Formation of the population composition of a stationary cell culture and the participation of nonproliferating cells with a doubled DNA content in regulating its density

The conditions of the cultivation of chick embryo diploid cells were alternated (prolonged maintenance with or without medium replacement, with or without consequent cell replating in fresh medium). In different times of culture growth, the cell DNA content was assessed by cytophotometry; the percentage of non-labeled mitoses after incubating the cells with 3H-thymidine and colcemide, as well as the cell density were determined. The phenomenon, detected earlier, of the accumulation of cells containing 4c DNA during the transition of the culture from logarithmic into the stationary phase of growth, was confirmed. These cells were shown to differ in their ability to survive in conditions of stationary culture and by proliferative potential. The fraction of cells reversibly arrested in G2-period was described, by which fraction the change of the cell population size is occurring after the decrease of its proliferation rate. The transitional stage is distinguished at the beginning of the stationary phase of culture growth. During this stage the stabilization of structural and numerical composition of the population is taking place.     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells

We studied the effect of 2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline(THDA), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells following 24 h exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A. THDA also caused a marked increase in apoptosis, which was associated with activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. These molecular alterations provide an insight into THDA-caused growth inhibition, G2/M arrest and apoptotic death of K562 cells.     

Investigations on the effects of cobalt-alkyne complexes on leukemia and lymphoma cells: cytotoxicity and cellular uptake

Cobalt-alkyne complexes represent a new class of antiproliferative drugs with high activity on cell lines derived from human solid tumors. These promising results encouraged us to evaluate also their effects against leukemia and lymphoma cells. For this purpose, we selected three cobalt complexes with (2-propyn-1-yl)acetylsalicylate (Co-ASS), 2-propyn-1-ol (Co-Prop) and diphenylacetylene (Co-Diph) ligands and investigated their growth inhibiting properties on the LAMA-84, K-562, SD-1 leukemia and U-937 lymphoma cell lines. The cobalt complexes showed high effects on LAMA-84 cells (IC(50)=7.7-16.8 microM) after 48 and 72 h of incubation, but were inactive (K-562, U-937) or low active (SD-1) on the other cell lines. The proliferation of SD-1 cells was reduced by Co-Prop (IC(50)=18.6 microM) and Co-Diph (IC(50)=7.5 microM) only after a 72 h exposure. The antiproliferative effects did not correlate with the accumulation of the drugs into the tumor cells. The time dependent uptake during 24 h determined by atomic absorption spectroscopy was comparably the same in sensitive LAMA-84 and insensitive K-562 cells.     

The effect of topoisomerase inhibitors on the expression of differentiation markers and cell cycle progression in human K-562 leukemia cells.

Treatment of human K-562-J leukemia cells for 1 h with the topoisomerase II-reactive drugs VP-16, VM-26, or mAMSA resulted in a dose-dependent inhibition of proliferation and in an increase in the percentage of cells staining positive for hemoglobin, a marker of erythroid differentiation. Staining for hemoglobin of up to about 60% of the cells was observed at 20 microM VP-16, 1 microM VM-26, and 8 microM mAMSA. Such treatment also caused a G2/M arrest in the cell cycle. Incubation of the cells with radiolabeled VP-16 indicated that the induced erythroid differentiation was not due to continuous cell exposure to a residual amount of the drug. VP-16-induced erythroid differentiation was also not affected by DNA, RNA, or protein synthesis inhibitors. Differentiation induction and the G2/M arrest evoked by VP-16, VM-26, and mAMSA were, however, reduced in the presence of novobiocin. Our results indicate that topo-reactive drugs that cause G2/M arrest in the K-562-J cell cycle can induce in these cells erythroid differentiation after a short and irreversible interaction with their target molecule(s).     

