Development of cutaneous amelanotic melanoma in the absence of a functional tyrosinase.

Lack of characteristic pigmentation and a wide range of clinical presentations account for the diagnostic challenge associated with amelanotic malignant melanoma. Experimental studies of this important human cancer have been hampered by the lack of an appropriate animal model. We previously described a transgenic mouse line (TG-3) that spontaneously develops pigmented cutaneous melanoma. F1 crosses were generated with TG-3 and several albino strains, and backcrosses were then made with the albinos. In the present report, we describe the restricted development and characterization of cutaneous amelanotic melanoma in these albino transgenic backcrosses. The incidence and behavior of melanoma in these mice were monitored. A high incidence (80-100%) of spontaneous amelanotic melanoma was observed in albino transgenic mice derived from backcrosses with A, AKR, FVB, and SJL strains. The lowest incidence (30%) was obtained in BALB/c-derived crosses. No tumors were observed in non-transgenic mice. Immunohistochemical and western blot analyses using antibodies against three melanocyte-specific markers of the tyrosinase family of proteins confirmed that the tumors were composed of amelanotic melanocytes. Furthermore, the presence of numerous premelanosomes observed by electron microscopy further supported the melanocytic origin of these tumors. Previous in vitro studies on human melanoma have suggested that cutaneous amelanotic melanoma was evolving from preexisting pigmented cutaneous melanoma. However, our results indicate that it can occur directly, as evidenced by the appearance of cutaneous amelanotic melanoma in the tyrosinase-deficient albino mice. These mice represent a potentially valuable model for studying the mechanistic, diagnostic, and therapeutic features of this highly malignant neoplasm.

High-level expression of Mpl in platelets and megakaryocytes is independent of thrombopoietin.

Thrombopoietin (TPO) is a hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production through binding to its receptor, Mpl, encoded by the c-mpl proto-oncogene. Circulating levels of TPO are regulated by receptor-mediated uptake and degradation. To better understand this mode of TPO regulation, we examined whether expression of Mpl was regulated by its ligand. Using RNase protection analysis, we found no differences in the levels of c-mpl transcripts in megakaryocytes (MKs) produced in vitro either in the presence or absence of TPO and in platelets (PLTs) obtained from mice hyperstimulated in vivo by ectopic secretion of TPO. Similarly, Western blot analysis of MKs produced in the presence or absence of TPO showed no difference in Mpl levels. Levels of Mpl, GpIIb, or P-selectin were virtually identical in platelet lysates obtained from normal, TPO knockout and mildly TPO-stimulated mice. In contrast, the expression of Mpl was significantly reduced in PLTs from severely thrombocythemic mice. These results show that TPO does not have a major effect on the transcription or translation of Mpl. However, they do suggest that an excess of circulating TPO can lead to the disappearance of Mpl from PLTs via catabolism.

Phenotypic and functional evidence for the expression of CXCR4 receptor during megakaryocytopoiesis.

The identification of stromal cell-derived factor (SDF)-1alpha as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1alpha during megakaryocytopoiesis. CD34(+) cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41(+) cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34(+) cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41(+) cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1alpha acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1alpha is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1alpha induced the migration of colony forming unit-megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1alpha as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1-related thrombocytopenia.

Inhibition of erythroid differentiation and induction of megakaryocytic differentiation by thrombopoietin are regulated by two different mechanisms in TPO-dependent UT-7/c-mpl and TF-1/c-mpl cell lines.

