Alcohol abuse has a potential association with unfavourable clinical course and brain atrophy in patients with status epilepticus.

AIM: To evaluate chronological changes on serial magnetic resonance imaging (MRI) examinations and clinical prognosis in patients with status epilepticus (SE), as well as the effect of alcohol abuse and heavy alcohol use on clinicoradiological findings. MATERIALS AND METHODS: This retrospective, single-centre study was approved by the institutional review board. Among 345 patients with seizures between January 2010 and October 2021, 27 patients with SE who had undergone both initial MRI (within a week after onset) and follow-up MRI (within 1 month after the initial MRI) were included. Five and three patients with concurrent or previous alcohol abuse and heavy alcohol-use history were included, respectively, and they were classified into the AL (Alcohol use) group. The remaining 19 patients were classified into the non-AL group. Two neuroradiologists independently evaluated both initial and follow-up MRI examinations of each patient; MRI findings were compared between the AL and non-AL groups using Fisher's exact test. In 15 patients, including four patients from the AL group, clinical information 6 months after the onset of SE was available; this information was compared between the two groups. RESULTS: Brain atrophy (5/8 versus 2/19, p=0.011; odds ratio, 12.29 [95% confidence interval, 1.32-189.2]) and unfavourable clinical course with uncontrollable seizures (3/4 versus 1/11, p=0.033; odds ratio, 30[1.43-638.19]) were significantly more frequent in the AL group than in the non-AL group. CONCLUSION: Among patients with SE, alcohol abuse and heavy alcohol-use history were associated with unfavourable seizure control and brain atrophy.

Dynamic Contrast-Enhanced MRI Parameters and Normalized ADC Values Could Aid Differentiation of Skull Base Osteomyelitis from Nasopharyngeal Cancer.

BACKGROUND AND PURPOSE: The skull base osteomyelitis sometimes can be difficult to distinguish from nasopharyngeal cancer. This study aimed to investigate the differences between skull base osteomyelitis and nasopharyngeal cancer using dynamic contrast-enhanced MR imaging and normalized ADC values. MATERIALS AND METHODS: This study included 8 and 12 patients with skull base osteomyelitis and nasopharyngeal cancer, respectively, who underwent dynamic contrast-enhanced MR imaging and DWI before primary treatment. Quantitative dynamic contrast-enhanced MR imaging parameters and ADC values of the ROIs were analyzed. Normalized ADC parameters were calculated by dividing the ROIs of the lesion by that of the spinal cord. RESULTS: The rate transfer constant between extravascular extracellular space and blood plasma per minute (Kep) was significantly lower in patients with skull base osteomyelitis than in those with nasopharyngeal cancer (median, 0.43 versus 0.57; P = .04). The optimal cutoff value of Kep was 0.48 (area under the curve, 0.78; 95% CI, 0.55-1). The normalized mean ADC was significantly higher in patients with skull base osteomyelitis than in those with nasopharyngeal cancer (median, 1.90 versus 0.87; P < .001). The cutoff value of normalized mean ADC was 1.55 (area under the curve, 0.96; 95% CI, 0.87-1). The area under the curve of the combination of dynamic contrast-enhanced MR imaging parameters (Kep and extravascular extracellular space volume per unit tissue volume) was 0.89 (95% CI, 0.73-1), and the area under the curve of the combination of dynamic contrast-enhanced MR imaging parameters and normalized mean ADC value was 0.98 (95% CI, 0.93-1). CONCLUSIONS: Quantitative dynamic contrast-enhanced MR imaging parameters and normalized ADC values may be useful in differentiating skull base osteomyelitis and nasopharyngeal cancer. The combination of dynamic contrast-enhanced MR imaging parameters and normalized ADC values outperformed each measure in isolation.

ADC for Differentiation between Posttreatment Changes and Recurrence in Head and Neck Cancer: A Systematic Review and Meta-analysis.

