Estrogen receptor profiles: changes in mouse and rat mammary tumors by treatment with selective estrogen receptor modifiers.

OBJECTIVE: The aim of this work was to analyze the effect of estradiol (E(2)), medroxyprogesterone and the two selective estrogen receptor modulators (SERMs) (tamoxifen (Tam) and raloxifene (Ral)) on the estrogen receptor (ER) conformers profile performed by size exclusion HPLC in relation to hormone dependence of mammary tumors. MATERIALS AND METHODS: Two types of mammary tumors were studied: tumors transplanted in BALB/c mice that are medroxyprogesterone acetate (MPA)-dependent for growth, and tumors induced in Sprague-Dawley rats by intraperitoneal injection of N-nitroso-N-methylurea (NMU). Tumors from mice treated with MPA, E(2), Tam or Ral and NMU-treated rats were analyzed and compared to that of control. RESULTS: The tumor conformer profiles were as follows: control and MPA-treated mice showed only one peak (oligomeric form); E(2)-treated mice also showed only one peak (dimer); Tam-treated mice showed one peak corresponding to a possible proteolytic fragment, and Ral-treated mice showed two peaks (oligomeric and a possible proteolytic fragment). On the other hand, NMU-induced mammary tumors from rats showed three peaks (oligomeric, monomeric and proteolytic). CONCLUSION: Our findings may indicate that SERMs affect the aggregation state of ER and thereby its ability to modulate genomic transcription mechanisms related to growth rate.

Effects on mouse uterus of three antitumoral drugs acting upon estrogen and progesterone receptors directly and through other transduction pathways.

Three antitumoral drugs, tamoxifen (Tam), medroxyprogesterone acetate (MPA), and 8-Cl-cyclic AMP (8Cl), were administered separately and in combination to normal adult mice in order to record their effects on uterus weight, on estrous cycle, and on two estrogen receptor (ER) and progesterone receptor (PgR) parameters, namely content and nucleo-cytoplasm distribution. Tam decreased uterus weight (49%) and total ER content (118+/-6 vs 328+/-20 fmol/mg protein in controls) but increased total PgR (1183+/-230 vs 743+/-52 fmol/mg protein in controls) and nuclear retention of ER and PgR. MPA down-regulated PgR content and increased uterus weight (36%), but failed to modify ER and PgR nuclear retention. The only parameter changed by 8Cl was nucleo-cytoplasm PgR distribution. Tam + MPA association produced the same results as Tam alone for ER and PgR nuclear retention, but receptor content was not significantly different from that of controls. Both drugs, administered separately, had opposite effects on PgR content; when both were acting concurrently, an algebraic addition of effects was observed, as if both transcription circuits were triggered independently. Remaining Tam effects, not modified by a combination with MPA, indicated the predominance of Tam on the corresponding parameters. When Tam and 8Cl were administered together, 8Cl counteracted the effect of Tam only on PgR content. When associated with MPA, 8Cl changed the effects of MPA on ER and PgR nuclear retention, whereas on receptor content, only that of ER was increased (502+/-47 vs 328+/-20 fmol/mg protein in controls). These crossed effects indicate that interrelations between different transduction pathways can affect certain functional circuits while sparing others. The possibility of acting pharmacologically upon different transcription pathways represents a novel approach to modify drug effects directed to specific transduction targets through cross-talk between their components.

Evaluation of a displacement assay with tamoxifen as prognostic indicator in breast-cancer patients with estrogen-receptor-positive tumors.

A displacement assay with tamoxifen, based on the relative binding affinity of tamoxifen and estradiol for the estrogen receptor (ER), was proposed in 1990 as prognostic indicator for breast-cancer patients. Validation of its predictive results in relation to the outcome of 73 patients with ER+ tumors is analyzed. ER, progesterone receptor (PgR) determinations and other conventional prognostic factors in relation to the displacement assay, were considered. Displacement assay results allowed ER+ tumors to be grouped as displaceable (D) or weakly displaceable (WD), with the implication that D tumors should respond better to tamoxifen (Tam) administration. Survival and disease-free interval curves showed highly significant differences between patients with ER+ D and ER+ WD tumors. For survival, including all tumor stages, 73.9% of patients were alive at 9 years after surgery in the group with D tumors and 37.0% in the group with WD tumors (p < 0.005); relative contribution of the different stages is analyzed. Addition of axillary-node number increased the prognostic significance of displacement categories for survival and disease-free interval. PgR determination as another ER functional expression failed to show significant differences for survival and disease-free interval between ER+ PgR+ and ER+ PgR- tumors. Thus, results from the displacement assay and from PgR determinations reflect 2 independent ER functional expressions. Displacement assay data appear as reliable prognostic indicators of breast-cancer outcome, and contribute to more appropriate treatment decisions in this pathology.

