Effect of the tumor promoter TPA on the distribution of PHA-stimulated human lymphocytes by cell cycle phases

Patterns of the cell cycle distribution in human peripheral blood lymphocytes, stimulated by PHA alone and PHA plus 12-o-tetradecanoylphorbol-13-acetate (TPA), were studied using DNA cytometry in different times after PHA stimulation. In the first period (nearly 3 days after PHA stimulation) TPA induces no significant differences in the characters under consideration, but in the later period, when the proliferation of the cultures stimulated by PHA alone is reducing, in other cultures stimulated by PHA plus TPA the percentage of cells in S-phase does not reduce, whereas the percentage of cells in G2-phase is rising, which may suggest that this phase is blocked. Concurrently the tetraploid cells are appearing. Accumulation of cells in G2-phase can be overcome by the application of chlorpromazine, which is known to inhibit the membrane-associated protein kinase C.     

Interleukin 2 mRNA induction in human lymphocytes: analysis of the synergistic effect of a phorbol ester and phytohemagglutinin

Induction of interleukin 2 (IL2) mRNA synthesis in human tonsillar lymphocytes was studied by quantifying the relative levels of IL2 mRNA in the lymphocytes stimulated under various conditions by the dot hybridization method. A remarkable increase of IL2 mRNA was induced by stimulation with phytohemagglutinin (PHA) in the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The kinetic study revealed that the IL2 mRNA level of the lymphocytes increased from 2 h of culture, reached a maximal level at 12 h, maintained a relatively high level until 48 h and then sharply decreased by 72 h after the stimulation. Inhibition experiments with actinomycin D showed that the increase was due to a transient synthesis of the mRNA after the stimulation, which almost stopped by 12-16 h. DNA synthesis and cell division were not necessary for the induction of IL2 mRNA production but the induction was inhibited by dexamethasone, showing that the production was mainly associated with the G1 phase of the cell cycle. Two-step culture experiments showed that prior exposure of the lymphocytes to TPA for 1 h at 37 degrees C resulted in a remarkable increase of IL2 mRNA on subsequent stimulation with PHA. This suggests that TPA induces certain changes in the biochemical pathway of signal transduction so that the cells can be triggered to express IL2 gene by subsequent stimulation with mitogen.     

Increase of uv-induced DNA repair synthesis during blast transformation of human lymphocytes.

UV-induced DNA repair synthesis, measured as unscheduled DNA synthesis, was studied in human peripheral lymphocytes in various phases of the cell cycle. Mitogen transformation of the lymphocytes was effected with phytohemagglutinin (PHA), and the stage in the cell cycle was determined by measuring the Feulgen DNA content and the dry mass in individual cells by cytophotometry. The initial rate of repair was determined by autoradiography after UV-light irradiation (19.2 J/m²) and incubation of the cells for 30 min with [³H]thymidine. When the cells progressed from the G0 to the G1 phase there was a 3-fold increase in the grain count. The correlation between the grain count and the dry mass indicated an increase in the initial rate of repair during the progression of cells from G0 to G2 phase. G2 cells were more heavily labelled than those in G1, but there did not seem to be any difference between these two phases as regards the relationship between grain count and DNA content. The results indicate that the initial rate of UV-induced DNA repair may differ in various phases of the lymphocyte cell cycle.     

Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.     

Alteration of cell cycle progression in human leukemia cell line (KOPM-28) induced by 12-o-tetradecanoylphorbol-13-acetate

Terminal cell differentiation usually results in an irreversible arrest in the G1 phase of the cell cycle and loss of cell renewal ability. Human promyelocytic leukemia HL-60 cells induced with 12-o-tetradecanoylphorbol-13-acetate (TPA) differentiate into monocytes/macrophages and accumulate in G1. We determined the effect of TPA on the growth kinetics of a human leukemia cell line (KOPM-28), which developed several of the characteristics of megakaryocytes in response to TPA, such as the surface antigen complex IIb/IIIa, platelet peroxidase and polyploidy. Cell growth was immediately and completely inhibited by TPA. Flow cytometric analysis of cellular DNA content revealed a gradual decrease in cells in G1 and an accumulation of cells in G2. These data suggest that TPA prolonged G1 and rapidly arrested the cells in G2. Synchronized cells were utilized to further analyze the rapid G2 arrest. Cells arrested with aphidicolin at the G1/S interphase were released, and the effects of TPA (added at different intervals) on cell cycle progression were examined 14 h after release. The results showed that TPA added at the end of the S phase, as well as at the G1/S interphase incompletely but distinctly arrested cells in G2. Moreover, G2 arrest was observed when TPA was added to cells released from a colcemid-induced G2/M block, suggesting that cells already in G2 were inhibited by TPA from moving through M to G1. Since some cells became multi-nucleated in the course of incubation with TPA, this G2 accumulation may have resulted at least in part from a prolongation of the phase or a transient G2 block. These changes in cell cycle progression induced by TPA may be characteristic of and/or related to megakaryocytic differentiation of hemopoietic precursor cells.     

