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PubMed1985

Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression.

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

Animals↗

PubMed1986

Minimal transcriptional enhancer of simian virus 40 is a 74-base-pair sequence that has interacting domains.

We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.

Animals↗

PubMed1986

Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.

Animals↗

PubMed1986

trans Activation of the simian virus 40 enhancer.

We describe experiments which demonstrated that the simian virus 40 (SV40) enhancer affects certain transcriptional units differently. We also found that a specific enhancer-transcriptional unit interaction can be regulated by trans-acting factors. Using transient assays, we examined the effects of the SV40 enhancer on herpesvirus thymidine kinase (tk) RNA levels when transcription was initiated either by the herpesvirus tk promoter or by an SV40 early promoter-tk fusion. We were unable to detect any effect of the enhancer on transcription from the tk promoter in CV-1 or HeLa cells. However, we found that the addition of T-antigen in trans allowed the enhancer to stimulate expression from the tk promoter. This induction by T-antigen did not require T-antigen-binding sites in cis and appeared to be an indirect effect. In contrast, tk expression from the SV40 early promoter fusion was greatly stimulated by the enhancer in CV-1 cells. Furthermore, in 293 cells the SV40 enhancer had only a marginal effect on the SV40 promoter-tk fusion, whereas it strongly stimulated tk expression from the tk promoter. Our results raise the possibility that the enhancer function may not show cell specificity per se; rather, the interaction between the enhancer and a specific gene may be responsible for cell specificity. We discuss these observations in terms of the SV40 early gene-to-late gene switch that occurs during SV40 lytic growth.

Animals↗

PubMed1987

Binding of a cellular protein to the gibbon ape leukemia virus enhancer.

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.

Animals↗

PubMed1989

Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene.

The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.

Animals↗

PubMed2002

Characterization of liver lesions with mangafodipir trisodium-enhanced MR imaging: multicenter study comparing MR and dual-phase spiral CT.

PURPOSE: To evaluate whether mangafodipir trisodium (Mn-DPDP)-enhanced magnetic resonance (MR) imaging surpasses dual-phase spiral computed tomography (CT) in differentiating focal liver lesions. MATERIALS AND METHODS: One hundred forty-five patients who had or were suspected of having focal liver lesions were included in a multicenter study and underwent dual-phase spiral CT and enhanced MR imaging. Image interpretations performed by independent experienced radiologists were compared with the final diagnosis that was based on all available clinical information (including histopathologic findings in 77 patients) and that was determined with consensus. Differences in classifications by using either enhanced MR imaging or dual-phase spiral CT were analyzed with the McNemar test, and receiver operating characteristic (ROC) curves were used to compare the diagnostic performance of enhanced MR imaging and dual-phase spiral CT. RESULTS: Lesion classification was correct in 108 (74%) patients with enhanced MR imaging and in 83 (57%) with dual-phase spiral CT (P =.001). Lesions were correctly classified as either malignant or benign in 123 (85%) patients with enhanced MR imaging and in 98 (68%) with dual-phase spiral CT (P =.001). Classification of lesions as either hepatocellular or nonhepatocellular was correct in 130 (90%) patients with enhanced MR imaging and in 93 (64%) with dual-phase spiral CT (P =.001). These differences remained when analyses were restricted to histopathologically confirmed diagnoses. Comparison of the ROC curves illustrated that enhanced MR imaging performance surpassed that of dual-phase spiral CT. CONCLUSION: Mn-DPDP-enhanced MR imaging is superior to dual-phase spiral CT in classification of focal liver lesions.

Adult↗

PubMed2003

Differential diagnosis of hepatic tumors by using contrast enhancement patterns at US.

