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Biomedical subjects

S Waxman

Publications and source records attributed to S Waxman.

At least 127 records · Page 7Linked to original sources
PubMed1987

Phosphate-induced phosphoribosylpyrophosphate elevations to assess deranged folate and purine nucleotide metabolism.

Phosphoribosylpyrophosphate (PRPP) levels increase several-fold in HL-60 cells adapted to folate deficiency either by continuous passage in folate-deficient medium or by short-term incubation with 10(-8) M methotrexate (MTX). The addition of folic acid (PteGlu) or 5-formyltetrahydrofolic acid (5-CHO-H4PteGlu) in the form of Leucovorin normalizes this effect. The reactions for measuring PRPP levels are time and temperature dependent and are influenced by PRPP-reacting substances in undialyzed serum. Inorganic phosphate (PO4), when added to the assay, markedly stimulates PRPP levels in HL-60 cells and can be used to stress folate-dependent PRPP utilization for purine synthesis. The integrity of the folate-dependent pathways of purine-synthesizing cells can be sensitively assessed by measurement of PRPP levels during a 2-hr assay in the presence of PO4 in medium free of folate but containing dialyzed serum. In HL-60 cells that are folate deficient or in the presence of MTX (as low as 2 X 10(-9) M), PO4-stimulated PRPP levels remain elevated due to ineffective utilization unless folate is added to the incubation mixture. The sensitivity of this PRPP assay to metabolically assess the integrity of folate-dependent reactions in purine synthesis is comparable to that of the deoxyuridine suppression assay. Inorganic phosphate can also be used to stimulate the incorporation of purine analogs, such as 6-mercaptopurine, into intact red blood cells which may have therapeutic implications for targeting drug delivery.

Cell Line↗
PubMed1986

Basic principles for utilizing combination differentiation agents.

The induction of differentiation in several tumor lines serves as a basis for a new approach to cancer treatment. In vitro studies in the mouse erythroleukemia (MEL) cell system have identified about 300 agents capable of inducing differentiation by mechanisms that remain to be elucidated. The design of differentiation therapy will depend on the specific tumor cell type, an effective time course, and the synergistic interaction among combinations of two or more inducers. The induction of differentiation may be followed by terminal cell division (TCD) or programmed cell death in several tumor cell systems. This mechanism for the destruction of tumor cells is one goal of differentiation therapy and differs from nonspecific cytotoxic therapy. To evaluate the effect of differentiation therapy, a clear distinction must be made between nonspecific cytotoxicity and the programmed TCD of induced cytodifferentiation. One possible parameter for assessing the commitment to TCD in the MEL cell system is a selective decrease in DNA ligase activity, which does not appear to occur following treatment with nonspecific cytotoxic agents. These biological and biochemical parameters should be helpful in designing agents capable of inducing TCD in vivo.

Animals↗
PubMed1985

Lack of evidence for activation of a serum factor in protease-induced differentiation of mouse erythroleukemia cells.

The addition of certain proteases to cultures of Friend virus-infected mouse erythroleukemia cells can induce up to 90% of the cells in culture to become hemoglobin-containing, as assessed by positive staining for benzidine (B+). Because the mechanism of this protease action is unknown, media components were studied as possible targets for protease activity. Aliquots of medium plus serum were incubated for various times with levels of protease sufficient to induce approximately 50% of the cells to the B+ state. Cells were added to protease-pretreated serum either before or after inactivation of the protease. In all cases, enzymatically active protease had to be present with the cells to induce B+ cells to form. Serum and other components of the medium pretreated with protease were inactive. Mouse erythroleukemia cells grown in the absence of serum were also induced by proteases to form B+ cells. These data imply that the inducing action of proteases cannot be passively transferred by protease-pretreated serum or medium nor is serum required for protease-mediated induction of B+ cells. Taken together, these conclusions suggest that the protease action is on the cells or on cellular products intimately associated with cells.

Animals↗
PubMed1985

The proportions of hemoglobin types induced in mouse erythroleukemia cells vary with the inducer or combination of inducers, the inducer concentration and the time of induction.

