene expression , clone (java method) , homology (biology) , cell culture , blot , southern blot , rna , genetics
In an effort to isolate genes involved in the progression of colonic cells leading to a carcinoma, we used as a model 2 rat colon‐carcinoma cell lines selected from the same tumor, differing by their tumorigenicity. When soluble, Triton‐X‐100 extracted, or cytoskeletal proteins from the progressive PROb cells and the regressive REGb cells were analyzed by SDS‐PAGE, minor differences were seen. Furthermore, mRNA‐cDNA hybridization analyses showed extensive homology between the 2 mRNA populations. Thus, the homology between the 2 clones is high at both the protein and the mRNA levels. A PROb cDNA library was hybridized with 32 P‐cDNA synthesized from PROb or REGb mRNA. The clones giving a stronger signal when hybridized with the homologous PROb probe were isolated. The specificity of each clone was confirmed by RNA blotting. Most of the positive clones showed a 2‐ to 3‐fold higher expression in PROb cells when compared with REGb cells. One clone (J 13) corresponded to an mRNA 7‐ to 10‐fold more abundant in PROb cells, and was further studied. No gene amplification was detected by Southern blot analysis, indicating that the difference in mRNA content was most likely due to an increased transcription of this gene in PROb cells. Sequencing of the cDNA showed high homology with the rat ferritin light sub‐unit. Over‐expression of ferritin in PROb cells as compared with REGb cells was confirmed at the protein level using specific antibodies.