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Biomedical subjects

R Baserga

Publications and source records attributed to R Baserga.

At least 127 records · Page 7Linked to original sources
PubMed1993

Simian virus 40 large tumor antigen is unable to transform mouse embryonic fibroblasts lacking type 1 insulin-like growth factor receptor.

Fibroblast cell lines were established from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 receptor for insulin-like growth factor I (IGF-I) and from their wild-type littermates. The cells from the wild-type embryos (W cells) grow in serum-free medium supplemented with platelet-derived growth factor, epidermal growth factor, and IGF-I, whereas the cells from Igf1r(-/-) embryos (R- cells) do not, although they grow at a reduced rate in 10% fetal calf serum. The simian virus 40 (SV40) large T antigen, expressed from a transfected plasmid, can transform W cells, which form foci in monolayer cultures and colonies in soft agar (anchorage-independent growth). In contrast, the SV40 large tumor antigen, although normally expressed from the transfected template, is unable to transform R- cells, which remain contact-inhibited and fail to grow in soft agar. The transformed phenotype is restored if the R- cells carrying the SV40 large tumor antigen are stably transfected with a plasmid expressing the human IGF-I receptor. These results demonstrate that signaling via the IGF-I receptor is an indispensable component of the SV40 transformation pathway. This conclusion is further supported from the results of antisense RNA experiments with tumor cell lines showing that interference with the function of the IGF-I receptor has a profound effect on anchorage-independent growth, even under conditions that only modestly affect growth in monolayers.

Animals↗
PubMed1993

Ethanol inhibits the autophosphorylation of the insulin-like growth factor 1 (IGF-1) receptor and IGF-1-mediated proliferation of 3T3 cells.

The effect of ethanol on cell proliferation was studied in Balb/c 3T3 cells and in stably transfected 3T3 cells constitutively overexpressing the human insulin-like growth factor 1 (IGF-1) receptor (p6 cells). Ethanol inhibited growth of both cell lines when they were cultured in serum-free medium supplemented with individual growth factors, i.e. platelet-derived growth factor and IGF-1 for 3T3 cells, and IGF-1 only for p6 cells. Increases in cell number were prevented in both cell lines even when ethanol was present exclusively during the period of IGF-1 stimulation. The inhibitory effect of ethanol was concentration-dependent, with a 30% inhibition observed at 10 mM ethanol. IGF-1 receptor tyrosine autophosphorylation was completely prevented by ethanol both in intact cells and in immunopurified IGF-1 receptor preparations. The binding of IGF-1 to its receptor on intact cells was unaffected by ethanol. Ethanol also inhibited the stimulation of IGF-1 receptor autophosphorylation and the corresponding growth of p6 cells induced by IGF-2. Transcription of c-myc, c-fos, and c-jun in response to IGF-1 was inhibited by ethanol. These findings demonstrate that ethanol at low concentrations markedly inhibits IGF-1 receptor autophosphorylation and IGF-1-mediated cell growth.

3T3 Cells↗
PubMed1993

IGF-1 receptor levels and the proliferation of young and senescent human fibroblasts.

We have investigated the role of the IGF-1 receptor in the proliferation of young and senescent human diploid fibroblasts. Using WI-38 cells, we have established the following: 1) both young and senescent cells have IGF-1 receptors, which can be autophosphorylated by IGF-1, the intensity of the autophosphorylation being roughly the same in both types of cells; 2) the levels of IGF-1 receptor mRNA are also similar in young and senescent cells; 3) both young and senescent cells have an absolute requirement for the IGF-1 receptor in order to be stimulated by either serum or SV40, respectively; 4) despite these similarities, young cells respond to IGF-1 (in combination with other growth factors) with DNA synthesis and mitosis, and senescent cells do not. We conclude that, although the IGF-1 receptor is still needed by senescent cells for a growth response to SV40, it is not, by itself, the determinant of senescence, at least in WI-38 cells.

