Emotional experience in patients with clinically isolated syndrome and early multiple sclerosis.

BACKGROUND AND PURPOSE: Evidence suggests that there are changes in the processing of emotional information (EP) in people with multiple sclerosis (MS). It is unclear which functional domains of EP are affected, whether these changes are secondary to other MS-related neuropsychological or psychiatric symptoms and if EP changes are present in early MS. The aim of the study was to investigate EP in patients with early MS (clinically isolated syndrome and early relapsing/remitting MS) and healthy controls (HCs). METHODS: A total of 29 patients without neuropsychological or psychiatric deficits and 29 matched HCs were presented with pictures from the International Affective Picture System with negative, positive or neutral content. Participants rated the induced emotion regarding valence and arousal using nine-level Likert scales. A speeded recognition test assessed memory for the emotional stimuli and for the emotional modulation of response time. A subgroup of participants was tested during a magnetic resonance imaging (MRI) session. RESULTS: Patients in the MRI subgroup rated the experience induced by pictures with positive or negative emotional content significantly more weakly than HCs. Further, these patients were significantly less aroused when watching the pictures from the International Affective Picture System. There were no effects in the non-MRI subgroup or effects on emotional memory or response times. CONCLUSIONS: Emotional processing changes may be present in early MS in the form of flattened emotional experience on both the valence and arousal dimensions. These changes do not appear to be secondary to neuropsychological or psychiatric deficits. The fact that emotional flattening was only found in the MRI setting suggests that EP changes may be unmasked within stressful environments and points to the potential yet underestimated impact of the MRI setting on behavioral outcomes.

Increasing medical students' engagement in public health: case studies illustrating the potential role of online learning.

BACKGROUND: The value of e-learning in medical education is widely recognized but there is little evidence of its value in teaching medical students about public health. Such evidence is needed because medical students' engagement with public health has been low. We present three recent case studies from UK medical schools to illustrate diverse ways in which online approaches can increase medical students' engagement with learning public health. METHODS: A comparative case study approach was used applying quantitative and qualitative data to examine engagement in terms of uptake/use amongst eligible students, acceptability and perceived effectiveness using an analytic framework based on Seven Principles of Effective Teaching. RESULTS: Across the three case studies, most (67-85%) eligible students accessed online materials, and rated them more favourably than live lectures. Students particularly valued opportunities to use e-learning flexibly in terms of time and place. Online technologies offered new ways to consolidate learning of key public health concepts. Although students found contributing to online discussions challenging, it provided opportunities for students to explore concepts in depth and enabled students that were uncomfortable speaking in face-to-face discussions to participate. CONCLUSIONS: E-learning can be applied in diverse ways that increase medical student engagement with public health teaching.

Sub-cellular force microscopy in single normal and cancer cells.

This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures.

Impact of sex on clinical outcome in early Multiple Sclerosis.

BACKGROUND: Previous evidence suggests sex differences in the clinical course of relapsing remitting multiple sclerosis (RRMS), but comprehensive early-stage prospective studies are lacking. We aim to quantify the impact of sex on clinical outcomes in early-stage RRMS. METHODS: Utilizing prospective cohort data, we assessed the impact of biological sex on time-to-relapse, disability progression (Expanded Disability Status Scale [EDSS]), extremity function (Nine-Hole Peg Test, Timed-25-food walk test), cognition (Paced Auditory Serial Addition Test, Symbol Digit Modalities Test), quality-of-life (Hamburg Quality of Life Questionnaire in Multiple Sclerosis, Short-Form-36), fatigue (Fatigue Severity Scale, Fatigue Scale for Motor and Cognitive functions), and depression (Beck Depression Inventory-II) in clinically isolated syndrome (CIS) or RRMS patients. Inclusion was within 12 months of symptom onset. Linear, negative binomial, mixed, and Cox models estimated male vs. female effects at the four-year follow-up including baseline-to-follow-up course. RESULTS: We included 149 patients (65.1 % female). Eighty-five completed four-year follow-up. No sex differences in time-to-relapse emerged (HR = 0.91;95 %CI = 0.53-1.58). Males had no increased risk of EDSS worsening (OR = 0.75;95 %CI = 0.21-2.35) compared to females. Similarly, minor/no sex differences emerged in other outcomes. CONCLUSIONS: Four years after first manifestation, neither disease activity (disability progression and relapse rate) nor patient-reported outcomes showed sex-related disparities in this early-MS-cohort. GOV IDENTIFIER: NCT01371071.

