Correction: The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation.

Since the publication of the above article, the authors have noted that the input data in Fig. 6E is incorrect. The correct data are included in the below Fig. 6E. The mistake does not affect the conclusions of the paper as the levels of input proteins remain similar between samples. We apologise for any inconvenience caused by this error.

RING finger protein 31 promotes p53 degradation in breast cancer cells.

The atypical E3 ubiquitin ligase RNF31 is highly expressed in human breast cancer, the most frequent neoplastic lethality among women. Here, RNF31 depletion in breast cancer cells in combination with global gene expression profiling revealed p53 (TP53) signaling as a potential RNF31 target. Interestingly, RNF31 decreased p53 stability, whereas depletion of RNF31 in breast cancer cells caused cell cycle arrest and cisplatin-induced apoptosis in a p53-dependent manner. Furthermore, RNF31 associated with the p53/MDM2 complex and facilitated p53 polyubiquitination and degradation by stabilizing MDM2, suggesting a molecular mechanism by which RNF31 regulates cell death. Analysis of publically available clinical data sets displayed a negative correlation between RNF31 and p53 target genes, including IGFBP3 and BTG1, consistent with RNF31 regulating p53 function in vivo as well. Together, our findings suggest RNF31 as a potential therapeutic target to restore p53 function in breast cancer.

The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation.

Estrogen receptor α (ERα) is initially expressed in the majority of breast cancers and promotes estrogen-dependent cancer progression by regulating the transcription of genes linked to cell proliferation. ERα status is of clinical importance, as ERα-positive breast cancers can be successfully treated by adjuvant therapy with antiestrogens or aromatase inhibitors. Complications arise from the frequent development of drug resistance that might be caused by multiple alterations, including components of ERα signaling, during tumor progression and metastasis. Therefore, insights into the molecular mechanisms that control ERα expression and stability are of utmost importance to improve breast cancer diagnostics and therapeutics. Here we report that the atypical E3 ubiquitin ligase RNF31 stabilizes ERα and facilitates ERα-stimulated proliferation in breast cancer cell lines. We show that depletion of RNF31 decreases the number of cells in the S phase and reduces the levels of ERα and its downstream target genes, including cyclin D1 and c-myc. Analysis of data from clinical samples confirms correlation between RNF31 expression and the expression of ERα target genes. Immunoprecipitation indicates that RNF31 associates with ERα and increases its stability and mono-ubiquitination, dependent on the ubiquitin ligase activity of RNF31. Our data suggest that association of RNF31 and ERα occurs mainly in the cytosol, consistent with the lack of RNF31 recruitment to ERα-occupied promoters. In conclusion, our study establishes a non-genomic mechanism by which RNF31 via stabilizing ERα levels controls the transcription of estrogen-dependent genes linked to breast cancer cell proliferation.

The kinase-inhibitory domain of p21-activated kinase 1 (PAK1) inhibits cell cycle progression independent of PAK1 kinase activity.

p21-activated kinase 1 (PAK1) is a mediator of downstream signaling from the small GTPases Rac and Cdc42. In its inactive state, PAK1 forms a homodimer where two kinases inhibit each other in trans. The kinase inhibitory domain (KID) of one molecule of PAK1 binds to the kinase domain of its counterpart and keeps it inactive. Therefore, the isolated KID of PAK1 has been widely used to specifically inhibit and study PAK function. Here, we show that the isolated KID induced a cell cycle arrest with accumulation of cells in the G1 phase of the cell cycle with an inhibition of cyclin D1 and D2 expression. This cell cycle arrest required the intact KID and was also induced by a mutated KID unable to block PAK1 kinase activity. Furthermore, the KID-induced cell cycle arrest could not be rescued by the expression of a constitutively active PAK1-T423E mutant, concluding that this arrest occurs independently of PAK1 kinase activity. Our results suggest that PAK1 through its KID inhibits cyclin D expression and thereby enforces a cell cycle arrest. Our results also call for serious precaution in the use of KID to study PAK function.

RANTES promotes growth and survival of human first-trimester forebrain astrocytes.

