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The objective of this study was to investigate the effects of oxysterols (OS), namely 5 alpha-hydroxy-6-ketocholestanol, 6-ketocholestanol and 25-hydroxycholesterol, on specific cell-mediated cytotoxicity by C57BL/6 spleen cells against P815-X2 (a DBA/2 mastocytoma) target cells. Cytolytic T lymphocytes (CTL) were generated by intraperitoneally injecting C57BL/6 mice with P815-X2 tumor cells 10 d prior to the cytotoxicity experiments. Preincubation of CTL with 10(-5) M 5 alpha-hydroxy-6-ketocholestanol and 6-ketocholestanol for 45 min in lipoprotein-depleted medium resulted in an inhibition of cytolytic activity (73 and 43%, respectively) as measured by 4-h 51Cr release. At a concentration of 5 x 10(-6) M, 5 alpha-hydroxy-6-ketocholestanol inhibited CTL activity by 65%, whereas 6-ketocholestanol did not elicit any inhibition. By contrast, 25-hydroxycholesterol did not inhibit CTL at either concentration, although it is known to be a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. When CTL were preincubated with OS in lipoprotein-replete medium, there was no inhibition of CTL activity at the respective concentrations. The results suggest that the inhibition of CTL activity upon short-term incubation with OS is not due to the inhibition of cholesterol synthesis, but may be due to the insertion of OS into the plasma membrane to replace cholesterol and alteration of membrane physical properties.

Some of the oxidation products of cholesterol (oxysterols) have profound effects on plasma membrane structure and function. The present studies were undertaken to determine the effects of oxysterols on NK cell-mediated cytotoxicity. When mouse spleen cells were preincubated with certain oxysterols, NK cell cytotoxicity was inhibited without loss of effector cell viability. The strongest inhibition was observed with oxysterols that are oxidized at the C-5, C-6, or C-7 positions of the sterol nucleus. Among these, 7 beta-hydroxycholesterol caused more inhibition than 7 alpha-hydroxycholesterol suggesting that the spatial orientation of the hydroxyl group in the beta-position results in a greater perturbation in plasma membrane structure than that oriented in the alpha-position. In contrast, oxysterols that are oxidized at the C-20 and C-25 positions that are located on the C-17 acyl chain had little or no inhibitory effect, suggesting that oxidation in the cholesterol nucleus which is situated closer to the phospholipid headgroups at the lipid bilayer-aqueous interface results in a more profound effect on the plasma membrane physical structure. These results suggest that the lytic function of NK cell is sensitive to alterations in the physical state of its plasma membrane induced by oxysterols.

The oxysterol content in normal and sickle red blood cell (RBC) membranes was assessed using thin-layer chromatography and capillary gas chromatography/mass spectrometry. Several more oxysterols were present in sickle RBCs compared to normal RBCs. Sickle RBC membranes had a higher concentration of 5 alpha,6 alpha-epoxycholesterol, 5 alpha-cholestane-3 beta,5,6 beta-triol, 7-ketocholesterol and 19-hydroxycholesterol than normal RBC membranes. The increased oxysterols in sickle RBC may be an effect of the increased oxidative stress which occurs in sickle RBC membranes. Physical characteristics of normal and sickle RBC membrane ghosts with and without inserted oxysterols were examined by Fourier transform infrared spectroscopy. The data are consistent with a greater sterol content in sickle cells compared to normal RBC membranes, and a possible oxysterol-cholesterol synergism.

Fourier transform infrared (FTIR) spectroscopy was used to examine the effect of oxysterol insertion into normal and sickle RBC membranes and the total lipid extracts of the membranes. Examination of the FTIR C-H stretch and fingerprint regions reveal that the insertion of 7 alpha- and 7 beta-hydroxycholesterol has the greatest effect on the fluidity of RBC membranes and lipid extracts. The results confirm the observation that sterol molecules are oriented in the membrane so that the 7 position is located in the phospholipid head group region at the lipid/water interface. The substitution of a keto for a hydroxy group at the number seven position decreases the effect of the sterol on membrane packing.

The effects of competitive inhibition of hydroxymethylglutaryl coenzyme A (HMG CoA) reductase by compactin on the in vitro proliferation of peripheral blood myeloid leukemia cells were studied using the cells from 45 patients with acute myeloid leukemia or chronic myelogenous leukemia in blast phase. The cells from 58% of these patients showed a dose-related inhibition of DNA synthesis when incubated with compactin. Unexpectedly, cells from 18% of the patients were resistant to the inhibitory effects of compactin on DNA synthesis and responded to the HMG CoA reductase inhibition with an actual increase in the incorporation of 14C-labeled thymidine into DNA. Another 18% of the patients studied displayed both inhibition and stimulation of DNA synthesis in a biphasic response depending on the particular concentration of compactin used. The maximum enhanced rates of cellular DNA synthesis were observed with lower compactin concentrations (5 x 10(-7) mol/L) than were required for maximum inhibition of DNA synthesis (10(-5) mol/L). Leukemia cells displaying a stimulated response to compactin had a significantly lower baseline DNA synthetic rate than did cells that showed an inhibitory response of DNA synthesis to compactin. There was no correlation between these cells' varying DNA synthetic response to compactin and measures of baseline HMG CoA reductase activity or acetate conversion to cholesterol. Whereas the observation of cellular DNA synthesis stimulation by HMG CoA reductase inhibition has not been observed in other mammalian cells and seems paradoxical, explanations may emerge in light of our growing knowledge concerning the importance of isoprenylation for the function of certain cell regulatory proteins.

