Tyrosine kinase inhibitor-induced differentiation of K-562 cells: alterations of cell cycle and cell surface phenotype.

Protein tyrosine kinase (PTK) inhibitor herbimycin A inhibited proliferation, induced accumulation of cells in the G0/G1 phase of the cell cycle and a marked increae of hemoglobin-producing human leukemic K-562 cells in vitro. The isoflavonoid PTK- and topoisomerase II inhibitor genistein produced a similar effect with the accumulation of cells in the G2/M phase of cell cycle. Genistein potentiated the effect of herbimycin A on the cell cycle (i.e. decreased the proportion of S-phase cells) and induced an increased proportion of hemoglobin-producing cells. Genistein, but not herbimycin A induced a marked increase in cell surface expression of CD15 (LewisX) antigen. Both of these agents down-regulated CD45 (leukocyte common antigen) and monocyte-associated CD14 antigen on K-562 cells. Neither genistein nor herbimycin A induced increased cell surface expression of glycophorin.

Alterations of cell surface antigens induced by placental isoform of ferritin in human carcinoma cell lines.

AIM: the ability of the breast cancer-associated placental acid isoform of ferritin (p43-PLF) to modulate cell surface expression of HLA, CD59 (protectin) and CD66 antigen (adhesion antigen related to CEA) was examined. METHODS: the expression of these antigens in human breast carcinoma cell lines BT-20, T47D and MDA-MB-468 was determined with the aid of flow cytometry and monoclonal antibodies. RESULTS: PLF induced a transient up-regulation followed by a down regulation of cell surface protectin (CD59 antigen) on the cell surface of T47D and to a lesser extent, BT-20 human breast carcinoma cell lines. Furthermore, PLF down-regulated cell surface expression of CEA-related CD66 antigen on both these cell lines. No PLF-induced alterations of protectin, CD66 antigen and HLA class I antigen were found on the MDA-MB-468 breast cancer cell line. CONCLUSIONS: breast cancer-associated p43 induces alterations of the expression of cell surface molecules in breast cancer cells which could have an effect on the modulation of cancer cell adhesive interactions.

Protein kinase inhibitor-induced alterations of drug uptake, cell cycle and surface antigen expression in human multidrug-resistant (Pgp and MRP) promyelocytic leukemia HL-60 cells.

Protein kinase inhibitors staurosporine and CGP 41251, a benzoylated derivative of staurosporine with selective PKC inhibitory activity, reversed the decreased rhodamine 123 uptake in HL-60/VCR (with Pgp-mediated drug resistance) but not in HL-60/ADR (MRP-mediated drug resistance) cells. CGP 41251 reversed the decreased rhodamine 123 uptake in HL-60/VCR cells more efficiently (when compared on a equimolar basis) than staurosporine. However, the protein tyrosine kinase inhibitor genistein unexpectedly modulated the decreased rhodamine 123 uptake in Pgp positive (HL-60/VCR) cells, but not in HL-60/ADR (MRP positive) cells. Cell surface phenotype of both HL-60 drug-resistant cell sublines was compared with that of the parental, drug-sensitive HL-60 cells. Both drug-resistant cell lines expressed markedly decreased levels of cell surface HLA class I antigen in comparison with the parental HL-60 cells. A similar decreased cell surface expression of HLA class II/DR on both drug-resistant, as well as of CD59 (protectin) on HL-60/ADR cells was found. Both protein kinase C inhibitors studied (staurosporine and CGP 41251) exhibited variable effects on cell surface antigen (HLA, ICAM-1, CD59) expression, suggesting complex interactions between PKC-dependent and -independent mechanisms in the regulation of surface antigen expression in these cell lines. Staurosporine differed from CGP 41251 in the cell cycle alterations induced in the HL-60 cell lines examined. Staurosporine induced the accumulation of cells in the G2/M phase of the cell cycle and the appearance of pre-G0 (apoptotic) cells in both examined drug-resistant cell lines. Staurosporine induced the appearance of cells with high DNA content in HL-60/ADR, but not in HL-60/VCR cells.

The protein kinase C inhibitor H7 blocks phosphorylation of stathmin during TPA-induced growth inhibition of human pre-B leukemia REH6 cells.