PURPOSE: To assess the accuracy of pattern-based classification of contrast material-enhanced wideband harmonic gray-scale ultrasonographic (US) images in the differential diagnosis of hepatic tumors. MATERIALS AND METHODS: A total of 183 hepatic lesions in 183 patients were studied; lesions included 116 hepatocellular carcinomas, 42 liver metastases, and 25 liver hemangiomas. After injection of a galactose-palmitic acid contrast agent, lesions were scanned with contrast-enhanced wideband harmonic gray-scale US in the arterial, portal venous, and late venous phases. The enhancement patterns were classified, and multiple logistic regression analysis was used to identify diagnostic patterns that enabled differentiation between hepatic tumors. RESULTS: Five enhancement patterns were found to be significant in predicting different hepatic tumors. In hepatocellular carcinomas, the presence of intratumoral vessels in the arterial phase and homogeneous or heterogeneous enhancement in the portal phase were the most typical patterns. In metastases, the absence of intratumoral vessels in the arterial phase and ring enhancement or a perfusion defect in the portal phase were the most typical patterns. In hemangiomas, the absence of intratumoral vessels in the arterial phase and peripheral nodular enhancement in the portal phase were the most typical patterns. The sensitivity, specificity, and accuracy of diagnosis based on combinations of enhancement patterns were, respectively, 94.8%, 94.0%, and 94.5% for hepatocellular carcinoma; 90.5%, 94.3%, and 93.4% for metastasis; and 88.0%, 99.4%, and 97.8% for hemangioma. CONCLUSION: Contrast-enhanced wideband harmonic gray-scale US is a useful tool for differentiating among the hepatic tumors studied.

Carcinoma, Hepatocellular↗

PubMed2004

Rheumatoid arthritis: epidural enhancement as an underestimated cause of subaxial cervical spinal stenosis.

PURPOSE: To assess the frequency and site of subaxial spinal canal stenosis due to enhancing tissue in patients with rheumatoid arthritis. MATERIALS AND METHODS: Data from 33 consecutive patients with rheumatoid arthritis were evaluated; these patients had undergone 1.5-T magnetic resonance imaging following gadolinium chelate administration, in combination with a frequency selective fat-suppression technique. Stenosis and enhancement were scored for each of six cervical spinal levels and were compared with results in a control population consisting of 16 patients with degenerative disease. Enhancement was scored as superficial or deep on the anterior and posterior sides from the cervical spinal cord. Differences between patient groups were tested by using the chi(2) test for trend and the Fisher exact test. RESULTS: No significant difference was found in the frequency or severity of subaxial stenosis between rheumatoid arthritis and degenerative disease. Deep epidural enhancement was observed more often with rheumatoid arthritis than with degenerative disease both anterior (25 of 33 patients vs seven of 16 patients, respectively; P <.001) and posterior (24 of 33 patients vs two of 16 patients, respectively; P =.001) to the spinal cord. Enhancing stenosing tissue in rheumatoid arthritis frequently occurred anterior and posterior at the same time and at the same level, with segmental cufflike extension of enhancing tissue around the dural sac. Stenosing tissue enhanced more frequently with rheumatoid arthritis than with degenerative disease (22 of 33 vs four of 16 patients, respectively; P =.008). CONCLUSION: In patients with rheumatoid arthritis, subaxial stenosis is frequently caused by enhancing epidural tissue. This enhancing tissue presumably represents pannus.

Adult↗

PubMed2004

Soft-tissue tumors: value of static and dynamic gadopentetate dimeglumine-enhanced MR imaging in prediction of malignancy.

PURPOSE: To prospectively evaluate static and dynamic gadopentetate dimeglumine-enhanced magnetic resonance (MR) imaging relative to nonenhanced MR imaging in differentiation of benign from malignant soft-tissue lesions and to evaluate which MR imaging parameters are most predictive of malignancy, with associated interobserver variability. MATERIALS AND METHODS: One hundred forty consecutive patients (78 male patients [median age, 51 years], 62 female patients [median age, 53 years]) with a soft-tissue mass underwent nonenhanced static and dynamic contrast material-enhanced MR imaging. Diagnosis was based on histologic findings in surgical specimens (86 of 140), findings at core-needle biopsy (43 of 140), or results of all imaging procedures with clinical follow-up (11 of 140). Multivariate logistic regression analysis was used to identify the best combination of MR imaging parameters that might be predictive of malignancy. Subjective overall performance of two observers was evaluated with receiver operating characteristic analysis. RESULTS: For subjective overall diagnosis, area under the receiver operating characteristic curve, a measure for diagnostic accuracy, was significantly larger for combined nonenhanced and contrast-enhanced MR imaging than it was for nonenhanced MR imaging alone, with no significant difference between observers. Multivariate analysis of all lesions revealed that combined nonenhanced static and dynamic contrast-enhanced MR imaging parameters were significantly superior to nonenhanced MR imaging parameters alone and to nonenhanced MR imaging parameters combined with static contrast-enhanced MR imaging parameters in prediction of malignancy. The most discriminating parameters were presence of liquefaction, start of dynamic enhancement (time interval between start of arterial and tumor enhancement), and lesion size (diameter). Results for extremity lesions were the same, with one exception: With dynamic contrast-enhanced MR imaging parameters, diagnostic performance of one observer did not improve. CONCLUSION: Static and dynamic contrast-enhanced MR imaging, when added to nonenhanced MR imaging, improved differentiation between benign and malignant soft-tissue lesions.

