Differences in the kinetic properties of thymidine kinase isoenzymes in unstimulated and phytohemagglutinin-stimulated human lymphocytes

Summary The two thymidine kinases, TK 1 and TK 2, found in phytohemagglutinin-stimulated human lymphocytes and the thymidine kinase, TK 2N, found in unstimulated human lymphocytes were purified and characterized. All three kinases had molecular weights between 70000 and 75000 which increased to 170000–200000 in the presence of 2 mM ATP.Studies on the kinetic properties of the enzymes with thymidine and ATP as the substrates and dTTP as the inhibitor showed clear differences between TK 1 and TK 2, but a close similarity between TK 2 and TK 2N. With thymidine as the variable substrate, TK 1 showed Michaelis-Menten kinetics, whereas TK 2 and TK 2N showed characteristic biphasic kinetics. With ATP as the variable substrate, all three enzymes showed positive cooperative kinetics, but TK 2 and TK 2N lost the cooperativity in the presence of dTTP. The results from inhibition studies showed, that dTTP was a cooperative inhibitor of TK 1 but a non-cooperative inhibitor of TK 2 and TK 2N.     

Modulation of SCE induction and cell proliferation by 2-mercaptoethanol in phytohemagglutinin-stimulated rat lymphocytes

2-Mercaptoethanol (2-ME) is used as a medium supplement to enhance the proliferation of lymphocytes culturedin vitro. In this study, we have examined the effects of 2-ME on cell growth and on SCE induction in cultures of unstimulated and phytohemagglutinin (PHA)-stimulated Fischer 344 rat lymphocytes. There were virtually no metaphases detected in cells cultured without PHA. In PHA-stimulated cultures, 2-ME decreased SCE-frequency but it enhanced SCE frequency in the presence of S to 12.5 µM bromodeoxyuridine (BRd U). Both mitotic and replication indices were increased in the PHA/2-ME system. The levels of incorporated exogenous thymidine, in the presence of 2-ME, were relatively low in unstimulated cells, suggesting that 2-ME is not mitogenic for T-cells. However, 2-ME enhanced PHA-induced response of T-cells as evidenced by increased levels of thymidine incorporation into cellular DNA. The growth promoting effects and the decrease in SCE frequency caused by 2-ME upon PHA stimulation indicate that 2-ME may alter the nature of interaction between PHA and cellular activating properties or the replicative processes.Abbreviations BRdU bromodeoxyuridine - FBS fetal bovine serum - SCE sister-chromatid exchanges - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - IL-2 interleukin-2 - 2-ME 2-mercaptoethanol - PBS phosphate buffered saline - PHA phytohemagglutinin - MI mitotic index - RI replication index - NADH nicotinamide adenine dinucleotide (reduced form)     

Volume regulation of human peripheral blood lymphocytes and stimulated proliferation of volume-adapted cells

Human peripheral blood lymphocytes exposed to hypotonic media (Ca/Mg-free, room temp.) first swell and then shrink. This shrinking response is characterized by a simple exponential with a half-time of 1.44±0.60 min (n=11) and its extent but not the half-time for a given hypotonicity is influenced by [K⁺]₀. Using K-selective electrodes, we observe a change in [K⁺]₀ when cells are diluted into hypotonic media. A half-time of 1.55±0.06 min (n=4) was obtained. A similar half-time was obtained by assay of total cell K using atomic absorption spectroscopy. At all osmolarities [K⁺]_i was decreased from control values and was constant as [K⁺]₀ was increased. Short-term incubation with ouabain (10⁻⁴ M) had no effect. Decreasing osmolarities progressively inhibited phytohemagglutinin-stimulated DNA synthesis, yet cell number and viability remained unaltered. Our evidence indicates that the volume response is mediated by a change in the passive permeability of the plasma membrane to K and/or to the accompanying anions, and that the consequently volume-adapted cells are growth-inhibited.     

The effect of aging on micronuclei frequency and proliferation in human peripheral blood lymphocytes

INTRODUCTION:

Increase in the instability of cellular genome with an increasing age is the result of an accumulation of cellular damage and mutations. This instability which might be observed as chromosome damage or chromosome losses can be measured by the micronucleus technique.

