Membrane associated events during proliferative inhibition of granulocyte precursors

A new way of assessing the significance of intracellular signals that may regulate cellular proliferation, would be to analyze possible 'second messengers' when proliferation is slowed down, rather than stimulated. Therefore, we examined proliferating mononuclear blood cells from leukaemic patients which had been exposed to an inhibitory ox leucocyte extract. The extract decreased 3H-thymidine incorporation in leukaemic cells in short-term cultures. The inhibition was not cell-line specific, but was nevertheless non-toxic and not due to endotoxin. The K+ flux into the leukaemic cells was assessed with 86Rb+, a K+ analogue. An inverse relationship was found between 86Rb+ uptake and 3H-thymidine incorporation. The increased 86Rb+ influx was probably due to leakage or exchange mechanisms other than the Na+/K+ membrane pump, as suggested by ouabain inhibition experiments. However, the long lag time (greater than 45 min) between addition of inhibitor and a marked increase in 86Rb+ uptake does not support a role for the K+ flux as an early mediator of the inhibitory signal.     

Indomethacin-induced augmentation of lymphoproliferative responses in patients with head and neck cancer

Augmentation of the phytohemagglutinin (PHA)-induced lymphoproliferation of peripheral blood mononuclear cells by indomethacin, a drug which blocks prostaglandin (PG) synthesis, was assessed in 37 patients with squamous cell carcinoma of the head and neck. Indomethacin enhanced the uptake of 3H-thymidine in stimulated cultures both from patients and normal individuals. However, because lymphoid cells from cancer patients were less reactive than those from normal controls, the proportionate increase in PHA-stimulated 3H-thymidine incorporation caused by indomethacin was greater in this population than in the normal population. The degree of enhancement induced by indomethacin did not correlate with the percent of esterase-positive mononuclear cells in the preparations. The amounts of PGE synthesized at 48 h by patients' or normal cells were similar. Cell populations that exhibited elevated levels of augmentation in the presence of indomethacin were approximately 3 times as sensitive to inhibition by 3 nM PGE2. The degree of augmentation detected in the presence of Ro-20-5720, which also prevents PG synthesis, was related to that produced by indomethacin. These results suggested that: the enhancing effect of indomethacin on lymphoproliferation in vitro was related to its inhibition of PG synthesis; and the sensitivity of lymphoid cells to inhibition by PGE2 was slightly, but significantly, increased in individuals with elevated augmentation values.     

Effect of cytochalasin D on DNA synthesis in cultured cells

The effect of cytochalasin D (CD), an agent specifically destroying actin cytoskeleton, on DNA replication of cultured mouse embryonic fibroblasts (MEF) and BALB/3T3 strain cells was studied. Incubation of normal cells with CD resulted in progressive inhibition of DNA synthesis: in the first 16-20 h the percentage of cells pulse-labelled with 3H-thymidine was similar to that in control cultures, on day 8 the percentage of labelled cells was 7-8 times lower than in the control. The transfer of cells into fresh medium upon 8-day incubation in the presence of CD resulted in the recovery of DNA synthesis. Similar curves of DNA synthesis inhibition in the presence of CD and of DNA synthesis recovery in fresh medium were observed both in mononuclear and binuclear cells. Thus, CD-induced reorganization of actin cytoskeleton can have an abrupt but reversible disturbing effect on normal cell cycle.     

Effect of thiopyrimidine ribonucleosides on DNA and RNA synthesis in synchronized mammalian cell cultures

The uptake of ³H-uridine into RNA and of ³H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either ³H-thymidine or ³H-uridine at selected times, and analyzed in autoradiographs. Incorporation of ³H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10⁻³ M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of ³H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis.     

