Jean-Claude Jardillier

Distinctive alterations of invasiveness, drug resistance and cell–cell organization in 3D-cultures of MCF-7, a human breast cancer cell line, and its multidrug resistant variant

Growth of human tumor cells as three-dimensional (3D) multicellular spheroids modifies their invasive properties. Here we study the differences in the biological features of MCF-7, a human breast cancer cell line, and its multidrug resistant variant (MDR-MCF-7) cultured as spheroids or as monolayers. Three-dimensional culture decreased the proliferative rate of both cell lines, reduced the drug sensitivity of MCF-7 cells and did not affect the resistance of MDR-MCF-7 cells. Transmission electron microscopic studies and intercellular junctions labeling showed that MCF-7 spheroids had a junctional system involving E-cadherin, tight-junctions and desmosomes. In MDR-MCF-7 cell spheroids, cell cohesion was mostly due to membrane interdigitations. MDR-MCF-7 cells, but not their parental counterpart, displayed a higher invasive potential when cultured as spheroids, as shown in the Boyden chamber assay. 3D-induced invasiveness was correlated with serine protease and plasminogen activator (PA) secretion. MCF-7 cells did not show any tendency to invade, whatever the mode of culture. These results show that 3D-cultures as spheroids distinctively altered structural features of parental and MDR-MCF-7 cells. In MCF-7 cells, 3D-culture increased cell–cell contacts and drug resistance; in MDR-MCF-7 cells, it induced invasive properties.

The antileukemic alkaloid fagaronine is an inhibitor of DNA topoisomerases I and II

The antileukemic alkaloid, fagaronine, is a potent differentiation inducer of various hematopoietic cell lines. We show here that fagaronine is a DNA base-pair intercalator with a K(app) of 2.1 x 10(5) M-1 for calf thymus DNA. Fagaronine inhibits the catalytic activity of purified calf thymus topoisomerase I as shown by relaxation of supercoiled plasmid DNA followed by electrophoresis in neutral as well as in chloroquine-containing gels. The catalytic activity of topoisomerase I is inhibited at concentrations above 30 microM. Fagaronine also inhibits the catalytic activity of purified calf thymus topoisomerase II at concentrations above 25 microM as shown by decatenation of kinetoplast DNA. Fagaronine stabilizes the covalent DNA-enzyme reaction intermediate (the cleavable complex) between topoisomerase I and linear pBR322 DNA at concentrations up to 1 microM. Further increase of the fagaronine concentration leads to a progressive decrease in the cleavable complex formation, which is totally inhibited at 100 microM. In contrast, up to 1 microM fagaronine has no effect on cleavable complex formation between purified calf thymus topoisomerase II and linear pBR322 DNA, whereas cleavable complex formation is inhibited at higher concentrations. Exposure to fagaronine results in an increase in DNA-protein complex formation in intact P388 murine leukemia cells. P388CPT5 cells, which have an altered topoisomerase I activity, are 4-fold resistant to the growth inhibitory effects of fagaronine compared to the parental cell line. Similarly, DC-3F/9-OH-E Chinese hamster fibrosarcoma cells, which have an altered topoisomerase II activity, are about 5-fold resistant to the growth inhibitory effects of fagaronine. We conclude that fagaronine is an inhibitor of both DNA topoisomerase I and II and propose that this might play a role in the cytotoxic activity.

Evidence for distinct regulation processes in the aclacinomycin- and doxorubicin-mediated differentiation of human erythroleukemic cells☆

Human erythroleukemic K 562 cells were induced to were induced to differentiate along the erythroid lineage by anthracycline antitumor drugs, such as aclacinomycin (ACLA) and doxorubicin (DOX). Subsequent stimulation of heme and globin synthesis led to a differential quantitative expression of hemoglobins. Gower 1 (epsilon2, zeta2) was the major type for ACLA and X (epsilon2, gamma2) for DOX. Although ACLA and DOX increased both the expression of gamma-globin and porphobilinogen deaminase mRNAs, striking differences were observed in the expression of erythropoietin receptor mRNAs and in erythroid transcription factors GATA-1 and NF-E2, known to play a key role in erythroid gene regulation. Indeed, ACLA induces an increase either in the binding capacity of GATA-1 and NF-E2 or in the accumulation of erythropoietin receptor, GATA-1 and NF-E2 transcripts. In contrast, their expression with DOX was not significantly modified compared to uninduced cells, except for a slight decrease in NF-E2 expression on day 3. In conclusion, these data show that: 1. increased expression of erythroid transcription factors and erythroid genes are associated only with ACLA treatment, and 2. although cytotoxicity of both ACLA and DOX is certainly dependent on DNA intercalation, regulation of differentiation processes by these two drugs involves distinct mechanisms.

Piperazine derivatives of butyric acid as differentiating agents in human leukemic cells

Abstract Butyric acid is a potent antineoplastic agent with a well-documented differentiation activity on a wide variety of tumor cells. However, its clinical development is strongly limited by its very short metabolic half-life. In this study we report on the in vitro effects of new original piperazine derivatives of butyric acid on the induction of differentiation and the growth inhibition of human erythroleukemia K562 cells and myeloid leukemia HL60 cells. 1-(2-hydroxyethyl) 4-(1-oxobutyl)-piperazine (HEPB) and [1-(2-hydroxyethyl) 4-(1-oxobutyl)-piperazine] butyrate (HEPDB) were efficient in acting on the differentiation and proliferation of both cell lines, whereas 1-phenyl 4-(1-oxobutyl)-piperazine (PPB) and 1-(3,4-methylene dioxybenzyl) 4-(1-oxobutyl)-piperazine (POB) acted only on proliferation rates. Such derivatives did not induce significant toxicity in mice. These preliminary results should enable, by the development of new series of piperazine derivatives, a better understanding of the mechanisms of action of butyric acid and its analogues on the coupling of growth and differentiation of neoplastic cells.