Adolescent↗

PubMed1994

Hepatic CT enhancement. Part I. Alterations in the volume of contrast material within the same patients.

PURPOSE: To evaluate the effects of alterations in the volume of contrast material on liver enhancement. MATERIALS AND METHODS: Thirty patients underwent repeat computed tomographic (CT) examinations within 6 months, the first with either 100, 150, or 180 mL of nonionic contrast material (2 mL/sec). In the second, only volume was altered. Liver attenuation was measured before and after contrast material administration, and enhancement was calculated for each image. Comparisons were made only within the same patients. RESULTS: Mean liver enhancement was greater with 150 mL compared with 100 mL beyond 55 seconds after injection. Mean peak enhancement was greater by 21 HU with 150 mL; mean time to peak enhancement was longer by 14 seconds. Mean liver enhancement was greater with 180 mL compared with 150 mL at all intervals beyond 80 seconds. Mean peak enhancement was greater by 22 HU; mean time to peak liver enhancement was longer by 24 seconds. CONCLUSION: Alterations in contrast material volume have substantial impact on liver enhancement and potentially tumor conspicuity. The optimal window of time for liver imaging after contrast material injection varies with the volume administered.

Adult↗

PubMed1998

Musculoskeletal tumors: does fast dynamic contrast-enhanced subtraction MR imaging contribute to the characterization?

PURPOSE: To prospectively analyze the value of fast, dynamic, subtraction magnetic resonance (MR) imaging in the characterization of musculoskeletal tumors. MATERIALS AND METHODS: In 175 consecutive patients with a musculoskeletal mass, dynamic contrast-enhanced subtraction MR imaging was performed after administration of 0.1 mmol per kilogram of body weight gadopentetate dimeglumine or gadoteridol. A turbo gradient-echo technique was used, with a temporal resolution of 1-3 seconds. The interval between arterial and early tumor enhancement, the pattern (peripheral or diffuse) of enhancement, and the progression of tumor enhancement, as visualized on time-signal intensity curves, were assessed. MR enhancement features were related to the histopathologic diagnoses. RESULTS: Differentiation of benign from malignant soft-tissue masses was possible with a sensitivity of 91% and specificity of 72% based on start of enhancement, a sensitivity of 73% and specificity of 97% based on peripheral or diffuse enhancement, and a sensitivity of 86% and specificity of 81% based on progression of enhancement. Benign bone tumors could not be accurately differentiated from malignant bone tumors on the basis of the three defined parameters (sensitivity, 63%-76%; specificity, 50%-76%). CONCLUSION: Dynamic, contract-enhanced, subtraction MR images may be useful to differentiate malignant from benign soft-tissue masses.

Adolescent↗

PubMed2000

Dynamic contrast-enhanced MR imaging of uterine cervical cancer: pharmacokinetic analysis with histopathologic correlation and its importance in predicting the outcome of radiation therapy.

PURPOSE: To investigate the histopathologic bases of different enhancement patterns on dynamic contrast material-enhanced magnetic resonance (MR) images of cervical cancer and to assess their importance in predicting the outcome of patients after radiation therapy. MATERIALS AND METHODS: Dynamic enhanced MR imaging and pharmacokinetic analyses were performed in 26 patients with cervical cancer who subsequently underwent hysterectomy and in 36 patients with cervical cancer who received radiation therapy. Histopathologic findings and clinical outcomes were correlated with results of dynamic MR imaging and pharmacokinetic analysis. RESULTS: On dynamic MR images of the surgical patients, areas with intense homogeneous enhancement showed increased permeability (k = 27.4 x 10(-3)) compared with areas with poor enhancement (k = 19.0 x 10(-3)). Well-enhanced areas were predominantly composed of cancer cell fascicles, whereas poorly enhanced areas were composed of fibrous tissue with scattered cancer cells. Radiation therapy was more effective in tumors with higher tissue permeability (k = 31.3 x 10(-3)) on dynamic MR images than in those with lower tissue permeability (k = 18.3 x 10(-3)). CONCLUSION: Areas of increased contrast enhancement are mainly composed of abundant cancer cell fascicles, whereas poorly perfused areas are composed of fibrous tissue with scattered cancer cells. Radiation therapy is more effective in well-enhanced tumors, resulting in improved local control.

