Lymphocyte activation: the dualistic effect of cAMP

The effects of exogenously added cyclic nucleotides on DNA synthesis have been investigated in human peripheral blood lymphocytes stimulated with phytohemagglutinin (PHA). At low doses of PHA the addition of exogenous cAMP resulted in an inhibition of DNA synthesis. At optimal or supraoptimal doses of PHA the addition of cAMP, db-cAMP, or 8-Br-cGMP resulted in enhancement of DNA synthesis. Measurement of cell associated cAMP and cGMP levels in lymphocytes exposed to PHA with or without exogenously added cAMP revealed a gradual increase in cAMP levels and a fluctuating decline in cGMP levels.     

Effects of in vivo exposure to antineoplastic drugs on DNA repair and replication in human lymphocytes

In peripheral blood lymphocytes of 12 nurses and 3 patients exposed to antineoplastic drugs we determined the ability to repair DNA after UV irradiation and DNA replicative synthesis after stimulation by PHA. In nurses the levels of unscheduled DNA synthesis and DNA replication were not different than in a control group, whereas in patients significant changes were observed during and after chemotherapy in the level of both types of DNA synthesis.     

A sodium requirement for mitogen-induced proliferation in human peripheral blood lymphocytes

Mitogen-stimulated DNA synthesis in human peripheral blood lymphocytes is dependent on extracellular Na. DNA synthesis was similarly inhibited in:

1. 1. Cells that were suspended in hypotonic media containing decreased extracellular Na.

2. 2. Cells that were suspended in media containing decreased Na and equimolar replacement with choline.

3. 3. Cells that were suspended in media containing decreased Na and equiosmolar replacement with mannitol.

A decreased PHA-induced DNA synthesis was observed at day 3 even when lymphocytes were exposed to low Na for only the first 3 h and then returned to normal levels of Na. Our studies of protein synthesis indicate that the effect of lowered extracellular Na on DNA synthesis and cell division is not due to an initial inhibition of overall protein synthesis. These data suggest that reduced external Na has a significant effect on some specific early event(s) (3 h) in lymphocyte mitogenesis.     

Effects of chlorambucil on human chromosomes

No significant amount of chromosomal damage was found in the 48-h cultures of lymphocytes of 18 patients who had been treated with the bifunctional alkylating agent chlorambucil (CBC). However, there was suggestive evidence of chromatid damage (i.e. of types attributable to damage during or after DNA synthesis in the cell cycle). In marrow cells of 3 patients given a single large dose of chlorambucil (equivalent to 2 days' normal treatment) there was also suggestive evidence of induced chromatide-type damage.Extensive series of in vitro experiments yielded evidence that (a) exposure of human lymphocytes over the whole period of culture showed chromatid-type damage; (b) this damage increased sharply from concentrations of 0.5 μg/ml to3.0 μg/ml; (c) although chromatide-type damage always predominated, there was suggestive evidence also of chromosome-type aberrations attributable to damage occuring in the G₀/G₁ period, although some or all of this could be attributed to “derived” chromatid damage; (d) even if lymphocytes were only exposed during the G₀ or G₁ periods of the cycle, damage was found in the subsequent metaphases and it was almost entirely of the chromatid type; (e) much more damage occurred in lymphocytes exposed for varying periods to the drugs after stimulation by phytohaemagglutinins than in those exposed in whole blood, or in medium before stimulation; (f) damaged occurred in lymphocytes exposed to the drug while in S but not exposed only when in G₂; (g) no evidence was found that unschaduled DNA synthesis during G₀ or G₁ was induced by the drug; (h) there appeared to be no delay caused by the drug in the time at which cells reached the first “S” phase in culture but there was some evidence consistent with prolongation of “S” in cells exposed in culture; (i) there was evidence that CBC alone could stimulate lymphocyte tto DNA synthesis, and that a few cells proceeded in the cycle to prophase, or even metaphase. However, there was a considerable amount of cell-killing during CBC-stimulated DNA synthesis.     

Increased frequency of sister-chromatid exchange and chromatid breaks in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.     

