Increased synergistic effect of EGF and IGF-I on DNA synthesis of cultured psoriatic keratinocytes.

BACKGROUND: DNA synthesis is significantly more stimulated in response to insulin-like growth factor I (IGF-I) in growth-arrested cultures of psoriatic than of nonpsoriatic keratinocytes. Epidermal growth factor (EGF), by itself only a weak stimulator, was recently found to cause a strong synergetic effect when added together with IGF-I to newborn human keratinocytes. OBJECTIVE: (1) To measure this effect in cultured adult psoriatic and nonpsoriatic keratinocytes, and (2) to examine whether the difference in DNA synthesis after IGF-I addition could be due to differences in the binding of this growth factor. METHODS: 3H-deoxythymidine incorporation and 125I-IGF-I binding studies. RESULTS: Psoriatic keratinocytes demonstrate a considerably stronger response to the combination of EGF and IGF-I than nonpsoriatic keratinocytes. This is apparently not caused by differences in binding of IGF-I. CONCLUSION: Differences in the proliferative rate of normal and psoriatic keratinocytes may be in part due to differences in signal transduction distal from the IGF-I receptor or in the cooperation of the EGF and the IGF-I receptor.

Studies on stimulation of DNA synthesis with epidermal growth factor and insulin-like growth factor-I in cultured human keratinocytes.

Epidermal growth factor (EGF) and insulin are the most widely used growth factors (GFs) in human keratinocyte cultures. Insulin is supposed to exert its growth-promoting activity in this system through the insulin-like growth factor-I (IGF-I) receptor. In order to obtain more information about the contribution of EGF and IGF-I to the proliferation of keratinocytes, the effect of each factor on DNA synthesis was studied with 3H thymidine incorporation in an otherwise GF-free system. Both factors are able to initiate DNA synthesis, DNA synthesis peaks 18-20 h after the addition of each factor, and neither factor influences significantly the binding of the other to the respective receptor. IGF-I is the more potent growth factor, and IGF-I-stimulated cells enter the S-phase regularly approximately 3 h earlier than EGF-stimulated keratinocytes (7-9 h vs 10-12 h). However, IGF-I-stimulated DNA synthesis can be completely turned off by the addition of the monoclonal antibody (mAb) to the EGF receptor LA1. This inhibition cannot be reversed by higher IGF-I concentrations, but only by the addition of EGF to the culture medium. These results may suggest the presence of two keratinocyte populations, one responding to EGF and one to IGF-I, with an additional signal from the EGF receptor, or they may be explained on the basis of only one cell population for which EGF acts as a "competence' factor and IGF-I as a "progression' factor.

Chromosomal damages by ethanol and acetaldehyde in Saccharomyces cerevisiae as studied by pulsed field gel electrophoresis.

We report on the effect of ethanol and acetaldehyde on yeast chromosomal DNA and on isolated DNA. Ethanol induced DNA single-strand breaks in repair deficient but not in repair proficient Saccharomyces cerevisiae. Acetaldehyde has a deleterious effect on chromosomal DNA in cells as well as on isolated DNA. The results presented support earlier data to show that ethanol is mutagenic via its first metabolite, acetaldehyde.

Effect of insulin-like growth factor-I/somatomedin C on thymidine incorporation in cultured psoriatic keratinocytes after growth arrest in growth factor-free medium.

Near confluent cultures of human keratinocytes will arrest in GO/G1 of the cell cycle when they are placed into growth factor (GF)-free medium with a Ca++ concentration below 0.1 mM for 4 days. Addition of certain growth factors will stimulate the cell to re-enter the growth cycle and move into the S-phase after 7-12 h. Insulin-like growth factor/somatomedin C (IGF-I) was found to be the most active factor in promoting 3H-dT incorporation among several growth factors evaluated in this system. This system was applied to keratinocytes cultured from uninvolved skin of psoriatic patients and keratinocytes from clinically healthy skin of nonpsoriatic control persons. 3H-dT incorporation in response to 50 ng IGF-I per ml medium was significantly more stimulated in psoriatic keratinocytes (7.0 +/- 0.41 fold) than in normal keratinocytes (2.9 +/- 0.49 fold). There was no significant difference between the age of donors or the time cells were in culture when the measurements were performed. The stimulatory effect became even more apparent when cultures were growth-arrested in the presence of heparin, which mainly causes a suppression of the 'background' incorporation of 3H-dT occurring in the absence of exogenous GF (psoriatic: 35.0 +/- 2.2 fold; normal: 9.3 +/- 2.1 fold). The increased response of psoriatic keratinocytes to IGF-I does not appear to be related to a difference in time required for the cells to traverse from the presumed restriction point in GO/G1 to the beginning of S (7-10 h) and the middle of S (approximately 19 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 does not stimulate DNA synthesis of cultured human keratinocytes growth-arrested in growth-factor-depleted medium.