BACKGROUND: Previous studies reported that the ADC values of recurrent head and neck cancer lesions are lower than those of posttreatment changes, however, the utility of ADC to differentiate them has not been definitively summarized and established. PURPOSE: Our aim was to evaluate the diagnostic benefit of ADC calculated from diffusion-weighted imaging in differentiating recurrent lesions from posttreatment changes in head and neck cancer. DATA SOURCES: MEDLINE, Scopus, and EMBASE data bases were searched for studies. STUDY SELECTION: The review identified 6 prospective studies with a total of 365 patients (402 lesions) who were eligible for the meta-analysis. DATA ANALYSIS: Forest plots were used to assess the mean difference in ADC values. Heterogeneity among the studies was evaluated using the Cochrane Q test and the I2 statistic. DATA SYNTHESIS: Among included studies, the overall mean of ADC values of recurrent lesions was 1.03 × 10-3mm2/s and that of the posttreatment changes was 1.51 × 10-3mm2/s. The ADC value of recurrence was significantly less than that of posttreatment changes in head and neck cancer (pooled mean difference: -0.45; 95% CI, -0.59-0.32, P < .0001) with heterogeneity among studies. The threshold of ADC values between recurrent lesions and posttreatment changes was suggested to be 1.10 × 10-3mm2/s. LIMITATIONS: Given the heterogeneity of the data of the study, the conclusions should be interpreted with caution. CONCLUSIONS: The ADC values in recurrent head and neck cancers are lower than those of posttreatment changes, and the threshold of ADC values between them was suggested.

Differentiation of Skull Base Chondrosarcomas, Chordomas, and Metastases: Utility of DWI and Dynamic Contrast-Enhanced Perfusion MR Imaging.

BACKGROUND AND PURPOSE: Differentiation of skull base tumors, including chondrosarcomas, chordomas, and metastases, on conventional imaging remains a challenge. We aimed to test the utility of DWI and dynamic contrast-enhanced MR imaging for skull base tumors. MATERIALS AND METHODS: Fifty-nine patients with chondrosarcomas, chordomas, or metastases between January 2015 and October 2021 were included in this retrospective study. Pretreatment normalized mean ADC and dynamic contrast-enhanced MR imaging parameters were calculated. The Kruskal-Wallis H test for all tumor types and the Mann-Whitney U test for each pair of tumors were used. RESULTS: Fifteen chondrosarcomas (9 men; median age, 62 years), 14 chordomas (6 men; median age, 47 years), and 30 metastases (11 men; median age, 61 years) were included in this study. Fractional plasma volume helped distinguish all 3 tumor types (P = .003, <.001, and <.001, respectively), whereas the normalized mean ADC was useful in distinguishing chondrosarcomas from chordomas and metastases (P < .001 and P < .001, respectively); fractional volume of extracellular space, in distinguishing chondrosarcomas from metastases (P = .02); and forward volume transfer constant, in distinguishing metastases from chondrosarcomas/chondroma (P = .002 and .002, respectively) using the Kruskal-Wallis H test. The diagnostic performances of fractional plasma volume for each pair of tumors showed areas under curve of 0.86-0.99 (95% CI, 0.70-1.0); the forward volume transfer constant differentiated metastases from chondrosarcomas/chordomas with areas under curve of 0.82 and 0.82 (95% CI, 0.67-0.98), respectively; and the normalized mean ADC distinguished chondrosarcomas from chordomas/metastases with areas under curve of 0.96 and 0.95 (95% CI, 0.88-1.0), respectively. CONCLUSIONS: DWI and dynamic contrast-enhanced MR imaging sequences can be beneficial for differentiating the 3 common skull base tumors.

Normalized Parameters of Dynamic Contrast-Enhanced Perfusion MRI and DWI-ADC for Differentiation between Posttreatment Changes and Recurrence in Head and Neck Cancer.