Fatigue in multiple sclerosis is closely related to sleep disorders: a polysomnographic cross-sectional study.

BACKGROUND: Sleep disorders can cause tiredness. The relationship between sleep disorders and fatigue in patients with multiple sclerosis (MS) has not yet been investigated systematically. OBJECTIVE: To investigate the relationship between fatigue and sleep disorders in patients with MS. METHODS: Some 66 MS patients 20 to 66 years old were studied by overnight polysomnography. Using a cut-off point of 45 in the Modified Fatigue Impact Scale (MFIS), the entire cohort was stratified into a fatigued MS subgroup (n=26) and a non-fatigued MS subgroup (n=40). RESULTS: Of the fatigued MS patients, 96% (n=25) were suffering from a relevant sleep disorder, along with 60% of the non-fatigued MS patients (n=24) (p=0.001). Sleep-related breathing disorders were more frequent in the fatigued MS patients (27%) than in the non-fatigued MS patients (2.5%). Significantly higher MFIS values were detected in all (fatigued and non-fatigued) patients with relevant sleep disorders (mean MFIS 42.8; SD 18.3) than in patients without relevant sleep disorders (mean MFIS 20.5; SD 17.0) (p<0.001). Suffering from a sleep disorder was associated with an increased risk of fatigue in MS (odds ratio: 18.5; 95% CI 1.6-208; p=0.018). CONCLUSION: Our results demonstrate a clear and significant relationship between fatigue and sleep disorders.

Inebilizumab in AQP4-Ab-positive neuromyelitis optica spectrum disorder.

Until recently, the use of preventative immunotherapy in neuromyelitis optica spectrum disorders (NMOSD) was based on observational studies and clinical experiences. Meanwhile, the first drugs, among others the monoclonal antibody inebilizumab, were approved for the treatment of aquaporin-4 (AQP4) antibody-positive NMOSD. Inebilizumab binds to the CD19 antigen on B cells and leads to B-cell depletion. The first two dosages of 300 mg inebilizumab are administered intravenously at an interval of 2 weeks followed by further infusions every 6 months. In the placebo-controlled pivotal phase II/III study N-MOmentum, inebilizumab significantly prolonged the time to a first adjudicated relapse in AQP4 antibody-positive patients compared with placebo. The most frequent side effects were infusion reactions, urinary and respiratory tract infections, and arthralgia. This review presents data on clinical and preclinical pharmacology, administration, safety aspects and clinical trials of inebilizumab.

A novel nuclear form of estradiol receptor in MCF-7 human breast cancer cells.

Nuclear estrogen receptor from MCF-7 cells undergoes a time-dependent, hormone-inducible transformation to a form that is less extractable from nuclei and less exchangeable with ligand. This receptor-modifying, intranuclear event is independent of receptor loss (processing) and appears associated with hormone responsiveness (progesterone-receptor induction) in these cells. The magnitude of receptor loss, however, is variable and apparently not a prerequisite for hormone action to induce progesterone receptor.

MR spectroscopy (MRS) and magnetisation transfer imaging (MTI), lesion load and clinical scores in early relapsing remitting multiple sclerosis: a combined cross-sectional and longitudinal study.

