To prepare for the DNA synthesis (S) phase of the cell cycle, transcription of many genes required for nucleotide biosynthesis increases. The promoters of several of these genes contain binding sites for the E2F family of transcription factors, and, in many cases, mutation of these sites abolishes growth-regulated transcription. The RNA levels of one family member, E2F1, increase about 15-fold at the G1/S-phase boundary and expression of E2F1 in quiescent cells activates transcription from some G1/S-phase-specific promoters, suggesting that E2F1 plays a critical role in preparing cells to enter S phase. To elucidate the signal transduction pathway leading to the activation of genes required for DNA synthesis, we are investigating the mechanism by which expression of E2F1 is regulated. To determine whether levels of E2F1 mRNA are controlled by changes in promoter activity, we have cloned and characterized the mouse E2F1 promoter. Sequence analysis revealed two sets of overlapping E2F-binding sites located between -12 and -40 relative to the transcription initiation site. We show that these sites bind cellular E2F and that an E2F1 promoter fragment can be activated up to 100-fold by coexpression of E2F proteins. We also show that the activity of this E2F1 promoter fragment increases -80-fold at the Gl/S-phase boundary and that this activation is, in part, regulated by Go-specific repression via the E2F sites. However, the E2F sites are not sufficient to mediate growth-regulated transcriptional activity; our results indicate that multiple DNA elements are required for transcription regulation of the E2F1 promoter at the G1/S-phase boundary.
Terminal differentiation of many cell types involves permanent withdrawal from the cell division cycle. The p18INK4c protein, a member of the p16/INK4 cyclin-dependent kinase (CDK) inhibitor family, is induced more than 50-fold during myogenic differentiation of mouse C2C12 myoblasts to become the predominant CDK inhibitor complexed with CDK4 and CDK6 in terminally differentiated myotubes. We have found that the p18INK4c gene expresses two mRNA transcripts-a 2.4-kb transcript, p18(L), and a 1.2-kb transcript, p18(S). In proliferating C2C12 myoblasts, only the larger p18(L) transcript is expressed from an upstream promoter. Work over the past decade with several model systems has identified a number of transcription factors that play a critical role in initiating a cascade of events leading to activation of lineage-specific genes and, ultimately, to conversion of precursor cells into functionally specialized cells. Coupled with this process is withdrawal of proliferating undifferentiated cells from the mitotic cell cycle at a specific point in G 1 phase to become permanently arrested, terminally differentiated cells. Myogenesis is a complex, multistep process in which determined muscle precursor cells first enter the differentiation pathway and then undergo phenotypic differentiation which is characterized by the expression of muscle structural genes before fusing to form multinucleated myotubes (reviewed in references 23, 32, and 35). Irreversible withdrawal from the cell cycle occurs after myogenin induction, and establishment of the postmitotic state is required for the expression of musclespecific contractile proteins. The MyoD family of basic helixloop-helix transcription factors regulates the determination and differentiation of muscle precursor cells and, in conjunction with the MEF2 family of MADS box transcription factors, also activates muscle structural genes. In contrast to the progress in understanding the mechanisms that regulate the initiation of differentiation and the subsequent expression of lineage-specific genes, little is known about how cell cycle arrest is initiated and maintained during terminal differentiation.Primary control of the eukaryotic cell cycle is provided by the activity of a family of serine/threonine protein kinases, CDKs (cyclin-dependent kinases; see two recent reviews in references 15 and 30). The enzymatic activity of a CDK is regulated by several mechanisms, including positively by the binding of a cyclin and negatively by the binding of a CDK inhibitor. In mammalian cells, there exist at least two distinct families of CDK inhibitors, represented by the two prototype CDK inhibitors p21 and p16 (31, 34). p21 (also variously known as CIP1, WAF1, SDI1, and CDKN1), first identified in normal human fibroblasts as a component of quaternary cyclin D-CDK complexes that also contain proliferating cell nuclear antigen, is a potent inhibitor of multiple cyclin-CDK enzymes. The p21 family contains two other related CDK inhibitor genes, p27 Kip1 and p57Kip2 . Expression of the p21 ge...
