Cytotoxic and DNA-inhibitory effects of iron chelators on human leukaemic cell lines

Cytotoxic and DNA synthesis inhibitory effects of several iron chelators with different physicochemical properties have been tested in four myeloid leukaemic cell lines (U937, K562, ML2 and HL60). The small lipophilic chelators 8-hydroxyquinoline, tropolone and omadine at 2 X 10(-5) M, caused substantial inhibition of labelled leucine and thymidine uptake into cells and also cell death following 4-h incubation. These effects were approximately 10-fold increased when the drugs were pre-incubated with equimolar amounts of iron. Iron alone and hydrophilic chelators e.g. desferrioxamine had insignificant cytotoxic and DNA synthesis inhibitory effects under the same conditions.     

Characterisation of seven human ovarian tumour cell lines.

Characteristics of a panel of seven human ovarian tumour cell lines are presented. Positive staining with HMFG2 and ultrastructural identification of desmosomes confirmed the epithelial nature of the cell lines. The lines showed wide variations in ploidy, doubling times and clonogenicity in soft agar. Both vimentin and keratin were equally expressed in five lines, one line showed strong preferential expression of keratin and one line showed preferential expression of vimentin. Karyotypic changes associated with ovarian cancer were identified in all the lines. Four of the seven cell lines showed loss of chromosome material distal to 11p13-15. These cell lines offer considerable potential for research into the biology and genetics of ovarian cancer.     

Loss of epithelial markers and acquisition of vimentin expression in adriamycin- and vinblastine-resistant human breast cancer cell lines.

We have previously observed that breast cancer cell lines could exhibit either epithelial or fibroblastic phenotypes as reflected by their morphologies and intermediate filament protein expression (C. L. Sommers, D. Walker-Jones, S. E. Heckford, P. Worland, E. Valverius, R. Clark, M. Stampfer, and E. P. Gelmann, Cancer Res., 49:4258-4263, 1989). Fibroblastoid, vimentin-expressing breast cancer cell lines are more invasive in vitro and in vivo (E. W. Thompson, S. Paik, N. Brunner, C. L. Sommers, G. Zugmaier, R. Clarke, T. B. Shima, J. Torri, S. Donahue, M. E. Lippman, G. R. Martin, and R. B. Dickson, J. Cell. Physiol., 150: 534-544, 1992). We hypothesized that a breast cancer cell with an epithelial phenotype could undergo a transition to a fibroblastic phenotype, possibly resulting in more invasive capacity. We now show that two Adriamycin-resistant MCF-7 cell lines and a vinblastine-resistant ZR-75-B cell line have undergone such a transition. Adriamycin-resistant MCF-7 cells express vimentin, have diminished keratin 19 expression, have lost cell adhesion molecule uvomorulin expression, and have reduced formation of desmosomes and tight junctions as determined by reduced immunodetection of their components desmoplakins I and II and zonula occludens (ZO)-1. Other MCF-7 cell lines selected for resistance to vinblastine and to Adriamycin and verapamil did not have these characteristics, indicating that drug selection does not invariably cause these phenotypic changes. In addition, to determine if vimentin expression in MCF-7 cells alone could manifest a fibroblastic phenotype, we transfected the full-length human vimentin complementary DNA into MCF-7 cells. Although vimentin expression was achieved in MCF-7 cells, it did not affect the phenotype of the cells in terms of the distribution of keratins, desmoplakins I and II, ZO-1, or uvomorulin or in terms of in vitro invasiveness. We conclude that vimentin expression is a marker for a fibroblastic and invasive phenotype in breast cancer cells but does not by itself give rise to this phenotype.     

