Cascades of transcriptional induction during human lymphocyte activation.

Lymphocyte activation is known to be associated with the induction of genes implicated in cytokine signaling and cellular proliferation. High-density microarrays offer the means to monitor global cellular expression profiles, temporal relationships between classes of transcripts, and alterations associated with human disease or immunosuppression. We sought to determine whether microarray analysis would accurately reflect the normal pattern of gene expression following human T cell activation, and whether the complex expression patterns identified could be analyzed to produce a functional profile of lymphocyte activation. We examined a time course of sequential expression profiles for 6,800 cellular transcripts in human lymphocytes activated with concanavalin A. Expression patterns were grouped using clustering analysis and validated using Northern blotting. Genes known to be induced following T cell activation were accurately identified, and the qualitative patterns of gene expression were well correlated between Northern and microarray analyses. Quantitative differences in gene expression levels were less well correlated between these two techniques. Expression profile analysis revealed the sequential induction of groups of functionally similar genes, whose temporal coregulation underscores known cellular events during T cell activation. This functional "fingerprint" of lymphocyte activation may prove useful for comparisons of lymphocyte responses under experimental conditions and in disease states.

E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis.

The retinoblastoma protein (pRB) and its two relatives, p107 and p130, regulate development and cell proliferation in part by inhibiting the activity of E2F-regulated promoters. We have used high-density oligonucleotide arrays to identify genes in which expression changed in response to activation of E2F1, E2F2, and E2F3. We show that the E2Fs control the expression of several genes that are involved in cell proliferation. We also show that the E2Fs regulate a number of genes involved in apoptosis, differentiation, and development. These results provide possible genetic explanations to the variety of phenotypes observed as a consequence of a deregulated pRB/E2F pathway.

The Wilms tumor suppressor WT1 encodes a transcriptional activator of amphiregulin.

WT1 encodes a zinc finger transcription factor implicated in kidney differentiation and tumorigenesis. In reporter assays, WT1 represses transcription from GC- and TC-rich promoters, but its physiological targets remain uncertain. We used hybridization to high-density oligonucleotide arrays to search for native genes whose expression is altered following inducible expression of WT1. The major target of WT1 was amphiregulin, a member of the epidermal growth factor family. The WT1(-KTS) isoform binds directly to the amphiregulin promoter, resulting in potent transcriptional activation. The in vivo expression profile of amphiregulin during fetal kidney development mirrors the highly specific pattern of WT1 itself, and recombinant Amphiregulin stimulates epithelial branching in organ cultures of embryonic mouse kidney. These observations suggest a model for WT1 as a transcriptional regulator during kidney differentiation.

Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1.

The breast cancer susceptibility gene BRCA1 encodes a protein implicated in the cellular response to DNA damage, with postulated roles in homologous recombination as well as transcriptional regulation. To identify downstream target genes, we established cell lines with tightly regulated inducible expression of BRCA1. High-density oligonucleotide arrays were used to analyze gene expression profiles at various times following BRCA1 induction. A major BRCA1 target is the DNA damage-responsive gene GADD45. Induction of BRCA1 triggers apoptosis through activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), a signaling pathway potentially linked to GADD45 gene family members. The p53-independent induction of GADD45 by BRCA1 and its activation of JNK/SAPK suggest a pathway for BRCA1-induced apoptosis.

Chromatin, TAFs, and a novel multiprotein coactivator are required for synergistic activation by Sp1 and SREBP-1a in vitro.

