Altered DNA-cleavage activity of topoisomerase II from WEHI-3B leukemia cells with specific resistance to ciprofloxacin.

In order to investigate the mechanisms of drug resistance arising in tumor cells, we investigated the capacity of fluoroquinolones to inhibit the in vitro growth of WEHI-3B monomyelocytic leukemia cells and then we established a variant of this line (currently maintained in the absence of drug). The line, named WEHI-3B/CPX, expresses a specific resistance to ciprofloxacin (CPX; resistance index=17.3+/-2.2), and does not show cross-resistance with other fluoroquinolones, camptothecin and topoisomerase II inhibitors such as doxorubicin, etoposide and teniposide. Although a little decrease in intracellular accumulation of CPX is observed in WEHI-3B/CPX cells, these cells do not express MDR or LRP markers, and the resistance is not circumvented by verapamil. Purified nuclear extracts from WEHI-3B and WEHI-3B/CPX cells were tested for topoisomerase I catalytic activity and checking in vitro topoisomerase I sensitivity to CPX and camptothecin inhibition, but no difference was observed. As the treatment with CPX showed that the resistant cell line suffers a significantly lower number of breaks in the DNA molecule we also addressed our investigations to the topoisomerase II-dependent DNA cleavage that, in the resistant clone, was found dramatically less susceptible to be enhanced by CPX both in pre-strand and post-strand DNA passage conditions. WEHI-3B/CPX cells do not express any character of multidrug resistance and represent a rare case of specific drug resistance to CPX. The specific resistance to CPX observed in these cells is related to a functional decrease of topoisomerase II cleavage activity. It could be consequent to a decreased binding affinity of CPX for the topoisomerase II--DNA complex or to a decreased affinity or specificity of topoisomerase II for its DNA cleavage sites.

Role of SR-4987 stromal cells in the modulation of doxorubicin toxicity to in vitro granulocyte-macrophage progenitors (CFU-GM).

Bone marrow stromal microenvironment is essential for the maintenance of the hematopoietic stem cell renewal both by cell-cell interaction and cytokine production. However, stromal cells also exhibit drug metabolizing activities and they may accumulate the drug and successively affect hematopoietic progenitors by a retarded release. Our study investigated the role of both primary culture of murine bone marrow stroma and established stromal cells (SR-4987) in modulating the "in vitro" toxic activity of Doxorubicin (DXR) against murine granulocyte-macrophage progenitors (CFU-GM). The main part of the study has been performed by a "in vitro" agar bilayer technique based on the CFU-GM assay performed over a feederlayer of stromal cells. The results suggest that bone marrow stromal cells play also an important role in decreasing the toxicity of Doxorubicin. Further SR-4987 stromal cells produce a Doxorubicin metabolite (not belonging to the series of metabolites described in literature) which is completely ineffective in inhibiting the growth of CFU-GM and the activity of topoisomerase I. Our data suggest that bone marrow stromal cells must be considered as a cell population having opposite pharmacological roles in modulating the drug toxicity on hematopoietic progenitors. In our model a mechanism of detoxification concerns the capacity of SR-4987 stromal cells to inactivate the drug. For a better prediction of drug hematotoxicity, it is very important to develop "in vitro" cell models able to discriminate between positive and negative modulation of drug toxicity that stromal cells can exert in the bone marrow microenvironment.

SR4987 and L1210 cell lines: two models in which cholera toxin susceptibility does not correlate with cAMP accumulation and ganglioside content.

The CT-mediated signaling mechanisms have been widely used as a tool for helping the knowledge of the more complex mechanisms regulating cell growth and proliferation in which gangliosides are involved as receptors and cAMP as second messenger. In the present study we compare the susceptibility of two murine cell lines (SR-4987 stromal cells and L1210 leukemic cells) to inhibitory effect of cholera toxin (CT) on cell growth and correlate their sensitivity to CT with ganglioside content and intracellular cAMP accumulation. The results indicate a very different response of the two cell lines to CT treatment. L1210 cells (which contain GM1a ganglioside) are sensitive to the inhibiting activity of CT (IC50 in the clonogenic assay = 10(-9) M) but no cAMP accumulation was observed after the treatment. SR-4987 cells (which lack GM1a) show a dramatic increase of intracellular cAMP without any inhibition of cell growth following the CT treatment until 10(-8) M. However, after SR4987 cells have incorporated GM1a they became susceptible to CT (with a IC50 value = 10(-11) M). The comparison of these results with our previous studies on WEHI-3B leukemia cells confirms the remarkable heterogeneity of cell sensitivity to the growth inhibition by CT by emphasizing that this inhibition is the final event of very different mechanisms in which CT binding to a specific ganglioside seems to be necessary and sufficient whereas cAMP accumulation may not be coupled with the antiproliferative effect of CT.

