Reactivity of B leukemic lymphocytes of patients with chronic lymphocytic leukemia to PWM and PHA

The ability of leukemic B lymphocytes to proliferate after in vitro stimulation with PWM and PHA was studied in 15 patients with chronic lymphocytic leukemia. Peripheral blood lymphocytes of five healthy subjects as well as purified normal B lymphocytes were used as controls. Leukemic lymphocytes of all donors expressed the same membrane phenotype, M receptor, and B7 and Ia antigens. The lymphocyte populations investigated were not completely free from myelomonocytic cells and contained small numbers of T lymphocytes. DNA synthesis was determined on days 3, 5, and 7 of culture by measuring the incorporation of tritiated thymidine. PWM-induced proliferation of leukemic B lymphocytes of nine patients was within normal limits, while the response of leukemic cells of six patients was very low. On the other hand, all CLL donors responded very well to PHA. Moreover, the response of leukemic B lymphocytes was significantly higher than the response of normal B cells. It was concluded that leukemic B lymphocytes of CLL patients are capable of proliferation after stimulation with PWM and PHA. The mechanisms underlying these responses to PWM and PHA are likely to be different.     

DNA synthesis and 3-hydroxy-3-methylglutaryl CoA reductase activity in PHA stimulated human lymphocytes: A comparative study of the inhibitory effects of some oxysterols with special reference to side chain hydroxylated derivatives

The effects of a series of oxygenated sterols on DNA synthesis and HMG-CoA reductase activity were tested in human lymphocytes. The cells were stimulated by PHA and cultured in cholesterol containing medium. The inhibitory effects of sterols on DNA synthesis were strictly related to the position and the configuration of the hydroxyl on the side chain, to the side chain conformation and integrity and to the structure of the sterol nucleus. The inhibition of HMG-CoA reductase activity was less dependent on these structural features since all the sterols tested were strong inhibitors. In our experimental conditions the inhibition of DNA synthesis was not related to the suppression of the HMG CoA reductase activity. The specificity of the structures required for DNA synthesis inhibition could be explained by the involvement of a specific hydroxysterol binding protein.     

Synthesis of sterol and phospholipid induced by the interaction of phytohemagglutinin and other mitogens with human lymphocytes and their relation to blastogenesis and DNA synthesis

Human peripheral blood lymphocytes (PBL) responded to phytohemagglutinin (PHA) and a variety of other mitogens by increased synthesis of sterol and phospholipid. This activity was established within 4–7 hr of the addition of mitogen and was dependent upon the binding of the ligand to the cell membrane. Sterol and phospholipid synthesis reached a peak at approximately 24 hr in association with blastogenic expansion of the lymphocyte membrane and initiation of DNA synthesis. Lipid synthesis and blast transformation occurred independently of replication of the genome since inhibition of DNA synthesis did not reduce the degree of blast transformation and lipid synthesis observed. However, inhibition of sterol synthesis using 20α-hydroxycholesterol resulted in decreased blastogenesis and DNA synthesis, demonstrating that early synthesis of lipid is important for these subsequent events. Human thymocytes responded to T-cell mitogens in the conventional manner as regards synthesis of lipid and blast transformation; however, they did not synthesize DNA. Possible reasons for this incomplete response are discussed. Several nonmitogenic agents which agglutinate lymphocytes were also found to initiate early increases in sterol and phospholipid synthesis, and the possible significance of this observation is considered.     

PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以[~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以[~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合     

Regulation of cholesterol biosynthesis at a stage after the 3-hydroxy-3-methylglutaryl-CoA reductase step, in normal and leukemic (L2C) guinea pig lymphocytes

Cholesterogenic activity in normal and leukemic guinea pig lymphocytes was measured by incorporation of labeled sodium acetate into cholesterol, after separation from other labeled metabolites. Our study is in agreement with the large difference previously found between the two kinds of cells at the 3-hydroxy-3-methylglutaryl-CoA reductase step, but it also shows that the difference is not as great as described earlier, when expressed in terms of the final product, cholesterol. This is mainly due to differences in the analytical methods. Our more detailed procedure showed a blockage of cholesterol synthesis in leukemic guinea pig lymphocytes (L2C cells) at the step of lathosterol (cholest-7-en-3 beta-ol) isomerization, and a higher plasma membrane permeability of these cells for sodium acetate, compared to normal cells. The lack of cholesterogenesis regulation by low density lipoproteins in L2C cells, previously reported after measuring 3-hydroxy-3-methylglutaryl-CoA reductase activity, was confirmed with regard to cholesterol itself, as well as the usual regulation of normal cells, which appeared to occur also at a post-hydroxymethylglutaryl-CoA step.     

