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Virchows Arch. B Cell Path. 31, 67 73 (1979)

9 by Springer-Verlag 1979

On Serum as a Mitogen in Intact Tissue Klas Norrby and Anders Grimvall Department of PathologyII and Department of Mathematics, Universityof Link6ping, S-581 85 Link6ping, Sweden

Summary. The mitogenic effect of serum in defined cells in organ-cultured normal intact membranous rat mesentery was studied. Serum caused a strictly concentration-dependent stimulation of proliferation in mesothelial-cell-like and fibroblast-like cells, whereas the sequence S-G2-mitosis was apparently unaffected by serum. The results support the concept that similar mitogenic mechanisms may operate in cell culture and in situ in an intact tissue. Key words: Serum - Mitogenesis - Organ-culture - Mesentery - Fibroblasts - Mesothelial cells.

Introduction

Serum is a potent mitogen in cell cultures. It is a complex mixture of salts, lipids, hormones, enzymes, and at least 60 peptides and proteins (Schultze and Heremans, 1966). It is therefore no wonder that serum may contain various mitogenic factors effective in different cell lines individually or in combination (Baserga, 1976; Holley, 1975; Pledger et al., 1977). Besides the effect of serum growth factors, other factors may modify the mitogenic reaction of cells in cell culture. Such factors include the size of the free surface area of individual cells and hence the number, availability, or mobility of surface receptors for the mitogenic factor(s) (Holtey, 1975), various microenvironmental features including pH, local deposits of catabolic products, the state of diffusion barriers (Holley, 1975; Stoker, 1973), the substrate area available per cell (Dulbecco and Elkington, 1973; Thrash and Cunningham, 1975), and limitation of any of several materials in the medium (Dulbecco and Elkington, 1975; Hassell and Engelhardt, 1977; Holley, 1975). Furthermore, cells can demonstrate varying affinities for different substrates, and the biological properties of cells can be Dr. Klas Norrby, Department of Pathology II, Universityof Link6ping, S-581 85 Link6ping, Sweden Send offprint requests to:

0340-6075/79/0031/0067/$01.40

68

K. Norrby and A. Grimvall

modified by the particular substrate with which they are in contact (Hodges, 1976). This is noteworthy since the plasma membrane seems to play a central initial role in mitogenesis (Holley, 1975), probably by regulating the influx of C a 2 + and/or M g 2 + (Berridge, 1975 ; Andersson and Norrby, 1977; McKeehan and Ham, 1978). The applicability of results regarding stimulation of proliferation obtained using cells in culture to conditions in vivo may be questioned. Cell membranes in cultured cells have relations quite different from those in organised tissue, where they are in contact with the surfaces of other cells, or intercellular connective tissue material. It is unlikely that the differentiation of surfaces into upper and lower areas with different properties, such as occurs in cell culture (DiPasquale and Bell, 1974), takes place in cells in a tissue in situ. It is therefore pertinent to study the mitogenic effect of serum in cells remaining in a tissue in situ. The aim of the investigation now reported was to study the effect of serum on the proliferation in microscopically identified cells in organ-cultured, normal, intact membranous mesentery.

Material and Methods Organ Culture of Mesentery We cultured rat mesenteric membranes from Sprague-Dawley rats killed at 9 a.m. for 52 h, using Eagle's minimum essential medium (MEM) and new-born-bovine serum (Flow Laboratories, Irvine, Scotland) (Norrby and Franz6n, 1979).

Proliferation Variables Microdensitometric Feulgen-DNA Analysis. DNA in randomly selected fibroblast-like and mesothelialcell-like nuclei was assessed with the aid of a Vickers M86 scanning microdensitometer (EnerbS.ck et al., 1976; Franz6n and Norrby, 1977; Norrby and Franz6n, 1979). The instrument was connected to a tape punch. This was fed to a PDP 8 minicomputer which constructed a DNA-frequencydistribution histogram. No DNA values beyond the 2n-4n range were found. Free Hand Estimation of the Fraction of G1 Cells. With free hand drawing of the boundary between G1 and S at the upper end of the modal distribution, the fraction of G1 cells tends to be systematically overestimated, owing to overlapping between G1 and S cells. In the present study we did not distinguish between fibroblast-like and mesothelial-cell-like cells. The measured population was thus a mixed population initially made up of about 52% fibroblast-like cells and about 48% mesothelial-cell-like cells (Franz6n and Norrby, 1977). The number of cells measured per specimen varied between 209 and 218 (mean 212, n=30).

Computer Analysis of DNA Histogramsfor the Estimation of the Fractions of G1, S and G2 Cells. We have also performed a computer analysis of the DNA-histograms and compared the results of this analysis with the free hand procedure. The mathematical model at the bottom of the computer analysis is similar to the one proposed by Macdonald (1975). We have assumed that all GI cells have the same DNA content m and all G2 cells the DNA content 2m. The DNA content of a randomly selected S cell was assumed to have a linear probability density between m and 2m. The measurement errors were assumed to be normal with a standard deviation proportional to the true DNA content. In probability language the model can be described as follows: The observed absorbance value Y of a randomly selected cell is the sum of a quantity X measuring the true DNA content of the cell and a measurement error e = X- rt, where r/is independent

Mitogenesis in Tissue Cells

69

of X and normal with mean 0 and standard deviation or. If the proportions of G1 and G2 cells are denoted by Pl and Pz, we have P{X=m}=pl

P{X=2m}=p2

Furthermore, the conditional probability density of X given m < X < 2m is of the form 1 X-m fm

Lihat lebih banyak...
Virchows Arch. B Cell Path. 31, 67 73 (1979)

9 by Springer-Verlag 1979

On Serum as a Mitogen in Intact Tissue Klas Norrby and Anders Grimvall Department of PathologyII and Department of Mathematics, Universityof Link6ping, S-581 85 Link6ping, Sweden

Summary. The mitogenic effect of serum in defined cells in organ-cultured normal intact membranous rat mesentery was studied. Serum caused a strictly concentration-dependent stimulation of proliferation in mesothelial-cell-like and fibroblast-like cells, whereas the sequence S-G2-mitosis was apparently unaffected by serum. The results support the concept that similar mitogenic mechanisms may operate in cell culture and in situ in an intact tissue. Key words: Serum - Mitogenesis - Organ-culture - Mesentery - Fibroblasts - Mesothelial cells.