The human pre-B acute lymphoblastic leukemia cell line REH6 was used to analyze the regulation of a ubiquitous intracellular phosphoprotein stathmin (Mr 19,000, pl = 5.6-6.2). We demonstrated by 32P-labeling that the short (1 h) treatment of the REH6 cells with the tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), resulted in a rapid phosphorylation of at least three (P1, P2 and P3) stathmin isoforms without an alteration of stathmin isoform expression. Furthermore, Western blot analysis with specific antiserum showed that the prolonged period (48 h) of TPA treatment partially reduced protein levels particularly of two (N2 and P2) stathmin isoforms. The potent and relatively specific protein kinase C (PKC) inhibitor, 1,(5-isoquinolinesulphonyl)2methylpiperasine dihydrochloride (H7), partially inhibited these TPA effects, whereas the specific calmodulin inhibitor R24571 (calmidazolium) had no effect upon these events. Our findings suggest that stathmin phosphorylation in REH6 cells could be in part mediated by PKC activation.

Expression and alterations of CD66 antigen by interferon-gamma in human breast carcinoma cell lines.

Monoclonal antibodies of the CD66/67 panel from the 5th Workshop on Leukocyte Antigens bound (as determined by flow cytometry) to the cell surface of human breast carcinoma cell lines BT-20 and MDA-MB-468. The molecular weight of antigenic polypeptides recognized by the CD66 monoclonal antibody F34-187 differed between the two examined breast carcinoma lines as follows: 50kDa, 95 kDa and 130 kDa polypeptides were expressed on both BT-20 and MCF-7 cell lines, while the major 160-180 kDa polypeptide was found only in MDA-MB-468 cells. IFN-gamma, all-trans retinoic acid and phorbol ester (TPA) induced up-regulation of CD66 antigen(s) recognized by the monoclonal antibody F34-187 (as determined by flow cytometry). Different alterations of CD66 antigenic polypeptides recognized by F34-187 monoclonal antibody, induced by interferon-gamma and indentified by immunoblotting, were found on BT-20, MCF-7 and MDA-MB-468 breast carcinoma cell lines.

PSC 833 induces apoptosis in drug-sensitive human leukemia cell line and modulates resistance to paclitaxel in its multidrug-resistant variant.

BACKGROUND: The non-immunosuppressive cyclosporine analog PSC 833 has been shown to reverse multidrug-resistance of neoplastic cells including the MDR-1 gene coded P-glycoprotein (P-gp)-mediated cells resistant to paclitaxel. MATERIALS AND METHODS: Apoptosis was demonstrated in drug-sensitive HL-60 and multidrug-resistant human promyelocytic leukemia HL-60/ADR (MRP) and HL-60/VCR (MDR-1) cells in vitro with the aid of flow cytometry, DNA analysis and western blotting. RESULTS: The techniques used herein determined accumulation of paclitaxel/PSC 833 induced apoptotic cells with sub-G0 (hypodiploid) DNA content and blocked in the G2/M phase of the cell cycle, internucleosomal DNA fragmentation, poly (ADP-ribose) polymerase cleavage, Bcl-2 modulation and Bax up-regulation, without any significant alterations in the levels of Bcl-xL, CD95/Fas or Fas-L proteins. CONCLUSION: Drug resistance modulator PSC 833 abolished the P-gp-mediated multidrug-resistance to paclitaxel and paclitaxel-induced apoptosis in human myeloid leukemia (HL-60/VCR) cells in vitro. Furthermore, PSC 833 alone induced apoptosis in parental drug-sensitive leukemia cells, but not in both multidrug-resistant sublines studied.

Resistance of human multidrug-resistant neoplastic cell lines to paclitaxel-induced-radiosensitization is reduced by the non-immunosuppressive cyclosporine analog SDZ PSC 833.

The non-immunosuppressive cyclosporine analog SDZ PSC 833 abolished the resistance of human multidrug resistant (MDR-1, P-gp) human promyelocyte leukemia HL-60/VCR cells in vitro to paclitaxel-induced cell cycle- and viability alterations, as well as resistance to paclitaxel-induced radiosensitization. Furthermore, SDZ PSC 833 abolished also the resistance of human multidrug-resistant ovarian A2780/ADR cells to paclitaxel-induced cell cycle alterations and reduced its resistance to paclitaxel-induced radiosensitization. In these multidrug-resistant ovarian carcinoma cells the supra-additive interaction between paclitaxel and SDZ PSC 833 pre-exposure and subsequent irradiation appeared at slightly higher paclitaxel concentrations (40-100 nM) compared to those required for a similar interaction in the parental drug sensitive A2780 cells (40-80 nM paclitaxel).

