Estrogen response elements alter coactivator recruitment through allosteric modulation of estrogen receptor beta conformation.

Estrogen receptor beta (ERbeta) activates transcription by binding to estrogen response elements (EREs) and coactivator proteins that act as bridging proteins between the receptor and the basal transcription machinery. Although the imperfect vitellogenin B1, pS2, and oxytocin (OT) EREs each differ from the consensus vitellogenin A2 ERE sequence by a single base pair, ERbeta activates transcription of reporter plasmids containing A2, pS2, B1, and OT EREs to different extents. To explain how these differences in transactivation might occur, we have examined the interaction of ERbeta with these EREs and monitored recruitment of the coactivators amplified in breast cancer (AIB1) and transcription intermediary factor 2 (TIF2). Protease sensitivity, antibody interaction, and DNA pull-down assays demonstrated that ERbeta undergoes ERE-dependent changes in conformation resulting in differential recruitment of AIB1 and TIF2 to the DNA-bound receptor. Overexpression of TIF2 or AIB1 in transient transfection assays differentially enhanced ERbeta-mediated transcription of reporter plasmids containing the A2, pS2, B1, and OT EREs. Our studies demonstrate that individual ERE sequences induce changes in conformation of the DNA-bound receptor and influence coactivator recruitment. DNA-induced modulation of receptor conformation may contribute to the ability of ERbeta to differentially activate transcription of genes containing divergent ERE sequences.

Allosteric modulation of estrogen receptor conformation by different estrogen response elements.

Estrogen-regulated gene expression is dependent on interaction of the estrogen receptor (ER) with the estrogen response element (ERE). We assessed the ability of the ER to activate transcription of reporter plasmids containing either the consensus vitellogenin A2 ERE or the imperfect pS2, vitellogenin B1, or oxytocin (OT) ERE. The A2 ERE was the most potent activator of transcription. The OT ERE was significantly more effective in activating transcription than either the pS2 or B1 ERE. In deoxyribonuclease I (DNase I) footprinting experiments, MCF-7 proteins protected A2 and OT EREs more effectively than the pS2 and B1 EREs. Limited protease digestion of the A2, pS2, B1, or OT ERE-bound receptor with V8 protease or proteinase K produced distinct cleavage products demonstrating that individual ERE sequences induce specific changes in ER conformation. Receptor interaction domains of glucocorticoid receptor interacting protein 1 and steroid receptor coactivator 1 bound effectively to the A2, pS2, B1, and OT ERE-bound receptor and significantly stabilized the receptor-DNA interaction. Similar levels of the full-length p160 protein amplified in breast cancer 1 were recruited from HeLa nuclear extracts by the A2, pS2, B1, and OT ERE-bound receptors. In contrast, significantly less transcriptional intermediary factor 2 was recruited by the B1 ERE-bound receptor than by the A2 ERE-bound receptor. These studies suggest that allosteric modulation of ER conformation by individual ERE sequences influences the recruitment of specific coactivator proteins and leads to differential expression of genes containing divergent ERE sequences.

Isolation of mouse TFIID and functional characterization of TBP and TFIID in mediating estrogen receptor and chromatin transcription.

TFIID is a general transcription factor required for the assembly of the transcription machinery on most eukaryotic promoters transcribed by RNA polymerase II. Although the TATA-binding subunit (TBP) of TFIID is able to support core promoter and activator-dependent transcription under some circumstances, the roles of TBP-associated factors (TAF(II)s) in TFIID-mediated activation remain unclear. To define the evolutionarily conserved function of TFIID and to elucidate the roles of TAF(II)s in gene activation, we have cloned the mouse TAF(II)55 subunit of TFIID and further isolated mouse TFIID from a murine FM3A-derived cell line that constitutively expresses FLAG-tagged mouse TAF(II)55. Both mouse and human TFIIDs are capable of mediating transcriptional activation by Gal4 fusions containing different activation domains in a highly purified human cell-free transcription system devoid of TFIIA and Mediator. Although TAF(II)-independent activation by Gal4-VP16 can also be observed in this highly purified human transcription system with either mouse or yeast TBP, TAF(II)s are strictly required for estrogen receptor-mediated activation independently of the core promoter sequence. In addition, TAF(II)s are necessary for transcription from a preassembled chromatin template. These findings clearly demonstrate an essential role of TAF(II)s as a transcriptional coactivator for estrogen receptor and in chromatin transcription.