Interleukin-1, a multifunctional cytokine, plays a central role in inflammatory processes. Several reports have appeared demonstrating that IL-1 stimulates growth of keratinocytes under certain experimental conditions, and we have shown previously that it can act as a strong stimulator of DNA synthesis in murine keratinocytes that have been growth-arrested by removal of growth factors (GF) from the medium for several days. Using the same assay system, we report here that, in contrast to cultured mouse keratinocytes, growth-arrested adult and newborn human keratinocytes do not respond to IL-1 with an increase in DNA synthesis. The experiments were performed with primary and secondary cells and cells propagated with and without feeder layer prior to the assay. A growth response to IL-1 in the absence of exogenous GF was only observed in non-growth-arrested human keratinocytes, i.e., when the cytokine was added for 24 to 48 h immediately after removal of other GF from the culture. Because keratinocytes continue to grow during this time anyway, although at a reduced rate, the additional growth observed with IL-1 could be explained as an enhancement of the growth-promoting effect by factors not previously completely removed from the culture. We would like to conclude from our results that, contrary to the findings in cultured mouse keratinocytes, IL-1 has no direct mitogenic effect on cultured human keratinocytes, but can still act as a growth promoter under certain conditions, apparently in concert with other GF.

Basic fibroblast growth factor and insulin-like growth factor I are strong mitogens for cultured mouse keratinocytes.

Basic fibroblast growth factor (bFGF), but not acidic fibroblast growth factor (aFGF), was found to be mitogenic for cultured mouse keratinocytes. A six-to-nine fold increase in 3H-thymidine (3H-dT) incorporation into the acid insoluble pool and a similar increase of the labeling index can be measured when bFGF, at a concentration between 1 and 10 ng/ml, is added to keratinocytes arrested in serum-free and growth factor-free medium with a Ca++-concentration below 0.1 mM. The half-maximal response is observed between 0.2 and 0.7 ng/ml. In the same culture system, insulin-like growth factor I/somatomedin C (IGF-I) and insulin act as mitogens. IGF-I shows half-maximal stimulation with 2-3 ng/ml, insulin with 100-500 ng/ml. Basic FGF, IGF-I and insulin can be classified as strong stimulators of DNA synthesis in mouse keratinocytes. In this regard they are comparable to epidermal growth factor, which shows a half-maximal stimulation at a concentration between 1.5-2 ng/ml. These results show that in addition to mesenchymal cells, FGF is a growth factor not only for neuroectodermal cells, but ectodermal cells in general. They further support the idea that the growth promoting effect of insulin on keratinocytes may be mediated by the IGF-I receptor.

Tyrocidine-induced modulation of the DNA conformation in Bacillus brevis.

Using the [3H]trimethylpsoralen photobinding method [Sinden, R.R., Carlson, J.O. & Pettijohn, D.E. (1980) Cell 21, 773-783], a decrease in unrestrained torsional tension of DNA was detected in Bacillus brevis cells when they had entered the sporulation phase. This decrease in superhelicity was found in cells which synthesized the peptide antibiotic tyrocidine and which were stimulated to sporulate. Fluctuations in superhelicity probably reflect a highly complicated picture of tension-relaxing and tension-inducing activities. Addition of tyrocidine to vegetative cells reduced by one-half the torsional tension from DNA, whereas ethidium bromide relaxes DNA completely. Cross-links between DNA and tyrocidine were introduced with ultraviolet light in vitro and in vivo indicating that the modulation of the DNA conformation in the cell may in fact be due to a DNA-tyrocidine interaction. In a growing B. brevis culture exogenous [3H]tyrocidine could only be photobound to DNA after the cells had entered the sporulation phase. Our results could mean that the peptide antibiotic tyrocidine is active in B. brevis on the DNA level as one regulatory factor controlling DNA functions.

The GTP pool in Bacillus brevis and its significance for sporulation.

The GTP pool of Bacillus brevis as well as that of other nucleotides is highly sensitive to all kinds of environmental changes like the cell transfer procedures or nutrient depletion of the cells. In growing cultures, as well as in cells transferred from rich to nitrogen-deficient medium, the nucleotide pool decreases significantly. This decrease is followed by the onset of sporulation only when cells are allowed to produce the peptide antibiotic tyrocidine or if tyrocidine is added to the culture. However, exogenous tyrocidine is active in triggering sporulation only when it is added within a short period of time immediately after shift down, that is when the nucleotide pool is observed to decrease.

A major factor contributing to epidermal proliferation in inflammatory skin diseases appears to be interleukin 1 or a related protein.

