Bovine mammary explant versus primary cell cultures: Effect of bovine somatotropin and insulinlike growth factor-I on DNA content and protein synthesis

Summary Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows (120 ± 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages. Explants and cells were cocultured with liver and adipose tissue in the presence of somatotropin, insulinlike growth factor-I, and somatotropin + insulinlike growth factor-I. Cellular DNA and milk proteins were assayed using fluorescent probes and flow cytometry. Media proteins were assayed by densitometer scanning of electrophoresis gel bands. DNA content of explant, confluent, and growing primary cells increased similarly through the 96 h incubation. DNA content in G₀G₁ phase was increased by: (a) insulinlike growth factor-I in explant cells; (b) somatotropin, insulinlike growth factor-I, and their combination in confluent primary cells; and (c) the combination of somatotropin and insulinlike growth factor in growing primary cells. Approximately 65% of explant and confluent primary cells were in the G₀G₁ or differentiated phase compared to 47% for the growing primary cells. Whey protein content and secretion were similar among cell types. Explant cells contained and secreted more β-casein than primary cells but secretion trends for β-casein and k-casein were similar after 48 h for both cell types. Results suggest that primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion.     

Cd++ inhibits EGF-induced DNA synthesis but not EGF induced myc mRNA accumulation in serum starved NRK-49F cells

Cd⁺⁺ inhibits EGF-induced ³H-thymidine incorporation in serum deprived NRK-49F cells in a dose dependent pattern. The underlying mechanisms for this inhibition are largely unknown. EGF-induced myc mRNA accumulation in NRK-49F cells and the effects of Cd⁺⁺ on this response were examined under conditions that result in partial or complete inhibition of EGF-induced DNA synthesis. It was found that doses of Cd⁺⁺ that inhibit EGF-induced DNA synthesis do not inhibit EGF-induced protein synthesis and myc mRNA accumulation. Cd⁺⁺ doses of 0.5 µM and 1 µM were found actually to increase EGF-induced myc mRNA accumulation and amino acid incorporation. These results show that the effect of Cd⁺⁺ on EGF-induced DNA synthesis is not due to inhibition of entrance into G₁, but rather that Cd⁺⁺ acts on events subsequent to myc accumulation; that is, events associated with either G₁ progression, entry into S or DNA synthesis.Abbreviations EGF Epidermal Growth Factor - ³HTdr Tritium thymidine - MeAIB Methylaminoisobutyrate     

The activities of thymidine metabolising enzymes during the cell cycle of a human lymphocyte cell line LAZ-007 synchronised by centrifugal elutriation

The activities throughout the cell cycle of thymidine kinase (EC 2.7.1.21), dihydrothymine dehydrogenase (EC 1.3.1.2), thymidine phosphorylase (EC 2.4.2.4) and dTMP phosphatase (EC 3.1.3.35) were measured in the Epstein-Barr virally transformed human B lymphocyte line LAZ-007. Cells were synchronised at different stages of the cell cycle using the technique of centrifugal elutriation. The degree of synchrony in each cycle-stage cell population was determined by flow microfluorimetric analysis of DNA content and by measurement of thymidine incorporation into DNA. The activity of the anabolic enzyme thymidine kinase was low in the G₁ phase cells, but increased many-fold during the S and G₂ phases, reaching a maximum after the peak of DNA synthesis, then decreasing in late G₂ + M phase. By contrast, the specific activities of the enzymes involved in thymidine and thymidylate catabolism, dihydrothymine dehydrogenase, thymidine phosphorylase and dTMP phosphatase remained essentially constant throughout the cell cycle, indicating that the fate of thymidine at different stages of the cell cycle is governed primarily by regulation of the level of the anabolic enzyme thymidine kinase and not by regulation of the levels of thymidine catabolising enzymes.     

Cessation of Nuclear DNA Synthesis in Differentiating Naegleria*

SYNOPSIS. DNA synthesis during growth and differentiation in Naegleria gruberi strain NEG populations has been studied. Autoradiography of cells labeled with [³H]thymidine revealed that grains are concentrated over the nuclei in logarithmically growing populations of cells, whereas in differentiating cells, grains are scattered over the cytoplasm; i.e. no significant nuclear labeling is detectable. It was established by MAK chromatographic analysis that [³H]thymidine is incorporated into double-stranded DNA in Naegleria and that the actual amount of incorporation in the logarithmically growing populations of cells is 20 times greater than that in differentiating cells. These results suggest that nuclear DNA synthesis is reduced markedly soon after the initiation of differentiation, while cytoplasmic DNA synthesis continues. It was established from cell cycle analysis that the approximate intervals of G₁, S, G₂, and M phases were 180, 183, 90, and 28 min, respectively. Hence, the reduction in the nuclear DNA synthesis in differentiating cells is not due to the inhibition of initiation of DNA replication, but rather to the termination of the DNA replicating process. Thus DNA synthesis is curtailed in the presence of RNA and protein synthesis which are required for differentiation.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Reduced inhibition of DNA synthesis and G2 arrest during the cell cycle of resistant HeLa cells in response tocis-diamminedichloroplatinum

