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Biomedical subjects

R Baserga

Publications and source records attributed to R Baserga.

At least 109 records · Page 6Linked to original sources
PubMed1995

Association of insulin receptor substrate 1 with simian virus 40 large T antigen.

Mouse embryo cells expressing a wild-type number of insulin-like growth factor I receptors (IGF-IR) (W cells) can be transformed either by simian virus 40 large T antigen (SV40 T) or by overexpressed insulin receptor substrate 1 (IRS-1), singly transfected. Neither SV40 T antigen nor IRS-1, individually, can transform mouse embryo cells with a targeted disruption of the IGF-IR genes (R- cells). However, cotransfection of SV40 T antigen and IRS-1 does transform R- cells. In this study, using different antibodies and different cell lines, we found that SV40 T antigen and IRS-1 are coprecipitated from cell lysates in a specific fashion, regardless of whether the lysates are immunoprecipitated with an antibody to SV40 T antigen or an antibody to IRS-1. The same antibody to SV40 T antigen, however, fails to coprecipitate another substrate of IGF-IR, the transforming protein Shc, and two other signal-transducing molecules, Grb2 and Sos. Finally, an SV40 T antigen lacking the amino-terminal 250 amino acids fails to coprecipitate IRS-1 and also fails to transform R- cells overexpressing mouse IRS-1. These experiments indicate that IRS-1 associates with SV40 T antigen and that this association plays a critical role in the combined ability of these proteins to transform R- cells. This finding is discussed in light of the crucial role of the IGF-IR in the establishment and maintenance of the transformed phenotype.

3T3 Cells↗
PubMed1995

The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene.

The insulin-like growth factors (IGFs) are important mitogens that exert their proliferative effects on cells via the type I IGF receptors (IGF-R). The IGFs also associate with IGF binding proteins (IGFBPs). IGF-inhibitory, IGF-stimulatory, and IGF-independent effects of IGFBPs on cell growth have been reported. We have asked whether the IGFBP-3 has an effect on cell growth, which is independent of its ability to bind IGF-I and thus inhibit the activation of the IGF-I receptor. For this purpose, we have used a fibroblast cell line (R- cells) derived from mouse embryos homozygous for a targeted disruption of the IGF-R gene. When compared with wild type cells (W), which bind IGF-I with high affinity and specificity, R- cells transfected with a mammalian expression vector containing the human (h) IG-FBP-3 cDNA were selected (R-/BP3) and found to express hIGFBP-3 mRNA (detected by Northern blots) and to secrete hIGFBP-3 protein [detected by Western ligand blotting (WLB), immunoblotting, and immunoprecipitation as well as immunofluorescence confocal microscopy]. Growth of five different R- cells, and 10-fold slower compared with W cells, grown under identical conditions. Confluent R- cells. These experiments show that the overexpression of IGFBP-3 has an inhibitory effect on cell growth which does not involve IGF binding to, or signal transduction via, the type I IGF-R. We conclude that the cellular production of IGFBPs serves as a negative regulator of cell proliferation which involves a cellular signaling pathway separate from the IGF-IGF-R system.

Animals↗
PubMed1995

Insulin-like growth factor-I receptor. Its role in cell proliferation, apoptosis, and tumorigenicity.

From the point of view of cell growth, the IGF-IR activated by its ligands has three important functions: (a) it is required for optimal growth both in vivo and in vitro, although some growth occurs even in its absence; (b) it is obligatory for the establishment and maintenance of the transformed phenotype and for tumorigenesis for several types of cells; and (c) it protects cells from apoptosis, both in vivo and in vitro. The IGF-I receptor does seem to occupy a central role in these processes. Whereas an overexpressed IGF-I receptor is mitogenic for IGF-I alone and is fully transforming and protects cells from apoptosis, the same cannot be said for overexpressed EGF and PDGF receptors (205, 206). These two receptors can neither induce growth or transform most cells lacking IGF-I receptors. The reversal of the transformed phenotype and the induction of apoptosis that occur when the levels of IGF-I receptors are artificially decreased also point out the essential role of the receptor in these three processes. An important distinction in this regard is that it is not so much an overexpressed IGF-I receptor that is important in transformation but the lack of it that does not allow the transformed phenotype. This distinction is extremely important if we wish to use the IGF-IR as an approach to therapeutic interventions. Returning to more basic questions, a mutational analysis of the IGF-I receptor has shown that specific domains are involved in its mitogenicity or its ability to facilitate transformation and that these two processes can be separated at the level of the receptor itself. This finding raises a crucial question: Is the transforming activity using a pathway that is separate from the mitogenic signaling pathway? Alternatively, is it simply a question of a quantitative effect? The answer to this question could be a very important contribution to the mechanism of transformation. Little is known about the mechanism(s) by which the IGF-I receptor protects cells from apoptosis; here again, some fundamental questions can be raised. Are there specific domains in the receptor for its antiapoptotic activity? Is this activity tied to mitogenesis and/or transformation? Which elements in the signal transduction pathway are involved in these three different functions of the IGF-I receptor? Although many problems are still unresolved, the last few years have seen a very rapid rise in the importance of the IGF-I receptor in both normal and abnormal growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Transforming potential of the insulin receptor substrate 1.