BACKGROUND AND PURPOSE: Differentiating recurrence from benign posttreatment changes has clinical importance in the imaging follow-up of head and neck cancer. This study aimed to investigate the utility of normalized dynamic contrast-enhanced MR imaging and ADC for their differentiation. MATERIALS AND METHODS: This study included 51 patients with a history of head and neck cancer who underwent follow-up dynamic contrast-enhanced MR imaging with DWI-ADC, of whom 25 had recurrences and 26 had benign posttreatment changes. Quantitative and semiquantitative dynamic contrast-enhanced MR imaging parameters and ADC of the ROI and reference region were analyzed. Normalized dynamic contrast-enhanced MR imaging parameters and normalized DWI-ADC parameters were calculated by dividing the ROI by the reference region. RESULTS: Normalized plasma volume, volume transfer constant between extravascular extracellular space and blood plasma per minute (K trans), area under the curve, and wash-in were significantly higher in patients with recurrence than in those with benign posttreatment change (P = .003 to <.001). The normalized mean ADC was significantly lower in patients with recurrence than in those with benign posttreatment change (P < .001). The area under the receiver operating characteristic curve of the combination of normalized dynamic contrast-enhanced MR imaging parameters with significance (normalized plasma volume, normalized extravascular extracellular space volume per unit tissue volume, normalized K trans, normalized area under the curve, and normalized wash-in) and normalized mean ADC was 0.97 (95% CI, 0.93-1). CONCLUSIONS: Normalized dynamic contrast-enhanced MR imaging parameters, normalized mean ADC, and their combination were effective in differentiating recurrence and benign posttreatment changes in head and neck cancer.

Pretreatment ADC Histogram Analysis as a Prognostic Imaging Biomarker for Patients with Recurrent Glioblastoma Treated with Bevacizumab: A Systematic Review and Meta-analysis.

BACKGROUND: The mean ADC value of the lower Gaussian curve (ADCL) derived from the bi-Gaussian curve-fitting histogram analysis has been reported as a predictive/prognostic imaging biomarker in patients with recurrent glioblastoma treated with bevacizumab; however, its systematic summary has been lacking. PURPOSE: We applied a systematic review and meta-analysis to investigate the predictive/prognostic performance of ADCL in patients with recurrent glioblastoma treated with bevacizumab. DATA SOURCES: We performed a literature search using PubMed, Scopus, and EMBASE. STUDY SELECTION: A total of 1344 abstracts were screened, of which 83 articles were considered potentially relevant. Data were finally extracted from 6 studies including 578 patients. DATA ANALYSIS: Forest plots were generated to illustrate the hazard ratios of overall survival and progression-free survival. The heterogeneity across the studies was assessed using the Cochrane Q test and I2 values. DATA SYNTHESIS: The pooled hazard ratios for overall survival and progression-free survival in patients with an ADCL lower than the cutoff values were 1.89 (95% CI, 1.53-2.31) and 1.98 (95% CI, 1.54-2.55) with low heterogeneity among the studies. Subgroup analysis of the bevacizumab-free cohort showed a pooled hazard ratio for overall survival of 1.20 (95% CI, 1.08-1.34) with low heterogeneity. LIMITATIONS: The conclusions are limited by the difference in the definition of recurrence among the included studies. CONCLUSIONS: This systematic review with meta-analysis supports the prognostic value of ADCL in patients with recurrent glioblastoma treated with bevacizumab, with a low ADCL demonstrating decreased overall survival and progression-free survival. On the other hand, the predictive role of ADCL for bevacizumab treatment was not confirmed.

Prognostic Factors of Stroke-Like Migraine Attacks after Radiation Therapy (SMART) Syndrome.

BACKGROUND AND PURPOSE: Prognostic factors of stroke-like migraine attacks after radiation therapy (SMART) syndrome have not been fully explored. This study aimed to assess clinical and imaging features to predict the clinical outcome of SMART syndrome. MATERIALS AND METHODS: We retrospectively reviewed the clinical manifestations and imaging findings of 20 patients with SMART syndrome (median age, 48 years; 5 women) from January 2016 to January 2020 at 4 medical centers. Patient demographics and MR imaging features at the time of diagnosis were reviewed. This cohort was divided into 2 groups based on the degree of clinical improvement (completely versus incompletely recovered). The numeric and categoric variables were compared as appropriate. RESULTS: There were statistically significant differences between the completely recovered group (n = 11; median age, 44 years; 2 women) and the incompletely recovered group (n = 9; median age, 55 years; 3 women) in age, months of follow-up, and the presence of steroid treatment at diagnosis (P = .028, .002, and .01, respectively). Regarding MR imaging features, there were statistically significant differences in the presence of linear subcortical WM susceptibility abnormality, restricted diffusion, and subcortical WM edematous changes in the acute SMART region (3/11 versus 8/9, P = .01; 0/11 versus 4/9, P = .026; and 2/11 versus 7/9, P = .022, respectively). Follow-up MRIs showed persistent susceptibility abnormality (11/11) and subcortical WM edematous changes (9/9), with resolution of restricted diffusion (4/4). CONCLUSIONS: Age, use of steroid treatment at the diagnosis of SMART syndrome, and MR imaging findings of abnormal susceptibility signal, restricted diffusion, and subcortical WM change in the acute SMART region can be prognostic factors in SMART syndrome.