We have examined the role of alpha and beta chemokines in the promotion of the ontogenetic development of the brain. RANTES was expressed preferentially in human fetal astrocytes in an age-dependent manner. Astrocytes from 5-week-old brains showed high proliferation and reduced survival, whereas 10-week-old astrocytes exhibited opposite effects. These effects were suppressed by anti-RANTES or anti-RANTES receptor antibodies and were enhanced by recombinant RANTES. RANTES induced tyrosine phosphorylation of several cellular proteins and nuclear translocation of STAT-1 in astrocytes. Interferon-gamma (IFN-gamma) was required for RANTES effects because RANTES induced IFN-gamma and only 10-week-old astrocytes expressed the IFN-gamma receptor. Blocking of IFN-gamma with antibody reversed the effects of RANTES, indicating that cytokine/chemokine networks are critically involved in brain development.

Integrin alpha(v)beta3 promotes M21 melanoma growth in human skin by regulating tumor cell survival.

Growth and dissemination of malignant melanoma has a profound impact on our population, and little is known concerning the mechanisms controlling this disease in humans. Evidence is provided that integrin alpha(v)beta3 plays a critical role in M21 melanoma tumor survival within human skin by a mechanism independent of its known role in angiogenesis. Antagonists of alpha(v)beta3 blocked melanoma growth by inducing tumor apoptosis. Moreover, M21 melanoma cell interactions with denatured collagen, a known ligand for alpha(v)beta3, caused a 5-fold increase in the relative Bcl-2:Bax ratio, an event thought to promote cell survival. Importantly, denatured collagen colocalized with alpha(v)beta3-expressing melanoma cells in human tumor biopsies, suggesting that alpha(v)beta3 interaction with denatured collagen may play a critical role in melanoma tumor survival in vivo.

Heart rate dependency of cardiac performance in heart failure patients treated with metoprolol.

AIMS: To investigate whether a low heart rate is necessary to maintain improvement in myocardial function after long-term treatment with a beta-blocker in patients with heart failure. METHODS AND RESULTS: Forty-eight patients with congestive heart failure were investigated: 30 patients with dilated cardiomyopathy participating in a placebo-controlled trial (15 on placebo, 15 on metoprolol), and 18 patients treated by metoprolol in an open protocol. Investigations of spontaneous heart rate and of matched paced heart rates were performed at baseline and after 3, 6 and 12 months of follow-up by radionuclide angiography. There were significant signs of improvement in systolic indices of the spontaneous heart rate in the metoprolol-treated group (peak ejection rate: 0.98 to 1.32 end-diastolic volume.s-1, P = 0.015) as compared to placebo (1.14 to 1.19 end-diastolic volume.s-1, not significant). Similar effects were observed during the matched paced heart rate (peak ejection rate: metoprolol 0.91 to 1.38 end-diastolic volume.s-1, P = 0.037; placebo 1.22 to 1.12 end-diastolic volume.s-1, not significant). No effects were observed in the early peak filling rate. Left ventricular volumes decreased during metoprolol treatment, both for the spontaneous heart rate and during matched pacing. CONCLUSIONS: These data imply that beta-blocker treatment improves the force-frequency relationship of myocardial performance. A lower heart rate is not necessary to maintain cardiac function on a short-term basis, once myocardial recovery has occurred.

Integrin alphavbeta3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis.

Angiogenesis depends on growth factors and vascular cell adhesion events. Integrins and growth factors are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic fibroblast growth factor (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least 20 h. The initial wave of ERK activity (5-120 min) was refractory to integrin antagonists, whereas the sustained activity (4-20 h) depended on integrin alphavbeta3, but not beta1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained alphavbeta3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked growth factor induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a growth factor receptor and integrin alphavbeta3.

Cell adhesion and angiogenesis.

Cell-adhesion mechanisms play a fundamental role during angiogenesis. This article summarizes the role of various cell-adhesive events in blood vessel formation, including general aspects of cell-matrix and cell-cell interactions. In particular, the authors discuss the role of integrin alphavbeta3 in vascular cell survival, proliferation and invasion during the complex process of angiogenesis.

Integrins, angiogenesis and vascular cell survival.

The interactions between integrins and the extracellular matrix have been identified as important regulators of vascular cell survival, proliferation and invasion during the complex process of blood vessel formation by angiogenesis.

Requirement of receptor-bound urokinase-type plasminogen activator for integrin alphavbeta5-directed cell migration.