The purpose of this study was to correlate magnetic resonance imaging (MRI)-based lesion load assessment with clinical disability in early relapsing remitting multiple sclerosis (RRMS). Seventeen untreated patients (ten women, seven men; mean age 33.0 +/- 7.9 years) with the initial diagnosis of RRMS were included for cross-sectional as well as longitudinal (24 months) clinical and MRI-based assessment in comparison with age-matched healthy controls. Conventional MR sequences, MR spectroscopy (MRS) and magnetisation transfer imaging (MTI) were performed at 1.5 T. Lesion number and volume, MRS and MTI measurements for lesions and normal appearing white matter (NAWM) were correlated to clinical scores [Expanded Disability Status Scale (EDSS), Multiple Sclerosis Functional Composite (MSFC)] for monitoring disease course after treatment initiation (interferon beta-1a). MTI and MRS detected changes [magnetisation transfer ratio (MTR), N-acetylaspartate (NAA)/creatine ratio] in NAWM over time. EDSS and lesional MTR increases correlated throughout the disease course. Average MTR of NAWM raised during the study (p < 0.05) and correlated to the MSFC score (r = 0.476, p < 0.001). At study termination, NAA/creatine ratio of NAWM correlated to the MSFC score (p < 0.05). MTI and MRS were useful for initial disease assessment in NAWM. MTI and MRS correlated with clinical scores, indicating potential for monitoring the disease course and gaining new insights into treatment-related effects.

MRI-Based Methods for Spinal Cord Atrophy Evaluation: A Comparison of Cervical Cord Cross-Sectional Area, Cervical Cord Volume, and Full Spinal Cord Volume in Patients with Aquaporin-4 Antibody Seropositive Neuromyelitis Optica Spectrum Disorders.

BACKGROUND AND PURPOSE: Measures for spinal cord atrophy have become increasingly important as imaging biomarkers in the assessment of neuroinflammatory diseases, especially in neuromyelitis optica spectrum disorders. The most commonly used method, mean upper cervical cord area, is relatively easy to measure and can be performed on brain MRIs that capture cervical myelon. Measures of spinal cord volume (eg, cervical cord volume or total cord volume) require longer scanning and more complex analysis but are potentially better suited as spinal cord atrophy measures. This study investigated spinal cord atrophy measures in a cohort of healthy subjects and patients with aquaporin-4 antibody seropositive neuromyelitis optica spectrum disorders and evaluated the discriminatory performance of mean upper cervical cord cross-sectional area compared with cervical cord volume and total cord volume. MATERIALS AND METHODS: Mean upper cervical cord area, cervical cord volume, and total cord volume were measured using 3T MRIs from healthy subjects (n = 19) and patients with neuromyelitis optica spectrum disorders (n = 30). Group comparison and receiver operating characteristic analyses between healthy controls and patients with neuromyelitis optica spectrum disorders were performed. RESULTS: Mean upper cervical cord area, cervical cord volume, and total cord volume measures showed similar and highly significant group differences between healthy control subjects and patients with neuromyelitis optica spectrum disorders (P < .01 for all). All 3 measures showed similar receiver operating characteristic-area under the curve values (mean upper cervical cord area = 0.70, cervical cord volume = 0.75, total cord volume = 0.77) with no significant difference between them. No associations among mean upper cervical cord cross-sectional area, cervical cord volume, or total cord volume with disability measures were found. CONCLUSIONS: All 3 measures showed similar discriminatory power between healthy control and neuromyelitis optica spectrum disorders groups. Mean upper cervical cord area is easier to obtain compared with cervical cord volume and total cord volume and can be regarded as an efficient representative measure of spinal cord atrophy in the neuromyelitis optica spectrum disorders context.

Z-DNA formation in the rat growth hormone gene promoter region.