Expression of B cell markers on SR-4987 cells derived from murine bone marrow stroma.

The murine cell line SR-4987 was originated in our laboratory from adherent cells of a long term bone marrow culture. SR-4987 cells do not express p21-ras and c-fms products on membrane whereas secrete M-CSF, evidence a fibroblast-like morphology and are vimentine positive. This line shows a very poor "in vitro" agar clonogenicity which is not modulated by the addition of different cytokines and growth factors (M-CSF, GM-CSF, G-CSF, IL-3, IL-7, alpha-TNF, PDGF, and EGF). On the contrary, a dramatic increase in clonogenicity is observed in the presence of bFGF. The RT-PCR investigation evidences the mRNA encoding for bFGF, IL-7, GM-CSF, and SCF (c-kit ligand). The analysis of CD antigen expression on SR-4987 cell membrane indicates a phenotype (CD5+, CD44+, 45R(B220)+, sIg+, 5'-nucleotidase+) that is consistent with a B cell feature. Our observations suggest that exogenous bFGF might represent an appropriate stimulus for inducing the SR-4987 cells proliferation also in the absence of cell-substrate anchorage. Further, they indicate that SR-4987 cells could represent a particular differentiation stage in which characters of "stromal cell" and "B cell" are coexpressed in agreement with the hypothesis of a common stromal-hematopoietic differentiation.

The different inhibiting effect of cholera toxin on two leukemia cell lines does not correlate with their toxin binding capacity.

The murine leukemia cell lines L1210 and WEHI-3B show a very different sensitivity to the cholera toxin (CT). The in vitro growth of L1210 is completely inhibited by 10(-8) M CT, while WEHI-3B growth shows the same inhibition at 10(-11) M. The analysis of membrane ganglioside pattern of the two cell lines shows that in L1210 cells the major component is the GM1a ganglioside while the monosialogangl oside fraction from WEHI-3B is entirely composed of gangliosides of the 'b' series among which GM1b is the more represented. The total cholera toxin binding capacity of the ganglioside extract from L1210 cells is more than hundred fold higher than that of WEHI-3B and this difference is also confirmed by the number of CT receptors/cell and by the binding of FITC-B subunit of CT on the cells. These surprising data are in conflict with the poor sensitivity to CT evidenced by L1210 compared to WEHI-3B cells. In order to clarify this discrepancy we investigated the cAMP accumulation, the cell viability and the clonogenicity of these two leukemia cell lines following the treatment with CT and forskolin (FRSK). The treatment of WEHI-3B cells with CT induces a dramatic increase of intracellular cAMP which highly correlates with cell death and the decrease of clonogenicity and this result is partially obtained by the treatment with FRSK. L1210 cells do not evidence significant cAMP accumulation neither with CT nor with FRSK treatment. These data suggest that the different inhibiting effect of CT on WEHI-3B and L1210 cells does not correlate with their different pattern of gangliosides and the related toxin binding capacity. Further they indicate that the growth inhibition of WEHI-3B cells is closely related with a cAMP-dependent cell killing mechanism, while the inhibition of L1210 growth (produced by high concentration of CT) is mediated by a cAMP independent mechanism.

In vitro short-term and long-term cytotoxicity of fluoroquinolones on murine cell lines.