Characterization of insulin-like growth factor I receptors on human erythroleukemia cell line (K-562 cells)

Specific insulin-like growth factor I (IGF-I) receptors on a human erythroleukemia cell line (K-562 cells) were identified and characterized. [125I]-IGF-I specifically bound to K-562 cells and the binding was displaced by unlabeled IGF-I in a dose dependent manner, and half maximal inhibition of the binding was observed at 7 ng/ml IGF-I. [125I]IGF-I binding to the cells was displaced by multiplication stimulating activity (MSA) and by porcine insulin, with potencies that were 10, and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present in the K-562 cells. When the cells were differentiated by hemin (40 microM), specific binding of [125I]IGF-I to the cells was decreased to 56.8 +/- 5.0% of that for undifferentiated cells. Furthermore, at physiological concentration of IGF-I stimulated thymidine incorporation into DNA and increased the number of cells. These data demonstrate that K-562 cells have specific receptors for IGF-I which may be functionally important for these cells, and that the IGF-I binding sites decrease with cell differentiation. This system might be useful in studying the interaction of IGF-I receptors.     

Selenium protection against mercury-induced apoptosis and growth inhibition in cultured K-562 cells

Selenium and mercuric chloride (MC) interactions regarding effects on cell growth and cell death have been studied. Human K-562 cells were pretreated or simultaneously treated with either selenite (5 or 50 μM) or selenomethionine (10 or 50 μM) and with MC (35 or 50 μM). The 35-μM MC treatments resulted in a clear inhibition of cell growth with no obvious difference between mercury-treated and mercury-selenium-treated cells. Furthermore, the apoptotic frequency was similar at all observations for all selenium treatments with 35 μM MC. In the simultaneously treated selenite and 50-μM MC combinations, a selenite-dependent protection was shown both by increased cell growth and by lower apoptotic frequency at 48 and 96 h of exposure. Both treatments with selenomethionine showed protection observed as an increased cell growth at 48 and 96 h and as decreased apoptotic frequency at 96 h of exposure.     

Serum-induced expression of metallothionein isoforms in K-562 cells

The metallothionein (MT) expression was studied in the hematopoietic precursor cell line K-562, after serum deprivation and reconstitution of the cells in medium with 10% (v/v) FCS. Serum deprivation for 72 h markedly downregulated the MT mRNA expression, only the isoforms most abundant in normal K-562 cells were clearly detectable. Within 1-1.5 h after serum supplementation however, a definite induction of MT mRNA was noticed, and all isoforms were induced. Forty-eight hours after serum stimulation, the MT mRNA expression of all isoforms decreased again. Also MT protein levels increased twofold 24 h after serum stimulation. These results suggest that MT has a function in the re-entry of resting cells into the cell cycle, this function however could not be assigned to a specific MT isoform. The induction of MT after serum stimulation was independent of protein synthesis, but dependent on phosphorylation.     

Natural killer cell-mediated antitumor reactivity of rhesus monkeys

We have analyzed natural killer (NK) cell-mediated antitumor activity or peripheral blood mononuclear cells of rhesus monkeys. All monkeys displayed significant NK cell cytolytic activity against the human tumor cell lines K-562, Daudi and CEM in a short-term (3 h) 51Cr-release assay. Similar to NK cells described in other species, the cytotoxic cells of monkeys were relatively nonadherent to nylon wool columns, exhibited low density after separation on discontinuous Percoll density gradients, and displayed large granular lymphocyte (LGL) morphology. Analysis of the mechanism of NK cell cytotoxicity of rhesus monkeys demonstrated that on the average, 7.1% (range: 3.1-13.2%) of lymphocytes bound to K-562 tumor, and that approximately 14.8% (range: 7.9-26.3%) of these tumor-binding cells (TBC) were cytolytically active. Examination of TBC on cytocentrifuge slides indicated that the majority of binders displayed LGL morphology. The cytotoxic reaction mediated by monkey NK cells exhibited Michaelis-Menten kinetics pattern; the maximum rate of lysis (Vmax) of K-562 was found to be 1-2 X 10(4) following 3 h of incubation. Using similar culture conditions, the recycling capacity of NK cells of this species was estimated at 2-6 times. Finally, it was observed that the NK cell activity of most monkeys could be potentiated following in vitro exposure to the biological response modifier, interleukin-2.     

A monoclonal antibody to human transferrin receptor which inhibits erythroid differentiation of human leukemic K-562 cells

In this paper we demonstrate that the 42/6 monoclonal antibody to human transferrin receptor (1) inhibits erythroid differentiation of human leukemic K-562 cells without affecting cell proliferation. Erythroid induction was monitored by benzidine-staining of K-562 cell suspensions and hemoglobin accumulation by cellulose acetate gel electrophoresis of post-mitochondrial cell lysates (4,5). Our results suggest that erythroid differentiation and cell growth require a different number of transferrin receptors.     