It is widely accepted that the neoplastic B cells from patients with chronic lymphocytic leukemia (CLL) respond poorly to common mitogens. The fungal metabolite cytochalasin B (0.5 micrograms/ml) is a weak mitogen for normal lymphocytes. However, when peripheral blood lymphocytes from 19 patients with CLL of B cell origin (B-CLL) were cultured with 0.5 micrograms cytochalasin B/ml, significant new DNA synthesis ( [14C]thymidine incorporation) occurred in 18. Stimulation indices with cytochalasin B varied widely (range = 1.9-28.2, mean +/- SD = 10.6 +/- 7.5; delta cpm range = 1,157-153,818; n = 26) but in 11 cases exceeded those seen with concanavalin A (Con A), phytohemagglutinin, or pokeweed mitogen. In all 11, the mitogenic response to cytochalasin B exceeded that to pokeweed mitogen, which is believed to be a T cell-dependent B cell mitogen. In three cases, the responses to cytochalasin B were 8.6, 3.5, and 2.3 times greater than those to Con A. As with other mitogens, the DNA synthetic response to cytochalasin B was time and dose dependent. Peak thymidine incorporation occurred at 72-88 h and declined thereafter. Significant mitogenic effects were observed with 0.1-5 micrograms cytochalasin B/ml with a peak at 0.5-2 micrograms/ml. Stimulated DNA synthesis was abolished by 1 mM hydroxyurea. Cells from two patients with B-CLL were separated by rosetting with sheep erythrocytes (E). Depletion of E-rosette-positive cells from the CLL cell population abolished the response to Con A but did not affect the response to cytochalasin B. Cytochalasin B is a potent mitogen for B-CLL cells and may be useful in cytogenetic studies of this often indolent neoplasm.

We have studied the effects of human alpha-fetoprotein (HAFP), isolated from the serum and ascitic fluid of a hepatoma-bearing patient, on the in vitro transformation of human peripheral blood lymphocytes by a variety of mitogenic stimuli. At a concentration of 2.5 mg/ml, HAFP inhibited the lymphocyte response to phytohemagglutinin, concanavalin A, and rabbit anti-human thymocyte serum, but failed to inhibit the response to pokeweed mitogen. HAFP was able to inhibit the one-way mixed lymphocyte culture at concentrations of 250-500 mug/ml, but failed to inhibit at 100 mug/ml. Exposure of lymphocytes to 2.2 mg/ml of HAFP for 18 hr did not result in significant lymphocytotoxicity, and such cells washed free of HAFP were fully capable of participating in the mixed lymphocyte culture. HAFP did not inhibit lymphocyte E-rosette formation. Fetal HAFP was more effective in inhibiting human lymphocyte responses than hepatoma HAFP. These experiments support the suggestion that HAFP plays an important immunoregulatory role during fetal development, possibly through the suppression of thymus-derived lymphocyte responses to antigenic stimuli; they also suggest that there are important differences in the biological properties of hepatoma and fetal HAFP.

The ability of human alpha-fetoprotein (HAFP) isolates to inhibit human lymphocyte transformation varies over 2-3 orders of magnitude. Since HAFP is known to bind fatty acids, we have examined the possible role of lipid binding in producing this variability. While certain oxygenated sterols are potent inhibitors of lymphocyte transformation, and while the kinetics of such inhibition precisely mimic those of HAFP, HAFP is, by contrast, a weak inhibitor of sterol synthesis in mitogen-stimulated human lymphocytes. Fatty acids (18:0, 18:1, 18:2, 18:3, 20:4, 22:6) and 10(-4) M have little or no effect upon lymphocyte transformation. The fatty acid content of seen HAFP isolates ranged from 2.3 to 8.0 mol fatty acid/mol HAFP and included 12:0, 14:0, 16:0 (the most abundant), 18:0, 20:4, and 22:0; no 22:6 was found. There was no correlation between the total or individual fatty acid contents of HAFPs and their ability to inhibit lymphocyte transformation. Lysooleoyl-, lysostearoyl-, and lysopalmitoyl-lecithin at 5 X 10(-5) M all inhibited lymphocyte transformation (30.1%, 52.1%, and 58.1%, respectively) and the latter was active at 6.3 X 10(-6) M. Exposure of HAFP to a 5-fold molar excess of [14C]-lysopalmitoyl lecithin resulted in significant (10%) binding to HAFP, while no significant binding of dipalmitoyl lecithin occurred under similar conditions. Analysis of lipid extracts of potent HAFP isolates by thin-layer chromatography failed to reveal the presence of phospholipids. Although HAFP can bind certain lipids, and although certain lipids inhibit lymphocyte transformation, we conclude that HAFP suppression of lymphocyte transformation cannot be attributed to the binding of hydrocortisone, prostaglandins, fatty acids, lysolecithins, or oxygenated sterol compounds.