The urokinase plasminogen activator (uPA) interacts with its cell surface receptor (uPAR), providing an inducible, localized cell surface proteolytic activity, thereby promoting cellular invasion. Evidence is provided for a novel function of cell surface-associated uPA.uPAR. Specifically, induction of cell surface expression of uPA. uPAR by growth factors or phorbol ester was necessary for vitronectin-dependent carcinoma cell migration, an event mediated by integrin alphavbeta5. Cell migration on vitronectin was blocked with either a soluble form of uPAR, an antibody that disrupts uPA binding to uPAR, or a monoclonal antibody to alphavbeta5. Moreover, plasminogen activator inhibitor type 2 blocked this migration event but did not affect adhesion, suggesting a direct role for uPA enzyme activity in this process and that migration but not adhesion of these cells is regulated by uPA.uPAR. Growth factor-mediated induction of uPA.uPAR on the carcinoma cell surface promotes a specific motility event mediated by integrin alphavbeta5, since cells transfected with the beta3 integrin subunit expressed alphavbeta3 and migrated on vitronectin independently of growth factors or uPA.uPAR expression. This relationship between alphavbeta5 and the uPA.uPAR system has significant implications for regulation of motility events associated with development, angiogenesis, and tumor metastasis.

Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin alphaVbeta3 during angiogenesis.

Induction of p53 activity in cells undergoing DNA synthesis represents a molecular conflict that can lead to apoptosis. During angiogenesis, proliferative endothelial cells become apoptotic in response to antagonists of integrin alphavbeta3 and this leads to the regression of angiogenic blood vessels, thereby blocking the growth of various human tumors. Evidence is presented that administration of alphavbeta3 antagonists during angiogenesis in vivo selectively caused activation of endothelial cell p53 and increased expression of the p53-inducible cell cycle inhibitor p21WAF1/CIP1. In vitro studies revealed that the ligation state of human endothelial cell alphavbeta3 directly influenced p53 activity and the bax cell death pathway. Specifically, agonists of endothelial cell alphavbeta3, but not other integrins, suppressed p53 activity, blocked p21WAF1/CIP1 expression, and increased the bcl-2/bax ratio, thereby promoting cell survival. Thus, ligation of vascular cell integrin alphavbeta3 promotes a critical and specific adhesion-dependent cell survival signal during angiogenesis leading to inhibition of p53 activity, decreased expression of p21WAF1/CIP1, and suppression of the bax cell death pathway.

Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3.

SUMMARY: Cellular invasion depends on cooperation between adhesive and proteolytic mechanisms. Evidence is provided that the matrix metalloproteinase MMP-2 can be localized in a proteolytically active form on the surface of invasive cells, based on its ability to bind directly integrin alpha v beta 3. MMP-2 and alpha v beta 3 were specifically colocalized on angiogenic blood vessels and melanoma cells in vivo. Expression of alpha v beta 3 on cultured melanoma cells enabled their binding to MMP-2 in a proteolytically active form, facilitating cell-mediated collagen degradation. In vitro, these proteins formed an SDS-stable complex that depended on the noncatalytic C-terminus of MMP-2, since a truncation mutant lost the ability to bind alpha v beta 3. These findings define a single cell-surface receptor that regulates both matrix degradation and motility, thereby facilitating directed cellular invasion.

Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin.

Angiogenesis plays a fundamental role in human breast tumor progression. In fact, recent findings indicate that vascular density is a prognostic indicator of breast cancer disease status. Evidence is presented that the integrin alpha v beta 3 is not only a marker of human breast tumor-associated blood vessels, but that it plays a significant role in human angiogenesis and breast tumor growth. To assess the role of alpha v beta 3-dependent angiogenesis in the progression of human breast cancer, we examined a SCID mouse/human chimeric model with transplanted full thickness human skin containing alpha v beta 3-negative human breast tumor cells. This tumor induced a human angiogenic response as measured by vascular cell immunoreactivity with monoclonal antibodies LM609 and P2B1 directed to human alpha v beta 3 and CD31, respectively. Intravenous administration of LM609 either prevented tumor growth or markedly reduced tumor cell proliferation within the microenvironment of the human skin. These LM609-treated tumors not only contained significantly fewer human blood vessels but also appeared considerably less invasive than tumors in control animals. These findings demonstrate that alpha v beta 3 antagonists may provide an effective antiangiogenic approach for the treatment of human breast cancer.