The role of the insulin receptor substrate 1 (IRS-1) in cellular transformation was studied in R- cells, which are 3T3-like fibroblasts derived from mouse embryos with a targeted disruption of the insulin-like growth factor I receptor gene. These cells cannot be transformed by oncogenes that readily transform cells originating from wild-type littermate embryos (or other 3T3-like cells). In the present study, we demonstrate that in R- cells, the overexpression of the functional IRS-1 protein was sufficient to induce a mitogenic response to insulin but did not promote transformation, as measured by colony formation in soft agar. The coexpression of IRS-1 and the SV40 T antigen, however, induced transformation. Conversely, expression of an antisense IRS-1 RNA reversed the transformed phenotype in wild-type cells carrying the T antigen. Since the type 1 insulin-like growth factor receptor, by itself, is fully transforming, we propose the hypothesis that the transforming competence of this receptor is based on at least two signaling pathways, one of which is IRS-1-dependent, whereas the other(s) can be substituted with the SV40 T antigen.

Animals↗
PubMed1994

Mitogenicity and transforming activity of the insulin-like growth factor-I receptor with mutations in the tyrosine kinase domain.

We have investigated the effect of mutations in tyrosines 1131, 1135, and 1136 of the human insulin-like growth factor-I receptor (IGF-IR) on the growth and transformation of mammalian cells. We have used for this purpose R- cells, which are 3T3-like fibroblasts derived from mouse embryos with a targeted disruption of the IGF-IR genes. These cells have no IGF-IR, do not grow in serum-free medium supplemented with the growth factors that sustain the growth of 3T3 cells, and cannot be transformed by simian virus 40 large tumor antigen or other oncogenes. The R- cells were transfected with plasmids expressing: 1) a wild type human IGF-IR cDNA; 2) a receptor with a triple mutation in the above mentioned tyrosines; and 3) receptors with single tyrosine mutations. Cells expressing the wild type or the single tyrosine mutants Y1 (Y1131F) and Y2 (Y1135F) grew in serum-free medium supplemented solely with IGF-I. Cells expressing the triple tyrosine mutant YF or the single mutant Y3 (Y1136F) failed to grow in response to IGF-I only. All mutants, though, failed to form colonies in soft agar, indicating that a fully functional IGF-IR is more critical for anchorage-independent growth than for monolayer growth. The triple mutant expression plasmid also functioned as a dominant negative, inhibiting the growth of wild type cells transformed by the simian virus tumor antigen.

3T3 Cells↗
PubMed1994

Growth inhibition of human melanoma cells in nude mice by antisense strategies to the type 1 insulin-like growth factor receptor.

The growth of human melanoma cells FO-1 in nude mice is strongly inhibited or even abrogated when the cells are stably transfected with a plasmid expressing an antisense RNA to the insulin-like growth factor 1 receptor (IGF-1R) RNA, which causes a marked reduction in the number of IGF-1 receptors. When a tumor arises after a long delay in nude mice, it can be shown that the tumor cells have lost the expression plasmid and that the number of IGF-1 receptors has returned to wild-type levels. The antisense effect is even more remarkable, since the growth of FO-1 melanoma cells in monolayers is not affected by the expression of the antisense RNA. Inhibition of tumorigenesis was also evident when FO-1 melanoma cells were treated with antisense oligodeoxynucleotides to the IGF-1R RNA prior to injection into nude mice. These results confirm in human cells that the IGF-1R plays a dominant role in transformation and tumorigenesis and that its effect on tumorigenesis is more profound than its effect on mitogenesis.

