Transcriptional repression by thyroid hormone receptors. A role for receptor homodimers in the recruitment of SMRT corepressor.

Nuclear hormone receptors, such as the thyroid hormone receptors (T3Rs) and retinoid X receptors (RXRs), are ligand-regulated transcription factors that control key aspects of metazoan gene expression. T3Rs can bind to DNA either as receptor homodimers or as heterodimers with RXRs. Once bound to DNA, nuclear hormone receptors regulate target gene expression by recruiting auxiliary proteins, denoted corepressors and coactivators. We report here that T3R homodimers assembled on DNA exhibit particularly strong interactions with the SMRT corepressor, whereas T3R.RXR heterodimers are inefficient at binding to SMRT. Mutants of T3R that exhibit enhanced repression properties, such as the v-Erb A oncoprotein or the T3Rbeta-Delta432 mutant found in human resistance to thyroid hormone syndrome, display enhanced homodimerization properties and exhibit unusually strong interactions with the SMRT corepressor. Significantly, the topology of a DNA binding site can determine whether that site recruits primarily homodimers or heterodimers and therefore whether corepressor is efficiently or inefficiently recruited to the resulting receptor-DNA complex. We suggest that T3R homodimers, and not heterodimers, may be important mediators of transcriptional repression and that the nature of the DNA binding site, by selecting for receptor homodimers or heterodimers, can influence the ability of the receptor to recruit corepressor.

Resistance to thyroid hormone (RTH) syndrome reveals novel determinants regulating interaction of T3 receptor with corepressor.

Thyroid hormone receptors (T3Rs) both repress and activate gene transcription by interacting with auxiliary factors denoted corepressors and coactivators. Resistance to thyroid hormone (RTH) syndrome in humans is manifested as a failure to respond properly to elevated circulating thyroid hormone. RTH syndrome has been mapped to T3Rbeta mutations that alter the transcriptional properties of the receptor, resulting in a dominant negative phenotype. We report here a characterization of a series of RTH mutant T3Rs that exhibit unusual interactions with corepressor. Two mutations in receptor helix 11 (delta430, delta432) greatly enhance the ability of the mutant receptors to bind to corepressor. A distinct mutation, V264D, in an 'omega loop' region of the receptor, impairs corepressor release but does not fully eliminate the ability to recruit coactivator. These mutations reveal novel determinants that regulate the interaction of the T3R with important ancillary cofactors, and that are disrupted in a human endocrine disease.

A conformational switch in nuclear hormone receptors is involved in coupling hormone binding to corepressor release.

Nuclear hormone receptors are ligand-regulated transcription factors that modulate gene expression in response to small, hydrophobic hormones, such as retinoic acid and thyroid hormone. The thyroid hormone and retinoic acid receptors typically repress transcription in the absence of hormone and activate it in the presence of hormone. Transcriptional repression is mediated, in part, through the ability of these receptors to physically associate with ancillary polypeptides called corepressors. We wished to understand the mechanism by which corepressors are recruited to unliganded nuclear hormone receptors and are released on the binding of hormone. We report here that an alpha-helical domain located at the thyroid hormone receptor C terminus appears to undergo a hormone-induced conformational change required for release of corepressor and that amino acid substitutions that abrogate this conformational change can impair or prevent corepressor release. In contrast, retinoid X receptors appear neither to undergo an equivalent conformational alteration in their C termini nor to release corepressor in response to cognate hormone, consistent with the distinct transcriptional regulatory properties displayed by this class of receptors.

Thyroid hormone resistance syndrome manifests as an aberrant interaction between mutant T3 receptors and transcriptional corepressors.

