The minimal replicator of Epstein-Barr virus oriP.

oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports the replication and stable maintenance of plasmids in human cells. oriP contains two essential components, called the DS and the FR, both of which contain multiple binding sites for the EBV-encoded protein, EBNA-1. The DS appears to function as the replicator of oriP, while the FR acts in conjunction with EBNA-1 to prevent the loss of plasmids from proliferating cells. Because of EBNA-1's role in stabilizing plasmids through the FR, it has not been entirely clear to what extent EBNA-1 might be required for replication from oriP per se, and a recent study has questioned whether EBNA-1 has any direct role in replication. In the present study we found that plasmids carrying oriP required EBNA-1 to replicate efficiently even when assayed only 2 days after plasmids were introduced into the cell lines 143B and 293. Significantly, using 293 cells it was demonstrated that the plasmid-retention function of EBNA-1 and the FR did not contribute significantly to the accumulation of replicated plasmids, and the DS supported efficient EBNA-1-dependent replication in the absence of the FR. The DS contains two pairs of closely spaced EBNA-1 binding sites, and a previous study had shown that both sites within either pair are required for activity. However, it was unclear from previous work what additional sequences within the DS might be required. We found that each "half" of the DS, including a pair of closely spaced EBNA-1 binding sites, had significant replicator activity when the other half had been deleted. The only significant DNA sequences that the two halves of the DS share in common, other than EBNA-1 binding sites, is a 9-bp sequence that is present twice in the "left half" and once in the "right half." These nonamer repeats, while not essential for activity, contributed significantly to the activity of each half of the DS. Two thymines occur at unique positions within EBNA-1 binding sites 1 and 4 at the DS and become sensitive to oxidation by permanganate when EBNA-1 binds, but mutation of each to the consensus base, adenine, actually improved the activity of each half of the DS slightly. In conclusion, the DS of oriP is an EBNA-1-dependent replicator, and its minimal active core appears to be simply two properly spaced EBNA-1 binding sites.

Comparison of the EBNA1 proteins of Epstein-Barr virus and herpesvirus papio in sequence and function.

The EBNA1 protein of Epstein-Barr virus (EBV) supports replication and maintenance of the circularized viral chromosome in cells that are latently infected. We have isolated, sequenced, and functionally characterized the EBNA1 gene of herpesvirus papio (HVP), an EBV-like virus that infects baboons. The amino acid sequences of EBNA1 of HVP and EBV are 56% identical, if the difference in the length of the glycine and alanine containing repetitive region, which is much shorter for HVP EBNA1, is omitted for the calculation. The key structural features of the DNA-binding/dimerization domain (the carboxyl-terminal domain) appear to have been conserved, as have amino acids in the two regions thought to be most critical for DNA binding. Most of the salient features of the amino-terminal two-thirds of EBNA1 (the amino-terminal domain), including a dearth of sequences predictive of alpha-helical or beta-sheet structures, are shared by the two sequences, although numerous gaps in this region were needed for alignment of the sequences. The amino-terminal fifty amino acids of EBNA1 of both EBV and HVP weakly resemble the amino terminus of rat ribosomal protein S2. Plasmids carrying oriP of either virus replicated stably in mammalian cells and supported efficient outgrowth of colonies under selection when supported by EBNA1 from either virus, although with each oriP there was a noticeable preference for EBNA1 to be from the same virus. HVP EBNA1 was less effective than EBV EBNA1 at activating the enhancer function of EBV oriP and under certain conditions was less effective than EBV EBNA1 at supporting maintenance of plasmids carrying EBV oriP. Results obtained with hybrid EBNA1 molecules indicated that differences in the amino-terminal and carboxyl-terminal domains, respectively, are primarily responsible for the differences in transcriptional activation and plasmid maintenance, respectively. The results showed that changes within EBNA1 can differentially alter its transcriptional and replicational activities.

Constitutive binding of EBNA1 protein to the Epstein-Barr virus replication origin, oriP, with distortion of DNA structure during latent infection.