Macrophage-mediated islet cell cytotoxicity in BB rats.

Islet cell killing mediated by natural killer cells and T-lymphocytes in diabetes-prone (DP) and diabetic BB rats has been described, but other killing mechanisms may also be involved. Histopathologic studies suggest that macrophages are the first immune cells to infiltrate islets. To determine if macrophages are the first cells mediating islet damage, macrophage-mediated cytotoxicity was evaluated in BB rats of different ages. Splenic macrophages isolated from DP rats at 33, 100, 120, and 140 days of age showed no enhanced islet killing compared with diabetes-resistant rats. Killing at diabetes onset (121 +/- 14 days) was markedly increased (43 +/- 9.3%) compared with age-matched diabetes-resistant controls (19 +/- 8.3%, P less than .001). Islet inflammation was monitored at all time points. At 120 and 140 days of age, 9 of 11 (82%) DP rats had insulitis, and cytotoxicity was increased in 6 of 11 (55%) rats, which is similar to the number of DP rats that progress to diabetes. At 100 days, 3 of 6 (50%) DP rats again showed diabetic levels of killing, even in the absence of insulitis. These data indicate that 1) islet inflammation is dissociated from clinical diabetes onset, 2) splenic macrophages may have islet-killing potential before islet inflammation, 3) macrophage-mediated islet killing is elevated in all animals immediately after diabetes onset, and 4) macrophages, in addition to natural killer cells and T-lymphocytes, are responsible for cell-mediated islet destruction and thus are candidates for the first cellular effector to result in islet killing.

Effects of adrenalectomy and glucocorticosteroid therapy on bone marrow T cells. Effect on T cell traffic and graft-versus-host (GVH) reactivity.

The effects of bilateral adrenalectomy (Ax) and glucocorticosteroid (GCS) treatment on the migratory behavior of circulating T cells in mice were evaluated by a 51Cr lymphocyte migration assay and two graft-versus-host (GVH) assays. The major new findings were that bilaterally adrenalectomizing a mouse effects it in two interrelated ways: 1) It decreases the accumulation of adoptively transferred 51Cr-labeled T cells to the bone marrow; and 2) it reduces the GVH reactivity of bone marrow cells. We also confirmed previous studies showing increases in the accumulation of T cells and increases in T cell-mediated GVH reactivity in the marrow of GCS-treated mice. We conclude that Ax has an opposite effect to that of GCS treatment on the intramarrow traffic of T cells and on T cell-mediated GVH reactivity of marrow cells.

Enhancement of humoral immune response against human lung elastin peptides.

When guinea pigs instead of rabbits were used as the host animals, 8--16 times higher antibody titers against human lung elastin peptides were produced with only 1/20 the amount of antigen per unit body weight. This corresponds to a 200-fold enhancement of the immune response.

Protection against fulminant sepsis in splenectomized mice by implantation of autochthonous splenic tissue.

Infection of splenectomized mice with Diplococcus pneumoniae, type III, resulted in a fourfold higher mortality rate than did infection of normal mice. Splenectomized animals were protected against fulminant, fatal sepsis by subcutaneous transplantation of autochthonous splenic tissue at the time of splenectomy. Animals with ectopic splenic tissue, and sham-splenectomized control mice, exhibited normal serum opsonin and leukophilic gamma-globulin activity, with respect to pneumococcus, that was lacking in splenectomized animals.

Antiserum to surface antigens as a marker for cultured rat lung endothelial cells (1).

Antibodies were developed in rabbits against an established line of endothelial cells from normal adult rat lung. Pre- and postimmunization sera were tested for antibody activity by the indirect immunofluorescence technique. Preimmunization serum failed to react with the endothelial cells, whereas the antibody titer of postimmunization serum from two rabbits was 1:512. Organ specificity and species specificity were assessed by absorbing the serum with packed dissociated cells from different organs of the rat and lung cells of other species. Only cells obtained from rat lung absorbed the antibodies completely. The antiserum showed some crossreactivity with the other cultured cells but the pattern of fluorescence was different. In the presence of complement the antiserum was found to be cytotoxic to cultured rat lung endothelial cells but not to the other crossreacting cells.

Depressed phagocytic function exhibited by polymorphonuclear leucocytes from chronically iron deficient rabbits.