Nuclear hormone receptors are hormone-regulated transcription factors that play critical roles in chordate development and homeostasis. Aberrant nuclear hormone receptors have been implicated as causal agents in a number of endocrine and neoplastic diseases. The syndrome of Resistance to Thyroid Hormone (RTH) is a human genetic disease characterized by an impaired physiological response to thyroid hormone. RTH is associated with diverse mutations in the thyroid hormone receptor beta-gene. The resulting mutant receptors function as dominant negatives, interfering with the actions of normal thyroid hormone receptors coexpressed in the same cells. We report here that RTH receptors interact aberrantly with a newly recognized family of transcriptional corepressors variously denoted as nuclear receptor corepressor (N-CoR), retinoid X receptor interacting protein-13 (RIP-13), silencing mediator for retinoid and thyroid hormone receptors (SMRT), and thyroid hormone receptor-associating cofactor (TRAC). All RTH receptors tested exhibit an impaired ability to dissociate from corepressors in the presence of thyroid hormone. Two of the RTH mutations uncouple corepressor dissociation from hormone binding; two additional RTH mutants exhibit an unusually strong interaction with corepressor under all hormone conditions tested. Finally, artificial mutants that abolish corepressor binding abrogate the dominant negative activity of RTH mutants. We suggest that an altered corepressor interaction is likely to play a critical role in the dominant negative potency of RTH mutants and may contribute to the variable phenotype in this disorder.

Epithelioid sarcoma with an 18q aberration.

Epithelioid sarcoma is a peculiar soft-tissue neoplasm of uncertain origin, which is characterized by an epithelioid morphology of tumor cells coexpressing epithelial (keratin) and nonepithelial (vimentin) antigens. We herein report a new cytogenetic abnormality with der(22)t(18;22)(q11;p11.2) in a case of epithelioid sarcoma that occurred in the elbow of a 75-year-old man. Histologically, the tumor demonstrated a multinodular proliferation of epithelioid cells, with positive immunostaining for keratin, epithelial membrane antigen (EMA), and vimentin. Cultured tumor cells obtained from fresh surgical materials were frozen in plastic ampules and stocked in a liquid nitrogen freezer. Six years after surgery, the cells were recovered from the freezer and utilized for both morphologic and cytogenetic analyses. These cultured cells both before and after the freezing exhibited essentially the same epithelioid morphology and immunophenotypes as those of the original tumor. A chromosome analysis, together with fluorescence in situ hybridization (FISH), demonstrated a 61-67 modal population, and a characteristic clonal abnormality with der(22)t(18;22)(q11;p11.2). Other clonal abnormalities included numerical (-3, -4, +7, -13, -14, -16, -18, +20, -22) and structural (8p+, 9p+, 12p+, i(21q)) aberrations. Some variant clones also demonstrated i(18q). Since the breakpoint at 18q11 is similar to that reported in synovial sarcoma, this finding may support the presence of a histogenetic relationship between epithelioid sarcoma and synovial sarcoma. Our study thus indicates that the storage of frozen cells is useful for both morphologic and cytogenetic analyses of soft tissue tumors.

[Criteria for the evaluating treatment on primary bone sarcoma].

The criteria for the evaluation of the treatment on primary bone sarcoma proposed by the Musculoskeletal Tumor Committee of the Japanese Orthopaedic Association (JOA) were accepted in 1993 by the Joint Committee for Cancer Therapy of the Japan Society for Cancer Therapy. The responses to treatment are classified as a complete response (CR), a partial response (PR), no change (NC), or as progressive disease (PD). It is a requirement that the condition of CR, PR, or NC continues for at least 4 weeks. The radiographical criteria are as follows: CR, the disappearance of the intramedullary lesions and the bone trabeculae recovering a normal appearance; PR, the circumscription of the extraosseous tumor and an appearance of sclerotic foci within the intramedullary lesion; NC, no changes noted in the extraosseous and intramedullary lesions; PD, an increase in the extraosseous or intramedullary lesion and/or the appearance of new lesions. Histopathological criteria are as follows: CR, no tumor cells appearing viable in any of the histologic sections; PR, greater than 90% tumor necrosis attributable to treatment; NC, 50 to 90% tumor necrosis and other secondary changes attributable to treatment; PD, less than 50% tumor necrosis. Responses in the clinical signs and symptoms including tumor size (or circumference of the extremities at the site of the tumor), pain and local heat are also graded as follows: PR, subsiding symptoms and/or decrease in the tumor size; NC, neither exacerbation nor decrease in the symptoms and no change in size; PD, the exacerbation of symptoms or an increase in size. Additionally, the serum alkaline phosphatase level can be used for evaluating the effect of the treatment on the osteosarcoma. The extent of the response to preoperative chemotherapy is a powerful predictor of patient survival.

