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Biomedical subjects

R Baserga

Publications and source records attributed to R Baserga.

At least 145 records · Page 8Linked to original sources
PubMed1992

The growth-stimulatory effect of simian virus 40 T antigen requires the interaction of insulinlike growth factor 1 with its receptor.

We have used a plasmid expressing a temperature-sensitive (ts) mutant of simian virus 40 (SV40) T antigen, stably transfected into 3T3 cells, to study the role of insulinlike growth factor 1 (IGF-1) and its receptor in T-antigen-mediated growth. While 3T3 cells do not grow in serum-free medium, in 1% serum, or with the sole addition of either platelet-derived growth factor (PDGF) or IGF-1, cells expressing the tsA T antigen (BALB 58 cells) grow at 34 degrees C in either PDGF or 1% serum but not in IGF-1. At the restrictive temperature (39.6 degrees C), these cells can only grow in 10% serum. We show that BALB 58 cells, at 34 degrees C, have a markedly increased expression of IGF-1 and IGF-1 mRNA and that their growth in 1% serum (at 34 degrees C) is inhibited by an antisense oligodeoxynucleotide to the IGF-1 receptor RNA. When this tsA plasmid is stably transfected into cells constitutively overexpressing the human IGF-1 receptor cDNA, the resulting cell lines show a constitutively phosphorylated IGF-1 receptor and grow in serum-free medium at 34 degrees C (but not at 39.6 degrees C). A functional SV40 T antigen also increases the expression of a plasmid in which the reporter luciferase gene is under the control of a rat IGF-1 promoter. We conclude (i) that the SV40 T antigen induces the expression of IGF-1 and IGF-1 mRNA, at least in part by a transcriptional mechanism, thus altering the growth factors requirements, and (ii) that, in BALB/c3t3 cells, the SV40 T antigen necessitates a functional IGF-1 receptor for its growth-stimulating effect in low serum (or PDGF).

3T3 Cells↗
PubMed1992

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.

3T3 Cells↗
PubMed1992

The double life of the IGF-1 receptor.

The IGF-1 receptor is expressed in many cell types, and its activation by its ligands is a required step for the proliferation of many cells in vivo and in vitro. In most cells in culture, requiring more than one growth factor for growth, the IGF-1 receptor can be found in one of two different modes: in the first mode, although it is autophosphorylated by its ligands and induces the expression of specific genes, it does not transmit a mitogenic signal. In the alternative mode, i.e., after priming with an unrelated growth factor, the IGF-1 receptor responds to its ligands with a mitogenic stimulus. This review examines briefly the possible alternatives to explain this different behavior, which is crucial to our understanding of the control of cellular proliferation.

Animals↗
PubMed1992

Constitutive expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor abrogates all requirements for exogenous growth factors.

BALB/c3T3 cells are exquisitely growth regulated and require both platelet-derived growth factor and insulin-like growth factor-1 (IGF-1) for optimal proliferation. BALB/c3T3 cells that constitutively express IGF-1 and elevated levels of IGF-1 receptor (IGF-1R) are capable of growth in serum-free medium without the addition of any exogenous growth factors. BALB/c3T3 cells overexpressing only the IGF-1R plasmid required IGF-1 or insulin for serum-free growth. Antisense oligodeoxynucleotides complementary to IGF-1R mRNA inhibited IGF-1-mediated cell growth. Under these conditions, neither the epidermal growth factor receptor nor phospholipase C gamma 1 was autophosphorylated. These findings indicate that constitutive expression of IGF-1 and IGF-1R allows 3T3 cells to grow in serum-free medium without addition of those exogenous growth factors that are required by the parent cell line.

3T3 Cells↗
PubMed1992

The insulin-like growth factor 1 receptor is required for the proliferation of hemopoietic cells.

We have investigated the role of the insulin-like growth factor one (IGF1) receptor and its relationship to the proto-oncogene c-myb in the growth of two types of hemopoietic cells: mitogen-stimulated human peripheral blood mononuclear cells and a human promyelocytic cell line (HL-60). Using the antisense strategy and the reverse transcriptase polymerase chain reaction (RT-PCR), we show that expression of the IGF1 receptor is required for the entry into S phase of both stimulated lymphocytes and HL-60 cells. The inhibition of DNA synthesis by antisense oligomers to the IGF1 receptor RNA is accompanied by an inhibition of the expression of the mRNA for a DNA synthesis gene, proliferating cell nuclear antigen (PCNA), the co-factor of DNA polymerase delta. Inhibition of c-myb expression results in a decrease in IGF1 receptor mRNA levels; on the other hand, inhibition of IGF1 receptor expression has no effect on c-myb mRNA levels. A tentative temporal relationship between these three genes (c-myb, IGF1 receptor, PCNA) is proposed.