Fluorescent double labeling of normal and malignant hematopoietic cells by monoclonal antibodies (FITC) and anthracycline cytostatic drug (Daunomycin): a cytometric technique for analysis of drug uptake in hematopoietic cell subpopulations.

A technique of simultaneous double labeling of normal and neoplastic hematopoietic cells with FITC-conjugated monoclonal antibodies directed to selectively expressed hematopoietic cell surface antigens (green fluorescence) and the anthracycline cytostatic drug (Daunomycin, red fluorescence) was described. Flow cytometric analysis of double labeled cells permitted anthracycline cell content determination in peripheral blood lymphocytes, granulocytes, monocytes from healthy donors, T- (MOLT-4), non-T, non-B (REH) and myelomonocytic (U-937) leukemic cell lines. After mixing peripheral blood lymphocytes from healthy individuals with cultured leukemic cells labeled on a restrictively expressed hematopoietic cell differentiation antigen (CALLA-CD10-, MHC class II-DR-antigen, a myelomonocytic differentiation antigen) detected by corresponding monoclonal antibodies (DGH-10-1-A9,Bra30, BraC8), the described technique allowed separate measurements of anthracycline content in leukemic cells vs. peripheral blood lymphocytes from healthy donors. Potential diagnostic aspects and research utilization of this technique are discussed.

Hematopoietic cell differentiation antigens (CD system 1997). Cancer research relevance.

The 6th International Workshop on Leukocyte Antigens (white cell differentiation antigens) continued the international cooperative effort aimed at characterization of all leukocyte cell surface antigens with respect to their biochemical properties, cell- and cell line expression, molecular and cellular function(s) and eventual disease relevance. Significantly, among 36 newly defined CD clusters identified with the aid of numerous monoclonal antibodies submitted to the workshop [17], 8 new clusters belonged to the endothelial section, 6 new CD clusters were identified within cytokine receptor section and 5 such clusters were characterized in the adhesion structure section, i.e. as antigens with the pattern of expression also on cells, tissues and cell lines outside the hematopoietic system (Tables 1-3). Minority of newly defined clusters appurtened to lineage-specific or non-lineage hematopoietic differentiation antigens (Table 4), i.e. 5 new CD clusters within myeloid section, 3 new clusters within both non-lineage and NK antigens, 2 T-cell antigens and one new CD cluster defined within both B-cell or platelet sections.

A simple technique for cell surface radioactive labeling of human and animal neoplastic cells: reductive methylation with formaldehyde and tritiated borohydride.

Avian sarcoma virus-, or 3,4-benzopyrene-transformed cultured rat cells and human leukemia or lymphoblastoid cell lines were radiolabeled by reductive methylation with formaldehyde and tritiated sodium borohydride--an application of a known technique for radiolabeling of soluble proteins. Optimal conditions for tritium incorporation into cell proteins with the aid of this technique were ascertained. Analysis of cell proteins tritium radiolabeled with the aid of this technique by acrylamide electrophoresis or by two-dimensional electrophoretic analysis allowed to disclose typical transformation-associated alterations in oncovirus-, or chemical carcinogen-transformed cells, as well as cell type-associated protein patterns in examined lymphoid cell lines. An individual protein (class II MHC antigen) radiolabeled by this technique has been identified as bimolecular complex p30,35 by immunoprecipitation with a monoclonal antibody recognizing this antigen; electrophoretic properties of immunoprecipitated antigen were identical to those observed after immunoprecipitation of the same antigen radiolabeled by sodium periodate/tritiated borohydride glycoprotein radiolabeling.

Differentiation of human myeloid leukemia cell lines induced by tumor-promoting phorbol ester (TPA). II. Electrophoretic patterns of metabolically- and cell surface radiolabeled proteins and glycoproteins.