Thrombopoietin (TPO) regulates megakaryocytic (MK) maturation and platelet production. Molecular and cellular mechanisms of the TPO-induced MK differentiation are not totally understood. In order to develop cellular models to study these mechanisms, we introduced c-mpl into UT-7 and TF-1 cells by means of a retroviral vector and compared the effects of TPO on these two cell lines. UT-7 and TF-1 cell lines are two factor-dependent leukemic cell lines with an erythroid and MK phenotype. They proliferate in response to IL-3, GM-CSF and EPO, but not to TPO. The erythroid differentiation of both cell lines can be markedly increased by EPO. Several UT-7/c-mpl and TF-1/c-mpl cell clones which express different levels of the c-mpl protein (Mpl) were obtained and all became TPO-dependent for their proliferation. The UT-7/c-mpl clones, but not the TF-1/c-mpl clones, were capable of undergoing MK differentiation in response to TPO. This was demonstrated by the increase in MK markers (GPIIb, GPIIIa, GPIb alpha, GPIX and vWF), the appearance of cytoplasmic alpha-granules, intracellular membranes resembling demarcation membranes which were immunologically labeled with an GPIIb/IIIa anti-antibody, and a small percentage of polyploid cells (8N and 16N). In contrast, TPO inhibited the erythroid program of differentiation (glycophorin A, beta-globin and EPO receptor) as well as the differentiative activity of EPO in both UT-7/c-mpl and TF-1/c-mpl clones. It is noteworthy that the differentiative effect of EPO in TF-1/c-mpl cells was associated with an increase in GATA-1 transcripts which was totally suppressed by TPO. Overall the effects of TPO are the same as those of phorbol myristate acetate (PMA) which also induces MK differentiation and inhibits erythroid differentiation. These results suggest that: (1) Mpl expression is necessary but not sufficient for induction of MK differentiation; and (2) induction of Mk differentiation and inhibition of erythroid differentiation by TPO involve different signaling pathways; the pathway involved in the inhibition of erythroid differentiation might be related to a downregulation of GATA-1 expression in TF-1 cells.

Endomitosis of human megakaryocytes are due to abortive mitosis.

During megakaryocyte differentiation, the promegakaryoblast (immature megakaryocyte) increases its ploidy to a 2(x) DNA content by a poorly understood process called endomitosis. This leads to the formation of a giant cell, the megakaryocyte (MK), which subsequently gives rise to platelets. In this report, we show that endomitosis of human MKs is due to abortive mitosis. Human MKs were obtained by a two-step purification of CD34(+) blood or marrow precursors followed by in vitro culture in the presence of MK growth factors. Microscopic examination shows that a large number of centrosomes (up to 32) and centrioles are present in polyploid MKs. After nocodazole treatment, more than 20% of the MK are blocked in a typical pseudo-metaphase. Both spontaneous and nocodazole-induced endomitosis are associated with a breakdown of the nuclear envelope and possess a complex mitotic spindle composed of several asters. Spindle microtubules radiate from each aster, creating a spherical structure. At metaphase, expression of the kinetochore phosphoepitope recognized by the 3F3/2 antibody is lost, and the sister chromatids segregate moving toward the spindle poles. After limited segregation, the chromosomes decondense and the nuclear envelope reforms in the absence of cytokinesis, isolating all chromosomes in a single nucleus. It has been proposed that endomitosis could be due to an abnormal CDK1 activity or an absence of cyclin B1. Our results show that cyclin B1 can be detected in all MKs, including those with a ploidy of 8N or more. The cyclin B1 staining colocalizes with the mitotic spindle. Using flow cytometry, the level of cyclin B1 increased until 8N, but remained identical in 16N and 32N MKs. Cell sorting was used to separate the MKs into a 2N/4N and >4N population. Both cyclin B1 and CDK1 could be detected in the endomitotic polyploid MKs using Western blot analysis, and a histone H1 kinase activity was associated with immunoprecipitated cyclin B1. We conclude that endomitosis of human MKs is due to abortive mitosis, possibly due to alterations in the regulation of mitotic exit.

Mpl ligand or thrombopoietin: biological activities.

