Diversity of Cellular Molecules in Human Cells Detected by Monoclonal Antibodies Reactive with c‐myc Proteins Produced in Escherichia coli

Abstract

Six clones of monoclonal antibodies, MYC‐1 to ‐6, were prepared by using two kinds of truncated c‐myc proteins, p23 and p42, produced in Escherichia coli as immunogens. Analysis with enzyme‐linked immunosorbent assays and immunoblotting assays with peptides produced in Escherichia coli showed that 5 clones of monoclonal antibodies, MYC‐1 to ‐4 and ‐6, were reactive with c‐myc protein encoded by exon 2. The remaining one clone, MYC‐5, was reactive with the portion of c‐myc protein encoded by exon 3. All monoclonal antibodies were also reactive with phosphorylated c‐myc protein produced by insect cells infected by the baculovirus expression vector with the human c‐myc gene. With immunoblotting assays using cellular lysates, MYC‐1 and ‐3 detected bands at the levels of 58 kDa and 60 kDa, MYC‐5 detected a band at 56 kDa and MYC‐6 detected bands at 68 kDa and 75 kDa. All of these bands were detectable in nuclear extracts of HL‐60 and Colo320, both of which have amplified c‐myc genes, and also the extract of RmycYl which is the c‐myc gene transfectant into 3Y1 rat cells. None of them was detectable in peripheral blood mononuclear cells and 3Y1, both of which lacked activated c‐myc genes. This indicates that these nuclear proteins are either c‐myc gene products or molecules closely related to the c‐myc gene. The remaining two clones, MYC‐2 and ‐4, detected a band at the level of 85 kDa in cytoplasmic extracts of all the above‐mentioned cells independent of the presence of the c‐myc gene. This suggests that 85 kDa protein might be irrelevant to the c‐myc gene. The 56 kDa protein was detectable by MYC‐5 in phytohemagglutinin‐stimulated peripheral blood mononuclear cells as well as leukemic cells of some patients.

Immunohistochemical Detection of c-myc Products in Colorectal Cancer and Proliferative Cell Rate

“…MYC-l was detected not only in tumor cells but also in stromal cells. MYC-l is a protein for the identification of the 58-and 60-kDa protein expressed by the human c-myc gene [15], In our western blotting study, single bands to molec ular weight 60 kDa were recognized.…”

“…Suzuki et al [14] showed, however, that p62 immunoreac tivity was detected in the cytoplasm regardless of the fixa tion. We used antisera (MYC-1) to two laids of truncated human c-myc protein in Escherichia coli as produced by Naoeet al [15]. The M YC-I used in this study was detect ed in the cytoplasm of tumor cells, but a few cells showed positively stained nuclei.…”

Expression of c-myc Gene Product in Gastric Carcinoma

“…MYC-I reacts specifically with c-myc protein encoded by exon 2. MYC-1 was confirmed to detect bands at the levels of 58 and 60 kDa, that were detectable in nuclear extracts of HL-60 and COLO320, both of which amplified c-myc genes [13].…”

“…In our previous report, the staining of c-myc gene product with monoclonal antibody was predominantly cytoplasmic despite the known nuclear location of p62c-myc from im munochemical studies [14][15][16][17][18]. Naoe et al [13] report ed that MYC-1 detected bands at the levels of 58 and 60 kDa, that were detectable in nuclear extracts of HL-60 and COLO320, both of which have amplified c-myc genes, and also in the extract of RmycYI which is the c-myc transfectant into 3YI rat cells. In the present study, however, staining with MYC-1 was predominantly cytoplasmic.…”

c‐myc over‐expression in human primary ovarian tumours: Its relevance to tumour progression

“…Frozen tissues were sectioned at 6 pm thickness and fixed in 6% neutral formalin. Monoclonal mouse antibody against C-MYC protein, which was produced in Escherichia coli, MYC-1 (IgG,) (Naoe et al, 1989) was kindly provided by Dr. Shiku (Nagasaki University, Japan). After application of biotinylated horse anti-mouse immunoglobulin, staining was carried out by Vectorstain ABC kit (Vector, Burlingame, CA).…”