Animals↗
PubMed1994

Introns determine the time of appearance of PCNA mRNA in 3T3 cells stimulated by growth factors.

In quiescent fibroblasts stimulated to proliferate, the mRNAs for several DNA synthesis genes increase sharply at the G1/S boundary. However, the corresponding hnRNAs often increase shortly after stimulation, several hours before the increase in mRNA levels. To investigate the delay that occurs between the increases in hnRNA and mRNA levels, we have used several constructs of the human PCNA gene stably transfected into Balb/c-3T3 cells. The 3' untranslated region of the PCNA gene can be replaced without affecting the time course of appearance of the mature RNA. However, when the introns are deleted, the mRNA levels are already high in quiescent cells and increase as cells progress through G1. These experiments define the introns of the human PCNA gene as responsible for the delay in the appearance of the PCNA mRNA that occurs in stimulated Balb/c-3T3 cells.

3T3 Cells↗
PubMed1994

Platelet-derived growth factor-induced expression of messenger RNA for the proliferating cell nuclear antigen requires a functional receptor for the insulin-like growth factor I.

Stimulation of quiescent fibroblasts or fibroblast-like cells with growth factors causes a sharp increase in the mRNA levels of several DNA synthesis genes. With most of these genes, the increase in mRNA levels requires at least 2 growth factors [usually platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I)], but the mRNA of the proliferating cell nuclear antigen (PCNA) gene is induced by PDGF only. Since PDGF is known to induce also expression of IGF-I and its receptor, we inquired as to whether the PCNA mRNA induction by PDGF depended on a functional IGF autocrine loop. Using R- cells, i.e., mouse embryo cells in which the IGF-I receptor genes have been disrupted by homologous recombination, we show here that the functional integrity of the IGF-I receptor is obligatory for the PDGF-induced increase in PCNA mRNA levels. However, PCNA pre-mRNA levels are increased by PDGF even in the absence of the IGF-I receptor. These experiments confirm the importance of the IGF-I receptor in the control of cellular proliferation in animal cells, and suggest that one of its functions may be in the processing of certain pre-mRNAs required for orderly cell cycle progression.

3T3 Cells↗
PubMed1994

Rat glioblastoma cells expressing an antisense RNA to the insulin-like growth factor-1 (IGF-1) receptor are nontumorigenic and induce regression of wild-type tumors.

Insulin-like growth factor-1 (IGF-1) and IGF-2 are critical regulators of cell proliferation. The growth-promoting action of both ligands is mediated by the type 1 IGF receptor (IGF-1R). We have investigated the role of the IGF-1R in the growth and tumorigenicity of rat C6 glioblastoma cells. For this purpose, antisense RNA to IGF-1R RNA was introduced into cells by either the addition of oligodeoxynucleotides or by transfection with plasmids that express antisense RNA to IGF-1R RNA. At low cell density, C6 cells grew slowly in serum-free medium and proliferated with the sole addition of IGF-1 or IGF-2. Both antisense IGF-1R oligodeoxynucleotides and stable transfection with a plasmid expressing an antisense IGF-1R RNA inhibited IGF-1-mediated growth in monolayers and clonogenicity in soft agar. Sense oligodeoxynucleotides and sense-expressing plasmid had no effect on either parameter. In stable antisense transfectants, tyrosine-phosphorylated IGF-1 receptors were not detectable, although they were easily detected in wild-type cells. When wild-type C6 cells were injected s.c. into syngeneic immunocompetent rats, tumors developed within 1 week. In contrast, stably transfected C6 cells overexpressing antisense IGF-1R RNA were nontumorigenic. Moreover, when C6 IGF-1R antisense cells were injected, subsequent tumor formation by wild-type C6 cells was completely prevented. Finally, injection of C6 IGF-1R antisense cells into rats carrying an established wild-type C6 tumor caused complete regression of the tumors. The results demonstrate the critical importance of the IGF-1R in glioblastoma cell growth, clonogenicity, and tumorigenicity. Although the mechanism is presently unknown, the fact that the injection of C6 cells expressing an antisense RNA to IGF-1R RNA leads to regression of already established wild-type C6 tumors suggests the possibility of practical applications.

Animals↗
PubMed1994

Growth regulation of human glioblastoma T98G cells by insulin-like growth factor-1 and its receptor.

