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K Helin

Publications and source records attributed to K Helin.

At least 37 records · Page 2Linked to original sources
PubMed1998

E2F-6: a novel member of the E2F family is an inhibitor of E2F-dependent transcription.

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 five 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 identification of a novel member of this family, which we refer to as E2F-6. E2F-6 shares significant 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-I 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 different 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 effects of coexpression of E2F-1 and DP-1. The inhibitory effect 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 S-phase, which further emphasizes the functional difference between E2F-6 and the previously known E2F family members.

Amino Acid Sequence↗
PubMed1998

E2F-1-induced p53-independent apoptosis in transgenic mice.

The E2F transcription factors are key targets for the retinoblastoma protein, pRB. By inactivation of E2Fs, pRB prevents progression to the S phase. To test proliferative functions of E2F, we generated transgenic mice expressing human E2F-1 and/or human DP-1. When the hydroxymethyl glutaryl coenzyme A reductase promoter was used to express DP-1, overexpression occurred in a variety of tissues and did not confer phenotypic changes. In contrast, expression of E2F-1 from the same promoter was obtained only in testicles, in which E2F-1 overexpression caused atrophy and sterility through a process involving increased apoptosis in the germinal epithelium. This effect was potentiated by simultaneous overexpression of DP-1. Testicular atrophy as a result of overexpression of E2F-1 and DP-1 is independent of functional p53, since p53-nullizygous transgenic mice overexpressing E2F-1 and DP-1 also suffered testicular atrophy.

Animals↗
PubMed1998

Regulation of cell proliferation by the E2F transcription factors.

Experimental data generated in the past year have further emphasized the essential role for the E2F transcription factors in the regulation of cell proliferation. Genetic studies have shown that E2F activity is required for normal development in fruitflies, and the generation of E2F-1(-/-) mice has demonstrated that individual members of the E2F transcription factor family are likely to have distinct roles in mammalian development and homeostasis. Additional mechanisms regulating the activity of the E2F transcription factors have been reported, including subcellular localization and proteolysis of the E2Fs in the proteasomes. Novel target genes for the E2F transcription factors have been identified that link the E2Fs directly to the initiation of DNA replication.

Animals↗
PubMed1998

Cell cycle-regulated expression of mammalian CDC6 is dependent on E2F.

The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The CDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.

Animals↗
PubMed1998

The promyelocytic leukemia gene product (PML) forms stable complexes with the retinoblastoma protein.

PML is a nuclear protein with growth-suppressive properties originally identified in the context of the PML-retinoic acid receptor alpha (RAR alpha) fusion protein of acute promyelocytic leukemia. PML localizes within distinct nuclear structures, called nuclear bodies, which are disrupted by the expression of PML-RAR alpha. We report that PML colocalizes with the nonphosphorylated fraction of the retinoblastoma protein (pRB) within nuclear bodies and that pRB is delocalized by PML-RAR alpha expression. Both PML and PML-RAR alpha form complexes with the nonphosphorylated form of pRB in vivo, and they interact with the pocket region of pRB. The regions of PML and PML-RAR alpha involved in pRB binding differ; in fact, the B boxes and the C-terminal region of PML, the latter of which is not present in PML-RAR alpha, are essential for the formation of stable complexes with pRB. Functionally, PML abolishes activation of glucocorticoid receptor-regulated transcription by pRB, whereas PML-RAR alpha further increases it. Our results suggest that PML may be part of transcription-regulatory complexes and that the oncogenic potential of the PML-RAR alpha protein may derive from the alteration of PML-regulated transcription.

Cell Division↗
PubMed1997

Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures.

Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative disorders caused by an expansion of a polyglutamine tract. It is characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. To understand the pathogenesis of SCA1, we examined the subcellular localization of wild-type human ataxin-1 (the protein encoded by the SCA1 gene) and mutant ataxin-1 in the Purkinje cells of transgenic mice. We found that ataxin-1 localizes to the nuclei of cerebellar Purkinje cells. Normal ataxin-1 localizes to several nuclear structures approximately 0.5 microm across, whereas the expanded ataxin-1 localizes to a single approximately 2-microm structure, before the onset of ataxia. Mutant ataxin-1 localizes to a single nuclear structure in affected neurons of SCA1 patients. Similarly, COS-1 cells transfected with wild-type or mutant ataxin-1 show a similar pattern of nuclear localization; with expanded ataxin-1 occurring in larger structures that are fewer in number than those of normal ataxin-1. Colocalization studies show that mutant ataxin-1 causes a specific redistribution of the nuclear matrix-associated domain containing promyelocytic leukaemia protein. Nuclear matrix preparations demonstrate that ataxin-1 associates with the nuclear matrix in Purkinje and COS cells. We therefore propose that a critical aspect of SCA1 pathogenesis involves the disruption of a nuclear matrix-associated domain.