Thrombopoietin (TPO) or Mpl ligand is the primary physiological regulator of platelet production. This cytokine is the most potent stimulator of the proliferation and differentiation of MK progenitor and precursor cells in vitro. It also acts additively or synergistically with several cytokines on progenitor cells from various hematopoietic lineages, including the primitive stem cells. The factor is an extremely potent thrombocytopoietic agent when administrated to normal animals, and it accelerates platelet and erythropoietic recovery in several models of myelosuppression. Phase I/II clinical trials are ongoing with no detectable adverse effects. Mpl ligand does not induce platelet aggregation, but it lowers the platelet sensitivity to physiological dose of agonists. In experimental mouse models, high and chronic dose of Mpl ligand results in myelofibrosis. TPO is constantly produced by the liver and the kidney; its plasmatic clearance occurs by binding to its receptor expressed on megakaryocytes and platelets. However, the full spectrum of the biological effects of this new cytokine is not fully understood, in particular its the role in the terminal stage of platelet production. In the near future, it is likely that new insights will be obtained in the physiopathological mechanisms underlying abnormal platelet production in human.

The Mpl-ligand is involved in the growth-promoting activity of the murine stromal cell line MS-5 on ES cell-derived hematopoiesis.

We investigated the ability of the murine stromal cell line MS-5 to enhance the hematopoietic potential of embryonic stem (ES) cells. The presence of increasing concentrations of MS-5 cells during the differentiation of ES cells into embryoid bodies (EBs) resulted in a positive dose effect on the efficiency of EB development. Moreover, the number of myeloid progenitors derived from EBs at days 6 and 10 of differentiation significantly increased. This increase resulted from an elevation of both the proportions of positive EBs (EBs containing at least one progenitor each) and the progenitor cell content per positive EB. The stimulatory activity of MS-5 cells affected all types of myeloid progenitors except erythroid progenitors, which were depressed. However, the relative numbers of ES-derived granulocyte-macrophage progenitors (colony-forming units granulocyte/macrophage [CFU-GM], -macrophage [CFU-M], and -granulocyte [CFU-G]) and of mixed cell colonies were unchanged. In contrast, the incidence of megakaryocytic progenitors (colony-forming units-megakaryocyte [CFU-MK]) was significantly increased, that of erythroid progenitors (burst-forming units-erythroid [BFU-E]) was concomitantly decreased, and the total numbers of both progenitor types remained constant. Addition of Mpl-ligand (Mpl-L; thrombopoietin) during the growth of EBs was found to mimic the effect of the MS-5 cell line on the output of progenitor cells. No effect of Mpl-L on the efficiency of EB formation was observed. In addition, supplementation of cultures with sufficient soluble Mpl to abrogate Mpl-L activity resulted in the reversion of the quantitative and qualitative effects of MS-5 cells on progenitor cell formation but not on the efficiency of EB formation. Together, these data indicate two major effects and two levels of action of the MS-5 cell line on hematopoietic differentiation of ES cells. First, the cell line acts before hematopoietic determination, promoting the plating efficiency of ES cells via mechanisms that remain to be clarified. Second, at a later stage of differentiation, the MS-5 cells promote hematopoiesis within EBs. Mpl-L appears to be one of the components that confer this latter ability on the MS-5 cell line.

Compared effects of Mpl ligand and other cytokines on human MK differentiation.

The discovery of the Mpl ligand (Mpl-L), also called thrombopoietin (TPO), has facilitated in vitro investigation of human megakaryocytopoiesis. By confocal microscopy, endomitosis appeared as abortive mitosis skipping late stages of mitosis. No telophase and cytokinesis were observed. A spherical multipolar spindle which limits chromatid segregation was observed. The nuclear envelope subsequently reformed isolating all chromatids in a single nucleus. Platelet shedding was ultrastructurally studied. Platelet release occurred after formation of long cytoplasmic extensions (proplatelet formation), constriction areas delineating platelet territories. Heterogeneity in platelet size may be determined by the length of these extensions. Pegylated-recombinant human megakaryocyte growth and development factor, a truncated form of Mpl-L, was the most efficient cytokine to produce proplatelet-bearing megakaryocytes (MKs) and platelets in vitro. However, functional platelets with a normal ultrastructure could be produced in the presence of a combination of other cytokines. Finally, we investigated whether the induction of MK differentiation by the MS-5 stromal cell lines is due to Mpl-L. MS-5 cells synthesized Mpl-L transcripts and a biologically active protein. When human CD34+ cells were grown in contact or noncontact cultures with MS-5 cells, MK differentiation was observed. Soluble Mpl receptor (sMpl-Fc) addition inhibited MK growth, suggesting that the MK-promoting activity was due to Mpl-L production. Marrow stromal cell lines derived from TPO-/- mice were also able to sustain MK growth. Despite the absence of any production of Mpl-L, the sMpl-Fc continued to inhibit MK differentiation. This result suggests that the sMpl has a direct inhibitory effect and may explain the divergent results in the literature concerning the precise role of Mpl-L on the MK terminal differentiation.