Depletion of 9L rat brain tumor cell polyamine content by treatment with d,l-α-difluoromethylornithine inhibits proliferation and the G1 to S transition

d,l-α-Difluoromethylornithine (DFMO), an irreversible inactivator of ornithine decarboxylase, inhibited 9L monolayer culture rat brain tumor cell proliferation at concentrations as low as 1 mM DFMO to about 25% of control growth when cells were seeded at an initial density of 5 × 10⁵/flask. DFMO reduced intracellular putrescine content to <5% of control by 8 h and spermidine content to <5 % of control by 48 h post-treatment. Cytostasis caused by 10 or 25 mM DFMO could both be reversed and blocked by addition of exogenous putrescine. Cells pretreated for 48 h with DFMO and then replated in its absence could not enter exponential growth until polyamine production resumed. Addition of exogenous putrescine at the time of replating allowed pretreated cells to resume exponential growth at the same time as controls. Flow cytometry revealed that the fraction of cells in G1 increased until polyamine accumulation resumed, implying the presence of a G1-S block. Within 6 h of replating, there was a decrease in the fraction of control cells in G1. These observations support the hypothesis that entry of 9L cells into S phase depends on an adequate intracellular pool of polyamines.     

Cell kinetics in the mouse esophageal epithelium in relation to the time of day

The kinetics of mouse esophageal epithelial cells was investigated throughout 90 h after a single injection of 3H-thymidine at 01 or at 13 h--the times of the peak and minimal magnitudes of the radioisotope index in the circadian rhythm of proliferation. The mitotic cycle parameters in the cells varied but insignificantly. For cells treated with 3H-thymidine at 01 h, T = 24.3 h, ts = 6 h, tG2 min = 1.5 h, tG2+ 1/2 M = 2.9 h and tG1+/2 M = 15.4 h; for those treated with 3H-thymidine at 13 h, T = 25.6 h, ts = 8.4 h, tG2 min = 1 h, tG2+ 1/2 M = 2.2 h, tG1+ 1/2 M = 15 h. Cells labeled at 01 h proliferated more actively for a long period of time as compared to those labeled at 13 h. The synchronism in undergoing several successive mitotic cycles was greater for cells labeled at the peak radioisotope index. The data obtained also suggest that the majority of cells enter the G0 phase after the completion of the first cycle. The duration of the G0 phase varies in different cell populations.     

Large-scale fragmentation of dna and the death of tumor cells by the action of the binary system ascorbic acid-metallocomplexes of cobalt in vitro

High-molecular-weight DNA fragments are the markers of the early stage of apoptosis induced in eukaryotic cells by cytotoxins of different nature. The dynamics of the development of large-scale DNA fragmentation in K-562 leukemia cells by the action of the antitumor drug, the binary system ascorbic acid--cobalt phthalocyanine within 48 h of incubation, which correspond to two periods of the doubling of cell number in growing control cultures, have been studied. It was shown that, within the first hours of incubation, hydrogen peroxide generated by the system induces the formation of DNA fragments from 2200 to 50 kbp long. Later on the cell death accompanied by a decrease in the content of fragmented DNA is observed. Within 24 h of incubation, part of fragmented DNA remains unrepaired; after 48 h of incubation, a delay or a slowed down proliferation of K-562 cells, which differ from control cells also by a high level of death and a higher content of high-molecular-weight DNA fragments, is observed.     

Dynamic immunological and cytochemical markers in the induced differentiation of the K-562 cell line

The possibility of TPA-induced differentiation of K-562 cell line was studied. Monoclonal antibodies against differentiating antigens of HAE erythroid lineage and against myelomonocytic ICO lineage raised in the USSR Cancer Research Centre were used. Changes in immunological and cytochemical indexes suggest that K-562 cell differentiation goes in the erythroid direction. The cells lost early differentiation and acquired late differentiation markers.     

