Loss of Cul1 results in early embryonic lethality and dysregulation of cyclin E.

The sequential timing of cell-cycle transitions is primarily governed by the availability and activity of key cell-cycle proteins. Recent studies in yeast have identified a class of ubiquitin ligases (E3 enzymes) called SCF complexes, which regulate the abundance of proteins that promote and inhibit cell-cycle progression at the G1-S phase transition. SCF complexes consist of three invariable components, Skp1, Cul-1 (Cdc53 in yeast) and Rbx1, and a variable F-box protein that recruits a specific cellular protein to the ubquitin pathway for degradation. To study the role of Cul-1 in mammalian development and cell-cycle regulation, we generated mice deficient for Cul1 and analysed null embryos and heterozygous cell lines. We show that Cul1 is required for early mouse development and that Cul1 mutants fail to regulate the abundance of the G1 cyclin, cyclin E (encoded by Ccne), during embryogenesis.

Multimodal imaging of pancreatic beta cells in vivo by targeting transmembrane protein 27 (TMEM27).

AIMS/HYPOTHESIS: Non-invasive diagnostic tools specific for pancreatic beta cells will have a profound impact on our understanding of the pathophysiology of metabolic diseases such as diabetes. The objective of this study was to use molecular imaging probes specifically targeting beta cells on human samples and animal models using state-of-the-art imaging modalities (fluorescence and PET) with preclinical and clinical perspective. METHODS: We generated a monoclonal antibody, 8/9-mAb, targeting transmembrane protein 27 (TMEM27; a surface N-glycoprotein that is highly expressed on beta cells), compared its expression in human and mouse pancreas, and demonstrated beta cell-specific binding in both. In vivo imaging was performed in mice with subcutaneous insulinomas overexpressing the human TMEM27 gene, or transgenic mice with beta cell-specific hTMEM27 expression under the control of rat insulin promoter (RIP-hTMEM27-tg), using fluorescence and radioactively labelled antibody, followed by tissue ex vivo analysis and fluorescence microscopy. RESULTS: Fluorescently labelled 8/9-mAb showed beta cell-specific staining on human and mouse pancreatic sections. Real-time PCR on islet cDNA indicated about tenfold higher expression of hTMEM27 in RIP-hTMEM27-tg mice than in humans. In vivo fluorescence and PET imaging in nude mice with insulinoma xenografts expressing hTMEM27 showed high 8/9-mAb uptake in tumours after 72 h. Antibody homing was also observed in beta cells of RIP-hTMEM27-tg mice by in vivo fluorescence imaging. Ex vivo analysis of intact pancreas and fluorescence microscopy in beta cells confirmed these findings. CONCLUSIONS/INTERPRETATION: hTMEM27 constitutes an attractive target for in vivo visualisation of pancreatic beta cells. Studies in mouse insulinoma models and mice expressing hTMEM27 demonstrate the feasibility of beta cell-targeted in vivo imaging, which is attractive for preclinical investigations and holds potential in clinical diagnostics.

VHL takes HIF's breath away.

Inactivation of the von Hippel-Lindau (VHL) tumour-suppressor protein (pVHL) is associated with the von Hippel-Lindau cancer syndrome and the majority of kidney cancers. New evidence suggests that pVHL has properties of an F-box protein that targets the alpha-subunits of hypoxia-inducible factor (HIF)-1 for oxygen-dependent ubiquitination.

Interaction between ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation.

The transcription factor E2F-1 is important in the control of cell proliferation. Its activity must be tightly regulated in a cell-cycle-dependent manner to enable programs of gene expression to be coupled closely with cell-cycle position. Here we show that, following its accumulation in the late G1 phase of the cell cycle, E2F-1 is rapidly degraded in S/G2 phase. This event is linked to a specific interaction of E2F-1 with the F-box-containing protein p45SKP2, which is the cell-cycle-regulated component of the ubiquitin-protein ligase SCFSKP2 that recognizes substrates for this ligase. Disruption of the interaction between E2F-1 and p45SKP2 results in a reduction in ubiquitination of E2F-1 and the stabilization and accumulation of transcriptionally active E2F-1 protein. These results indicate that an SCFSKP2-dependent ubiquitination pathway may be involved in the downregulation of E2F-1 activity in the S/G2 phase of the cell cycle, and suggest a link between SCFSKP2 and cell-cycle-dependent gene control.

p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells.