[New concepts on hormone dependence in breast cancer]. / Nuevos conceptos sobre hormonodependencia en el cáncer de mama.

The type of hormone dependence in mammary neoplasias is usually defined by the presence or absence of estrogen and progesterone receptors. At present, new advances in the knowledge related to the functionality of these receptors are changing our previous concepts. Estrogen receptors classified as negative by biochemical or immunocytochemical methods because of deletions or mutations in their ligand-binding domain, are still able to regulate the expression of genes related to cellular proliferation. Receptors defined as positive, may present other defective domains with disappearance or distortion of their transcriptional function. As a result, regulation of the cellular proliferative process is distorted and the tumoral growth seems autonomous, as if the receptors were absent. The modular organization of the receptor molecule allows a relative functional independence of the constitutive domains. Functional assays to evaluate receptor behavior under different experimental or clinical situations are necessary. A displacement assay with tamoxifen, for studying the relative binding affinity of tamoxifen and estradiol for the estrogen receptor contributes to a more appropriate use of this antiestrogen in mammary oncology. Conformational changes and mutations in one or several of these genomic molecules may after the transcriptional message with repercussion on cellular proliferation. In this way, antiprogestinic agents can show progestin agonistic effects when combined with cAMP analogues; on the other hand, opposite effects on cellular growth by cAMP analogues can be observed according to the type of hormone dependency (autonomous or dependent) of the tumors. Modulation of steroid receptor transcriptional activity is also achieved through non-transcriptional proteins associated to the receptor molecule. These proteins are then potential targets for the pharmacological regulation of the transcription message. Resistance to antihormone treatments in breast cancer is a dominant feature in the evolution of this malignancy. It cannot be attributed to the presence or absence of steroid receptors when only defined by their quantitative variations.

Opposite effect of a cAMP analogue on tumoral growth related to hormone dependence of a murine mammary tumor.

Interactions among transcription factors are one of the mechanisms that regulate gene expression. Through protein-protein associations different signaling pathways become connected and the message triggered by each of the molecules involved can modify other related routes. In a murine mammary tumor induced by medroxyprogesterone acetate (MPA), further treatment with this agent showed a different response on tumor growth. In one group of tumors, growth rate was increased (hormone dependent, HD), whereas in the other group the progestin agent failed to modify the rate of tumor development (hormone autonomous, HA). Progesterone receptors (PgR) and estrogen receptors (ER) were expressed in both groups. Administration of 8-CI-cAMP, a cAMP analogue, stimulated tumor growth in the HD subline and inhibited growth in the HA subline. Simultaneous administration of 8-CI-cAMP and MPA resulted in suppression of inhibitory 8-CI-cAMP action in the HA tumor subline attributable to changes in molecular configuration of protagonic members of each signaling pathway, whereas in the HD subline growth was additive as if each of the pathways were acting separately. MPA induced down regulation of PgR in both tumor sublines and up regulation of ER in the C4-HD subline. The effect of 8-CI-cAMP alone or associated with MPA was more complex and variations in PgR and ER content by themselves are insufficient to explain changes in tumoral growth. A model consistent with our experimental findings is presented.

Estrogen receptor isoforms and progestin hormone dependence in a mouse mammary tumor model.

The close interaction between receptors and other transcription factors suggests that their corresponding transducing signals can trigger functional and structural changes in other related molecules. The effect of a progestinic agent, medroxyprogesterone acetate (MPA), on some of the estrogen-receptor (ER) parameters was studied in 2 murine mammary tumor sublines with different progestin hormone dependence for their respective growth. The relative binding affinity of estradiol and tamoxifen for the ER, the receptor content and the ER isoforms studied by HPLC were determined in the hormone-autonomous (HA) and the hormone-dependent (HD) tumor sublines. In the HA subline administration of MPA did not modify the tumor growth rate, whereas this was accelerated in the HD subline. The ER content was clearly increased in the HD tumor subline, but not in the HA subline, compared with the untreated controls. In contrast, the E2 and tamoxifen relative binding affinity for the ER and the isoform profiles were affected by MPA treatment in the HA, but not in the HD tumor subline. The functional change (decrease in relative binding affinity) can be attributed to the appearance of a lower-molecular-size ER isoform under the progestinic treatment. Modifications in one receptor molecule by the action of ligands corresponding to another type of receptor show the interconection between transcription factors and the necessity of broadening conventional concepts regarding hormone dependence in mammary tumorigenesis.

