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Renato Baserga

Publications and source records attributed to Renato Baserga.

At least 19 recordsLinked to original sources
PubMed2007

Downregulation of the upstream binding factor1 by glycogen synthase kinase3beta in myeloid cells induced to differentiate.

The upstream binding factor 1 (UBF1), one of the proteins that regulate the activity of RNA polymerase I, is downregulated in 32D myeloid cells induced to differentiate into granulocytes, either by the type 1 insulin-like growth factor (IGF-1) or the granulocytic colony stimulating factor (G-CSF). Downregulation of UBF1 is largely due to protein degradation, while mRNA levels are not affected. Inhibition of UBF1 degradation by lithium chloride (LiCl)and lactacystin suggest a role of glycogen synthase kinase beta (GSK3beta) in a proteasome-dependent degradation of UBF. GSK3beta phosphorylates in vitro and in vivo the UBF protein, which has five putative motifs for phosphorylation by GSK3beta. Elimination and/or mutations of these motifs stabilize the UBF1 protein even in cells induced to differentiate. Conversely, a stably transfected, constitutively active GSK3beta accelerates the downregulation of UBF1. We show further that activation of the differentiating protein C/EPBalpha in 32D cells transformed by the oncogenic BCR/ABL protein causes downregulation of UBF1. Finally, inhibition of differentiation of myeloid cells by a dominant negative mutant of Stat3 stabilizes the UBF1 protein, while rapamycin-induced differentiation of myeloid cells downregulates UBF1 levels. Taken together, our results indicate that the induction of granulocytic differentiation in 32D murine myeloid cells causes the degradation of UBF1, via GSK3beta and the proteasome pathway.

Animals↗
PubMed2006

A mechanism for cell size regulation by the insulin and insulin-like growth factor-I receptors.

Deletion of the type 1 insulin-like growth factor receptor (IGF-IR) or of the insulin receptor substrate-1 (IRS-1) genes in animals causes a 50% reduction in body size at birth. Decrease in body size is due to both a decreased number of cells and a decreased cell size. Deletion of the insulin receptor (InR) genes results in mice that are normal in size at birth. We have used 32D-derived myeloid cells to study the effect of IGF-IR and InR signaling on cell size. 32D cells expressing the IGF-IR and IRS-1 are almost twice as large as 32D cells expressing the InR and IRS-1. A mechanism for the difference in size is provided by the levels of the upstream binding factor 1 (UBF1), a nucleolar protein that participates in the regulation of RNA polymerase I activity and rRNA synthesis and therefore cell size. When shifted to the respective ligands, UBF1 levels decrease in cells expressing the InR and IRS-1, whereas they remain stable in cells expressing the IGF-IR and IRS-1. The expression of the IGF-IR and IRS-1 is crucial to the stability of UBF1.

Animals↗
PubMed2006

Expression profiles of micro RNA in proliferating and differentiating 32D murine myeloid cells.

32D cells are murine myeloid cells that grow indefinitely in Interleukin-3 (IL-3). In these cells, the type 1 insulin-like growth factor (IGF-I) and granulocytic-colony stimulating factor (G-CSF) induce differentiation to granulocytes. 32D cells do not express insulin receptor substrate-1 (IRS-1) or IRS-2, docking proteins of the IGF-I receptor. Ectopic expression of IRS-1 in these cells inhibits differentiation, the cells become IL-3 independent and IGF-1 dependent and can form tumors in mice. 32D and 32D-derived cells offer a good model in which to study the expression profiles of Micro Rna (miR) related to sustained proliferation or differentiation. We present here the data obtained with miR micro-arrays and identify the miR that are regulated by IGF-1 or G-CSF and are associated with either differentiation or indefinite cell proliferation of 32D murine myeloid cells.

Animals↗
PubMed2005

Functional significance of type 1 insulin-like growth factor-mediated nuclear translocation of the insulin receptor substrate-1 and beta-catenin.