Electrophoretic patterns of cell surface proteins and cell surface sialogalactoproteins from human myeloid leukemia cell lines HL-60, ML-1, ML-2 and ML-3 before and after induction of morphological differentiation by 12-O-tetradecanoylphorbol-13-acetate (TPA), have been compared. Minor quantitative cell surface protein alterations associated with TPA-induced differentiation have been observed as follows: decrease of a 150-160k glycoprotein on some TPA-induced myeloid cell lines, increase of a 50k glycoprotein and alteration of a 95k glycoprotein associated with the induction of differentiation by TPA. Electrophoretic patterns of 35S-methionine metabolically radiolabeled cell proteins revealed minor but distinct quantitative differences in several proteins, such as increased labeling of 180k, 160k and 130k proteins and decreased expression of 83k, 76k and 63k proteins on TPA-induced HL-60 cells. Two-dimensional electrophoretic analysis of 35S-methionine metabolically radiolabeled cell proteins revealed at least 17 proteins more intensively labeled and 13 proteins with decreased intensity of labeling on TPA-induced HL-60 cells compared to uninduced HL-60 cells.

Cell surface alterations of avian sarcoma virus B77- and 3,4-benzo(a)pyrene-transformed rat fibroblasts. I. Cell surface proteins characterized by two-dimensional electrophoresis.

Plasma membrane proteins of avian sarcoma virus B77-, 3,4-benzo(a)pyrene- and spontaneously transformed rat fibroblasts were metabolically- and cell surface radiolabeled and analyzed by electrophoresis under denaturing conditions (SDS-PAGE) or by two-dimensional electrophoresis. Most extensive series of protein alterations on transformed cells was detected by two-dimensional electrophoresis of cell surface proteins radiolabeled by lactoperoxidase catalyzed radioiodination: a fibronectin-like 220k protein with two isoelectric forms (pI 5.9 and 6.2-6.6), a 180k protein of pI 6.2-6.6 and two proteins of 48k and 50k with pI approximately 5.4-all decreased on examined transformed cells. In addition to these proteins, a series of proteins more markedly detectable on transformed cells was found: a 110-120k (pI 5.6) protein found particularly on 3,4-benzo(a)pyrene- and spontaneously transformed cells, a series of proteins in the region of molecular weights 46-54k with pI 5.5-6.0, two proteins of 35k (pI 6.2 and 6.4) and two further, basic proteins of 38k (pI approximately 7.2 and 7.8). Some of these changes were visualized also by further techniques for radiolabeling of proteins, i.e. reductive methylation of cell surface proteins, metaboling radiolabeling by 3H-leucine followed by plasma membrane isolation, as well as by protein staining after two-dimensional electrophoresis of isolated plasma membrane proteins. A 70k and a 120k radioiodinated proteins were immunoprecipitated from all transformed cells and, to a markedly lesser extent from untransformed fibroblasts by the immune serum against rat type-C endogenous virus. A similar 70k protein was immunoprecipitated from avian sarcoma virus B77-transformed cells also by the antiserum against Friend murine leukemia virus envelope glycoprotein gp70.

Cell surface alterations of avian sarcoma virus B77-and 3,4-benzo(a)pyrene-transformed rat fibroblasts. II. Cell surface glycoproteins characterized by two-dimensional electrophoresis.

Cell surface proteins of avian sarcoma virus B77-, 3,4-benzo(a)pyrene- and spontaneously transformed rat fibroblasts were tritium-radiolabeled by mild periodate oxidation followed by tritiated sodium borohydride reduction and by galactoseoxidase/NaB3H4 technique. Radiolabeled cell surface sialoglycoproteins were analyzed by electrophoresis under denaturing conditions (SDS-PAGE), or by two-dimensional electrophoresis (isoelectric focusing, SDS-PAGE). Following major glycoproteins were quantitatively decreased on all examined transformed cells: a 220k glycoprotein with pI of approximately 6.2, a 180k glycoprotein with pI of approximately 6.0-6.2, a 110k glycoprotein (pI 6.2). A series of further sialoglycoproteins was quantitatively increased on all examined transformed cells, as follows: a markedly increased 70k glycoprotein (pI approximately 4.8) and a 120k glycoprotein (pI 5.0-5.2) increased more markedly on onco-virus-transformed and spontaneously transformed cells. Further sialogalactoprotein (28k, pI 5.8) was visualized as increased on all examined transformed cells only by galactoseoxidase/NaB3H4 technique. A 70k sialoglycoprotein was immunoprecipitated from transformed cells by the immune serum against Friend murine leukemia virus envelop glycoprotein gp70 and, to a markedly lesser extent, from untransformed cells. A similar glycoprotein, together with an another 120k glycoprotein were precipitated from transformed cells and to a minor extent from untransformed cells also by an antiserum against endogenous rat type-C virus.