A nitroxide-sterol derivative potently modifies cholesterol biosynthesis by normal and neoplastic guinea pig lymphocytes

Leukemic guinea pig lymphocytes (L₂ C) synthesise cholesterol in vitro at a forty-fold greater rate than normal cells. Equilibration (18 h) with lecithin or lecithin-cholesterol liposomes, respectively, enhances or suppresses sterol manufacture by normal lymphocytes but does not influence sterol production by L₂ C cells. In contrast, $\text{> 5·10}{}^9$ molecules/cell of a nitroxide-derivative of androstane, (17 β-hydroxy-4′,4′-dimethylspiro [5 α-androstan-3,2′-oxazolidin]-3′-yloxyl), commonly used as a membrane spin-probe, drastically inhibit sterol production by both normal and leukemic cells (maximum within 2 h). At $\text{< 5·10}{}^9$ molecules/cell, this sterol stimulates cholesterol synthesis. 25-Hydroxycholesterol at low concentrations also stimulates sterol manufacture, whereas high concentrations are also inhibitory in both cell types.     

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.     

Effects of removing PHA from cultures of PHA-stimulated lymphocytes

The proliferative capacity of PHA-stimulated lymphocytes following removal of PHA from the cultures was investigated. Lymphocytes were incubated with different PHA concentrations for 3 or 24 h and were then cultured in fresh medium with or without PHA in the original concentration. Cell proliferation was measured by incorporation of ³H-TdR. The effect of removing PHA was found to vary with the PHA concentration used for stimulation. Thus removal of PHA at 3 and 24 h from cells stimulated with half the optimal and at 3 h from cells stimulated with optimal PHA concentrations inhibited thymidine incorporation almost completely. Removal at 24 h from the latter cells resulted in a moderately decreased thymidine incorporation, whereas no decrease was seen after the removal of PHA from cells stimulated with twice the optimal concentration. When the cells were stimulated with very high PHA concentrations (20 × optimal), removal of PHA even resulted in an increased thymidine incorporation, a phenomenon that most probably has to do with the utilization of exogenous thymidine being inhibited by high PHA concentrations.The decreased thymidine incorporation after removal of low PHA concentrations was due to a reduction in the number of cells entering the proliferation cycle as well as to a decreased multiplication of cells already in DNA synthesis. This shows that PHA stimulates the cells even after they have initiated DNA synthesis. Various explanations for the results are discussed.     

Linkage of the isoprenoid biosynthetic pathway with induction of DNA synthesis in mouse lymphocytes. Effects of compactin on mitogen-induced lymphocytes in serum-free medium

Concanavalin A induction of DNA synthesis in mouse spleen lymphocytes cultured in serum-free medium was shown to be very sensitive to inhibition by compactin (ML-236B), a specific competitive inhibitor of hydroxymethylglutaryl-CoA reductase. As low as 0.1 microM compactin could give 98% inhibition of mitogen induction of a 5.10(6) cells/ml culture. This inhibition could be reversed completely by addition of exogenous mevalonate, but could not be reversed by either exogenous cholesterol or isopentenyladenine. Oxygenated sterol inhibition of mitogen-induced DNA synthesis could be reversed by cholesterol or by mevalonate, whereas cyclic AMP inhibition could not be reversed by either compound. These results suggest that endogenous cholesterol production is a necessary but not sufficient factor co-ordinated with mitogen-induced DNA synthesis, and that the presence of some additional product of mevalonate metabolism is involved also. Isopentenyladenine, though, did not have as significant effect of alleviating any of the above inhibitions. Since mevalonate could not relieve cyclic AMP inhibition, but could overcome compactin inhibition, cyclic AMP inhibition cannot be explained as due only to blockage of mevalonate production.     