The promoter selectivity factor Sp1 often cooperates with other enhancer-binding proteins to activate transcription. To study the molecular underpinnings of these regulatory events, we have reconstituted in vitro the synergy observed in vivo between Sp1 and the sterol-regulated factor SREBP-1a at the low density lipoprotein receptor (LDLR) promoter. Using a highly purified human transcription system, we found that chromatin, TAFs, and a novel SREBP-binding coactivator activity, which includes CBP, are all required to mediate full synergistic activation by Sp1 and SREBP-1a. The SREBP-binding domain of CBP inhibits activation by SREBP-1a and Sp1 in a dominant-negative fashion that is both chromatin- and activator-specific. Whereas recombinant CBP alone is not sufficient to mediate activation, a human cellular fraction containing CBP can support high levels of chromatin-dependent synergistic activation. Purification of this activity to near homogeneity resulted in the identification of a multiprotein coactivator, including CBP, that selectively binds to the SREBP-1a activation domain and is capable of mediating high levels of synergistic activation by SREBP/Sp1 on chromatin templates. The development of a reconstituted chromatin transcription system has allowed us to isolate a novel coactivator that is recruited by the SREBP-1a activation domain and that functions in concert with TFIID to coordinate the action of multiple activators at complex promoters in the context of chromatin.

SREBP transcriptional activity is mediated through an interaction with the CREB-binding protein.

The sterol regulatory element binding proteins (SREBP-1 and -2) activate transcription of genes whose products are involved in the cellular uptake and synthesis of cholesterol. Although considerable effort has been exerted to define the events regulating the levels of active SREBP, little is known about the transcriptional cofactors mediating SREBP function. In an unbiased search for potential coactivators of SREBP, we isolated a protein of 265 kD from HeLa cells that directly bound SREBP-1 and SREBP-2. Peptide sequencing and Western blot analysis established that the 265-kD protein was CBP (CREB-binding protein), a recently identified transcriptional coactivator. The putative activation domain of SREBP was shown to bind specifically to amino-terminal domains of recombinant CBP and p300 (a CBP-related protein). Moreover, transfection studies demonstrated that CBP enhances the ability of SREBP to activate transcription of reporter genes in HeLa cells. Together, these data suggest that CBP mediates SREBP transcriptional activity, thus revealing a new step in the biochemical pathway regulating cholesterol metabolism.

Topological control of p21WAF1/CIP1 expression in normal and neoplastic tissues.

The p53-regulated gene product p21WAF1/CIP1 is the prototype of a family of small proteins that negatively regulate the cell cycle. To learn more about p21WAF1/CIP1 regulation in vivo, monoclonal antibodies were developed for immunohistochemistry. These revealed that p21WAF1/CIP1 expression followed radiation-induced DNA damage in human skin in a pattern consistent with its regulation by p53. A detailed comparison of the human, rat, and mouse p21WAF1/CIP1 promoter sequences revealed that this induction was probably mediated by conserved p53-binding sites upstream of the transcription start site. In unirradiated tissues, p21WAF1/CIP1 expression was apparently independent of p53 and was observed in a variety of cell types. Moreover, there was a striking compartmentalization of p21WAF1/CIP1 expression throughout the gastrointestinal tract that correlated with proliferation rather than differentiation. As epithelial cells migrated up the crypts, the Ki67-expressing proliferating compartment near the crypt base ended abruptly, with the coincident appearance of a nonproliferating compartment expressing p21WAF1/CIP1. In colonic neoplasms, this distinct compartmentalization was largely abrogated. Cell cycle inhibitors are thus subject to precise topological control, and escape from this regulation may be a critical feature of neoplastic transformation.

Interactions between p53 and MDM2 in a mammalian cell cycle checkpoint pathway.

Normal p53 function is required for optimal arrest of cells in the G1 phase of the cell cycle following certain types of DNA damage. Loss of this cell cycle checkpoint may contribute to tumor development by increasing the number of genetic abnormalities in daughter cells following DNA damage. The MDM2 protein is an endogenous gene product that binds to the p53 protein and is able to block p53-mediated transactivation of cotransfected reporter constructs; thus, interactions between MDM2 and p53 in this checkpoint pathway following ionizing irradiation were examined. Though increases in p53 protein by DNA damage were not abrogated by MDM2 overexpression, increased levels of MDM2, resulting either from endogenous gene amplification or from transfection of an exogenous expression vector, were associated with a reduction in the ability of cells to arrest in G1 following irradiation. In addition, expression of endogenous MDM2 was enhanced by ionizing irradiation at the level of transcription in a p53-dependent fashion. These observations demonstrate that MDM2 overexpression can inhibit p53 function in a known physiologic pathway and are consistent with the hypothesis that MDM2 may function in a "feedback loop" mechanism with p53, possibly acting to limit the length or severity of the p53-mediated arrest following DNA damage.