The F-box protein p45SKP2 is the substrate-targeting subunit of the ubiquitin-protein ligase SCFSKP2 and is frequently overexpressed in transformed cells. Here we report that expression of p45SKP2 in untransformed fibroblasts activates DNA synthesis in cells that would otherwise growth-arrest. Expression of p45SKP2 in quiescent fibroblasts promotes p27Kip1 degradation, allows the generation of cyclin-A-dependent kinase activity and induces S phase. Coexpression of a degradation-resistant p27Kip1 mutant suppresses p45SKP2-induced cyclin-A-kinase activation and S-phase entry. We propose that p45SKP2 is important in the progression from quiescence to S phase and that the ability of p45SKP2 to promote p27Kip1 degradation is a key aspect of its S-phase-inducing function. In transformed cells, p45SKP2 may contribute to deregulated initiation of DNA replication by interfering with p27Kip1 function.

Casein kinase II is a predominantly nuclear enzyme.

Casein kinase II (CK II) has been implicated in regulating multiple processes related to cell growth, proliferation, and differentiation. To better understand the function(s) and regulation of this ubiquitous kinase, it is important to know its subcellular distribution. However, this issue has been the subject of contradictory reports. In this study, we have used indirect immunofluorescence microscopy and cell fractionation to study the subcellular distribution of all three subunits of chicken CK II, alpha, alpha', and beta. We examined primary chick embryo fibroblasts, virally transformed chicken hepatoma cells, as well as HeLa cells transiently transfected with cDNAs encoding chicken CK II subunits. We found that each of the three CK II subunits was located predominantly in the cell nucleus, irrespective of the cell type analyzed or the procedure used for cell fixation. No major differences were detected in the subcellular distributions of individual CK II subunits, and no evidence was obtained for subunit redistributions during interphase of the cell cycle. During mitosis, the bulk of the enzyme was dispersed throughout the cell, though a fraction of all three subunits was associated with the mitotic spindle. Biochemical studies based on mechanical enucleation of chicken cells confirmed the predominantly nuclear location of all three CK II subunits. Finally, immunoblotting experiments were carried out to study the expression of CK II subunits. A survey of different adult chicken tissues revealed substantial tissue-specific differences in the levels of CK II protein, but no evidence was obtained for pronounced tissue specificity in the expression of individual CK II subunits. These results strongly suggest that CK II functions primarily in regulating nuclear activities, and that the two catalytic subunits, alpha and alpha', may carry out overlapping functions.

Binding to DNA and the retinoblastoma gene product promoted by complex formation of different E2F family members.

The E2F family of transcription factors functions in the control of the mammalian cell cycle. Here it is shown that two family members, E2F-1 and DP-1, form specific heterodimers in vivo, a process that enhances DNA binding, transactivation, and the binding of the retinoblastoma gene product. These results suggest that heterodimerization regulates E2F function and contributes to cell cycle control.

Sporadic human renal tumors display frequent allelic imbalances and novel mutations of the HRPT2 gene.

Inactivation of the HRPT2 gene encoding parafibromin was recently linked to the familial hyperparathyroidism-jaw tumor syndrome. Patients with this syndrome carry an increased risk of parathyroid and renal tumors. To determine the relevance of HRPT2 for sporadic renal tumors, clear cell, papillary and chromophobe renal cell carcinomas as well as oncocytomas and Wilms tumors were analysed for HRPT2 gene alterations. Loss of heterozygosity (LOH) of HRPT2 was found in seven of 56 (12.5%) clear cell, three of 14 (21%) papillary, six of 10 (60%) chromophobe renal cell carcinomas, three of eight (38%) oncocytomas and four of 10 (40%) Wilms tumors. In addition, two novel HRPT2 point mutations, causing K34Q and R292K changes in parafibromin, were detected in one clear cell carcinoma and one Wilms tumor, respectively. These tumors displayed LOH of the remaining wild-type allele, but interestingly no von Hippel-Lindau (VHL) mutation. Functional analysis revealed that the K34Q mutant species of parafibromin is, unlike wild-type protein, defective in suppressing cyclin D1 expression in vivo. Taken together, these results suggest that renal cancer-associated mutations in parafibromin occur in the absence of VHL mutation, which in turn may contribute to constitutively elevated cyclin D1 expression and abnormal cell proliferation.