Myelofibrosis: experimental models and human studies.

Thrombopoietin (TPO) is the central regulator of megakaryocytopoiesis and thrombocytopoiesis. Preclinical data and human studies have so far shown that the recombinant molecule is safe to administer and associated with very little toxicity. Nevertheless, different experimental animal models have revealed that a chronic exposure to very high doses of TPO could result in myeloproliferative syndromes with a spectrum of pathological features in common with human idiopathic myelofibrosis (PMF). A number of investigators have researched whether TPO or its receptor Mpl were involved in the pathogenesis of human myeloproliferative syndromes which are also characterized by a predominant megakaryocytic involvement, in PMF and primitive essential thrombocythemia. In both diseases, megakaryocyte (MK) progenitors develop autonomously in serum-deprived cultures. This spontaneous MK development is also observed at limiting dilution demonstrating that MK escape the normal regulatory controls. Furthermore, this abnormal MK proliferation and maturation is neither due to an autocrine stimulation by TPO nor by point mutation or deletion in the coding region of the c-mpl gene. This paper will review the data that have been reported to date on the effects of an overexpression of Mpl ligand and related molecules on the induction of experimental myelofibrosis and highlight recent insights into the pathogenesis of PMF.

High thrombopoietin production by hematopoietic cells induces a fatal myeloproliferative syndrome in mice.

To evaluate the effects of long-term, high-dose exposure to thrombopoietin (TPO), lethally irradiated mice were grafted with bone marrow cells infected with a retrovirus carrying the murine TPO cDNA. Mice were studied for 10 months after transplantation. In plasma, TPO levels were highly elevated (10(4) U/mL) throughout the course of the study. All mice developed a lethal myeloproliferative disorder evolving in two successive phases. During the first phase (7-9 weeks posttransplant), platelet and white blood cell (WBC) counts rose four- and ten-fold, respectively, whereas hematocrits decreased slightly to 29% +/- 3%. The WBC were mainly mature granulocytes, but myeloid precursor cells were invariably observed as well as giant platelets with an irregular granule distribution. The striking features were a massive hyperplasia of megakaryocytes and granulocytes in the spleen and bone marrow and a hypoplasia of erythroblasts in bone marrow. Total numbers of megakaryocyte colony-forming cell, burst-forming unit-erythroid, and granulocyte macrophage colony-forming cells were increased but colony-forming unit-erythroid numbers decreased. From 10 weeks posttransplant and thereafter, WBC, platelets, and red blood cell numbers declined dramatically. The absolute numbers of progenitor cells were very low in the spleen and bone marrow, but sharply increased in the blood and peritoneal cavity. Extramedullary hematopoiesis was observed in several organs. Histologic sections of the spleen and bones revealed severe fibrosis and osteosclerosis. The mean survival time was 7 months posttransplant and mice died with severe pancytopenia. Notably, two mice died between 3 and 4 months posttransplant with a leukemic transformation. This disorder was transplantable into secondary recipients who developed an attenuated form of the disease similar to the one previously described (Yan et al, Blood 86:4025, 1995). Taken together, our data show that high and persistent TPO production by transduced hematopoietic cells in mice results in a fatal myeloproliferative disorder that has a number of features in common with human idiopathic myelofibrosis.

Thrombopoietin (Mpl-ligand) and the regulation of platelet production.