Phosphatase isoenzymes as bone metastasis markers in prostatic carcinoma.

Bone alkaline phosphatase (b-ALP) and tartrate resistant acid phosphatase (tr-ACP) are markers of the activity of osteoblasts and osteoclasts, respectively. We have already shown that the serum activity of these isoenzymes was elevated in breast cancer patients with bone metastasis (BM); we show here that the serum activity of b-ALP and tr-ACP were also elevated in prostate cancer patients with BM. Specificity and sensitivity of b-ALP for BM were 0.90 and 0.75, respectively; and for tr-ACP, 0.60 and 0.60, respectively. The accuracy of b-ALP as a BM marker was higher than the accuracy of usual markers of prostatic carcinoma (tartrate labile ACP [tl-ACP], prostatic acid phosphatase [PAP] and prostate specific antigen [PSA]). The highest value predictive of a positive bone scan was obtained with b-ALP (0.88); this increased to 0.97 when b-ALP was coupled with PAP.

Expression of mdr1 gene in human breast primary tumors and metastases

SummaryExpression of mdr1 gene has been evaluated in 34 tumor samples obtained from breast cancer patients who were classified according to their treatment, and clinical follow-up. No gene amplification was found. mdr1-RNA was never detected in 29 primary breast tumors including 5 samples from patients previously treated by 6 courses of 5-fluorouracil, epirubicin, cyclophosphamide (FEC). On the other hand, mdr1-RNA expression was detected in 1 local recurrence and 2 out of 3 metastases, all of them being treated and exhibiting a poor evolution. A second, untreated local recurrence remained negative. Clinical follow-up for 7 to 48 months in patients receiving chemotherapy showed that absence of mdr1-RNA could not be an accurate factor of satisfactory response to chemotherapy. But, all the patients with detectable mdr1-RNA exhibited a poor evolution and response to treatment. In conclusion, evaluation of mdr1-RNA seemed to be of little interest in primary breast tumors. However, the concomitant presence of an mdr1-RNA and a metastatic phenotype could give a new insight into the relationship between invasive and resistance properties of cancer cells. Such situations would need to be analyzed very carefully for a better utilization of chemotherapy.

The antileukemic alkaloid fagaronine and the human K 562 leukemic cells: Effects on growth and induction of erythroid differentiation☆

In view of new antitumor compounds which could exert their therapeutic effect through a combination of cell growth inhibition and cell maturation, we describe here the effects of a novel antileukemic alkaloid, fagaronine, on the growth and the induction of hemoglobin synthesis in the K 562 cell line. We found that fagaronine, after 3 days, reduces in a concentration dependent relationship the cell growth rate without lethality and this effect on the cell growth is irreversible. Reducing the cell growth rate by 50% (IC50 = 3 X 10(-6)M) is sufficient to induce an optimal amount of hemoglobin synthesis (75% benzidine-positive cells, 13-15 pg hemoglobin/cell) after 4 days of culture. Considering the variation of the total intracellular protein content during the response, it appears that fagaronine stimulated mainly hemoglobin synthesis, and to a lesser extent non-hemoglobin proteins. These results suggest that the novel antileukemic alkaloid, fagaronine, can be considered as a potent inducer of differentiated-associated properties in the human K 562 leukemic cells.

Effect of fagaronine on cell cycle progression of human erythroleukemia K562 cells

Fagaronine (Fine) is a novel antileukemic drug extracted from Fagara xanthoxyloides Lam. (Rutaceae). In an attempt to know more about its mechanism of action we describe here its inhibitory activity on cell division, 3H-thymidine incorporation and on cell cycle progression. Fine inhibits cell proliferation of K562 cells by 50% at a concentration of 3 x 10(-6) mol/l at day 4. It stimulates incorporation of labelled macromolecular thymidine on day 1, but decreases incorporation on days 2, 3 and 4. Fine induces a cell accumulation in G2 and late-S phases. This accumulation (i) increases with Fine concentration, but a complete blockade is not observed, (ii) reaches a plateau after approximately 48 h, (iii) is reversible, whereas we have previously shown that cell growth inhibition and differentiation were not reversible.

A technique for laser raman spectroscopic studies of isolated cell populations

A new method has been developed for the study of cell populations by laser Raman spectroscopy. Resonance Raman spectra of a small number of living or fixed tumor cells (ca 5000) treated with the antitumor agent adriamycin have been successfully obtained. This new approach leads to a better understanding of the molecular interactions between drug and biological targets, i.e., nuclear DNA at the cellular level.

Benzophenanthridine and furoquinoline accumulation in cell suspension cultures of Fagara zanthoxyloides

Abstract In vitro cell culture of Fagara zanthoxyloides is reported for the first time. The cell suspension cultures produced fagaronine, but the production of this metabolite was lost after several subcultures, whereas furoquinoline accumulation remained high and constant.