Exploring the Possible Impact of Unbalanced Open-Label Drop-In of Glucose-Lowering Medications on EXSCEL Outcomes.

BACKGROUND: EXSCEL (Exenatide Study of Cardiovascular Event Lowering) assessed the impact of once-weekly exenatide 2 mg versus placebo in patients with type 2 diabetes mellitus, while aiming for glycemic equipoise. Consequently, greater drop-in of open-label glucose-lowering medications occurred in the placebo group. Accordingly, we explored the potential effects of their unbalanced use on major adverse cardiovascular events (MACE), defined as cardiovascular death, nonfatal myocardial infarction or nonfatal stroke, and all-cause mortality (ACM), given that some of these agents are cardioprotective. METHODS: Cox hazard models were performed by randomized treatment for drug classes where >5% open-label drop-in glucose-lowering medication occurred, and for glucagon-like peptide-1 receptor agonists (GLP-1 RAs; 3.0%) using three methodologies: drop-in visit right censoring, inverse probability for treatment weighting (IPTW), and applying drug class risk reductions. RESULTS: Baseline glucose-lowering medications for the 14 752 EXSCEL participants (73.1% with previous cardiovascular disease) did not differ between treatment groups. During median 3.2 years follow-up, open-label drop-in occurred in 33.4% of participants, more frequently with placebo than exenatide (38.1% versus 28.8%), with metformin (6.1% versus 4.9%), sulfonylurea (8.7% versus 6.9%), dipeptidyl peptidase-4 inhibitors (10.6% versus 7.5%), SGLT-2i (10.3% versus 8.1%), GLP-1 RA (3.4% versus 2.4%), and insulin (13.8% versus 9.4%). The MACE effect size was not altered meaningfully by right censoring, but the favorable HR for exenatide became nominally significant in the sulfonylurea and any glucose-lowering medication groups, while the ACM HR and p-values were essentially unchanged. IPTW decreased the MACE HR from 0.91 (P=0.061) to 0.85 (P=0.008) and the ACM HR from 0.86 (P=0.016) to 0.81 (P=0.012). Application of literature-derived risk reductions showed no meaningful changes in MACE or ACM HRs or P values, although simulations of substantially greater use of drop-in cardioprotective glucose-lowering agents demonstrated blunting of signal detection. CONCLUSIONS: EXSCEL-observed HRs for MACE and ACM remained robust after right censoring or application of literature-derived risk reductions, but the exenatide versus placebo MACE effect size and statistical significance were increased by IPTW. Effects of open-label drop-in cardioprotective medications need to be considered carefully when designing, conducting, and analyzing cardiovascular outcome trials of glucose-lowering agents under the premise of glycemic equipoise. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01144338.

Increased warm ischemia time during vessel harvest decreases the primary patency of cryopreserved conduits in patients undergoing lower extremity bypass.

OBJECTIVE: Autologous vein is the preferred conduit for lower extremity bypass. However, it is often unavailable because of prior harvest or inadequate for bypass owing to insufficient caliber. Cryopreserved cadaveric vessels can be used as conduits for lower extremity revascularization when autogenous vein is not available and the use of prosthetic grafts is not appropriate. Many studies have shown that donor characteristics influence clinical outcomes in solid organ transplantation, but little is known regarding their impact in vascular surgery. The purpose of this study was to examine the effects donor variables have on patients undergoing lower extremity bypass with cryopreserved vessels. METHODS: The tissue processing organization was queried for donor blood type, warm ischemia times (WITs), and serial numbers of cryopreserved vessels implanted at a single center from 2010 to 2016. The serial numbers were then matched with their respective patients using the institutional Clinical Data Repository and patient data were obtained from the Clinical Data Repository and chart review. Primary outcomes were primary patency of the bypass conduits and limb salvage. Time to loss of patency was evaluated using Kaplan-Meier methods and a Cox proportional hazards model determined risk-adjusted predictors of patency and limb salvage. RESULTS: Sixty patients underwent lower extremity bypass with 65 cryopreserved vessels (23 superficial femoral arteries, 41 saphenous veins, 1 femoral vein). Thirty-eight procedures were reoperations. There were 21 inflow, 44 outflow, and 44 infrainguinal procedures. Preexisting comorbidities did not differ significantly between those who lost patency and those who did not. The mean WIT among the entire cohort was 892.3 ± 389.1 minutes (range, 158.0-1434.0 minutes). The median follow-up was 394 days. Kaplan-Meier analysis demonstrated an overall 1-year primary patency rate of 51%. Primary patency at 1 year was 67% and 41% for inflow and outflow procedures, respectively, and did not differ significantly between the two groups (P = .15). Donor-to-recipient ABO incompatibility was not associated with loss of primary patency. The 1-year amputation-free survival was 74%. Primary patency significantly decreased with each hourly increase in WIT on risk-adjusted analysis (hazard ratio, 1.1; P = .02). CONCLUSIONS: Higher cryopreserved vessel WIT was associated with increased risk-adjusted loss of primary patency in this cohort. At 1 year, the overall primary patency was 51% and amputation-free survival was 74%. Vascular surgeons should be aware that WIT may affect outcomes for lower extremity bypass.