PTEN expression in PTEN-null leukaemic T cell lines leads to reduced proliferation via slowed cell cycle progression

The balance of activities between the proto-oncogene phosphoinositide 3-kinase (PI3K) and the tumour suppressor gene PTEN has been shown to affect cellular growth and proliferation, as well as tumorigenesis. Previously, PTEN expression in the PTEN-null Jurkat T cell leukaemia line was shown to cause reduced proliferation without cell cycle arrest. Here, we further these investigations by determining the basis for this phenomenon. By BrdU pulse-chase and cell cycle arrest and release assays, we find that PTEN expression reduced proliferation by slowing progression through all phases of the cell cycle. This was associated with reduced levels of cyclins A, B1 and B2, cdk4, and cdc25A and increased p27KIP1 expression. Apoptosis played no role in the antiproliferative effect of PTEN, since only marginal increases in the rate of apoptosis were detected upon PTEN expression, and inhibitors of effector caspases did not restore proliferative capacity. Active Akt blocked the antiproliferative effects of PTEN, indicating that PTEN mediates its effects through conventional PI3K-linked signalling pathways. Similar results were obtained from a different PTEN-null leukaemia T cell line, CEM. Together, these results show that PTEN expression in leukaemic T cells leads to reduced proliferation via an apoptosis-independent mechanism involving slower passage through the cell cycle.     

Characterisation of four merkel cell carcinoma adherent cell lines

We have previously described the establishment of a number of cell lines from Merkel cell carcinoma (MCC), also known as small cell cancer of the skin or neuroendocrine carcinoma of the skin. These cells, all of which grew as suspension cultures, were found to resemble small cell lung cancer (SCLC) lines types 1, 2 and 3 by their morphology and growth characteristics. We now report 4 more MCC cell lines which resemble the SCLC type 4 cell lines in that they grow as adherent monolayers. These MCC lines would belong to the variant subgroup as they no longer express most neuroendocrine markers, grow at low cell density and have population doubling times of 1–5 days in contrast to the MCC suspension lines which have doubling times of 6–12 days. MCC 14/1 and MCC 14/2 were established from the same metastatic node and would appear to represent 2 clones of the tumour which differ in morphology, histochemical markers and DNA content. We present details of the morphology, DNA content and immunohistochemistry of these 4 lines and com-pare their growth patterns with those of SCLC and MCC lines which grow in suspension. © 1995 Wiley-Liss, Inc.     

Characterisation of taurine uptake in human KB MDR and non-MDR tumour cell lines in culture

Studies on taurine transport in MDR (multidrug resistant) and non-MDR KB cancer cells show that both cell types contain Na(+)- and Cl(-)-dependent high-affinity and low-affinity transport systems selective for beta-amino acids and a taurine diffusion component. Good buffers, such as HEPES or MOPS, interfered with taurine uptake. The basal taurine Vmax measured in isoosmotic medium represented 1/5 of taurine captured by uptake in KB non-MDR, but was negligible in KB MDR cells. High-and low- affinity uptake systems were reduced by medium hyperosmolarity in both cell types. Although properties of low-and high-affinity transport systems were similar in both cell types, Vmax (but not Km) were reduced in MDR compared to non-MDR cells. Taurine uptake was unaffected by chemotherapeutic agents (doxorubicin, vinblastine) or MDR revertants (verapamil). Taurine did not affect cell proliferation of MDR or non-MDR cells nor did it alter the inhibitory effect of doxorubicin or vinblastine cell proliferation.     

Modulation of T leukaemic cell phenotype with phorbol ester

A panel of monoclonal antibodies and other markers (e.g., terminal deoxynucleotidyl transferase, sheep erythrocyte rosettes, peanut agglutinin) have been used in conjunction with flow cytometry and biochemical analysis to monitor the induction of maturation in human thymic (T) leukaemic cell lines by phorbol ester (TPA). Seven cell lines underwent multiple phenotypic alterations in response to TPA but were unresponsive to synthetic thymic hormones (TP5, FTS) or to other compounds (e.g. DMSO, retinoic acid) which induce maturation in other types of leukaemia. The changes parallel those observed in normal T-cell differentiation and partly reflect alterations in glycosyl transferase activity, altered synthesis of proteins and regulation of cell surface receptors (for transferrin) associated with rapid growth and metabolism. These studies further illustrate the reversibility of maturation arrest in human leukaemia and provide support for the view that leukaemia may involve regulatory defects in the coupling of proliferation and maturation. Induction or promotion of terminal differentiation in leukaemic equivalents of T-cell precursors may provide a convenient system for the study of biochemical and molecular events involved in T-cell development and diversification.     