Diagnostic Role of Diffusion-Weighted and Dynamic Contrast-Enhanced Perfusion MR Imaging in Paragangliomas and Schwannomas in the Head and Neck.

BACKGROUND AND PURPOSE: Distinguishing schwannomas from paragangliomas in the head and neck and determining succinate dehydrogenase (SDH) mutation status in paragangliomas are clinically important. We aimed to assess the clinical usefulness of DWI and dynamic contrast-enhanced MR imaging in differentiating these 2 types of tumors, as well as the SDH mutation status of paragangliomas. MATERIALS AND METHODS: This retrospective study from June 2016 to June 2020 included 42 patients with 15 schwannomas and 27 paragangliomas (10 SDH mutation-positive and 17 SDH mutation-negative). ADC values, dynamic contrast-enhanced MRI parameters, and tumor imaging characteristics were compared between the 2 tumors and between the mutation statuses of paragangliomas as appropriate. Multivariate stepwise logistic regression analysis was performed to identify significant differences in these parameters. RESULTS: Fractional plasma volume (P ≤ .001), rate transfer constant (P = .038), time-to-maximum enhancement (P < .001), maximum signal-enhancement ratio (P < .001) and maximum concentration of contrast agent (P < .001), velocity of enhancement (P = .002), and tumor characteristics including the presence of flow voids (P = .001) and enhancement patterns (P = .027) showed significant differences between schwannomas and paragangliomas, though there was no significant difference in ADC values. In the multivariate logistic regression analysis, fractional plasma volume was identified as the most significant value for differentiation of the 2 tumor types (P = .014). ADC values were significantly higher in nonhereditary than in hereditary paragangliomas, while there was no difference in dynamic contrast-enhanced MR imaging parameters. CONCLUSIONS: Dynamic contrast-enhanced MR imaging parameters show promise in differentiating head and neck schwannomas and paragangliomas, while DWI can be useful in detecting SDH mutation status in paragangliomas.

Assessment of MR Imaging and CT in Differentiating Hereditary and Nonhereditary Paragangliomas.

BACKGROUND AND PURPOSE: Head and neck paragangliomas have been reported to be associated with mutations of the succinate dehydrogenase enzyme family. The aim of this study was to assess whether radiologic features could differentiate between paragangliomas in the head and neck positive and negative for the succinate dehydrogenase mutation. MATERIALS AND METHODS: This single-center retrospective review from January 2015 to January 2020 included 40 patients with 48 paragangliomas (30 tumors positive for succinate dehydrogenase mutation in 23 patients and 18 tumors negative for the succinate dehydrogenase mutation in 17 patients). ADC values and tumor characteristics on CT and MR imaging were evaluated by 2 radiologists. Differences between the 2 cohorts in the diagnostic performance of ADC and normalized ADC (ratio to ADC in the medulla oblongata) values were evaluated using the independent samples t test. P < .05 was considered significant. RESULTS: ADCmean (1.07 [SD, 0.25]/1.04 [SD, 0.12] versus 1.31 [SD, 0.16]/1.30 [SD, 0.20]× 10-3 mm2/s by radiologists 1 and 2; P < .001), ADCmaximum (1.49 [SD, 0.27]/1.49 [SD, 0.20] versus 2.01 [SD, 0.16]/1.87 [SD, 0.20] × 10-3 mm2/s; P < .001), normalized ADCmean (1.40 [SD, 0.33]/1.37 [SD, 0.16] versus 1.73 [SD, 0.22]/1.74 [SD, 0.27]; P < .001), and normalized ADCmaximum (1.95 [SD, 0.37]/1.97 [SD, 0.27] versus 2.64 [SD, 0.22]/2.48 [SD, 0.28]; P < .001) were significantly lower in succinate dehydrogenase mutation-positive than mutation-negative tumors. ADCminimum, normalized ADCminimum, and tumor characteristics were not statistically significant. CONCLUSIONS: ADC is a promising imaging biomarker that can help differentiate succinate dehydrogenase mutation-positive from mutation-negative paragangliomas in the head and neck.

