Loss and reappearance of transferrin receptors in human leukemic cell lines

Serum transferrin (the iron binding protein) exerts its iron carrier function at the cell surface after binding to the appropriate receptor (TrR). In this work it is demonstrated that differentiating agents induce loss of TrR from the surface of three leukemic cell lines (Molt-3, HL-60 and K-562). Loss of TrR correlates with change in morphology and induction of phenotypic markers of the differentiated cells. Removal of the differentiating agent from the culture is followed by reexpression of TrR on the cell surface. The data presented in this paper suggest that TrR may play a regulatory role in cell differentiation and malignant transformation.     

Correlation of membrane glucocorticoid receptor levels with glucocorticoid-induced apoptotic competence using mutant leukemic and lymphoma cells lines

We have studied the presence and functional implications of membrane glucocorticoid receptor (mGR) in several wild type (WT) and mutant mouse lymphoid cell lines (nuclear transfer decrease, NT(-); nuclear transfer increase, NT(i); and receptorless, R(-)). Direct fluorescent antibody staining revealed large aggregates of mGR-specific fluorescing antigens in the plasma membrane of the WT and mGR-enriched (mGR(++)) S-49 cells. While R(-) cells totally lacked mGR, this receptor level was low in NT(-) and NT(i) groups. FACS analysis corroborated these results, showing a approximately 4-10-fold difference between the highest mGR levels (mGR(++)) and the R(-) and NT(i) cells. Membrane extracts were analyzed for mGR by immunoblotting. Multiple receptor forms, ranging in M(r) from 94,000 to > 200,000, were observed in the WT cells, while only smaller peptides (85,000-94,000) were found in NT(-) cells. No detectable immunoreactive bands were identified in either membrane or cytosol immunoprecipitates of NT(i) and R(-) cell groups. Within 48 h post dexamethasone exposure > 98% of WT and mGR(++) S-49 cells underwent apoptosis, compared to 0-30% in the mutant cells, albeit the total receptor number is two to three times higher in NT(i) compared to WT. These results suggest a better correlation between the quantity and quality of mGRs (rather than total cellular GRs) and the ability of glucocorticoids (GCs) to lyse lymphoid cells.     

Accessory function and interleukin 1 production by human leukemic cell lines

Highly purified T lymphocytes do not proliferate in response to mitogens, unless adherent HLA-DR-positive monocytes are added to the culture. This accessory function (AF) of monocytes requires the release of interleukin 1 (IL 1). Cells from three human leukemic cell lines, K562, HL60, and U937, could very efficiently replace monocytes in a 72-hr mitogen-induced T cell proliferation assay. The AF was clearly related to precise maturational stages of these cells; the hematopoietic precursor K562 cells spontaneously exerted high AF, but lost this property when treated with differentiation inducers. On the contrary, the promyelocytic HL60 cells and the "histiocytic" U937 cells exhibited no spontaneous AF, but acquired this property when induced to differentiate along the granulocytic and/or monocytic pathway. Three leukemic cells could not only stimulate T cells to proliferate and produce IL 2 in the presence of mitogens, but also under appropriate culture conditions these cells could produce IL 1, which could not be distinguished from normal human monocyte derived IL 1 by gel filtration and isoelectric focusing. Moreover, analysis of phenotypic markers revealed that AF and production of IL 1 could be demonstrated in different cell types and therefore are not restricted to the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL60 cells. Thus, the expression of the DR antigens is not required for AF and IL 1 production in response to mitogens. These three human leukemic cell lines will provide convenient sources of human IL 1.     

The role of iron in the growth of human leukemic cell lines

The growth requirements of three human leukemic cell lines (K 562, HEL, U937) have been studied in the absence of serum. For growth in serum-free medium, the cells require insulin, transferrin, and albumin. Two highly water-soluble iron salts, ferric ammonium citrate and ferric ammonium sulfate, may completely replace transferrin for supporting the growth of these cell lines. Similar results were obtained when mitogen-stimulated lymphocytes were grown in serum-free media. Iron containing compounds, such as hemin or hemoglobin, were also able to replace transferrin. Experiments using 42/6 monoclonal antibody strongly suggest that free-iron salts are taken up by the cells by a mechanism that is completely independent from transferrin-receptors.     

Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines

We have examined the effects of estrogen and progestin agonist and antagonist ligands on regulation of progesterone receptor (PR) protein and mRNA levels in a variety of human breast cancer cell lines. By Northern blot analysis, using human PR cDNA probes, PR mRNA in T47D and MCF-7 cells appears as five species of approximately 11.4, 5.8, 5.3, 3.5, and 2.8 kilobases. PR mRNA species are not detected in the PR protein-negative breast cancer cell lines MDA-MB-231 and LY2. T47D cells contain high levels of PR mRNA and protein (detected by hormone binding assay or Western blot analysis), and the PR protein and mRNA content of T47D cells are reduced to about 10% of the control level within 48 h of treatment with 10 nM promegestone; 17, 21-dimethyl-19-nor-pregna-4,9-diene-3, 20-dione (R5020) or 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione (ORG2058), both potent progestins. In contrast, treatment of T47D cells with the antiprogestin 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]-17 alpha-(1-propynyl)-estra- 4, 9-dien-3-one) (RU38486) reduces PR protein and mRNA levels only transiently. PR protein and mRNA are virtually undetectable in control MCF-7 cells grown in the absence of estrogens. When estradiol is administered to MCF-7 cells, the PR mRNA and protein levels increase gradually and proportionately (10- or 40-fold, respectively, in 3 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of macrophage activating factor by human leukemic T cell lines

Human leukemic T cell lines were tested for their ability to produce a macrophage activating factor. When mouse peritoneal macrophages were cultured for 48 hr in the presence of culture supernatants from cell lines HPB-ALL, CCRF-CEM, or MOLT-4, glucose oxidation via the hexose monophosphate pathway was enhanced by five to seven fold. Culture supernatants from cell line HPB-MLT stimulated the oxidation to a lesser extent. However, cell line CCRF-HSB-2 was essentially inactive as a producer. The active supernatants also stimulated the release of hydrogen peroxide from macrophages, whereas the inactive one did not. Since treatment of the cell lines with 12-o-tetradecanoyl phorbol acetate or phytohemagglutinin had little effect on the production of the factor except HPB-ALL, the cell lines seemed to secrete the factor constitutively. The stimulatory effect was dose-dependent and evident at a concentration as low as a 1/80 dilution. The factor was resistant to heat treatment at 100 C for 20 min, nondialysable and sensitive to protease digestion. The activating factor could be partially purified by anion exchange and gel filtration chromatographies.     

Biological effects of glucocorticoid hormones on two rat colon adenocarcinoma cell lines

Glucocorticoid hormones are thought to play a role in carcinogenesis as they regulate cell differentiation and proliferation. We have investigated the effect of dexamethasone on two cell lines derived from a colon carcinoma, which differ by their tumorigenicity. Dexamethasone was found to inhibit growth of both the progressive (PROb) and the regressive clone (REGb). Upon glucocorticoid treatment, PROb cells were found to secrete an additional M_r 40,000 protein. The synthesis and the release in the culture medium of this protein is stimulated specifically by glucocorticoid agonists, and not by other steroid hormones. The anti-glucocorticoid RU 38486 is ineffecient and suppresses the induction of this protein by dexamethasone. Induction is sensitive to actinomycin D, suggesting that regulation may be related to an alteration of the rate of mRNA synthesis. The cellular effect of glucocorticoid hormones being mediated through a specific soluble receptor, we have characterized this protein. The PROb cells contained more specific glucocorticoid-binding sites ( 170,000 sites per cell) than the regressive ones (REGb cells; 100,000 sites per cell). In both clones, the receptor was associated with the M_r 90,000 heat shock protein to yield large complexes (Stokes radius R_s 7.5 nm), which were dissociated to the same extent upon heat- and salt-treatment. The steroid- and DNA-binding unit of the receptor, characterized under denaturing conditions using an anti-receptor monoclonal antibody, was found to be more degraded in the PROb cell line.     

Determination of eight elements in six human cancer cell lines and two human normal cell lines by PIXE

Gastric, hepatic, and pulmonary cancer cell lines, and the third passage of normal gastric and pulmonary cell lines were analyzed by proton induced X-ray emission (PIXE) method. The contents of element Sr, Ca, Fe, Zn, P, K, Cu, and As in the cell lines were determined. The Sr, Ca, Fe, Zn, and As contents in cancer cell lines were significantly lower than those in the normal cell lines (p less than 0.05), whereas there were no significant differences for the P, K, and Cu contents (p greater than 0.1). The results suggest that the need of some essential elements has been diminished in cancer cell proliferation.     

Diploid and haploid states of the glucocorticoid receptor gene of mouse lymphoid cell lines

A glucocorticoid-sensitive mouse thymoma line, W7, is compared to the mouse lymphoma line S49 which has been extensively used in studies of steroid action. Glucocorticoid-resistant variants are known to arise spontaneously at high rate from S49 (3.5 × 10^?6 per cell per generation) and at a frequency orders of magnitude lower in the case of W7 (<3 × 10^?9). The receptors of both cell lines have the same affinity for dexamethasone (K_d = 1.3 ± 0.3 × 10^?8 M), but W7 cells contain twice the amount of glucocorticoid receptors present in S49 and are measurably more sensitive than S49 cells to dexamethasone. By selection for resistance to low concentrations of dexamethasone, derivatives of W7 have been isolated which are similar to S49 in that they have a higher resistance than the parental W7 line and approximately half the receptor content. Moreover, like S49, the partially resistant variants of W7 give rise to fully resistant derivatives at a high rate (2 × 10^?6 per cell per generation). These results suggest that a structural gene (r) coding for the receptor is present in two functional copies in W7 (r^?,+), but in only one functional copy (r^+/?) in partially resistant derivatives of W7 and in S49. The gene dosage effect observed in these pseudodiploid lines indicates that the receptor gene, r, is autosomal, and that the inactivation of the r gene is a recessive genetic event. Consequences of the homozygous and heterozygous states of the receptor locus are discussed.     