Adenocarcinoma↗

PubMed2001

Carotid artery stenosis: prospective comparison of CT, three-dimensional gadolinium-enhanced MR, and conventional angiography.

PURPOSE: To prospectively compare gadolinium-enhanced magnetic resonance (MR) angiography and computed tomographic (CT) angiography with digital subtraction angiography (DSA) for use in detecting atheromatous stenosis and plaque morphology at the carotid bifurcation. MATERIALS AND METHODS: Forty-four carotid arteries (in 22 patients) were analyzed by using CT angiography, enhanced MR angiography, and DSA. CT and enhanced MR angiograms were reconstructed with maximum intensity projection and multiplanar volume reconstruction. The following four features were analyzed: degree of stenosis on the basis of North American Symptomatic Carotid Endarterectomy Trial criteria, length of stenosis, luminal surface, and presence of ulcers. RESULTS: There was significant correlation between CT angiography, enhanced MR angiography, and DSA for degree and length of stenosis. With enhanced MR angiography and CT angiography, degree of stenosis was underestimated in two of 44 cases. No case of overestimation with CT angiography was found. Severe internal carotid artery stenoses were detected with high sensitivity and specificity: 100% and 100%, respectively, with CT angiography; 93% and 100%, respectively, with enhanced MR angiography. Luminal surface irregularities were most frequently seen at CT angiography. With CT angiography and enhanced MR angiography, more ulceration was detected than with DSA. CONCLUSION: There was a significant correlation between CT angiography, enhanced MR angiography, and DSA in evaluation of carotid artery stenosis. Enhanced MR angiography or CT angiography can be used to adequately evaluate carotid stenosis.

Aged↗

PubMed2001

Imaging time after Gd-DTPA injection is critical in using delayed enhancement to determine infarct size accurately with magnetic resonance imaging.

BACKGROUND: In patients with acute myocardial infarction (MI), delayed enhancement is seen in MRI 5 to 7 minutes after gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) injection, and the enhancement occurs in regions that later show recovery of function. However, in a canine model of acute MI, delayed enhancement 20 to 30 minutes after injection only occurs in necrotic regions and not in surrounding, reversibly injured myocardium. The objective of the present study was to determine (1) if the size of the enhanced region varies with time after Gd-DTPA injection and (2) if and when the size of the enhanced region corresponds to the true infarct size. METHODS AND RESULTS: The left coronary artery was occluded in 15 Lewis rats for 30 minutes (n=9) or 2 hours (n=6); this was followed by reperfusion. MRI scans were performed 48+/-2 hours after-MI. Midventricular short-axis images were obtained continuously for 40 minutes after Gd-DTPA injection (0.3 mmol/kg). The sizes of enhanced regions at each time were determined by threshold analysis and compared with triphenyltetrazolium chloride-stained sections of the excised rat heart. In all animals, the enhanced region overestimated infarct size (28+/-5%) immediately after the injection of Gd-DTPA, although it then gradually receded to match the size of the infarct. The time required for enhancement to accurately determine infarct size was significantly different between 2-hour infarcts (16+/-2 minutes) and 30-minute (26+/-4 minutes) infarcts (P<0.05). CONCLUSIONS: In reperfused acute MI, accurate determination of infarct size by delayed enhancement MRI requires imaging at specific times after Gd-DTPA injection, and this time varies with the duration of occlusion.

Animals↗

PubMed2006

Functional characterization of polymorphisms in the kidney enhancer of the human renin gene.

The renin gene is regulated by an enhancer located 2.6 kb upstream of the transcription start site in the mouse and 11 kb upstream in humans. Despite extensive sequence conservation, the mouse renin enhancer is transcriptionally more active than the human renin enhancer. We report that the mechanism accounting for this is a result of sequence variation in the promoter proximal half-site of a retinoic-acid response element present in the enhancer. This sequence difference also prompted us to search for naturally occurring polymorphisms in the renin enhancer among normal and hypertensive human subjects. We sequenced the kidney enhancer from 90 samples derived from the Coriell Polymorphism Discovery Resource and 95 severely hypertensive Caucasian and African-American individuals. A single relatively frequent polymorphism (7, 2, and 7%, respectively in the Coriell, African-American, and Caucasian) was identified in the enhancer, one nucleotide downstream of the promoter distal half-site of the retinoic-acid response element. This variant was transcriptionally silent in transfection assays performed in renin-expressing As4.1 cells, a model of renal juxtaglomerular cells. A singleton polymorphism in the promoter was also identified in a single African-American individual. This polymorphism was located between binding sites for CBF1 and homeobox D10 but was also transcriptionally silent either in the presence or absence of the enhancer. Our study demonstrates the presence of silent polymorphisms in the renin promoter and enhancer, thus underscoring the critical importance of performing functional analyses before initiating expensive clinical studies seeking association between polymorphisms and complex diseases such as hypertension.