MRI Findings of Immune Checkpoint Inhibitor-Induced Hypophysitis: Possible Association with Fibrosis.

BACKGROUND AND PURPOSE: Hypophysitis is one of the well-known adverse effects of immune checkpoint inhibitors. Immune checkpoint inhibitor-induced hypophysitis frequently causes irreversible hypopituitarism, which requires long-term hormone replacement. Despite the high frequency and clinical significance, characteristic MR imaging findings of immune checkpoint inhibitor-induced hypophysitis have not been established. In the present study, we aimed to review and extract the MR imaging features of immune checkpoint inhibitor-induced hypophysitis. MATERIALS AND METHODS: This retrospective international multicenter study comprised 20 patients with melanoma who were being treated with immune checkpoint inhibitors and clinically diagnosed with immune checkpoint inhibitor-induced hypophysitis. Three radiologists evaluated the following MR imaging findings: enlargement of the pituitary gland and stalk; homogeneity of enhancement of the pituitary gland; presence/absence of a well-defined poorly enhanced area and, if present, its location, shape, and signal intensity in T2WI; and enhancement pattern in contrast-enhanced dynamic MR imaging. Clinical symptoms and hormone levels were also recorded. RESULTS: Enlargement of the pituitary gland and stalk was observed in 12 and 20 patients, respectively. Nineteen patients showed poorly enhanced lesions (geographic hypoenhancing lesions) in the anterior lobe, and 11 of these lesions showed hypointensity on T2WI. Thyrotropin deficiency and corticotropin deficiency were observed in 19/20 and 12/17 patients, respectively, which persisted in 12/19 and 10/12 patients, respectively, throughout the study period. CONCLUSIONS: Pituitary geographic hypoenhancing lesions in the anterior lobe of the pituitary gland are characteristic and frequent MR imaging findings of immune checkpoint inhibitor-induced hypophysitis. They reflect fibrosis and are useful in distinguishing immune checkpoint inhibitor-induced hypophysitis from other types of hypophysitis/tumors.

Differential use of CREB binding protein-coactivator complexes.

CREB binding protein (CBP) functions as an essential coactivator of transcription factors that are inhibited by the adenovirus early gene product E1A. Transcriptional activation by the signal transducer and activator of transcription-1 (STAT1) protein requires the C/H3 domain in CBP, which is the primary target of E1A inhibition. Here it was found that the C/H3 domain is not required for retinoic acid receptor (RAR) function, nor is it involved in E1A inhibition. Instead, E1A inhibits RAR function by preventing the assembly of CBP-nuclear receptor coactivator complexes, revealing differences in required CBP domains for transcriptional activation by RAR and STAT1.

Transcription factor-specific requirements for coactivators and their acetyltransferase functions.

Different classes of mammalian transcription factors-nuclear receptors, cyclic adenosine 3',5'-monophosphate-regulated enhancer binding protein (CREB), and signal transducer and activator of transcription-1 (STAT-1)-functionally require distinct components of the coactivator complex, including CREB-binding protein (CBP/p300), nuclear receptor coactivators (NCoAs), and p300/CBP-associated factor (p/CAF), based on their platform or assembly properties. Retinoic acid receptor, CREB, and STAT-1 also require different histone acetyltransferase (HAT) activities to activate transcription. Thus, transcription factor-specific differences in configuration and content of the coactivator complex dictate requirements for specific acetyltransferase activities, providing an explanation, at least in part, for the presence of multiple HAT components of the complex.

Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors.

