Cell type-dependent modulation of the dominant negative action of human mutant thyroid hormone beta 1 receptors

BACKGROUND: Mutations in the ligand-binding domain of the thyroid hormone receptor beta (TR beta) gene cause the syndrome of resistance to thyroid hormone (RTH). The clinical phenotype results from the antagonism of the normal TR alpha and the non-mutated TR beta alleles by the TR beta 1 mutants, via a dominant negative effect. There is, however, marked heterogeneity of organ resistance within and among kindreds with RTH. This study examines the potential role of cell type in modulating the dominant negative potency of human TR beta 1 (h-TR beta 1) mutants. MATERIALS AND METHODS: Transient transfections were performed in HeLa and NIH3T3 cells, using a wild type (WT) and three naturally occurring mutant h-TR beta 1 constructs, and three natural thyroid hormone response elements (TREs). Immunocytochemistry was performed to detect levels of TR beta 1 expression in these two cell types. In order to determine how TR beta 1 interacts with other cellular partners, gel-shift analyses using HeLa and NIH3T3 nuclear extracts were performed. RESULTS: Transfection studies using WT h-TR beta 1 in HeLa and NIH3T3 cells, showed that the 3,3',5-triiodothyronine (T3)-induced transactivation of the different TREs varied between cell types. Unlike the non-T3-binding h-TR beta 1 mutant, PV, mutants ED and OK displayed the expected T3-induced dose responsiveness in these two cell types. For each TRE examined, the magnitude of the dominant negative effect varied between the cell types. The levels of receptor expression in HeLa and NIH3T3 cells were identical, as determined by immunocytochemistry. Gel-shift analyses showed differences in the formation of hetero- and homodimers depending on both the cell type and TRE motif. CONCLUSIONS: The cell type in which a mutant receptor operates affects the relative amounts of hetero- and homodimers. Together with the nature of the mutation and the TRE-motif, this could modulate the dominant negative action of mutant receptors in different tissues, which, in turn, could contribute to the variable phenotypic characteristics of RTH.     

Comparison of sonography, sonohysterography, and hysteroscopy for evaluation of abnormal uterine bleeding

We compared transvaginal sonography, sonohysterography, and diagnostic hysteroscopy in the evaluation of abnormal uterine bleeding, Sixty-eight women 40 or older with abnormal uterine bleeding were assigned to undergo either transvaginal sonography or sonohysterography. All subjects then had diagnostic hysteroscopy and endometrial biopsy. Patients with abnormal findings underwent operative hysteroscopy or definitive therapy. Transvaginal sonography, sonohysterography, and diagnostic hysteroscopy revealed a sensitivity of 95%, 90%, and 78%, and a specificity of 65%, 83%, and 54%, respectively. The average cost for transvaginal sonography of sonohysterography was $195 and the cost for diagnostic hysteroscopy was $675. Transvaginal sonography and sonohysterography are cost-effective alternatives and more sensitive diagnostic tests than office diagnostic hysteroscopy.     

Conditional immortalization of human endometrial stromal cells with a temperature-sensitive simian virus 40

The study of immortalization and other alterations associated with neoplastic transformation of endometrial stromal cells is important to understanding the development of uterine sarcomas and mixed tumors. Because stromal cells are important regulators of associated epithelial cells, alterations in the regulation of stromal cell proliferation that influence epithelial cells may also contribute to the development of endometrial carcinomas. To study immortalization and associated phenotypic and genetic alterations of human endometrial stromal cells, cultures were transfected with a plasmid containing an ori-, temperature-sensitive mutant SV40, A209 (tsSV40). Morphologically transformed colonies were selected and propagated at the permissive temperature until they entered 'crisis'. In contrast to human fibroblasts, every clone tested was immortalization competent. The frequency of immortalization was approximately 1 x 10(-6). One uncloned and six cloned cell lines escaped from crisis and appear to be immortal. Two clones, M4 and B10T1, were selected for further study. Immortalization is conditional; proliferative arrest occurs at the restrictive temperature for large T antigen function. Furthermore, withdrawal of the large T antigen results in expression of the senescent phenotype of enlarged, flattened cells. Colony-forming efficiency at the restrictive temperature was undetectable. Immortalization is also associated with several genetic alterations. The DNA content of tsSV40 transfected cells was either diploid or tetraploid in the precrisis stage of proliferation, but became aneuploid upon immortalization. Several structural rearrangements of chromosomes were detected in the immortalized stromal cells which differ from those found in SV40 immortalized fibroblasts. Although their capacity for anchorage-independent proliferation (AIP) is variable, tsSV40-immortalized endometrial stromal cells have a higher capacity for AIP than their tsSV40-transfected progenitor cells in the period of proliferation prior to 'crisis'.     