In a previous study, we demonstrated three variants of human alphafetoprotein by crossed immunoelectrophoresis. In addition, we correlated the capacity of alpha-fetoprotein isolates from various hepatoma and fetal sources to suppress human lymphocyte transformation in vitro with the relative proportion of the electronegative variant, HAFP-3, present in each isolate. We have now isolated alpha-fetoprotein from the serum, ascitic fluid, and saline extract of tumor from a single hepatoma patient and from a homogenate of fetal livers. When tested for their capacity to inhibit human lymphocyte transformation in vitro, tumor and fetal liver alphafetoprotein were found to be extremely potent, serum alphafetoprotein had intermediate potency, and ascitic fluid alpha-fetoprotein was the least potent. Analysis of these isolates by crossed immunoelectrophoresis confirmed the correlation between the proportion of HAFP-3 and the immunosuppressive potency of each isolate. In addition, analysis of these isolates by isoelectric focusing in polyacrylamide gels containing 8 M urea revealed further evidence of microheterogeneity; at least six molecular variants were apparent. The proportion of one of these variants, termed HAFP-3a, in each isolate was correlated with the immunosupressive potency of the isolate. The sialic acid content of the various alpha-fetoprotein isolates did not vary significantly. Our data suggest that a postsynthetic modification of alphafetoprotein occurs, probably after secretion, which reduces immunosuppressive potency by converting the active electronegative species to an inactive electropositive form. This modification probably involves a charged moiety other than sialic acid on the molecule.

Deviations from the normal of alpha-fetoprotein (AFP) concentrations in fetal serum, amniotic fluid, maternal serum and adult human serum can be explained by understanding the normal physiology and the pathophysiology of AFP synthesis and metabolism. AFP is the prototype of oncofetal markers. Emphasis is given to the usefulness of elevated serum AFP levels on the diagnosis and management of primary hepatomas and tumors of germ cell origin. The ability to detect neural tube defects early in gestation by monitoring maternal serum and amniotic fluid AFP concentrations is discussed.

We have previously shown that human alpha-fetoprotein (HAFP) inhibits the response in vitro of human lymphocytes to a variety of mitogenic stimuli. In this report we present evidence that HAFP preparations containing dimeric and trimeric forms are no more inhibitory than HAFP monomeric isolates. HAFP isolates of low inhibitory potency cannot impede the capacity of high-potency HAFP to inhibit lymphocyte transformation. Since lymphocyte responses to phytomitogens or anti-human thymocyte antiserum cannot be totally suppressed by increasing doses of HAFP, we conclude that human lymphocyte preparations contain a population of lymphocytes resistant to the inhibitory action of HAFP.

Oxygenated sterol compounds are potent inhibitors of sterol and DNA synthesis in mammalian cells. We studied the effects of oxygenated sterols on human marrow granulocytic progenitor cells in vitro (CFU-C). 25-Hydroxycholesterol was found to be a potent inhibitor of sterol synthesis in marrow mononuclear cells, with 50% inhibition occurring at approximately 10(-7) M. This compound, as well as 6-ketocholestanol, 7-ketocholesterol, and 20 alpha-hydroxycholesterol, also demonstrated marked inhibition of CFU-C proliferation. The latter effect, which was not a result of direct cytoxicity of the compounds, was reversible by cholesterol, but not by mevalonic acid. We conclude that inhibition of sterol synthesis by oxygenated sterol compounds may be insufficient to explain their suppression of CFU-C proliferation.

Mevalonic acid (5 x 10(-4)-1 x 10(-2) M) stimulates DNA synthesis, morphologic transformation and cell cycling in peripheral blood human lymphocytes. Other organic acid anions which serve as cholesterol and mevalonate precursors are devoid of such effects. Both ML-236B and 25-hydroxycholesterol, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, inhibit concanavalin A-induced lymphocyte transformation, but only the inhibition by ML-236B can be overcome by exogenous mevalonate. In contrast, only 25-hydroxycholesterol inhibits mevalonate-induced lymphocyte DNA synthesis. The effects of mevalonic acid on lymphocytes cannot be reproduced by isopentenyl adenine or isopentenyl adenosine. Unregulated endogenous cellular synthesis of mevalonic acid may contribute to uncontrolled growth in certain malignant cell lines.

Human alpha-fetoprotein (HAFP) isolated by immunoadsorbent column was shown to suppress the mitogenic response of human lymphocytes to phytomitogens, antihuman thymocyte antiserum, and the mixed lymphocyte culture. HAFP isolated from the sera and ascitic fluid of five hepatoma patients, and from fetal liver, varied in biological potency over three orders of magnitude. Extended agarose gel electrophoresis and crossed immunoelectrophoresis demonstrated three molecular species of HAFP. Quantitation of the three species revealed a correlation between the relative amount of the most negatively charged species and biological potency. Treatment of HAFP with neuraminidase to remove completely sialic acid residues did not alter the biological potency, but converted the three species to two species having slower electrophoretic mobilities. We conclude that differences in sialic acid content are only partly responsible for the microheterogeneity demonstrated by HAFP, and that variability in another charged moiety is also present. Variation in the relative proportions of the different molecular species of HAFP may be important in the regulation of its immunosuppressive properties.