Calf serum- and cell surface proteins released from cultured avian sarcoma virus-transformed and untransformed rat fibroblasts.

Cell surface and calf serum proteins were released in vitro from cultured virus-transformed, chemically transformed and normal rat embryo fibroblasts in 0.2 M urea containing serum-free culture medium. Fibronectin (LETS protein) was found in considerably higher amount in medium from normal rat embryo fibroblasts than in that from transformed cells. Major calf serum protein released from cultured normal and transformed rat cells corresponded by its electrophoretic mobility to calf serum albumin. No substantial differences in electrophoretic patterns of calf serum proteins released from normal and transformed cells into the medium were found. Time course of release of serum proteins from cell surface was measured quantitatively with the aid of radioiodinated Staphylococcus aureus protein A radioimmune assay. More than 50% of cell bound calf serum proteins remained on cell surface after 24 hours of incubation in serum-free medium.

Phorbol ester-induced modulation of cell surface antigens on U-937 cells in protein-free medium: effect of protein kinase and calmodulin inhibitors.

Phorbol ester (PMA) induced decrease in cell surface expression of CD4 antigen was potentiated on human monoblastoid cell line U-937 cultured and induced in protein-free culture medium. Down-regulation of the CD4 antigen was partially inhibited by the isoquinoline protein kinase inhibitor H7 and unaffected by the calmodulin inhibitor R24571 (calmidazolium). PMA-induced increase of the monocyte-associated CD14 antigen was dependent the presence of fetal bovine serum (FBS) in culture medium and partially abolished by calmodulin inhibitor R24571. H7 inhibitor partially abrogated this up-regulation in 4 out of 8 induction experiments. PMA-induced down-regulation of MHC class I antigen was found in both FBS-supplemented and protein-free culture media. This effect was partially inhibited by both H7 and R24571 inhibitors.

Complement-fixing soluble antigen from rat lymphoma (C58NT)D. Preliminary characterization of the antigen and some positive human sera.

A soluble complement-fixing antigen prepared from the Gross virus-induced rat lymphoma (C58NT)D reacted in complement-fixation with the sera of some patients with different malignancies (particularly acute leukemia) and with the sera of some healthy individuals. Results of gel filtration of crude (C58NT)D antigen on Sephadex G 200 indicate that the antigenic activity is eluted with the void volume of the column, i. e. in fractions corresponding to proteins of low molecular weight. Gel filtration of some positive patient sera indicate that complement-fixing activity of tested sera is associated with fractions corresponding to IgM and/or IgG.

Cell surface protein and glycoprotein electrophoretic patterns in some human hemopoietic malignancies.

Surface proteins radiolabeled by lactoperoxidase catalyzed radioiodination and/or sialoglycoproteins tritium-labeled by sodium metaperiodate/NaB3H4 technique, obtained from neoplastic cells of more than 50 patients with malignant hemopoietic disease have been analyzed with acrylamide gradient gel electrophoresis under denaturing conditions (SDS-PAGE). Electrophoretic protein and glycoprotein patterns were essentially similar within the group of examined chronic lymphocytic leukemia (CLL) patients with major surface glycoproteins gp44 and gp29,35. Glycoprotein gp29,35 corresponds by its electrophoretic mobility to Ia-like (HLA-DR antigen), which was identified on some CLL cells also by immunoprecipitation with a conventional anti-Ia antigen serum and subsequent electrophoretic analysis of immuno-precipitated antigen. Electrophoretic patterns of cell surface proteins and glycoproteins of CLL cells were markedly different from those of acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cells which possessed a characteristic major cell surface glycoprotein gp95 (with relative molecular weight of 95-105k). This glycoprotein was markedly decreased or absent on examined CLL cells. Electrophoretic glycoprotein patterns revealed variations in the expression of several glycoproteins, as well as in the electrophoretic mobility of major glycoprotein gp95 also within the examined groups, particularly in acute myeloblastic leukemia.