Intensity-modulated radiotherapy at high-volume centers improves survival in patients with esophageal adenocarcinoma receiving trimodality therapy.

The standard of care trimodality therapy for resectable locally advanced esophageal adenocarcinoma is complex and necessitates multidisciplinary care and expertise. In this work, it is hypothesized that facility clinical volume and utilization of intensity-modulated radiotherapy (IMRT) may influence outcomes. The National Cancer Data Base was queried for patients with cT1-4-N0-3 M0 esophageal adenocarcinoma undergoing trimodality therapy from 2004 to 2013 (n = 2445). All patients received chemoradiation followed by esophagectomy at a Commission on Cancer facility. The facility volume was categorized into tertiles: high-volume centers (HVCs) in the highest 25th percentile of cases per year, intermediate-volume centers (IVCs) with the next highest 25th percentile of cases, and low- and very low-volume centers (LVCs) in the lowest 50th percentile. Overall survival (OS) was estimated using Kaplan-Meier methods and Cox proportional hazard regression. Propensity score matching to balance patient characteristics between volume centers was performed. Subgroup analysis was done comparing IMRT versus 3D conformal radiotherapy. The median follow-up was 26 months. Treatment at an HVC (hazard ratio 0.63, 95% CI 0.49-0.81, P < 0.001) was found to be independently associated with improved overall survival in multivariable analysis. Three-year OS was 58.4%, 46.2%, and 47.5% for HVCs, IVCs, and LVCs, respectively (P < 0.001). Patients at HVCs were more likely to receive IMRT over 3D chemoradiation (CRT; OR 3.45, 95% CI 2.4-5.0, P < 0.001). Patients treated using IMRT at HVCs had improved OS compared to those treated at IVCs or LVCs (HR 0.68, 95% CI 0.52-0.90, P < 0.01), while patients treated with 3D CRT at HVCs had no survival advantage over those at IVCs or LVCs (P = 0.28). Patients with locally advanced esophageal adenocarcinoma treated with IMRT and at HVCs appear to have improved survival.

The thyroid: review of imaging features and biopsy techniques with radiologic-pathologic correlation.

Knowledge of the normal and abnormal imaging appearances of the thyroid gland is essential for appropriate identification and diagnosis of thyroid lesions. Thyroid nodules are often detected incidentally at computed tomography, magnetic resonance imaging, and positron emission tomography; however, ultrasonography (US) is the most commonly used imaging modality for characterization of these nodules. US characteristics that increase the likelihood of malignancy in a thyroid nodule include microcalcifications, solid composition, and central vascularity. Nuclear scintigraphy is commonly used for evaluation of physiologic thyroid function and for identification of metabolically active and inactive nodules. When fine-needle aspiration biopsy (FNAB) of a lesion is indicated based on clinical and radiologic features, appropriate US-guided biopsy technique and careful cytologic analysis are crucial for making the diagnosis. FNAB and core biopsy are the two percutaneous techniques used to obtain a specimen, with the latter technique being indicated following nondiagnostic or indeterminate FNAB. Specimen adequacy and diagnostic accuracy vary due to several factors, including location of aspiration and biopsy technique used. The radiologist must have a basic knowledge of thyroid disease, be familiar with specimen processing, and recognize the cytologic and radiologic appearances of thyroid lesions, all of which will facilitate the management of these lesions. Online supplemental material is available for this article.

Brief ampakine treatments slow the progression of Huntington's disease phenotypes in R6/2 mice.