AIM:

The aim of this study was to investigate the effect of aging and oxidative stress induced by non-toxic levels of H₂O₂ on micronuclei induction and their relationship to cell proliferation in human peripheral blood lymphocytes.

MATERIALS AND METHODS:

Healthy volunteers with different ages were choosen. Spontaneous and H₂O₂ induced micronuclei frequencies were measured in peripheral blood lymphocytes of 30 volunteers by the micronucleus method.

RESULTS:

Spontaneous micronuclei frequencies increased first then started to decrease after 50 years of age. This biphasic response was significantly higher than micronucleus (MN) frequencies induced by H₂O₂ (P < 0.05), which followed the similar shape of response to increasing ages with lower frequencies. Proliferative capacity of cells either treated with H₂O₂ or not did not differ with an increasing age giving similar responses.

CONCLUSION:

These results indicate biphasic character of chromosome damage; first increase and decrease after 50 years with an increasing age. But this change pattern was not correlated with the steady state of proliferation capacity of cells through an increasing age. Decreases in H₂O₂-induced MN frequencies compared to spontaneous MN frequencies may be inducing an apoptosis by H₂O₂ treatment leading to underscoring damaged cells.     

Effects of 50-Hertz electromagnetic fields on proliferation and on chromosomal alterations in human peripheral lymphocytes untreated or pretreated with chemical mutagens

Cultivation of human peripheral lymphocytes (HPL) in the presence of 50Hz electromagnetic fields (EMFs) does not alter the spontaneous frequencies of sister-chromatid exchanges (SCE) and of chromosomal aberrations (CA), but leads to an enhancement of the cell cycle progression of HPLs in vitro. Pretreatment of HPLs with trenimon (TRN), diepoxybutane (DEB), or methylnitrosournea (MNU) in the G₀ phase of the cell cycle results in dose-dependent elevations of the SCE frequencies. In some cases culturing of HPLs pretreated with MNU or TRN in the presence of EMFs led to significantly higher frequencies of SCEs when compared to cells cultivated in the absence of EMDs. Since we did not use multiple fixation times these data may rather result from differential influences on HPL subsets than from EMF exposure.     

Anti-phospholipase C antibodies inhibit the lectin-induced proliferation of human lymphocytes

A novel approach was used to assess the role of phosphoinositide hydrolysis in the mitogenic action of phytohemagglutinin (PHA) or concanavalin A (ConA). The treatment of human peripheral blood leukocytes (PBL) with monospecific antibodies against phospholipase C (PLC) produced a dose-dependent inhibition (up to 100%) of PHA (10 g/ml) or ConA (25 g/ml) proliferative effects. Thus, the activation of membrane-bound PLC is asine-qua-non condition for lectin-induced proliferation of T lymphocytes. The key-role of PLC versus protein kinase C (PKC) is stressed by the fact that the inhibition of PKC with Hidaka's compound H-7 (40 M) produced only a partial blockade (about 25%) of lectin mitogenic effect.To whom correspondence should be addressed.     

连翘酯苷对小鼠脾脏淋巴细胞体外增殖与分泌功能的影响

目的分析连翘酯苷（FS）对小鼠脾脏T和B淋巴细胞增殖、分泌NO和TNF-α的影响,初步探讨其免疫调节作用机制。方法无菌操作分离小鼠脾脏,制备脾脏细胞并用含10%胎牛血清的RPMI 1640培养,在培养液中分别加入刺激剂刀豆蛋白（ConA）和脂多糖（LPS）以及不同浓度40、80、160μg/mL的FS共培养不同时间,采用MTT法检测T和B淋巴细胞的吸光度变化,ELISA和Griess法分别检测细胞分泌TNF-α和NO的水平。结果低浓度和中浓度FS对ConA诱导T淋巴细胞24 h和48 h后细胞增殖和存活率明显提高,诱导时间延长至72 h后FS明显抑制细胞转化;低浓度FS对LPS诱导脾脏B淋巴细胞24 h后细胞增殖和生存率显著提高;FS促进小鼠脾脏T和B淋巴细胞分泌NO;FS促进B淋巴细胞分泌TNF-α,中浓度FS促进T淋巴细胞分泌TNF-α而高浓度反而抑制其分泌。此外,FS对环磷酰胺（CY）处理小鼠的脾脏淋巴细胞体外增殖有明显影响,对细胞NO分泌影响不显著。结论结果提示FS可能通过影响小淋巴细胞增殖和细胞因子分泌而调节免疫细胞功能。     

