Regulation of fibronectin biosynthesis by dexamethasone, transforming growth factor beta, and cAMP in human cell lines.

The regulation of fibronectin (FN) biosynthesis by dexamethasone (a synthetic glucocorticoid), forskolin (an activator of adenylate cyclase), and transforming growth factor beta (TGF-beta) was examined in six human cell lines. Dexamethasone treatment produced the largest increase in FN biosynthesis in the fibrosarcoma cell line, HT-1080 (approximately 45-fold). This seems to result from a dexamethasone-mediated increase in FN mRNA stability which increases the message half-life from approximately 11 to 26 h. The relative instability of FN mRNA in the fibrosarcoma (t1/2 11 h) compared to normal fibroblasts (70 h) appears to result from the particular transformed phenotype of the HT-1080 cells. Forskolin and TGF-beta increase the rate of FN gene transcription in most of the cell lines. These effects (four- to six-fold) occur rapidly and do not require protein synthesis in the responsive cell lines which include normal fibroblasts. However, in the fibrosarcoma (HT-1080), a surprisingly large induction (20-30-fold) is observed and this induction is different from that in the normal fibroblasts and the other cell lines in that both protein synthesis and a lag period are required. Synergism is seen with dexamethasone and either forskolin or TGF-beta in HT-1080 cells increasing the rate of FN biosynthesis approximately 200-fold to a level similar to normal fibroblasts. This seems to result from a combination of FN mRNA stabilization (dexamethasone) and increased transcription (forskolin and TGF-beta).

Forskolin inducibility and tissue-specific expression of the fibronectin promoter.

The mechanism of cyclic AMP (cAMP) induction of fibronectin (FN) in HT-1080 and JEG-3 cells differs (D. C. Dean, R. F. Newby, and S. Bourgeois, J. Cell Biol. 106:2159-2170, 1988). In the fibrosarcoma cell line HT-1080, induction requires both protein synthesis and a lag period of 12 to 24 h. In the choriocarcinoma cell line JEG-3, protein synthesis is not required and induction peaks before 24 h, declining thereafter. We show that the FN promoter is transcribed in vitro and that the transcripts initiate at the proper site. Based on transfection experiments with these cells and FN promoter constructions, a cAMP-responsive element (CRE) was identified between -157 and -188 base pairs upstream of the human FN gene. This sequence also conferred cAMP inducibility in both cell lines on the herpesvirus thymidine kinase promoter when it was placed upstream of a thymidine kinase-chloramphenicol acetyltransferase fusion gene. DNase I protection analysis and gel retardation experiments revealed that the CRE was bound by a protein(s) that was present in both HT-1080 and JEG-3 cells as well as in NIH 3T3 cells. Multiple protein-CRE complexes were resolved by gel retardation with extracts of both cell lines. Forskolin treatment of these cells did not alter qualitatively or quantitatively the pattern of CRE-binding proteins that was observed. The FN promoter was at least 10 times more active in HT-1080 than in JEG-3 cells, even though in JEG-3 cells both the rate of FN biosynthesis and the level of accumulated FN mRNA were greater than those in HT-1080 cells. The difference in promoter activity in HT-1080 and JEG-3 cell was mediated by sequences that were located between positions -510 and -56. Deletion of the FN promoter from positions -510 to -56 resulted in an ~30-fold decrease in promoter activity when this construction was transfected into HT-1080 cells, and similar results were observed in NIH 3T3 cells; however, less than a 2-fold effect was observed in JEG-3 cells. Results of these studies suggest that there is some degree of tissue specificity of FN gene expression and reveal that cAMP induction is mediated, in part, by the same element (CRE) in both HT-1080 and JEG-3 cells.

Correlation between glucocorticoid receptor and cytolytic response of murine lymphoid cell lines.

By fusion between murine thymoma lines which are either homozygous (r+/r+ or r-/r-) or hemizygous (r+/r-) for the glucocorticoid receptor structural gene r, hybrid clones have been obtained which carry one, two three, or four copies of the r+ allele. These hybrids contain different amounts of normal glucocorticoid receptor as the result of a r+ gene dosage effect. Measurements of the cytolytic response of these hybrids to dexamethasone indicate a tight correlation between receptor content and sensitivity.

Glucorticoid resistance in murine lymphoma and thymoma lines.