Thrombopoietin is the primary physiological regulator of platelet production. TPO stimulates both early and late megakaryocyte (MK) progenitors, and acts additively or synergistically with several cytokines on various progenitors including the most primitive stem cells. Much has been learn about the physiology of the endogenous TPO production and its regulation. TPO is constantly produced by the liver and kidney; its plasmatic clearance occurs by binding to the Mpl receptor expressed on MKs and platelets. TPO binding is followed by internalization and catabolism. The factor is a potent thrombopoietic agent in vivo and accelerates platelet recovery in several models of marrow suppression. Phase I clinical trials have begun. The lack of adverse effects suggests that TPO may join other cytokines in the clinical armamentarium. Nevertheless, several important areas of basic or clinical haematology remain to be explored to fully understand the biology of this new cytokine. For example, is TPO needed for the terminal stage of platelet formation? What genetic events are involved in the process of endomitosis? What are the physiopathological mechanisms underlying thrombocytopenia or thrombocytemia in human?

Constitutive expression of Mpl ligand transcripts during thrombocytopenia or thrombocytosis.

Mpl ligand (thrombopoietin [TPO]) is the physiological regulator of platelet production. In mice, mRNA encoding the Mpl ligand (Mpl-L) is predominantly found by Northern blot analysis in the liver and kidney. To investigate the mode of regulation of the Mpl-L gene, we have developed several experimental models of severe thrombocytopenia differing in their kinetics and an opposite model of chronic thrombocytosis. Northern analysis performed at various times after induction of a thrombocytopenic state demonstrates that, whatever the number of circulating platelets, no change in Mpl-L mRNA level occurs in liver and kidney. By ribonuclease protection assays, we analyzed the ratios between mRNAs coding for the wild-type Mpl-L form and various splice variants encoding inactive or nonsecreted Mpl-L proteins. No modification in levels of these various isoforms was detected confirming the data of a previous report. Because the highest level of Mpl-L bioactivity in sera was observed only in mice with drastically reduced numbers of both platelets and megakaryocytes, these results further suggest that not only platelets, but also megakaryocytes, must be involved in the regulation of the level of circulating Mpl-L. In addition, we show that no downregulation of wild-type Mpl-L mRNA and no change in the ratio of Mpl-L mRNA isoforms were detected in mice in which a chronic thrombocytosis was induced. Together, these different models extend and further confirm that the regulation of Mpl-L does not occur at a transcriptional level or by a modulation in the ratios of Mpl-L mRNA isoforms.

[Mpl ligand (thrombopoietin) and platelet regulation]. / Le ligand de Mpl (thrombopoïétine) et la régulation plaquettaire.

After 35 years of research, the physiological regulator of platelet production has been isolated and its gene cloned. This discovery originates from studies performed with the myeloproliferative leukemia virus (MPLV), a murine retrovirus which induces an acute myeloproliferative syndrome in adult mice. MPLV carries in its genome the v-mpl oncogene which corresponds to a truncated form the c-mpl proto-oncogene. c-mpl encodes a cytokine receptor (Mpl-R) belonging to the hematopoietin receptor superfamily. Among the hematopoietic cell lineages, Mpl-R is preferentially expressed on late megakaryocyte progenitors, megakaryocytes and platelets. The ligand for Mpl-R, called Mpl-L or TPO or MGDF or megapoietin, is a glycosylated hormone of 352 amino acids in human which comprises two domains: the N-terminus domain shares 50% similarity with erythropoietin and is responsible for the biological activity; the C-terminus part is required for secretion. Notwithstanding its major action on megakaryocytopoiesis and thrombocytopoiesis, Mpl-L also potentiates the action of other cytokines on several hematopoietic lineages. Mpl-L/TPO/MGDF, the homeostatic regulator of platelet production, might be a useful therapeutical cytokine to treat thrombocytopenia induced in patients by chemotherapy.

Progesterone receptor expression in human saphenous veins.

