- Email: [email protected]
Hormones and chemotherapy: To combine or not to combine
1634
CHARACTERIZATIONOF SALT-EXTRACTED NUClEAR ANTIESTROGEN RECEPTOR CO)I(PLEXES. M.A.Miller*? A,Mullick and B.S.Katzenellenbogen, Dept. of Physiology
and Biophysics,
Univ. of Illinois,
Urbana,
Illinois 61801 USA.
As part of our studies on the mechanism of antiestrogen (AE) antagonism of MCF-7 cell growth, we found that nuclear salt-extracted antiestrogen receptor complexes (AE-ERn) sediment on high salt sucrose gradients as a stable 5.5 S complex versus an unstable 5.5 S complex when bound to estrogen. To further characterize the subunit nature of AE-ERn, we have employed density labeling and chemical ~ro~~~~~~~';g,mi~~ra~~~fiptor turnover studies, cells were exposed to dense ( H for different time periods and then labeled with jH-A;. Analysis of the AE-ERn on sucrose gradients shows that the normal density 5 S peak shifts to a broader peak at 4-6 h, and by 8 h sediments as a sharp, more dense This density shift time course is the same as that seen for the species. 4 S urea-disaggregated AE-ERn receptor form, suggesting that the 5 S AE-ERn is composed of two species which turnover at the same rate (t1/2=4 sediments as a h). AE-ERn chemically crosslinked with 2-iminothiolane, 5 S species on 3 M urea-containing sucrose gradients hile noncrosslinked receptors are 4 S. SDS-PAGE analysis of crosslinked 'i; H-tamoxifen aziridine labeled ERn reveals a species of Mr lZO,OOO-130,000 while the non~rosslinked or crosslinked but mercaptan cleaved receptor is 62,000. Therefore, the 5 S AE-ERn complex appears to consist of two Mr 60,000species which turnover with similar half-lives. These data are consistent with the 5 S AE-ERn being a homodimer of ER molecules rather than ER with a dissimilar protein. (Supported by NIH CA18119 and CA31870).
K18 TO COMBINE OR NOT TO COMBINE. P.P. Carbone HORMONES AND CHEMOTHERAPY: USA Wisconsin Clinical Cancer Center, University of Wisconsin-Madison, Mechanisms of cell kill for breast cancer cells by cytotoxic drugs These usually involve interrupting have been defined in the laboratory. The mechanisms of resistance to DNA, RNA, and/or protein synthesis. cytotoxic agents are not as well defined, but many experimental models In contrast little is known in the lab or the exist to test hypotheses. clinic of how breast cancer cells die following ablative or additive The demonstration that the estrogen receptor assay endocrine therapies. results predict responders is very important, yet there is clinical evidence that responses to second therapies occur regularly following Despite these undefined problems, clinifailure on the first hormone. cians, almost from the beginning, began to put the two treatments together. Unfortunately the approaches What have we learned from these trials? have included relatively few controlled trials. They can be characterized as combined studies a) utilizing ablative therapies, b) additive treatments, c) combinations of endocrines, and more recently d) the attempt to stimulate cells with endocrine agents to enhance the effectiveness of The former three kinds of trials all use the hormonal cytotoxics. These same studies all hypothesize agents to add to the effects of drugs. that there are a mixture of hormone and endocrine sensitive cells and Clearly if the endocrine the modalities are used as independent effecters. sensitive cells are slowed or arrested in their growth a corollary would be that the two modalities would counteract each other. Data will be reviewed to develop workable approaches for future studies.
CHARACTERIZATIONOF SALT-EXTRACTED NUClEAR ANTIESTROGEN RECEPTOR CO)I(PLEXES. M.A.Miller*? A,Mullick and B.S.Katzenellenbogen, Dept. of Physiology
and Biophysics,
Univ. of Illinois,
Urbana,
Illinois 61801 USA.
As part of our studies on the mechanism of antiestrogen (AE) antagonism of MCF-7 cell growth, we found that nuclear salt-extracted antiestrogen receptor complexes (AE-ERn) sediment on high salt sucrose gradients as a stable 5.5 S complex versus an unstable 5.5 S complex when bound to estrogen. To further characterize the subunit nature of AE-ERn, we have employed density labeling and chemical ~ro~~~~~~~';g,mi~~ra~~~fiptor turnover studies, cells were exposed to dense ( H for different time periods and then labeled with jH-A;. Analysis of the AE-ERn on sucrose gradients shows that the normal density 5 S peak shifts to a broader peak at 4-6 h, and by 8 h sediments as a sharp, more dense This density shift time course is the same as that seen for the species. 4 S urea-disaggregated AE-ERn receptor form, suggesting that the 5 S AE-ERn is composed of two species which turnover at the same rate (t1/2=4 sediments as a h). AE-ERn chemically crosslinked with 2-iminothiolane, 5 S species on 3 M urea-containing sucrose gradients hile noncrosslinked receptors are 4 S. SDS-PAGE analysis of crosslinked 'i; H-tamoxifen aziridine labeled ERn reveals a species of Mr lZO,OOO-130,000 while the non~rosslinked or crosslinked but mercaptan cleaved receptor is 62,000. Therefore, the 5 S AE-ERn complex appears to consist of two Mr 60,000species which turnover with similar half-lives. These data are consistent with the 5 S AE-ERn being a homodimer of ER molecules rather than ER with a dissimilar protein. (Supported by NIH CA18119 and CA31870).
K18 TO COMBINE OR NOT TO COMBINE. P.P. Carbone HORMONES AND CHEMOTHERAPY: USA Wisconsin Clinical Cancer Center, University of Wisconsin-Madison, Mechanisms of cell kill for breast cancer cells by cytotoxic drugs These usually involve interrupting have been defined in the laboratory. The mechanisms of resistance to DNA, RNA, and/or protein synthesis. cytotoxic agents are not as well defined, but many experimental models In contrast little is known in the lab or the exist to test hypotheses. clinic of how breast cancer cells die following ablative or additive The demonstration that the estrogen receptor assay endocrine therapies. results predict responders is very important, yet there is clinical evidence that responses to second therapies occur regularly following Despite these undefined problems, clinifailure on the first hormone. cians, almost from the beginning, began to put the two treatments together. Unfortunately the approaches What have we learned from these trials? have included relatively few controlled trials. They can be characterized as combined studies a) utilizing ablative therapies, b) additive treatments, c) combinations of endocrines, and more recently d) the attempt to stimulate cells with endocrine agents to enhance the effectiveness of The former three kinds of trials all use the hormonal cytotoxics. These same studies all hypothesize agents to add to the effects of drugs. that there are a mixture of hormone and endocrine sensitive cells and Clearly if the endocrine the modalities are used as independent effecters. sensitive cells are slowed or arrested in their growth a corollary would be that the two modalities would counteract each other. Data will be reviewed to develop workable approaches for future studies.