Animals↗
PubMed1997

Induction of DNA synthesis and apoptosis are separable functions of E2F-1.

The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E2F-1, suggesting that it may be mediated through alleviation of E2F-dependent transcriptional repression. These results indicate that E2F-1 can show independent cell cycle progression and apoptotic functions, consistent with its putative role as a tumor suppressor.

Apoptosis↗
PubMed1997

Loss of the retinoblastoma protein-related p130 protein in small cell lung carcinoma.

The retinoblastoma gene family consists of the tumor suppressor protein pRB and its two relatives p107 and p130. These proteins have been implicated in the regulation of cell cycle progression, in part, through inactivation of members of the E2F transcription factor family. Overexpression of pRB, p107, or p130 leads to growth arrest in the G1 phase of the cell cycle, and this arrest is abolished by complex formation with the adenovirus E1A, human papilloma virus E7, or simian virus 40 T oncoproteins. Inactivation of pRB by gross structural alterations or point mutations in the RB-1 gene has been described in a variety of human tumors, including retinoblastomas, osteosarcomas, and small cell lung carcinomas. Despite the structural and functional similarity between pRB, p107, and p130, alterations in the latter two proteins have not been identified in human tumors. We have screened a panel of 17 small cell lung carcinoma cell lines for the presence of functional p107 and p130 by evaluating their ability to form complexes with E1A in vitro. In the GLC2 small cell lung carcinoma cells no p130 protein was detected. The loss of the p130 protein is the result of a single point mutation within a splice acceptor sequence in the GLC2 genomic DNA. This mutation eliminates exon 2, leading to an in-frame stop codon, and no detectable protein is produced. These data are, to our knowledge, the first to describe the loss of p130 as a consequence of a genetic alteration, suggesting that not only pRB but also the other members of the family may contribute to tumorigenesis, providing a rationale for the observation that the DNA tumor viruses selectively target all the members of the retinoblastoma protein family.

Carcinoma, Small Cell↗
PubMed1997

Cyclin E-induced S phase without activation of the pRb/E2F pathway.

In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk6 and, possibly, cyclin E-dependent Cdk2 positively regulate the G1- to S-phase transition, by phosphorylating the retinoblastoma protein (pRb), thereby releasing E2F transcription factors that control S-phase genes. Here we performed microinjection and transfection experiments using rat R12 fibroblasts, their derivatives conditionally overexpressing cyclins D1 or E, and human U-2-OS cells, to explore the action of G1 cyclins and the relationship of E2F and cyclin E in S-phase induction. We demonstrate that ectopic expression of cyclin E, but not cyclin D1, can override G1 arrest imposed by either the p16INK4a Cdk inhibitor specific for Cdk4 and Cdk6 or a novel phosphorylation-deficient mutant pRb. Several complementary approaches to assess E2F activation, including quantitative reporter assays in live cells, showed that the cyclin E-induced S phase and completion of the cell division cycle can occur in the absence of E2F-mediated transactivation. Together with the ability of cyclin E to overcome a G1 block induced by expression of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs, these results provide evidence for a cyclin E-controlled S phase-promoting event in somatic cells downstream of or parallel to phosphorylation of pRb and independent of E2F activation. They furthermore indicate that a lack of E2F-mediated transactivation can be compensated by hyperactivation of this cyclin E-controlled event.

Animals↗
PubMed1997

p27KIP1 blocks cyclin E-dependent transactivation of cyclin A gene expression.

Cyclin E is necessary and rate limiting for the passage of mammalian cells through the G1 phase of the cell cycle. Control of cell cycle progression by cyclin E involves cdk2 kinase, which requires cyclin E for catalytic activity. Expression of cyclin E/cdk2 leads to an activation of cyclin A gene expression, as monitored by reporter gene constructs derived from the human cyclin A promoter. Promoter activation by cyclin E/cdk2 requires an E2F binding site in the cyclin A promoter. We show here that cyclin E/cdk2 kinase can directly bind to E2F/p107 complexes formed on the cyclin A promoter-derived E2F binding site, and this association is controlled by p27KIP1, most likely through direct protein-protein interaction. These observation suggest that cyclin E/cdk2 associates with E2F/p107 complexes in late G1 phase, once p27KIP1 has decreased below a critical threshold level. Since a kinase-negative mutant of cdk2 prevents promoter activation, it appears that transcriptional activation of the cyclin A gene requires an active cdk2 kinase tethered to its promoter region.

3T3 Cells↗
PubMed1997

Induction of S-phase entry by E2F transcription factors depends on their nuclear localization.