Effects of constant and cycling hot environments on mitogen-stimulated proliferation of peripheral blood lymphocytes from sows and litters

1. 1. Lymphocytes from sows maintained in a constant hot environment (32°C) showed reduced proliferative responses to mitogens PHA (P < 0.02) and PWM (P < 0.01) in comparison to sown maintained in a constant cool environment (21°C). In the piglets the hot constant temperature slightly reduced (P < 0.05) proliferative responses of lymphocytes to PHA.

2. 2. No significant effects of a cycling hot environment (27–32°C) were found for any proliferative responses of lymphocytes from sows and litters.

3. 3. In the constant hot environment, serum cortisol concentrations were significantly reduced in the sows (P < 0.0001) while no differences in serum cortisol concentrations were found in the litters.

     

Deoxycytidylate deaminase activity in non-stimulated and phytohemagglutinin-stimulated human lymphocytes,and in leukemic cells

Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic leukemia and acute monocytic leukemia has been characterized in regard to the substrate, dAMP and the allosteric regulators dCTP and dTTP. The enzymes exhibited sigmoidal initial velocity versus dCMP concentration whereas in the presence of the activator, dCTP, Michaelis-Menten kinetics were obtained.At saturating substrate concentrations dTTP acted as an allosteric inhibitor of the enzyme isolated from non-stimulated as well as from stimulated lymphocytes. However, the enzymes isolated from the leukemic cells had lost the allosteric regulation by dTTP.At low substrate concentrations the competitive inhibitor, dAMP, activated all the enzymes. This activation was abolished in the presence of dCTP which indicates that dAMP might be involved in the regulation of dCMP deaminase activity and thus influence the dCTP and dTTP pools under physiological conditions.Abbreviations dCMP deaminase deoxycytidylate deaminase - PHA Phytohemagglutinin - ALL acute lymphoblastic leukemia - CLL chronic lymphocytic leukemia - AMOL acute monocytic leukemia - WBC white blood cells     

Effect of human beta-defensin-3 on the proliferation of fibroblasts on periodontally involved root surfaces

Human beta-defensin-3 (HBD-3) has versatile antibacterial activity against oral bacteria and can promote the proliferation of fibroblasts. The goal of the present study was to investigate the effect of HBD-3 on attachment and proliferation of periodontal ligament cells (PDL) onto the periodontitis affected root surfaces. PDL cells were seeded onto healthy and diseased root specimens with scaling and root planing (SRP), SRP & HBD-3 (100 ng/ml), or SRP & HBD-3 (200 ng/ml) treatment for 1, 3, and 7 days incubation. The results showed that HBD-3, especially in the 200 ng/ml group, significantly promoted fibroblast attachment and proliferation onto the diseased root surfaces. The cell number in the HBD-3 group was much greater than in the group treated with SRP alone. On day 7, the cells in the HBD-3 were well-spread and formed a network similar to those on the surfaces of the healthy root specimens. These results suggest that HBD-3 could play an important role in antibacterial activity and fibroblast proliferation, thus promoting periodontal regeneration. Meanwhile, HBD-3 might act as a potent regeneration-promoter in infectious diseases.     

Effects of thyroid hormones on human breast cancer cell proliferation

The involvement of estrogens in breast cancer development and growth has been well established. However, the effects of thyroid hormones and their combined effects with estrogens are not well studied. We investigated the response of human breast cancer cells to thyroid hormone, particularly the role of T3 in mediating cell proliferation and gene expression. We demonstrated that 17β-estradiol (E2) or triiodothyronine (T3) promoted cell proliferation in a dose-dependent manner in both MCF-7 and T47-D cell lines. The E2- or T3-dependent cell proliferation was suppressed by co-administration of the ER antagonist ICI. We also demonstrated that T3 could enhance the effect of E2 on cell proliferation in T47-D cells. Using an estrogen response element (ERE)-mediated luciferase assay, we determined that T3 was able to induce the activation of ERE-mediated gene expression in MCF-7 cells, although the effects were much weaker than that induced by E2. These results suggest that T3 can promote breast cancer cell proliferation and increase the effect of E2 on cell proliferation in some breast cancer cell lines and thus that T3 may play a role in breast cancer development and progression.     

