We have cloned two distinct mouse heat shock transcription factor genes, mHSF1 and mHSF2. The mHSF1 and mHSF2 open reading frames are similar in size, containing 503 and 517 amino acids, respectively. Although mHSFI and mHSF2 are quite divergent overall (only 38% identity), they display extensive homology in the DNA-binding and oligomerization domains that are conserved in the heat shock factors of Saccharomyces cerevisiae, Kluyveromyces lactis, Drosophila, tomato, and human. The ability of these two mouse heat shock factors to bind to the heat shock element (HSE) is regulated by heat. mHSFI is expressed in an in vitro translation system in an inactive form that is activated to DNA binding by incubation at temperatures >41~ the same temperatures that activate heat shock factor DNA binding and the stress response in mouse cells in vivo. mHSF2, on the other hand, is expressed in a form that binds DNA constitutively but loses DNA binding by incubation at >41~ Both mHSFI and mHSF2 are encoded by single-copy genes, and neither is transcriptionally regulated by heat shock. However, there is a striking difference in the levels of mHSFI mRNA in different tissues of the mouse.

The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their transactivation domains. Seven members of the E2F transcription factor family have been isolated so far, and we were interested in investigating the possible contribution of the various E2Fs to cell cycle control. By presenting the results of the generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4 and microinjection of expression plasmids for all members of the E2F family, we demonstrate here that the pRB-associated E2Fs (E2F-1, E2F-2, and E2F-3) all induce S phase in quiescent rat fibroblasts when expressed alone. In contrast, the p107/p130-associated E2Fs require the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G 1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G 1 arrest mediated by the p16 INK4 tumor suppressor protein, and E2F-1 was shown to override a G 1 block mediated by a neutralizing antibody to cyclin D1. The p16 INK4-induced G 1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simultaneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G 1 progression, is sufficient to induce S-phase entry, and overcomes a p16-mediated G 1 block, and since E2F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p16-cyclin D-pRB pathway. Furthermore, our data suggest that the two subsets of E2Fs are regulated by distinct mechanisms and/or that they have distinct functions in cell cycle control. Since E2F-4 and E2F-5 cannot promote S-phase entry by themselves, our results may provide an explanation for the apparent lack of aberrations in p107 or p130 in human cancer.Entry into and progression through the mammalian cell cycle are highly regulated processes, which at the molecular level involve a number of positively and negatively acting proteins. Many data suggest that among the negative regulators, the prototypic tumor suppressor, pRB, is crucial for proper cell cycle control (for a review, see reference 60). The gene for pRB, RB-1, was the first mammalian tumor suppressor gene to be cloned, and as such it has attracted much attention. Mutations in the RB-1 gene have been found not only in retinoblastomas but also in a variety of human tumors such as osteosarcomas, small-cell lung carcinomas, prostate carcinomas, and cervical cancers (60). The identification of RB-1 mutations in a diversity of human tumors was the first indication that pRB may have a more global regulatory role in cell proliferation. This indication has since been substantiated by a large number of experiments, and in particular it has been demonstrated that overexpression or microinjection of wild...

The E2F family of transcription factors are essential for the regulation of genes required for appropriate progression through the cell cycle. Five members of the E2F family have been previously reported, namely E2F1-5. All ®ve are key elements in transcriptional regulation of essential genes, and they can be divided into two functional groups, those that induce S-phase progression when overexpressed in quiescent cells (E2Fs 1 ± 3), and those that do not (E2Fs 4 ± 5). Here, we describe the identi®cation of a novel member of this family, which we refer to as E2F-6. E2F-6 shares signi®cant homology with E2Fs 1 ± 5, especially within the DNA binding, heterodimerization and marked box domains. Unlike E2Fs 1 ± 5, E2F-6 lacks a transactivation and a pocket protein binding domain, hence, forms a unique third group within the E2F family. E2F-6 is a nuclear protein that can form heterodimers with the DP proteins (both DP-1 and DP-2) in vitro and in vivo. Our results show that the complex formed between E2F-6 and the DP proteins, possesses high DNA binding activity, displaying a preference for a TTTCCCGC E2F recognition site, which is slightly dierent to the E2F consensus site derived from the E2 promoter (TTTCGCGC). In contrast to the other members of the E2F family, ectopic expression of E2F-6 inhibits transcription from promoters possessing E2F recognition sites rather than activating transcription. In addition, overexpression of E2F-6 suppresses the transactivational eects of coexpression of E2F-1 and DP-1. The inhibitory eect of E2F-6 is dependent on its DNA binding activity and its ability to form heterodimers with the DPs. Interestingly, ectopic expression of E2F-6 leads to accumulation of cells in S-phase. Our data suggest that E2F-6 expression delays the exit from S-phase rather than inducing Sphase, which further emphasizes the functional dierence between E2F-6 and the previously known E2F family members.

The retinoblastoma (pRb)-related p130 pocket protein is a regulator of cell growth and differentiation, and a candidate tumour suppressor. Both pRb and p130 operate through interactions with cellular proteins, including the E2F transcription factors. While such interactions are controlled by phosphorylation of multiple sites of pRb, regulation of p130 remains poorly understood. We now identify 22 in vivo phosphorylation sites of p130, targeted by diverse kinases, and present evidence for three cyclin-dependent kinase 4(6) [Cdk4(6)] speci®c phosphorylations, which appear critical for controlling the growth-restraining activity of p130. When expressed in U2OS cells, the phosphorylation-de®cient mutant p130 DCdk4 , in which the Cdk4 speci®c sites were mutated to alanine residues, imposed a more sustained G 1 arrest than a constitutively active pRb DCdk , known to repress all cellular E2F activity. Experiments using p130 DCdk4 and another phosphorylation-de®cient mutant, p130 PM19A , with 19 phosphorylation sites mutated, revealed that the p130-imposed G 1 block re¯ects cooperative growth-suppressive effects of phosphorylation-regulated E2F binding and phosphorylation-independent sequestration of cyclin E(A)±Cdk2 through the N-terminal cyclin binding motif of p130.

Background. Lemierre syndrome is characterized by head/neck vein thrombosis and septic embolism usually complicating an acute oropharyngeal bacterial infection in adolescents and young adults. We described the course of Lemierre syndrome in the contemporary era. Methods. In our individual-level analysis of 712 patients (2000-2017), we included cases described as Lemierre syndrome if these criteria were met: (i) primary site of bacterial infection in the head/neck; (ii) objectively confirmed local thrombotic complications or septic embolism. The study outcomes were new or recurrent venous thromboembolism or peripheral septic lesions, major bleeding, all-cause death and clinical sequelae. Results. The median age was 21 (Q1-Q3: 17-33) years, and 295 (41%) were female. At diagnosis, acute thrombosis of head/neck veins was detected in 597 (84%) patients, septic embolism in 582 (82%) and both in 468 (80%). After diagnosis and during inhospital follow-up, new venous thromboembolism occurred in 34 (5.2%, 95% CI 3.8-7.2%) patients, new peripheral septic lesions became evident in 76 (11.7%; 9.4-14.3%). The rate of either was lower in patients who received anticoagulation (OR: 0.59; 0.36-0.94), higher in those with initial intracranial involvement (OR: 2.35; 1.45-3.80). Major bleeding occurred in 19 patients (2.9%; 1.9-4.5%), and 26