Base Sequence↗
PubMed1992

The role of the promoter in the expression of the PCNA gene.

G1-specific temperature-sensitive (ts) mutants of the cell cycle arrest in G1 after serum stimulation at the restrictive temperature. Under these conditions, the RNA levels of late growth-regulated genes (such as DNA polymerase alpha, PCNA, thymidine kinase, and core histones) are markedly decreased or even undetectable, while early growth-regulated genes (for instance, c-myc) are normally expressed, and certain promoters are actually super-induced. We have used the human PCNA gene transfected into TK-ts13 cells (a G1-specific ts mutant) to investigate whether the inhibition of gene expression caused by this type of growth inhibition occurs at a transcriptional or post-transcriptional level. Constructs were made in which the 5' and 3' flanking sequences of the human PCNA gene were replaced by the corresponding elements of the SV40 T antigen coding gene. Using these constructs and data from run-on assays and RT-PCR, we conclude that the failure of expression of the PCNA gene in G1-arrested TK-ts13 cells occurs at the transcriptional level.

Animals↗
PubMed1991

The protooncogene c-myb increases the expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor messenger RNAs by a transcriptional mechanism.

The protooncogene c-myb is the cellular equivalent of the viral transforming oncogene v-myb. When human c-myb is constitutively expressed in Balb/c3T3 cells it abrogates their absolute requirement for insulin-like growth factor 1 (IGF-1). We show now, in two different cell lines, that the constitutive expression of the protooncogene c-myb causes an increase in both IGF-1 and IGF = 1 receptor mRNA levels. This increase in mRNA levels is due, at least in part, to an increase in the rate of transcription since, by run-on assay, cells carrying the human c-myb cDNA show a 3-fold increase in transcriptional rates in comparison to the control parent cell lines. The increased expression of IGF-1 receptor mRNA also results in an increased number of IGF-1 binding sites per cell. Although some oncogenes have been described that are homologous to growth factors, or growth factor receptors, c-myb seems to represent a novel way of oncogene action inasmuch as it increases the expression of both a growth factor receptor and its ligand, thus establishing a quasi-autocrine mechanism which modifies the growth factor requirements of the cell and its growth regulation.

3T3 Cells↗
PubMed1991

Senescent human fibroblasts have a post-transcriptional block in the expression of the proliferating cell nuclear antigen gene.

The product of the proliferating cell nuclear antigen (PCNA) gene is the co-factor of DNA polymerase delta, which is required for cellular and viral DNA replication. Its steady-state mRNA levels are growth-regulated in young human diploid fibroblasts (HDF) as well as in many other cell types. In senescent HDF, PCNA mRNA is not detectable. However, the PCNA gene is transcribed in senescent HDF as efficiently as in young cells. Furthermore, PCNA hnRNA is easily detectable by reverse transcriptase-polymerase chain reaction in both senescent and young HDF, and the levels are essentially similar. These results indicate that in senescent HDF which are incapable of synthesizing cellular DNA, one of the genes coding for a protein of the DNA-synthesizing apparatus is still transcribed, but the product fails to be processed into mature mRNA.

Cell Cycle↗
PubMed1991

Effect of the SV40 T antigen on the posttranscriptional regulation of the proliferating cell nuclear antigen and DNA polymerase-alpha genes.