DNA repair after gamma irradiation in lymphocytes exposed to low-frequency pulsed electromagnetic fields

The effect of exposure to extremely low-frequency pulsed electromagnetic fields (EMFs) on DNA repair capability and on cell survival in human lymphocytes damaged in vitro with gamma rays was studied by two different micromethods. In the first assay, which measures DNA repair synthesis (unscheduled DNA synthesis, UDS), lymphocyte cultures were stimulated with phytohemagglutinin (PHA) for 66 h and then treated with hydroxyurea (which blocks DNA replication), irradiated with 100 Gy of 60Co, pulsed with [3H]thymidine ([3H]TdR), and then exposed to pulsed EMFs for 6 h (the period in which cells repaired DNA damage). In the second assay, which measures cell survival after radiation or chemical damage, lymphocytes were first irradiated with graded doses of gamma rays or treated with diverse antiproliferative agents, and then stimulated with PHA, cultured for 72 h, and pulsed with [3H]TdR for the last 6 h of culture. In this case, immediately after the damage induced by either the radiation or chemicals, cultures were exposed to pulsed EMFs for 72 h, during which cell proliferation took place. Exposure to pulsed EMFs did not affect either UDS or cell survival, suggesting that this type of nonionizing radiation--to which humans may be exposed in the environment, and which is used for both diagnostic and therapeutic purposes--does not affect DNA repair mechanisms.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

SCE evaluations in human lymphocytes after G0 exposure to mitomycin C Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions

We conducted a series of experiments designed to determine whether DNA damage induced in G₀ lymphocytes by mitomycin C (MMC) would be expressed as sister-chromatid exchanges during the second and third post-treatment cell cycles. Lymphocytes from normal donors were exposed to MMC for 2 h prior to culture in the presence of phytohemagglutinin. MMC-treated and control cells were subsequently exposed to bromodeoxyuridine (BrdUrd) for the entire culture period (i.e. 48 h or 72 h) or for the terminal 24 h of 72-h cultures. We observed a 3–4-fold increase in SCEs in MII metaphases from lymphocytes treated with MMC and cultured in the presence of BrdUrd for the entire culture period. In contrast, in replicate cultures of MMC-treated lymphocytes that were exposed to BrdUrd for the terminal 24 h only, the SCE frequency in uniformly harlequinized metaphases was not significantly different from that observed in control cultures. We interpret these data as providing evidence that MMC-induced lesions (or alterations) in the DNA of G₀ lymphocytes are probably expressed as SCEs during the first period of mitogeninduced DNA synthesis, and that these lesions do not persist and give rise to SCEs in subsequent cell divisions.     

Reactivity of human lymphocytes to aggregated and nonaggregated PPD-tuberculin

PPD-tuberculin was adsorbed to bentonite and the effect of this preparation on induction of DNA synthesis in cultures of human lymphocytes was compared to that of ordinary soluble PPD. At high PPD concentrations the two preparations gave rise to comparable responses. At lower concentrations the bentonite-PPD always stimulated stronger lymphocyte responses than soluble PPD. Lymphocytes exposed to bentonite-PPD became activated earlier than cells confronted with soluble PPD. It is suggested that the findings are caused by a more efficient interaction between particle-bound as compared to soluble antigen and lymphocytes having low affinity receptors for the antigen.     

Effects of arsenic on DNA synthesis in human lymphocytes stimulated by phytohemagglutinin

Effects of arsenic on DNA synthesis in human lymphocytes were biphasic: either trivalent (arsenic trioxide and sodium arsenite) or pentavalent (sodium arsenate) arsenic compounds at very low concentrations enhanced DNA synthesis in human lymphocytes stimulated by phytohemagglutinin (PHA), whereas higher concentrations inhibited DNA synthesis. There were differences among individual susceptibities to arsenic-induced DNA synthesis. Either stimulating or inhibiting effects of trivalent arsenic on DNA synthesis in PHA-stimulated lymphocytes were always stronger than those of pentavalent arsenic. It was also shown that both trivalent and pentavalent arsenic could be rapidly taken up into the human lymphocytes, and immediately stimulated or inhibited DNA synthesis. A possible dual effect of arsenic at very low concentrations as both comutagen and inhibitor of mutagenesis is discussed.     

Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes

An early event in death of interphase lymphocytes exposed in vivo or in vitro to low doses of gamma-irradiation is the degradation of DNA into nucleosome-sized fragments. Induction of fragmentation required RNA and protein synthesis because actinomycin D and cycloheximide, respectively, are able to inhibit DNA fragmentation in irradiated lymphocytes. Studies adding cycloheximide and actinomycin D at various times postirradiation suggest that once the metabolic process is initiated within an individual cell it proceeds to completion. The reversible RNA synthesis inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole inhibits DNA fragmentation in irradiated thymocytes. When this drug is removed after 6 hr, irradiated thymocytes proceed to fragment their DNA; this suggests that an inducing "signal" that is not simply mRNA persists within the irradiated cell for at least 6 hr after irradiation. In contrast to mitogen-activated T and B lymphoblasts, resting T and B cells show significant DNA fragmentation after exposure to 100 to 500 rad. At 2000 rad, all of the splenic subpopulations die rapidly via a different mechanism. By studying the mechanism of DNA fragmentation induced during the interphase death of lymphocytes, we hope to understand better the extreme sensitivity of resting lymphocytes to radiation and what may be the common final pathway of programmed cell death.     