The relative amounts of hemoglobin (Hb) major and Hb minor accumulated during induction of erythrodifferentiation in mouse erythroleukemia (MEL) cells were studied. The ratio of major to minor was found to depend not only upon the inducer tested (as reported previously by others), but also upon the concentration of the inducer and the time of exposure to the inducer as well as the specific cell line of MEL cells studied. At concentrations required for optimal induction of differentiation, certain agents led to the accumulation of predominantly Hb major, but suboptimal concentrations of the same inducers led to predominantly Hb minor accumulation. After a relatively short induction time (2 da) utilizing a given inducer either the level of Hb minor was higher than that of Hb major or the levels of the two Hb's were approximately equal, but after longer induction periods (3-7 da) Hb major was more abundant than Hb minor. In addition, it was found that the three proteases tested induced predominantly Hb minor. The addition of suboptimal concentrations of low molecular weight inducers acted synergistically with a given protease to produce a high yield of Hb-containing cells. When these agents were added singly they induced relatively low Hb major/Hb minor ratios, but when a low molecular weight inducer was added together with a protease in a "synergistic" combination, elevated ratios were induced. The proportions of hemoglobin types induced in MEL cells may be related in part to the intensity of the induction response. In view of these data, classifications of inducers based solely on the ratios of Hb types produced must be guarded.

Acetamides↗
PubMed1985

Differential binding of folates by rat renal cortex brush border and basolateral membrane preparations.

The binding of radioactive 5-methyltetrahydrofolate and folic acid was found to be greater in brush border than in basolateral membrane preparations of rat renal cortex. This appeared to be due to an increased amount of a specific folate binding protein in the brush border membrane preparations as compared to those of the basolateral membrane. The binding was saturable and inhibited by nonradioactive folic acid and, therefore, a specific, rather than nonspecific process. The Km's for folic acid binding in brush border and basolateral membrane preparations were similar and involved a single high-affinity binding site. In contrast, methotrexate was found to bind equally well to both brush border and basolateral membrane preparations. Moreover, folic acid binding was not inhibited by an equimolar amount of methotrexate. A folate binding protein could be extracted from either membrane preparation with 1% Triton X-100 and, to a lesser extent, with 0.6 M NaCl. These different extraction procedures resulted in different apparent molecular weights for folate binding protein (greater than 160,000 for Triton X-100-extracted samples and 40,000 for NaCl-extracted samples). The membrane preparation pellets remaining after NaCl extraction were able to rebind tritiated folic acid and also the 40,000-Da folate binding protein. On the other hand, membrane preparations extracted with Triton X-100 lost the ability to bind folic acid or the 40,000-Da folate binding protein. These differences in molecular weight and rebinding capacity may be explained by the existence of a receptor for folate binding protein which was extracted by Triton X-100, but not by NaCl. The greater concentration of folate binding protein in the renal tubule cell brush border membrane preparations as compared to those from basolateral membranes ascribes, for the first time, a functional role for folate binding protein in the renal reabsorption of folates which is required to prevent loss of folate in the urine and perhaps in the membrane transport of folates in general.

Animals↗
PubMed1985

Protease induction of hemoglobin synthesis but not terminal cell division in K562 cells.

Several protease preparations of varied specificity increased hemoglobin levels in K562 cells. These are the first enzymes shown to stimulate this process in these cells. Hemin, at a concentration at which it did not act as a potent inducer of hemoglobin production, was found to synergistically stimulate induction by proteases. As seen in some other cell types, six different protease preparations also stimulated K562 cell yield. Hemin did not enhance the protease stimulation of cell yield, but was, instead, slightly inhibitory. Trypsin was one of the most potent inducers of the proteases tested. A combination of trypsin with a "synergistic" concentration of hemin did not decrease the size of K562 cells during induction of hemoglobin production, suggesting that these cells were not irreversibly differentiated nor induced to terminal cell division by this treatment. This was supported, although not proven, by an assay that demonstrated no progressive decrease in the rate of cell multiplication associated with the induction of hemoglobin synthesis.

