Control of DNA synthesis in tissue culture cells

Summary Eukaryotic DNA is functionally divided into thousands of replicons, each of which may be duplicated at a characteristic time within the DNA synthetic (S) period. Our approach toward an understanding of the molecular mechanisms which control orderly eukaryotic DNA synthesis has been: (a) to devise a method of cell synchrony in a suitable tissue culture system wherein all cells in the population enter and traverse the S period with a high degree of synchrony; (b) to determine, utilizing this system, precisely when during the S period critical events and macromolecular syntheses occur; and (c) to examine, by polyacrylamide-gel electrophoresis, the spectrum of proteins which become associated with chromatin during the S period in such a way as to suggest their involvement with DNA synthesis. Possible mechanisms for control are discussed based on the results presented here. Presented in the formal symposium on Mechanisms of Cellular Control at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. The work reported in this communication was supported by NCI Grant CA 18612 to A.B.P.     

A simple method for analysis of DNA histograms in human myeloid cells perturbed by stimulators of DNA synthesis

In the past, phase fractionation has been used as a determinate of the level of stimulation of cells perturbed by stimulators and inhibitors of DNA synthesis. This method has, in our hands, proven to be insensitive and unreliable with human myeloid cells. A new method of analysis is described in this paper which involves utilization of the magnitude of the slope of a line fit to the mid-portion of S phase as an index of the level of stimulation of cultured human myeloid cells. This method is fast, reliable and simple to implement on an inexpensive microcomputer.     

Regulation of connective tissue collagenase production: stimulators from adult and fetal epidermal cells

We have examined the ability of primary adult rabbit skin cells to regulate collagenase production in vitro. Dermal cells constitutively produce collagenase in culture, and enzyme production by these cells can be influenced by epithelial cells. Co-culture with skin epidermal cells resulted in more enzyme production by dermal cells, whereas co- culture with corneal epithelial cells yielded less enzyme activity. Connective tissue cells from a different source, cornea, also produced collagenase when co-cultured with skin epidermal cells, although the stromal cells alone made no enzyme. The drug cytochalasin B had very little influence on collagenase production by dermal cells, either alone or in co-culture with epidermal cells, but did significantly potentiate enzyme production by corneal stromal cells responding to epidermal effector molecules. Epidermal-cell-conditioned medium from both fetal and adult rabbit skin was a potent source of stimulators (apparent mol wt 20,500 and 55,000) of connective-tissue-cell collagenase production. Stimulator production by epidermal cultures was cell density dependent. Optimal production of stimulators occurred in adult cultures containing 10(6) epidermal cells/ml of medium, and in fetal cultures containing 10(5) cells/ml. Inhibitors of connective tissue cell enzyme production were not detected in conditioned medium from either adult or fetal epidermal cells.     

DNA repair in mammalian nerve cells. I. DNA synthesis in the neocortex of rats induced by gamma irradiation

A study was made of the DNA synthesis in cerebral cortex of rats, aged 14 and 60 days, after gamma-irradiation in vivo in a dose of 7 Gy, the 3H-thymidine incorporation into DNA being determined.137 Cs-radiation induces additional DNA synthesis in the neocortex tissue and in neurons. In the cortex of 14 day-old rats, the induced DNA synthesis stops 2 hours after irradiation, whereas in the cortex of 60 day-old rats and in neurons of rats of both the age groups DNA synthesis is proceeding for 3-3.5 hours. Specificity of DNA reparation processes in non-dividing cells is discussed.     