The influence of cisplatin, an anticancer agent, on DNA synthesis and cell cycle progression of a cisplatin-resistant cell line was investigated. Cell cycle analysis using flow cytometry showed that cytotoxic concentrations of cisplatin caused a transient inhibition of parental HeLa cells at S phase, followed by accumulation at G₂ phase. In contrast, the resistant cells progressed through the cell cycle without being affected by the same treatment. However, cell cycle distributions were the same in the resistant and the parental cells at IC₅₀, the drug concentration inhibiting cell growth by 50%. Studies using a [³H]thymidine incorporation technique also demonstrated a transient inhibition of DNA synthesis in HeLa cells by cisplatin; such inhibition was greatly reduced in the resistant cells. These data argue for the hypothesis that the inhibition of DNA synthesis is important in determining cisplatin-induced cytotoxicity. In addition, the accumulation of cells at G₀/G₁ by serum starvation was not effective in the resistant cells compared to the parental cells, suggesting that the control of cell cycle exiting is also altered in the resistant cells. Taken together, these results support the notion that alterations in cell cycle control, in particular G₂ arrest, are important in determining the sensitivity or resistance of mammalian cells to cisplatin and may have a role in clinical protocols.     

Control of Ehrlich cell division by zinc

The nutritional requirement for zinc in the proliferation of normal and malignant cells has been demonstrated in a number of animal studies. A distinction is made between the effect of zinc status upon the host during carcinogenesis and tumor growth. The present studies focus on the Ehrlich ascites tumor in mice fed a semipurified zinc-deficient diet along with defined concentration of zinc in the drinking water. This model of zinc deficiency is compared with others in which chelating agents are used to create zinc-deficient conditions or the microorganismEuglena gracilis is examined in a defined zinc-deficient medium. It is reported here that Ehrlich cells remain quiescent for several weeks in severely deficient mice, suggesting their restriction to a G₁ or G₀ state of the cell cycle. The kinetics of thymidine and uridine uptake and incorporation into DNA and RNA in Zn-normal and Zn-deficient tumors is consistent with the inhibition of thymidine kinase and DNA polymerase in the Zn-deprived system, but with little effect on RNA synthesis. The concentration of metabolites of these labeled nucleosides in Ehrlich cells is also consistent with a primary effect upon thymidine kinase. Although the ascites fluid Zn is depressed in Zn deficiency, total cellular zinc and its distribution among cell fractions is not significantly affected. It is suggested that these effects are specific in nature and not the result of a general lack of zinc for zinc metalloproteins and other binding sites in the cell.     

Adaptation of thymidine utilization to changing rates of DNA synthesis in the cell cycle

Summary In synchronous cultures of P-815 murine mastocytoma and of Chinese hamster ovary (CHO) cells, the relative contribution of exogenous thymidine to DNA synthesis was studied by comparing rates of (³H)thymidine incorporation with the rate of DNA synthesis as derived from incorporation of (³H)thymidine (10^–5 m) in the presence of amethopterin. In synchronous P-815 cultures, time-dependent variations of DNA synthesis rates were in close agreement with those of (³H)thymidine incorporation rates at concentrations of the precursor ranging from 5 × 10^–8 to 10^–5 m. Similarly, in synchronous CHO cell cultures prepared by two different methods, time-dependent changes in DNA synthesis rate were almost identical with those of the rate of incorporation of (³H)thymidine supplied at 5 × 10^–8 m. Thus, at a given thymidine concentration in the medium, the proportion of thymine residues in DNA that were derived from exogenous thymidine remained nearly constant, even though rates of cellular DNA synthesis underwent pronounced changes. This indicates that in the synchronous culture systems used, utilization of exogenous thymidine is efficiently adapted to changing rates of DNA synthesis.In partial fulfillment of the requirements for the degree of Ph.D. by G.G.M.     