The DCC gene: structural analysis and mutations in colorectal carcinomas.

DCC is a candidate tumor-suppressor gene encoding a protein with sequence similarity to cell adhesion molecules such as N-CAM. A set of overlapping YAC clones that contains the entire DCC coding region was isolated. Studies of this YAC contig showed that the DCC gene spans approximately 1.4 Mb. For elucidation of exon-intron structure, lambda phage clones containing all known coding sequences were isolated from a genomic library. These clones were used to demonstrate the existence of 29 DCC exons, and the sequences of the exon-intron boundaries were determined for each. Twenty-three polymorphic markers from chromosome 18 were then studied in a panel of primary colorectal tumors that had lost some, but not all, of chromosome 18. In most of these tumors, the region that was lost included DCC. Finally, Southern blot and PCR-based approaches were used to search for subtle mutations in several DCC exons. One tumor that had a point mutation in exon 28 was found, resulting in a proline to histidine substitution. A second tumor with a point mutation in intron 13 was also found. The regional map and genomic structure of DCC should provide the means to more extensively study DCC gene alterations and protein function in normal and neoplastic cells.

Discerning the function of p53 by examining its molecular interactions.

Of the many genes mutated on the road to tumor formation, few have received as much attention as p53. The gene has come to occupy center stage for the simple reason that it is more frequently altered in human tumors than any other known gene, undergoing mutation at a significant rate in almost every tumor type in which it has been studied. This association between p53 mutation and tumorigenesis has spurred a flurry of research attempting to delineate the normal function of p53 and, by extension, the role of p53 mutation in tumor formation. At the cellular level, p53 has been shown to suppress growth. Recent efforts to further discern the function of p53 have centered on the underlying molecular basis for this growth suppression. In particular, research has focused on the identification of cellular molecules (specifically DNA and proteins) with which the p53 protein associates. p53 has now been shown to bind DNA in a sequence-specific manner, and mounting evidence suggests that p53 acts as a transcription factor, perhaps regulating the expression levels of genes involved in the inhibition of cell growth. The logical next step in understanding p53 function involves the resolution of two questions: (1) what are the physiological transcriptional targets of p53, and (2) what cellular proteins regulate or mediate the ability of p53 to modulate transcription? Some initial clues to these puzzles are now emerging, and these form the subject of this review.

In vivo cloning of PCR products in E. coli.

This report describes an efficient method to clone PCR products exploiting endogenous Escherichia coli enzymatic activities. PCR products are engineered to contain terminal sequences identical to sequences at the two ends of a linearized vector. PCR products and vector DNA are then simply co-transfected into E. coli strain JC8679, obviating the requirement for enzymatic treatment of the PCR product or in vitro ligation. The high rate of homologous recombination in this strain results in efficient incorporation of the insert into the vector, a process we refer to as in vivo cloning (IVC).

p53 Mutation and MDM2 amplification in human soft tissue sarcomas.

The p53 and MDM2 genes were analyzed in 24 human soft tissue sarcomas (11 malignant fibrous histiocytomas and 13 liposarcomas). Alterations of p53, consisting of point mutations, deletions, or overexpression, were detected in one-third (8 of 24) of the sarcomas. MDM2 gene amplification was detected in another 8 tumors, but no tumor contained an alteration of both genes. Monoclonal antibodies reactive with the human MDM2 gene product were developed, and immunohistochemical analysis revealed nuclear localization and overexpression of MDM2 in those tumors with amplified MDM2 genes. These data support the hypothesis that p53 and MDM2 genetic alterations are alternative mechanisms for inactivating the same regulatory pathway for suppressing cell growth.

Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53.