The interaction of insulin-like growth factors (IGFs) with the IGF-1 receptor is an important step in the control of cell proliferation and development. In particular, IGF-1 and IGF-2 are key regulators of central nervous system development, and may modulate the growth of glial tumors. We have investigated the growth factor regulation of the human glioblastoma cell line T98G. These cells growth arrested in serum-free medium at 34 degrees C, despite their secretion of substantial amounts of bioactive IGF-1. To be stimulated to divide, growth-arrested cells required the addition of platelet-derived growth factor (PDGF) or its equivalent, 1% serum. Cell proliferation in serum-free medium could also be obtained by shifting the cells to a temperature of 39.6 degrees C. Treatment of growth-arrested cells with PDGF or temperature shift was accompanied by a transient increase in the expression of the mRNA for the IGF-1 receptor. Transfection with a plasmid constitutively expressing the full cDNA for the human IGF-1 receptor allowed autonomous growth in serum-free medium at 34 degrees C. By contrast, growth induction by growth factors or temperature shift was abrogated by transfection of the cells with a plasmid expressing a 300 bp segment of mRNA antisense to the IGF-1 receptor mRNA. Cloning in soft agar was also inhibited by expression of antisense IGF-1 receptor mRNA. These results demonstrate that the IGF-1 receptor is strictly required for the growth of T98G glioblastoma cells. Moreover, the autocrine interaction of IGF-1 with its receptor regulates both autonomous and anchorage-independent growth of these cells.

Base Sequence↗
PubMed1994

Platelet-derived growth factor increases the activity of the promoter of the insulin-like growth factor-1 (IGF-1) receptor gene.

Stimulation by platelet-derived growth factor (PDGF) is known to increase the number of IGF-I binding sites in cells in culture. We show here that PDGF also increases the levels of IGF-1 receptor mRNA. Using cell lines stably transfected with an expression plasmid in which the reporter luciferase gene is under the control of the rat IGF-1 receptor gene promoter, we find that PDGF increases the activity of this promoter. A short IGF-1 receptor gene promoter, comprising about 100 base pairs of the sequence immediately upstream of the initiation of transcription site, is sufficient for a response to the stimulatory action of PDGF. These results suggest that an increase in RNA levels and in promoter activity may play an important role in the increase in IGF-1 receptor levels that occurs after stimulation by PDGF.

3T3 Cells↗
PubMed1994

The role of the IGF-I receptor in the growth and transformation of mammalian cells.

Recent developments in the molecular biology of the insulin-like growth factor I (IGF-I) receptor have clarified its role in cellular growth and transformation. Although cells homozygous for a targeted disruption of the IGF-I receptor genes can grow in serum-supplemented medium, the IGF-I receptor is required for optimal growth, and is required equally in all phases of the cell cycle. The receptor plays an even more stringent role in cellular transformation and tumorigenicity, which seem to be dependent on its normal expression in several cell types. The expression of both the IGF-I receptor and its ligands is regulated by other growth factors (especially PDGF and EGF), by oncogenes (like SV40 T antigen and c-myb) and by tumour suppressor genes (like WT1 and RB). The picture emerging from these studies is that several transforming agents may exert their growth promoting effects through the direct or indirect activation of the IGF autocrine loop.

Animals↗
PubMed1994

Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.

Animals↗
PubMed1994

A functional insulin-like growth factor I receptor is required for the mitogenic and transforming activities of the epidermal growth factor receptor.

When wild-type mouse embryo cells are stably transfected with a plasmid constitutively overexpressing the epidermal growth factor (EGF) receptor (EGFR), the resulting cells can grow in serum-free medium supplemented solely with EGF. Supplementation with EGF also induces in these cells the transformed phenotype (growth in soft agar). However, when the same EGFR expression plasmid is introduced and overexpressed in cells derived from littermate embryos in which the insulin-like growth factor I (IGF-I) receptor genes have been disrupted by homologous recombination, the resulting cells are unable to grow or to be transformed by the addition of EGF. Reintroduction into these cells (null for the IGF-I receptor) of a wild-type (but not of a mutant) IGF-I receptor restores EGF-mediated growth and transformation. Our results indicate that at least in mouse embryo fibroblasts, the EGFR requires the presence of a functional IGF-I receptor for its mitogenic and transforming activities.