Daily, systemic injections of a positive AMPA-type glutamate receptor modulator (ampakine) have been shown to reduce synaptic plasticity defects in rodent models of aging and early-stage Huntington's disease (HD). Here we report that long-term ampakine treatment markedly slows the progression of striatal neuropathology and locomotor dysfunction in the R6/2 HD mouse model. Remarkably, these effects were produced by an ampakine, CX929, with a short half-life. Injected once daily for 4-7 weeks, the compound increased protein levels of brain-derived neurotrophic factor (BDNF) in the neocortex and striatum of R6/2 but not wild-type mice. Moreover, ampakine treatments prevented the decrease in total striatal area, blocked the loss of striatal DARPP-32 immunoreactivity and reduced by 36% the size of intra-nuclear huntingtin aggregates in R6/2 striatum. The CX929 treatments also markedly improved motor performance of R6/2 mice on several measures (rotarod, vertical pole descent) but did not influence body weight or lifespan. These findings describe a minimally invasive, pharmacologically plausible strategy for treatment of HD and, potentially, other neuropathological diseases.

Recurrent ependymoma with cartilaginous metaplasia in an adult: report of a rare case and review of literature.

Cartilaginous metaplasia in ependymomas is an uncommon phenomenon and is hypothesized to be due to metaplasia of the mesenchymal supportive elements or arising from the neoplastic glial cells. Most of the previous cases reported have occurred in children less than 10 years of age. The present report discusses an unusual case of ependymoma with cartilaginous metaplasia in a 21-year-old male. A brief review on the histogenesis of cartilaginous metaplasia is also provided.

Lip Repositioning, Aesthetic Crown Lengthening, and Gingival Depigmentation: A Combined Approach for a Gummy Smile Makeover.

This clinical report describes the successful use of aesthetic crown lengthening, lip-repositioning technique as well as gingival depigmentation for the reduction of excessive gingival display and dark gums, respectively. Lip-repositioning technique was performed with the main objective of reducing gummy smile by limiting the retraction of elevator muscles (e.g., zygomaticus minor, levator anguli oris, orbicularis oris, and levator labii superioris) during smiling, thereby restricting the upper lip from shifting apically while smiling. This technique includes removing a strip of mucosa from the maxillary labial and buccal vestibule, creating a partial-thickness flap between mucogingival junction and upper lip musculature, and suturing the lip mucosa with mucogingival junction, resulting in a narrow vestibule and restricted muscle pull, thereby reducing gingival display. The results obtained with lip repositioning for the treatment of gummy smile are substantial and it is a simple and effective procedure, well accepted by patients. Proper case selection is important for using this procedure.

Changes in Serum Calcitonin Concentrations, Incidence of Medullary Thyroid Carcinoma, and Impact of Routine Calcitonin Concentration Monitoring in the EXenatide Study of Cardiovascular Event Lowering (EXSCEL).

OBJECTIVE: Increases in serum calcitonin, a tumor marker for medullary thyroid carcinoma (MTC), have been associated with glucagon-like peptide 1 receptor agonist use in some preclinical studies. We report calcitonin changes in exenatide-treated and placebo-administered participants and MTC incidence in the EXenatide Study of Cardiovascular Event Lowering (EXSCEL) and consider the impact of within-trial calcitonin monitoring. RESEARCH DESIGN AND METHODS: EXSCEL participants were randomized 1:1 to once-weekly exenatide 2 mg or placebo. Serum calcitonin was measured at baseline (with trial medication discontinued if >40 ng/L) and annually thereafter (with trial medication discontinued if ≥50 ng/L). Median calcitonin concentrations were calculated at each time point, and thyroid malignancies were collected prospectively. Data regarding follow-up after an elevated calcitonin were collected retrospectively. RESULTS: At baseline, 52 (30 exenatide and 22 placebo) participants had calcitonin >40 ng/L, and during follow-up an additional 23 participants (15 exenatide and 8 placebo) had calcitonin ≥50 ng/L in the intention-to-treat population. Median calcitonin concentrations were similar between treatment groups at baseline with no increase over time. Confirmed MTC occurred in three participants (2 exenatide and 1 placebo), all of whom had significantly elevated baseline calcitonin values (413, 422, and 655 ng/L). CONCLUSIONS: During a median 3.2 years' follow-up, no change in serum calcitonin was seen with exenatide therapy. The three confirmed cases of MTC all occurred in participants with markedly elevated baseline calcitonin levels, measured prior to trial medication administration. Regular calcitonin monitoring identified no additional cases of MTC, suggesting no benefit of routine calcitonin monitoring during exenatide treatment.