刺参酸性粘多糖对人宫颈癌Hela细胞株增殖的影响

目的:研究刺参酸性粘多糖(SJAMP)对人宫颈癌Hela细胞株增殖的影响,并探讨其可能的作用机制.方法:体外培养Hela细胞;采用四甲基偶氮唑蓝比色法(MTT法)测定SJAMP对Hela细胞的增殖抑制率;免疫细胞化学法检测SJAMP对Hela细胞内突变型P53、细胞周期抑制蛋白P21表达的影响.结果:SJAMP在体外抑制Hela细胞生长呈时效和量效关系;与对照组比,各SJAMP浓度组均能影响细胞周期调控因子p53、p21蛋白的表达:降低p53蛋白的表达(p＜0.05),刺激p21蛋白的表达(p＜0.05).结论:SJAMP可能通过调整细胞周期调节蛋白而在体外对宫颈癌Hela细胞起到生长抑制作用.     

Effects of rGM-CSF on leukemia cell proliferation and on the incorporation of cytosine arabinoside into DNA.

In vitro studies of the effects of recombinant granulocyte macrophage-colony stimulating factor (rGM-CSF) on freshly obtained human leukemia cells were conducted to determine if there is a relationship between the effects of this growth factor on the proliferative characteristics of leukemia cells and on their incorporation of cytosine arabinoside (araC) into DNA. While rGM-CSF was found to be able to stimulate both leukemia cell proliferation and araC incorporation, for individual leukemia specimens there was no consistent relationship among these effects. In some specimens proliferation was stimulated without an increase in araC incorporation. The reverse was also observed. These studies demonstrate the difficulty in identifying assays capable of predicting the clinical effects of growth factors on leukemia cells in patients since the effect in vitro vary with the assay.     

Micronucleus frequency and proliferation in human lymphocytes after exposure to herbicide 2,4-dichlorophenoxyacetic acid in vitro and in vivo

Widespread use of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its association with non-Hodgkin’s lymphoma (NHL) and other cancers has raised public concern. Here, micronucleus (MN) formation has been used as a biomarker of genotoxicity, and replicative and mitotic indices (MIs) as biomarkers of cell cycle kinetics in human lymphocytes. Cells were cultured either as whole blood or isolated lymphocytes and treated with pure or commercial forms of 2,4-D at doses between 0.001 and 1 mM for 48 h. Exposure to 2,4-D produced a minimal increase in MN in whole blood and even smaller one in isolated lymphocyte cultures. This induction took place only at levels approaching cytotoxicity and was accompanied by a significant inhibition of replicative index (RI). At a low (0.005 mM) dose of commercial 2,4-D, a small, marginally significant increase in RI (12–15%) was found in two independent sets of experiments (P=0.052). Additionally, we found that lymphocyte RI was more affected by commercial 2,4-D containing 9.4% of the chemically pure 2,4-D, than with an equal concentration of the latter suggesting that other ingredients present in the commercial pesticide may be responsible or may enhance the effect of 2,4-D. Mitotic index, however, did not show any significant change with either commercial or pure 2,4-D. The lymphocytes of 12 male applicators exposed solely to 2,4-D during a 3-month period had a significantly higher RI than the same group prior to exposure and than a control group (P<0.01), in accordance with the in vitro finding of increased RI at low doses.     

Effect of pH shifts on radiosensitivity of human lymphocytes irradiated in the G2 stage

Experiments were carried out using human lymphocytes in order to test the effect of pH shifts on radiosensitivity of cells irradiated in the G₂ stage. In our culture conditions constant variations in medium pH over the range of 7.4–6.8 were observed as a function of incubation time after PHA stimulation; in addition the pH of the medium was adjusted in parallel cultures over the range of 5.9–8.2. Then we exposed the cultures with different pH to a treatment of X-rays delivered 2 h before fixation.

The pH of the medium was found to greatly affect the yield of induced chromatid aberrations.     