Base Sequence↗
PubMed1993

Inhibition of growth of prostatic cancer cell lines by peptide analogues of insulin-like growth factor 1.

We have investigated three prostatic cancer cell lines, PC-3, DU-145, and LNCa.FGC, and found that all three cell lines can grow in serum-free medium without the addition of exogenous growth factors. All three cell lines produce substantial amounts of insulin-like growth factor 1 (IGF-1) that is secreted in the medium and they all display constitutively autophosphorylated IGF-1 receptors; two of the cell lines overexpress IGF-1 receptor RNA. The growth of all three cell lines is inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA or by peptide analogues of IGF-1 that compete with IGF-1 binding to its receptor. Our results indicate that these three cell lines grow by an autocrine loop in which the overproduced IGF-1 activates its receptor. Interference with the activation of the receptor leads to cessation of growth.

Base Sequence↗
PubMed1993

Use of an antisense oligonucleotide to inhibit expression of a mutated human procollagen gene (COL1A1) in transfected mouse 3T3 cells.

A series of antisense oligonucleotides were developed to inhibit specifically expression of a mutated exogenous gene for collagen without inhibiting expression of an endogenous gene for the same protein. The test system consisted of mouse NIH 3T3 cells that were stably transfected with an internally deleted construct of the human gene for the pro alpha 1(I) chain of type I procollagen [Olsen et al. (1991) J. Biol. Chem. 266, 1117]. The target site was a region at the 3' end of exon 1 and the first few nucleotides of intron 1 of the exogenous human gene that differed in sequence by nine nucleotides from the sequence of the endogenous mouse gene. Expression of the two genes was assayed by Western blot with cross-reacting antibodies and by steady-state levels of mRNAs. None of the oligonucleotides were effective in concentrations up to 25 microM when administered without any carrier. However, when administered with 5 or 10 micrograms/mL lipofectin, one of the oligonucleotides in concentrations of 0.1-0.2 microM inhibited expression of the exogenous gene from 50% to 80% without significant inhibition of expression of the endogenous gene. Also, a missense version of the same oligonucleotide had no significant effect, and the inhibition observed with the most effective oligonucleotide was abolished by a single base change. Time course experiments indicated that, after a 4-h treatment, inhibition appeared at 8 h and persisted for at least 22 h.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1993

Gene regulation by IGF-I.

Constitutive overexpression of the insulin-like growth factor-I (IGF-I) receptor makes 3T3 cells capable of growing in serum-free medium supplemented solely with IGF-I to the total exclusion of the platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors, the other two receptors that are required for the growth of 3T3 cells. Pietrzkowski et al. (Cell Growth Differ 92:199-205, 1992) found that these cells, overexpressing the IGF-I receptor, can also be induced to grow by the addition of EGF. We have ascertained that the mechanism by which EGF induces growth in these cells is through the induction of substantial amounts of IGF-I RNA and IGF-I. In 3T3 cells overexpressing the EGF receptor, a functional IGF-I receptor is still required for growth. We have also found, using a temperature-sensitive mutant of the SV40 large T antigen, that the levels of IGF-I mRNA and of IGF-I secreted in the medium are markedly increased by a functional T antigen. Using an IGF-I promoter driving a reporter luciferase gene, we have determined that this increase in the expression of IGF-I by T-antigen occurs, at least in part, at a transcriptional level. It seems, therefore, that transformation by SV40 reduces the requirement for growth factors by increasing the production of IGF-I.

3T3 Cells↗
PubMed1993

The role of the IGF-1 receptor in the stimulation of cells by short pulses of growth factors.

Balb/c 3T3 cells require prolonged stimulation by serum or growth factors to enter DNA synthesis. However, in p6 cells, a derivative cell line from 3T3 cells which constitutively over-express the insulin-like growth factor-1 (IGF-1) receptor, a serum pulse of only 1 h is sufficient for maximal stimulation. Furthermore, maximal stimulation of DNA synthesis is also obtained when 3T3 cells, serum-stimulated for only 1 h, are subsequently incubated with IGF-1. Our results indicate that short pulses of growth factors render 3T3 cells capable of responding to IGF-1, either by increasing the number of IGF-1 receptors or by providing a new substrate for the activated receptor.