Provision of cholesterol to lymphocytes by high density and low density lipoproteins. Requirement for low density lipoprotein receptors

The capacity of lipoprotein fractions to provide cholesterol necessary for human lymphocyte proliferation was examined. When endogenous synthesis of cholesterol was blocked, proliferation of mitogen-stimulated normal human lymphocytes was markedly inhibited unless an exogenous source of sterol was supplied. All lipoprotein fractions with the exception of high density lipoprotein subclass 3 were able to provide cholesterol for lymphocyte proliferation. Each of the lipoprotein subfractions capable of providing cholesterol was also able to regulate endogenous sterol synthesis in cultured human lymphocytes. Provision of cholesterol by lipoproteins required the interaction of apolipoprotein B or apolipoprotein E with specific receptors on normal lymphocytes. Apolipoprotein modification by acetylation or methylation, which markedly reduced the ability to regulate sterol biosynthesis, also diminished the capacity of lipoproteins to provide cholesterol. In addition, depletion of apolipoprotein B- and apolipoprotein E-containing particles from high density lipoprotein decreased its ability to suppress cholesterol synthesis and prevented it from providing cholesterol to proliferating lymphocytes. Monoclonal antibodies directed against the receptor-recognition sites on apolipoprotein B and apolipoprotein E were used to define the specific apolipoproteins required for the provision of cholesterol to lymphocytes by the various lipoprotein fractions. The antibody to apolipoprotein B inhibited cholesterol provision by both low density lipoprotein (LDL) and other lipoprotein fractions. The antibody to apolipoprotein E did not decrease provision of cholesterol by LDL but did inhibit the capacity of other fractions to provide cholesterol. In addition, a monoclonal antibody against the ligand binding site on the LDL receptor inhibited provision of cholesterol to normal lymphocytes by all lipoproteins. Finally, lymphocytes lacking LDL receptors were unable to obtain cholesterol from any lipoprotein fraction. These studies demonstrate that LDL receptor-mediated interaction with apolipoprotein B or apolipoprotein E is essential for the provision of cholesterol to normal human lymphocytes from all lipoprotein sources.     

Purine nucleotide metabolism in phytohemagglutinin-induced human T lymphocytes

The comprehensive studies of purine nucleotide metabolism were done in nonstimulated and phytohemagglutinin (PHA)-stimulated human peripheral blood T lymphocytes. Nonstimulated lymphocytes synthesize nucleotides in two alternative pathways: via biosynthesis de novo and salvage pathways. Although synthesis of triphosphonucleosides in unstimulated lymphocytes was the predominant pathway, interconversion of monophosphonucleosides was also active. Exposure of cells to PHA affects differently various pathways of nucleotide metabolism. The most marked changes observed were rapid activation of purine salvage within minutes after exposure to PHA, and significant increase of 5-phosphoribosyl-1-pyrophosphate levels. In addition, significant increases were found in de novo purine biosynthesis, nucleotide interconversions, and RNA and DNA synthesis, whereas catabolism of nucleotides remained unchanged. These results indicate that PHA activation of T lymphocytes causes a rapid synthesis of nucleotides which may be required immediately for increases in energy metabolism and later as the precursors of nucleic acid synthesis.     

Sterol synthesis is up-regulated in cholesterol-loaded pigeon macrophages during induction of cholesterol efflux.