Replication of the circular, 170 kb genome of Epstein-Barr virus (EBV) during latent infection is performed by the cellular replication machinery under cell-cycle control. A single viral protein, EBNA1, directs the cellular replication apparatus to initiate replication within the genetically defined replication origin, oriP, at a cluster of four EBNA1 binding sites, referred to here as the physical origin of bidirectional replication, or OBR. A second cluster of EBNA1 binding sites within oriP, the 30 bp repeats, serves an essential role as a replication enhancer and also provides a distinct episome maintenance function that is unrelated to replication. We examined the functional elements of oriP for binding by EBNA1 and possibly other proteins in proliferating Raji cells by generating in vivo footprints using two reagents, dimethylsulfate (DMS) and KMnO4. We also employed deoxyribonuclease I (DNase I) with permeabilized cells. The in vivo and permeabilized cell footprints at the EBNA1 binding sites, particularly those obtained using DMS, gave strong evidence that all of these sites are bound by EBNA1 in asynchronously dividing cells. No consistent evidence was found to suggest binding by other proteins at any other sites within the functional regions of oriP. Thymines at symmetrical positions of the OBR within oriP were oxidized when cells were treated with permanganate, suggestive of bends or other distortions of DNA structure at these positions; binding of EBNA1 in vitro to total DNA from Raji cells induced reactivity to permanganate at identical positions. The simplest interpretation of the results, which were obtained using asynchronously dividing cells, is that EBNA1 binds to its sites at oriP and holds the OBR in a distorted conformation throughout most of the cell cycle, implying that replication is initiated by a cellular mechanism and is not limited by an availability of EBNA1 for binding to oriP.

Plasminogen activator content of gynecological tumors and their metastases.

The plasminogen activator content of surgically excised gynecological tumors was measured with an azocaseinolytic assay. Ovarian (10 primary, 18 metastases) and uterine (5 primary and 6 metastases) tumors showed similar mean activator activities (21 CTA units/g tissue) mainly of the urokinase type with similar wide variations in each group. About 44% (14 of 32) of the tissues placed in short term organ culture were shown to produce and secrete urokinase activity. Results of the plasminogen activator activity found in the patient tumors or produced by the tumors during culture in the absence or presence of some drugs indicate a wide range of individual variations in sensitivity to these agents.

Plasminogen activator content of human tumor and adjacent normal tissue measured with fibrin and non-fibrin assays.

Plasminogen activators are enzymes which convert the zymogen to plasmin, the physiological enzyme for dissolving fibrin. There are two different physiological activator enzymes, urokinase (UK) and tissue plasminogen activator (t-PA, also known as vascular activator). The most striking difference in the behavior of the two activators is the ability of fibrin to augment the activity of t-PA but not of UK. Since tumor and normal tissues have been shown to contain different ratios of UK:t-PA, it can be anticipated that their comparative activator activities measured with fibrinolytic assays would yield different results from those measured with non-fibrin tests. This study was designed to test the validity of earlier conclusions that: (a) the measured activity of t-PA is augmented in fibrinolytic assays when compared with a non-fibrin assay based on azocaseinolysis; and (b) this difference could explain the failure of some laboratories using fibrinolytic assays to detect a difference in activator activity between tumor and normal tissues or to find more activity in the normal tissue. Azocaseinolytic and fibrinolytic (fibrin plate) assays were used to measure activator activity in a series of 14 normal-tumor tissue pairs. Using azocasein tests, cancer tissues were found to contain significantly higher median activities than normal tissues [Wilcoxon test, P less than 0.05; 13.8 versus 3.7 Committee on Thrombolytic Agents (CTA) units/g tissue, respectively], whereas no significant difference was found with fibrin assays (43.5 versus 69.0 CTA units/g tissue, respectively). Of total activator activity, the median percentage of UK was significantly higher in tumor (95%) than in normal tissue (58%). In addition, using azocaseinolysis it was found that the median UK activity was significantly higher in tumor (12.1 CTA units/g) relative to normal (3.51 CTA units/g) tissues, whereas no difference was found for t-PA. To explain these results in tumor and normal tissues, a mathematical model was derived to describe the difference between azocasein and fibrin assays for both purified plasminogen activator enzymes and activator enzymes in tissue extracts. The model fits the data well, confirming in a quantitative manner the hypotheses of the study. In addition, the study revealed that the azocaseinolytic assay was able to measure the full potential activator activity of purified pro-urokinase enzyme. Pro-urokinase activity could not be measured with standard fibrinolytic assays. These results show the importance of selection and interpretation of plasminogen activator assays in studies dealing with malignant transformation.

Plasminogen activators in human malignant melanoma.

Metastatic malignant melanomas from 16 patients, extracted with Triton X-100, were analyzed for plasminogen activator activity by azocaseinolysis . In 6 cases tumor explants were set up also in short-term organ culture, and the rate of plasminogen activator secretion into the culture medium was determined. Both the extractable activator content [8.66 +/- 7.8 "Committee on Thrombolytic Agents" (CTA) U/g tissue] and the activator secretion rates (0.90 +/- 1.6 CTA U/g/hr) were low in comparison with values for other human tumors. In addition to the activity, the type of plasminogen activator also was determined by immunoinhibition with goat antihuman urokinase antibody in the azocaseinolytic assay, as well as by sodium dodecyl sulfate (SDS) gel electrophoresis followed by zymography on fibrin-agar, in the presence and absence of antibody. On the average, 77% of the activator activity was of the urokinase type in the extracts, and 90% in the culture fluids. Immunoperoxidase reaction for the detection of urokinase showed this enzyme to be localized mainly in the cell membrane of the melanoma cells; stromal elements showed no specific staining. These results are of interest in view of the findings made recently by investigators in several laboratories that in all but one of the melanoma cell cultures derived from metastatic human tumors, only the vascular type ("tissue activator") was cell associated or was secreted into the culture medium. The possible reasons for this discrepancy are discussed.