Acute coronary syndrome patients with depression have low blood cell membrane omega-3 fatty acid levels.

OBJECTIVE: To determine the extent to which levels of membrane eicosapentaenoic (EPA)+docosahexaenoic acids (DHA) (the omega-3 index) were associated with depression in patients with acute coronary syndrome (ACS). Depression is associated with worse cardiovascular (CV) outcomes in patients with ACS. Reduced levels of blood cell membrane omega-3 (n-3) fatty acids (FAs), an emerging risk factor for both CV disease and depression, may help to explain the link between depression and adverse CV outcomes. METHODS: We measured membrane FA composition in 759 patients with confirmed ACS. The analysis included not only EPA and DHA but also the n-6 FAs linoleic and arachidonic acids (LA and AA). Depressive symptoms were measured with the Patient Health Questionnaire-9 (PHQ). Multivariable linear regression was used to adjust for demographic and clinical characteristics. RESULTS: There was a significant inverse relationship between the n-3 index and depressive symptoms (PHQ) in the fully adjusted model (p = .034). For every 4.54% point rise in the n-3 index, there was a 1-point decline in depressive symptoms. In contrast to the n-3 FAs, membrane levels of the n-6 FAs LA and AA were not different between depressed and nondepressed ACS patients. CONCLUSION: We found an inverse relationship between the n-3 index and the prevalence of depressive symptoms in patients with ACS. Therefore, this study supports the hypothesis that reduced n-3 FA tissue levels are a common and potentially modifiable link between depression and adverse CV outcomes.

Chordoid glioma: report of two rare examples with unusual features.

BACKGROUND: Chordoid glioma, a rare tumour of the third ventricle, represents a distinct clinico-pathologic entity. Thirty nine examples have been described in the literature, mostly in females and in the third ventricle. The clinical presentation is variable but they tend to occur mostly in adults. There is only one report of a chordoid glioma in a 12 year old child. FINDING: This paper describes two examples of chordoid glioma in a seven year old female child and a 70 year old male respectively. Radiologically, the paediatric chordoid glioma was located in the juxtaventricular region in the occipital horn of the lateral ventricle and was of mixed density whereas the adult patient had a typical third ventricle location with homogenous contrast enhancement. Gross total surgical removal was achieved in both but the adult patient died post-operatively due to intra ventricular bleeding and bacterial meningitis. The younger patient is doing well at the last follow up two years post-operatively. Microscopically, both showed the classic morphology of chordoid glioma. Ultrastructural examination was suggestive of ependymal differentiation. CONCLUSION: The younger age and unusual location are some of the rare features which need documentation and have not been described earlier. We propose that chordoid glioma is a variant of an ependymoma (WHO grade II) which arises from tanycytes and should be included in the WHO classification of brain tumors.

Fatal subacute necrotising brainstem encephalitis in a young man due to a rare parasitic (Balamuthia) infection.

We describe a case of brainstem inflammation in a young man which at first defied diagnosis. However, after his death, and notwithstanding our inability to find a cause at autopsy, we did not give up. After sending paraffin blocks to the Centers for Disease Control in Atlanta, Georgia, USA, they suggested the diagnosis of Balamuthia (amoebic) infection.