[Intra-arterial combination chemotherapy using both regimen selected by chemosensitivity test and caffeine to increase effect for osteosarcoma in extremity].

We treated three patients with osteosarcoma in extremity with intra-arterial combination chemotherapy using a regimen selected by succinate dehydrogenase inhibition chemosensitivity test, and used caffeine to enhance its effect. In two patients with osteosarcoma in distal diametaphysis of the radius and extraskeletal osteosarcoma in sole of the foot, the effect of intra-arterial combination chemotherapy was increased and followed by the functional limb salvage procedure. But in one patient with osteosarcoma in the proximal tibia, the effect of pre-operative intra-arterial combination chemotherapy could not be increased, so amputation was required. He was changed to a post-operative intra-venous combination chemotherapy regimen selected by chemosensitivity test of the surgical material. All patients have been continuously disease-free at five to twelve months after surgery.

Sympathetic denervation of the epicardial border zone in the genesis of dispersion of refractoriness and arrhythmogenesis in a 7-day-old canine myocardial infarction model.

BACKGROUND: The purpose of this study was to clarify whether sympathetic denervation occurs in the infarcted heart and contributes to the dispersion of the effective refractory period (ERP) and arrhythmogenesis. METHODS: ERP was measured at 47 epicardial sites in 13 dogs with 7-day-old infarctions after proximal ligation of the left anterior descending artery. To delineate the sympathetic innervation, the effects of ansae subclaviae stimulation (ASS), norepinephrine infusion, and prazosin infusion on ERP were tested. RESULTS: The per cent change in ERP (delta ERP) induced by ASS was significantly lower at test sites where the surviving epicardial myocardial thickness (Th) was 2 mm or less than at those with a Th of more than 2 mm and the normal zone. Eleven out of 179 sites (6.1%) overlying the infarct showed no ERP change after ASS. ASS paradoxically prolonged ERP at 29 sites (16.2%). In contrast, norepinephrine infusion produced a greater delta ERP in the infarct zone than in the normal zone. Prazosin shortened ERP at sites where ASS prolonged it, but had no effect at sites where ASS shortened ERP. ASS increased both the degree of ERP dispersion and inducibility of ventricular tachycardias or ventricular fibrillation (VT/VF), whereas norepinephrine increased VT/VF inducibility despite a reduction in ERP dispersion. CONCLUSIONS: We conclude that heterogeneous sympathetic denervation contributed to a prolongation and dispersion of ERP in the surviving epicardium overlying the infarct. Furthermore, a supersensitive response to norepinephrine with resultant ERP shortening and a paradoxical ERP prolongation during ASS caused by alpha-receptor mechanisms that may be related to increased electrical instability were observed.

[Post-operative intra-arterial chemotherapy for high grade malignant musculoskeletal sarcoma].

Twenty-nine patients with high grade malignant musculoskeletal sarcoma were treated with chemotherapy. Intra-arterial chemotherapy was performed post-operatively for patients with local recurrence, patients with poor response to pre-operative chemotherapy or patients with intra-lesional or marginal surgical margin. The local recurrence rate in patients with intra-arterial chemotherapy (8.8%) was lower than in those with intravenous chemotherapy (37.5%). Patients with post-operative intra-arterial chemotherapy showed no local recurrence. Of the 29 patients except stage 3 with intra-arterial chemotherapy, the metastatic rate in patients with intra-arterial chemotherapy (41.4%) was lower than in chemotherapy patients with intravenous chemotherapy (83.3%). The rate in the post-operative group (28.6%) was lower than in the pre-operative group (42.9%) or pre- and post-operative group (50.0%). The ten-year survival curve of patients given intra-arterial chemotherapy (51.0%) was higher than in patients on intravenous chemotherapy (17.9%). Among the 34 patients who underwent intra-arterial chemotherapy, the survival rate in the other pre- and post-operative groups and the post-operative group (55.6%) was higher than in the pre-operative group and pre- and post-operative group (49.8%).

Immature teratoma of the uterine fundus.