Functional aspect of a phosphopeptide derived from calf thymus nuclei and DNA

Phosphopeptides (PPs) isolated from highly purified calf thymus DNA (N-DNA) and extracted from calf thymus nuclei were fractionated, and the effect of one PP fraction on DNA replication has been examined. In the absence of inhibitors, the increasing PP concentration caused a linear decrease of 3H-thymidine uptake in L5178Y cells. If PP fraction was mildly hydrolysed with 1NHCl, the decrease in uptake was much steeper. The studies in which the inhibitors were used revealed that by the addition of the unhydrolysed PP fraction the inhibition of 3H-thymidine uptake by alpha-amanitin could be completely overcome, and that the inhibition by puromycin was reduced to 65-77% of the control. With puromycin, there was a gradual decrease of 3H-thymidine uptake with PP concentration above 3 mg/ml. The PPs gave an increase in incorporation of 3H-thymidine even after removal of alpha-amanitin and puromycin; thus, it is suggested that there is no direct interaction of either inhibitor with PP in the cell. Data on the utilization of 3H-cytidine for the synthesise of new DNA suggest that PP fraction might cause an acceleration of DNA replication.     

Sodium orthovanadate stimulation of DNA synthesis in Nakano mouse lens epithelial cells in serum-free medium

Quiescent cultured Nakano mouse lens cells incubated for 40 hours with sodium orthovanadate incorporated 3H-thymidine at an accelerated rate; the greatest response occurred at 20 microM vanadate, whereas by 2 microM an incorporation rate equivalent to unstimulated cells was noted. Microscopic examination of the cells revealed that those exposed to concentrations of vanadate greater than 100 microM had lysed by the end of the 40-hour incubation. Reduction in vanadate exposure time to 1 hour caused the cells to incorporate the greatest amount of 3H-thymidine at a vanadate concentration of 200 microM to 500 microM. Half-maximum incorporation of 3H-thymidine (after a 40-hour incubation) was induced by a 2-hour incubation with 20 microM vanadate. Studies with insulin showed that while 20 ng/ml insulin alone did not increase 3H-thymidine incorporation, 20 ng/ml insulin in combination with 20 microM vanadate resulted in a significant increase in 3H-thymidine uptake over cells exposed to only vanadate. Insulin alone will increase cell number and insulin with vanadate are synergistic in the stimulation of DNA synthesis, but the two together show no further increase in cell number over that produced by insulin alone. Thus, vanadate can increase progression from G1/G0 to S-phase, but cannot stimulate cells to divide. Studies designed to detect DNA damage and repair rather than S-phase DNA synthesis demonstrated that vanadate was not causing increased 3H-thymidine uptake by damaging DNA. Cell counts revealed that vanadate, while able to induce DNA synthesis, does not induce mitosis. Autoradiography and equilibrium sedimentation experiments demonstrated that gene amplification was not occurring. A known vanadate exchange inhibitor blocked the ability of vanadate to increase 3H-thymidine incorporation which is consistent with the idea that cellular internalization of vanadate is required for this effect to be seen. 86Rb+ uptake experiments demonstrate that the vanadate concentration inducing 50% inhibition of (Na+, K+)ATPase is nearly two orders of magnitude more concentrated that vanadate concentrations shown capable of inducing 3H-thymidine uptake. This strongly suggests that (Na+, K+)ATPase inhibition is not the central mechanism by which DNA synthesis is stimulated by vanadate.     

Phosphonoacetic acid: inhibition of transformation of human cord blood lymphocytes by Epstein-Barr virus.

Phosphonoacetic acid disodium salt (PAA) inhibited the transformation of human cord blood lymphocytes by Epstein-Barr virus (EBV) at concentrations of 50-100 microgram/ml. At these concentrations, PAA had no effect on the multiplication of EBV transformed human lymphoblastoid cells or on the survival of human cord blood lymphocytes. The transformation of human cord blood lymphocytes by the B95-8 strain of EBV was measured by 3H-thymidine uptake, 5 days or more after infection. The degree of inhibition of transformation was correlated with the relation between the input of EBV and the concentration of PAA in the experiment. PAA inhibited the transformation even when added 24 h after EBV infection, but had no effect when added 48 h after EBV infection. The inhibitory effect of PAA could be overcome by its removal and normal 3H-thymidine uptake was restored even after 6 days of inhibition. The specificity of the inhibitory effect on EBV induced transformation of human cord blood lymphocytes is discussed.     