Closed-line integral optimization edge detection algorithm and its application in equilibrium radionuclide angiocardiography.

UNLABELLED: Automatic evaluation of left ventricular (LV) function using equilibrium radionuclide angiocardiography requires an edge detection algorithm to correct and reproducibly delineate the left ventricle. Available algorithms, usually based on differentiation of a radial profile, generally suffer from low precision due to low signal-to-noise ratios and overlapping structures, for example, the left atrium. METHODS: An edge detection algorithm was developed based on the assumption that the LV border can be defined as the maximum, normalized, closed-line integral of a closed curve in a vector field derived by image differentiation. It is further assumed that the closed curve can be described by a Fourier expansion with a limited number of harmonics. Regions of interest (ROIs) generated by this algorithm were compared with ROIs generated by an algorithm based on a combination of thresholding and second-order derivatives. RESULTS: This algorithm delineates the left ventricle and gives results more closely related to ROIs generated manually than the algorithm combining thresholding and the second-order derivative. Our algorithm can also handle the problem of overlapping structures, as demonstrated in phantom simulations. CONCLUSION: The concept of a maximum, normalized closed-line integral will improve the delineation of the LV in an equilibrium radionuclide angiocardiography study. The problem of overlapping structures is overcome by this algorithm because it takes into consideration global edge information.

The RG2 rat glioma model.

The ethylnitrosourea-induced cell line RG2 grows very well in infinite cell culture in vitro, and provides a simple, reproducible glioma model when inoculated into the brains of syngeneic Fischer 344 rats. We have used this tumor model in a series of therapy studies. We here report our experiences of the untreated (= tumor bearing control) animals, e.g. in terms of the techniques employed and also the growth, histology and effects upon the blood-brain barrier of the tumors. Weight loss as a measure of systemic effects during tumor development is also described. The RG2 model has considerable potential as a suitable tool for experimental neuro-oncology.

Increased expression of and sensitivity to transforming growth factor-alpha: a promotive role during rat liver carcinogenesis.

The influence of the tumor promoter 2-acetylaminofluorene (2-AAF) on cell proliferation and on the epidermal growth factor receptor (EGFR) system was assessed in normal and nodular rat livers. DNA replication in vivo was inhibited below the detection level after 8d of dietary 2-AAF treatment of previously unexposed rats. The 2-AAF-induced growth inhibition was accompanied by downregulation of the number of epidermal growth factor (EGF)-binding sites and decreased levels of EGFR transcripts, whereas no changes in the transforming growth factor-alpha (TGF-alpha) mRNA levels were observed. The persistent liver nodules generated by intermittent 2-AAF-feeding had a 30- to 35-fold higher replicating cell fraction than normal liver. Treatment with 2-AAF in vivo reduced the replicating cell fraction to one third in nodules after 14 d of 2-AAF treatment. The initial EGFR mRNA levels and number of EGF binding sites in nodules before 2-AAF administration was about 605 that of control livers and was slightly reduced by 2-AAF feeding. The levels of EGFR mRNA after 14 d of 2-AAF feeding were thus similar in the nodules and in the 2-AAF-treated control livers, whereas the fraction of proliferating cells in nodules after the 2-AAF treatment was much larger than in normal liver. The TGF-alpha mRNA level in the nodules was found to be 1.4-fold and in malignant hepatomas 1.7-fold the level in normal liver. Primary hepatocytes isolated from control livers were four to five times more sensitive to replicative stimulation with EGF than with TGF-alpha, whereas nodular cells responded at lower concentrations than control cells and equally well to both EGF and TGF-alpha. We conclude that the decreased amounts of EGFR in the nodular cells with respect to proliferative stimulation could be more than compensated for by elevated synthesis of TGF-alpha combined with an increased TGF-alpha sensitivity. Collectively, these changes implicate TGF-alpha in sustaining cell proliferation during chemically induced rat liver carcinogenesis.

The coupling between transforming growth factor-alpha and the epidermal growth factor receptor during rat liver regeneration.