Sex differences in adrenocortical structure and function. XI

Summary Adrenal glands from orchectomized and ovariectomized rats, with and without replacement therapy, and also from intact controls of both sexes, were examined by autoradiography with ³H-thymidine. The labelling index after 1 or 2 nucleoside injections was higher in the zona glomerulosa of females than in male rats, while no differences were found in the fascicular and reticular zones. Orchiectomy increased the labelling index in the fascicular and reticular zones, an effect prevented by testosterone. Ovariectomy did not change the labelling index, while estradiol lowered it in the zona glomerulosa. Duration of the S phase was longer in the zona fasciculata cells of males than in females. Both orchiectomy and testosterone shortened this phase in cells of the zona fasciculata and zona reticularis. Ovariectomy prolonged the S phase in the zona fasciculata and shortened this time in the reticular zone, an effect reversed by estradiol.In the glomerular and fascicular zones, cell cycle time was longer in males than in females. Orchiectomy shortened this time in all adrenocortical zones, an effect reversed by testosterone. Ovariectomy shortened cell cycle time in the glomerular and reticular zones and prolonged it in the zona fasciculata; these effects were reversed by estradiol. Turnover rate in adrenocortical cells was markedly higher in females than in males, a difference due to testosterone which markedly decreased turnover rate.     

Effect of sodium butyrate on induction of ornithine decarboxylase activity in phytohemagglutinin-stimulated lymphocytes

Effect of sodium butyrate on DNA synthesis and the induction of ornithine decarboxylase (EC 4.1.1.17), a rate-limiting enzyme of polyamine biosynthesis, was studied in phytohemagglutinin(PHA)-stimulated bovine lymphocytes. Millimolar concentrations of butyrate completely inhibited the incorporation of [³H] thymidine into the acid-insoluble fraction and reversibly suppressed the induction of ornithine decarboxylase. Other shortchain fatty acids were much less active than butyrate. These results suggest that the suppression of ornithine decarboxylase activity may be one of the reasons for the inhibition of DNA synthesis with butyrate in bovine lymphocytes, because our previous experimental results have shown that the induction of ornithine decarboxylase closely correlates with the DNA synthesis in growth-stimulated cells.     

Adaptive response in irradiated human lymphocytes: radiobiological and genetical aspects

The adaptive response (AR) in human lymphocytes in different experimental protocols was investigated. The AR was found to be present in cells pre-exposed to 3 cGy of X-rays in G0, G1 and S phase as well as with tritiated water (4 muCi/ml) when the 'challenge' dose was given in G2. There was no AR after prior exposure of the cells in S phase to secondary irradiation from 70 GeV protons. The AR was not observed after preliminary X-irradiation of the lymphocytes in G0 and G1 and 'challenge' irradiation in G1. Cells from 6 patients with Down's syndrome were tested. At least 5 of them did not show the AR. The AR is considered to be a phenomenon of the antimutagenic aftereffect.     

Lentivirus mediated silencing of Ubiquitin Specific Peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro

Background

Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells.

Results

In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G₂/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells.

Conclusions

All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0006-y) contains supplementary material, which is available to authorized users.     

Time-resolved fluorescence spectroscopy of hematoporphyrin-derivative in human lymphocytes

This paper reports on time-resolved microfluorimetric measurements on hematoporphyrin-derivative (HpD)-treated lymphocytes. HpD is at present widely used as a tumor-locating and photosensitizing drug. It is therefore of great importance to study the extent to which the HpD uptake process depends on cell functional and structural properties. Time-resolved fluorescence measurements in single cells are very useful in this respect, since they give information on the content of fluorescent molecules through fluorescence peak-intensity, and, indirectly, on the binding properties through the fluorescence decay times. In particular, we studied the dependence of HpD fluorescence on the cellular functional state. To this end, we performed in-cell fluorescence measurements on human lymphocytes, both in quiescent conditions and in the pre-replicative phase, after stimulation with phytohemagglutinin (PHA). We found a higher HpD content in stimulated lymphocytes. Moreover, we found a spectral band around 575 nm, corresponding to a particular porphyrin species, in which the differences between normal and stimulated lymphocytes are more striking. The porphyrin species emitting in this band seems to play a role in the specific interaction of HpD with tumors, since a similar emission band has also been found in tumor cells containing HpD.