Modulation of endothelial cells growth induced by heparin

We have previously shown that heparin was bound and internalized by cultured human endothelial cells. In this study, we have investigated the effect of heparin on endothelial cells growth. We found that heparin inhibited 3H-thymidine uptake as well as actual cell growth in a dose-dependent manner in the presence of low concentrations of human serum. Inhibition was maximal at 1% serum concentration and was abolished at 10%. Chasing experiments supported the role of membrane-bound heparin in this inhibition. Low molecular weight heparin fractions, or pentosan polysulfate, were equally effective in inhibiting 3H-thymidine uptake. On the other hand, the simultaneous addition of heparin and ECGS was synergic in stimulating 3H-thymidine uptake. These results suggest a modulatory role of heparin in endothelial cells growth.     

Effect of folinate on thymidine uptake by Pediococcus cerevisiae

Uptake of (3)H-thymidine by resting cells of Pediococcus cerevisiae was found to be energy- and temperature-dependent. The pH optimum was between 6.5 and 8.0, and after 2 min of incubation most of the radioactivity was found in the deoxyribonucleic acid (DNA) fraction. Iodoacetate at a concentration of 10(-2)m caused a 50% inhibition of uptake. Preincubation of resting cells for 10 min with folinate (10(-3)mu mole/ml) diminished the (3)H-thymidine uptake by 75%. In growing cells, the folinate-induced inhibition was still more striking. Deoxyuridine augmented the folinate effect, whereas fluorodeoxyuridine and aminopterin or amethopterin abolished it. Preincubation with folinate did not interfere with the uptake of (3)H-amethopterin, and thus the inhibitor did not compete for uptake sites within the cell. The role of these inhibitors in reversing the folinate effect is discussed. Cells preincubated with folinate showed an increased incorporation of (14)C-uracil into DNA, presumably after prior conversion to thymidylate. We concluded that the folinate effect was due to stimulation of de novo thymidylate synthesis with concomitant inhibition of the uptake of external thymidine.     

Immunoregulatory properties of human esophageal tumor extract.

Noncytotoxic aqueous extracts of esophageal tumors (TEx) inhibit the spontaneous uptake of 3H-thymidine by human peripheral blood mononuclear cells (PBM). TEx also inhibits mitogen- and antigen-induced PBM blastogenesis, mitogenesis, 3H-thymidine uptake, and 3H-leucine incorporation. Inhibition is reversible and is mediated by a heat-labile protein with a m.w. of approximately 70 to 80,000 daltons. Inhibitory activity is not due to trivial binding of mitogen or neucleotide by the inhibitor, nor is it entirely tumor specific since similar but quantitatively less activity is extractable from adjacent normal esophageal mucosa. Assorted cell surface membrane phenomena such as contact inhibition of in vitro endothelial cell proliferation, lymphocyte E rosette formation, and cap formation are unaffected by TEx. In contrast, immunoglobulin synthesis by pokeweed mitogen-stimulated PBM is enhanced by TEx. The potential importance of the local production of immunoregulatory agents like TEx in the immunologic control of tumor cell growth is discussed.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

The effect of photosensitizing dyes on the 3H-thymidine incorporation of cells grown in vitro

The photodynamic inactivation of ³H-thymidine incorporation in mouse embryo (ME) and mouse L cells by acridine orange (AO), methylene blue (MB) or neutral red (NR) has been studied by estimating the number of nuclei capable of incorporating ³H-thymidine during a 24 h period following light exposure. In the dark NR and AO reduced the number of ME-nuclei incorporating ³H-thymidine but MB caused an increase in non-scheduled DNA synthesis. The dark effect on L cells was less but the photoinactivation of thymidine uptake was proportionally greater in these cells. Polyoma virus was shown to be capable of growing in cells whose thymidine uptake was reduced or completely stopped by photoinactivation with NR. However, if the NR damage was very great, or when AO was used to photosensitize cells, the synthesis of viral DNA was interfered with.     