Role of G proteins and ERK activation in hemin-induced erythroid differentiation of K562 cells

Heterotrimeric G proteins which couple extracellular signals to intracellular effectors play a central role in cell growth and differentiation. The pluripotent erythroleukemic cell line K562 that acquires the capability to synthesize hemoglobin in response to a variety of agents can be used as a model system for erythroid differentiation. Using Western blot analysis and RT-PCR, we studied alterations in G protein expression accompanying hemin-induced differentiation of K562 cells. We demonstrated the presence of G(alpha s), G(alpha i2) and G(alpha q) and the absence of G(alpha i1), G(alpha o) and G(alpha 16) in K562 cells. We observed the short form of G(alpha s) to be expressed predominantly in these cells. Treatment of K562 cells with hemin resulted in an increase in the levels of G(alpha s) and G(alpha q). On the other hand, the level of G(alpha i2) was found to increase on the third day after induction with hemin, followed by a decrease to levels lower of those of uninduced cells. The mitogen-activated protein kinase ERK1/2 pathway is crucial in the control of cell proliferation and differentiation. Both Gi- and Gq-coupled receptors stimulate MAPK activation. We therefore examined the phosphorylation of ERK1/2 during hemin-induced differentiation of K562 cells. Using anti-ERK1/2 antibodies, we observed that ERK2 was primarily phosphorylated in K562 cells. ERK2 phosphorylation increased gradually until 48 h and returned to basal values by 96 h following hemin treatment. Our results suggest that changes in G protein expression and ERK2 activity are involved in hemin-induced differentiation of K562 cells.     

Apoptosis induced by 3,7-dinitrodibenzobromonium salts in K562 cells

We evaluated the inhibitory effect of 3,7-dini-trodibenzobromonium salts (cBr) on the proliferation of human chronic myelogenous leukemia K562 cell by trypan blue exclusion test and MTT colorimetric assay.The degree of DNA damage in K562 cells treated with cBr,was detected by isotopic tracer method (3H-TdR).The morphological changes of these K562 cells were examined by fluorescence and electron microscopy.Biochemical characteristics of K562 cells were detected by flow cytome-try and 3H-thymidine incorporation assay.Findings indi-cated that cBr could significantly inhibit cell proliferation and result in DNA damage of K562 cells,cBr is a new type of immunostimulant and can induce cell apoptosis.     

The lack of correlation between the rate of rRNA transport from nucleoli into cytoplasm and the duration of G1 and G2 phases in root meristem cells

In root meristems of 3 species (Secale cereale L., Vicia faba L. subsp. minor, Allium cepa L.) the durations of cell cycles and their phases were calculated using 3H-thymidine labelling. In the above species and in Helianthus annuus L. (parameters of the cell cycle determined earlier) the G1 and G2 phase durations were different: G1 + 1/2 M from 3 h to 6.1 h, G2 + 1/2 M from 1.1. h to 8.3 h, depending on the species. The rate of rRNA transport from nucleoli into cytoplasm during recovery after cold treatment was calculated from our data presented earlier. The results indicate that in 4 species studied there is no correlation (at P = 0.05) between the rate of rRNA transport and the duration of G1 and G2 phases.     

No effect of extremely low-frequency magnetic field observed on cell growth or initial response of cell proliferation in human cancer cell lines

An effect on the tumor promotion process, as represented by accelerated cell growth, has been indicated as one example of areas that demonstrate the possibility of biological effects of extremely-low frequency magnetic fields. We, therefore, exposed the five cell lines (HL-60, K-562, MCF-7, A-375, and H4) derived from human tumors to a magnetic field for 3 days to investigate the effects on cell growth. Prior to exposure or sham exposure, the cells were precultured for 2 days in low serum conditions. The number of growing cells was counted in a blind manner. To investigate the effect on the initial response of cell proliferation, two cell lines were synchronized in G1 phase by serum starvation and then exposed to a magnetic field for 18 h (H4 cells) or 24 h (MCF-7 cells), both with and without serum stimulation. The rate of DNA synthesis, taken as a measure of the cell proliferation, was determined by following the incorporation of [(3)H]-thymidine into the DNA. Three different magnetic field polarizations at both 50 and 60 Hz were used: linearly polarized (vertical); circularly polarized; and an elliptically polarized field. Magnetic field flux densities were set at 500, 100, 20 and 2 microT (rms) for the vertical field and at 500 microT (rms) for the rotating fields. No effect of magnetic field exposure was observed on either cell growth or the initial response of cell proliferation.     