Previous work has shown that the transcriptional regulator beta-catenin can translocate to the nuclei when cells are stimulated with the type 1 insulin-like growth factor (IGF-1). We show by immunocoprecipitation and by confocal microscopy that beta-catenin binds to and co-localizes with the insulin receptor substrate-1 (IRS-1), a docking protein for both the insulin and the IGF-1 receptors. IRS-1 is required for IGF-1-mediated nuclear translocation of beta-catenin, resulting in the activation of the beta-catenin target genes. IGF-1-mediated nuclear translocation of beta-catenin is facilitated by the nuclear translocation of IRS-1. Both IRS-1 and beta-catenin are recruited to the cyclin D1 promoter, an established target for beta-catenin, but only IRS-1 is recruited to the ribosomal DNA (rDNA) promoter. UBF proteins (known to interact with both IRS-1 and beta-catenin) are also detectable in the cyclin D1 and rDNA promoters. These results indicate that IRS-1 (activated by the IGF-1 receptor) is one of several proteins that regulate the subcellular localization and activity of beta-catenin. The ability of IRS-1 to localize to both RNA polymerase II (with beta-catenin) and RNA polymerase I-regulated promoters suggest an explanation for the effect of IRS-1 on both cell growth in size and cell proliferation. This possibility is supported by the demonstration that enforced nuclear localization of IRS-1 causes nuclear translocation of beta-catenin and transformation of normal mouse embryo fibroblasts (colony formation in soft agar).

3T3 Cells↗
PubMed2005

24p3 in differentiation of myeloid cells.

24p3 is a secreted lipocalin that has been variously related to apoptosis, proliferation, and the neutrophil lineage of blood cells. We have investigated the expression of 24p3 mRNA and protein in myeloid cell lines induced to differentiate by insulin-like growth factor 1 (IGF-1) and the granulocytic-colony simulating factor (G-CSF). Both these growth factors, which cause myeloid cells to differentiate into granulocytes, induced a marked increase in the expression of both 24p3 protein and mRNA. The mRNA especially appeared early after the cells were induced with either IGF-1 or G-CSF, at a time when the cells were still proliferating and are morphologically undifferentiated. 24p3 can be considered an early marker of granulocytic differentiation.

Acute-Phase Proteins↗
PubMed2005

A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells.

The tandem affinity purification (TAP) tag technique has been used with success to identify under nondenaturing conditions protein complexes in yeast. The technique can be used in mammalian cells, but we found that the original technique does not yield enough recovery for the identification of proteins when mammalian cells growing in monolayer have to be used. We present here a modified TAP tag technique that allows sufficient recovery of proteins from mouse fibroblasts growing in monolayer cultures. The recovery allows protein identification by mass spectrometry.

Affinity Labels↗
PubMed2005

The insulin-like growth factor-I receptor as a target for cancer therapy.

This review examines the rationale for targeting the insulin-like growth factor (IGF)-I receptor in the therapy of human tumours and their metastases. The rationale is based on two crucial findings: 1) in experimental animals, normal cells are only partially affected by the deletion of the IGF-I receptor, whereas tumour cells undergo apoptosis when the IGF-I receptor is downregulated; and 2) cells with a deleted IGF-I receptor are refractory to transformation by viral and cellular oncogenes. This review focuses on the mechanisms underlying the experimental findings, and discusses the possibility of extrapolating the results obtained in animals to the cure of human tumours.

Animals↗
PubMed2004

Regulation of upstream binding factor 1 activity by insulin-like growth factor I receptor signaling.

The upstream binding factor 1 (UBF1) is one of the proteins in a complex that regulates the activity of RNA polymerase I, which controls the rate of ribosomal RNA (rRNA) synthesis. We have shown previously that insulin receptor substrate-1 (IRS-1) can translocate to the nuclei and nucleoli of cells and bind UBF1. We report here that activation of the type I insulin-like growth factor receptor (IGF-IR) by IGF-I increases transcription from the ribosomal DNA (rDNA) promoter in both myeloid cells and mouse fibroblasts. The increased activity of the rDNA promoter is accompanied by increased phosphorylation of UBF1, a requirement for UBF1 activation. Phosphorylation occurs on a number of UBF1 peptides, most prominently on the highly acidic, serine-rich C terminus. In myeloid cells (but not in mouse embryo fibroblasts) IRS-1 signaling stabilizes the levels of UBF1 protein. These findings demonstrate that IGF-IR signaling can increase the activity of UBF1 and transcription from the rDNA promoter, providing one explanation for the reported effects of the IGF/IRS-1 axis on cell and body size in animals and cells in culture.

3T3 Cells↗
PubMed2004

Role of pescadillo in the transformation and immortalization of mammalian cells.