tk-ts13 cells are G1-specific temperature-sensitive mutants of the cell cycle that arrest in G1 at the restrictive temperature. In these cells the mRNAs for early growth-regulated genes (for instance, c-myc) are inducible by serum at both permissive and restrictive temperatures. In contrast, the mRNAs for late growth-regulated genes [such as histones, proliferating cell nuclear antigen (PCNA), and DNA polymerase-alpha] are not detectable at the restrictive temperature, although they are normally induced by serum at the permissive temperature. Despite the absence of their mRNAs at the restrictive temperature, transcription rates for DNA polymerase-alpha, PCNA, and histone H3 are the same in serum-deprived cells and in cells that are serum stimulated at either the permissive or the restrictive temperature. Since the half-lives of the mRNAs are not substantially different at the two temperatures, the conclusion is that in tk-ts13 cells the mRNA levels of these late growth-regulated genes are regulated at a posttranscriptional level, presumably during hnRNA processing. When serum-deprived tk-ts13 cells carrying a stably integrated SV40 T antigen-coding gene (T-neo cells) are stimulated with serum, they are capable of one additional round of DNA replication at the restrictive temperature. At 20 h after stimulation of T-neo cells, the mRNAs for the late growth-regulated genes are detectable at the restrictive temperature in amounts not substantially different than those at the permissive temperature. Transcription rates in T-neo cells are increased for histone H3 (in comparison to tk-ts13 cells) but not for PCNA and DNA polymerase-alpha. The presence of the T antigen does not seem to seriously affect the half-lives of the mature mRNAs. The conclusion is that the presence of the SV40 T antigen in tk-ts13 cells promotes the appearance of mature mRNAs for DNA polymerase-alpha and PCNA. These experiments suggest that T antigen, in this instance, may intervene either directly or indirectly at a posttranscriptional level in the regulation of the steady state mRNA levels of certain cellular genes.

Animals↗
PubMed1991

Growth regulated expression of B-myb in fibroblasts and hematopoietic cells.

The B-myb cDNA has extensive sequence similarities to the c-myb proto-oncogene, but, at variance with c-myb, it is expressed in cells other than hematopoietic cells. In this paper, we show that (1) B-myb is expressed in mouse, human, and hamster fibroblasts; (2) B-myb mRNA levels are growth-regulated in both fibroblasts and peripheral blood mononuclear cells; (3) by its mode of growth regulation (peak of expression, behavior in G1-specific temperature sensitive (ts) mutants and in the presence of cycloheximide), B-myb can be classified, like c-myb, thymidine kinase, PCNA, and others, as a late growth-regulated gene; (4) B-myb mRNA levels decrease when HL-60 cells are induced to differentiate; and (5) the increase in mRNA levels in serum-stimulated cells is only partially explained by an increase in rate of transcription. The possibility that the B-myb gene may be the equivalent in fibroblasts and epithelial cells of the c-myb proto-oncogene of hematopoietic cells is discussed.

Animals↗
PubMed1991

Characterization of an enhancer-like structure in the promoter region of the proliferating cell nuclear antigen (PCNA) gene.

The steady-state mRNA levels of the proliferating cell nuclear antigen (PCNA) gene depend on the length of its promoter. A promoter extending from the HpaII restriction site at -210 from the cap site to the cap site itself is very active, while a -45 promoter (AatII restriction site) is very weak. We now show that the sequences between -73 and -45 of the human PCNA promoter contain an enhancer-like sequence that markedly increases the levels of PCNA mRNA. This sequence has characteristics of an enhancer, having an enhancing function also when placed away from the native position in the 5' flanking sequence. The increase in mRNA levels that occurs after serum stimulation, however, is independent of the enhancer. Synthetic promoters were also constructed containing mutations in the -73 to -45 sequence and these mutants completely lost their ability to drive the transcription of a heterologous cDNA. Nuclear proteins were shown to bind to this sequence, both by gel shift and by methylation interference analysis. We conclude that the levels of PCNA mRNA are controlled, in part, by a structure located in the 5' flanking sequence of the gene, but that this enhancer-like structure does not play a role in the serum regulation of the mRNA levels.

Animals↗
PubMed1991

Constitutively expressed c-myb abrogates the requirement for insulinlike growth factor 1 in 3T3 fibroblasts.

The proto-oncogene c-myb, whose expression is usually limited to cells of the hematopoietic lineages, can be expressed in fibroblasts if placed under the control of a constitutive promoter, such as the simian virus SV40 early promoter. 3T3 cells carrying a constitutively expressed human c-myb were found to grow in 1% serum or in a serum-free, platelet-derived growth factor-supplemented medium, whereas the parent cell line, BALB/c 3T3, needed insulinlike growth factor 1 (IGF-1) in addition to platelet-derived growth factor for growth. myb-carrying cells, however, could not grow in platelet-poor plasma. In fibroblasts, therefore, a constitutively expressed c-myb can abrogate the requirement for platelet-poor plasma or IGF-1. When 3T3 cells constitutively expressed both c-myc and c-myb, they could grow in serum-free medium without added growth factors. The ability of c-myb to abrogate in fibroblasts the IGF-1 requirement seems to be due to its ability to induce overexpression of IGF-1, as indicated by an increase in steady-state levels of IGF-1 mRNA. These results have some important implications; for instance, they suggest a commonality of pathways for entry into S phase in different cell types and the possibility of a myb-like or myb-equivalent gene product of critical importance for entry of fibroblasts into S phase.