The short-term and long-lasting changes in DNA and RNA synthesis in the cerebral cortex cells of animals subjected to hypoxia and nerve tissue transplantation]

The DNA and RNA synthesis in the cells of the brain cortex of intact rats and animals subjected to hypoxia, hypoxia with subsequent transplantation or by the local brain injury has been investigated. The DNA synthesis changes insignificantly in the case of hypoxia, it enhances slightly in the area of the injury and increases much more after transplantation. The RNA synthesis decreases considerably immediately after hypoxia and decreases much more 120 days later. Using the ultracentrifuge method it has been found that under the effect of hypoxia the number of nervous cells decreases, the number of glial cells does not change. The local injury in the nervous tissue enhances abruptly the synthesis in neurons and glial cells in the hypoxia-exposed animals, the embryonic nervous tissue transplantation normalizes the number of neurons in the specimens under study and the RNA synthesis in the neurons and glial cells.     

Complement-dependent induction of DNA synthesis and cell proliferation in human liver connective tissue cells in vitro

Summary Liver connective tissue cells (LCTC) isolated from patients with fibrotic livers have morphological and biochemical characteristics of myofibroblasts. We have examined the proliferation of LCTC derived from normal livers and from livers with fibrosis of different etiologies, as well as proliferation of skin fibroblasts. We have compared proliferation rates in the presence of fresh human serum and heat-inactivated serum. While skin fibroblast and LCTC from normal liver showed no difference, proliferation of LCTC from fibrotic livers was markedly decreased in the presence of heat-inactivated serum. We demonstrate that the native complement component C1 is a factor involved in the induction of DNA synthesis and proliferation of LCTC isolated from fibrotic livers. We propose that native C1, acting probably in cooperation with other growth factors, is involved in the expansion of connective tissue cells during the development of liver fibrosis.     

DNA synthesis in cytokinin-autotrophic tobacco cells

DNA isolated from various Nicotiana tabacum cell types, differing in their degree of hormone autotrophy and incubated in the presence of bromodeoxyuridine (BrdUrd), was analyzed by isopycnic CsCl gradient centrifugation. All cell types incorporate BrdUrd into DNA in such a way that hybrid DNA is formed with 60–80% of thymine (Thy) residues replaced by bromouracil (BrUra) in the newly synthesized strand. This DNA is not replicated further under ordinary culture conditions. Whereas in normal hormone-dependent cells this state is final and cells necrotize, in tumor (cytokinin-auxin autotrophic) and cytokinin-autotrophic cells a mechanism is induced leading to the reduction of BrUra content in DNA. As a result a decrease in the buoyant density (in CsCl) of BrUra DNA can be observed. In the case of cytokinin-autotrophic cells supplemented with kinetin, the buoyant density of the whole DNA decreases gradually to the value of that of unsubstituted DNA, but specific radioactivities of different DNA fractions reflect the retention of the pyrimidine ring of BrUra in DNA. This is interpreted as debromination of DNA in situ. The process can be inhibited by fluorodeoxyuridine (FdUrd) and deoxycytidine (dCyd). Moreover, FdUrd (but not dCyd) allows replication of hybrid DNA in tumor cells in such a way that HH DNA with all Thy residues replaced by BrUra is formed. For cytokinin-autotrophic cells FdUrd and kinetin are required. In hormone-dependent cells replication of hybrid DNA cannot be induced under any conditions. Most of these conclusions complement our previous findings that BrdUrd tolerance in hormone-autotrophic tobacco cells in hormone controlled. It is postulated that a modulation of thymidylate synthetase specificity is one factor affecting the level of BrUra substitution in DNA. The possibility of cytokinins being involved in the control of DNA synthesis is discussed.Abbreviations BrdUrd 5-bromo-2-deoxyuridine - BrUra 5-bromouracil - dCyd 2-deoxycytidine - FdUrd 5-fluoro-2-deoxyuridine - dThd thymidine - Thy thymine - EDTA Na₂-ethylenedia-minotetraacetate - IAA idole-3-acetic acid (auxin) - SDS Na-dodecylsulphate - LL, HL, HH DNA light-light (unsubstituted), heavy-light (unifilarly BrUra substituted), heavy-heavy (bifilarly BrUra substituted) DNAs, respectively     