The thymoma line W7 contains 30,000 dexamethasone-binding sites per cell and gives rise to glucocorticoid-resistant variants at a frequency less than 1.6 X 10(-9); the lymphoma S49 contains one-half that amount of receptor, is resistant to low concentrations of dexamethasone, and gives rise to fully resistant variants at high frequency. These results suggest that S49 is functionally haploid (r+/-) for a gene coding for the receptor while W7 appears diploid for that locus (r+/+). Derivatives of the W7 (r+/+) line, selected for resistance to low concentrations of dexamethasone, have the same properties as S49. These putative W7's (r+/-) give rise to the same types of receptor variants as S49 and in the same proportion; 80 to 90% are "receptorless" (r-) while 10 to 20% are "nuclear transfer defective" (nt-). A total of 127 variants resistant to high concentrations of dexamethasone were derived from the W7 (r+/+) line after mutagenesis. All are receptor variants but N-methyl-N'-nitro-N-nitrosoguanidine and ethyl methanesulfonate induce only 60 to 70% r- variants, confirming the presence of two r+ alleles in the parental line. Ultraviolet light induces a higher proportion (87%) of r+ variants, as expected from the introduction of breaks and deletions in DNA.

Membrane permeability as a determinant of dexamethasone resistance in murine thymoma cells.

The variant MS23 of the murine thymoma cell line W7 , selected for growth at low concentrations of dexamethasone (7.5 nM), is cross-resistant to various unrelated drugs, including colchicine. By stepwise selection in combinations of dexamethasone and colchicine at increasing concentrations, we have isolated a series of variants with increased resistance to dexamethasone and cross-resistance to puromycin, colchicine, daunomycin, gramicidin, and vincristine. Surprisingly, resistance to triamcinolone acetonide, a glucocorticoid structurally related to dexamethasone, did not develop. Assays for specific dexamethasone and triamcinolone acetonide binding sites in variant cell extracts reveal that the glucocorticoid receptors of these variants are unchanged as compared to the W7 parental line. However, whole cell binding assays yielded reduced apparent affinity for dexamethasone in the MS23 variant and drastically reduced dexamethasone binding after selection for increased resistance. We demonstrate that this is due to reduced drug uptake. Procaine, a membrane-active anesthetic, potentiates uptake of puromycin and dexamethasone in the variants. The variants are stable, and karyotypic analysis did not reveal double minute chromosomal structures. These results demonstrate that permeability can be a rate-limiting step in steroid hormone action and is the basis for dexamethasone resistance in these variants.

Expression of an mdr gene is associated with a new form of resistance to dexamethasone-induced apoptosis.

A variant, MS23, of murine thymoma W7 cells, previously selected for its resistance to low concentrations of dexamethasone, is cross-resistant to unrelated drugs such as puromycin and colchicine. We report here that transcription of the mouse mdr1 gene is activated and P-glycoprotein is expressed in MS23 cells. Moreover, additional variants with increased resistance to dexamethasone and other drugs can be isolated from MS23 by stepwise selections in dexamethasone and colchicine. In one such variant (S7CD-5), the mdr1 gene is amplified and the mdr protein overexpressed. These variants have classical mdr characteristics: they accumulate reduced concentrations of drugs (including dexamethasone), and both drug sensitivity and intracellular accumulation can be restored by verapamil. The variants are most resistant to glucocorticoids with both 11- and 17-hydroxyl groups. The results indicate that we have identified a new form of glucocorticoid resistance, one associated with expression of the mouse mdr1 P-glycoprotein.

Relationship between the "cognitive triad" from the personality inventory for children and an extended Halstead-Reitan neuropsychological battery.

The relationships between the Achievement, Intellectual Screening, and Development scales from the Personality Inventory for Children (PIC) and a comprehensive neuropsychological battery were examined in a sample of 49 children with histories of behavioral problems, learning problems, or both but without known neurological impairments or pervasive developmental disorders. Using stepwise multiple regression, the PIC scales predicted global neuropsychological functions moderately well, particularly verbal IQ and achievement. More specific neuropsychological functions were not significantly predicted. The Developmental scale from the PIC consistently outperformed the other two PIC "cognitive-triad" scales, and the latter made no statistically significant additional contribution in predicting neuropsychological functions after the contribution of the Development scale was taken into account.

Use of the restricted academic task in ADHD dose-response relationships.

Behavioral assessment techniques have been shown to make a significant contribution in the evaluation of stimulant medication response in children with attention-deficit/hyperactivity disorder. This article examines the role of a behavioral measure consisting of standardized observation of a child while he or she performs an academic-like task (Restricted Academic Task). This technique is reliable and valid for repeated administrations across medication dosages and placebo. It can assist in optimizing medication dosage for individual children, given its sensitivity to dosage effects. It also allows the capture of medication-related changes in an area of functioning that is not directly assessed by parent and teacher judgments and ratings. In this way the Restricted Academic Task may have greater ecological validity than the results of other, more traditional in-clinic measures. Finally, given the idiosyncratic nature of stimulant medication effects on individual children, it contributes to the explication of a specific child's behavioral dose-response relationship.