Addition of dimethyl sulfoxide or phorbol myristate acetate (PMA) to HL-60 cell cultures induces granulocytic or monocyte-macrophage differentiation, respectively, in HL-60 cells. Dimethyl sulfoxide-induced granulocyte differentiation in HL-60 cells is associated with a decrease in cellular 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and with a decrease in the incorporation of [14C]acetate and mevalonate into products of the cholesterol biosynthetic pathway. PMA-induced monocyte-macrophage differentiation in HL-60 cells is associated with a rapid and profound fall in cell proliferation but nonetheless is accompanied by a dose-dependent increase in cellular HMG-CoA reductase activity and [14C]acetate incorporation into the cholesterol biosynthetic pathway. In addition, PMA induces an increase in [14C]mevalonate incorporation into cholesterol and its precursors, suggesting that post-HMG-CoA reductase events in cholesterol biosynthesis are also enhanced. Mature peripheral blood human monocytes possess an active cholesterol biosynthetic pathway, whereas mature human granulocytes are almost entirely lacking in the ability to synthesize post-squalene products. Our results with HL-60 cells indicate that this divergence in sterol-synthesizing ability between two cell lineages, which normally also derive from a common stem cell, can be observed as an early event in the differentiation process.

Freshly isolated hairy cells from the peripheral blood of patients with hairy cell leukemia (HCL) synthesize 3-5-fold greater amounts of cholesterol, lanosterol, and squalene from [1-14C]-acetate than do normal human peripheral blood mononuclear cells under basal conditions of culture (i.e., in the presence of low-density lipoprotein). HCL cells also exhibit an eightfold increase in the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase. These changes cannot be ascribed to increased rates of cellular proliferation in the HCL cells, nor are they a consequence of an increased rate of loss of newly synthesized cholesterol into the culture medium. The increased rate of cholesterol biosynthesis in HCL cells may result in an increase in their total cellular cholesterol content, as well as in an increase in their plasma membrane cholesterol:phospholipid molar ratio. These changes, in turn, are probably responsible for some of the clinical manifestations of this disease.

We have examined the effect of different variables such as tissue culture media, with or without various supplements, lymphocyte isolation techniques, lymphocyte contamination by autologous red blood cells and platelets, and lymphocyte numbers, on the requirement for serum during phytohaemagglutinin (PHA) induced DNA synthesis in human lymphocytes. At all mitogen doses tested, we have found that dialysable constituents of serum enrich the ability of all tissue culture media to support lymphocyte DNA synthesis; however, human lymphocytes display an absolute requirement for nondialysable macromolecular constituents of serum in order to synthesize DNA.

ML-236B is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the key regulatory enzyme in the sequence that catalyzes the conversion of acetate to mevalonic acid in cholesterol biosynthesis. This compound caused marked inhibition of human bone marrow granulocyte progenitor cell (CFU-C) proliferation, the 50% inhibitory concentration (IHD50) being 2.0 X 10(6)M. Inhibition of colony formation was reversed by mevalonic acid but not by cholesterol. ML-236B also inhibited DNA synthesis and acetate incorporation into cholesterol in marrow mononuclear cells (IHD50 = 5.6 x 10(6)M and 3.2 x 10(7)M, respectively). No inhibition of mevalonate incorporation into cholesterol was observed. These results differ from those observed with 25-hydroxycholesterol, another inhibitor of HMG CoA reductase. The latter compound also inhibited CFU-C proliferation and cholesterol biosynthesis from acetate; inhibition of colony formation was reversed by cholesterol but not by mevalonic acid. In addition, 25-hydroxycholesterol inhibited cholesterol synthesis from mevalonic acid precursor. We conclude that: (1) ML-236B is a potent inhibitor of CFU-C proliferation, DNA synthesis, and cholesterol biosynthesis from acetate precursor in marrow mononuclear cells; (2) the effects of ML-236B are completely reversed by mevalonic acid but not by cholesterol, suggesting that mevalonic acid per se or one or more of its nonsterol products are critical for cell growth; (3) the inhibitory effects of 25-hydroxycholesterol on CFU-C proliferation and cholesterol biosynthesis are not solely a result of its inhibition of HMG CoA reductase, but are due in part to inhibition of enzymatic steps distal to mevalonic acid in the sterol synthetic pathway; and (4) mevalonic acid and cholesterol are independent requirements for CFU-C proliferation and differentiation in vitro.

The phytohemagglutinin mitogenic proteins derived from Phaseolus vulgaris comprise a class of five glycoproteins that are isomeric tetramers composed of varying proportions of two different subunits (L and R). Within the native tetramer, the L subunit is a potent leukoagglutinin and mitogen that lacks hemagglutinating properties, whereas the R subunit is a potent hemagglutinin with little or no mitogenic activity. The subunits have been isolated in homogeneous form by isoelectric focusing in 8 M urea. Previous work has shown that they have equal molecular weights and differ in amino-acid sequence from residues 1-7, but are identical in positions 8-24 [(1973) J. Exp. Med. 138, 939-951]. We now report amino-acid composition studies which reveal striking similarities between the subunits. Both lack methionine and cysteine. The twelfth residue in each subunit is a glycosylated asparagine, with the identical carbohydrate composition in each. The last three residues of the subunits, as determined by carboxypeptidase A digestion, are identical. Tryptic peptide mapping of the succinylated phytohemagglutinin subunits reveals a high degree of similarity. We conclude that the substantial difference in biological properties among the tetrameric phytohemagglutinin mitogens is a result of relatively restricted differences in the primary structure of their constituent subunits.

Peripheral blood mononuclear cells from a patient with Sézary syndrome which lacked E-rosette-forming ability and surface immunoglobulins, and which displayed a markedly depressed response to a variety of mitogens, were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) on 3 occasions. The first peripheral blood sample (smooth) differed significantly from two later samples (moderate numbers of microvilli) when surface characteristics were examined by SEM; these differences were confirmed by TEM. The Sézary syndrome cells in this patient may be related to a T lymphocyte which has lost certain surface markers and mitogen response characteristics through a process of de-differentiation.