Proteolysis and the G1-S transition: the SCF connection.

Temporal control of ubiquitin-proteasome mediated protein degradation is critical for normal G1 and S phase progression. Recent work has shown that central to the temporal control mechanism is a relationship between newly identified E3 ubiquitin protein ligases, designated SCFs (Skp1-cullin-F-box protein ligase complexes), which confer substrate specificity on ubiquitination reactions and the activities of protein kinases that phosphorylate substrates destined for destruction at specific sites, thereby converting them into preferred targets for ubiquitin modification catalyzed by SCFs. The constituents of SCFs are members of evolutionary conserved protein families. SCF-based ubiquitination pathways may play a key role in diverse biological processes, such as cell proliferation, differentiation and development.

Pocket protein-independent repression of urokinase-type plasminogen activator and plasminogen activator inhibitor 1 gene expression by E2F1.

Expression of genes of the plasminogen activator (PA) system declines at the G(0)/G(1)-S-phase boundary of the cell cycle. We found that overexpression of E2F1-3, which acts mainly in late G(1), inhibits promoter activity and endogenous expression of the urokinase-type PA (uPA) and PA inhibitor 1 (PAI-1) genes. This effect is dose dependent and conserved in evolution. Mutation analysis indicated that both the DNA-binding and transactivation domains of E2F1 are necessary for this regulation. Interestingly, an E2F1 mutant lacking the pRB-binding region strongly repressed the uPA and PAI-1 promoters. An E2F-mediated negative effect was also observed in pRB and p107/p130 knockout cell lines. This is the first report that E2F can act as a repressor independently of pocket proteins. Mutation of AP-1 elements in the uPA promoter abrogated E2F-mediated transcriptional inhibition, suggesting the involvement of AP-1 in this regulation. Results shown here identify E2F as an important component of transcriptional control of the PA system and thus provide new insights into mechanisms of cellular proliferation.

Induction of hepatocyte proliferation and liver hyperplasia by the targeted expression of cyclin E and skp2.

Cells in culture become competent to replicate in the absence of growth factor after progressing beyond the late G1 restriction point, suggesting that a set of genes expressed during G1 phase is sufficient to trigger completion of the cell cycle. However, this has not been demonstrated in an in vivo system. In this study, we examined whether transfection of genes associated with the G1/S transition could trigger hepatocyte replication. Co-transfection of cyclin E and skp2 synergistically promoted cell cycle progression in cultured primary hepatocytes in the absence of mitogen or in the presence of growth inhibitors. Furthermore, transfection of hepatocytes in vivo with cyclin E and skp2 promoted abundant hepatocyte replication and hyperplasia of the liver. These studies confirm that transfection with a small number of genes can trigger proliferation of quiescent hepatocytes in vivo, and suggest that therapies to enhance liver regeneration by targeting cell cycle control genes may be feasible.

Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells.

The ErbB2 receptor tyrosine kinase is overexpressed in a variety of human tumours. In order to understand the mechanism by which ErbB2 mediates tumour proliferation we have functionally inactivated the receptor using an intracellularly expressed, ER-targeted single-chain antibody (scFV-5R). Inducible expression of scFv-5R in the ErbB2-overexpressing SKBr3 breast tumour cell line leads to loss of plasma membrane localized ErbB2. Simultaneously, the activity of ErbB3, MAP kinase and PKB/Akt decreased dramatically, suggesting that active ErbB2/ErbB3 dimers are necessary for sustained activity of these kinases. Loss of functional ErbB2 caused the SKBr3 tumour cells to accumulate in the G1 phase of the cell cycle. This was a result of reduction in CDK2 activity, which was mediated by a re-distribution of p27Kip1 from sequestering complexes to cyclin E/CDK2 complexes. The level of c-Myc and D-cyclins, proteins involved in p27KiP1 sequestration, decreased in the absence of functional ErbB2. Ectopic expression of c-Myc led to an increase in D cyclin levels, CDK2 activity and resulted in a partial G1 rescue. We propose that c-Myc is a primary effector of ErbB2-mediated oncogenicity and functions to prevent normal p27Kip1 control of cyclinE/CDK2.