As the obligate member of most nuclear receptor heterodimers, retinoid X receptors (RXRs) can potentially perform two functions: cooperative binding to hormone response elements and coordinate regulation of target genes by RXR ligands. In this paper we describe allosteric interactions between RXR and two heterodimeric partners, retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs); RARs and PPARs prevent and permit activation by RXR-specific ligands, respectively. By competing for dimerization with RXR on response elements consisting of direct-repeat half-sites spaced by 1 bp (DR1 elements), the relative abundance of RAR and PPAR determines whether the RXR signaling pathway will be functional. In contrast to RAR, which prevents the binding of RXR ligands and recruits the nuclear receptor corepressor N-CoR, PPAR permits the binding of SRC-1 in response to both RXR and PPAR ligands. Overexpression of SRC-1 markedly potentiates ligand-dependent transcription by PPARgamma, suggesting that SRC-1 serves as a coactivator in vivo. Remarkably, the ability of RAR to both block the binding of ligands to RXR and interact with corepressors requires the CoR box, a structural motif residing in the N-terminal region of the RAR ligand binding domain. Mutations in the CoR box convert RAR from a nonpermissive to a permissive partner of RXR signaling on DR1 elements. We suggest that the differential recruitment of coactivators and corepressors by RAR-RXR and PPAR-RXR heterodimers provides the basis for a transcriptional switch that may be important in controlling complex programs of gene expression, such as adipocyte differentiation.

Transcriptional activation by NF-kappaB requires multiple coactivators.

Nuclear factor-kappaB (NF-kappaB) plays a role in the transcriptional regulation of genes involved in inflammation and cell survival. In this report we demonstrate that NF-kappaB recruits a coactivator complex that has striking similarities to that recruited by nuclear receptors. Inactivation of either cyclic AMP response element binding protein (CREB)-binding protein (CBP), members of the p160 family of coactivators, or the CBP-associated factor (p/CAF) by nuclear antibody microinjection prevents NF-kappaB-dependent transactivation. Like nuclear receptor-dependent gene expression, NF-kappaB-dependent gene expression requires specific LXXLL motifs in one of the p160 family members, and enhancement of NF-kappaB activity requires the histone acetyltransferase (HAT) activity of p/CAF but not that of CBP. This coactivator complex is differentially recruited by members of the Rel family. The p50 homodimer fails to recruit coactivators, although the p50-p65 heterodimeric form of the transcription factor assembles the integrator complex. These findings provide new mechanistic insights into how this family of dimeric transcription factors has a differential effect on gene expression.

Autocrine growth factor in defined serum-free medium of human salivary gland adenocarcinoma cell line HSG.

Human salivary gland adenocarcinoma cell line HSG secretes an epidermal growth factor (EGF)-like molecule and contains EGF receptors. Growth of HSG cells is inhibited by glucocorticoid. We have identified that the growth inhibition by glucocorticoid is induced by the reduced secretion of the EGF-like molecule and that addition of anti-human EGF antibody to the culture specifically inhibits the growth of HSG cells, suggesting that autocrine secretion is involved in the growth of HSG cells. To prove that autocrine secretion functions in glucocorticoid-regulated growth of the HSG cell line, we purified the EGF-like molecule from serum-free, defined medium conditioned by the HSG cells and examined the growth-stimulatory effect of the purified molecule. The cultivation of HSG cells in serum-free defined medium, which contains insulin (10 micrograms/ml) and transferrin (10 micrograms/ml) only as proteinaceous components, resulted in establishment of a new cell line (HSG-SF) which had different morphological features from the parental HSG cell line. HSG-SF cells were found to have basically the same responsiveness to glucocorticoid as parental HSG cells. Parental HSG cells secreted high molecular weight EGF-like molecules (Mr 46,000 and 57,000), which were recognized by specific antibody to low molecular weight human EGF (Mr 6,201). From conditioned, serum-free medium of HSG-SF cells, an EGF-like molecule (Mr 46,000) was purified by using an anti-human EGF antibody-coupled Sepharose CL-4B column. This EGF-like molecule induced a maximal increase (36%) in incorporation of [3H]thymidine into DNA of parental HSG cells as well as low molecular weight human EGF. These observations demonstrate that growth of the HSG cell line is regulated by autocrine secretion.

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line.

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG.

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.

Retinoic acid receptor in subclone of human salivary gland adenocarcinoma cell line HSG and effect of retinoic acid on cellular growth.