In order to investigate the role of neutrophils in the DNA synthetic response of human peripheral blood lymphocytes to mevalonic acid, we obtained preparations of both cells each free of cross-contamination by the other. Purified lymphocytes respond poorly to mevalonic acid, but their response can be significantly enhanced by one half their number of neutrophils. Preincubation of lymphocytes with neutrophils for 24 hr, even in the absence of mevalonic acid, further increases the lymphocyte response. We have been unable to demonstrate the production by granulocytes of either an intracellular or extracellular mevalonate-derived growth factor that in turn stimulates lymphocytes. Granulocytes preexposed to mevalonate do not acquire the ability to stimulate lymphocyte DNA synthesis in the absence of mevalonate. Our experiments suggest that neither neutrophil lysosomal enzymes nor reactive oxygen species generated by neutrophils are responsible for the help neutrophils provide. Normal E rosette-positive lymphocytes fail to respond to mevalonate, whereas E rosette-negative cells do. The mevalonate response of normal E rosette-negative cells is enhanced by the presence of granulocytes in contrast to B cell-chronic lymphocytic leukemia cells that synthesize DNA briskly in response to mevalonic acid in the absence of neutrophil help. These observations add to our knowledge of the relationship between mevalonate metabolism and the regulation of cellular DNA synthesis and mitosis.

When preincubated with certain oxygenated sterol compounds in lipoprotein-depleted serum [20% (vol/vol)], human polymorphonuclear leukocytes show inhibition of chemotaxis toward the synthetic dipeptide N-formylmethionylphenylalanine without alteration of random movement or loss of cell viability. These effects can occur at sterol concentrations as low as 6.25 microM and after as little as 5 min of preincubation, but they are increased at higher concentrations and longer preincubation times. The inhibition can be almost completely reversed by preincubation in lipoprotein-replete serum [human AB serum, 20% (vol/vol)] and may be partially corrected by addition of free cholesterol (0.125 mM) to the medium. These effects are unlikely to be due to inhibition of cellular sterol synthesis, competition for chemotaxin membrane binding sites, or deactivation of the leukocytes but they may be a consequence of insertion of the sterol molecule into the leukocyte plasma membranes.

Human peripheral blood lymphocytes, in the presence of human neutrophils, initiate DNA synthesis and cell cycling when exposed to mevalonic acid. The ability of lymphocytes to respond in this manner is a radiosensitive property of cells, whereas the help provided by neutrophils is maintained despite their exposure to x-irradiation. Other organic acid anions, including precursors of mevalonic acid biosynthesis and a variety of products of mevalonate metabolism, fail to initiate DNA synthesis when added to human lymphocytes. Because only the metabolically active R(--) enantiomer of mevalonic acid initiates lymphocyte DNA synthesis, we presume that physiological pathways of mevalonate metabolism are involved. The response to mevalonic acid of ML-236B (compactin)-inhibited lymphocytes is increased, and the threshold concentration of meyalonate at which lymphocyte DNA synthesis first appears is decreased, when the cells are cultured in lipoprotein-containing (as opposed to lipoprotein-depleted) medium. The response to mevalonic acid of lymphocytes cultured in lipoprotein-depleted medium can be enhanced by addition to the cultures of low density lipoprotein but not by addition of high density lipoprotein. Based upon the flux diversion hypothesis of mevalonate metabolism, these observations suggest that a nonsterol product of mevalonate metabolism may be responsible for the initiation of lymphocyte DNA synthesis by mevalonic acid.

Oxygenated sterol compounds (OSC), when incubated for 1 hr with human erythrocytes in lipoprotein-depleted medium at concentrations of 0.625-5 X 10(-5) M, are inserted into the cell membrane and remain there despite subsequent washing of the cells. The insertion results in expansion of the surface area of the red cell ghost membrane, an increase in critical hemolytic volume, and as a consequence, in diminished osmotic fragility of the erythrocytes. This effect is seen with echinocyte-forming as well as with non-echinocyte-forming OSC. Erythrocytes treated with OSC do not differ from control cells with respect to their mean cell volume (MCV) in isotonic solution, water content, ion fluxes, and filterability through polycarbonate filters. The shift of the osmotic fragility curve toward lower NaCl concentrations is proportional to the amount of OSC inserted into the red cell membrane. 7 beta-Hydroxycholesterol, 22-ketocholesterol, and 20 alpha-hydroxycholesterol are the most potent inhibitors of osmotic lysis. The effect of OSC on osmotic fragility is diminished if the erythrocytes are incubated in a lipoprotein-containing medium; free cholesterol, however, does not change this effect. Various progesterones also protect red cell from osmotic lysis, but only if the erythrocytes are directly exposed to the compounds present in the hypotonic NaCl solutions used for measurement of their osmotic fragility. Progesterones do not remain in the membrane after the cells have been washed. The OSC are also capable of correcting the osmotic fragility curve of red cells from patients with hereditary spherocytosis. These experiments may suggest an approach to the pharmacologic treatment of hereditary spherocytosis.