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Background

DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.

Aim

To investigate the effect of red blood cells on H₂O₂-induced DNA damage and repair in human peripheral blood mononuclear cells.

Methods

DNA breaks were induced in peripheral blood mononuclear cells by H₂O₂ in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by ³H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.

Results

Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.

Conclusion

Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.     

Recovery of Pisum root meristems after mitotic-inhibitory treatments with 3H-thymidine. Inhibition of cell-cycle progression by unincorporated 3H-thymidine.

Primary root meristems of Pisum sativum recover form a 3H-thymidine-induced reduction in mitotic activity once the roots are no longer exposed to exogenous 3H-thymidine. Cells arrested in G2 during 3H-thymidine treatment apparently do not divide for at least 16 hours after treatment, whereas cells remaining in G1 and S do divide and thereby account for recovery. Recovery occurs only when meristems are no longer exposed to exogenous (i.e. unincorporated) 3H-thymidine, suggesting that cytoplasmic irradiation from unincorporated 3H-thymidine prevents cellular recovery from 3H-thymidine-induced inhibition of cell progression through the mitotic cycle. Concentrations of 14C-thymidine which result in cytoplasmic irradiation nearly equivalent to that achieved with 3H-thymidine, but much lower levels of nuclear irradiation, also prevent recovery from 3H-thymidine-induced inhibition of mitotic activity, but do not alone produced such inhibition. These results support the contention that cytoplasmic irradiation prevents recovery from the effects of nuclear irradiation. Unincorporated 3H-thymidine also prevents recovery from sucrose deprivation in stationary phase G2 cells which have not incorporated 3H-thymidine into nuclear DNA.     

The flavonoid component isorhamnetin in vitro inhibits proliferation and induces apoptosis in Eca-109 cells

Isorhamnetin is one member of flavonoid components which has been used in the treatment of heart disease. Recently the in vitro anti-cancer effect of isorhamnetin on human esophageal squamous carcinoma cell line Eca-109 was investigated in our lab. When Eca-109 cells were in vitro exposed to the graded doses of isorhamnetin (0-80 microg/ml) for 48 h, respectively, isorhamnetin exhibited cytostatic effect on the treated cells, with an IC(50) of 40+/-0.08 microg/ml as estimated by MTT assay. Inhibition on proliferation by isorhamnetin was detected by trypan blue exclusion assay, clone formation test, immunocytochemical assay of PCNA and (3)H-thymidine uptake analysis. Cell cycle distribution was measured by FCM. It was found that the viability of Eca-109 cells was significantly hampered by isorhamnetin. Compared with the negative control group, the treated group which was exposed to isorhamnetin had increased population in G(0)/G(1) phase from 74.6 to 84 while had a significant reduction in G(2)/M phase from 11.9 to 5.8. In addition to its cytostatic effect, isorhamnetin also showed stimulatory effect on apoptosis. Typical apoptotic morphology such as condensation and fragmentation of nuclei and blebbing membrane of the apoptotic cells could be observed through transmission electron microscope. Moreover, the sharp increase in apoptosis rate between the control and treated group were detected by FCM from 6.3 to 16.3. To explore the possible molecular mechanisms that underlie the growth inhibition and apoptosis stimulatory effects of isorhamnetin, the expressions of six proliferation- and death-related genes were detected by FCM. Expressions of bcl-2, c-myc and H-ras were downregulated whereas Bax, c-fos and p53 were upregulated. However, the in vivo experiments were required to further confirm the anti-cancer effects of isorhamnetin. In conclusion, isorhamnetin appears to be a potent drug against esophageal cancer due to its in vitro potential to not only inhibit proliferation but also induce apoptosis of Eca-109 cells.     