BACKGROUND: Clinical and epidemiological observations regarding varicose veins, such as their predominance in women and the occurrence of venous stasis during sex-hormone therapy, the luteal phase of the menstrual cycle, and pregnancy, suggest a sex hormone-dependency of this venous pathology. In the present study, analysis of steroid receptors was used to determine if these effects were due to a direct hormonal action on the saphenous vein. METHODS AND RESULTS: Biopsy samples were obtained from patients undergoing stripping removal of varicose saphenous veins. Patients were men (n = 5) and premenopausal (n = 15) or postmenopausal (n = 10) women. Progesterone receptors (PR) and estrogen receptors (ER) were determined by both enzyme immunoassay (EIA) and immunocytochemistry by use of monoclonal antibodies. Ninety percent of the biopsy samples showed PR positivity by EIA (range, 5 to 53 fmol/mg cytosol protein). When present, PR staining was observed in the cell nuclei of the tunica media and the subendothelial layer (neointima). No significant variation was observed in the PR content of different regions within the same saphenous vein. In contrast, no ER or extremely low levels of ER (< 5 fmol/mg cytosol protein) were detected by EIA in 25 of 30 varicose biopsy samples. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze PR and ER mRNAs in biopsy samples that were PR positive/ER negative. With primers to the hormone-binding region encoded by PR mRNA, a RT-PCR product of the expected size was detected and its identity confirmed by Southern blot by use of a PR cDNA probe. In contrast, no RT-PCR product could be detected by use of primers to the DNA-binding domain, the hinge region, and the ligand-binding domain encoded by ER mRNA. CONCLUSIONS: These results indicate that human saphenous veins from both sexes express PR, as previously described for arterial blood vessels. This observation suggests that progesterone acts directly on these veins via a classic receptor-mediated pathway.

Phosphorylation sites in ligand-induced and ligand-independent activation of the progesterone receptor.

Steroid hormone receptors are phosphoproteins that undergo hyperphosphorylation upon binding of hormone. The mechanism and the role of this reaction remain poorly understood. Two-dimensional analysis of ligand-free progesterone receptor (PR) tryptic digests showed the existence of seven main phosphopeptides. Incubation of the cells with the progestin R5020 led to a global increase in the levels of PR phosphorylation. However, the same phosphopeptides were seen, and their levels of labeling relative to each other were unchanged. A similar result was observed after incubation of cells with the antiprogestin RU486. The antiprogestin ZK98299 demonstrated only half of the activity of RU486 in terms of receptor hyperphosphorylation, but the same phosphopeptides, proportionally labeled to the same extent, were observed by chromatography electrophoresis. Ligand-induced DNA binding did not play a role in receptor hyperphosphorylation since the mutant delta 547-592, which is devoid of the first zinc finger region, exhibited the same phosphopeptides, labeled to the same extent, as did wild-type receptor after incubation of cells with hormone. These results suggest that the same kinase(s) act in vivo on ligand-free and on agonist or antagonist-bound progesterone receptor. Binding of different ligands produces different conformational changes in the ligand binding domain of the receptor which enhance, to varying extents, affinity of the receptor for the kinase(s). The DNA binding region also plays a role in the interaction with the kinase(s), although binding to DNA per se is not necessary for the hyperphosphorylation of the receptor to take place.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific binding of progesterone receptor to progesterone-responsive elements does not require prior dimerization.

Steroid-hormone receptors undergo, prior to binding to DNA, a hormone-dependent dimerization. It is generally accepted that this dimerization is indispensable for the high-affinity binding of hormone receptor to hormone-responsive elements. Using a progesterone-receptor mutant with the complete steroid-binding domain deleted (positions 663-930), with or without the epitope required for binding the monoclonal antibody Let 126, we have shown that this receptor species was unable to undergo dimerization in solution. However, this mutant retained a high affinity (60-70% of the affinity of the wild-type receptor) for the progesterone-responsive elements of the mouse-mammary-tumor-virus long-terminal-repeat promoter and for a consensus palindromic progesterone-responsive element, as measured by both DNase-I protection experiments and gel-shift experiments. This mutant also increased gene transcription. Thus, at least in the case of the progesterone receptor, prior dimerization is dispensable for receptor binding to regulatory DNA elements and for subsequent transcription activation.