Biomedical subjects

R Baserga

Publications and source records attributed to R Baserga.

At least 91 records · Page 5Linked to original sources

PubMed1996

The role of Grb2 in the growth and transformation of mouse embryo cells.

An overexpressed insulin-like growth factor I receptor (IGF-IR) allows cells to grow in IGF-I only and to form colonies in soft agar. Conversely, cells with a targeted disruption of the IGF-IR genes, R- cells, are refractory to transformation by several oncoproteins and growth factor receptors, that readily transform their wild type counterparts, W cells. Grb2 is an SH2-SH3 domains protein that links tyrosine kinase receptors to ras signalling. In order to determine its role in mitogenesis and transformation, we have transfected a plasmid expressing Grb2 into R- and W cells, and their derivatives already expressing the SV40 large T antigen. In addition, we have used loss-of-function mutants of Grb2 to inquire whether they would act as dominant negatives. Our results show that: (1) an overexpressed Grb2 cannot replace the IGF-IR in IGF-I-mediated mitogenesis; (2) Grb2 also fails to transform either W or R- cells; (3) Grb2 and SV40 T antigen, singly transfected, cannot transform R- cells, but can do so when combined; and (4) SH3 domain mutants of Grb2 act as dominant negatives, causing reversion of the transformed phenotype. We conclude that Grb2 is necessary but not sufficient for transformation.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Inhibition of rat C6 glioblastoma tumor growth by expression of insulin-like growth factor I receptor antisense mRNA.

The expression of insulin-like growth factor I receptor (IGF-IR) antisense mRNA inhibits the growth of C6 rat glioblastoma cells both in vitro and in vivo [Cancer Res (1994) 54:2218]. Moreover, the injection of C6 cells expressing an antisense mRNA to the IGF-IR into syngeneic rats prevents subsequent wild-type tumorigenesis and induces regression of established tumors. For the study of immune function in syngeneic rats, C6 cells expressing either IGF-IR sense or IGF-IR antisense mRNA were injected and splenic lymphocyte function analyzed in vitro after 2 weeks. Cytotoxic, CD8+ lymphocytes from animals injected with IGF-IR antisense cells, but not from those treated with IGF-IR sense cells, proliferated in vitro in response to wild-type C6 cells. Wild-type C6 cells or IGF-IR-sense-RNA-expressing cells rapidly formed tumors upon subcutaneous injection into athymic nude mice. IGF-IR antisense cells were weakly tumorigenic, exhibiting a six- to tenfold increase in tumor latency. Injection of IGF-IR antisense C6 cells mildly delayed the development of wild-type tumors, and did not induce the regression of established wild-type C6 tumors in athymic nude mice. Thus, these findings demonstrate the stimulation of a cellular immune response in rats following the injection of IGF-IR antisense cells. However, studies of athymic nude mice indicate that expression of IGF-IR antisense mRNA also inhibits C6 cells tumorigenicity by additional mechanisms.

Animals↗

PubMed1996

Controlling IGF-receptor function: a possible strategy for tumor therapy.

The insulin-like growth factor-1 receptor (IGF-IR) is one of several growth-factor receptors that regulate the proliferation of mammalian cells. However, its ubiquitousness and certain unique aspects of its function make IGF-IR an ideal target for therapeutic interventions against abnormal growth, with very little effect on normal cells.

Animals↗

PubMed1996

Regression of C6 rat brain tumors by cells expressing an antisense insulin-like growth factor I receptor RNA.

We have previously shown that C6 cells expressing an antisense insulin-like growth factor I receptor (IGF-IR) RNA are no longer tumorigenic in syngeneic rats, protecting them from subsequent subcutaneous tumor challenge and causing regression of established subcutaneous tumors. In the present study, we have investigated the efficacy of this strategy on intracerebrally implanted C6 rat glioblastoma cells. We demonstrate that C6 cells expressing an antisense IGF-IR RNA implanted for 24 h in the subcutaneous tissue of the rats are able to elicit an anti-tumor response in the brain, leading to complete brain tumor regression and long-term survival of the rats. These findings suggest the possibility of therapeutic intervention in human gliomas.

Animals↗

PubMed1995

Different effects on mitogenesis and transformation of a mutation at tyrosine 1251 of the insulin-like growth factor I receptor.