Human peripheral blood leukocytes can stimulate G1(G0)-arrested mouse skin keratinocytes to enter the cell cycle again and synthesize DNA at the maximum rate 15-20 hr later. This growth-promoting activity is released by the monocyte fraction and is shown to have characteristics that have been reported for interleukin 1 (IL-1). Pure IL-1 is active in stimulating keratinocyte cultures as was shown with recombinant human IL-1. An IL-1-like protein released by monocytes-macrophages could explain the hyperproliferative epidermis found in certain types of inflammatory skin diseases.

DNA-supercoiling is affected in vitro by the peptide antibiotics tyrocidine and gramicidin.

Tyrocidine, a peptide antibiotic produced by Bacillus brevis (ATCC 8185), relaxes superhelical DNA in a biphasic manner and induces 'packaging' of the DNA at higher concentrations. This was concluded from studies using the sensitive 4,5',8-trimethylpsoralen photobinding technique [Sinden, R. R., Carlson, J. O. & Pettijohn, D.-E. (1980) Cell 21, 773-783]. Relaxed DNA is not affected by tyrocidine whereas linearized molecules become packaged. The linear gramicidin synthesized by the same strain reverses the tyrocidine-induced relaxation as well as the packaging, an observation which might be of biological relevance.

BSC-1 growth inhibitor/type beta transforming growth factor is a strong inhibitor of thymocyte proliferation.

Growth inhibitor/type beta transforming growth factor purified from BSC-1 cells and human platelets is shown to strongly inhibit the proliferation of Con A-stimulated mouse thymocytes. The inhibition can be achieved with growth inhibitor/type beta transforming growth factor concentrations approximately equal to 1/10th those necessary to inhibit keratinocyte cultures. The inhibitory effect in thymocyte cultures can be reversed by the addition of interleukin 2. These findings suggest that growth inhibitor/type beta transforming growth factor is a naturally occurring immunoregulator.

Peptide antibiotics and sporulation: induction of sporulation in asporogenous and peptide-negative mutants of Bacillus brevis.

Mutants of Bacillus brevis ATCC 8185 were isolated which were unable to produce detectable amounts of either tyrocidine or linear gramicidin, or both peptide antibiotics. Tyrocidine-negative mutants (BM5, BM21, BM44) sporulated normally. Gramicidin-negative mutants (BM2, BM24) were oligosporogenous, and mutants unable to produce both peptides (S18, S19) were asporogenous. Addition of tyrocidine and/or gramicidin to asporogenous mutants in rich medium did not stimulate sporulation. However, these mutants formed normal spores after being transferred to nitrogen-free medium and upon the addition of tyrocidine. It was demonstrated that nutrient broth has a suppressive effect on tyrocidine-induced sporulation of S18. The tyrocidine-negative mutant BM44, sporogenous in rich medium, could sporulate under nitrogen deprivation only if supplemented with tyrocidine. The significance of the peptide antibiotics for a regulatory role in sporogenesis of B. brevis is discussed.

Stimulation of DNA synthesis in cultured mouse epidermal cells by human peripheral leukocytes.

Primary epidermal cells derived from the skin of newborn mice can be arrested in G1(G0) of the cell cycle when kept in serum-free medium with 0.06 mM Ca++ for 4 days. These cells can be restimulated to grow by the addition of Chelex 100-treated inactivated fetal calf serum, as shown by liquid scintillation counting and autoradiography after [3H]-thymidine incorporation and by counting mitotic figures. This system may be useful in studying growth-promoting and -inhibiting substances in vitro, and it is shown here that leukocytes from peripheral human blood are able to stimulate the DNA synthesis of epidermal cells although the effect is smaller than that observed with serum. The stimulation was not inhibited by pretreatment of leukocytes with mitomycin C, excluding the possibility that the increased incorporation of radioactivity in mixed epidermal-leukocyte cultures is due to increased DNA synthesis in leukocytes stimulated by epidermal cells. Therefore, this system may also be helpful in the investigation of the role of leukocytes in the hyperproliferation of the epidermis.

Induction of sporulation in Bacillus brevis. 1. Biochemical events and modulation of RNA synthesis during induction by tyrocidine.

Under conditions of severe nitrogen starvation, brought about by nutritional shift-down, Bacillus brevis ATCC 8185 was unable to sporulate unless supplemented with the peptide antibiotic tyrocidine. The induction of sporulation was highly specific for tyrocidine and required only very low concentrations of the peptide (5 microM). Tyrocidine-induced sporulation was accompanied by the typical sporulation-specific events (e.g. extracellular protease production and dipicolinate synthesis) as well as the formation of linear gramicidin. The addition of tyrocidine produced acute inhibition of RNA synthesis that was followed by a limited activation of transcription near the time of onset of linear gramicidin synthesis, when the first sporulation-specific changes were observed. These results provide direct evidence for a role of tyrocidine in sporulation of B. brevis and suggest that the action of the peptide antibiotic may involve the control of transcription. Such a notion is supported by earlier studies on the effects of tyrocidine and linear gramicidin on purified RNA polymerase.

