P6 Cells Research Articles

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.

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.

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.

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.

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.

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.

Involvement of plasmids in total degradation of chlorinated biphenyls.

Acinetobacter sp. strain P6 has previously been reported to utilize biphenyl (BP) and chlorinated BPs, with accumulation of corresponding chlorobenzoic acids. Arthrobacter sp. strain M5 was isolated as a contaminant in the culture of Acinetobacter sp. strain P6 growing on 4-chlorobiphenyl and showed properties similar to P6 in the degradation of chlorinated BPs. Both strains harbored an identical plasmid of 53.7 megadaltons. These strains spontaneously lost the ability to utilize BP and 4-chlorobiphenyl with high frequency (4 to 8%) after overnight growth in nutrient broth. The BP- derivatives could not regain the BP-assimilating ability (reversion frequency, less than 10(-9) per cell per generation) but retained the plasmid with small, detectable deletions. BP+ P6 cells grown on BP or benzoate oxidized BP and 2,3-dihydroxybiphenyl and produced meta cleavage compounds from the latter compound (lambda max, 434 nm) and also from catechol (lambda max, 375 nm) through the meta pathway. On the other hand, benzoate-grown BP- segregants totally lost the BP-metabolizing activities and oxidized catechol through the ortho pathway. A combined culture of the chlorinated BP-dissimilating P6 or M5 strain (harboring the putative 53.7-megadalton plasmid specifying conversion of chlorobiphenyls to chlorobenzoic acids) and genetically constructed mono- or dichlorobenzoate-utilizing pseudomonads (harboring plasmids encoding complete utilization of mono- or dichlorobenzoates) allowed greater than 98% utilization of mono- and dichlorobiphenyls, with the liberation of equivalent amounts of chloride ions.

Phenotypic properties of a large-cell, radiation-resistant strain of Escherichia coli.

Mean cell volumes, total amount of deoxyribonucleic acid (DNA), number of nuclear bodies, and X-ray responses were determined for exponential cultures of Escherichia coli P6, a large-cell radiation-resistant mutant strain, and for E. coli 82/r, the parent strain of P6, growing in three different media at varying growth rates. Combined results comparing cultures in the same growth medium indicated that exponentially growing P6 cells are 2,5 ± 0.2 times larger, contain 3.6 ± 0.3 times more DNA, and 1.8 ± 0.1 times more nuclear bodies than comparable 82/r cells. Individual P6 nuclear bodies contained 2.01 + 0.13 times as much DNA as 82/r nuclear bodies in comparable cultures, suggesting that each P6 nuclear body may contain two complete 82/r genomes. All P6 cultures gave sigmoidal X-ray survival curves with extrapolation numbers averaging 2.0 and with limiting slopes smaller in absolute value than the slopes of the exponential survival curves obtained for 82/r cultures. P6 cells appeared to be no more efficient than 82/r cells in enzymatic dark repair of X-ray or ultraviolet damage, suggesting that the greater X-ray resistance of P6 cells may be associated with the doubled DNA content of each P6 nuclear body.