The wild type insulin-like growth factor I (IGF-I) receptor has both mitogenic and transforming activities. We have examined the effect of point mutations at tyrosine residues 1250 and 1251 on these two properties of the receptor. For this purpose, we stably transfected plasmids expressing mutant and wild type receptors into R- cells, which are 3T3-like cells, derived from mouse embryos with a targeted disruption of the IGF-I receptor genes, and therefore devoid of endogenous IGF-I receptors. A tyrosine to phenylalanine mutation of either the 1250 or 1251 residue, or both, has no effect on the ability of the receptor to transmit a mitogenic signal. However, the tyrosine 1251 mutant receptor and the double mutant have lost the ability to transform R- cells (colony formation in soft agar), even when the receptors are expressed at very high levels, while the Y1250F mutant is fully transforming. These experiments show that the 1251 tyrosine residue is required for the transforming activity of the IGF-I receptor.

3T3 Cells↗

PubMed1995

Mutant IGF-I receptors as dominant negatives for growth and transformation.

The insulin-like growth factor I receptor (IGF-IR) plays a crucial role in cell growth, transformation and protection from apoptosis. We have transfected several mutant IGF-IRs into C6 rat glioblastoma cells, in order to determine whether they can act as dominant negatives. We find that some of them can act as dominant negatives in growth assays (monolayer or soft agar), but that none of those examined can induce apoptosis in C6 cells.

Analysis of Variance↗

PubMed1995

Correlation between apoptosis, tumorigenesis, and levels of insulin-like growth factor I receptors.

We have investigated whether there is a quantitative relationship between the insulin-like growth factor I receptor (IGF-IR), the extent of apoptosis in vivo, and tumorigenesis. C6 rat glioblastoma cells were treated with increasing concentrations of antisense oligodeoxynucleotides to the IGF-IR RNA. The extent of apoptosis in vivo is correlated to the decrease in IGF-IR levels and, in turn, tumorigenesis in nude mice is correlated to the fraction of surviving cells. In syngeneic rats, a host response leads to complete inhibition of tumorigenesis. These findings establish, for the first time on a quantitative basis, the relationship between IGF-IR levels and the extent of apoptosis, as well as the relationship between the initial apoptotic event and the time of appearance of transplantable tumors.

Animals↗

PubMed1995

The insulin-like growth factor I receptor protects tumor cells from apoptosis in vivo.

The role of the insulin-like growth factor I receptor (IGF-IR) in programmed cell death has been investigated in vivo in a biodiffusion chamber, where the extent of cell death could be determined quantitatively. We found that a decrease in the number of IGF-IRs causes massive apoptosis in vivo in several transplantable tumors, either from humans or rodents. Conversely, an overexpressed IGF-IR protects cells from apoptosis in vivo. We also show that the same conditions that in vitro cause only partial growth arrest with a minimum of cell death, induce in vivo almost complete cell death. We conclude that the IGF-IR activated by its ligands plays a very important protective role in programmed cell death, and that its protective action is even more striking in vivo than in vitro.

3T3 Cells↗

PubMed1995

Loss of the metastatic phenotype in murine carcinoma cells expressing an antisense RNA to the insulin-like growth factor receptor.

The ability of malignant cells to form metastases in secondary sites remains a major obstacle to the curative treatment of cancer. Previously, we identified type 1 insulin-like growth factor (IGF-1) as a paracrine mitogen for highly metastatic murine carcinoma, H-59 cells. Here the role of IGF-1 and its receptor (IGF-1R) in metastasis was further investigated using H-59 cells transfected with a plasmid vector expressing IGF-1R cDNA in the antisense orientation. The transfectants had a markedly reduced expression of IGF-1R and lost the ability to respond to IGF-1 in vitro. When injected in vivo, either directly into the microvasculature of the liver or lung (experimental metastasis) or s.c. to allow the growth of primary local tumors (spontaneous metastasis), these cells did not give rise to any metastases under conditions which allowed wild-type or control transfectants to form multiple hepatic and pulmonary metastases. The results demonstrate that the IGF-1R can play a critical role in the regulation of carcinoma metastasis.

Animals↗

PubMed1995

Effect of a mutation at tyrosine 950 of the insulin-like growth factor I receptor on the growth and transformation of cells.