Human alpha-fetoprotein has been isolated from the serum and ascitic fluid of a patient with hepatoma by a combination of immunoadsorbent column chromatography and Sephadex G-150 gel filtration. Human alpha-fetoprotein is a sialylated glycoprotein with an estimated molecular weight of 67 500, composed of a single-chain polypeptide of approximately 580 amino acid residues and 3.6% carbohydrate. It is a negatively charged protein with an acid isoelectric point (pH 4.57). In addition to the monomeric form of alpha-fetoprotein, we have identified human alpha-fetoprotein polymers, including dimeric and trimeric forms, which dissociate to the monomer only upon exposure to disulfide-reducing reagents, implying that their formation is dependent upon intermolecular disulfide bonds. These polymers are found in human alpha-fetoprotein isolated by isoelectric focusing in both the major (pI 4.57) and minor (pI 5.2) alpha-fetoprotein fractions. The first 17 residues of the NH2-terminal amino acid sequence of the hepatoma-derived human alpha-fetoprotein have been identified. Fetal alpha-fetoprotein is indistinguishable from hepatoma alpha-fetoprotein by several criteria, including immunoelectrophoresis, acryalmide gel electrophoresis, and proclivity for dimerization.

25-Hydroxycholesterol, 20 alpha-hydroxycholesterol, 7 alpha-hydroxycholesterol, and 5 alpha-hydroxy-6-ketocholestanol, when added to cultures of human lymphocytes in lipoprotein-depleted medium (LPDM) at a concentration of 2.5 x 10(-6) M, inhibit E-rosette formation with sheep red blood cells. 20 alpha-Hydroxycholesterol, 7 alpha-hydroxycholesterol, and 5 alpha-hydroxy-6-ketocholestanol are more potent inhibitors than 25-hydroxycholesterol. The inhibitory effect of 5 alpha-hydroxy-6-ketocholestanol on E-rosette formation appears after 15 min of exposure; with the other three compounds, an exposure time of 18 hr is necessary. The inhibitory effect of E-rosette formation can be abolished by addition of free cholesterol, low-density lipoprotein, or high-density lipoprotein to the LPDM or by incubation of the cells in normal AB serum, but not by the addition of mevalonic acid to the LPDM. These observations suggest that the capacity of oxygenated sterol compounds (OSC) to inhibit E-rosette formation is independent of their inhibitory effect on sterol synthesis. It is possible that OSC inhibit E-rosette formation as a consequence of their insertion into the lymphocyte membrane as cholesterol analogues.

A glycopeptide obtained from tryptic digestion of citraconylated leukoagglutinating (L-subunit) phytohemagglutinin was isolated and purified. The composition of its ten amino acid and seven hexose residues, when compared to a previous analysis of the NH2-terminal amino acid sequence, indicated that the glycopeptide was derived from residues 11 through 20, with the oligosaccharide unit linked N-glycosidically to asparagine at the twelfth position. Structural studies involving a combination of alpha-mannosidase digestion, periodate oxidation, and permethylation, in conjunction with computerized mass-spectrometric and masschromatographic techniques, suggested a branched sequence of five mannose and two glucosamine residues similar to that found in many mammalian glycoproteins.

Five pure isolates of human alpha-foetoprotein (HAFP) from adults with tumours of the li er or stomach, as well as HAFP isolated from foetal liver, inhibit in vitro human lymphocyte transformation induced by phytomitogens, anti-human thymocyte serum, and the mixed lymphocyte culture. Foetal HAFP produces 50% inhibition at concentrations of 1-5 mug/ml. The HAFPs isolated from tumour-bearing adults are 1-3 orders of magnitude less potent (50% inhibition achieved at approximately 20, 130, 500, and 2000 mug/ml, respectively). In order to achieve maximum inhibition HAFP must be present at the time of mitogen addition; pre-exposure of lymphocytes to HAFP, followed by washing, does not result in lymphocyte suppression. The inhibiting effect of HAFP cannot be overcome by a ten-fold increase in mitogen concentration implying that HAFP does not act by simple competition with the lymphocyte membrane for the mitogen combining site. HAFP may play an immunoregulatory role during foetal development.

Human alpha-fetoprotein (HAFP) was demonstrated in clusters of atypical cells of an extensive Bowen's disease involving the pubic area. Frozen sections were studied by indirect immunofluorescence using monospecific rabbit anti-HAFP antiserum or purified rabbit anti-HAFP antibody followed by staining with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit gamma-globulin and by a direct technique using FITC-labeled rabbit anti-HAFP Fab2 fragments. Sections of fetal liver served as positive controls and sections of adult human skin and liver were used as negative controls. Preabsorption of the anti-HAFP antibodies with purified HAFP blocked the fluorescence. This finding adds Bowen's disease to the list of premalignant disorders which possess HAFP as an oncofetal marker of their malignant potential. HAFP might possibly enhance malignant behavior through interference with cellular immunosurveillance mechanisms.

The heterogeneity of human alpha-fetoprotein has been studied by analytical isoelectric focusing in polyacrylamide gel slabs in the presence of 8 M urea. Six major isoelectric variants could be identified over a pH range of 6.0--6.2. Verification of their identity was achieved by crossed immunoelectrophoresis into agarose gel containing monospecific antiserum to human alpha-fetoprotein. Complete desialylation of the protein did not abolish the heterogeneity; a complex pattern of major alpha-fetoprotein bands persisted over a more alkaline pH range. We have been able to correlate the pattern of alpha-fetoprotein heterogeneity seen following extended agarose gel electrophoresis with that obtained during isoelectric focusing in the presence of urea. The quantity of certain alpha-fetoprotein charge isomers in various alpha-fetoprotein isolates may be important in considering certain biological functions of this protein.