3T3 Cells↗
PubMed1993

Basic fibroblast growth factor stimulates DNA synthesis in cells overexpressing the insulin-like growth factor-I receptor.

Insulin-like growth factor-I (IGF-I), by itself, cannot sustain the growth of BALB/c 3T3 cells, but requires the cooperation of other growth factors, such as platelet-derived growth factor or epidermal growth factor. In 3T3 cells constitutively overexpressing the human IGF-I receptor, called p6 cells, IGF-I by itself is fully mitogenic. We show here that p6 cells are also stimulated to enter DNA synthesis by the sole addition of basic fibroblast growth factor (bFGF), which, by itself, is incapable of stimulating parental 3T3 cells. Although bFGF does not bind directly to the IGF-I receptor, it induces its autophosphorylation. Stimulation of p6 cells by bFGF is not inhibited by an antibody to the IGF-I receptor that inhibits IGF-I-mediated DNA synthesis, and IGF-I is not detectable in the medium of bFGF-treated p6 cells. Stimulation cannot be explained by an increased number of FGF receptors, because p6 cells actually have slightly fewer FGF receptors than parental BALB/c 3T3 cells. Basic FGF also stimulates DNA synthesis in 3T3 cells overexpressing a mutant IGF-I receptor that does not autophosphorylate in response to IGF-I and has lost its mitogenic potential. Although we were unable to demonstrate directly that bFGF causes transphosphorylation of the IGF-I receptor, we conclude that in cells overexpressing the IGF-I receptor, bFGF can stimulate DNA synthesis either by an unknown mechanism or through transphosphorylation of the IGF-I receptor.

3T3 Cells↗
PubMed1993

Oncogenes, growth factors and control of the cell cycle.

Recent advances in the field of cell cycle progression have extended our knowledge of the mechanism(s) of cellular proliferation and have placed it on a solid molecular basis. This survey highlights some of these findings, selecting the most intriguing in the hope of providing new insights on cell cycle controls. We have considered two large groups of factors involved in cell cycle control: extracellular (environmental signals, ie growth factors, and their cellular receptors) and intracellular (oncogenes and anti-oncogenes), indicating which, to us, may be the most promising areas of investigation. These include the events occurring after the IGF1 receptor is activated by its ligand, the role of anti-oncogenes in preventing the activation of growth factor receptors, the mechanism(s) by which some proto-oncogenes may short circuit the cell cycle and the use of peptide analogues and the antisense strategy to study and to regulate cellular proliferation.

Animals↗
PubMed1992

Inhibition of cellular proliferation by peptide analogues of insulin-like growth factor 1.

The activation of the insulin-like growth factor 1 (IGF-1) receptor by its ligand plays a central role in the growth of most cell types. We have used the techniques of computational chemistry in order to design and synthesize several novel analogues of IGF-1. These analogues were able to inhibit the autophosphorylation of the IGF-1 receptor as well as the growth of several different cell types, including prostate carcinoma cells and SV40-transformed cells. Additionally, we have found that D-amino acid analogues of these peptides are apparently resistant to the proteolytic degradation that occurs in the presence of whole sera. Consequently, these analogues seem to show great potential both as probes of the structure/function activities of the IGF-1 signalling pathway and as novel clinical strategies in controlling abnormal cellular growth.

3T3 Cells↗
PubMed1992

Inhibition of cell cycle progression by antisense oligodeoxynucleotides.