Plasminogen activator content and secretion in explants of neoplastic and benign human prostate tissues.

The plasminogen activators of surgically excised prostate cancers (43 specimens) and benign prostatic hyperplasias (33 specimens) were extracted with an acetate:arginine:detergent buffer, and the activities were quantitated with azocaseinolytic tests. Immunoinhibition with anti-urokinase antibody served to distinguish between activator types. The mean activator activities (total; urokinase type; and nonurokinase type) of the neoplastic group were about 2 times higher (p less than 0.05) than that of the benign prostatic hyperplasia tissues. Each group had more non-urokinase-type activator activity relative to urokinase type. Studies with autopsy tissues (13 specimens) revealed that different anatomical compartments of the prostate contain about the same mean activator activity, indicating that the site of origin of the disease did not influence the results. Immunoperoxidase staining for urokinase revealed its presence in the tumor cells and, to a lesser extent, in the epithelial elements of some benign ducts and glands but not in the connective tissue. The secretion and synthesis of activator activities were monitored in short-term (approximately 120 hr) organ cultures (serum-free media) of 21 neoplastic tissues. On the average, about 12 times more activity (approximately 80% as urokinase type) was recovered in the media and postculture tissue extracts than was present in preculture tissues. Similar results were observed with 10 benign prostatic hyperplasia specimens. Wide individual variations were present in both groups (1.5 to 322 times more activity than the initial values). Except for one case, urokinase-type activity was secreted continuously, while nonurokinase was secreted only initially in quantities similar to that present in preculture tissue extracts. After culture, tissue explants contained higher quantities of both activator activities than were present initially. Dexamethasone (10 or 100 microM) decreased secretion and/or synthesis of activators by about 70%. This human organ culture model appears to be a reproducible system for individual tissues and may prove to be a valuable tool for further studies.

Plasminogen activator secretion of human tumors in short-term organ culture, including a comparison of primary and metastatic colon tumors.

The secretion of plasminogen activator by explants of 92 human malignant tumors was studied in short-term organ culture. Where possible, adjacent normal tissue of the presumed origin of the tumors was also studied. The study included adenocarcinomas of the lung, colon, prostate, breast, and stomach and different types of sarcomas. In addition to the measurement of secretion rates, all tissues were quantitatively extracted to determine the amount of cell-bound enzyme. Both culture fluids and extracts were analyzed with respect to the type of plasminogen activator they contained by immunoinhibition with goat immunoglobulin G formed against purified human urinary urokinase sodium dodecyl sulfate:gel electrophoresis followed by zymography. The study yielded the following conclusions: (a) the measurement of plasminogen activator secretion rates gives a much sharper differentiation between malignant and normal tissues than does the amount of extractable enzyme; (b) the enzymes secreted in short-term organ culture are, in the great majority of the cases studied, of the urokinase type, even when a large fraction of the activator contained in the tissue is of the vascular type; (c) the secretion rates of metastatic tumors of the colon are much lower than those of the primary ones; (d) immunoperoxidase staining of tissue sections reveals that urokinase is localized predominantly in the tumor cells. The low secretion rates of metastatic tumors, probably a reflection of this property in the original cell that gave rise to the metastatic focus, could be of advantage to circulating cancer cells. Such cells would not dissolve the microthrombus thought to be essential for the arrest of cancer cells in the capillaries of target organs.

Improved medium for extraction of plasminogen activator from tissue.

Quantitation of plasminogen activators present in tissue may depend to a large extent on the extraction procedure used to solubilize the enzymes. Potassium thiocyanate solution is known to be an efficient solubilizer, but it can inhibit assay systems other than fibrin plates. An equally effective acetate-detergent extractant is reported here which can be used with the highly sensitive azocaseinolytic assay procedure. The results indicate that a three-fold increase in activator activity can be extracted from selected tissues relative to that previously reported for a phosphate-detergent extractant. The extraction medium contains 75 mM K acetate, 0.3 M NaCl, 0.1 M L-arginine, 10 mM EDTA, 0.25% Triton X-100, final pH 4.2.