An exceedingly rare case of extragonadal immature teratoma, which occurred primarily in the uterus, is described. The tumor developed into the pelvic cavity from the uterine fundus and consisted of ectodermal, mesodermal and endodermal derivatives. There were also significant amounts of immature elements; immature neuroepithelium with brisk mitotic activity, immature mesenchymal tissue, immature cartilage, immature striated muscle and immature hepatic tissue. Histologically, it was classified as a grade 3 immature teratoma. Treatment consisted of total simple hysterectomy followed by 2 courses of combination chemotherapy with vincristine, actinomycin D and cyclophosphamide (VAC). The patient was well and without evidence of recurrence at 5 years post-operatively.

Malignant fibrous histiocytoma. Proliferative compartment and heterogeneity of "histiocytic" cells.

To elucidate the precise origin and characteristics of the proliferating cells in malignant fibrous histiocytoma (MFH), the authors analyzed 33 MFH tumors, using immunohistochemical techniques with a panel of 12 antibodies. All three types of MFH cells (spindle cells, polygonal cells, and bizarre giant cells) stained positively for mesenchymal antigens (FU3 and vimentin) but did not stain for macrophage/histiocyte markers (HAM 56 and CD68). Therefore, the MFH cells may not represent true histiocytes, although they may be mesenchymal-derived cells behaving as "facultative histiocytes" with superficial resemblance to actual histiocytes. Normal histiocytes in the stroma tested positive for macrophage/histiocyte antigens; the most common cells were HAM 56-positive cells constituting 30-80% of nonneoplastic stromal cells, followed by those positive for CD68 (10-50%), Mac 387 (less than 2%), and S-100 protein (less than 1%). Our results indicate the presence of heterogeneity of "histiocytic" cells in MFH. Proliferating-cell nuclear antigen (PCNA) was expressed not only in the spindle and polygonal MFH cells but also in the bizarre giant cells. These findings suggest that all three types of MFH cells participate in the proliferative compartment of MFH. Uneven PCNA staining of the irregular nuclear segments of the bizarre giant cells may result in abnormal DNA synthesis, possibly contributing to the marked diversity of nuclear morphology in MFH. Touton-type and osteoclast-like giant cells did not stain for PCNA but stained positively for histiocytic markers. Therefore, these giant cells may lack proliferative activity and probably result from normal histiocytes fusing together.

Malignant fibrous histiocytoma. A tumor of facultative histiocytes showing mesenchymal differentiation in cultured cell lines.

The histogenesis of malignant fibrous histiocytoma (MFH) is controversial. To elucidate the cellular origin and characteristics of this neoplasm, the authors analyzed cell lines grown from 17 patients (15 soft tissue MFH and 2 bone MFH) by using light and electron microscopy, immunocytochemistry, enzyme cytochemistry, and functional tests for receptors for the Fc portion of immunoglobulin (Fc receptors) and immunophagocytosis. Each culture exhibited a storiform/pleomorphic pattern with mixed cellular populations consisting of spindle cells, polygonal cells, and bizarre giant cells; these morphologic features corresponded to the histologic characteristics of the primary tumors. The cells in each MFH line displayed histiocytic functional markers such as lysosomal enzymes, Fc receptors and immunophagocytosis. However, these cells differed from monocyte-derived macrophages (histiocytes) in immunoreactivity; the MFH cells expressed a mesenchymal antigen (FU3) distributed among perivascular cells and fibroblasts but demonstrated no positive reactions with Leu-M1 (CD15) and Leu-M3 (CD14), which recognize the cells of the monocyte-macrophage lineage. In conclusion, these findings suggest that MFH is not a tumor of true histiocytes but of facultative histiocytes showing mesenchymal differentiation in vitro. Chromosomal analysis performed in one MFH line demonstrated abnormal karyotypes; the modal chromosome number was 58, with 5 marker chromosomes.

Quantitative indices of dispersion of refractoriness for identification of propensity to re-entrant ventricular tachycardia in a canine model of myocardial infarction.