We have used the antisense strategy to study the role of certain genes in cell cycle progression. In particular, we used antisense oligodeoxynucleotides to study: (1) the role of the IGF-1 receptor in the control of cell proliferation; and (2) the sequence of gene expression during the cell cycle. Our results can be summarized as follows: (1) the activation of the IGF-1 receptor by its ligand, IGF-1, is an obligatory step in the proliferation of fibroblasts and hemopoietic cells; and (2) the expression of DNA synthesis genes, such as PCNA, DNA polymerase alpha, and cdc2, is dependent on the expression of previous genes. A tentative temporal order is: c-myc > c-myb > IGF-1 receptor > DNA synthesis genes.

Animals↗
PubMed1992

Cyclin D1 messenger RNA is inducible by platelet-derived growth factor in cultured fibroblasts.

In fibroblasts in culture, the levels of cyclin D1 mRNA are growth regulated. In mouse and in human fibroblasts, both serum and platelet-derived growth factor increase cyclin D1 mRNA levels with similar kinetics of induction. Insulin-like growth factor 1 by itself does not induce cyclin D1 expression, and an antisense oligodeoxynucleotide to the insulin-like growth factor 1 receptor RNA does not affect the growth-regulated levels of cyclin D1 mRNA.

3T3 Cells↗
PubMed1992

A conserved region in intron 1 negatively regulates the expression of the PCNA gene.

The Proliferating Cell Nuclear Antigen (PCNA) gene is a growth-regulated gene, whose expression is under the control of both transcriptional and posttranscriptional mechanisms. In previous work, it was shown that the 73 bp immediately upstream of the CAP site and intron 4 are major regulatory elements. We show here that intron 1 also plays a role in determining the levels of PCNA mRNA. Specifically, we show: 1) deletion of intron 1 increases the expression of PCNA mRNA in serum-deprived cells; 2) a 35 bp sequence in intron 1, containing a reverse CCAAT element specifically binds proteins from nuclear extracts; 3) this intron 1 sequence inhibits the expression of a co-tranfected human PCNA gene in transient expression assays suggesting that it competes for positive transcription factors; 4) mutations in the CCAAT region of the 35bp intron 1 probe abrogate both its protein-binding capacity and its ability to inhibit the expression of a co-transfected wt PCNA gene; and 5) the CCAAT region of human intron 1 is highly conserved in the mouse gene. We conclude that the reverse CCAAT region of intron 1 is a negative regulatory element of PCNA gene expression, and hypothesize that its inhibitory effect is abolished when certain protein(s) bind to it and that inhibition is restored if these proteins are competed out by an homologous sequence.

Antigens, Neoplasm↗
PubMed1992

A cryptopromoter is activated in the proliferating cell nuclear antigen gene of growth arrested cells.

We have examined the regulation of the proliferating cell nuclear antigen gene (PCNA) in a hamster fibroblast cell line (tk-ts13) which is temperature sensitive for growth. These tk-ts13 cells, at the restrictive temperature, are growth arrested in the G1 phase of the cell cycle. The cells were stably transfected with a full length human PCNA gene, and the resulting cell lines (K525 cells) were analyzed. We find that, in growth arrested K525 cells, a cryptopromoter is activated in the transfected human PCNA gene. The cryptopromoter resides in intron 4 which is necessary for proper regulation of the PCNA gene. Removal of this intron leads to increased expression of PCNA in cells which have entered the G0 state. An Alu sequence residing in intron 4 is implicated as the promoter element which is active during growth arrest.

Animals↗
PubMed1992

The role of the IGF1 receptor in the regulation of cdc2 mRNA levels in fibroblasts.

The levels of cdc2 mRNA increase when quiescent cells are stimulated by growth factors. In BALB/c 3T3, both platelet-derived growth factor and insulin-like growth factor 1 (IGF-1) are required to increase cdc2 mRNA levels. In p6 cells, which constitutively overexpress the IGF-1 receptor, IGF-1 is sufficient. The importance of the IGF-1/IGF-1 receptor interaction in regulating the levels of cdc2 mRNA was further confirmed by showing that an antisense oligodeoxynucleotide to the IGF-1 receptor RNA inhibited the IGF-1-mediated increase.

3T3 Cells↗