Polyamine depletion results in impairment of polyribosome formation and protein synthesis before onset of DNA synthesis in mitogen-activated human lymphocytes

Lymphocytes, exposed to mitogens in culture, show enhanced protein and RNA synthesis before the onset of DNA synthesis. Inhibition by DL-alpha-difluoromethylornithine of polyamine synthesis in phytohaemagglutinin-activated human lymphocytes resulted in a suppression of protein synthesis, which was evident before the initiation of DNA synthesis. The mitogen-induced increase in the incorporation of [3H]thymidine into DNA was subsequently inhibited in parallel with the activity of thymidine kinase in the polyamine-depleted cells. Ultraviolet absorbance measurement of the ribosomes after sucrose gradient centrifugation revealed a suppression of polyribosome formation that coincided with the decrease in the rate of protein synthesis. The disturbance in the polysomal profiles did not appear to be due to a shortage of mRNA, since the synthesis of poly(A)-rich mRNA was reduced less than that of rRNA after inhibiting polyamine synthesis. Entry of both the pre-existing and newly synthesized ribosomal subunits into polysomal structures was found to be impaired. These results thus suggest an important role for polyamines in the initiation step of protein synthesis.     

Expression of the p53 protein during the cell cycle of human peripheral blood lymphocytes

We have investigated the role of the cellular p53 protein in the induction of growth in size and cell DNA replication in human peripheral blood lymphocytes (PBL) and in monocyte/macrophage-depleted lymphocyte (MDL) cultures stimulated with phytohemagglutinin (PHA). Our results show that in human lymphocytes exposed to PHA, the induction of p53 protein synthesis and accumulation correlates with the extent of cellular DNA replication, rather than with growth in size. Moreover, the induction of p53 is dependent on the presence of the T-cell mitogen, Interleukin-2. A monoclonal antibody to Interleukin-2 receptors (anti-Tac) inhibits PHA-stimulated cellular DNA synthesis, and this inhibition is correlated with a reduction in the percentage of p53-positive cells. We conclude from this work that the p53 protein is a cell cycle-dependent gene whose expression can be regulated by different mitogens in different cell types.     

Cytogenetic effects of hexavalent chromium in Bulgarian chromium platers

The aim of the present study was to evaluate the genotoxic effects of hexavalent chromium (Cr(VI)) in vivo in exposed Bulgarian chromium platers by using classical cytogenetic and molecular cytogenetic analyses of peripheral lymphocytes and exfoliated buccal cells. No significant difference was observed between the exposed workers and the controls with regard to the frequency of cells with chromosome aberrations (CAs) using conventional Giemsa staining and in the frequency of sister chromatid exchanges (SCEs). However, there was a significant increase in the number of cells with micronuclei (MN) in peripheral lymphocytes from chromium exposed workers as compared to the controls. In the buccal cells from these workers, this increase was even more pronounced. Cytosine arabinoside (AraC), an inhibitor of DNA synthesis and repair, was found to significantly increase the levels of MN in vitro in the lymphocytes of both groups. The increase was more expressed in the lymphocytes of chromium exposed workers. Both centromere positive (C(+)) as well as centromere negative (C(-)) MN were observed by the fluorescence in situ hybridization (FISH) technique in both of the cell types studied. No difference between C(+) and C(-) MN frequencies was found in the lymphocytes as well as in the buccal cells. Thus, Cr(VI) appears to have both clastogenic as well as aneugenic effects in humans.     

Stimulation of sterol and DNA synthesis in leukemic blood cells by low concentrations of phytohemagglutinin

The response of leukemic cells from AKR/J mice to phytohemagglutinin (PHA) was compared with that of normal lymphocytes. PHA stimulated first cholesterol synthesis and then DNA synthesis in both lymphocytes and leukemic cells. The neoplastic cells were, however, much more sensitive to PHA, requiring less time and a lower concentration of the lectin for optimum stimulation as compared to lymphocytes. In fact, the amount of PHA which was required to activate lymphocytes to proliferate, as measured by increases in sterol and DNA synthesis, was inhibitory to leukemic cells. The basal level of cholesterol synthesis and the induction of cholesterol synthesis following PHA activation were depressed in lymphocytes and leukemic cells by treatment with 25-hydroxycholesterol and 7-ketocholesterol. These two oxygenated derivatives of cholesterol are known to be potent and specific inhibitors of sterol synthesis. Blockage of sterol synthesis by these reagents also abolished PHA-activated DNA synthesis in lymphocytes and leukemic cells. The results support the hypothesis that the synthesis of cholesterol is an important event leading to cell proliferation.     