Transcriptional and post-transcriptional regulation of hepatic expression of the epidermal growth factor receptor (EGF-R) and its autocrine ligand, transforming growth factor-alpha (TGF-alpha), were analyzed during liver regeneration. The EGF-R mRNA levels were about twofold induced at 3 h after hepatectomy, caused by transcriptional activation. This was immediately followed by a decrease, reaching a low at half the initial level after 18 h, due to a decreased transcriptional rate. TGF-alpha mRNA expression was detected in normal liver using solution hybridization analysis. Concurrent with the decrease in EGF receptors, an increase of the TGF-alpha mRNA level occurred, starting at 6 h after hepatectomy and peaking at twice the initial TGF-alpha mRNA level after 12-24 h. For TGF-alpha, however, no increase in the rate of gene transcription could be detected. TGF-alpha and EGF competed for binding to the same hepatic receptor in normal as well as in regenerating liver. TGF-alpha bound to a similar number of binding sites as EGF in both control and 18-h posthepatectomy livers, but with 4-5 times lower affinity than EGF. At 18 h posthepatectomy, the number of binding sites was reduced to about 55% for both ligands. When the subcellular distribution of endocytosed 125I-labeled TGF-alpha was compared with that of 125I-labeled EGF, no differences were observed, and furthermore, no changes were observed in the subcellular distribution of 125I-labeled TGF-alpha during liver regeneration. The distinct and coordinate regulation of the two interactors, EGF-R and TGF-alpha, suggests that the EGF-receptor system may be functionally involved in the different phases of the prereplicative growth stimulatory process during liver regeneration.

Trisomy 7 and sex chromosome loss need not be representative of tumor parenchyma cells in malignant glioma.

We describe the cytogenetic findings in short-term cultures from 40 malignant gliomas, all of which had at least one clone with a simple numerical chromosome aberration. More than one aberrant clone was found in 17 tumors. The most frequent changes were loss of a gonosome (sole aberration in 38 clones), trisomy 7 (sole aberration in four clones), and combinations thereof (the aberrations +7 and -X or -Y were found together as the only changes in four clones). Clones with solitary trisomies for other autosomes--3, 5, 6, and 18--were seen in five tumors. Clones with structural rearrangements were found in nine tumors. The bands most commonly involved were lp36, 7p22, 9p22, 17p13, and 19q13. An extra copy of chromosome 7 was seen as part of a structurally abnormal clone in five tumors. In one case, trisomy 7 and even tetrasomy 7 were found in clones with simple numerical changes, but not in the clone with structural rearrangements. Likewise, the clonal loss of a gonosome was in six tumors, with structural abnormalities not present in the structurally aberrant clones; on the other hand, in two clones with structural aberrations a sex chromosome had been lost. The combined findings indicate that loss of a sex chromosome and trisomy 7 should not be seen as tumor-specific aberrations in gliomas. Instead, both glioma parenchyma cells and nonneoplastic cells in brain tumors may have a propensity to acquire extra copies of chromosome 7 and to lose gonosomes.

Thoracic epidural anesthesia improves global and regional left ventricular function during stress-induced myocardial ischemia in patients with coronary artery disease.

The aim of the present investigation was to study the effects of high thoracic epidural anesthesia (TEA), including the cardiac sympathetic segments, on ischemic ST-segment changes and left ventricular global and regional wall motion abnormalities. Ten patients with a two- or three-vessel coronary artery disease, all treated with the beta-adrenergic blocker metoprolol because of severe stable angina pectoris, performed two identical exercise stress tests, the first without TEA (control exercise) and the second with TEA (TEA exercise). Before each stress test, intravenous metoprolol was given to achieve maximal or near maximal beta-adrenoceptor blockade. Systolic and diastolic arterial pressures (radial artery cannula), heart rate, and rate-pressure product, as well as global and regional ejection fractions, using equilibrium radionuclide angiography in the left anterior oblique projection, were measured at rest and during maximal exercise. ST-segment analysis (V3 or V5) was performed, and the regional wall motion score was calculated at control exercise and TEA exercise. Intravenous metoprolol or intravenous metoprolol plus TEA at rest did not cause any significant changes of any of the variables. During TEA exercise, systolic arterial pressure, diastolic arterial pressure, and rate-pressure product, but not heart rate, were significantly lower compared to control exercise. The global and anterolateral ejection fractions were significantly higher (52.8% versus 46.5% and 53.2% versus 46.0%, respectively, P less than 0.05), and the regional wall motion score was significantly lower (8.8 versus 11.8, P less than 0.01) during TEA exercise than during control exercise. ST-segment depression was significantly lower during TEA exercise (-1.03 versus -1.84 mV, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)