NOVEL AND SENSITIVE DETECTION SYSTEMS FOR THE VITAMIN-D-RECEPTOR - INSITU-REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION AND IMMUNOGOLD CYTOCHEMISTRY

The receptor for the active metabolite of vitamin D, 1,25(OH)(2)D-3, k nown as the vitamin D receptor (VDR), belongs to the steroid hormone n uclear receptor superfamily. We have developed novel methods for detec tion of VDR mRNA and protein within a human promyelomonocytic cell lin e, HL-60. Using the newly developed technique of in situ-reverse trans criptase-polymerase chain reaction (IS-RT-PCR), low levels of VDR mRNA could be amplified and demonstrated unequivocally within these cells, and also within a human kidney proximal tubule cell line, CL-8. Use o f a novel immunogold cytochemical technique has allowed clear and sens itive detection of VDR protein expression within the HL-60 cells. Furt her development of IS-RT-PCR has allowed us to apply this technique to tissue sections. We have shown clear amplification of VDR transcripts within sections of formalin-fixed paraffin-embedded human kidney and liver. These techniques mill be useful to localise specifically the VD R within cell types that contain low levels of mRNA and protein, and w ill permit further investigation of the role played by 1,25(OH)(2)D-3 in cellular regulatory mechanisms.