Modalities of synthesis of Ki67 antigen during the stimulation of lymphocytes

The antibody Ki67 is currently used to evaluate the proliferative fraction of solid tumors and some hematological malignancies. We have used phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes as a model to study the entry of quiescent cells into cell cycle and to follow their progress to the next cycle. Flow cytometric analysis of lymphocyte samples stained with the antibody Ki67 and a DNA marker has allowed us to follow the expression of Ki67 antigen (Ki67 Ag) as a function of the position of the cells in the cell cycle. The use of drugs blocking the stimulated lymphocytes in different phases of the cell cycle permitted us to demonstrate that Ki67 Ag expression started from the beginning of the first S phase. The level of Ki67 Ag increased during S phase until mitosis, when its expression was maximal. After division, the cells in G1 phase showed a decrease in Ki67 Ag expression (possibly corresponding to degradation) until they reentered S phase, when the level of Ki67 Ag increased again. The results confirm that the expression of Ki67 Ag is related to the proliferative state of the cells and suggest that it may be used to determine the proliferative cell fraction in hematopoietic tissues.     

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.     

Endogenous blockage and delay of the chromosome cycle despite normal recruitment and growth phase explain poor proliferation and frequent edomitosis in Fanconi anemia cells.

BrdU-Hoechst flow cytometry was employed to study the proliferation kinetics of blood lymphocytes from patients with Fanconi anemia (FA). Compared to controls, untreated FA lymphocytes show normal response to PHA stimulation, normal G0/G1 exit rates, and normal first S-phase durations. The G2 phase of the first cell cycle, however, is severely prolonged, and 24% of the recruited population become arrested during the first chromosome cycle (S, G2/M phases). The delay suffered during G2 appears to be compensated in part by a subsequent G1 phase duration that is unusually short for postnatal human cells (3.7 +/- 0.5 hrs). In analogy to what has been observed in other cell systems after experimental delays of the chromosome cycle, we therefore postulate that at least some FA cells enter their second growth phase without prior completion of the delayed chromosome cycle. Renewed replication would ensue in such cells without prior passing through mitosis and cytokinesis, leading to endoreduplication, which is a frequent finding in the FA syndrome.     

12-O-tetradecanoylphorbol-13-acetate induces transient cell cycle arrest in G1 and G2 in metastatic melanoma cells: inhibition of phosphorylation of p34cdc2.

The growth of Demel human metastatic melanoma cells was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) and other nonphorbol tumor promoters including palytoxin and okadaic acid. Using flow cytometry, we have demonstrated that the cells arrested growth in G1 and G2 phases of the cell cycle. Detailed analysis of the kinetics of the growth arrest in unsynchronized cells showed that (a) the growth arrest was transient and peaked 16-20 h following addition of TPA; (b) effects of TPA on cell growth began within 1-2 h after the addition; and (c) cells completed S phase and arrested in G2. In addition, TPA induced a pronounced morphological change, which peaked by 1 h and gradually subsided over 24 h. In populations of cells synchronized in G1 using lovastatin, (a) addition of TPA blocked the onset of DNA synthesis up to the end of G1; (b) the lag between addition of the drug and onset of DNA synthesis was less than 30 min; and (c) addition of TPA at the end of G1 prevented the increased phosphorylation of p34cdc2, as determined by immunoprecipitation. The experiments reported here show that TPA transiently blocked the proliferation of Demel melanoma cells at the G1-S border and in G2, thus preventing cells from progressing through the cell cycle. These experiments suggest that pathways involving protein kinase C interact with and rapidly alter the molecular pathways involving p34cdc2 which regulate the onset of DNA synthesis and the G2-M transition.     