Animals↗
PubMed1991

Effect of the myb gene product on expression of the PCNA gene in fibroblasts.

Tk-ts13 cells are BHK-derived fibroblasts that have a G1-specific temperature sensitive (ts) mutation so that the cells arrest in the G1 phase of the cell cycle at the restrictive temperature of 39.6 degrees. We have introduced into these cells a plasmid carrying the human c-myb cDNA under the control of the early SV40 promoter. The resulting cell line, ts13 myb cells, express the c-myb RNA and protein and, when serum-stimulated, they undergo one round of DNA replication even at the restrictive temperature. Under the same conditions, the parent cell line (tk-ts13 cells) are blocked in G1 and fail to replicate DNA. We have investigated the expression of two late growth-regulated genes, PCNA and histone H3, in tk-ts13 and in ts 13 myb cells, both at permissive and restrictive temperature. At the permissive temperature of 34 degrees, the mRNA levels for PCNA and histone H3 increase markedly after serum stimulation in both cell lines, reaching a peak at the time of DNA synthesis. At the restrictive temperature of 39.6 degrees, the mRNA's for PCNA, and histone H3 are detectable in serum-stimulated ts13 myb cells while they are not detectable in the parent tk-ts13 cell line. Run-on transcription assays indicate that these 2 genes are transcribed in tk-ts13 cells equally well at permissive and nonpermissive temperatures, and that the presence of the myb product does not significantly increase their rates of transcription. The stability of the respective mRNA's is roughly the same at either temperature and in both types of cells. These results indicate: (1) a constitutively expressed c-myb gene product confers to fibroblasts the ability of temporarily by-passing a ts block in the G1 phase of the cell cycle and (2) the myb product, under these conditions, regulates the mRNA levels of PCNA and histone H3 either directly or indirectly by a post-transcriptional mechanism.

Animals↗
PubMed1991

A new growth-regulated complementary DNA with the sequence of a putative trans-activating factor.

A new complementary DNA (cDNA) clone has been isolated by differential screening of a cDNA library. The cognate RNA of this clone, called SC1, is growth regulated in human, mouse, and hamster cell lines. Its kinetics of growth regulation (time of increase in mRNA levels, sensitivity to cycloheximide, behavior in G1-specific temperature-sensitive mutants) classify the SC1 gene as a late growth-regulated gene, like the histone genes and the genes coding for the proteins of the DNA synthesis apparatus. By run-on assay, there is a modest increase in transcriptional rates after serum stimulation, which is not sufficient to explain the sharp increase in mRNA levels. The SC1 gene localizes to human chromosome 6p21-22. In bacteria, the SC1 cDNA clone makes a protein of Mr 39,000, in agreement with the putative reading frame. The amino acid sequence derived from the cDNA sequence indicates a previously unknown gene with a domain strongly suggestive of a trans-activating domain. The SC1 gene can be considered as coding for a possible new trans-activating factor that could play an important role in the transcription of genes required for the later stages of cell cycle progression.

Amino Acid Sequence↗
PubMed1990

The human fibroblast adenine nucleotide translocator gene. Molecular cloning and sequence.

The human adenine nucleotide translocator multigene family consists of three transcribed genes and at least seven pseudogenes. We report the cloning and sequencing of the gene expressed in human fibroblasts. Like the other two transcribed genes, it has four exons. The 5'-flanking region of the fibroblast translocase gene contains a functional promoter. The promoter has a TATA motif beginning 30 base pairs upstream of the transcription initiation site and five potential SP1 binding sites, but lacks a canonical CCAAT box immediately upstream of the TATA sequence. Differences among the sequence motifs of the 5'-flanking region of the three human translocase genes could account for differences in their cell type-specific and proliferation-associated expression.

Base Sequence↗