Structure and developmental expression of chicken nucleolin and NO38: coordinate expression of two abundant non-ribosomal nucleolar proteins.

We report the complete primary structures of two major chicken non-ribosomal nucleolar proteins known as nucleolin/C23 and NO38/B23, respectively. By comparison with homologous proteins from other species, this sequence information contributes to the identification of evolutionarily conserved motifs that may be relevant to the function and subcellular distribution of the two proteins. Using cDNA probes and monoclonal antibodies, we have also studied the expression of nucleolin and NO38 in the course of chicken embryogenesis. In all tissues examined, Northern analyses revealed single hybridization signals for nucleolin (at 3.0 kb) and NO38 (at 1.6 kb), and no evidence was obtained for multiple protein products. In total embryos between days 3 and 11 after egg laying, nucleolin and NO38 mRNA and protein levels decreased in parallel (2-5-fold), suggesting transcriptional down-regulation of expression. Coordinate expression of nucleolin and NO38 was observed also when examining individual tissues at various stages of development. Interestingly, however, there was no consistent correlation between relative mRNA and protein levels. In particular, several adult tissues contained exceedingly low levels of either nucleolin or NO38, despite the presence of large amounts of corresponding mRNAs. From these results we conclude, first, that the expression of nucleolin and NO38 is controlled coordinately, and, second, that regulation is likely to involve both transcriptional and posttranscriptional mechanisms.

Hypertriglyceridemia as a possible risk factor for prostate cancer.

We retrospectively studied anthropometric and laboratory parameters (including serum triglycerides, cholesterol), as well as comedication in 504 patients diagnosed with prostate cancer between January 1997 and August 2002 at a single referral center, and compared these patients with 565 age-matched patients with benign prostatic hyperplasia. A positive correlation was found between serum triglycerides and prostate cancer (odds ratio: 1.148/mmol/l; 95% confidence interval (CI) 1.003-1.315; P<0.05) after correcting for age, body mass index, diabetes and comedication with statins. Hypertriglyceridemia may increase the risk of prostate cancer, and the prognostic relevancy of serum triglycerides should be studied prospectively.

Metabolic stabilization of p27 in senescent fibroblasts correlates with reduced expression of the F-box protein Skp2.

When mortal human cells reach their finite lifespan, they enter an irreversible G1 growth arrest status referred to as senescence. Growth suppression of senescent cells can be explained by the accumulation of several growth-suppressive proteins, acting on mitogenic signal transduction and cell cycle regulation, respectively. We show here that the cdk inhibitor p27(KIP1), which is involved in several forms of G1 checkpoint control, accumulates in senescent cells. Whereas, the rate of p27 synthesis is reduced, accumulation of p27 is accompanied by an increase of the metabolic stability in senescent cells. p27 is a substrate for ubiquitin-mediated proteolysis, and its stabilization in senescent cells correlates with a deregulation of the p27-specific E3 ubiquitin ligase referred to as the SCF complex. Whereas, the Skp1 component of the SCF complex is overexpressed in senescent fibroblasts, the abundance of the F-box protein Skp2 is strongly reduced. In contrast to our findings with p27, the synthesis of the cell cycle regulators p21 and cyclin D1 is increased in senescent cells; however, both proteins are also highly unstable in these cells.

pVHL co-ordinately regulates CXCR4/CXCL12 and MMP2/MMP9 expression in human clear-cell renal cell carcinoma.