The complete DNA sequence of the 1.7 kilobase pairs (kbp) 5' of the rat growth hormone gene (rGH) has been determined and analyzed for Z-DNA-forming potential. Regions of alternating purine-pyrimidine (APP) sequences located between -1047 and -986 [(GT)31], between -445 and -433 bp, and between -426 and -403 bp relative to the rGH RNA transcription initiation site were identified and shown to form Z-DNA in negatively supercoiled plasmids by two-dimensional gel electrophoresis. Free-energy calculations indicated that Z-DNA forms most readily in the proximal Z-DNA regions. Diethyl pyrocarbonate footprinting of physiologically supercoiled plasmid DNA confirmed the presence of Z-DNA from -444 to -404 bp spanning the two most proximal APP sequences and a short non-APP sequence in between. DNA sequence analysis also predicted a region of DNA curvature near this proximal Z-DNA region. Formation of Z-DNA in the distal Z-DNA region consisting of a (GT)31 repeat was constrained at physiological plasmid superhelical densities. This may be related to the presence of DNA sequences (-1584 to -1559) 512 bp upstream of (GT)31 that undergo cruciform formation and thereby utilize the available free energy. Removal of 580 bp containing the cruciform region resulted in Z-DNA formation within (GT)31, thus demonstrating that deletion mutations can exert topological changes at a distance within the rGH 5'-flanking region. Methylation of two specific cytosines in the rGH 5'-flanking DNA that have been associated with inhibition of rGH promoter activity had no effect on Z-DNA formation. No evidence for DNA secondary structure formation within the rGH second exon-intron or 3'-flanking region was observed. We conclude that the rGH 5'-flanking region undergoes secondary-structure formation at physiological superhelical densities, thus providing a potential mechanism(s) for modulating rGH activity.

Antidepressants for depressed elderly.

BACKGROUND: Depression is a relatively common experience in older adults. The syndrome is associated with considerable distress, morbidity and service commitment. Approximately two thirds of patients presenting with severe forms will respond to antidepressant treatment and the last twenty years has witnessed a great increase in the number of these drugs. Older, frail people are particularly vulnerable to side effects. OBJECTIVES: The aims of this review were to examine the efficacy of antidepressant classes, to compare the withdrawal rates associated with each class and describe the side effect profile of antidepressant drugs for treating depression in patients described as elderly, geriatric, senile or older adults, aged 55 or over. SEARCH STRATEGY: The Cochrane Collaboration Depression, Anxiety and Neurosis Controlled Trials Register (CCDANCTR-Studies) was searched (2003-08-13). Reference lists of relevant papers and previous systematic reviews were hand searched for published reports and citations of unpublished studies. SELECTION CRITERIA: Only randomised controlled trials were included. Trials had to compare at least two active antidepressant drugs in the treatment of depression. DATA COLLECTION AND ANALYSIS: Reviewers extracted data independently. In examining efficacy, the reviewers assumed that people who died or dropped out had no improvement. Withdrawal rates irrespective of cause and specifically due to side effects were compared between drug classes. Relative risk (RR) for dichotomous data and weighted mean difference for continuous data were calculated with 95% confidence intervals (CI). Qualitative side effect data were reported in terms of ratios of side effects and percentage of patients experiencing specific side effects. MAIN RESULTS: A total of 29 trials provided data for inclusion in the review. We were unable to find any differences in efficacy when comparing classes of antidepressants. However, as the trials contained relatively small numbers of patients, these findings may be explained by a type two error. Tricyclic antidepressants (TCAs) compared less favourably with selective serotonin reuptake inhibitors (SSRIs) in terms of numbers of patients withdrawn irrespective of reason (RR: 1.24, CI 1.04, 1.47) and number withdrawn due to side effects (RR: 1.30, CI 1.02, 1.64). Subgroup analyses demonstrated that TCA related antidepressants had similar withdrawal rates to SSRIs irrespective of reason of withdrawal (RR: 1.49, CI 0.74, 2.98) or withdrawal due to side effects (RR: 1.07, CI 0.43, 2.70). The qualitative analysis of side effects showed a small increased profile of gastro-intestinal and neuropsychiatric side effects associated with classical TCAs. AUTHORS' CONCLUSIONS: Our findings suggest that SSRIs and TCAs are of the same efficacy. However, we have found some evidence suggesting that TCA related antidepressants and classical TCAs may have different side effect profiles and are associated with differing withdrawal rates when compared with SSRIs. The review suggests that classical TCAs are associated with a higher withdrawal rate due to side effect experience, although these results must be interpreted with caution due to the relatively small size of the review and the heterogeneity of the drugs and patient populations.

Vasopressin: action on WRK-1 rat mammary tumor cells.