Animals↗
PubMed1984

Abnormal isoelectric focusing patterns of serum galactosyltransferase activity in patients with liver neoplasia.

Serum galactosyltransferase activity (GT) has been studied using isoelectric focusing chromatography in normal subjects, in patients with non-neoplastic liver diseases, and patients with liver neoplastic diseases (hepatoma and liver metastases) using isoelectric focusing chromatography. Freshly obtained serum was applied to an isoelectric focusing column containing Ampholine (pH 4-6) and the fractions were assayed using asialo-agalactofetuin as the galactose acceptor. Three peaks of GT activity were found in sera from normal subjects at pH 4.8, 4.95, and 5.1. In contrast, sera from patients with hepatoma or with gastrointestinal adenocarcinoma with liver metastases contained only two peaks of GT activity (pI 4.75 and 4.95). Sera from patients with non-neoplastic liver diseases had similar GT isoelectric focusing patterns as those from normal subjects. These results suggest that the isoelectric focusing patterns of serum GT activity may be useful in characterizing human liver neoplasia.

Adenocarcinoma↗
PubMed1983

G-actin and F-actin levels at different stages of mouse erythroid differentiation.

Monomeric (G-) actin and filamentous (F-) actin levels were determined in Triton X-100 extracts prepared from mouse erythroid cells at various stages of differentiation. G-actin and F-actin were found in the Triton-soluble fraction and in the Triton-insoluble fraction, respectively. G-actin levels in untreated and dimethyl sulfoxide-treated (differentiated) erythroleukemia cells, reticulocytes, and erythrocytes were 48, 33, 2.8, and 0.37 microgram/mg protein, respectively, and F-actin levels were 17, 35, 45, and 59 micrograms/mg protein, respectively. G-actin/F-actin ratios were successively lower in cells representing the more mature stages of development.

Actins↗
PubMed1983

Effects of dexamethasone and phorbol myristate acetate on the induction of differentiation in mouse erythroleukemic cells by dimethyl sulfoxide, proteases, and other compounds.

Lines DS-19 and 5-86, each derived from line 745, when tested for their responses to various inducers and inhibitors of differentiation, shared some characteristics, but differed in others. In particular, DS-19 was markedly induced to differentiate by actinomycin D whereas 5-86 was only slightly affected. The patterns of the ability of PMA to influence induction and cell multiplication by various inducing agents differed in the two lines. The pattern of DEX inhibition of differentiation was similar in the two lines. Notably, DEX markedly inhibited induction due to all of the inducers tested except protease V8, actinomycin D, PGE1, and butyrate and its fatty acid analogues that were tested. DEX stimulated growth during its inhibition of induction by DMSO and many other inducers, but reduced cell multiplication in the presence of butyrate. PMA inhibited induction by most of the inducers tested in DS-19 cells except for some of the fatty acids. The inhibition by PMA generally was accompanied by cytotoxicity in DS-19 cells, but not in 5-86 cells. PMA markedly inhibited differentiation by only 5 of the inducing agents tested in 5-86 cells, but was not as cytotoxic in this line. Proteases, which have been shown to stimulate both MEL cell differentiation and growth, are inhibited with respect to their effects on differentiation, but not with respect to those on growth by DEX and/or PMA. DEX did not have the same effect on induction stimulated by the two proteases studied. Many of these findings indicate that at least some effects by inducers on cell multiplication in this system are not inextricably linked to differentiation. It is hoped that the further study of induction by proteases, which have known enzymatic activities, as well as of inhibitors of induction, will shed light on the molecular mechanism(s) of action of DMSO and other low molecular weight inducers.

Animals↗
PubMed1983

Biotransformation of homofolate from a growth inhibitor to a growth-promoting derivative.