[Transcription factors and proliferative diversity of neoplastic cells]. / Factores de transcripción y diversidad proliferativa de las células neoplásicas.

Cancer is a multistage process with sequential steps: initiation, promotion, progression. However, the distinction between the agents that can trigger any of these processes is less clear, depending on doses, biological stage of the tissue, molecular interactions, genomic and somatic mutation, etc. Thus, steroid receptors, classically considered as promoters, could be acting as initiators under different alterations. On the other hand, chemical carcinogens that can produce DNA damage leading to cellular transformation, in lower dosage are able to alter nuclear proteins which in turn favor the action of other agents triggering the initiation process. Transcription factors (TF) are nuclear proteins with particular structural configurations for their interaction with DNA. There are families of TF with specificity for certain nucleotide sequences called "response elements". The association TF-DNA response elements modulate the genomic transcription and synthesis of proteins, many of which are related to cellular proliferation. The transcriptional message can also be influenced by TF protein-protein interactions, besides their association with the response elements. This "cross-talk" or "side regulation" becomes an important genomic regulatory mechanism. Certain biochemical signals acting on particular proteins (receptors, enzymes) can then trigger biological effects attributable to other proteins. Another transcriptional regulatory mechanism is the cytoplasm-nucleus shuttling of TF according to the cellular replication rhythm and other metabolic cellular requirements. These recent advances open new vistas for the pharmacological attack to the proliferative diversity of neoplastic cells.

Factores de transcripción y diversidad proliferativa de las células neoplásicas. / [Transcription factors and proliferative diversity of neoplastic cells]

Cancer is a multistage process with sequential steps: initiation, promotion, progression. However, the distinction between the agents that can trigger any of these processes is less clear, depending on doses, biological stage of the tissue, molecular interactions, genomic and somatic mutation, etc. Thus, steroid receptors, classically considered as promoters, could be acting as initiators under different alterations. On the other hand, chemical carcinogens that can produce DNA damage leading to cellular transformation, in lower dosage are able to alter nuclear proteins which in turn favor the action of other agents triggering the initiation process. Transcription factors (TF) are nuclear proteins with particular structural configurations for their interaction with DNA. There are families of TF with specificity for certain nucleotide sequences called [quot ]response elements[quot ]. The association TF-DNA response elements modulate the genomic transcription and synthesis of proteins, many of which are related to cellular proliferation. The transcriptional message can also be influenced by TF protein-protein interactions, besides their association with the response elements. This [quot ]cross-talk[quot ] or [quot ]side regulation[quot ] becomes an important genomic regulatory mechanism. Certain biochemical signals acting on particular proteins (receptors, enzymes) can then trigger biological effects attributable to other proteins. Another transcriptional regulatory mechanism is the cytoplasm-nucleus shuttling of TF according to the cellular replication rhythm and other metabolic cellular requirements. These recent advances open new vistas for the pharmacological attack to the proliferative diversity of neoplastic cells.

Characterization of rat uterine estrogen receptors in vivo.

In vivo binding of [3H]estradiol ([3H]E2) in the rat uterus was performed by an intraluminal perfusion of the ligand for different time periods. In this way the binding takes place in the intact organ before processing the tissue. In 10 min, with 10 nM [3H]E2 apparent saturation or steady state incorporation of the [3H]E2 was achieved with a similar distribution of the label between cytosol and nuclear fractions. In vitro, the subcellular localization of the estrogen receptor (ER) is influenced by the extent of tissue damage. With the intact organ the ER subcellular distribution approaches that of the in vivo perfusion. With increasing [3H]E2 in the perfusate it was possible to obtain a "saturation" curve and to derive the kinetic parameters. For cytosol: Kd 16 nM; Bmax 235 fmol/mg prot. For nucleus: Kd 2.7 nM; Bmax 103 fmol/mg prot. To follow the time course of the ER movement in vivo, "pulse and wait" experiments were designed. Both uterine horns were perfused for 1 min. One of the horns was immediately processed (0 time) and the other was left in place after the perfusion for different periods. At 0 time 90% of the bound label appeared in the cytosol. At 5, 15 and 30 min, the label in the cytosol decreased and that of the nucleus increased approx. to 50%. Thus, translocation of the bound label from cytosol to nucleus was apparent. The role of the cytoplasm-nucleus ER traffic in the regulation of gene transcription by estrogens is discussed.