Production of plasminogen activators by human T-cell leukaemia virus-transformed human T cell lines

Six human T cell lines HAMA, KUN, KAN, TCL-Haz, TCL-Ter, and TCL-Mor, which were transformed by a retrovirus, human T-cell leukaemia virus (HTLV), constitutively produced plasminogen activators (PAs) in culture supernatants. The amount of PAs produced varied among the cell lines. The PAs were distinguished by immunochemical analysis between two types: urokinase (UK)-type and non-UK-type. KUN, TCL-Ter, and HAMA mainly produced UK-type PA, whereas the other cell lines produced both types. Thus, HTLV-transformed T cell lines differ in the quality and quantity of the PAs they produce. The PAs in the culture supernatants of each cell line were separated into several mol. w forms on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results indicate that the same cell line produces PAs of different mol. wt. PA production by these cell lines was affected by treatment with phorbol miristate acetate, concanavalin A, and phytohaemagglutinin; the effects were substantially different in each cell line. The data described here indicate that HTLV-transformed T cell lines constitutively produce PAs which are very heterogeneous in both quality and quantity.     

Role of Smac in human leukaemic cell apoptosis and proliferation

Smac (or DIABLO) is a recently identified, novel proapoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac functions by eliminating the caspase-inhibitory properties of the inhibitors of apoptosis proteins (IAP), particularly XIAP. In this study, we stably transfected both full-length (FL) and mature (MT) Smac genes into the K562 and CEM leukaemic cell lines. Both FL and MT Smac transfectants increased the sensitivity of leukaemic cells to UV light-induced apoptosis and the activation of caspase-9 and caspase-3. Purified cytosol from the mature Smac transfectants, or the addition of human recombinant Smac protein or N-7 peptide into nontransfected cytosol, showed an increased sensitivity to cytochrome c-induced activation of caspase-3. The mature Smac enhanced the susceptibility of both K562 and CEM cells to TRAIL-induced apoptosis. Overexpression of the mature Smac protein also inhibited proliferation, as detected by reduced colony formation and Ki-67 expression in leukaemic cells. Cell cycle analysis revealed that Smac transfectants displayed significant G0/G1 arrest and reduction in 5-bromo-2'-deoxyuridine (BrdU) incorporation. Smac sensitized human acute myeloid leukaemia blasts to cytochrome c-induced activation of caspase-3. However, Smac failed to overcome Apaf-1-deficiency-mediated resistance to cytochrome c in primary leukaemic blasts. In summary, this study reveals that Smac/DIABLO exhibits a potential role in increasing apoptosis and suppressing proliferation in human leukaemic cells. Importantly, it also indicates that it is crucial to evaluate the levels of Apaf-1 and XIAP proteins in patient samples before using Smac peptide therapy in the treatment of human leukaemia.     

Characterisation of novel human lung carcinoma cell lines selected for resistance to anti-neoplastic analogues of staurosporine