STUDY OBJECTIVE: To identify propensity to re-entry in a canine model of 7 day old myocardial infarction, the sensitivity and specificity of five indices of dispersion of refractoriness (ERP) were investigated. DESIGN: With an epicardial patch electrode containing 47 electrodes, ERP was measured by S1-S2 method at each site overlying the infarct. ERP range, maximum adjacent dispersion, difference between mean ERPS of the infarct and normal zones, standard deviation (sigma) of the mean ERP, and sigma/mean ERP X 100 were calculated. EXPERIMENTAL MATERIAL: 42 dogs were divided into three groups; 20 dogs with epicardial functional block on induction of sustained ventricular tachycardia or fibrillation, 10 dogs with inducible ventricular tachycardia or fibrillation but without epicardial functional block, and 12 control dogs without ventricular tachycardia, fibrillation or block. MEASUREMENTS AND MAIN RESULTS: All five indices were significantly greater in the 20 dogs with ventricular tachycardia or fibrillation than in control dogs. A receiver operating characteristic curve analysis of the five indices showed that sigma was the most sensitive and specific index for discriminating these 20 dogs. The sensitivity and specificity of a sigma value greater than 14 ms (the mean value plus two SD of the control dogs) were 70% and 100%, respectively.

Electrophysiologic effects of E-4031, a class III antiarrhythmic agent, on re-entrant ventricular arrhythmias in a canine 7-day-old myocardial infarction model.

Effects of E-4031, a class III antiarrhythmic agent, on re-entrant ventricular arrhythmias were studied in eight dogs with a 7-day-old myocardial infarction. Epicardial mapping and local refractory periods were obtained using 47-channel bipolar electrodes attached to the epicardium. The induction of sustained ventricular tachycardia by programmed electrical stimulation was not suppressed by i.v. infusion of E-4031 at 1 microgram/kg/min, but was suppressed markedly by infusion at 10 micrograms/kg/min in six of seven dogs. During the infusion of E-4031 at 10 micrograms/kg/min, epicardial conduction velocity in the normal ventricle did not change (0.7 +/- 0.12 to 0.71 +/- 0.13 m/sec, n = 6), whereas slowed conduction in the infarct zone improved (0.58 +/- 0.10 to 0.77 +/- 0.13 m/sec, n = 6). E-4031 at 10 micrograms/kg/min prolonged effective refractory periods (ERP) in the normal zone (139 +/- 8 to 164 +/- 18 msec, P less than .01, n = 8), nontransmural infarct zone (145 +/- 7 to 177 +/- 15 msec, P less than .01, n = 8) and transmural infarct zone (156 +/- 14 to 191 +/- 22 msec, P less than .01, n = 8). The degrees of ERP prolongation were almost equal in all zones. On epicardial mapping, the areas of longer ERP and delayed conduction were observed to become inexcitable after the administration of E-4031. These results demonstrated that E-4031 effectively prevented the induction of re-entrant ventricular tachycardia in canine myocardial infarction model, and suggested that E-4031 rendered re-entrant circuits inexcitable by marked ERP prolongation in both normal and infarct zones.

Electrophysiological and anatomical substrates for late potential recorded by signal averaging in seven-day-old myocardial infarction in dogs.

A filtered QRS (fQRS) was recorded by signal averaging in 7-day-old myocardial infarction (MI) in dogs to detect late potential (LP). The criteria for the LP included a duration of fQRS (D) greater than or equal to 60 msec and a voltage in the last 15 msec (V15) less than or equal to 10 microV. These parameters were determined from the control data from 15 dogs without infarction (D: 45 to 60 msec and V15: 12.0 to 83.6 microV). On the seventh day of infarction, the D had increased from 53.5 +/- 4.7 to 62.2 +/- 9.6 msec (P less than 0.05) and the V15 decreased from 38.6 +/- 19.5 to 18.4 +/- 16.0 microV (P less than 0.01). Of 23 dogs, 14 met the LP criteria (group A) and 9 did not (group B). Sustained ventricular tachycardia (SVT) was induced in 12 group A dogs and in none of the group B dogs. The delayed epicardial activation (DEA) was recorded after the end of QRS at 5.1 +/- 4.7 sites in group A dogs and 1.3 +/- 1.8 sites in group B dogs (P less than 0.05). The maximum value of epicardial activation time was more prolonged in group A than in group B (70.0 +/- 28.3 vs 44.4 +/- 9.8 msec, P less than 0.01). The area of MI was more extensive in dogs with DEA than those without (24.9 +/- 5.8% vs 10.3 +/- 9.0% of the total left ventricular weight, P less than 0.01). In 72 of 90 sites with DEA, the thickness of the surviving epicardial muscle was less than or equal to 1 mm. The sensitivity and specificity of the criteria for LP in detecting DEA were 71.4% and 55.6%, and 100% and 81.8% for predicting inducibility of SVT. It was thus concluded that LP, reflected the DEA, was identified from infarct areas of slow conduction within a reentry circuit of SVT.