Previous studies have indicated that the presence of an E2F site is not sufficient for G 1 /S phase transcriptional regulation. For example, the E2F sites in the E2F1 promoter are necessary, but not sufficient, to mediate differential promoter activity in G 0 and S phase. We have now utilized the E2F1 minimal promoter to test several hypotheses that could account for these observations. To test the hypothesis that G 1 /S phase regulation is achieved via E2F-mediated repression of a strong promoter, a variety of transactivation domains were brought to the E2F1 minimal promoter. Although many of these factors caused increased promoter activity, growth regulation was not observed, suggesting that a general repression model is incorrect. However, constructs having CCAAT or YY1 sites or certain GC boxes cloned upstream of the E2F1 minimal promoter displayed E2F site-dependent regulation. Further analysis of the promoter activity suggested that E2F requires cooperation with another factor to activate transcription in S phase. However, we found that the requirement for E2F to cooperate with additional factors to achieve growth regulation could be relieved by bringing the E2F1 activation domain to the promoter via a Gal4 DNA binding domain. Our results suggest a model that explains why some, but not all, promoters that contain E2F sites display growth regulation.E2F plays an important role in regulating gene expression at the G 1 /S phase transition in the mammalian cell cycle. Promoters in which E2F sites contribute to transcriptional regulation are found in genes involved in DNA synthesis (such as dihydrofolate reductase, DNA polymerase ␣, and thymidine kinase) as well as in genes that are involved in cell cycle control (such as B-myb, several cyclins, and cdc2). E2F-mediated regulation of many of these genes leads to differential expression in G 0 and S phase, causing low promoter activity in quiescent cells and increased activity at the G 1 /S phase boundary (1). However, certain promoters contain E2F sites that do not confer growth-regulated transcriptional activity (2, 3). Several models have been put forth to explain why certain E2F sites mediate growth regulation and others do not.One such model is based on the fact that E2F is a family of transcription factors of which seven members have been characterized to date. E2F1-5 can heterodimerize with either DP1 or DP2 to create functional E2F activity (4, 5). Some of the E2Fs are present only at certain stages of the cell cycle (such as E2F1), whereas others are constitutively present (such as E2F4). Also, E2F activity is regulated by the Rb family of proteins (4 -9). E2F1-3 preferentially bind Rb, whereas E2F4 and E2F5 mainly bind p107 and p130. Thus, it is possible that growth-regulated promoters have a different composition of E2F protein complexes bound to the promoter DNA than do non-growth-regulated promoters. This model, however, cannot account for results obtained from deletion analyses of growthregulated promoters. For example, we have shown that deletion o...
The aim of this study was to compare the efficacy and safety of laparoscopic primary closure of the common bile duct (CBD) combined with percutaneous transhepatic cholangiographic drainage (PTCD) and laparoscopic choledocholithotomy with T-tube placement for the treatment of CBD stones. Between January 1991 and July 2002, 50 patients with choledocholithiasis and a CBD diameter larger than or equal to 1 cm underwent laparoscopic CBD explorations. The study group consisted of 10 patients undergoing laparoscopic primary closure of the CBD combined with PTCD. The control group consisted of 40 patients undergoing laparoscopic choledocholithotomy with T-tube placement. Parameters were compared statistically. The study group showed higher female/male ratio (6/4 vs 8/32, P = 0.02), less stone numbers (1.90 ± 0.88 vs 3.40 ± 1.65, P = 0.0078), shorter operation time (138 ± 37 minutes vs 191 ± 75 minutes, P = 0.014), and shorter postoperative stays (7 ± 3 days vs 10 ± 3 days, P = 0.0013). It seems that laparoscopic primary closure of the CBD combined with PTCD can shorten the operation time and postoperative stays as compared with laparoscopic choledocholithotomy with T-tube placement for the treatment of CBD stones.
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