WRK-1, a cell line in long-term culture derived from a 7, 12-dimethylbenz[a]anthracene-induced rat mammary tumor, responds to physiologic concentrations of vasopressin with increased precursor incorporation into phospholipids and with increased protein accumulation. Because vasopressin has been reported to be a potent mitogen for Hela cells and 3T3 cells, a study was conducted to determine whether it could act as a mitogen for WRK-1 cells. Under no conditions was a clear-cut mitogen response to vasopressin demonstrated.

Prolonged retention of estradiol by human breast cancer cells in tissue culture.

Conditions are described under which prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for three hr with a physiological concentration of [3H]estradiol (3 to 5 nM) and then were washed with 3 successive exchanges of medium 3, 17, and 24 or 48 hr following incubation with [3H]estradiol. The total wash period was 78 hr. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture; (a) the concentration of [3H]estradiol and [3H]estradiol metabolites in the media washes; (b) the intracellular concentration of [3H]estradiol and [3H]estradiol metabolites; and (c) the time course of estradiol-enhanced rates of radiolabeled thymidine incorporation. The [3H]estradiol concentration in the final medium wash was approximately 0.05 nM. The total intracellular concentration of tritium was about 50 nM prior to wash and 9 nM following 78 hr of wash. The intracellular concentration of specifically bound [3H]estradiol was initially 18 nM, and after 78 hr of wash, it was 2.8 nM. After 48 hr of wash, nearly all specifically bound [3H]estradiol was present in the nucleus. Following incubation of the cells with 5 nM estradiol and an identical wash procedure, estrogenic activity as measured by a stimulation of thymidine incorporation was observed throughout the 78 hr monitored. When 10(-6) M tamoxifen or 10(-7) M unlabeled estradiol was included in the medium washes, the washout of nonspecific binding was unaffected; however, specifically bound [3H]estradiol was essentially eliminated within 24 hr. When bovine serum albumin was included in the medium washes, total, nonspecific, and specific [3H]estradiol binding was reduced in a parallel and dose-dependent fashion. After 48 hr, cells washed with medium containing 3.5 or 7% bovine serum albumin contained one-tenth of the [3H]estradiol present in cells washed with medium alone. We conclude that medium exchanges alone do not effectively remove estradiol from MCF-7 cells, and suggest that estrogen retention by estrogen-responsive cells may mask in vitro assessments of such responsiveness in this and other systems. Inclusion of bovine serum albumin in the washes may alleviate this problem.

Inhibition of human breast cancer cell proliferation in tissue culture by the neuroleptic agents pimozide and thioridazine.

Permanent cell culture lines derived from human breast cancer tissue are important experimental models in the study of human breast cancer cell proliferation. In the present work, pimozide, thioridazine, W-13, and W-12 were shown to inhibit MCF-7 human breast cancer cell growth. The 50% inhibition concentration values determined in two proliferation assays, [3H]thymidine incorporation and cell number, were in close agreement for each compound tested. The order of potency for growth inhibition in the presence of 2% stripped calf serum was pimozide (Ki 2 microM) greater than thioridazine (Ki 5 microM) greater than W-13 (Ki 15 microM) greater than W-12 (Ki 39 microM). Similar concentrations of these compounds blocked estradiol-induced growth of MCF-7 cells, but estrogen receptor (ER) interactions do not seem to be involved. Pimozide and thioridazine had no effect on the estradiol binding properties of the MCF-7 ER, nor did pimozide interfere with the induction of progesterone receptors by estradiol. Furthermore, pimozide also inhibited incorporation of [3H]thymidine into MCF-7 cells stimulated by polypeptide hormones in serum-free medium. The Ki for pimozide in serum-free medium alone, 0.46 microM, was similar to that determined in the presence of insulin (0.42 microM), insulin-like growth factor I (0.54 microM), and epidermal growth factor (0.43 microM). The effects of pimozide on breast cancer cell growth were not limited to the MCF-7 cell line. Pimozide also blocked cell growth and [3H]thymidine incorporation into the ER-positive T47D and ZR75-1B human breast cancer cell lines and the ER-negative human breast cancer cell line, MDA-MB-231. Although numerous mechanisms of action of pimozide and thioridazine have been identified, both drugs are calmodulin antagonists at drug concentrations that inhibit breast cancer cell growth in vitro. Inhibition of MCF-7 cell growth by the selective calmodulin antagonists W-13 and W-12 is consistent with a role for calmodulin antagonism in the broad growth-inhibitory properties of pimozide. We conclude that pimozide and thioridazine may be useful in the control of estradiol- and polypeptide hormone-induced growth of ER-positive and ER-negative human breast tumors.