Homofolic acid (HF) was found to inhibit the growth of Lactobacillus casei ATCC 7469 in 100 ng/ml or higher concentrations in the presence of folic acid (PGA) on short (24-48 h) incubation. HF blocked the uptake of 3HPGA into L. casei cell suspensions. On prolonged (3-5 days) incubation HF (greater than 10 ng/ml) promoted growth of L. casei both in the presence and absence of PGA. No growth was obtained when both PGA and HF were omitted. Thus, HF was apparently biotransformed from an inhibitory analog into a growth-promoting HF derivative. Indeed, sterile filtrates from HF-grown cultures supported growth of L. casei in a folate-free medium on short incubation, supporting the notion that a folate-like active compound was formed from HF on prolonged incubation with L. casei. In the presence of homopteorate (10-60 micrograms/ml) no transformation of HF into folate-active compounds took place.

Biotransformation↗
PubMed1983

Proteases act synergistically with low molecular weight inducers to stimulate mouse erythroleukemia cell differentiation.

Proteases stimulate mouse erythroleukemia (MEL) cell differentiation and multiplication. The stimulation of differentiation is synergistically increased by low concentrations of dimethyl sulfoxide. Synergism of other low molecular weight inducers with representative proteases, alpha-chymotrypsin and protease V8, was tested. Hemin. hypoxanthine, actinomycin D, aminonucleoside of puromycin, hexamethylene bisacetamide, and 5-azacytidine were also found to act synergistically with this protease to augment MEL cell hemoglobin production, but not cell multiplication. Fatty acids (acetate, propionate, butyrate, isobutyrate, and valerate), prostaglandins A1 and E1, amino acids, and amino acid analogs and metabolites did not act synergistically with chymotrypsin. Some physiologic amino acids were found to be weak inducers. Several of the low molecular weight inducers also acted synergistically with protease V8 in inducing differentiation, and, as with chymotrypsin, did not act synergistically in stimulating cell multiplication. Like chymotrypsin, protease V8 did not act synergistically with butyrate. The earlier finding that proteases, but not low molecular weight inducers, stimulate cell multiplication during the induction of differentiation was confirmed. Carboxypeptidase A also was found to be an inducer.

Amino Acids↗
PubMed1983

Glycosyltransferase alterations are cell type related when human promyelocytic leukemia (HL-60) cells are treated with various inducers of differentiation.

We have assayed glycosyltransferase activities during the granulocytic and macrophage-like differentiation of human promyelocytic leukemia (HL-60) cells. Functional granulocytic differentiation was assayed by the decarboxylation of 2-deoxyglucose in addition to nitroblue tetrazolium reduction. Dimethylsulfoxide (DMSO) treated HL-60 cells, induced to granulocytic differentiation, had higher 2-deoxy-glucose decarboxylation activity, and contained less sialyltransferase (ST), more fucosyltransferase (FT), and more N-acetylglucosaminyltransferase (NGT) activities than untreated cells. HL-60 cells treated with another granulocytic differentiator, retinoic acid, also had higher 2-deoxyglucose decarboxylation activity, and contained less ST, more FT, and more NGT activities than untreated cells. In contrast, cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reported to differentiate HL-60 to macrophage-like cells, but did not show an increased level of 2-deoxyglucose decarboxylation activity, but contained more galactosyltransferase (GT) and FT activities as compared to untreated cells. These findings suggest that the alterations of glycosyltransferase levels during the differentiation of precursor cells may not depend upon different inducers, but are characteristic of the phenotypic expression of the mature cell type.

Cell Line↗
PubMed1983

Inhibition of amino acid transport by cis-diamminedichloroplatinum(II) derivatives in L1210 murine leukemia cells.