Introduction

Serum is a potent mitogen in cell cultures. It is a complex mixture of salts, lipids, hormones, enzymes, and at least 60 peptides and proteins (Schultze and Heremans, 1966). It is therefore no wonder that serum may contain various mitogenic factors effective in different cell lines individually or in combination (Baserga, 1976; Holley, 1975; Pledger et al., 1977). Besides the effect of serum growth factors, other factors may modify the mitogenic reaction of cells in cell culture. Such factors include the size of the free surface area of individual cells and hence the number, availability, or mobility of surface receptors for the mitogenic factor(s) (Holtey, 1975), various microenvironmental features including pH, local deposits of catabolic products, the state of diffusion barriers (Holley, 1975; Stoker, 1973), the substrate area available per cell (Dulbecco and Elkington, 1973; Thrash and Cunningham, 1975), and limitation of any of several materials in the medium (Dulbecco and Elkington, 1975; Hassell and Engelhardt, 1977; Holley, 1975). Furthermore, cells can demonstrate varying affinities for different substrates, and the biological properties of cells can be Dr. Klas Norrby, Department of Pathology II, Universityof Link6ping, S-581 85 Link6ping, Sweden Send offprint requests to:

0340-6075/79/0031/0067/$01.40

68

K. Norrby and A. Grimvall

modified by the particular substrate with which they are in contact (Hodges, 1976). This is noteworthy since the plasma membrane seems to play a central initial role in mitogenesis (Holley, 1975), probably by regulating the influx of C a 2 + and/or M g 2 + (Berridge, 1975 ; Andersson and Norrby, 1977; McKeehan and Ham, 1978). The applicability of results regarding stimulation of proliferation obtained using cells in culture to conditions in vivo may be questioned. Cell membranes in cultured cells have relations quite different from those in organised tissue, where they are in contact with the surfaces of other cells, or intercellular connective tissue material. It is unlikely that the differentiation of surfaces into upper and lower areas with different properties, such as occurs in cell culture (DiPasquale and Bell, 1974), takes place in cells in a tissue in situ. It is therefore pertinent to study the mitogenic effect of serum in cells remaining in a tissue in situ. The aim of the investigation now reported was to study the effect of serum on the proliferation in microscopically identified cells in organ-cultured, normal, intact membranous mesentery.

Material and Methods Organ Culture of Mesentery We cultured rat mesenteric membranes from Sprague-Dawley rats killed at 9 a.m. for 52 h, using Eagle's minimum essential medium (MEM) and new-born-bovine serum (Flow Laboratories, Irvine, Scotland) (Norrby and Franz6n, 1979).

Proliferation Variables Microdensitometric Feulgen-DNA Analysis. DNA in randomly selected fibroblast-like and mesothelialcell-like nuclei was assessed with the aid of a Vickers M86 scanning microdensitometer (EnerbS.ck et al., 1976; Franz6n and Norrby, 1977; Norrby and Franz6n, 1979). The instrument was connected to a tape punch. This was fed to a PDP 8 minicomputer which constructed a DNA-frequencydistribution histogram. No DNA values beyond the 2n-4n range were found. Free Hand Estimation of the Fraction of G1 Cells. With free hand drawing of the boundary between G1 and S at the upper end of the modal distribution, the fraction of G1 cells tends to be systematically overestimated, owing to overlapping between G1 and S cells. In the present study we did not distinguish between fibroblast-like and mesothelial-cell-like cells. The measured population was thus a mixed population initially made up of about 52% fibroblast-like cells and about 48% mesothelial-cell-like cells (Franz6n and Norrby, 1977). The number of cells measured per specimen varied between 209 and 218 (mean 212, n=30).

Computer Analysis of DNA Histogramsfor the Estimation of the Fractions of G1, S and G2 Cells. We have also performed a computer analysis of the DNA-histograms and compared the results of this analysis with the free hand procedure. The mathematical model at the bottom of the computer analysis is similar to the one proposed by Macdonald (1975). We have assumed that all GI cells have the same DNA content m and all G2 cells the DNA content 2m. The DNA content of a randomly selected S cell was assumed to have a linear probability density between m and 2m. The measurement errors were assumed to be normal with a standard deviation proportional to the true DNA content. In probability language the model can be described as follows: The observed absorbance value Y of a randomly selected cell is the sum of a quantity X measuring the true DNA content of the cell and a measurement error e = X- rt, where r/is independent

Mitogenesis in Tissue Cells

69

of X and normal with mean 0 and standard deviation or. If the proportions of G1 and G2 cells are denoted by Pl and Pz, we have P{X=m}=pl

P{X=2m}=p2

Furthermore, the conditional probability density of X given m < X < 2m is of the form 1 X-m fm

Somos uma comunidade de intercâmbio. Por favor, ajude-nos com a subida ** 1 ** um novo documento ou um que queremos baixar:

OU DOWNLOAD IMEDIATAMENTE