Mevalonic acid is capable of initiating DNA synthesis, morphologic transformation, and cell division in cultures of human peripheral blood lymphocytes. Pure populations of lymphocytes respond poorly to mevalonic acid, but their response can be enhanced by peripheral blood neutrophils. Addition of cytochalasin B (0.5-1.0 micrograms/ml), but not cytochalasin A, to mixed neutrophil-lymphocyte cultures enhances the response of lymphocytes to both Con A and mevalonate, but the increment in mevalonate-induced DNA synthesis (+343%) far exceeds the enhancement which cytochalasin B produces in the Con A response (+24%). As a consequence, the DNA synthetic response in mevalonate (10(-2) M) containing cultures averages 39% of the response to an optimal dose of Con A. The cytochalasin B-enhanced response of mixed neutrophil-lymphocyte cultures to mevalonate is abolished by prior X irradiation of the lymphocytes, or their pretreatment with mitomycin C, as well as by the addition of hydroxyurea to the cultures but is not altered by prior X irradiation or mitomycin C pretreatment of the neutrophil helper population. These experiments suggest that the enhancing effect of cytochalasin B in the response of mixed neutrophil-lymphocyte cultures to mevalonic acid results from its ability to potentiate a time-dependent conditioning effect on lymphocytes which neutrophils exert. The conditioning effect of neutrophils cannot be achieved by cell-free neutrophil lysosomal enzymes released by exocytosis, and reactive oxygen species potentially generated by neutrophils are not involved. Attempts to demonstrate the production by neutrophils of a soluble mediator induced by mevalonate in the presence of cytochalasin B were without success. In the presence of cytochalasin B, only the metabolically active R(-) enantiomeric form of mevalonate is capable of initiating DNA synthesis in mixed neutrophil-lymphocyte cultures. The cytochalasin B-potentiated response of mixed neutrophil-lymphocyte cultures to mevalonic acid is preferentially displayed in cultures containing E-rosette-negative, as opposed to E-rosette-positive, lymphocytes. These observations add to the growing knowledge about the relationship between mevalonate metabolism, DNA synthesis, and cell replication.

The insertion of oxygenated sterol compounds into human red blood cell membranes as well as the consequent transformation of the red cells to an echinocyte shape and the expansion of the membranes are impeded by the presence of serum lipoproteins in the incubation medium. All density classes of human serum lipoproteins bind oxygenated sterol compounds, and lipoproteins can act as acceptors of oxygenated sterols previously inserted into red cells. Since oxygenated sterols have been reported to be atherogenic, the modulating and possibly protective effects of serum lipoproteins on oxygenated sterol-induced derangement of cell membrane structure and function may provide a useful model for further study.

The peripheral blood lymphoid cells of five patients with Sézary syndrome (SS) were examined with respect to their surface membrane characteristics and their response to mitogens. These cells showed markedly defective mitogenic responses to a broad dose range of phytohaemagglutinin (PHA), pokeweed mitogen, concanavalin A, and a rabbit antihuman lymphocyte antiserum (ATS), when compared with normal human lymphocytes. SS lymphoid cells (three patients studied) also displayed diminished or nearly absent capacity to form rosettes with unsensitized sheep erythrocytes (E-rosettes), and lacked surface immunoglobulin determinants. Despite their poor mitogenic response to ATS, they were as susceptible as normal lymphocytes to ATS-induced, complement mediated cytotoxicity. By comparison with lymphocytes from patients with chronic lymphocytic leukaemia, however, SS lymphoid cells showed decreased susceptibility to leukoagglutination by PHA. By way of contrast, three patients with mycosis fungoides having normal-appearing peripheral blood lymphocytes showed normal lymphocyte responses to mitogens, as well as normal proportions of E-rosette forming and surface immunoglobulin-bearing lymphocytes. These studies demonstrate that the SS lymphoid cell may, in some cases, lack surface properties and mitogen response characteristics of both B- and T-lymphocytes.

Mevalonic acid can stimulate leukemic cells from some patients with B cell chronic lymphocytic leukemia (CLL) to enter the cell cycle in vitro and to synthesize DNA. Unlike normal human peripheral blood lymphocytes, which require the presence of granulocytes for a maximal DNA synthetic response to mevalonic acid, CLL cells do not require a helper cell. Only the physiologically active R(-) enantiomer of mevalonic acid is active in initiating DNA synthesis, and no stimulatory effect is noted after addition of the precursors of mevalonic acid or of its known products. The mitogenic responses to mevalonic acid measured in CLL cells from 35 patients varied widely (range of stimulation indices, 1.2-18.6), but the DNA synthetic response of CLL cells from 9 patients to mevalonic acid exceeded those seen with such common lymphocyte mitogens as concanavalin A, phytohemagglutinin, poke-weed mitogen, or a phorbol ester. The mevalonate-responsive CLL cell was enriched in the E(-), M(+) rosette subpopulation. When CLL cells were incubated in lipoprotein-depleted serum-containing medium, the presence of low density lipoprotein (LDL) cholesterol increased their sensitivity to mevalonic acid transformation, suggesting that a nonsterol product of mevalonic acid metabolism may be involved. Mevalonic acid, which is essential for the growth of cells programmed to divide or stimulated to divide by mitogens, can by itself initiate cell replication in relatively inert G0 phase normal and neoplastic lymphocyte populations.

