Aurelian Radu

PTEN Induces Cell Cycle Arrest by Decreasing the Level and Nuclear Localization of Cyclin D1

ABSTRACT PTEN is a tumor suppressor frequently inactivated in brain, prostate, and uterine cancers that acts as a phosphatase on phosphatidylinositol-3,4,5-trisphosphate, antagonizing the activity of the phosphatidylinositol 3′-OH kinase. PTEN manifests its tumor suppressor function in most tumor cells by inducing G1-phase cell cycle arrest. To study the mechanism of cell cycle arrest, we established a tetracycline-inducible expression system for PTEN in cell lines lacking this gene. Expression of wild-type PTEN but not of mutant forms unable to dephosphorylate phosphoinositides reduced the expression of cyclin D1. Cyclin D1 reduction was accompanied by a marked decrease in endogenous retinoblastoma (Rb) protein phosphorylation on cyclin D/CDK4-specific sites, showing an early negative effect of PTEN on Rb inactivation. PTEN expression also prevented cyclin D1 from localizing to the nucleus during the G1- to S-phase cell cycle transition. The PTEN-induced localization defect and the cell growth arrest could be rescued by the expression of a nucleus-persistent mutant form of cyclin D1, indicating that an important effect of PTEN is at the level of nuclear availability of cyclin D1. Constitutively active Akt/PKB kinase counteracted the effect of PTEN on cyclin D1 translocation. The data are consistent with an oncogenesis model in which a lack of PTEN fuels the cell cycle by increasing the nuclear availability of cyclin D1 through the Akt/PKB pathway.

The carboxy-terminal fragment of inhibitor-2 of protein phosphatase-2A induces Alzheimer disease pathology and cognitive impairment.

Development of rational therapeutic treatments of Alzheimer disease (AD) requires the elucidation of the etiopathogenic mechanisms of neurofibrillary degeneration and β‐amyloidosis, the two hallmarks of this disease. Here we show, employing an adeno‐associated virus serotype 1 (AAV1)‐induced expression of the C‐terminal fragment (I2CTF)of I2PP2A, also called SET, in rat brain, decrease in protein phosphatase 2A (PP2A) activity, abnormal hyperphosphorylation of tau, and neurodegeneration; littermates treated identically but with vector only, i.e., AAV1‐enhanced green fluorescent protein (GFP), served as a control. Furthermore, there was an increase in the level of activated glycogen synthase kinase‐3β and enhanced expression of intraneuronal Aβ in AAV1‐I2CTF animals. Morris water maze behavioral test revealed that infection with AAV1‐I2CTF induced spatial reference memory and memory consolidation deficits and a decrease in the brain level of pSer133‐CREB. These findings suggest a novel etiopathogenic mechanism of AD, which is initiated by the cleavage of I2PP2A, producing I2CTF, and describe a novel disease‐relevant nontransgenic animal model of AD.—Wang, X., Blanchard, J., Kohlbrenner, E., Clement, N., Linden, R. M., Radu, A., Grundke‐Iqbal, I., Iqbal, K. The carboxy‐terminal fragment of inhibitor‐2 of protein phosphatase‐2A induces Alzheimer disease pathology and cognitive impairment. FASEB J. 24, 4420–4432 (2010). www.fasebj.org

Paired-Type Homeodomain Transcription Factors Are Imported into the Nucleus by Karyopherin 13

ABSTRACT We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin β family member Karyopherin 13 (Kap13). Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen. Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation. Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay. Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain. Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted. The paired-type homeodomain transcription factor family includes more than 20 members. All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13. We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13.

Expression of follicle-stimulating hormone receptor by the vascular endothelium in tumor metastases.