Recent evidence has shown that the insulin-like growth factor I (IGF-I) receptor plays a major role in the establishment and maintenance of transformation. To identify domains in the IGF-I receptor that are necessary for transformation, the tyrosine at residue 950 of the human IGF-I receptor cDNA was mutated to phenylalanine, and the plasmid expressing the mutant receptor was stably transfected into R- cells, which are mouse embryo fibroblasts with a targeted disruption of the type 1 receptor for the insulin-like growth factors. At variance with the wild-type receptor, the Y950 mutant receptor has lost its ability to transmit an IGF-I-mediated mitogenic signal or to transform R- cells. These experiments show, for the first time, that tyrosine 950 of the IGF-I receptor is necessary for its mitogenic and transforming activities.

3T3 Cells↗

PubMed1995

The insulin-like growth factor I receptor: a key to tumor growth?

The insulin-like growth factor I receptor (IGF-IR) belongs to the family of transmembrane tyrosine kinase receptors, like the receptors for platelet-derived growth factor, the epidermal growth factor, insulin, and others. Genetic evidence has shown that the IGF-IR is required for optimal growth in vitro and in vivo. Even more important, however, have been recent findings from several laboratories clearly showing that the IGF-IR is an absolute requirement for the establishment and maintenance of the transformed phenotype, both in vivo and in vitro and in several cell types. These findings indicate that the IGF-IR plays a central role in the mechanism of transformation and, as such, could be a preferred target for therapeutic interventions.

Animals↗

PubMed1995

Insulin-like growth factor I (IGF-I) and the IGF-I receptor prevent etoposide-induced apoptosis.

The interaction of insulin-like growth factors (IGF) with the IGF-I receptor promotes cell proliferation and survival. We examined the role of the IGF-I receptor as a possible direct inhibitor of apoptosis induced by the topoisomerase I inhibitor etoposide. When exposed to this agent, BALB/c 3T3 cells that constitutively overexpress the human IGF-I receptor (p6 cells) arrested in S phase and subsequently underwent apoptosis as determined by the appearance of a pre-G1 apoptotic peak when studied by flow cytometry and the characteristic internucleosomal fragmentation of DNA. The addition of IGF-I markedly inhibited etoposide-induced apoptosis in a concentration-dependent manner. IGF-I was not mitogenic in the presence of etoposide. IGF-I was less effective in preventing apoptosis in parental BALB/c 3T3 cells and had no effect on etoposide-induced cell killing of mouse embryo fibroblasts that have a targeted disruption of the IGF-I receptor gene. These results demonstrate an important role for the IGF-I receptor as an inhibitor of apoptosis, independent of its mitogenic actions.

3T3 Cells↗

PubMed1995

Insulin-like growth factors and cancer.

The insulin-like growth factor (IGF) family of peptides, binding proteins, and receptors are important for normal human growth and development and are involved in the specialized functions of most physiologic systems. Most members of the IGF system are expressed by different cancer cells and may play an important role in the propagation of these malignancies. New therapies aimed at modulating various components of the IGF system could affect the progression and metastasis of cancer.

Animals↗

PubMed1995

Insulin-like growth factor I receptor is required for the mitogenic and transforming activities of the platelet-derived growth factor receptor.

R- cells are 3T3-like cells derived from mouse embryos in which the insulin-like growth factor I (IGF-I) receptor (IGF-IR) genes have been disrupted by targeted homologous recombination. These cells cannot grow in serum-free medium supplemented by the growth factors that sustain the growth of other 3T3 cell lines, and cannot be transformed by oncogenes that easily transform wild type mouse embryo cells. We have used these cells to study the role of the IGF-IR in the growth and transformation of cells overexpressing the platelet-derived growth factor (PDGF)-beta beta receptor. We report that an overexpressed PDGF-beta beta receptor fails to induce mitogenesis or transformation in cells lacking the IGF-IR, while capable of doing so in cells expressing the IGF-IR. We conclude that the ability of the activated PDGF-beta beta receptor to stimulate cell proliferation and transformation requires a functional IGF-IR.

Animals↗

PubMed1995

Dissociation of mitogenesis and transforming activity by C-terminal truncation of the insulin-like growth factor-I receptor.