Animals↗
PubMed1994

Ethanol inhibits insulin-like growth factor-1-mediated signalling and proliferation of C6 rat glioblastoma cells.

BACKGROUND: Alcohol consumption during pregnancy often results in disorders of fetal development (Fetal Alcohol Syndrome). The brain appears to be particularly vulnerable, and alcohol abuse during pregnancy is probably the most common cause of acquired mental retardation. We therefore studied the in vitro effects of ethanol on insulin-like growth factor-1 (IGF-1)-mediated proliferation of rat C6 glioblastoma cells. EXPERIMENTAL DESIGN: The proliferation of C6 rat glioblastoma cells was measured in serum-free medium supplemented with specific growth factors in the presence or absence of ethanol. The effect of ethanol on IGF-1 receptor and insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation was determined by immunoprecipitation and Western blotting, as was the phosphatidylinositol 3-kinase content within IRS-1 immunoprecipitates. RESULTS: C6 cells grew slowly in serum-free medium and proliferated in response to IGF-1. Ethanol, at physiologically tolerated concentrations, markedly inhibited the growth of C6 cells in response to IGF-1, but had no effect on the proliferative rate in the presence of platelet-derived growth factor or 1% fetal bovine serum. Inhibition of cell proliferation was evident when ethanol was only present during a 1-hour pulse of IGF-1. Cell growth in the presence of IGF-2 was also prevented by ethanol. The inhibition of IGF-1-mediated cell proliferation was accompanied by abrogation of IGF-1 receptor tyrosine autophosphorylation. Ethanol also interfered with the IGF-1-induced tyrosine phosphorylation of IRS-1, and the association of phosphatidylinositol-3 kinase with IRS-1. CONCLUSIONS: The data indicate that physiologically relevant concentrations of ethanol inhibit the responses of glial cells to IGF-1, including IGF-1 receptor autophosphorylation, IRS-1 and phosphatidylinositol-3 kinase activation, and cell growth.

Amino Acid Sequence↗
PubMed1994

An E2F binding sequence negatively regulates the response of the insulin-like growth factor 1 (IGF-I) promoter to simian virus 40T antigen and to serum.

The promoter of the Insulin-like growth factor I (IGF-I) gene is activated by the Simian Virus 40 large T antigen (SVLT), and one of the elements responding to SVLT activation has been localized to a short 124 bp immediately upstream of the first initiation of transcription site. This short promoter contains an E2F binding site, that, in gel shifts, binds a protein complex, but only when the promoter activity is reduced or absent. A mutation in the E2F binding site deregulates the activity of the promoter, which becomes active even in those conditions in which the wild type promoter is inactive. By using antibodies in gel retardation analyses, we can show that the different protein complexes include, at least, the following proteins: E2F, cyclin A and p107. We conclude that the short IGF-I promoter is negatively regulated by an E2F binding site that complexes with several proteins. Our data suggest that disaggregation of these complexes by the action of SVLT (or other activators) increases expression from the promoter, thus establishing a link between the regulation of cell proliferation by growth factors and the E2F-associated proteins.

3T3 Cells↗
PubMed1994

The role of the insulin-like growth factor I receptor in the transformation by simian virus 40 T antigen.

Balb/c 3T3 cells transformed by the tsA58 temperature-sensitive (ts) mutant of SV40 large T antigen, BalbA58 cells, grow in 1% serum at the permissive temperature of 34 degrees C but fail to grow at the restrictive temperature of 39.6 degrees C. Although incapable of growing, BalbA58 cells, in low serum at 39.6 degrees C, still synthesize DNA and tend to accumulate in the G2 phase of the cell cycle. Growth in 1% serum at 39.6 degrees C resumes if BalbA58 cells are treated with insulin-like growth factor I (IGF-I). By using cells overexpressing the IGF-I receptor, and cells with a targeted disruption of the IGF-I receptor genes, we show that: 1) the activation of the IGP-I receptor by its ligand(s) plays a major role in the ability of the SV40 large T antigen to promote growth in low serum; and 2) the IGF-I receptor plays a role in the progression of cells not only through G1, but also through the S and G2 phases of the cell cycle. These findings, together with other recent findings from the literature, suggest that one of the mechanisms by which oncogenes and tumor suppressor genes regulate cell growth is through the modulation of growth factors and their receptors.

3T3 Cells↗