The extent to which cholesterol synthesis is modulated in macrophage foam cells by changes in cholesterol influx and efflux was determined using thioglycollate-elicited peritoneal macrophages from normal and cholesterol-fed White Carneau (WC) and Show Racer (SR) pigeons. In peritoneal macrophages from normocholesterolemic pigeons, sterol synthesis from [(14)C]-acetate was down-regulated by more than 90% following incubation in vitro with beta-VLDL. Sterol synthesis was increased when the cellular free cholesterol concentration was decreased in response to stimulation of cholesterol efflux with apoHDL/phosphatidylcholine vesicles and cyclodextrin. Peritoneal macrophages isolated from hypercholesterolemic pigeons were loaded with cholesterol to levels similar to foam cells from atherosclerotic plaques (375-614 microg/mg cell protein), and had an extremely low rate of sterol synthesis. When cholesterol efflux was stimulated in these cells, sterol synthesis increased 8 to 10-fold, even though the cells remained grossly loaded with cholesterol. Cholesterol efflux also stimulated HMG-CoA reductase activity and LDL receptor expression. This suggests that only a small portion of the total cholesterol pool in macrophage foam cells was responsible for regulation of sterol synthesis, and that cholesterol generated by hydrolysis of cholesteryl esters was directed away from the regulatory pool by efflux from the cells. When the increase in sterol synthesis was blocked with the HMG-CoA reductase inhibitor mevinolin, there was no difference in the cholesterol content of the cells, or in the mass efflux of cholesterol into the culture medium.Thus, under these conditions, the increase in cholesterol synthesis during stimulation of cholesterol efflux does not appear to contribute significantly to the mass of cholesterol in these macrophage foam cells. Whether a similar situation exists in vivo is unknown.     

Characterization of Low Density Lipoprotein Receptors in Freshly Isolated Leukemic Guinea Pig Lymphocytes (L2C)

Abstract

The present study shows that L₂C leukemic guinea pig lymphocytes have 10 times as many low density lipoprotein (LDL) receptors per cell as normal lymphocytes. The affinity of these receptors is higher for guinea pig LDL than for human LDL. In contrast to normal cells, in which the degradation of the receptor-bound LDL is quite efficient, the leukemic cells only degraded a small fraction of these same receptor-bound LDL. Thus, the internalization index was nearly 4 times higher in the normal cells than in the leukemic cells.

In L₂C cells, cholesterol homeostasis derived 38% of its cholesterol input from receptor-mediated degradation of LDL and 62% from cholesterol synthesis, whereas in normal cells, these fractions were 97% and 3% respectively     

Lymphocyte reactivity in pregnant women and newborn infants.

The mitotic response to phytohaemagglutinin (PHA) was determined in lymphocytes of mothers and their newborn infants obtained at delivery and seven days later by measuring the rate of 125 I-idoxuridine uptake into DNA in lymphocytes cultured in their own plasma and after washing and resuspension in fetal bovine serum. There was no difference in the unstimulated counts of maternal lymphocytes taken at delivery, whether unwashed or washed, compared with those from nonpregnant controls. With PHA stimulation the mitotic response of the maternal lymphocytes cultured in their own plasma was reduced compared with that of the control lymphocytes but washed maternal cells showed a similar response to the controls. These findings suggest that the reduced lymphocyte mitotic response to PHA in pregnancy is due to a plasma inhibitory factor This inhibition was not evident in maternal blood taken seven days after delivery. DNA synthesis in unstimulated cultures from newborn infants at birth and seven days after birth was greater than that in adult control cultures. With PHA stimulation the mitotic response of cord-blood lymphocytes cultured in their own plasma paralleled that of control lymphocytes but washed newborn cells showed a greater response. Thus plasma suppression similar to that observed in the mother seems also to affect infants at birth. This inhibition was not demonstrable in blood taken from infants of 7 days.     

Incorporation of L Cell Sterols into Vesicular Stomatitis Virus

The incorporation of host cell sterol into vesicular stomatitis virus can be effectively studied in an L cell system. The end product of de novo sterol synthesis in the L cell is desmosterol, and as the concentration of cholesterol in the medium is increased the cells incorporate the exogenous cholesterol and the synthesis of desmosterol decreases. L cells which contained desmosterol as their sole sterol produced virus whose sterol content was similarly composed of only desmosterol. Virus grown in L cells which had a constantly changing sterol ratio also contained a mixture of cholesterol and desmosterol, but the virus was found to be more enriched in cholesterol than in the L cells in which it was grown. Viral stability, growth, and plaquing efficiency were tested and found not to be affected by the alteration of its sterol composition, i.e., by partially or completely replacing cholesterol with desmosterol.     

A further contribution on nucleoli of human lymphocytes.