The tumour-suppressor gene p53 is inactivated in most human malignancies either by missense mutations or by binding to oncogenic proteins. In human soft tissue sarcomas, inactivation apparently results from MDM2 gene amplification. MDM2 is an oncogene product that may function by binding to p53 and inhibiting its ability to activate transcription. Here we show that, when expressed in Saccharomyces cerevisiae, human MDM2 inhibits human p53's ability to stimulate transcription by binding to a region that nearly coincides with the p53 acidic activation domain. The isolated p53 activation domain fused to another DNA-binding protein is also inactivated by MDM2, confirming that MDM2 can inhibit p53 function by concealing the activation domain of p53 from the cellular transcription machinery.

Amplification of a gene encoding a p53-associated protein in human sarcomas.

Despite extensive data linking mutations in the p53 gene to human tumorigenesis, little is known about the cellular regulators and mediators of p53 function. MDM2 is a strong candidate for one such cellular protein; the MDM2 gene was originally identified by virtue of its amplification in a spontaneously transformed derivative of mouse BALB/c cells and the MDM2 protein subsequently shown to bind to p53 in rat cells transfected with p53 genes. To determine whether MDM2 plays a role in human cancer, we have cloned the human MDM2 gene. Here we show that recombinant-derived human MDM2 protein binds human p53 in vitro, and we use MDM2 clones to localize the human MDM2 gene to chromosome 12q13-14. Because this chromosomal position appears to be altered in many sarcomas, we looked for changes in human MDM2 in such cancers. The gene was amplified in over a third of 47 sarcomas, including common bone and soft tissue forms. These results are consistent with the hypothesis that MDM2 binds to p53, and that amplification of MDM2 in sarcomas leads to escape from p53-regulated growth control. This mechanism of tumorigenesis parallels that for virally-induced tumours, in which viral oncogene products bind to and functionally inactivate p53.

Relation between anterograde blood flow through a coronary artery and the size of the perfusion bed it supplies: experimental and clinical implications.

The relation between anterograde blood flow through a coronary artery and the size of the perfusion bed it supplies is not known. Accordingly, the left circumflex coronary artery was cannulated and perfused with arterial blood in 12 open chest mongrel dogs. In Group I dogs (n = 7), the goal was to correlate the size of the perfusion bed with the magnitude of anterogradely derived myocardial blood flow. The size of the perfusion bed was measured with use of two-dimensional myocardial contrast echocardiography, whereas anterograde myocardial blood flow was determined by injecting radiolabeled microspheres directly into the artery. In Group II dogs (n = 5), the goal was to study the effects of altering coronary blood flow on both anterogradely and collateral vessel-derived myocardial flow within the perfusion bed. In these dogs, microspheres were injected directly into both the coronary artery and the left atrium at each flow rate. In Group I dogs, the left circumflex perfusion bed size, as defined by myocardial contrast echocardiography, decreased at lower anterograde myocardial blood flow rates. The change in perfusion bed size occurred at the lateral zones. There was a linear relation between the normalized perfusion bed size and the normalized anterograde myocardial blood flow: y = 0.45x + 54.2 (p less than 0.001, r2 = 0.77). These results were substantiated in Group II dogs, in which the size of the perfusion bed was approximated with use of radiolabeled microspheres. The size of the perfusion bed was most affected when anterograde myocardial blood flow decreased to less than approximately 33% of normal. At the lowest flow rates, there was a linear relation between anterograde blood flow versus the fraction of the left circumflex flow derived anterogradely: y = 2.41x + 0.22 (p less than 0.001, r2 = 0.90). The lower the level of anterograde flow, the greater was the blood flow derived from remote vessels. It is concluded that the size of the area perfused by a coronary artery is significantly influenced by the magnitude of anterograde blood flow through that artery. These findings may have important implications in experimental and clinical models of myocardial ischemia.

Scrambled exons.