Electrophoretic analysis of radiolabeled cell surface proteins and glycoproteins of some human hemopoietic cell lines.

Cell surface proteins and glycoproteins of several human lymphoblastoid- and neoplastic hemopoietic cell lines were radiolabeled by lactoperoxidase catalysed iodination and by sodium periodate/tritiated sodium borohydride. Electrophoretic patterns of radiolabeled proteins and glycoproteins obtained by electrophoresis under denaturing conditions (SDS-PAGE) were essentially similar for all examined lymphoblastoid cell lines, with a characteristic group of major radiolabeled glycoproteins gp44 and 24,31. Characteristic, individually different and distinguishable patterns of radiolabeled proteins were observed in different neoplastic hemopoietic cell lines: T-leukemia cell line MOLT 3, erythroleukemic cell line K562, pre-B cell leukemia line NALM 6, and a promyelocytic leukemia cell line HL-60. Cell surface proteins of HL-60 promyelocytic leukemia cell line displayed some similarities to those of another myeloid leukemia cell line ML 3. Examined Burkitt lymphoma cell lines were similar to lymphoblastoid cell lines with some minor differences. Glycoprotein gp44, markedly labeled on lymphoblastoid cell lines, was absent on Burkitt lymphoma cell line Daudi. Electrophoretic patterns of cell surface proteins of blast cells from a few patients with leukemia examined simultaneously with the cell lines are described and discussed.

Two-dimensional analysis of metabolically and cell surface radiolabeled proteins of some human lymphoid and myeloid leukemia cell lines. I. 35S-methionine labeled, lactoperoxidase radioiodinated and 3H-reductively methylated proteins.

Cell surface and cytoplasmic polypeptides of some human hemopoietic cell lines were radiolabeled by three different techniques: 35S-methionine metabolic radiolabeling, lactoperoxidase catalyzed radioiodination and reductive methylation with formaldehyde and tritiated sodium borohydride. Two-dimensional patterns of 35S-methionine labeled proteins revealed more than 500 separated protein spots with essentially similar general aspects of all examined cell lines, sharing major polypeptides such as those with electrophoretic mobilities of major cytoskeletal proteins. 125I-lactoperoxidase radio-iodinated cell surface protein patterns contained about 30 separated protein macromolecules with some marked differences between lymphoid versus myeloid lines and also between individual cell lines. Two-dimensional patterns of tritiated membrane polypeptides consisted of approximately 200 separated protein spots, some prominent of them appeared with electrophoretic mobilities of major cytoskeletal proteins being common to both 35S-methionine and reductive methylation patterns. Two-dimensional patterns of 35S-methionine labeled and cell surface radioiodinated proteins immunoprecipitated by monoclonal antibodies Bra30 and HL39, recognizing MHC class II antigens, appeared as bimolecular complex of larger acidic and smaller basic chains structurally corresponding to the expected two-dimensional patterns of MHC class II antigens.

Two-dimensional analysis of metabolically and cell surface radiolabeled proteins of some human lymphoid and myeloid leukemia cell lines. II. Glycosylated and phosphorylated proteins.

Cell surface glycoproteins, radiolabeled by sodium metaperiodate/tritiated borohydride technique and cell phosphoproteins, metabolically radiolabeled by 32P-orthophosphate were analyzed by two-dimensional electrophoretic analysis in some myeloid and lymphoid leukemia cell lines. Some markedly expressed major glycoproteins were predominant in some of cell lines (such as 95k and 100k glycoproteins with marked charge heterogeneity in non-T, non-B acute lymphoblastic leukemia cell lines NALM 6 and NALM 16), but markedly quantitatively reduced in other examined cell line, such as lymphoblastoid cell line UHKT 34/2. 32P-orthophosphate radiolabeled phosphoprotein two-dimensional patterns of examined lymphoid leukemia cell lines were essentially similar, with some minor differences, in examined lymphoid and myeloid leukemia cell lines, such as marked expression of a series of large phosphoproteins in the molecular weight range 80-100k in lymphoid cell lines and almost complete absence of these phosphoproteins on examined myeloid leukemia cell lines. Another configuration of acidic phosphoproteins (30-35k) exhibited individual cell line variability and differences between both individual myeloid leukemia cell lines and between lymphoid and myeloid cel lines examined.