Inhibition of rat growth hormone promoter activity by site-specific DNA methylation.

The effect of methylation on rat growth hormone (rGH) promoter activity was determined in GH3 cells by measuring rGH-Neo and rGH-CAT fusion gene expression with or without prior in vitro treatment with the site-specific DNA methyltransferases, M-BsuE and M-HhaI. To assay for rGH-promoter-specific effects of DNA methylation, RSV-Neo and RSV-CAT activities with or without M-BsuE, M-HhaI and M-HpaII treatment were measured in parallel cultures of GH3 cells. GH1-Neo and RSV-Neo fusion gene expression was inhibited by in vitro methylation from 44 to 83% as measured by the number of Geneticin-resistant GH3 cell colonies. Methylation of the GH1 promoter by M-BsuE exhibited some selective inhibition of Neo expression as determined by colony numbers, although extensive methylation of non-promoter DNA in GH1-Neo and RSV-Neo by M-HhaI and M-HpaII also inhibited Neo expression. Southern blot analysis of genomic DNA isolated from the Geneticin-resistant GH3 cells indicated that Geneticin-resistance was accompanied by demethylation of the BsuE (ThaI) sites in stably incorporated GH1-Neo DNA but not RSV-Neo DNA. Transient expression of the CAT gene in GH3 cells was selectively inhibited by 60% upon methylation of two BsuE (ThaI) sites in the GH1 promoter of GH1-CAT by M-BsuE. The data demonstrate, for the first time, to our knowledge, a direct effect of DNA methylation on the activity of the rat growth hormone promoter.

Kinetic alterations in estrogen receptors associated with estrogen receptor processing in human breast cancer cells.

After nuclear translocation of estrogen receptors in MCF-7 human breast cancer cells, a processing takes place resulting in a 30-70% decline in the number of estradiol-binding sites measured in nuclear extracts. We have investigated the mechanism of estrogen receptor processing and obtained evidence that multiple events are involved. We confirm, as others have shown previously, that processing involves a decrease in the amount of estradiol binding in MCF-7 cells. In addition, evidence is provided for the generation of a rapidly dissociating population of estradiol-binding sites as an early event in processing. There is a single, slowly dissociating population of estrogen binding sites when MCF-7 cells are exposed to estradiol in the presence of actinomycin D, an inhibitor of receptor processing. One hour after the addition of sufficient estradiol to induce receptor processing, an additional, more rapidly dissociating population of estrogen binding sites is detected. When cells are exposed to estradiol and ethidium bromide, a drug which shares many actions with actinomycin D, but does not inhibit receptor processing, the rapidly dissociating population of estradiol-binding sites is again observed. Significantly, the loss of estradiol-binding sites from MCF-7 cells associated with processing between 1 and 6 h of estradiol exposure, occurs exclusively from the rapidly dissociating population of sites. Whole cell equilibrium-binding assays were performed with MCF-7 cells after 30 min or 5 h of estradiol exposure to determine whether the detected changes in estradiol dissociation reflected affinity changes in a subpopulation of estrogen-binding sites. Although the number of sites detected per cell varied with the assay method employed, binding to a single saturable class of higher affinity sites is always observed. High affinity estradiol-binding sites were reduced by 45% after a 5-h incubation with estradiol in both assay methods. The loss of estradiol binding during processing may therefore be explained by the conversion of certain high affinity estrogen receptors to a rapidly dissociating form which then fails to rebind hormone, or undergoes subsequent reactions that destroy hormone binding activity. Additionally, after 6 h of exposure to estradiol, the remaining receptor-bound estradiol dissociates from intact cells with a rate increased by 50% over that seen from the slow dissociating receptors present at 1 h.(ABSTRACT TRUNCATED AT 400 WORDS)

A survey of human breast cancer sensitivity to growth inhibition by calmodulin antagonists in tissue culture.