The uptake of cis-diamminedichloroplatinum(II) (cisplatin) has been studied in the L1210 murine lymphoid leukemia cell line. Labeled cisplatin and its aquated derivatives were resolved by high-performance liquid chromatography on a strong cationic exchange column. After 10 min of incubation of cisplatin with the cells, the major portion of the non-protein-bound platinum was in the form of cisplatin. However, a portion of this platinum was converted with time to a derivative which coeluted with the monoaquo derivative of cisplatin. With the appearance of this derivative, there was a concomitant inhibition of sodium-dependent amino acid transport as measured by the uptake of aminoisobutyric acid and methionine. Furthermore, the exposure of L1210 cells to a preparation of predominantly aquated product(s) of cisplatin inhibited amino acid uptake following a brief (2-min) incubation, whereas measurable inhibition of amino acid uptake by cisplatin required a longer preincubation period. This inhibition of aminoisobutyric acid and methionine was dependent on the concentration of platinum. Aminoisobutyric acid and methionine were shown to be concentrated in L1210 cells in the presence of sodium ions, and competition experiments suggest similar uptake systems. Since L1210 cells are methionine-auxotrophic leukemic cells, inhibition of essential amino acid transport by cisplatin may be a mechanism of cytotoxic action.

Aminoisobutyric Acids↗
PubMed1982

Evaluation of X-methyl folate preparations as possible folic acid antagonists.

Crude X-methyl folate and two purified fractions (purified X-methyl folate and 9-methyl folate) were evaluated as possible folic acid antagonists. Antifolate activity was measured by the deoxyuridine suppression assay, direct measurement of dihydrofolate reductase and morphological changes characteristic of folate deficiency. Cytotoxicity was evaluated by cell growth curves. These drugs were studied in freshly obtained human normal evaluated by cell growth curves. These drugs were studied in freshly obtained human normal bone marrow and leukemia cells as well as in three established cell lines HL-60 (human promyelocytic leukemia), CEM (human T lymphocytes), and L1210 (murine lymphoid leukemia). Purified X-methyl folate and 9-methyl folate at concentrations up to 2X10(-5) M failed to induce cell toxicity or inhibit deoxyuridine suppression of 3H-thymidine into DNA. Dihydrofolate reductase was not inhibited by 9-methyl folate but was partially inhibited by purified X-methyl folate. Crude X-methyl folate (2X10(-1) M) is a weak folate antagonist as compared to methotrexate since it requires a 1,000-fold higher concentration to inhibit cell growth and dihydrofolate reductase activity. In addition, the crude X-methyl folate contains a folate-like fraction since it will correct the abnormal deoxyuridine suppression in folate-depleted cells. None of the drugs tested were able to induce myeloid differentiation in the HL-60 cells. The data suggest that 9-methyl folate would not be active as a folate antagonist antitumor agent and that purified and crude X-methyl folate in large concentrations will inhibit folate metabolism. Additional studies are needed to remove the folate-like activity in the crude material in order to further establish the antitumor effects of the crude X-methyl folate.

Cell Differentiation↗
PubMed1982

Characterization of galactosyltransferase released from human hepatoma cells.

A human hepatoma cell line (SK-H-MA) released a large amount of sialyltransferase (ST) and galactosyltransferase (GT) into the culture medium, whereas cells derived from normal human liver (Chang) released a large amount of GT but very little ST. The characteristics of hepatoma GT were studied since an abnormal GT isoenzyme has been associated with human gastrointestinal neoplasms. Both hepatoma and Chang medium GT activities had an absolute requirement for MnCl2 (25 mmol/l) and a broad optimal pH between 6.5 and 7.0, and were not affected by 0.1% Triton X-100. These two enzyme preparations were inhibited to the same extent by N-acetylglucosamine and N-acetylgalactosamine, while N-acetylglucosamine was 100 times more potent than N-acetylgalactosamine. Various nucleotides inhibited both enzyme activities equally well. Uracil-containing nucleotides were better inhibitors than thymine-containing nucleotides, and other nucleotides were only slightly inhibitory. The most effective inhibitor was UDP. More of the GT activity in hepatoma medium (65%) as compared to Chang medium (35%) bound to concanavalin A-Sepharose, and was eluted with 2.5% alpha-methylmannoside. These results suggest that the GTs from hepatoma and Chang media are not different in their enzymatic activity but may differ in their carbohydrate contents, which may be another manifestation of the neoplastic nature of the hepatoma cell line.

Animals↗