We have investigated the mitogenic and transforming ability of an IGF-I receptor with a 108-amino-acid C-terminal truncation in R- cells, which are 3T3-like cells derived from mouse embryos in which the IGF-I receptor genes have been disrupted by targeted homologous recombination. R- cells stably transfected with expression plasmids encoding either a wild-type or a truncated human IGF-I receptor were capable of growing in serum-free medium supplemented solely with IGF-I. This response was observed over a wide range of receptor levels. R- cells overexpressing the wild-type IGF-I receptor also formed colonies in soft agar, and colony formation was augmented by coexpression of the SV40 large T antigen. However, all the examined clones of R- cells expressing the truncated IGF-I receptor exhibited a dramatically impaired ability to grow in soft agar, even in the presence of the T antigen. The inability to form colonies in soft agar was not due to a quantitative impairment of signal transduction, because: (1) SV40-transformed cells with a physiological level of the wild-type IGF-I receptor did not respond to IGF-I with cell proliferation, but grew in soft agar; (2) R- cells stably transfected with both a truncated receptor and T antigen, on the contrary, responded with mitogenesis to IGF-I but could not form colonies in soft agar; (3) some clones with the truncated receptor expressed levels of receptor roughly 100-fold the level of wild-type cells; and (4) several parameters of IGF-I receptor signal transduction were not impaired in cells stably transfected with a truncated receptor. Furthermore, overexpression of an activated ras in cells with the truncated IGF-IR did not restore their ability to proliferate under anchorage-independent conditions. We conclude that the 108 amino acids of the IGF-I receptor are not essential for a mitogenic response to IGF-I, but are required for transformation (as assessed by the ability to grow in soft agar), indicating that these two functions can be dissociated at an intramolecular level. Moreover, although ras (activated) certainly plays a role in transformation, the transforming activity of the IGF-IR also requires signaling elements that are ras-dependent.

3T3 Cells↗

PubMed1995

Stabile D-peptide analog of insulin-like growth factor-1 inhibits smooth muscle cell proliferation after carotid ballooning injury in the rat.

Restenosis after angioplasty is believed to result from stimulation of smooth muscle cells (SMC) by various growth-promoting factors as a consequence of endothelial injury. In this study we have tested the hypothesis that insulin-like growth factor-1 (IGF-1)/IGF-1 receptor (IGF-1R) interaction is a rate-limiting step for SMC replication by blocking this interaction with a synthetic D-amino acid peptide structurally resembling the D-domain of IGF-1. After rat carotid artery denudation, semiquantitative PCR analysis demonstrated a significant elevation of IGF-1, platelet-derived growth factor B, transforming growth factor beta 1, and epidermal growth factor mRNAs 10 days after endothelial injury, concomitantly with the induction of intimal SMC proliferation and intimal thickening. Administration of 10-30 micrograms.kg-1.day-1 of D-analog of IGF-1, devoid of proteolytic degradation in body fluids, reduced intimal SMC replication by 60-70%. The peptide also inhibited [3H]TdR incorporation and [3H]glycine incorporation in cultured SMCs by 60-80%, whereas a "scrambled" control peptide consisting of the same amino acids had no effect. The results suggest that IGF-1/IGF-1R interaction is a rate-limiting step for SMC replication. Blocking of this interaction with stabile D-peptide analog of IGF-1 at the level of IGF-1R may offer an entirely new approach for the prophylaxis and treatment of restenosis after cardiac revascularization procedures.

Amino Acid Sequence↗

PubMed1995

The GC factor regulates the expression of the insulin-like growth factor-I receptor.

We have transfected a plasmid expressing the transcriptional regulator GC Factor (GCF) into cell lines and have found that the GCF: 1 causes a decrease in the levels of insulin-like growth factor I receptor (IGF-IR) mRNA; 2 causes a decrease in the number of IGF-IRs; and 3 represses the activity of the IGF-IR promoter. In addition, we show that the regulation of IGF-IR expression by GCF plays a physiological role in the control of cellular proliferation in vitro.

Animals↗

PubMed1995

Failure of the bovine papillomavirus to transform mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I receptor genes.

Mouse embryo cells with a targeted disruption of the insulin-like growth factor I receptor (IGF-IR) genes (R- cells) are refractory to transformation by the simian virus 40 large T antigen and/or an activated and overexpressed Ras, both of which readily transform cells from wild-type littermate embryos and other 3T3-like cells. R- cells are also refractory to transformation induced by overexpressed epidermal growth factor receptor and platelet-derived growth factor receptor beta. Since the platelet-derived growth factor receptor beta is required for transformation by bovine papillomavirus, we inquired whether the IGF-IR was also required for transformation by bovine papillomavirus E5 oncoprotein. We show here that R- cells are refractory to transformation by E5; reintroduction into R- cells of a human IGF-IR restores the susceptibility to transformation.

Animals↗