BackgroundThe Follicle Stimulating Hormone receptor (FSHR) is expressed by the vascular endothelium in a wide range of human tumors. It was not determined however if FSHR is present in metastases which are responsible for the terminal illness.MethodsWe used immunohistochemistry based on a highly FSHR-specific monoclonal antibody to detect FSHR in cancer metastases from 6 major tumor types (lung, breast, prostate, colon, kidney, and leiomyosarcoma ) to 6 frequent locations (bone, liver, lymph node, brain, lung, and pleura) of 209 patients.ResultsIn 166 patients examined (79%), FSHR was expressed by blood vessels associated with metastatic tissue. FSHR-positive vessels were present in the interior of the tumors and some few millimeters outside, in the normally appearing tissue. In the interior of the metastases, the density of the FSHR-positive vessels was constant up to 7 mm, the maximum depth available in the analyzed sections. No significant differences were noticed between the density of FSHR-positive vessels inside vs. outside tumors for metastases from lung, breast, colon, and kidney cancers. In contrast, for prostate cancer metastases, the density of FSHR-positive vessels was about 3-fold higher at the exterior of the tumor compared to the interior. Among brain metastases, the density of FSHR-positive vessels was highest in lung and kidney cancer, and lowest in prostate and colon cancer. In metastases of breast cancer to the lung pleura, the percentage of blood vessels expressing FSHR was positively correlated with the progesterone receptor level, but not with either HER-2 or estrogen receptors. In normal tissues corresponding to the host organs for the analyzed metastases, obtained from patients not known to have cancer, FSHR staining was absent, with the exception of approx. 1% of the vessels in non tumoral temporal lobe epilepsy samples.ConclusionFSHR is expressed by the endothelium of blood vessels in the majority of metastatic tumors.

Age-associated reduction of nuclear protein import in human fibroblasts.

Age-dependent decreases in the protein concentrations of the nucleocytoplasmic transport factors karyopherin alpha2, CAS, and RanBP1 were found by comparing fibroblast cultures obtained from young, mature, and old human donors. Karyopherin beta1 levels do not change with age and present very little variation among donors. The decrease in the concentration of transport factors is accompanied by a reduction in the protein import rate in fibroblasts from old donors, as detected by a change in the intracellular localization of a test transport substrate that shuttles between the cytoplasm and the nucleus. Measurements of concentrations of the same import factors in organs and tissues of old mice revealed a decrease of CAS in kidney, lung, and spleen. The import reduction in old age is expected to lead to impaired activity of proteins whose functions depend on timely import into the nuclei.

Karyopherin β2B participates in mRNA export from the nucleus

Transport of macromolecules between the cell nucleus and cytoplasm occurs through the nuclear pores and is mediated by soluble carriers known as karyopherins (Kaps), transportins, importins, or exportins. We report that Kap β2B (transportin-2) forms complexes with the mRNA export factor TAP in the presence of RanGTP, as shown by coimmunoprecipitation from HeLa cells. The interaction strictly depends on the presence of RanGTP. In digitonin-permeabilized cells, Kap β2B mediates TAP-GFP export from the nuclei in the presence of RanGTP. A TAP mutant that does not coimmunoprecipitate with Kap β2B is also not exported by Kap β2B. In the permeabilized cells assay, TAP is also exported independently of Kap β2B by direct interaction with nucleoporins, in agreement with previous reports. The export rate is, however, significantly lower than the Kap β2B-mediated pathway. Both Kap β2B and TAP are present and enriched in the poly(A)+ RNA complexes isolated from HeLa cell nuclear lysates. Poly(A)+ RNA strongly accumulates in the nuclei of HeLa cells treated with Kap β2B short interfering RNA, indicating that Kap β2B is involved in the export of at least a large proportion of the mRNA species. The export of β-actin and GAPDH mRNA is also inhibited, whereas 28S RNA is not affected. The data support the conclusion that Kap β2B participates directly in the export of a large proportion of cellular mRNAs, and TAP connects Kap β2B to the mRNAs to be exported.