Loss of pVHL function, characteristic for clear-cell renal cell carcinoma (ccRCC), causes increased expression of CXCR4 chemokine receptor, which triggers expression of metastasis-associated MMP2/MMP9 in different human cancers. The impact of pVHL on MMP2/MMP9 expression and their relationship to CXCR4 and its ligand CXCL12 in ccRCC is unclear. By using reverse transcription PCR, immunofluorescence and immunohistochemistry, strong mRNA and protein expression of CXCR4, CXCL12, MMP2, MMP9 and MMP inhibitors TIMP1 and TIMP2 was found in VHL-null 786-O ccRCC cells. Loss of CXCR4/CXCL12 expression after restoration of VHL function in these cells was accompanied by a significant reduction of MMP2 and MMP9 expression, whereas neither TIMP1 nor TIMP2 expression was affected. Using real-time PCR analysis, higher MMP2 (p = 0.0134) and MMP9 (p = 0.067) mRNA expression levels were detected in primary ccRCC with strong CXCR4 compared to cases with weak CXCR4 expression. There was no association between CXCR4 and TIMP1 or TIMP2 mRNA expression. MMP2 protein expression data obtained by immunohistochemistry on a tissue microarray uncovered positive cytoplasmic staining in 290/380 (76%) primary ccRCCs. Co-expression of CXCR4 and MMP2 was found in 282 of these tumours (74%). Our in vitro and in vivo data strongly indicate that pVHL coordinately regulates expression of metastasis-associated genes CXCR4/CXCL12 and MMP2/MMP9 but the exact molecular mechanism of this regulation remains to be determined. Co-expression of CXCR4 and CXCL12, as demonstrated in VHL-null 786-O cells, might enable ccRCC progression and metastatic dissemination by autocrine receptor stimulation, even in the absence of exogenous CXCL12.

[mRNA expression analysis of metastatic markers in clear-cell renal cell carcinoma]. / mRNA expressionsanalyse von metastasierungsmarkern in klarzelligen nierenzellkarzinomen.

Deregulated expression of Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Metalloproteinases (TIMPs) is an important pre-requisite for metastatic processes in a variety of human tumor types including renal cell cancer. Own previous cDNA microarray studies demonstrated differential expression of several MMPs and TIMPs in normal renal tissue and renal cancer cell lines. In order to analyze MMP/TIMP expression in primary clear-cell renal cell carcinoma (ccRCC) tissues we have determined the mRNA abundance of MMP-2, MMP-9, TIMP-1 and TIMP-2 by RT-PCR in 29 ccRCC and 7 normal renal tissues. Compared to normal renal tissue, expression of MMP-2 and TIMP-2 was significantly reduced in 16 and 12 of 29 ccRCCs, respectively. In contrast, MMP-9 expression was significantly increased in 11 of 29 ccRCCs. No difference was seen for TIMP-1 transcription levels. Because expression of the metastasis-associated CXCR4 chemokine receptor is increased and associated with poor tumour-specific survival in ccRCC we also compared MMP/TIMP and CXCR4 expression in the given tissue samples. Expression of TIMP-1 and TIMP-2 did not correlate with CXCR4 expression levels, whereas mRNA expression of MMP-2 and MMP-9 was significantly higher in tumours with strong CXCR4 expression (p = 0.04 and p = 0.01, respectively). These preliminary results suggest the involvement of CXCR4, MMP-2, and MMP-9 in renal cancer progression.

Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates.

In vertebrates, entry into mitosis is accompanied by dephosphorylation of p34cdc2 kinase on threonine 14 (Thr14) and tyrosine 15 (Tyr15). To examine the role of these residues in controlling p34cdc2 kinase activation, and hence the onset of mitosis, we replaced Thr14 and/or Tyr15 by non-phosphorylatable residues and transfected wild-type and mutant chicken p34cdc2 cDNAs into HeLa cells. While expression of wild-type p34cdc2 did not interfere with normal cell cycle progression, p34cdc2 carrying mutations at both Thr14 and Tyr15 displayed increased histone H1 kinase activity and rapidly induced premature mitotic events, including chromosome condensation and lamina disassembly. No phenotype was observed in response to mutation of only Thr14, and although single-site mutation at Tyr15 did induce premature mitotic events, effects were partial and their onset was delayed. These results identify both Thr14 and Tyr15 as sites of negative regulation of vertebrate p34cdc2 kinase, and they suggest that dephosphorylation of p34cdc2 represents the rate-limiting step controlling entry of vertebrate cells into mitosis.