Improving the reading comprehension of children with dysphonetic and dyseidetic dyslexia using story grammar.

Five 8- to 10-year-old children with dysphonetic and dyseidetic dyslexia were given instruction in reading comprehension using a story grammar strategy in which story instruction was differentially designed to match the simultaneous or sequential mental processing strengths of each dyslexia subtype. A multiple baseline, single subject experimental design and statistical analyses indicated that the experimental treatments yielded statistically and clinically significant improvements in the proportion of qualitatively important story elements recalled by the subjects, when compared to baseline traditional remedial instruction. The results suggested that students with dyslexia can increase their reading comprehension with training in metacognitive strategies. The question of whether the results were attributable to the subtype-matched methods per se or to strategy training in general, as well as a number of methodological issues, is being explored in subsequent research.

Confirmatory factor analysis of four general neuropsychological models with a modified Halstead-Reitan battery.

Four theoretical factor models for a modified Halstead-Reitan battery were formulated, drawing from previous work by Swiercinsky, Royce and co-workers, Christensen and Luria, and Lezak. The relative explanatory power of these four models for this particular battery in an adult neuropsychiatric population was examined using confirmatory factor analysis. None of the models was shown to fit adequately in an absolute sense, but three of them represented substantial, statistically reliable improvements over a null model of mutual independence, and a clear pattern of relative fit was observed. Further improvements were achieved by modifying the best fitting initial model in several ways. A cross-validation with an independent sample supported the results of the model development step. Tentative theoretical and clinical implications for the overall organization of the neuropsychological abilities measured by this battery were drawn, and recommendations were made for further application of this method in neuropsychological research.

Comparative evaluation of two MMPI short forms with chemical abusing inpatients.

Studied several novel analyses of the correspondence between two popular MMPI short forms and the full MMPI, including multivariate profile analyses and analyses of change scores. Pre- and posttreatment MMPIs were collected from 100 veterans in a treatment program for alcohol and drug problems. Scores for the Faschingbauer Abbreviated MMPI and the MMPI-168 were extracted from full MMPI protocols. Each short form was compared separately with the full form in several individual-case-oriented and group-data-oriented analyses. Significant differences between full and both short form profiles were found. It was concluded that neither short form is recommended for individual case descriptions, that significant problems arise in using these short forms for group descriptive or comparative research, and that these short forms show moderately acceptable correspondence with full MMPIs over time. It appears that short MMPIs continue to show serious deficits in reliability, i.e., in correspondence with full MMPIs, in spite of recent attempts to demonstrate validity somewhat independent of the full MMPI.

Regulation of fibronectin biosynthesis by glucocorticoids in human fibrosarcoma cells and normal fibroblasts.

When treated with the synthetic glucocorticoid dexamethasone, HT1080 human fibrosarcoma cells show changes in morphology, adhesion, and the extracellular matrix. Dexamethasone treatment results in a tenfold increase in the rate of fibronectin biosynthesis in HT1080 cells and a twofold increase in untransformed, normal human fibroblasts. Maximal induction levels are attained within one cell generation, while decay of the response requires several cell cycles. Pulse-chase studies showed that most of the newly synthesized fibronectin is secreted into the medium. The glucocorticoid antagonist, RU-486, blocks the dexamethasone-induced changes but does not alter the basal rate of fibronectin production. Therefore, fibronectin biosynthesis appears to be controlled by two distinct mechanisms--one, regulating basal rates of fibronectin production, which is transformation-sensitive and glucocorticoid-independent; and another, which is mediated by the glucocorticoid receptor, resulting in elevated rates of fibronectin biosynthesis upon dexamethasone treatment both in normal fibroblasts and in HT1080 cells.

A further report on a case of Floating-Harbor Syndrome in a mother and daughter.

We present the most extensive neuropsychological and language assessment yet reported of patients diagnosed with Floating-Harbor Syndrome (FHS), a rare genetic condition characterized by dysmorphid figures, short stature, and speech-onset delay. This is also the second reported occurrence of both a mother and daughter with FHS. Whereas the child demonstrated gross deficits in verbal expression, speech and language problems were largely ameliorated in the mother. Neuropsychological assessment also revealed a strikingly similar pattern of cognitive problems additional to language dysfunction, including difficulties with attention, mathematical, and visuospatial abilities. A mood disorder continued to be quite disabling for the mother.