Effects of EGF and PMA on the growth and proliferation of IEC-6 cells

Proliferation of an epithelial line (IEC-6) derived from the crypts of rat jejunum was induced with epidermal growth factor (EGF). EGF enhanced synthesis of protein, RNA, and DNA in a dose-dependent manner. Protein synthesis increased within 6-12 hours of exposure to EGF and remained elevated for 72 hours. Maximal 3H-thymidine incorporation occurred 48 hours after addition of EGF. The stimulatory effect of EGF on 3H-thymidine incorporation was two-fold greater in serum-free media than in media containing fetal calf serum (FCS). In contrast to EGF, phorbol-12-myristate-13-acetate (PMA) decreased 3H-thymidine uptake by IEC-6 cells and had no effect on either protein synthesis or RNA synthesis. EGF did not alter protein kinase-C activity in IEC-6 cells whereas PMA induced enzyme activity: activity was translocated from cytosol to membrane. Moreover, the EGF-associated increase in 3H-thymidine uptake was not altered by amiloride. These data suggest protein kinase-C activation may not be involved in the proliferation of IEC-6 cells.     

Effect of prostaglandins on 3H-thymidine uptake into human epidermal cells in vitro.

Prostaglandins A2, B1, E1, E2, F1alpha and F2alpha were added to cultures of human epidermal cells (keratinocytes) for 24 hours at 37 degrees C, and the effects on 3H-thymidine uptake into DNA was measured. At 70 mu/ml all prostaglandins tested except PGF2alpha inhibited the uptake of 3-thymidine greater than 50%. However, at 35 mug/ml, PGA2 and PGB1 were the only two prostaglandins to show significant inhibition, 96% and 51% respectively. At 17.5 mug/ml only PGA2 caused substantial inhibition, 68%. In order to determine if the PGA2 action was mediated by membrane receptors propranolol, phentolamine, metiamide and prostynoic acid were added in conjunction with PGA2. None of the above receptor antagonists were able to reduce the PGA2-induced inhibition of 3H-thymidine uptake. These results indicate that the pre-incubation of human keratinocytes with prostaglandins for 24 hours results in a decrease of 3H-thymidine incorporation in DNA. The precise mechanism of action is unknown at this time.     

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.     

The effect of 3H-thymidine on the proliferation of in vitro cultured mammalian cells

The effect of 3H-thymidine on the proliferation of Chinese hamster cells (clone V79) was studied. Following 3H-thymidine application the proliferation of cells (studied on the basis of plating efficiency) was found to be diminished, the drop being dependent on radioactivity (2-20 kBq/ml cultivation medium), the time of application (2-20 h) and specific activity of 3H-thymidine added. Exogenous macromolecular DNA was able to repair, to an important degree the radiotoxic effect of 3H-thymidine on V79 cells by a mechanism other than the mere reduction of specific activity of 3H-thymidine.     

A DELAY IN THE G2 PERIOD OF CULTURED HUMAN LEUCOCYTES AFTER TREATMENT WITH RUBIDOMYCIN (DAUNOMYCIN)

Human leucocytes were cultured for 3 days at 37°C, and during this time treated with rubidomycin (also known as daunomycin) for periods up to 48 hr. The effects of this treatment were studied by examining mitotic indices, uptake of ³H-thymidine, and patterns of DNA content (measured by microdensitometry on Feulgen-stained cells). A low concentration of rubidomycin (0.1 μg/ml) caused accumulation of cells in the G₂ period, which in turn resulted in a decrease in the mitotic index. A secondary effect was a slight drop in ³H-thymidine uptake after 12 hr. Higher doses (up to 10 μg rubidomycin per ml) caused an inhibition of DNA synthesis, with accumulation of unlabelled cells between G₂ and G₂. The probable mode of action of rubidomycin, as presented by earlier authors, is the intrusion of the drug molecule between DNA strands, forming a complex with DNA, and hindering its normal folding. This is discussed with respect to the present findings.