Endothelial follicle stimulating hormone receptor in primary kidney cancer correlates with subsequent response to sunitinib

Sunitinib is an anti‐angiogenic receptor tyrosine kinase inhibitor used to treat advanced metastatic renal cell carcinoma and other types of cancer. Sutent is effective in only approximately 70% of clear cell renal cell carcinoma (CCRCC) patients, has significant adverse side effects and no method is available to predict which patients will not respond. Our purpose was to explore the possibility of introducing an effective prediction method based on a marker of the tumour vasculature, the follicle stimulating hormone receptor (FSHR). Fifty patients diagnosed with advanced metastatic CCRCC have been subjected to surgery for removal of the primary tumour and were subsequently treated with sunitinib. After three months of therapy the patients were categorized as ‘responsive’, ‘stable’ or ‘non‐responsive’ based on the RECIST guidelines. The blood vessel density and the percentage of FSHR‐positive vessels were determined by immunofluorescence on sections from the primary tumours removed by surgery, prior to the sunitinib treatment. The percentage of FSHR‐stained vessels was on average fivefold higher for the patients who responded to the treatment in comparison with the stable group and almost eightfold higher than in the non‐responsive group. The percentage allowed the detection of responders with 87–100% sensitivity and specificity. No significant differences were detected in the total density of vessels among the three groups. The data suggest that FSHR expression levels in the blood vessels of CCRCC primary tumours can be used to predict, with high sensitivity and specificity, the patients who will respond to sunitinib therapy.

An approach to increased polyplex gene delivery by peptides selected from a phage display library

Phage display libraries were screened for peptides to be incorporated in nonviral gene delivery vehicles. Cells in culture were incubated with heptamer random peptide libraries displayed on M13 bacteriophages in three to five copies per phage. Surface-adherent phages were removed or inactivated and the cells were fractionated in a nuclear pellet and supernatant. Bacteriophages from each of the two fractions were amplified and reincubated with the cells. Three successive rounds of selection were performed. Eighteen sequenced clones revealed 14 different sequences. Two sequences were homologous to segments of the HIV gp120 protein. For three sequences, the corresponding synthetic peptides were generated and attached via avidin-biotin to polylysine-condensed plasmid DNA containing a reporter gene. The addition of the peptides led to 8-14 times increase in the expression of the reporter.

Oligonucleotide microarray data mining: search for age-dependent gene expression

Information on gene expression in colon tumors versus normal human colon was recently generated by an oligonucleotide microarray study. We used the associated database to search for genes that display age-dependent variations in expression. Statistically significant evidence was obtained that such genes are present in both the tumor and normal tissue databases. Besides the analysis of all genes included in the database, three subsets of genes were analyzed separately: genes controlled by p53, and genes coding for ribosomal proteins and for nuclear-encoded mitochondrial proteins. Among the genes controlled by p53 some show an age-dependent change in expression in tumor tissues, in the sense compatible with an activation of p53 at higher age. A decreased expression of some ribosomal genes at advanced age was detected both in tumor and normal tissues. No significant age-dependent expression could be detected for genes encoding mitochondrial proteins.

The cleavage of thyroid-stimulating hormone receptor is dependent on cell-cell contacts and regulates the hormonal stimulation of phospholipase c.

Thyroid‐stimulating hormone receptor (TSHR) consists of a hormone‐binding extracellular subunit and a seven‐transmembrane spanning subunit that interacts with the G proteins Gαs and Gαq. The two subunits, generated by proteolytic cleavage of a single polypeptide chain, are held together by disulphide bridges. The receptor is completely cleaved in thyroid tissue, while in cultured cells (thyrocytes and non‐thyroid cells) the cleaved and uncleaved forms coexist. The reasons for these divergent data are not understood. Here we provide an explanation by showing that cleavage depends on cell–cell contacts. An almost complete cleavage was observed in confluent cells, while in sparse cells most of the receptor was in the uncleaved form. We also show that coupling of TSHR to Gαq (as measured by inositolphosphate generation) is markedly reduced when the receptor is not cleaved. In contrast, coupling to Gαs [as measured by cyclic adenosine 3′,5′‐monophosphate (cAMP) synthesis] is unaffected by cleavage of the receptor. These results suggest that the cell–cell contacts are necessary for cleavage of the receptor, which acts as a regulatory step in inositolphosphate production via phospholipase C activation. The latter observation was confirmed using cells that express the uncleavable mutant TSHR‐Δ50‐NET, for which the TSH‐stimulated inositolphosphate production was completely abolished.