The production of chronic iron deficiency in rabbits was associated with impaired phagocytosis of opsonized Staphylococcus aureus by their polymorphonuclear leucocytes. These cells had a reduced ability to ingest the bacteria and the rate of ingestion could not be stimulated by autologous serum leucophilic gamma-globulin.

Immunological impairment and susceptibility to infection after splenectomy.

Asplenic patients or those who have undergone splenectomy are prone to overwhelming and often fatal sepsis, which is sometimes associated with disseminated intravascular coagulopathies. Although several pathogens are involved, the most common organism found in these subjects is the pneumococcus, and the infections respond poorly to antibiotic therapy.

Experimental cancer immunotherapy: comparison of tumor rejection if F344 rats given live Mycobacterium bovis (Strain BCG) and killed Corynebacterium parvum.

F344 rats received grafts of syngeneic 13762 mammary adenocarcinoma cells previously admixed with either living BCG of killed Corynebacterium parvum administered sc or intradermally (id). Animals given id transplants of tumor cells admixed with either BCG or killed C. parvum exhibited tumor growth for an average of 10 days, then regression in size and rejection of the tumor nodules. Lesions were found in rats given sc transplants of tumor cells admixed with the killed microorganism for an average of 13 days with the same results. When live BCG was added to the sc transplants, accelerated rates of tumor growth and early death were noted, compared with the group receiving tumor cells alone sc. Suppressed rates of tumor growth and prolonged survival were observed in the groups receiving id inoculations of tumor cells followed by treatment with killed C. parvum administered weekly ip or id 1 cm away and around the growing tumor. On the other hand, weekly treatment of BCG injected either ip or id 1 cm away and around the growing tumor resulted in accelerated rates of tumor growth and early death. Animals exhibiting C. parvum of BCG-mediated tumor rejection displayed tumor-specific protection to sc challenge injections of the cell line initially used, but they died with growing tumors and metastases when challenged with tumor cells of an antigenically different line syngeneic to F344 RATS. Microscopic examination of histologic sections of tumors formed from id inoculations of tumor cells admixed with either BCG or killed C. parvum revealed a nonspecific infiltrate of macrophages and polymorphonuclear leukocytes in the tumor, whereas sections of tumors formed from sc grafts of cells admixed with killed C. parvum revealed a specific organized infiltrate of mostly macrophages around the tumor follicles.

Rejection of mammary adenocarcinoma cell tumors in DBA/2 mice immunocompromised by thymectomy and treatment with antithymocyte serum.

Syngeneic mammary adenocarcinoma cells were mixed with killed Corynebacterium parvum organisms and were then transplanted subcutaneously into groups of normal and immunocompromised mice. The tumors formed at the site of injection exhibited normal growth for approximately 12 days followed by rapid and lasting rejection in both normal and immunocompromised animals. The control mice were protected against reinjections of 10-4 to 10-8 cells of the same line of tumor cells. The immunocompromised mice exhibited protection to reinjection of 10-4 cells of the tumor line initially employed. Microscopic examination of histologic sections of 10-day-old tumors in immunocompromised mice arising from administration of tumor cells and C.parvum mixtures revealed infiltration of only macrophages whereas those from normal animals exhibited both macrophages and lymphocytes. The data suggested that primary rejection of these tumors in immunocompromised mice may be the result of macrophage activity together with humoral immunity. These studies also revealed that 59 to 75% of immunocompromised mice exhibit protection against reinjection of the same line of tumor cells if twice-weekly treatment (i.p.) with killed C. parvum was included after inoculation of tumor cells mixed with C. parvum vaccine. These mice, unlike their saline-treated peers, exhibit delayed hypersensitivity reactions which are associated with proliferation of theta antigen-bearing lymphocytes. They also have intact tumor-directed humoral immune reactions.

Quantification of CEA in human gastrointestinal cancers grown in in vitro long-term organotype culture.

Two malignant tumors of the human gastrointestinal tract (obtained surgically) have been cultivated in vitro in long-term organotypic tissue culture. Of these line AZ110 (maintained for 6 years) originated from a primary adenocarcinoma of the ascending colon, whereas line Z200 (grown for 14 years) originated from a hepatic metastatic nodule of a gastric adenocarcinoma. Extracts of these tumors and comparable surgical specimens were evaluated quantitatively for carcinoembryonic antigen (CEA) using radioimmunoassay. Both cultured lines exhibited CEA levels that were somewhat higher than comparable surgical specimens.