The murine and human homologs of the zebrafish pescadillo protein (Pes1 and PES1, respectively) play important roles in ribosome biogenesis and DNA replication. We investigated the effect of Pes1 on the growth of mouse embryo (3T3-like) fibroblasts and conditionally immortalized human fibroblasts expressing the SV40 T antigen (AR5 cells). Increased expression of Pes1 causes transformation of mouse and human fibroblasts in culture (colony formation in soft agar). Although Pes1 can replace the SV40 T antigen in inducing colony formation in soft agar, it cannot substitute the T antigen in the immortalization of human fibroblasts, indicating that it distinguishes between the two functions. As the biological effects of Pes1 are similar to those of the insulin receptor substrate-1 (IRS-1), we investigated the interactions of Pes1 with IRS-1 itself and with the SV40 T antigen. The Pes1 protein (which localizes to the nuclei and nucleoli of cells) interacts with both IRS-1 and the SV40 T antigen, and markedly decreases the interaction of T antigen with p53. Taken together, these results suggest mechanisms for the ability of Pes1 to transform cells, and its failure to immortalize them.

3T3 Cells↗
PubMed2004

Deletion of the pleckstrin and phosphotyrosine binding domains of insulin receptor substrate-2 does not impair its ability to regulate cell proliferation in myeloid cells.

32D IGF-I receptor (IR) cells are IL-3-dependent myeloid cells that can be induced to differentiate into granulocytes by IGF-I. Like the parental 32D cells, 32D IGF-IR cells do not express the insulin receptor substrate (IRS)-1 or IRS-2. We investigated the effect of ectopic expression of IRS-2 in 32D IGF-IR cells. Expression in these cells of a wild-type IRS-2 inhibits IGF-I-induced differentiation, and the cells grow indefinitely in the absence of IL-3. We also investigated the effect of a mutant IRS-2 lacking both the pleckstrin (PH) and the phosphotyrosine-binding (PTB) domains, which are known to bind to the IR. The partial differentialPHPTB IRS-2 is fully as capable as the wild-type IRS-2 (and wild-type IRS-1) to stimulate the growth and inhibit the differentiation of 32D IGF-IR cells. In contrast, an IRS-1 protein lacking the same PH and PTB domains is completely inactive in blocking differentiation and stimulating IL-3-independent growth of 32D IGF-IR cells. The partial differentialPHPTB IRS-2 protein is dependent for its effect on an activated IGF-IR, is cytoplasmic, binds to the beta-subunit of the IGF-IR, and requires for its action the presence of phosphatidylinositol 3-kinase binding sequences. These experiments show that the PH and PTB domains of IRS-2 (but not IRS-1) are dispensable for the IGF-I/IRS-2-mediated growth of 32D myeloid cells. Our results also indicate that IRS-2 (either wild type or partial differentialPHPTB) is capable of inhibiting the differentiation of 32D cells.

Animals↗
PubMed2004

Control of cell size through phosphorylation of upstream binding factor 1 by nuclear phosphatidylinositol 3-kinase.

The insulin-like growth factor I/insulin receptor substrate 1 axis controls, in a nonredundant way, approximately 50% of cell and body size in animals from Drosophila to mice and in cells in culture. Although other factors may also intervene, cell size is strongly dependent on ribosome biogenesis, which is under the control of RNA polymerase I activity. We have previously shown that insulin receptor substrate 1 (IRS-1) translocates to the nuclei and nucleoli, where it binds to the upstream binding factor (UBF) 1, a regulator of RNA polymerase I activity. Activation of UBF1 requires its phosphorylation. However, IRS-1 is not a kinase, and we searched for an intermediate kinase that can phosphorylate UBF1. We demonstrate here that IRS-1 binds also to the phosphatidylinositol 3-kinase (PI3-K) subunits in nuclear extracts, and that the p110 subunit of PI3-K directly phosphorylates and activates UBF1, an exclusively nucleolar protein. The interaction of IRS-1, PI3-K, and UBF1 in the nucleoli provides one of the mechanisms for the effects of IRS-1 on cell and body size.

3T3 Cells↗
PubMed2004

Role of pescadillo and upstream binding factor in the proliferation and differentiation of murine myeloid cells.

