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Although a majority of malignant testicular Leydig cell tumors induced in the mouse by chronic estrogenization remain dependent upon estrogen stimulation for growth during early transplant generations, we have observed tumors of two lines, no longer growth dependent upon estrogen, that regressed when hosts bearing palpable tumor grafts were given the same dosage of diethylstilbestrol that had induced the original tumors. Both estrogen-"dependent" and -"responsive" tumors were found to possess a similar estrogen receptor system. The present study compares light and electron microscopic changes occurring during regression and determines the ultimate outcome of the process under these seemingly opposite endocrine conditions. The individual neoplastic cells of the dependent tumors decreased in size, mitochondria with typical tubular cristae rapidly converted to a fully condensed configuration, and endoplasmic reticulum, both rough and smooth, as well as polyribosomes gradually disappeared. A few dormant, RNA-depleted tumor cells always remained, however. After 5 months of dormancy, mitotic activity was induced in many of these cells in 2 to 3 days by reinstituting estrogen administration. This activity began prior to conversion of the mitochondria to an orthodox configuration, to the accumulation of cytochemically demonstrable RNA, or to the appearance of RNA-containing organelles. These observations suggest that at least many of the dormant tumor "stem" cells had been blocked in G2. Contrariwise, the cytoplasmic volumes of the cells of regressing estrogen-responsive tumors increased with a considerable accumulation of lipid droplets, while alterations of the cytoplasmic organelles were much less marked, the mitochondria retaining their pretherapy morphology. Biochemical studies confirmed the fact that, although DNA synthesis ceased within a few days. RNA synthesis was maintained at a near normal level, at least during the first month of tumor regression, during which time the RNA to DNA ratio increased significantly. After 2 months or more of a sustained complete remission, no tumor cells could be found at the transplantation sites, and removing the estrogenic stimulus did not result in tumor regrowth. In short, the treatment had completely obliterated the cancer. It is concluded, therefore, that the molecular events that result in tumor regression from these diametrically opposite endocrine therapies must differ significantly. Both bring about an abrupt cessation of mitotic activity in the neoplastic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

An estrogen-responsive mouse Leydig cell tumor line (Tumor 124958) has been shown to contain only a low-affinity binder for estradiol in the cytosol fraction. This differed from the putative estrogen receptor in terms of its hormone-binding specificity as well as affinity. In addition, the possibility that an estrogen receptor-like molecule exists in the nuclei even without hormonal stimuli was examined using purified nuclei. Scatchard plot analyses showed that these nuclei possessed a large amount of estrogen binder having a high affinity for estradiol and diethylstilbestrol. The content of this nuclear binding component was not diminished by using molybdate, a potent inhibitor for receptor activation, and in vitro incubation of collagenase-dispersed cells with estradiol did not cause significant increase in the number of nuclear binding sites when compared with the values obtained by direct incubation of isolated nuclei with estradiol. These results support the view that this nuclear estrogen binder is not due to artificial migration of the cytosol receptor into nuclei during homogenization. The characterization of this nuclear binding component under cell-free conditions revealed that its affinity for estradiol in Mg2+-containing buffer was temperature dependent (Kd 3 nM at 30 degrees and 12 nM at 0 degrees) without significant alteration in the number of maximum binding sites. Introduction of a chelating agent (ethylenediaminetetraacetate) into the buffer system abolished the temperature effect on the affinity, resulting in high affinity for estradiol at both low and high temperatures. These Mg2+ and temperature effects were reversible. In addition, when compared with putative nuclear estrogen receptors, this nuclear binding was observed to be relatively resistant to high salt or micrococcal nuclease treatments in relation to solubilization from nuclei. However, trypsin digestion was found to result in a marked decrease in the nuclear binding sites, indicating that this unique nuclear binding component contains a protein unit(s). These results suggest the possibility that this tumor line contains a unique unoccupied nuclear estrogen binder which might be able to transmit estrogen signals to tumor cell nuclei with regard to tumor growth.