Nucleolar silver-staining patterns related to cell cycle phase and cell generation of PHA-stimulated human lymphocytes

Silver staining (Ag-I) was used to investigate changes in the nucleolar structure of PHA-stimulated human lymphocytes through the phases of the cell cycle, G1, S and G2. Ag-I patterns and cell cycle phases of individual cells were assessed by sequential silver staining, Feulgen staining, DNA microdensitometry and 3H-thymidine autoradiography. The morphology and number of Ag-I nucleoli in a particular cell depended upon the phase of the cell cycle reached and on the number of generations the cell had passed through in culture. Resting, unstimulated cells usually had one small silver positive nucleolus. During blast transformation, the silver stained nucleoli increased in number and size, and then fused to form one very large, rounded or irregular-shaped nucleolus which was present through all cell cycle phases of the first reproductive cycle. Many lymphocytes developed a band-shaped nucleolus during their first S phase in culture. Lymphocytes at all cell cycle stages of the second and third generations after PHA-stimulation had multiple nucleoli whose combined areas approximated that of the single large nucleolus observed in first generation cells.     

Lamprey buccal gland secretory protein-2(BGSP-2) inhibits human T lymphocyte proliferation

Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We identified a buccal gland secretory protein-2 (BGSP-2) from a buccal gland cDNA library of Lampetra japonica. The full-length BGSP-2 gene was cloned and the recombinant BGSP-2 protein was generated. The role of BGSP-2 on lymphocyte proliferation was studied by examining its effects on human T lymphocytes. We found that lamprey BGSP-2 was able to effectively block the proliferation of T cells in vitro by inducing G_1/S cell cycle arrest. Furthermore, it inhibited the proliferation of human T lymphocytes stimulated by phytohemagglutinin (PHA) at a minimum concentration of 0.1μg/ml. Our data suggest that lamprey BGSP-2 is able to block the mitosis of human T lymphocytes at the G1/S point, and has the potential of anti-proliferative effect on PHA-activated T lymphocytes.     

Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle

The effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12–24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30–36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110^Rb phosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34^cdc2 synthesis, and cyclin A synthesis. In control cultures, p110^Rb phosphorylation was detected within 24 hr of PHA stimulation; p34^cdc2 and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c-fos, IL-2, and IL-4 mRNAs expression, and IL-2 receptor (p55) expression, were only marginally affected, if at all, in PHA-stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL-2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL-2/IL-2 receptor interaction but prior to p110^Rb phosphorylation and other key regulatory events signaling G₁/S transition. © 1993 Wiley-Liss, Inc.     

Differential effects of synthesized 2'-oxygenated chalcone derivatives: modulation of human cell cycle phase distribution

Ten structurally related 2'-oxygenated chalcone derivatives, bearing either hydroxy and/or methoxy substituents on the A and B rings, were synthesized through Claisen-Schmidt condensation. The synthesis procedure was relatively easy and had an acceptable yield. The in vitro cytotoxicities of these compounds against the human tumor cells such as Jurkat, U937 cells, and normal cells PHA stimulated PBMCs were investigated. Among those, compounds 1 (IC50 = 2.5 microM), 2 (1.7 microM), and 8 (3.2 microM) showed potent inhibitory activity toward Jurkat cell line. In parallel, compounds 1 (6.7 microM), 2 (1.5 microM), and 10 (5.3 microM) showed the highest activity against U937 cell line. However, the chalcones also inhibit the PHA stimulated PBMCs cells, but the IC50 values were relatively high when compared to the tumor cell line values. Studies were also on the effect of synthesized chalcones on the cell cycle phase distribution. In Jurkat cell line, compounds 7 and 9 showed the highest activity and the most striking effect in reduction of the percentage of cells in the S phase, which was associated with an increase of cells in G2/M phase. In U937 cell line, compound 3 increased the proportion of cells in the G0/G1 phase and reduced the proportion in S phase. In contrast, compounds 1, 9, and 10 showed a decrease effect on the percentage of cells in S phase and an increase effect on the percentage of cells in the G2/M phase of the cell cycle. Whereas in the case of PHA stimulated PBMCs, compounds 1, 4, 8, and 10 increased the percentage of cells in G2/M phase, which was associated with a decrease effect in the S phase of the cell cycle.     

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.     

