Nucleic Acids Research, 1993, Vol. 21, No. 23 5360-5365
© 1993
MOLECULAR BIOLOGY |
Modified binding of proteins from calcitonin-negative tumor cells to the neuroendocrine-specific CANNTG motif of the calcitonin gene
Section of Endocrinology, Department of Medical Specialties, The University of Texas M.D.Anderson Cancer Center 1515 Holcombe Blvd, Houston, TX 77030, USA
Received September 1, 1993. Revised October 11, 1993. Accepted October 11, 1993.
Transcription of the calcitonin (CT) gene in the medullary thyroid carcinoma (MTC) cell line TT is modulated by a neuroendocrine-specific enhancer fragment (nucleotides - 965 to - 905) containing two CANNTG motifs (E2 and E3) and an Ets-like response element. To determine the cell-specific component of this fragment, oligonucleotides containing the individual elements were inserted in front of a minimal CT promoter and tested for reporter protein production in CT-positive (TT) and -negative (RO-D81 and HeLa) cells. In TT cells, using two copies of E2 or four copies of Ets increased minimal promoter activity a 20–40 fold. Using two copies of E3 had no effect on minimal promoter activity. In CT-negative MTC cells (RO-D81), the Ets response element was active but the two copies of E2 were not. Similar results were obtained with the non-neuroendocrine cell-line HeLa. I therefore concluded that E2 was the cell-type-specific component of the enhancer. An E2-specific binding protein was detected in both MTC cell lines but not in HeLa. This protein had different mobility and DNAbinding specificity in CT-positive TT cells and CTnegative RO-D81 cells. In conclusion, the CAGCTG motif of E2 modulated the cell-specific transcription of the CT gene, and its inactivation in CT-negative MTC cells correlated with modifications in its binding proteins.