Immunologic studies on hamsters infected with Entamoeba histolytica.

The serologic and cell-mediated immune responses of hamsters exposed to 2 strains of Entamoeba histolytica (HM-1 and HM-19) were evaluated by a series of in vitro tests. The pathogenicity of the 2 strains was evaluated in terms of their ability to produce liver abscesses and spleen enlargement. Antibody response was evaluated by the indirect hemagglutination test. The cellular immune response was assayed by increased DNA synthesis by lymphocytes and migration inhibition of macrophages.     

Effect of 5-fluoro-2′-deoxyuridine and hydroxyurea on the phytohemagglutinin-induced increase of thymidine kinase,replicative DNA polymerase,deoxycytidylate deaminase and CDP reductase activities in human lymphocytes

Summary The inhibitors of DNA synthesis, 5-fluoro-2-deoxyuridine and hydroxyurea, caused an inhibition of thymidine kinase, replicative DNA polymerase and CDP reductase activities in stimulated lymphocytes when they were exposed to the inhibitors during the early transformation period (0–17 hr). However, the enzyme activities were unaffected when the inhibitors were added to cells stimulated for more than 17 hr. As opposed to these enzymes the deoxycytidylate deaminase activity was unaffected by the inhibitors during the entire transformation period (0–28 hr). This indicates a close regulatory mechanism in lymphocytes between DNA synthesis and induction of enzymes involved in DNA replication. The inhibitory mechanism exerted by the inhibitors is for the moment unknown. It might be independent of the well-known inhibition of the target enzymes, thymidylate synthetase and ribonucleoside diphosphate reductase, since there was no immediate apparent correlation in time between depletion of the pool sizes and the inhibition of the enzyme activities.     

The cytogenetic, proliferative and viability effects of four bacteriophages on human lymphocytes

Certain bacteriophages have been found in live virus vaccines, while a few others have been associated with disease states. Some of these phages have produced abnormal growth of eukaryotic tissue cultures. For this reason bacteriophages phiX-174, MS2, T2 and an isolate from live virus vaccines, phiV-1, were incubated with human cell cultures for examination of chromosomal effects, cell proliferation and viability. Mitogen-stimulated lymphocytes and human embryonic kidney tissue cultures showed no increase in chromosomal abnormalities for high doses of phage-infected versus control cultures. Tritiated-thymidine uptake, correlated with mitotic indices for phage-treated lymphocyte cultures, indicated a reduction in cell division, while 51-chromium release studies showed no cell death occurring in these cultures. This suggested that inhibition of DNA synthesis was occurring in some cells. The presence of phage in the supernate of cells that were exposed to phage suggested the possibility of phage attachment to the plasma membranes of lymphocytes, which may in turn affect the suppression of DNA synthesis.     

Changes in the spontaneous DNA-synthesizing activity in the peripheral blood lymphocytes of irradiated animals

In lymphocytes of sheep exposed to 52 and 103 mC/kg radiation revealed was an increase in spontaneous incorporation of 3H-thymidine into DNA. A change in spontaneous DNA-synthesizing activity in lymphocytes of exposed animals was accompanied by the increase in the total protein content of the peripheral blood lymphocytes.     

INHIBITORY EFFECTS OF MITOGENS ON ADENOIDAL LYMPHOCYTES IN VITRO

Cultures of human adenoidal lymphocytes exposed briefly to either phytohemagglutinin (PHA), Staphylococcus filtrate (Staph-F), concanavalin-A(Con-A), or pokeweed mitogen (PWM) incorporate increased amounts of thymidine earlier than replicate cultures exposed continuously to the mitogens. These effects can begin in the first 24 hr of culture and are seen maximally between 36 and 72 hr. Once a blastogenic response is established, PHA or PWM can diminish that response. Inhibition with PWM requires that the initial stimulation was with this mitogen, while PHA can inhibit blastogenesis to both PHA and PWM-stimulated cells. Because these mitogens can have a paradoxical effect on adenoidal lymphocytes, being capable of both initiating and inhibiting DNA synthesis, this phenomena should be kept in mind when such systems are utilized for the evaluation of antigens and drug effects.