Decreased DNA synthesis in mammalian cells : After exposure to deoxyadenosine

The effect of a pre-treatment with 2 mM deoxyadenosine on DNA synthesis in bovine liver cells in vitro was investigated. The incorporation of ³H-deoxythymidine was strongly depressed after 1 h treatment and recovered only gradually during the following 4 to 6 h. Addition of 50 μM deoxyguanosine during this time effectively counteracted the depression. An autoradiographic study revealed that the depression was due to a decrease in thymidine incorporation per cell rather than a decrease of the proportion of thymidine-incorporating cells. It is concluded from these results that the rate of DNA synthesis is decreased during the initial hours after removal of the deoxy-adenosine because of a continued deficiency of dGTP. This conclusion is supported by the appearance of a high proportion of ³H-label in replicating intermediates after 1 h of incorporation which is otherwise only observed after pulse labelling for 5 to 10 min.     

Stimulation of DNA synthesis and 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 δ-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.     

Irradiation of synchronized cell cultures

Summary When HeLa cells are synchronized by the double thymidine block method, it is shown that the mitotic delay produced by 450 R X-rays is longer when cells are irradiated in G₂ than in G₁ but longer still when they are irradiated in the early part of S. DNA synthesis, measured by³H-Thymidine incorporation, is not reduced considerably (± 25%) and only for a few hours when 600 R are given at this time. A more pronounced inhibition occurs when irradiation is given later in S. There is also no decrease in incorporation of³H-orotic acid in RNA when the cells are irradiated in S or in G₂. These results are discussed and it is concluded that the long mitotic delays cannot he due to a modification of nucleic acids metabolism.Paper read at the 6th Annual Meeting of the European Society for Radiobiology, Interlaken, 5.–8. June, 1968. Round Table: Radiation Effectsin vitro andin vivo. Correlations and Discrepancies.     

In vitro studies of the effect of intermittent compressive forces on cartilage cell proliferation.

Intermittent compressive (IC) forces (96 mm Hg, 0.3 Hz) inhibit by 35–60% the serum stimulated increase in ornithine decarboxylase activity (ODC) in chick embryo epiphyseal cartilage cells and rat chondrosarcoma cells. IC had no effect on mouse fibroblast L-cells ODC. The dose-response pattern of the IC effect indicated an all-or-none response with a threshold at 80 mm Hg, a pressure roughly equivalent to the in vivo weight bearing force. The k_m of the cartilage cell ODC, measured at four hours, was about 0.1 mM and was not affected by IC. The V_max, on the other hand, was significantly reduced by IC which is consistent with less enzyme or non-competitive inhibition. IC also produced a significant increase in cAMP levels in both cartilage explants and isolated cells in the presence and absence of serum and a significant reduction in ³H-thymidine incorporation into DNA. The findings show that cellular cAMP, on one hand, and ODC and DNA synthesis, on the other hand, change in opposite directions following exposure to serum and/or IC. Investigation of the IC effect on DNA synthesis in serum-deprived synchronized cartilage cells revealed that IC reduced the number of cells going into S but did not lengthen the G₁ phase. Exposure to IC early in G₁ (0–13 hours) produced the full effect, whereas IC application between 13 to 24 hours (pre S) had no effect. IC had no effect on ³H-thymidine incorporation in L-cells.     

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts: I. Temporal relationships between HMG-CoA reductase activity,sterol biosynthesis and thymidine incorporation into DNA

Temporal relationships between hydroxymethylglutaryl-CoA reductase activity, biosynthesis of C₂₇ sterols, and [³H]thymidine incorporation into DNA were studied in a rat embryo fibroblast cell line synchronized by double thymidine block and cultured in cholesterol-containing medium. Cyclic variations of HMG-CoA reductase activity and C₂₇ sterols occurred, with two maxima in S and G₂M phases; the relative shortness of the G₁ phase (3 h) in these cells could be responsible for the shift of sterol synthesis in the S phase. No noticeable variation of the individual C₂₇ sterols was observed during the entire cell cycle. In each experiment, there was a good linear correlation between HMG-CoA reductase activity and C₂₇ sterol synthesis, but from one experiment to another, a given level of enzymatic activity led to varying levels of [2-¹⁴C]acetate incorporation into sterols. In our experimental conditions, total HMG-CoA reductase activity is measured, and the preceding observation could be explained by a varying degree of phosphorylation of the enzyme depending on the metabolic state of the cells at the start of the experiment. The cyclic variations of the enzyme activity seem to be due more to increased synthesis at given times of the cycle than to periodic dephosphorylation. We question the existence of a relationship between cell division and cyclic sterol synthesis occurring in cells cultured in cholesterol-containing medium.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Assessment of interleukin 1 and interleukin 2 effects on cycling and noncycling murine thymocytes