13C-NMR investigation of protein synthesis during apoptosis in human leukemic cell lines

In order to evaluate the role of protein synthesis in apoptosis, ¹³C-NMR has been used to study the levels of protein synthesis in three different human leukemic cell lines in the presence and absence of dexamethasone-induced apoptosis. Measurements were done on one dexamethasone-sensitive (CEM-C7-14) and two different dexamethasone-resistant variants (CEM-4R4 and CEM-ICR27-4). The incorporation of ¹³C-labeled amino acids into cellular proteins, which reflects the level of new protein synthesis, was monitored by ¹³C-NMR spectroscopy. In the absence of dexamethasone, the level of protein synthesis was found to be significantly different among the three cell lines. Dexamethasone caused a significant reduction (≅60–87%) in the level of protein synthesis in dexamethasone-sensitive CEM-C7-14 cells, while having no significant effect on protein synthesis in dexamethasone-resistant CEM-4R4 cells. Dexamethasone treatment caused a significant enhancement of the level of protein synthesis in the CEM-ICR27-4 cells. Synthesis of proteins was found to occur during apoptosis, albeit at a low level, suggesting a role for the synthesis of specific proteins in the mechanism of apoptosis. J. Cell. Physiol. 181:147–152, 1999. © 1999 Wiley-Liss, Inc.     

In vitro modulation of leucocyte-function-associated antigen-1 expression on two leukemic cell lines

Summary Leukocyte-function-associated antigen-1 (LFA-1) expression on two widespread tumor cell lines: K562 (an erythroleukemia) and MOLT-4 (a T leukemia), was investigated using two monoclonal antibodies specific for the chain of this surface antigen, and flow cytometry analysis. When K562 cells are in the exponential phase of growth, they display very low levels of LFA-1. By contrast, cells from the plateau phase exhibit a strong labelling, which disappears rapidly when they are allowed to resume division by changing the culture medium. Using the same experimental conditions, we failed to detect any LFA-1 expression on MOLT-4 cells. However, after stimulation of these cells by phorbol myristate acetate, we observed a significant labelling, which occurred within 2 days of treatment. The LFA-1 expression disappears progressively after removal of the phorbol ester. From these results it may be concluded that (a) LFA-1 expression can vary considerably according to the culture conditions, (b) the expression of this antigen on the surface of non-expressing variants can be induced by phorbol ester, and (c) in both cases, the change in expression can be reversed completely by replacing the culture medium or by removing phorbol myristate acetate from it. Abbreviations used: LFA-1, leukocyte-function-associated antigen-1; PMA, phorbol myristate acetate; TNBS, trinitrobenzenesulfonic acid     

Externalization of transferrin receptor in established human cell lines

The externalization of transferrin receptors was found in established human tumor cell lines at the rate of 10-35 ng/hour/10(6) cells, when they were incubated with transferrin at 37 degrees C. This externalization is inhibited by lowering the incubation temperature to 4 degrees C or eliminating the ligand from the culture medium. Metabolic inhibitors such as sodium azide, colchicine, cytochalasin B and chloroquine also decreased the rate of externalization. Almost 95% of released transferrin receptors were precipitated by centrifugation at 100,000 x g for 30 min, suggesting that transferrin receptor is externalized into the medium as a vesicular form.     

Selective cytotoxicity of marine algae extracts to several human leukemic cell lines

Extracts from 8 species of marine algae which showed selective cytotoxicity in our previous screening program, were further examined for cytotoxic spectra to five human leukemic cell lines. The extract from a red alga, Amphiroa zonata exhibited strong cytotoxicity to all human leukemic cell lines tested and murine leukemic cells L1210 at the final concentrations from 15 to 375 μg ml−¹. Then the cytotoxicity was not found in normal human fibroblast HDF and murine normal cells NIH-3T3. The active extract fraction from this alga was soluble in higher polar organic solvents and water and heat-stable. The extract from a brown alga Dilophus okamurae with weak selective cytotoxic activity to L1210 cells exhibited not only strong cytotoxicity to L1210, but also to human leukemic cells, HL60 and MOLT-4 at 50 μg ml-¹. While, the extract from a green alga, Cladophoropsis vaucheriaeformis with most selective cytotoxic activity, did not show cytotoxicity to any human leukemic cell lines tested at 50 μg ml-¹. However, this extract showed strong cytotoxicity to two human leukemic cell lines and NIH-3T3 at 100 μg ml−¹. Thus, it was considered that a red alga, Amphiroa zonata might be suitable natural source for development of anti-cancer agents without side-effect. This revised version was published online in June 2006 with corrections to the Cover Date.