Retinoic acid (RA) binding has been detected in the nuclei of a subclone (CL-1) of human submandibular adenocarcinoma cell line HSG conditioned to grow in a serum-free defined medium. Competition assay confirmed the specificity of the RA binding. Scatchard analysis showed the binding molecule to have a high affinity and low capacity. From the analyses by gel-filtration and glycerol density gradient centrifugation, the nuclear binding molecule appears to be distinct from cellular RA binding protein (CRABP) in terms of molecular weight. Furthermore, immunoblotting analysis revealed a band (Mr 47,000) reactive with specific antibody to RA receptor (RAR) alpha in the gel containing the nuclear fraction of CL-1 cells. Northern blotting analysis with specific cDNA probes revealed the expression of RAR alpha and RAR gamma in CL-1 cells. These results indicate that CL-1 cells express two types of RAR subtype, suggesting that these receptor molecules may mediate biological effects of RA. Treatment of CL-1 cells with RA resulted in an increase in the incorporation of [3H]thymidine into TCA-insoluble materials. The maximal increase was observed at 10(-6) M around 48 h. Previously, we demonstrated the autocrine growth of HSG cells mediated by epidermal growth factor (EGF) receptors and EGF-like molecules (Kurokawa et al. (1989) Cancer Res. 49, 5136-5142) and showed that RA had no significant effect on the secretion of the EGF-like molecule. RA induced an increase in [125I]EGF binding to CL-1 cells. The increase in the EGF binding was maximal at 24 h at 10(-6) M RA. RA also increased the amount of [3H]leucine-labeled EGF receptor dose-dependently. No significant change was observed in total protein synthesis of CL-1 cells by treatment with RA. These results suggest that RA stimulates the growth of CL-1 cells by increasing EGF receptor levels.

Glucocorticoid regulates secretion of epidermal growth factor in the human salivary gland adenocarcinoma cell line.

The treatment of a human submandibular gland adenocarcinoma cell line (HSG cell line) for 48 h with triamcinolone acetonide (TA; 1-100 nmol/l) reduced the secretion of epidermal growth factor (EGF) in a closely related manner to a maximum of 66%. The reduction in the level of EGF secreted resulted in the suppression of DNA synthesis in the HSG cells to a similar extent. When the cells were incubated with TA and exogenous human EGF (hEGF), DNA synthesis was 1.7-fold higher than that without added hEGF. The removal of EGF by the addition of hEGF antibody reduced DNA synthesis in HSG cell cultures to the same extent as did TA. These results suggest that the growth inhibition of HSG cells by TA is due to the reduction in the amount of EGF secreted.

Mechanism of replenishment of androgen receptors in cytosol of mouse submandibular gland.

Female mice were used to examine the process of depletion and replenishment of cytosolic androgen receptors in submandibular glands, and to investigate the effects of cycloheximide and actinomycin D on these processes. The dose-dependence of receptor depletion and replenishment in the cytosolic fraction, and that of receptor accumulation in the nuclear fraction were investigated. Almost 100% depletion was revealed 1 h after the injection of testosterone propionate at a dose of 500 or 50 micrograms testosterone/100 g body weight. With a 5 micrograms dose, depletion of cytosolic receptors was not complete and replenishment proceeded rapidly compared with that which occurred with the 50 or 500 micrograms dose. The nuclear receptor level increased 1 h after injection of testosterone, and the raised level was gradually reduced to the pretreatment level with all doses. However, the time required for this return to pretreatment level was dependent on the dose of testosterone. The change in the levels of cytosolic and nuclear androgen receptors following injection of testosterone was parallel to the level of circulating androgen. To determine the requirements for transcriptional and translational events in the replenishment process, cycloheximide and actinomycin D were given in vivo. The process of replenishment of cytosolic receptors was inhibited by the injection of cycloheximide. However, actinomycin D exerted no inhibitory effect on receptor replenishment. Neither cycloheximide nor actinomycin D had any effect on the nuclear receptor level until 6 h after the injection of testosterone. Cycloheximide or actinomycin D alone had no effect on the cytosolic or nuclear receptor level. These results suggest that receptor replenishment involves protein synthesis.