Preparation and standardization of U.S. Standard Pertussis Vaccine, Lot No. 11

Novel glutathione (GSH) analogs, previously shown to inhibit glutathione S-transferase (GST) activity at about 1 microM in vitro, were tested for their ability to potentiate the killing of cultured tumor cells by chemotherapeutic drugs. When tested at doses up to 200 microM, the analogs were neither toxic nor capable of potentiating drug toxicity unless the diethyl ester (DEE) form was used for treatment of the cells. HPLC analysis revealed rapid internalization of the DEE and intracellular conversion to a monoethyl ester form that accumulated in the cell, followed by a more gradual loss of the second ester to generate the active parent form. For the four GSH analogs tested, the ability of the DEE forms to potentiate chlorambucil (CMB) toxicity in HT-29 human colon adenocarcinoma cells strongly correlated with the in vitro ability of the parent form to inhibit recombinant human P1-1. This isozyme is the dominant form of GST present in HT-29 cells. Of the four analog DEEs tested, gamma-glutamyl-S-(benzyl)cysteinyl-R(-)-phenyl glycine (TER 117) DEE was the most effective in potentiating CMB toxicity in several cell lines: HT-29, HT4-1 (HT-29 subclone), SKOV-3 ovarian carcinoma, and SK VLB (vinblastine-resistant variant of SKOV-3) cells. gamma-Glutamyl-S-(octyl)cysteinyl-glycine (TER 143) DEE potentiated mitomycin C (MTC) toxicity in HT4-1 and SK VLB cells while TER 117 DEE did not. TER 117 DEE enhanced melphalan effects on xenografts of HT4-1 in mice to a similar extent as that achieved with the previously described nonspecific GST inhibitor, ethacrynic acid. Taken together, our results indicate that cell-permeable analogs of GSH can potentiate cytotoxicity of common chemotherapeutic drugs and this effect has a strong positive correlation with the ability of the analogs to inhibit specific GST isozymes.     

Peptide targeting and delivery across the blood-brain barrier utilizing synthetic triglyceride esters: design, synthesis, and bioactivity

As an approach to the development of therapeutically useful peptide pharmaceuticals that can penetrate the blood-brain barrier, we have designed and demonstrated the application of a carrier-targeting system. We have developed a prodrug design strategy that is designed to utilize membrane-bound enzymes whereby release of a bioactive peptide from a highly lipophilic triglyceride peptide-carrier is achieved in situ, thus attaining high localized concentrations of the bioactive peptide. Following localization of such a system, normal peptidase and lipase action is utilized to release the active peptide (deltorphin II) intact and in high concentration. At present, the exact mechanisms are unclear, but the observed results in which analgesia is observed following peripheral administration suggest that the active peptide is able to cross the blood-brain barrier and sustain prolonged periods of analgesia as determined by antinociception tests by release of the bioactive peptide. In vitro tests of binding and bioactivity by the peptide conjugate show essentially no potency in either target or control analogues, but potent antinociceptive effects are observed following peripheral administration.     

Overexpression of eukaryotic initiation factor 4E (eIF4E) in breast carcinoma

BACKGROUND: Eukaryotic initiation factor 4E (eIF-4E) is a 25-kilodalton phosphoprotein that binds specifically to mRNA as the initial step for mRNA translation. An elevated level of eIF4E has been associated with the up-regulation of various protooncogene products. Transfection of cell lines by viral vectors with eIF4E overexpression has resulted in malignant transformation. The objective in this study was twofold: to examine benign and malignant breast specimens for eIF4E expression, and to determine whether eIF4E overexpression may have prognostic potential. METHODS: Western blot analysis was performed on benign and malignant breast specimens using anti-eIF4E rabbit antiserum. Quantification was accomplished by developing blots with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate and densitometry. Confirmation of eIF4E overexpression at the cellular level was performed using immunohistologic staining in situ. RESULTS: The authors examined 112 breast specimens for eIF4E protein expression. Of the 52 benign breast specimens examined, none showed eIF4E overexpression. All 12 ductal carcinoma in situ specimens were found to overexpress eIF4E in the intermediate range (mean elevation: 2.5-fold). Of the 48 breast carcinoma specimens examined, all had eIF4E elevation at levels of 3-30-fold (mean: 10.5 +/- 0.9-fold). Charts from 39 patients with Stage I, II, and III breast carcinoma were reviewed. In ten patients with eIF4E overexpression of < sevenfold, there was no recurrence or death from breast carcinoma. In the 29 breast carcinoma patients with > or = 7-fold eIF4E overexpression, 9 patients had breast carcinoma recurrences and 5 had died from disease at last follow-up. The median follow-up in this study was 34.5 months. CONCLUSIONS: Overexpression of eIF4E was observed in malignant breast specimens but not in normal or benign breast tissues. In patients with breast carcinoma, the group with high eIF4E overexpression (> or = 7-fold) experienced a worse clinical outcome (higher recurrences and death) compared with the group with low eIF4E overexpression (< 7-fold).