Chromatographic separation of eight progesterone receptor isoforms in human breast tumors, and detection by radioligand and monoclonal antibodies. Association with hsp90 and hsp70 heat shock proteins.
It has been shown previously that untransformed molybdate-stabilized breast cancer progesterone receptor (PR) complexes can be dissociated by KCl (0.4 to 1M) into eight different intermediate forms, or isoforms (680-600-361-224-193-119-88-52 kDa), separated by high performance size exclusion chromatography and with the use of computer assisted smoothing and deconvolution procedures (H Cren et al [1993] J Chromatogr 615, 23-36). The purpose of this work was to study the constitution of each isoform by using different monoclonal antibodies (mabs) raised against PR-A/B (JZB39 and KD68), against PR-B (PR6 and KC 146), and against hsp90 and hsp70 heat shock proteins (9D2 and Ab72, respectively). The differential recognition of nontransformed molybdate-stabilized PR isoforms by either radioligand (RLA, 3H-Org2058) or by an enzyme immunoassay (PgR-EIA Abbott) showed the presence of two different PR isoforms in the non-dissociated PR heteropolymeric peak eluted from a TSK-3000 SW column. After PR dissociation by 0.4 M KCl and interaction of PR isoforms with the different mabs, the presence of PR-A, PR-B, hsp90 and hsp70 was studied. Results showed that hsp90 was present in isoforms 1 (680 kDa), 2 (600 kDa) and 3 (361 kDa) exclusively, whereas hsp70 remained strongly bound to isoforms 4 (224 kDa) and 5 (193 kDa). Isoforms 6 (119 kDa) reacted with PR6 antibody and represented the PR-B protein, whereas isoform 7 (88 kDa) represented PR-A protein. Isoform 8 (52 kDa) was not detected by mabs and represented a truncated form of PR. Detection of isoform 1 either by RLA or by EIA showed ratios EIA/RLA approximately 1 or 2, and these values suggested that this heteropolymeric form may contain a dimeric structure. From these observations, a model is proposed for the composition of each PR isoform obtained from dissociation of breast tumor PR.
Laboratoire d'analyses isotopiques, centre François-Baclesse, Caen, France.
E
E
Lemoisson
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H
Cren
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J
Goussard
D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006801:Humans; D011869:Radioligand Assay; D011980:Receptors, Progesterone / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D011980:Receptors, Progesterone