Cultured human lymphocytes were investigated by means of light as well as electron microscopic procedures to provide more information on the structural organization of their nucleoli. The transformation of ring shaped nucleoli to nucleoli with less or more distinct nucleolonemata in PHA stimulated cells was characterized by a marked increase of granular RNP components in number indicating the activation of their production. This phenomenon seems to be related not only to the activation of the ribosomal RNA synthesis but also to its processing. The appearance of fibrillar RNP components in the central area of the ring shaped nucleoli apparently represents the first sign of the nucleolar RNA synthesis in these cells. The proportion of fibrillar and granular nucleolar RNP comonents in PHA inresponsive lymphocytes was similar to that in lymphocytes from patients with the usual type of lymphocytic leukemia. The intranucleolar chromatin areas appeared to be larger in PHA stimulated lymphocytes but the proportion of these areas to the nucleolar body did not show substantial difference as compared to the resting cells.     

X-irradiation of equine peripheral blood lymphocytes stimulated with phytohaemagglutinin in vitro.

Small lymphocytes were isolated from the peripheral blood of horses and incubated at 37 degrees C in Eagle's medium supplemented with 20 per cent foetal calf serum. The addition of phytohaemagglutinin (PHA) to the cultures resulted in: increased RNA and protein synthesis; the enlargement of the small lymphocyte into a lymphoblast-like cell; the initiation of DNA synthesis, and cell division. When survival was measured 24 hours after X-irradiation by means of phase-contrast microscopy, the lymphoblast-like cell was much more radio-resistant (D0 = 250 rad) than the small lymphocyte (D0 = 20 rad). This increase in radioresistance, however, was not observed until 12-24 hours after PHA treatment. To investigate which of the changes occurring during the transformation of the small lymphocyte was responsible for the increased resistance to irradiation, the percentage of cells surviving irradiation was compared with the percentage of cells incorporating significant amounts of 3HTdR, 3H-UR, or 3H-leucine at the time of irradiation. For this comparison, a dose of 100 rad was used because 100 rad killed essentially all of the small lymphocytes, but less than 35 percent of the cells which had become radioresistant from the PHA treatment. The results indicated that the increase in radioresistance was not associated with DNA synthesis, but instead correlated with the increase in RNA and protein synthesis which the cells had attained at the time of irradiation.     

Inhibition par les lipoprotéines de haute densité HDL2 et HDL3 de la synthèse du DNA et des stérols des lymphocytes humains stimulés par la concanavaline A.

Lipoproteins HDL₂ and HDL₃ inhibit DNA synthesis and sterol synthesis in human Con A-stimulated lymphocytes cultured in a medium supplemented with 20 per cent lipoprotein deficient serum. On the basis of the amount of proteins added, HDL₂ is more efficient on DNA and sterol synthesis than HDL₃ and less efficient than LDL. However, on the basis of the amount of cholesterol added, the inhibition of sterol synthesis induced by these three lipoproteins is not significantly different. At all concentrations of these three lipoproteins, the inhibition of sterol synthesis is higher than the inhibition of DNA synthesis.     

Sodium periodate stimulation of normal and chronic lymphocytic leukemia lymphocytes : II. The use of neuraminidase and trypsin to probe the nature of the response

Sodium periodate stimulated normal and leukemic lymphocytes to undergo DNA synthesis and cell division. The role exerted by cell surface components in this response was investigated by pre-treatment or post-treatment of cells with either neuraminidase or trypsin. Both enzymes substantially reduced the sodium periodate response of normal and leukemic lymphocytes when they were added after the mitogen. In normal lymphocytes when the treatment sequence was reversed (enzyme before sodium periodate), neuraminidase had no effect while trypsin greatly potentiated the action of this mitogen. In leukemic lymphocytes, however, neuraminidase reduced the response, whereas trypsin had little or no effect. In comparison with another mitogen, the response of normal and leukemic lymphocytes to phytohemagglutinin was not significantly altered by pre-treatment with neuraminidase or trypsin. Collectively, these results imply that significant differences exist in the cell surface of these two types of lymphocytes and that the oxidation of cell surface components other than sialic acid are involved in the response of these cells to sodium periodate.