Using a sensitive assay for RNA expression, we identified several abnormally spliced transcripts in which exons from a candidate tumor suppressor gene (DCC) were scrambled during the splicing process in vivo. Cloning and sequencing of PCR-amplified segments of the abnormally spliced transcripts showed that exons were joined accurately at consensus splice sites, but in an order different from that present in the primary transcript. Four scrambled transcripts were identified, each involving a different pair of exons. The scrambled transcripts were found at relatively low levels in a variety of normal and neoplastic cells of rodent and human origin, primarily in the nonpolyadenylated component of cytoplasmic RNA. These results demonstrate that the splicing process does not always pair sequential exons in the order predicted from their positions in genomic DNA, thus creating a novel type of RNA product.

Assessment of regional myocardial blood flow with myocardial contrast two-dimensional echocardiography.

It was hypothesized that regional myocardial blood flow could be measured using myocardial contrast echocardiography. Accordingly, arterial blood was perfused into the coronary circulation in 16 dogs. In Group 1 dogs (n = 8), blood flow to the cannulated left circumflex artery was controlled with use of a roller pump, whereas in Group 2 dogs (n = 8) blood flow to the left anterior descending coronary artery was controlled by a hydraulic occluder placed around it. Sonicated microbubbles (mean size 4 microns) were used as the contrast agent. In Group 1 dogs the microbubbles were injected subselectively into the left circumflex artery, whereas in Group 2 dogs they were injected selectively into the left main coronary artery and two-dimensional echocardiographic images were recorded. Computer-generated time-intensity curves were derived from these images and variables of these curves correlated with transmural blood flow measured with radiolabeled microspheres. A gamma-variate function (y = Ate-alpha t) best described the curves, and alpha (a variable of curve width) correlated well with transmural blood flow at different flow rates in all Group 1 and Group 2 dogs (mean r = 0.81 and 0.97, respectively). Other variables of the curve width also correlated well with myocardial blood flow, but peak intensity had a poor correlation with myocardial blood flow in both groups of dogs (r = 0.39 and r = 0.63, respectively). When data from all dogs were pooled, Group 1 dogs still showed good correlation between variables of curve width and myocardial blood flow (r = 0.81); Group 2 dogs did not (r = 0.45). The difference between the two sets of dogs was related to the site of contrast agent injection. It is concluded that measurement of the transit time of microbubbles through the myocardium with two-dimensional echocardiography accurately reflects regional myocardial blood flow. Although injection of contrast agent selectively into the left main coronary artery only allows measurement of relative flow, it may be feasible to measure absolute flow by injecting contrast agent subselectively into a coronary artery. Myocardial contrast echocardiography may, therefore, offer the unique opportunity of simultaneously assessing regional myocardial perfusion and function in vivo.

Relationship between left ventricular systolic function and plasma clearance of 169Yb-DTPA in normal and ischemic dogs.

UNLABELLED: Plasma clearance of 169Yb-DTPA, a heavy chelate metal excreted only by glomerular filtration, was measured serially in a canine model to determine whether the clearance of this substance correlates with left ventricular systolic function. Fourteen anesthetized open-chest dogs were studied. Six dogs (group I) were used to estimate the clearance of 169Yb-DTPA in the setting of normal left ventricular function. Eight dogs (group II) were used to estimate clearance of 169Yb-DTPA in the presence of ischemic left ventricular dysfunction. Cardiac output, mean arterial pressure, and plasma levels of 169Yb-DTPA were measured serially. In group II dogs, area at risk for necrosis was defined using myocardial contrast echocardiography. The change in clearance of 169Yb-DTPA in group II dogs correlated well with change in cardiac output and area at risk (r = 0.80 and -0.93, respectively) and was significantly different from group I dogs (p less than 0.01). However, changes in clearance of 169Yb-DTPA in the group II dogs did not correlate well with change in mean arterial pressure (r = 0.26). IN CONCLUSION: (1) clearance of 169Yb-DTPA is constant over time in the absence of ischemic left ventricular dysfunction and derangement of other hemodynamic parameters; (2) changes in clearance of 169Yb-DTPA correlate well with changes in systolic pump function and left ventricular area at risk in the setting of ischemic left ventricular dysfunction, and (3) clearance of 169Yb-DTPA is not related to mean arterial pressure.