Cardiovascular outcomes with glucagon-like peptide-1 receptor agonists in patients with type 2 diabetes: a meta-analysis.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) receptor agonists are effective glucose-lowering drugs. Findings from cardiovascular outcome trials showed cardiovascular safety of GLP-1 receptor agonists, but results for cardiovascular efficacy were varied. We aimed to examine overall cardiovascular efficacy for lixisenatide, liraglutide, semaglutide, and extended-release exenatide. METHODS: In this systematic review and meta-analysis, we analysed data from eligible trials that assessed the safety and efficacy of GLP-1 receptor agonists compared with placebo in adult patients (aged 18 years or older) with type 2 diabetes and had a primary outcome including, but not limited to, cardiovascular mortality, non-fatal myocardial infarction, and non-fatal stroke. We searched PubMed and MEDLINE without language restrictions up to Sept 18, 2017, for eligible trials. We did a meta-analysis of available trial data using a random-effects model to calculate overall hazard ratios (HRs) for cardiovascular efficacy outcomes and odds ratios for key safety outcomes. FINDINGS: Of 12 articles identified in our search and screened for eligibility, four trials of cardiovascular outcomes of GLP-1 receptor agonists were identified: ELIXA (lixisenatide), LEADER (liraglutide), SUSTAIN 6 (semaglutide), and EXSCEL (extended-release exenatide). Compared with placebo, GLP-1 receptor agonist treatment showed a significant 10% relative risk reduction in the three-point major adverse cardiovascular event primary outcome (cardiovascular mortality, non-fatal myocardial infarction, and non-fatal stroke; HR 0·90, 95% CI 0·82-0·99; p=0·033), a 13% RRR in cardiovascular mortality (0·87, 0·79-0·96; p=0·007), and a 12% relative risk reduction in all-cause mortality (0·88, 0·81-0·95; p=0·002), with low-to-moderate between-trial statistical heterogeneity. No significant effect of GLP-1 receptor agonists was identified on fatal and non-fatal myocardial infarction, fatal and non-fatal stroke, hospital admission for unstable angina, or hospital admission for heart failure. Overall, no significant differences were seen in severe hypoglycaemia, pancreatitis, pancreatic cancer, or medullary thyroid cancer reported between GLP-1 receptor agonist treatment and placebo. INTERPRETATION: Our findings show cardiovascular safety across all GLP-1 receptor agonist cardiovascular outcome trials and suggest that drugs in this class can reduce three-point major adverse cardiovascular events, cardiovascular mortality, and all-cause mortality risk, albeit to varying degrees for individual drugs, without significant safety concerns. GLP-1 receptor agonists have a favourable risk-benefit balance overall, which should allow the choice of drug to be individualised to each patient's needs. FUNDING: Amylin Pharmaceuticals (AstraZeneca).

Rumination syndrome: pathophysiology, diagnosis, and treatment.

BACKGROUND: Rumination syndrome is a functional gastrointestinal disorder characterized by effortless and repetitive regurgitation of recently ingested food from the stomach to the oral cavity followed by either re-swallowing or spitting. Rumination is thought to occur due to a reversal of the esophagogastric pressure gradient. This is achieved by a coordinated abdominothoracic maneuver consisting of a thoracic suction, crural diaphragm relaxation and an increase in intragastric pressure. Careful history is important in the diagnosis of rumination syndrome; patients often report "vomiting" or "reflux" and the diagnosis can therefore be missed. Objective testing is available with high resolution manometry or gastroduodenal manometry. Increase in intra-gastric pressure followed by regurgitation is the most important characteristic to distinguish rumination from other disorders such as gastroesophageal reflux. The mainstay of the treatment of rumination syndrome is behavioral therapy via diaphragmatic breathing in addition to patient education and reassurance. PURPOSE: The purpose of this review was to critically appraise recent key developments in the pathophysiology, diagnosis and therapy for rumination syndrome. A literature search using OVID (Wolters Kluwer Health, New York, NY, USA) to examine the MEDLINE database its inception until May 2016 was performed using the search terms "rumination syndrome," "biofeedback therapy," and "regurgitation." References lists and personal libraries of the authors were used to identify supplemental information. Articles published in English were reviewed in full text. English abstracts were reviewed for all other languages. Priority was given to evidence obtained from randomized controlled trials when possible.

Predicting loco-regional recurrence risk in T1, T2 breast cancer with 1-3 positive axillary nodes postmastectomy: Development of a predictive nomogram.

BACKGROUND: Role of postmastectomy radiotherapy (PMRT) in early breast cancer with 1-3 positive axillary nodes is still controversial. Hence, there is a need to identify subgroup of patients who have sufficiently high risk of disease recurrence to benefit from PMRT. AIM: The aim is to evaluate clinical outcomes of patients postmastectomy having pathological T1-T2 tumors with 1-3 positive axillary lymph nodes (LNs) treated with adjuvant systemic therapy and develop a predictive nomogram. MATERIALS AND METHODS: Data collected retrospectively from eligible patients from 2005 to 2011. Kaplan-Meier survival analysis was used for all time-to-event analysis. Various known clinical and pathological risk factors were correlated with outcome using uni- and multi-variable analysis in SPSS version 21. All comparisons were two-tailed and P < 0.05 were considered statistically significant. The nomogram to predict the risk of loco-regional control (LRC) was developed using least absolute shrinkage and selection operator shrinkage model in hdnom.io software. RESULTS: 38/242 (15.7%) patients had recurrent disease at loco-regional (10 patients), distant sites (22 patients) and simultaneous loco-regional and distant sites (6 patients) at a median follow-up 59.5 (range 4-133) months. Five years estimate of LRC, distant disease-free survival (DFS), DFS, cause-specific survival and overall survival was 87.8%, 85.4%, 84.2%, 93.1%, and 91.5%, respectively. Pathological tumor size, margin status, LN ratio as continuous variables and grade and triple negative breast cancer status as categorical variables were the risk factors included in the model for building nomogram. CONCLUSION: The nomogram developed based on institutional data can be a valuable tool in guiding adjuvant PMRT depending on the risk of 5 years loco-regional recurrence.