When mouse thymocytes are stimulated with PHA, the proliferative response is very low, unless the culture medium is enriched with interleukin 1 (IL-1)- or interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analyses show, however, that PHA stimulation generates a significant number of cells with increased RNA content (transition from the G₀ to G₁ phase of the cell cycle). If IL-2 is added to such cultures, the activated cells complete their process of RNA synthesis and then enter the S phase. The use of IL-2-containing culture medium thus permits one to obtain a high correlation between the number of g₁ cells and [³H]thymidine incorporation (r = 0.97). Enrichment with IL-1-containing supernatants also results in a statistically significant correlation (r = 0.68), but the regression lines are markedly different for the two interleukins (s = 20.3 for IL-2 and s = 9.2 for IL-1), when analyzed after 48 hr of incubation. These observations suggest that the G₁ phase must be divided into two subcompartments, G_1a and G_1b, the G_1a-G_1b transition being an IL-2-dependent event. If the number of G_1b cells is used to establish correlations with [³H]thymidine incorporation, all values fall on the same regression line, regardless of culture conditions and of the addition of interleukins. It is concluded that IL-2 regulates lymphocyte proliferation at the level of RNA synthesis (G_1a-G_1b transition) rather than that of DNA synthesis (G₁-S transition).     

Nucleic acids methylation of synchronized BHK 21 HS 5 fibroblasts during the mitotic phase

The methylation of nucleic acids has been investigated during the cell cycle of an asparagine dependent strain of transformed fibroblasts (BHK 21 HS 5). The synchrony was carried out by a partial asparagine starvation of cells for 24 hours. The amino acid supply induced all cells to enter synchronously the G₁ phase. Methylation and DNA synthesis were respectively measured by pulsed [methyl-¹⁴C] methionine and [methyl-³H] thymidine incorporation. DNA methylation followed a biphasic pattern with maximal methyl incorporations during both S phase and mitosis. A partial desynchronisation induced the S phase of the second cycle to proceed before all the cells have achieved their division. Hydroxyurea was used in order to inhibit the DNA synthesis of cells entering the second cell cycle, which might interfer with the mitosis of the first one. The inhibitor was added either at the first beginning of cell division or during all the G₁ phase. In both conditions it suppressed ³H thymidine incorporation of the second cycle. However, mitosis took place and methylations occurred as in previous experiments. The DNA methylation of the mitotic phase in the first cell cycle could thus be dissociated from the classical post-synthetic DNA maturation and did not correspond to any DNA methylation appearing in the course of the second cell cycle.     

Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase α and thymidine kinase activities, as well as a high rate of incorporation of [³H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [³H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G₁ peak of DNA distribution had a significant amount of incorporated [³H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [³H]thymidine.     

Role of calcium in the insulin-dependent stimulation of DNA synthesis in mouse mammary gland in vitro

The role of extracellular Ca2+ in the control of DNA synthesis in mouse mammary tissue was studied using mammary gland explants maintained under chemically defined conditions in vitro. Chelation of calcium with ethyleneglycol-bis-(beta-aminoethyl ether) or omission of Ca2+ from the incubation media substantially reduced both basal and insulin-stimulated incorporation of [3H]thymidine into DNA. Addition of calcium to the Ca2+-deficient media restored DNA synthesis; other divalent cations could not be substituted for calcium. Insulin reduced by 5-fold the calcium concentration required to achieve half-maximal stimulation of DNA synthesis in explants, thus indicating that the Ca2+-related process may be involved in the mechanism by which insulin exerts its effect on cell multiplication. Evidence is presented that in mammary gland explants, calcium does not stimulate DNA synthesis by action on the thymidine pool size. Neither calcium nor insulin showed any effect on the activity of thymidine kinase in the mammary gland explants. On the other hand, calcium ions were shown to be necessary to maintain the activity of DNA polymerase-alpha, the enzyme involved in nuclear DNA replication.     

Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum     

Lethal effects of fluorodeoxyuridine on cultured mammalian cells at various stages of the cell cycle

The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10^?5 M FUdR was very low (0.0001–0.0003) whether the drug was administered during early G₁, late G₁, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10^?7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.