Purified human alpha-fetoprotein (HAFP) from five patients with hepatoma, one with gastric carcinoma, one with an embryonal cell tumor, and from fetal liver has demonstrated immunosuppressive potencies in vitro which vary over three orders of magnitude. A reversible association of HAFP with the cell surface and a predominant effect on T cells are suggested. No evidence of complex formation between HAFP and mitogen has been found. The microheterogeneity of HAFP has been detailed with crossed immunoelectrophoresis and isoelectric focusing in polyacrylamide gels containing 8M urea, and the immunosuppressive potency of HAFP isolated from a given source can be correlated with the proportion of certain HAFP species contained in it.

Cholesterol synthesis from acetate was studied in leukaemic cells from 20 patients with acute nonlymphocytic leukaemia. Marked differences in the rates of cholesterol biosynthesis were noted among three morphologically distinct types of leukaemia. As leukaemic cells differentiated along myeloid (acute promyelocytic) or monocytoid (acute myelomonocytic) pathways, their cholesterol-synthetic rates diverged and approached those of their respective mature cellular counterparts, the neutrophil or the peripheral blood monocyte. Enhanced sterol synthesis in leukaemic cells could not be explained by more rapid efflux of membrane cholesterol to the environment. In addition, the different rates of cholesterol biosynthesis in leukaemic-cell subgroups did not correlate with differences in their rates of cellular DNA synthesis. The normal divergence of sterol-synthesizing capacity found between mature neutrophils and monocytes develops at an early stage of differentiation and is detectable even in leukaemic cells.

Fetuin, the bovine alpha-fetoprotein, contains glycopeptide sequences similar to those found on red cells. As a result, it is capable of strong physical interaction with the phytohemagglutinin isomitogens (H-PHAP) which possess two or more R (red cell binding) subunits as part of their tetrameric structures. Fetuin shows little or no interaction with L-PHAP, a phytohemagglutinin made up of four L subunits which also lack red cell affinity. Despite these differences fetuin is able to inhibit both H- and L-PHAP-induced lymphocyte transformation and is also capable of inhibiting the mitogenic effects of pokeweed mitogen, concanavalin A, antithymocyte antiserum, and the one-way mixed lymphocyte culture. In the case of L-PHAP, the inhibitory effect of fetuin is proportional to the intensity of the mitogenic stimulus. The inhibitory effects of fetuin upon lymphocyte transformation may result from perturbation or "blindfolding" of the cell membrane in a manner analogous to other immunosuppressive serum alpha-globulins. Alpha-Fetoproteins may play an immunoregulatory role during fetal development.

Alkaline phosphatase [orthophosphoricmonoester phosphohydrolase (alkaline pH optimum), EC 3.1.3.1] purified from a Burkitt lymphoma cell line (Daudi) and Moloney-virus-induced murine leukemia (YAC) showed unique catalytic properties in substrate specificity and inhibition by cysteamine-S-phosphate. It migrated on polyacrylamide gel electrophoresis in a single activity band. Alkaline phosphatase with similar properties was found in several human lymphoblastoid cell lines, in chronic lymphatic leukemic cells, in organs of leukemic mice, and in sera of patients with certain lymphoproliferative disorders.

Fourier transform infrared spectroscopy revealed that insertion of 20 alpha-hydroxycholesterol into human erythrocyte membranes (10% of total membrane sterol) immobilized the lipid acyl chains to a degree equivalent to enriching total membrane cholesterol by 50% (Rooney, M.W., Lange, Y. and Kauffman, J.W. (1984) J. Biol. Chem. 259, 8281-8285). Raman spectroscopy showed that the amount of acyl chain rotamers was not significantly altered by the presence of 20 alpha-hydroxycholesterol, indicating that acyl chain immobilization was limited to an inhibition of lateral motion. The presence of 20 alpha-hydroxycholesterol may synergistically enhance the acyl-chain-immobilizing behavior of membrane cholesterol. In addition, protein helical structure was not altered by 20 alpha-hydroxycholesterol. The insertion of 7 alpha-hydroxycholesterol into erythrocyte membranes resulted in an increase in protein helical structure which was comparable to that observed for erythrocyte membranes enriched with pure cholesterol by 50%. However, both acyl chain mobility and conformation were unchanged. These results suggest a synergistic behavior between oxysterols and cholesterol in modifying erythrocyte membrane packing.

Fourier Transform Infra-red and Raman Spectroscopies indicate that 7 alpha-hydroxycholesterol and 7-ketocholesterol have a diminished capacity to condense (increase the packing order of) fluid-state dipalmitoylphosphatidylcholine (DPPC) acyl chains when compared with the effects of cholesterol and the other oxidized sterols studied. DPPC head groups were also more ordered by 7-ketocholesterol over the temperature range 10 degrees - 70 degrees C. Primary effects of these sterols appear to be associated with the hydrophillic regions of the DPPC bilayer, although packing arrangements with acyl chains are also involved. Phosphate and acyl chain ester groups were observed to possess a packing order which was invariant which indicates that these may be the target groups in the interaction with 7-ketocholesterol. A surprising observation was the synergistic amplification of the effects of 7-ketocholesterol by the presence of cholesterol in the DPPC bilayer.