Induction of sporulation in Bacillus brevis. 2. Dependence on the presence of the peptide antibiotics tyrocidine and linear gramicidin.

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2-amino-3-hydroxy-3-phenylpropanoic acid [Mach, B., Reich, E., and Tatum, E. L. (1963) Proc. Natl Acad. Sci. USA, 50, 175-181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L-tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen-limited cultures. When either tyrocidine or linear gramicidin was added together with L-tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L-tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics.

Functions of the peptide antibiotics tyrocidine and gramicidin. Induction of conformational and structural changes of superhelical DNA.

The peptide antibiotic tyrocidine which is produced by Bacillus brevis and is probably involved in sporogenesis, unwinds superhelical plasmids in vitro at low peptide: DNA ratios, as found by gel electrophoresis. At higher peptide concentrations, the DNA is packed tightly leading to apparent nuclease stability of the complex and inhibition of RNA synthesis. The addition of the linear gramicidin, another peptide antibiotic synthesized by the same bacterial strain, partially restores transcription by breaking down the tightly packed DNA X peptide complex. The complexed DNA, after nuclease digestion, is retained on a nitrocellulose filter, but loses its affinity for the filter in the presence of gramicidin. The results are discussed with respect to possible functions of the two peptides within in the cell.

Harman and norharman: induction of sister-chromatid exchanges in human peripheral lymphocytes in vitro and interaction with isolated DNA.

Harman, but not norharman, induced sister-chromatid exchanges in human peripheral lymphocytes in vitro. Transcription of isolated DNA in vitro was inhibited by both compounds, harman being more effective than norharman. A filter-binding assay with isolated DNA showed that harman binds more effectively to the DNA than norharman.

Increased phosphatidylinositol synthesis in rat embryo fibroblasts after growth stimulation and its inhibition by delta-hexachlorocyclohexane.

When resting rat embryo fibroblasts are stimulated to grow, a substantial increase in phosphatidylinositol synthesis can be observed. This increase cannot be explained by increased glucose uptake or glycolysis. delta-Hexachlorocyclohexane having the same configuration as myo-inositol, inhibits phosphatidyl inositol synthesis as well as DNA synthesis and mitosis, but has no effect on phosphatidyl choline synthesis. When delta-hexachlorocyclohexane is added to fibroblast cultures during the first hours after stimulation, a delay of DNA synthesis and mitosis compared to uninhibited cultures can be observed. Since delta-hexachlorocyclohexane also inhibits the uptake of nucleotides, hexoses and amino acids, it is suggested that phosphatidylinositol is necessary for the proper functioning of those receptors and carriers which are an essential part of the early cellular processes after growth stimulation, and this role of phosphatidyl-inositol may explain its increased turnover in growing cells. The increased phosphatidylinositol synthesis could not be associated to one of the subcellular fractions. When cells were labeled with [32P]orthophosphate during the first 10 min after growth stimulation and were subsequently separated into cellular fractions such as nuclei, mitochondria, plasma membranes and microsomes, no significant differences in radioactivity of phosphatidylinositol among those fractions could be observed.

Stimulation of DNA synthesis any myo-inositol incorporation in mammalian cells.

Phosphatidylinositol (PI) synthesis and its role in controlling the cell cycle has been investigated using fibroblasts and liver cells in culture. PI synthesis as measured by incorporation of [3H]-myo-inositol into trichloroacetic acid precipitable material during 0--60 min after serum or growth factor stimulation of serum-starved cells is increased in primary fetal rat liver cells, rat embryo fibroblasts, and 3T3 mouse cells. In contrast, growth stimulation of 3T3 cells and hepatocytes rendered quiescent in G1 by amino acid starvation is not accompanied by increased incorporation of [3H[-myo-inositol into trichloroacetic acid precipitable material. This suggests that those cells might be arrested at a different point in G1 than cells arrested by serum depletion. Inhibition of PI synthesis by variation of-hexachlorocyclohexane (HCH), a steric analog of myo-inositol, during early times (e.g., 0--4 hr) after growth stimulation, reversibly blocks initiation of DNA synthesis in 3T3 cells. The results support the idea that increased PI synthesis in response to growth stimulation in the cell types studied here is a prerequisite for progression through G1 and subsequent entry into S phase.