Post-transcriptional regulation of the DNA damage-inducible gadd45 gene in human breast carcinoma cells exposed to a novel retinoid CD437.

The biologically active synthetic retinoid CD437 (6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene, AHPN) and different human breast carcinoma (HBC) cell lines were used to examine the possible mechanism(s) of gadd45 induction. Northern blot analysis of mRNA isolated from MCF-7, MDA-MB-468 and MDA-MB-231 HBC cell lines demonstrated a progressive increase in the 1.4 kb gadd45 transcript after exposure to 1 microM CD437. Western blot analysis showed increased gadd45 protein levels in MDA-MB-468 HBC cells following exposure to CD437. CD437 increased gadd45 mRNA levels by approximately 20-fold in MDA-MB-468 cells, however, the transcriptional activity was increased approximately 2-3-fold as demonstrated by the human gadd45 promoter-luciferase reporter construct and nuclear run-off assays. Sublines of MDA-MB-468 HBC cells expressing stably integrated GADD45 cDNA fragments were obtained and CD437-dependent induction of GADD45 analyzed. We report that approximately 300 nt located in the 5"-untranslated region (5"-UTR) of gadd45 mRNA are involved in the CD437-dependent 4-fold enhanced stability of gadd45 transcripts. MDA-MB-468 cells were stably transfected with either a plasmid having a CMV promoter-driven rabbit beta-globin gene or plasmids having a CMV promoter-driven chimeric gadd45 5"-UTR-rabbit beta-globin gene, where the entire gadd45 5"-UTR (from +1 to +298) or a 45 bp subfragment of the gadd45 5"-UTR (from +10 to +55) was positioned at the 5"-end of the rabbit beta-globin gene. CD437 was found to up-regulate expression of both the chimeric gadd45 -rabbit beta-globin transcripts, suggesting that cis element(s) involved in the CD437-dependent enhanced stability of gadd45 mRNA are contained in the 45 nt of the 5"-UTR of the gadd45 mRNA.

H19 gene overexpression in atypical multidrug-resistant cells associated with expression of a 95-kilodalton membrane glycoprotein.

The multidrug resistance phenotype of human breast carcinoma MCF-7/AdrVp cells is characterized by overexpression of a 95-kilodalton membrane glycoprotein (p95), accompanied by a marked reduction in intracellular anthracycline accumulation, without overexpression of P-glycoprotein or the multidrug resistance protein. We discovered that the mRNA of the H19 gene is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells or drug-sensitive MCF-7/AdrVp revertant cells. H19 is an imprinted gene with an important role in fetal differentiation, as well as a postulated function as a tumor suppressor gene. Another p95-overexpressing multidrug-resistant cell line, human lung carcinoma NCI-H1688, also displays high levels of H19 mRNA. In contrast, several multidrug-resistant cell lines that overexpress P-glycoprotein or the multidrug resistance protein do not have higher levels of H19 mRNA than their drug-sensitive counterparts. This is the first report of H19 gene overexpression accompanying any form of drug resistance. The association of H19 and p95 gene expression in drug resistance warrants further study.

Transcriptional repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 gene mediated by cis elements present in the 3'-untranslated region.