The staurosporine analogues CGP 41251, UCN-01 and Ro 31-8220 are specific inhibitors of protein kinase C (PKC). CGP 41251 and UCN-01 exert anti-neoplastic activity against human tumours grown in rodents, and CGP 41251 reverses multidrug resistance. The hypothesis was tested that these agents can induce drug resistance and alter cellular levels of target kinases. Human-derived A549 lung carcinoma cells were exposed for 6 months to CGP 41251, UCN-01 or Ro 31-8220 at gradually increasing concentrations. Cells acquired resistance against these agents, 4.3-fold against CGP 41251 (A549/CGP cells), 4.0-fold against UCN-01 (A549/UCN cells) and 14-fold against Ro 31-8220 (A549/Ro cells). Cells were neither collaterally cross-resistant towards the PKC inhibitors nor resistant against the growth-inhibitory properties of 12-O-tetradecanoylphorbol-13-acetate. However, cross-resistance was observed in A549/CGP cells against staurosporine (13-fold) and in A549/Ro cells against doxorubicin (26-fold). All 3 cell types expressed multidrug resistance–associated protein, and A549/Ro cells expressed P-glycoprotein, as adjudged by Western blot analysis. Phorbol ester–stimulated PKC activity in these cells was decreased by between 57% and 96% compared to wild-type A549 cells. Levels of the PKC isoenzymes α and θ in all 3 resistant cell types and of PKC-ϵ in A549/UCN cells were concomitantly reduced. Cells regained drug sensitivity after culture in the absence of drug for 6 (A549/Ro cells), 5 (A549/CGP cells) and 1 (A549/UCN cells) months. Our results suggest the following features of this type of anti-signalling drug: (i) they can induce drug resistance, (ii) they may be potentially useful in combination because of the lack of cross-resistance between them and (iii) they can down-regulate PKC, which may have pharmacological or toxicological consequences. Int. J. Cancer 73:763–768, 1997. © 1997 Wiley-Liss, Inc.     

Characterisation of high-level cisplatin-resistant cell lines established from a human hepatoma cell line and human KB adenocarcinoma cells: cross-resistance and protein changes.

Human liver carcinoma cells (BEL-7404) and human KB adenocarcinoma cells were selected by stepwise increases in cisplatin. Drug sensitivity assays indicated that the IC50 value for 7404-CP7.5 cells was 49 micrograms ml-1 cisplatin, 111-fold higher than for the parental hepatoma cells. The IC50 value for KB-CP10 cells was 38 micrograms ml-1 cisplatin, which is 1152-fold higher than for the parental KB cells. The 7404-CP7.5 cells were cross-resistant to methotrexate (39 x), 5-fluorouracil (23 x) and 6-mercaptopurine (13 x), but were sensitive to drugs which are known substrates for the multidrug transporter (P-glycoprotein), including colchicine, vinblastine and actinomycin D. Similar cross-resistance patterns were observed for KB-CP10 cells. No evidence of DNA amplification or expression of the MDR1 gene was found. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed increases in 52 kDa protein(s) in both the soluble cytosolic and crude membrane fractions in 7404-CP(r) cells and in KB-CP(r) cells. The amount of 52 kDa protein was proportional to the degree of resistance of the 7404-CP(r) cells to cisplatin. Two-dimensional gel analysis demonstrated that two polypeptides of molecular mass 52 and 50 kDa were overexpressed in the membrane fractions in both 7404-CP20 and KB-CP20 cells. Using amino acid microsequencing and Western blotting, major 52 kDa protein was identified as the mitochondrial heat shock protein hsp60. Two-dimensional gels of [35S]methionine-labelled polypeptides showed many other changes, including reduction in soluble proteins of approximately 57 kDa molecular weight in KB-CP20 cells, and of 35 kDa in both 7404-CP20 and KB-CP20 cells. These results suggest that alterations of certain proteins occur commonly in cisplatin-resistant cells, particularly proteins of molecular weight 52 and 50 kDa.     

Galectin-1 gene expression and methylation state in human T leukemia cell lines

Galectin-1 has been demonstrated to be a mediator of T-cell apoptosis acting on activated T-cells and, in a selective manner, on different T leukemia cell lines. Here we show that the sensitivity to galectin-1 is associated with repression of the endogenous galectin-1 gene whereas non-sensitive cells express high levels of galectin-1. Repression of galectin-1 gene in sensitive cells is associated with hyper-methylation of the promoter region. Transient treatment of non-expressing cells with the demethylating agent 5-azacytidine led to irreversible demethylation and subsequent reactivation of galectin-1 gene.     