Animals↗

PubMed1996

Functional identification of genes up- and down-regulated by glucocorticoids in AtT-20 pituitary cells using an enhancer trap.

The AtT-20/D1 mouse pituitary tumor cell line has been used to study glucocorticoid regulation of POMC. We have used an enhancer trap to determine whether other glucocorticoid-regulated genes exist in AtT-20 cells. An enhancer trap is a recombinant construction containing a selectable marker driven by a promoter that has been weakened by removal of its enhancers so that the transfected trap is only expressed if it comes under the influence of an endogenous enhancer. For a selectable marker, we used a fusion gene coding for hygromycin phosphotransferase (Hy) and herpes simplex thymidine kinase. Thus, expression of this gene conferred hygromycin resistance and ganciclovir sensitivity. Suppression resulted in ganciclovir resistance and hygromycin sensitivity. An enhancerless promoter was produced using a truncated, transcriptionally inactive, form of the POMC promoter. AtT-20/D1 cells were transfected with this construct and cultured in medium containing hygromycin to kill any cells not expressing the Hy gene. The survivors were cultured in medium containing ganciclovir and dexamethasone and cloned. Clones in which the transgene was down-regulated by dexamethasone survived and were designated AtT-20/NET (for negative enhancer trap). Northern blot analysis confirmed that the transgene was down-regulated by dexamethasone as expected and that in at least one instance, suppression of the transgene was more complete than suppression of the full-length POMC promoter. Southern blot analysis after restriction enzyme digestion showed that each cell clone contained a single copy of the transgene, and PCR analysis of the promoter region showed that insertion had occurred in two unique sites in at least two cell clones. Another plasmid construct was prepared that contained the selectable gene but lacked any promoter elements. After transfection of AtT-20 cells with this vector, up-regulated enhancers were trapped by selection in hygromycin and dexamethasone followed by ganciclovir alone and designated AtT-20/PET cells (for positive enhancer trap). Up-regulation of the selectable gene in AtT-20/PET cells was confirmed by Northern blot analysis of dexamethasone-treated cells. In summary, glucocorticoid-regulated enhancers have been identified in AtT-20/D1 cells by an enhancer trap strategy that uses sequential selection under conditions that test whether the transgene is active. These results indicate that in addition to the well characterized, down-regulated POMC gene, there are other glucocorticoid-regulated genes in AtT-20/D1 cells that are both up-regulated and down-regulated by glucocorticoids.

Animals↗

PubMed1993

Delayed computed tomography contrast enhancement patterns in biopsy proven cases.

After a stereotactic brain biopsy of intracranial lesions, many neurosurgeons routinely obtain a noncontrast computed tomographic scan to rule out hemorrhage. We have observed there is often a significant delayed contrast enhancement on this postoperative scan, and the pattern of this enhancement frequently differs from the preoperative pattern. To assess these changes and to determine if they are related to lesion pathology or other clinical factors, the pre- and postoperative scans of 89 patients who underwent stereotactic biopsies were reviewed. Despite an average time delay of 153 minutes between scans, 81% of the lesions enhanced similarly or better on the postbiopsy image. One lesion enhanced only on the postoperative image, and in two cases, new lesions were seen postoperatively. Ring and other enhancing lesions tended to fill in with time, becoming homogeneous. The area of contrast enhancement increased in 83%. The borders of the lesions tended to become less sharp with time. No patterns of delayed enhancement, which give histologically definitive diagnostic information, were found. The tendency toward an increased area of contrast enhancement with time suggests that computed tomographic scans taken at the time of contrast injection may not show the true extent of the lesion. For patients undergoing computed tomography-guided stereotactic biopsies or radiosurgery of poorly enhancing lesions, the localization scan might be delayed after the administration of contrast medium to improve resolution and target selection.

Adult↗