We compared the ability of N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13), a calmodulin antagonist, to inhibit the growth of seven human breast cancer cell lines in tissue culture, to determine whether drug sensitivity was related to estrogen receptor (ER) status, tamoxifen resistance (tamr), or levels of calmodulin activity. We examined three ER+ (estrogen receptor-positive) cell lines (MCF-7, ZR-75-1B, and T47D), two ER+/tamr lines (LY2 and RR), and two ER- (estrogen receptor-negative) cell lines (MDA-MB-231 and MDA-MB-435). There was no difference in the inhibition of cell growth by W-13 in MCF-7 cells and the two tamr MCF-7 cell derivatives, LY2 and RR. In addition, the sensitivity to W-13 did not appear to be related to ER status. Although the mean Ki of the five ER+ cell lines (31 microM) was somewhat higher than the mean Ki of the two ER- cell lines (23 microM), the two cell lines most sensitive to W-13 were the ER+ T47D cells (Ki 15 microM) and the ER- MDA-MB-435 cells (Ki 10 microM). Calmodulin activity was measured in three representative cell lines, MCF-7, LY2, and MDA-MB-435. Calmodulin levels were higher in the most sensitive cell line (MDA-MB-435, 2.7 ng calmodulin/micrograms protein) than in the two less sensitive cell lines, MCF-7 and LY2 (1.3 and 1.6 ng calmodulin/micrograms protein, respectively). However, the MCF-7, LY2, and MDA-MB-435 cells were equally sensitive to another specific calmodulin antagonist, calmidazolium. We conclude that neither ER status, tamoxifen resistance, nor levels of calmodulin activity predict the sensitivity of human breast cancer cell lines to growth inhibition in tissue culture by calmodulin antagonists.

Dexamethasone control of growth hormone mRNA levels in GH3 pituitary cells is cycloheximide-sensitive and primarily posttranscriptional.

To clarify the mechanism of growth hormone (GH) gene activation by glucocorticoids in GH3 pituitary cells, GH mRNA accumulation in nuclear and cytoplasmic compartments was measured in the presence and absence of cycloheximide. In dexamethasone-treated cells, levels of GH mRNA were increased in the nucleus by 6 h and in the cytoplasm by 12 h. Dexamethasone treatment caused a 5- to 24-fold rise in total GH mRNA levels by 48-72 h. The differential elevation of nuclear levels of GH mRNA relative to the amount of cytoplasmic GH mRNA persisted for 48 h. A transient accumulation of GH mRNA in the nucleus was followed by a brief rise in cytoplasmic GH mRNA levels in GH3 cells treated simultaneously with dexamethasone and cycloheximide. In GH3 cells pretreated for 2 h with cycloheximide, the rise in nuclear and cytoplasmic GH mRNA levels mediated by dexamethasone was blocked completely. Levels of glucocorticoid receptor were unaffected by cycloheximide. These data suggest that the stimulation of GH mRNA levels by glucocorticoids is initiated within the nucleus and that cycloheximide-sensitive events are essential for this stimulation to occur. To assess the importance of GH gene transcriptional activation by glucocorticoids, nuclear transcription run-on reactions and assays of GH promoter activity in an aminoglycoside 3'-phosphotransferase (Neo) fusion gene within stably transformed GH3 cells were performed. Evidence for a weak, transient transcriptional activation of the GH gene by dexamethasone in nuclear run-on assays was obtained. Consistent with this idea, a 30-72 h exposure to dexamethasone raised levels of Neo mRNA in GH-Neo GH3 cell transformants by less than or equal to 2-fold. We conclude that glucocorticoid stimulation of GH mRNA in GH3 cells requires ongoing protein synthesis and can occur largely independently of GH gene transcriptional activation.