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 plays a major role in the induction of G1 cell cycle arrest following DNA damage and is known to be regulated by p53-dependent and -independent pathways. Here, we show that p21WAF1/CIP1 transcription is also regulated, independently of p53, by the cis elements that are located downstream of the transcription start site. A cDNA fragment of approximately 180 bp, located 260 bases 3' to the translation termination codon of p21WAF1/CIP1 cDNA, was cloned in both the sense and antisense orientations downstream of the CMV promoter, upstream of the SV40 promoter, and both upstream and downstream of the p21WAF1/CIP1 promoter in the plasmids carrying the luciferase reporter gene. The constructs were transiently transfected in human breast carcinoma cells MCF-7 and MDA-MB-468 and a Syrian hamster smooth muscle cell line DDT1MF2 and were found to elicit 2-3-fold or higher repression of luciferase activities. By using overlapping deletions of the above 180-bp fragment, we identified a 48-bp subfragment that contains putative cis element(s) that participate in the transcriptional repression of the p21WAF1/CIP1 gene. The overlapping subfragments bind, in vitro, to specific proteins present in the nuclear extracts of MDA-MB-468 and DDT1MF2 cells. We, therefore, propose that additional mechanism(s) exist that regulate expression of the cellular p21WAF1/CIP1 and may contribute to p21WAF1/CIP1-dependent control of the cell cycle.

Estradiol regulation of the human retinoic acid receptor alpha gene in human breast carcinoma cells is mediated via an imperfect half-palindromic estrogen response element and Sp1 motifs.

Estrogen receptor (ER)-positive human breast carcinoma (HBC) cell lines express significantly higher levels of retinoic acid receptor alpha (RAR alpha) (isoform 1) mRNA than ER-negative HBCs. Estradiol enhances RAR alpha mRNA expression in different ER-positive HBCs by 2-3-fold, which in turn results in increased sensitivity of ER-positive HBCs to the growth inhibitory effects of retinoic acid. To investigate the regulatory mechanisms of estradiol-mediated enhancement of RAR alpha mRNA expression, the functional promoter for the human RAR alpha isoform 1 was cloned and used to assess estradiol-mediated promoter-dependent enhancement of firefly luciferase reporter gene activity in transiently transfected ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231) HBCs. Deletional promoter constructs were obtained to further delineate the promoter region responsible for estradiol-mediated enhancement of promoter activity. Here, we present evidence that approximately 130 bp of the promoter fragment preceding the transcriptional start site are responsible for estradiol-mediated enhancement of hRAR alpha gene expression. The estradiol-mediated enhancement is dependent on ER binding. Further deletional analysis showed that a promoter sequence of 42 base pairs, located approximately 100 bases upstream of the transcriptional start site, contains elements for estradiol-mediated enhancement. Specific deletion of either the Sp1 motif or mutations in the imperfect half-palindromic estrogen response element motif of this fragment abolish its estradiol responsiveness in transient transfections.

Posttranscriptional regulation of p21WAF1/CIP1 expression in human breast carcinoma cells.

p21WAF1/CIP1 plays a major role in the induction of G1 arrest following DNA damage. Although p21WAF1/CIP1 expression is regulated by the tumor suppressor p53, induction of p21WAF1/CIP1 expression through p53-independent pathways has been described in numerous cell types. In this report, we describe the mechanism by which the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induces p21WAF1/CIP1 in breast carcinoma cells possessing either a wild-type (MCF-7 cells) or mutated (MDA-MB-468 cells) p53. Exposure of MDA-MB-468 cells to this retinoid results in an approximately 10-fold increase in p21WAF1/CIP1 mRNA levels, whereas less than a 2-fold increase in p21WAF1/CIP1 gene transcription was observed as indicated by transient transfection experiments utilizing a p21WAF1/CIP1 promoter firefly luciferase reporter gene construct and nuclear run-off studies. We found similar results in the MCF-7 cells (Z-M. Shao et al., Oncogene, 11: 493-504, 1995). We have now found that while enhancing p21WAF1/CIP1 gene transcription minimally, this retinoid increases p21WAF1/CIP1 mRNA stability by 3-fold in both cell types. We also demonstrate that approximately 1.5 kb of the 3' untranslated region causes enhanced instability of p21WAF1/CIP1 mRNA. The retinoid-dependent increase in p21WAF1/CIP1 mRNA stability is accompanied by an increase in p21WAF1/CIP1 protein expression, as indicated by Western blot experiments utilizing anti-p21WAF1/CIP1 monoclonal antibody. This increase in p21WAF1/CIP1 is subsequently followed by the onset of programmed cell death in both cell types. Thus, CD437 is a novel retinoid which enhances p21WAF1/CIP1 mRNA levels through stabilization of the message regardless of the p53 status of the cell.