Radiation protection of stimulated human lymphocytes by nicotinamide

Nicotinamide (NA) when added to human lymphocytes in vitro together with a mitogen, protected against the inhibition by gamma and UV radiation of stimulated cell growth. When stimulated by phytohemagglutinin (PHA), concanavalin A (Con A) or pokeweed mitogen (PWM) maximum protection has been observed with approximately 1 mM NA (dose reduction factor of 2-3). To obtain protection the cells had to be stimulated immediately after irradiation in the presence of NA. It is suggested that the intracellular level of NAD+ may be rate limiting for excision repair in human lymphocytes irradiated in the G0 phase. This level is presumably increased by exogenously supplied NA, leading to enhanced repair of DNA damage and increased survival.     

Desferoxamine blocks IL 2 receptor expression on human T lymphocytes

Thymidine uptake by PHA-stimulated human lymphocytes is reduced in the presence of 100 microM or greater concentrations of the iron-chelating agent desferoxamine (DF). We assessed expression of IL 2 receptor, 4F2 and Ia antigens, IL 2 production, and cell cycle progression by blood mononuclear cells (MNC) stimulated by PHA in the presence or absence of DF to determine whether the lack of T cell proliferation was a manifestation of inhibition of an earlier activation event. Tac antigen expression on PHA-stimulated MNC was inhibited by DF throughout 8 days of culture, and those cells which were positive had a low density of Tac antigen as compared with controls without DF. Expression of other activation antigens, 4F2 and Ia, was not impaired by DF. The supernatants of the DF-containing and control cultures contained equivalent IL 2 activity, as measured on the HT-2 cell line. Cell cycle analysis of these cultures shows that the addition of DF at the beginning of culture blocks most cells from undergoing G0 to G1 transition, whereas later addition of DF arrests the progression of the T cell blasts through the cell cycle. Separation of cells cultured with PHA and DF into Tac+ and Tac- subsets showed that progression from G0 to G1 was restricted to the former subset. These results suggest that interference with IL 2 receptor expression might contribute to the block in mitogen-induced proliferation caused by DF.     

Enrichment of viable lymphocytes in defined cycle phases by sorting on the basis of pulse width of axial light extinction

A correlation between cell size and cycle state is demonstrated for mitogen stimulated murine lymphocytes. Cell cycle analysis was performed using multiparameter cytofluorometric analysis of acridine orange stained cells. Cell size was determined by measuring "time of flight" of axial light extinction. Cells with diameters of 5.0 - 6.0 micron are shown to be predominately in G0. Among stimulated cells, those 6.0 - 7.0 micron in diameter are predominately early and late G1 (80%) with some S phase cells. Cells in G2 + M were found to predominate among lymphocytes with diameters of 7.5 - 12 micron. This approach provides a method for isolation of lymphocytes in specific phases of the cell cycle which are suitable for subsequent functional studies.     

Alterations of cell cycle kinetics and vimentin expression in TPA-treated, asynchronous MPC-11 mouse plasmacytoma cells

Vimentin expression throughout the cell cycle has been analyzed at the single-cell level in asynchronously growing MPC-11 cells using multiparameter flow cytometry. We have previously shown that these cells normally lack detectable amounts of intermediate filament proteins. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), cell proliferation ceases and large quantities of the intermediate filament protein vimentin are synthesized and accumulate in most of the cells. In the present study, the short-term effect of TPA on distribution of cells within the cell cycle was a depletion in early S phase followed by a depletion in mid- and late S phase. In parallel, the G1-phase fraction increased significantly. In addition, a delay in progression through G2/M phase was observed. These data strongly suggest an inhibition of progression of cells through the cell cycle in G1 phase as the primary event on cell cycle kinetics elicited by TPA. Vimentin accumulation could be detected by flow cytometry as early as 2 h after TPA addition; at this time, the percentage of vimentin-positive cells was highest in G2/M phase. Prolonged TPA treatment induced vimentin accumulation in cells of all cell cycle phases. However, even at later times, the G1-phase population consisted of two subpopulations with low and high vimentin content, respectively. The fraction of cells which displayed a higher level of vimentin probably represents those G1-phase cells which previously had undergone cell division in the presence of TPA. Our data indicate that TPA-induced vimentin synthesis is regulated in a cell cycle-dependent manner and is maximally induced in cells which have passed a putative cell cycle restriction point in G1 phase.