Short-term and long-term cytotoxicity of four fluoroquinolones (ciprofloxacin, rufloxacin, ofloxacin, lomefloxacin) on 7 established murine cell lines (WEHI-3B, L1210, EL4, P388D1, 32DC13, L929, SR-4987) by a microtiter MTT assay have been studied. In short-term cytotoxic test (24 hr), cell lines with a high proliferating cell rate (as leukemias) showed a greater sensitivity to quinolones than other cell lines. In long-term cytotoxic test (7 days) no different sensitivity was observed among the cell lines. In short-term test ciprofloxacin and rufloxacin were more toxic than lomefloxacin and ofloxacin whereas in the long-term test the activity of the four quinolones was similar. Ratio between IC50 on cell lines and MIC50 against gram negative bacteria evidenced remarkable differences when short-term or long-term cytotoxic tests were considered. The results confirm toxic activity of quinolones on mammalian cells evidencing that the sensitivity to quinolones, in short-term cytotoxic test, correlates with the doubling time of cell population. The results further suggest that long-term cytotoxic tests measure better the antiproliferating activity of quinolones providing a more powerful assay to investigate their in vitro toxicity.

Establishment and characterization of a new murine cell line (SR-4987) derived from marrow stromal cells.

A new murine cell line designated as SR-4987 was established by treating a long-term bone marrow culture with the supernatant from Y-1 cells which actively produce viral C-particles (MuLV). The line showed a fibroblast-like morphology and its mesodermal origin was confirmed by immunocytochemical staining. Flow cytometric analysis of DNA index evidenced a tetraploid number of chromosomes whereas cell cycle analysis showed 34.8% of cells in S phase and 60.7% in G1. In vitro growth studies demonstrated a population doubling time of 14.7 h, a good plating efficiency (52.3%) and a very poor agar clonogenic capacity (0.6%). SR-4987 was tumorigenic only in syngeneic mice in which sarcomas were induced. The line produced M-CSF in the culture supernatant whereas G-CSF, IL-3 and GM-CSF were not detected. Studies are in progress to assess the production of other cytokines and to verify if same autocrine growth factor is involved in the control of SR-4987 proliferation. Our line provides a further model of stromal cells for studying the interaction between hemopoietic progenitors and their microenvironment, as well as to study factors produced by stromal cells acting as modulators of proliferation and differentiation of related cell populations.

Inhibition of murine leukemia (WEHI-3B and L1210) proliferation by cholera toxin B subunit.

Cholera holotoxin produces both stimulation and inhibition of the growth of different cell populations. These opposite effects were both attributed to the enzymatic activity of the subunit A that activates adenylate cyclase, increasing the intracellular level of cAMP. We observed that the B subunit of cholera toxin produced by itself an inhibition of the 'in vitro' growth of two murine leukemia cell lines (L1210 limphoid leukemia and WEHI-3B myelomonocytic leukemia). The sensitivity of WEHI-3B cells towards cholera toxin was about 5000-times higher than that of the L1210 cells, whereas the two leukemias showed an identical sensitivity to the B subunit (IC50 = 5.10(-10) M for L1210 and 10(-10) M for WEHI-3B). The inhibition produced by the B subunit was neutralized by GM1 and in a minor degree by type II gangliosides. The two leukemias showed a remarkable difference in their gangliosides contents (L1210 cells contained GM1 (80.6%) and GM2 (19.4%), while WEHI-3B cells contained GM1 (28.2%), Fuc-GM1 (44.9%) and a band (26.9%) with a chromatographic mobility between GD1a and GD1b). The inhibition could be explained by a competitive mechanism between the B subunit and some autocrine factor binding GM1-containing receptors. Our data strengthen the suggestion to consider gangliosides as very important pleiotropic biomodulators.

Effect of fluoroquinolones on the in-vitro proliferation of myeloid precursor cells.

The effects of three fluoroquinolones (ofloxacin, pefloxacin and rufloxacin) on the in-vitro proliferation of murine myeloid cells were studied. Their activity was compared with that of nalidixic acid and novobiocin. Therapeutic concentrations of quinolones do not affect the physiological course of myelopoiesis. Only very high concentrations of drug (greater than 70 mg/l) affect bone marrow cell growth producing a dose-dependent inhibition. Because quinolones are active on topoisomerase II from eukaryotic cells they can modulate mammalian cell growth.