Neuroectodermal tumor of bone. Evidence for neural differentiation in a cultured cell line.

A new cell line was established from a "neuroectodermal tumor of bone" affecting the right scapula of an 18-year-old man. The original neoplasm had dense proliferation of small round cells with abundant glycogen content and numerous Homer-Wright rosettes. The culture showed proliferation of small spindle cells with uniform oval nuclei and slender cytoplasmic processes. When the culture reached maximum density, rosette-like structures similar to those in the original tumor were formed. Under the influence of N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dibutyryl cAMP), the cultured cells expressed these rosette-like structures even in the lower cell concentration. Electron microscopy revealed that the cultured cells treated with dibutyryl cAMP contained high-density granules, well-developed microtubules, and abundant 10-nm filaments. By immunocytochemistry, neuron-specific enolase (NSE), and N-myc oncogene product were detected in the cultured cells as well as the original tumor. These results indicated the neuroectodermal origin of some of the small round cell tumors of bone.

[Post-operative intra-arterial chemotherapy in patients with malignant bone and soft tissue tumors].

Fourteen patients with bone and soft tissue sarcoma were treated with post-operative intra-arterial chemotherapy. Three drugs (Adriamycin, vincristine, carboquone or THP-adriamycin, cisplatin, vindesine) or two drugs (cisplatin, vindesine) were used post-operatively for patients with local recurrence, patients with poor response of pre-operative intra-arterial chemotherapy or patients with intra-lesional or marginal surgical margin. Of the 14 patients treated with post-operative intra-arterial chemotherapy, 8 (57.1%) were continuously disease-free (7.1%) were disease-free after treatment of lung metastasis, 2 (14.3%) had a local recurrence and/or multiple distant metastasis, and 3 (21.4%) died with multiple distant metastases. The rate of local recurrence was 14.3%. The Kaplar-Meier disease free survival curves showed 59.6%). Evaluation of limb function were excellent or good in 9 (69.2%) of 13 patients treated with limb-saving procedures.

Malignant fibrous histiocytoma in the thoracic region--a clinico-pathologic investigation.

Seven cases of malignant fibrous histiocytoma (MFH); 3 originating in the thoracic region, one which was considered to arise from the lung parenchyma, and 4 which were metastatic to the lungs, are presented herein. Six of these patients underwent surgical excision and analysis under light and electron microscopy revealed the lesions of MFH to be composed of two cell types; a fibroblast-like cell and a histiocyte-like cell. The latter showed histologically characteristic growth in a so-called storiform pattern. In all patients adjuvant chemotherapy was performed, although in only one patient did it prove temporarily effective. Despite the fact that the prognosis of MFH in the thoracic region is poor, the suggested therapy for longer survival is resection with postoperative combination chemotherapy including the use of sensitive anticancerous agents.

Anterior rib strut grafting for the treatment of malignant lesions in the thoracic spine.

Anterior stabilization during thoracotomy, using the patient's own ribs, was carried out seven times in six subjects with malignant lesions of the thoracic vertebrae. The primary malignancy was lung carcinoma in four cases, thyroid carcinoma in one case, and alveolar soft tissue sarcoma in one. Resection of the lung tumor and spinal surgery were carried out simultaneously. Four of the six patients had complete paraplegia and two had partial paraplegia prior to the operation. After the decompression and anterior stabilization, three subjects responded well and three responded poorly because of respiratory insufficiency.