低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制研究

目的初步探讨低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制。方法甲基纤维素集落形成实验检测低浓度丰加霉素对人白血病K562细胞集落形成能力的影响;CCK-8法检测低浓度丰加霉素对K562细胞的生长抑制率;AnnexinV／PI双染流式细胞仪检测低浓度丰加霉素作用下的K562细胞凋亡率;PI单染流式细胞仪检测药物作用后细胞的周期分布改变;Western免疫印迹和实时定量PCR检测周期相关分子表达水平变化。结果低浓度丰加霉素对人白血病K562细胞具有较强的集落形成抑制作用;可明显抑制K562细胞的生长,呈时间一剂量依赖性;尽管短时间（48h）的药物处理仅出现轻度的细胞凋亡和周期阻滞,但10nmol/L和30nmol/L的丰加霉素长时间（7d）作用后,K562细胞G0／G1期比例分别是（62．3±1．7）％和（76．9±0．7）％,与对照组（38．9±1．1）％相比差异具有高度统计学意义（P〈0．01）;低浓度丰加霉素长时间作用后诱导K562细胞周期相关分子P16蛋白水平和转录水平的高表达。结论丰加霉素在低浓度,长时间作用于人白血病K562细胞后,具有较强的集落形成抑制和生长抑制作用,此作用可能与诱导细胞周期相关分子p16高表达,导致细胞G0／G1期阻滞有关。     

Transforming growth factors beta slow down cell-cycle progression in a murine interleukin-2 dependent T-cell line

Transforming growth factors beta (TGF-beta) inhibit the growth of a variety of cell types, including lymphocytes. The immunosuppressive effects of TGF-beta have been attributed to the interference of these molecules with the interleukin-2 (IL-2)-driven component of lymphocyte proliferation. In order to elucidate in more detail the effects of TGF-beta on IL-2-induced proliferation, we investigated the effects of porcine transforming growth factor beta 1 and 2 (pTGF-beta 1 and 2) on the IL-2-driven proliferation of a murine IL-2-dependent T-lymphocyte line (CTLL). The results showed that pTGF-beta 1 and 2 decreased 3H-thymidine incorporation in CTLL cells in a dose-dependent fashion (maximum decrease of 75-85%). Combined-time kinetic analysis of the effects of pTGF-beta on 3H-thymidine incorporation, cell growth, and cell-cycle distribution (monitored as DNA content distribution) revealed that, in the first 48 h of culture, pTGF-beta 1 increased the doubling time from 11.4 to 19.2 h without significantly affecting the cell-cycle distribution of CTLL cells. After 96 h of culture in the presence of pTGF-beta 1, cells started to accumulate in G0/G1, although at this time point 30% of the pTGF-beta 1-treated cells were still in S-G2/M. Furthermore, during the first 48 h, neither the expression of the 55 kd chain of the IL-2 receptor (IL-2R) nor the expression of the transferrin receptor (TfR) was affected by TGF-beta. After 72 h of culture in the presence of pTGF-beta 1, the expression of the IL-2R and TfR was decreased. The data suggest that in CTLL cells TGF-beta initially slows the progression of cells in all phases of cell cycle. In addition, the initial TGF-beta-mediated decrease of IL-2-induced 3H-thymidine incorporation and cell proliferation in CTLL cells is not due primarily to downregulation of the IL-2R and/or TfR.     

DNA synthesis in human duodenal biopsies maintained in organ culture

Incorporation of 3H-thymidine during organ culture was studied in duodenal biopsies from 14 patients. Pulse-label at various intervals disclosed active incorporations during the first 2 h in culture. Labelling index declined to low levels at 3-4 h. Thereafter incorporation increased again and persisted throughout the rest of the culture period of 11 h. The DNA synthesis rate of crypt cells between 4 and 11 h in culture was calculated in 5 patients after pulse-label and continuous labelling of explants in parallel culture. The rate of entry into DNA synthesis was about 24 cells per 1,000 crypt cells per hour in flat, coeliac biopsies, versus 9-13 in controls, Gluten did not influence DNA synthesis rate, whereas wheat germ lectin inhibited DNA synthesis. Counting of the total number of mitoses and labelled fraction of mitoses disclosed active crypt cell renewal in flat, coeliac biopsies. In normal-appearing biopsies no mitoses were labelled, indicating delayed exit from S-phase or long duration of G2-phase in these explants.