The schedule-dependent effects of etoposide in leukaemic cell lines: a function of concentration and duration

PURPOSE: Etoposide is a commonly used anticancer agent that is highly schedule-dependent. The in vitro activity of etoposide (0-10 microM) was investigated in a panel of leukaemic cell lines. METHODS: Cells were cultured with etoposide in drug schedules of equal exposure duration (ED, durationxconcentration), and the effects of drug exposure on cell parameters, including cell cycle distribution, were assessed over an 8-day period. RESULTS: Proliferation assays indicated a concentration- and duration-dependent induction of cell death by etoposide in CEM and HL60 cells, and flow cytometric analysis indicated that this cell kill was by apoptosis. Efficacy was also dependent upon schedule, with more cell kill seen in schedules of longer duration. As an example, accumulative percent cell kill resulting from a continuous exposure to 0.05 microM etoposide was significantly greater than that in cultures involving either a 4-day exposure to 0.1 microM or a single-day exposure to 0.4 microM etoposide (193.4+/-15.9% vs 125.2+/-5.4% vs 42.3+/-5.9%, respectively; P<0.001 in all cases; equi-ED 0.4 microM.days). Efficacy was also dependent upon the ED of the schedule. At very low concentrations, the initial enhancement of cytotoxicity mediated by increasing duration would gradually and paradoxically be lost in the more protracted schedules (e.g. accumulative percent cell kill 66.4+/-7.4%, 158.3+/-12.0% and 40.1+/-6.0% with 100 n M for 2 days, 33 n M for 6 days and 25 n M for 8 days, respectively; P<0.001 in all cases; equi-ED 0.2 microM x days). CONCLUSIONS: Our results confirm the schedule-dependency of etoposide in vitro, highlighting the importance of total duration of drug exposure in determining cytotoxicity, and emphasizing the requirement to achieve a cytotoxic concentration in longer exposures. It is therefore crucial to ensure that etoposide regimens used clinically involve doses that are effectively cytotoxic.     

Exposure to low concentrations of etoposide reduces the apoptotic capability of leukaemic cell lines.

The use of topoisomerase inhibitors has been associated with the development of secondary malignancies, suggesting that these agents can induce DNA damage that may be persistent. We have investigated the effect of short exposures (>3 days) to low etoposide concentrations (LC-etoposide, 0.01-0.04 microM) on the ability of leukaemic cells to initiate apoptosis. Results showed that although LC-etoposide had no effect on cell growth characteristics, the pre-culture of cells with LC-etoposide conferred resistance to subsequent exposure to cytotoxic concentrations of etoposide (0.3 microM etoposide in HL60 on day 3: %V: 95.2 +/- 1.6% vs 60.3 +/- 12.1% in control cells with no pre-culture, and %A: 5.1 +/- 0.2 vs 19.0 +/- 0.7%; P < 0.001). This effect was still observed 4 weeks after the initial drug exposure. Associated with these observations was a three-fold increase in genetic instability and a reduction in induced bax protein levels. The anti-cytotoxic effect was also shown to be specific to topoisomerase II (topo II) inhibitors, as the pre-culture of cells with a low doxorubicin concentration also induced resistance, while low cisplatin concentrations did not. The persistence of these alterations in cellular processes following an initial exposure to topo II inhibitors suggests a DNA-based mechanism, and highlights the existence of drug/target interactions even at very low drug concentrations.     

Surface glycoprotein patterns of normal and malignant human lymphoid cells. I. T cells T blasts and leukemic T cell lines.

Exposed surface glycoproteins of resting and in vitro activated human T lymphocytes and leukemic T-cell lines were labelled by the galactose oxidase-tritiated sodium borohydride method. The labelled glycoproteins were separated by polyacrylamide slab gel electrophoresis and visualized by autoradiography. The basic glycoprotein patterns of the T lymphocytes and T blasts were found also in the leukemic T cells. The glycoprotein pattern of the T-cell lines was easily distinguishable from that of other hematopoietic cell lines. The findings suggest that: (1) surface glycoprotein analysis might be useful for the identification of cell lines and for the differential diagnosis of hematopoietic malignancies; and (2) cells of cultured T lines may be used for the identification and preparation of T lymphoid differentiation antigens and perhaps also tumor-associated surface molecules.