Rate of DNA synthesis in binucleate cells

Summary The microspectrophotometric study of the amount of DNA in the course of the interphase of synchronous binucleate cells, experimentally induced in the roots of the onion (Allium cepa), at 30° C, showed that the rate of DNA synthesis does not appear to be constant throughout the interphase, being greater at the beginning and end of the S period and slower during the middle part of the period.We found that the G ₁ period occupies from 0% at 10% of the cell cycle, the S period about 81% and G ₂ period plus mitosis about 14%.Contrary to our expectations, highly significant differences were found in the DNA content of the two nuclei of more than 1% of the binucleate cells, which suggests that DNA synthesis may be asynchronous in the two nuclei within a common cytoplasm.     

Silatranes as stimulators of the development of granulation tissue

Experiments on rats were made to study the effect of I-(chloromethyl) silatrane and I- (ethoxy) silatrane and that of triethanolamine and chloromethyl triethoxysilane on biochemical parameters of granular-fibrous tissue. The preparations were applied to a wound defect in the form of liniments. Silatranes were found to stimulate proliferation of the cells and to increase their biosynthetic activity, to favour accumulation of collagen and non-collagenic proteins, and reduction of the inflammatory phenomena. The silicon-containing fragment of the silatrane molecule chloromethyl triethoxysilane produced an analogous but less marked action. Triethanolamine containing no silicon was found to be as less active. These data indicate that silicon entering the silatrane grouping is of great importance for the occurrence of its biological activity.     

DNA repair in mammalian nerve cells. II. DNA synthesis in neurons, induced by gamma-irradiation of isolated rat neocortex slices

DNA synthesis was studied in the neocortex neurons of 0-, 14- and 60-day old rats after gamma-irradiation in vitro of isolated slices of the neocortex on determining 3H-thymidine incorporation into DNA. Gamma-irradiation (20 Grays) increases levels of DNA synthesis for all the age groups of animals examined. The induced DNA synthesis in neurons of newborn rats is higher than that in 14- and 60-day old animals. Specificity of DNA synthesis in the process of nerve cell differentiation is discussed.     

Fluorometric quantification of DNA in cells and tissue

The validation of a simple and rapid DNA solubilization procedure is described. Quantitative extraction of intact, polymerized DNA was achieved by cell lysis or tissue homogenization in an ammonium hydroxide-Triton X-100 solution. The solubilization procedure inactivates endogenous DNAase and increases the fluorescence-enhancement activity of the extracted DNA, thereby eliminating the need for enzyme treatment or exposure to high salt solutions. The extracts can be utilized directly in a sensitive fluorescence-enhancement assay with bisbenzimidazole (Hoechst 33258) reagent. Estimates of DNA cell content were unaffected by the number of cells lysed or the volume of lysate employed in the assay. In all cases, the solubilized DNA estimates were linear and parallel to the bovine DNA standard. The optimum range for estimation of DNA in this assay is 5-150 ng. In addition, estimates of DNA obtained with this method and the standard diphenylamine assay were in excellent agreement. This simple, one-step DNA extraction procedure can be utilized in conjunction with Hoechst reagent to obtain quantitative estimates of DNA levels in cell or tissue extracts.     

Radiation-stimulated DNA synthesis in cultured mammalian cells

A type of DNA synthesis in mammalian cells that is stimulated by ultraviolet light has been studied by means of radioautography and density gradient centrifugation. The characteristics of this synthesis are: (a) it is not semiconservative; (b) it is enhanced by the presence of 5-bromodeoxyuridine in the DNA molecule; (c) the degree of stimulation is dose dependent; (d) there is less variability in the rate of incorporation of H³-thymidine during this synthesis than during normal DNA synthesis; (e) it occurs in cells that are not in the normal DNA synthesis phase (G₁ and G₂ cells). This kind of synthesis has been found in cultured cell lines from five different species; however, in some strains, the presence of bromouracil in the DNA is required before it can be demonstrated by radioautography.