The E2F transcription factors are essential for regulating the correct timing of activation of several genes whose products are implicated in cell proliferation and DNA replication. The E2Fs are targets for negative regulation by the retinoblastoma protein family, which includes pRB, p107, and p130, and they are in a pathway that is frequently found altered in human cancers. There are five members of the E2F family, and they can be divided into two functional subgroups. Whereas, upon overexpression, E2F-1, -2, and -3 induce S phase in quiescent fibroblasts and override G1 arrests mediated by the p16INK4A tumor suppressor protein or neutralizing antibodies to cyclin D1, E2F-4 and -5 do not. Using E2F-1 and E2F-4 as representatives of the two subgroups, we showed here, by constructing a set of chimeric proteins, that the amino terminus of E2F-1 is sufficient to confer S-phase-inducing potential as well as the ability to efficiently transactivate an E2F-responsive promoter to E2F-4. We found that the E2F-1 amino terminus directs chimeric proteins to the nucleus. Surprisingly, a short nuclear localization signal derived from simian virus 40 large T antigen could perfectly substitute for the presence of the E2F-1 amino terminus in these assays. Thus, nuclearly localized E2F-4, when overexpressed, displayed biological activities similar to those of E2F-1. Furthermore, we showed that nuclear localization of endogenous E2F-4 is cell cycle regulated, with E2F-4 being nuclear in the G0 and early G1 phases and mainly cytoplasmic after the pRB family members have become phosphorylated. We propose a novel mechanism for the regulation of E2F-dependent transcription in which E2F-4 regulates transcription only from G0 until mid- to late G1 phase whereas E2F-1 is active in late G1 and S phases, until it is inactivated by cyclin A-dependent kinase in late S phase.

Animals↗
PubMed1997

The role of angiotensin receptors in cardiovascular diseases.

As an antihypertensive regimen, angiotensin I-converting enzyme (ACE) inhibition appears to have an antiproliferative cardiovascular effect that is not caused by blood pressure reduction alone. On the other hand, ACE inhibition has been shown to induce neocapillarization in hypertrophied myocardium. The possible mechanisms behind these beneficial cardiovascular effects of ACE inhibition are the suppression of angiotensin II formation and the potentiation of bradykinin. Angiotensin II receptor antagonism appears to have a similar antiproliferative effect on myocardium and vascular smooth muscle as ACE inhibition. This suggests that the antiproliferative action of both regimens is due only to the reduction of the pressor and growth effects of angiotensin II, or that both regimens have an additional, similarly effective antiproliferative action. Recently, knowledge about angiotensin II receptors has almost exponentially expanded. The two main classes of angiotensin II receptors, type 1 and 2 (AT1 and AT2), have been shown to belong to the same receptor family. However, their signal transduction and function seem to differ totally. The function and signal transduction of AT1 are to a large extent known. All the well-known physiological and pathophysiological effects of angiotensin II have been attributed to AT1. On the other hand, AT2 has quite recently been shown to mediate antiproliferation and differentiation at least in some tissues and cells, e.g. in vascular endothelial cells and some cells of neuronal origin. This review highlights the recent findings on angiotensin II receptors, and discusses the mechanisms behind the beneficial cardiovascular effects of interfering with the renin-angiotensin system.

Animals↗
PubMed1997

Endothelin-1 in heart and pulmonary tissue in experimental heart failure.

We studied the effect of congestive heart failure (CHF) on the tissue levels of endothelin 1 (ET-1) and of the mRNA of prepro-ET-1 in the rat. Congestive heart failure was produced by ligation of the left coronary artery. The rats were killed 3 weeks after the ligation. Congestive heart failure was determined by elevated plasma atrial natriuretic peptide and by a myocardial infarction scar covering more than 30% of the left ventricular circumference. Both ET-1 and prepro-ET-1 mRNA were measured in cardiac ventricles and lungs by radioimmunoassay and quantitative polymerase chain reaction. The rats with CHF had an increased heart weight/body weight ratio and elevated plasma atrial natriuretic peptide levels. In rats with CHF, as compared to controls, the concentration of ET-1 was elevated 3.6-fold in pulmonary tissue and 1.4-fold in the right ventricle, respectively, but not in the left ventricle. The concentration of mRNA showed a similar pattern, but due to methodological limitations this was assumed to represent a rough estimate only. The enhanced production of ET-1 in the right ventricle and in the pulmonary tissue may be of pathophysiological importance for the development of high pulmonary vascular resistance in CHF.

Animals↗
PubMed1996

Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression.

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 ED2Fs (E2F-1, E2F-2, and E2F-3) all induce S phase in quiescent rate 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 G1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G1 arrest mediated by the p16INK4 tumor suppressor protein, and E2F-1 was shown to override a G1 block mediated by a neutralizing antibody to cyclin D1. The p16INK4-induced G1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simulataneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G1 progession, is sufficient to induce S-phase entry, and overcomes a p16-mediated G1 block, and since E12F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p126-cyclin D-pRB pathway. Furthermore, our date 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.

Animals↗