Plasminogen activator content of neoplastic and benign human prostate tissues; fibrin augmentation of an activator activity.

The plasminogen activator content of the extracts of excise prostate cancers (25 specimens) was determined with an azocasein assay and found to be on the average 1.7 times higher than that of extracts of excised prostate benign hyperplasias (29 specimens). Both groups contained the same average percentage of human urokinase type activator (approximately 45%) as determined by the inhibition of activity when anti-human urokinase antibody was included in the assay system. The two types of activators were partially purified and found to have distinctly different properties. The most striking difference was the large augmentation of activity o the non-urokinase enzyme in fibrinolysis. The implications of an enhanced fibrinolysis relative to azocaseinolysis (or other) is discussed, particularly with respect to its importance in the quantitation and characterization of activators by different investigators. Highly purified urokinase-like activator was found to be similar to commercial urokinase preparation with respect to molecular weight, isoelectric point, inhibition by the antibody, and inhibition by placenta inhibitor.

Plasminogen activator content of human colon tumors and normal mucosae: separation of enzymes and partial purification.

Plasminogen activator content was determined quantitatively in extracts of 23 pairs of surgically removed colon tumors and adjacent normal mucosa specimens. The activator content averaged 4.4 times higher in the tumor samples than in the corresponding normal tissue. Polyps removed with the adenocarcinomas gave values intermediate between those for tumors and those for the normal mucosae. The enzyme content of the group of tumors that showed invasive propagation or metastatic spread was significantly higher (P < 0.05) than was the enzyme content of the group not manifesting these conditions. Activator activity of the tumor extracts was completely inhibited by rabbit antibody formed against human urokinse. The activity of the normal mucosae was variably inhibited, suggesting the presence of several kinds of activator in normal tissues. The activator from the normal tissues could be separated into a completely refractory fraction and a completely inhibitable fraction by means of an affinity column made of Sepharose-linked, rabbit antiurokinase antibody. The activator, eluted from this column with 1 M acetic acid in 0.5 M NaCl (pH 2.2), was highly purified and had an isoelectric point of 8.6, as does authentic urokinase. The details of the isoelectric profile of the two, however, differed. The data are discussed in relation to earlier studies on the fibrinolytic system in colon cancer.

Content and characterization of plasminogen activators in human lung tumors and normal lung tissue.

The plasminogen activator content of surgically removed lung cancers, as well as that of adjacent normal lung tissue has been determined quantitatively. Optimum conditions for the quantitative extraction have been worked out using a modification of the technique described by Nagy et al. (Int. J. Cancer, 19:614-620, 1977) which utilizes a buffered solution of the non-ionic detergent Triton X-100. Plasminogen activator was significantly elevated in the tumors (2.5- to 4.3-fold over the normal lung, depending on sample selection and other criteria), although individual variations between tumors were extremely large. No significant correlation was found between the histopathological character of the tumors and the activator content, or between invasiveness and activator content. Using rabbit antibody formed against purified urokinase as an inhibitor of activator action, it was found that lung tumors contain a urokinase-like enzyme as the predominant plasminogen activator, while the activator content of the adjacent normal lung tissue consists of some urokinase-like enzyme, but mostly of an enzyme which is not inhibited by the antibody. The urokinase-like activator has been purified approximately 20,000-fold from lung tumors by the combination of two affinity chromatographic procedures, and was compared with purified urokinase with a molecular weight of 55,000 on all criteria used, the lung tumor activator was identical to urokinase.

Tetracycline resistance in Escherichia coli isolates from hospital patients.

Hospital isolates of Escherichia coli resistant to tetracycline (TC) were studied to identify mechanisms which regulate TC resistance levels and ability to transfer TC resistance. Antibiotic resistance patterns, resistance levels to TC, and ability to transfer TC resistance were determined for the isolates. Similar data were obtained for the transferable plasmids after transfer to several new host strains of E. coli. Of the 110 isolates, 50% were able to transfer TC resistance by conjugation. There was a nearly linear relationship between the minimum inhibitory concentration (MIC) of TC for the hospital strains and the percentage of strains at a given MIC that could transfer TC resistance. The strains that were simultaneously resistant to tetracycline, streptomycin, and ampicillin had relatively high MICs of TC and high ability to transfer TC resistance. These results and surveys of TC-resistant E. coli by others suggest that TC resistance levels and transmissibility may be influenced by other resistance markers. The isolates which did not transfer TC resistance by conjugation were tested for the presence of TC resistance plasmids by mobilization or by transformation with deoxyribonucleic acid from the isolates. Evidence for plasmid-mediated TC resistance was found in 92 (84%) of the 110 hospital strains.