Pescadillo (PES1) and the upstream binding factor (UBF1) play a role in ribosome biogenesis, which regulates cell size, an important component of cell proliferation. We have investigated the effects of PES1 and UBF1 on the growth and differentiation of cell lines derived from 32D cells, an interleukin-3 (IL-3)-dependent murine myeloid cell line. Parental 32D cells and 32D IGF-IR cells (expressing increased levels of the type 1 insulin-like growth factor I [IGF-I] receptor [IGF-IR]) do not express insulin receptor substrate 1 (IRS-1) or IRS-2. 32D IGF-IR cells differentiate when the cells are shifted from IL-3 to IGF-I. Ectopic expression of IRS-1 inhibits differentiation and transforms 32D IGF-IR cells into a tumor-forming cell line. We found that PES1 and UBF1 increased cell size and/or altered the cell cycle distribution of 32D-derived cells but failed to make them IL-3 independent. PES1 and UBF1 also failed to inhibit the differentiation program initiated by the activation of the IGF-IR, which is blocked by IRS-1. 32D IGF-IR cells expressing PES1 or UBF1 differentiate into granulocytes like their parental cells. In contrast, PES1 and UBF1 can transform mouse embryo fibroblasts that have high levels of endogenous IRS-1 and are not prone to differentiation. Our results provide a model for one of the theories of myeloid leukemia, in which both a stimulus of proliferation and a block of differentiation are required for leukemia development.

Animals↗
PubMed2003

The IGF-1 receptor in cancer biology.

The type 1 insulin-like growth factor receptor (IGF-1R) plays an important role in the establishment and maintenance of the transformed phenotype. It also has a strong antiapoptotic activity and has a significant influence on the control of cell and body size. Downregulation of the IGF-1R leads to massive apoptosis of cancer cells. These characteristics make it an attractive target for anticancer therapy.

Animals↗
PubMed2003

Intracellular redistribution of nuclear and nucleolar proteins during differentiation of 32D murine hemopoietic cells.

We have investigated the intracellular localization of four proteins in murine hemopoietic 32D and 32D-derived cells during exponential growth and after induction of differentiation. The four proteins studied were the insulin receptor substrate-1 (IRS-1), the ID2 protein, nucleolin, and the upstream binding factor (UBF), all of which are involved directly or indirectly in the differentiation program. These four proteins were found to be predominantly nuclear (and/or nucleolar) during exponential growth, as expected. In three models of induced differentiation along the granulocytic pathway, IRS-1, ID2, and nucleolin shifted in part to the cytoplasm, where their levels eventually decreased. UBF also disappeared during differentiation, but we could not detect a cytoplasmic shift in this protein. These experiments indicate that induction of granulocytic differentiation in 32D and 32D-derived cells is accompanied by intracellular redistribution of proteins. This nucleo-cytoplasmic shuttle may play a significant role in the changes in gene expression that occur during differentiation.

Active Transport, Cell Nucleus↗
PubMed2003

Nuclear translocation of insulin receptor substrate-1 by the insulin receptor in mouse embryo fibroblasts.

Translocation of the insulin receptor substrate-1 (IRS-1) to the nuclei has been reported to occur in cells stimulated by insulin-like growth factor-1 (IGF-I) or expressing certain viral and cellular oncogenes. We show here that insulin can also induce nuclear translocation of IRS-1 in mouse embryo fibroblasts (MEF), that do not express the type 1 insulin-like growth factor receptor (IGF-IR). Only the A isoform of the insulin receptor (IR) can induce IRS-1 nuclear translocation, which is significant when the receptor is over-expressed. At physiological receptor levels, translocation occurs only in a fraction of cells, and only at high concentrations of ligand.

Active Transport, Cell Nucleus↗
PubMed2003

Signaling differences from the A and B isoforms of the insulin receptor (IR) in 32D cells in the presence or absence of IR substrate-1.

The A isoform of the insulin receptor (IR) is frequently overexpressed in cancer cells and is activated by IGF-II as well as by insulin, whereas the B isoform is predominant in differentiated tissues and responds poorly to IGF-II. The IR substrate-1 (IRS-1), a docking protein for the IR, is known to send a mitogenic signal and to be a powerful inhibitor of cell differentiation. We have investigated the biological effects of the two IR isoforms in parental 32D hemopoietic cells, which do not express IRS-1, and in 32D-derived cells in which IRS-1 is ectopically expressed. The effects of the two isoforms on cell survival, differentiation markers and nuclear translocation of IRS-1 were compared. The results confirm that the A isoform responds to IGF-II and preferentially sends mitogenic, antiapoptotic signals, whereas the B form, poorly responsive to IGF-II, tends to send differentiation signals.

Acute-Phase Proteins↗