Proton nuclear magnetic resonance spectroscopy reveals cellular lipids involved in resistance to adriamycin and taxol by the K562 leukemia cell line.

Proton nuclear magnetic resonance spectroscopy was performed on whole cells to study lipids and metabolites in Adriamycin- and Taxol-resistant K562 cells expressing multidrug resistance (MDR) and their sensitive counterparts. With one-dimensional spectra, both resistant cell lines showed lower fatty acid methylene:methyl ratios and higher choline:methyl ratios than sensitive cells. Using two-dimensional COSY spectra, a decrease in the glutamine content was evidenced in resistant cells. When these cells were maintained in culture medium without the drug, the fatty acid signals were partially recovered. Adriamycin-resistant K562 cells were also treated for 4 days with a high dose of verapamil, a MDR-reversing agent. The nuclear magnetic resonance spectra of verapamil-treated cells also showed partial recovery of fatty acid signals. These results could be paralleled with the reversion of the resistant phenotype, as evidenced by measuring the inhibiting concentration of Adriamycin and vinblastine in K562adr cells cultured without the drug or after short-term exposure to verapamil. Conversely, P-glycoprotein and mRNA expression and DNA amplification of the mdr gene were not modified when compared to resistant cells, suggesting that the MDR phenotype could be partially reversed independently of the mdr gene amplification and expression. These results demonstrate the role of lipids in the resistance phenomenon.

In vitro treatment with retinoids or the topoisomerase inhibitor, VP-16, evidences different functional apoptotic pathways in acute promyelocytic leukemic cells.

Understanding the mechanisms inherent to malignant cell eradication is a major determinant for cancer therapy. Recent data have demonstrated that apoptosis may be one of the mechanisms through which both cytotoxic and differentiating drugs may eliminate malignant cells. Treatment of acute promyelocytic leukemia (APL) by all-trans retinoic acid (ATRA) is the first model of differentiation therapy allowing achievement of more than 90% complete remission (CR). However, disease-free survival (DFS) is short if patients are not subsequently treated with chemotherapy. In order to address the question of APL cells' elimination during ATRA therapy, we studied phenotypic and molecular features of 14 APL cases relative to cell survival in primary culture in the presence or absence of ATRA. Compared to other acute myeloid leukemia (AML) subtypes, APL cells in short-term suspension culture present a better survival rate (P < 0.001). After incubation with ATRA, cell survival was not altered and was correlated with a concomitant absence of apoptosis, despite a significant decrease of the BcL-2 protein in APL differentiated cells. Indeed, after 6 days of culture, only 3 +/- 0.5% of APL cells exhibit morphological features of apoptosis after ATRA treatment compared to 30 +/- 5% in HL-60-treated cells. Treatment of APL cells with 9-cis RA, 13-cis RA or analogs of RAR alpha or RXR alpha also failed to induce apoptosis. Treatment of either APL or ATRA-differentiated APL cells with 40 microM etoposide resulted in DNA fragmentation and morphological changes characteristic of apoptosis in 23 +/- 5% cells after only 20 h of treatment and 68 +2- 3% after 48 h suggesting that other pathways of apoptosis are still functional in APL cells. Though these in vitro data cannot fully represent the mechanism of cell death and cell elimination in vivo, they clearly indicate that ATRA alone may not induce leukemic clone eradication by apoptosis correlating with the persistence of minimal residual disease and constant relapse after CR obtained with ATRA alone.

Selective induction of apoptosis in myeloid leukemic cell lines by monoacetone glucose-3 butyrate.

Butyric acid is a potent cell growth inhibitor and differentiation inducer. Our previous studies have shown that MAG=3but, a monosaccharide ester of butyric acid, used at 1 mM, induces apoptosis in the HL-60 cell line. We report here that this drug can also induce apoptosis in the U-937 leukemic cell lines whereas the myeloblastic KG1 and the NB4 promyelocytic leukemic cell lines were refractory to induction of apoptosis. In order to determine what can trigger cells to undergo apoptosis, cell cycle analysis, induction of differentiation and p53, c-myc and Bcl-2 expression was studied. Apoptosis was correlated to an arrest of cell growth in the G1 phase of the cell cycle and to an induction of differentiation through the monocytic pathway in HL-60 and U-937 cells. Time course studies demonstrated DNA fragmentation after few hours incubation with the drug, while morphological signs appeared later (days 2 or 3). Northern blot analysis and flow cytometric studies have shown that cell death induced by MAG=3but was not associated to an overexpression of c-myc and p53. However, in the HL-60 cells, BCL-2 protein expression was decreased after MAG=3but treatment, corroborating the apoptosis observed.

Butyric acid and its monosaccharide ester induce apoptosis in the HL-60 cell line.

Butyric acid is a potent cell growth inhibitor and differentiation inducer (1-4). However, the short plasma half-life of this fatty acid limits its potential therapeutic use (5,6). The recent synthesis of several monosaccharide esters of butyric sodium salt (BuONa) with a prolonged plasma half-life and similar biological properties as the sodium salt opens new perspectives (7-12). We report here that the human myeloid HL-60 cell line can be induced to apoptosis when cultured with one of these esters, monoacetone glucose 3-butyrate (MAG = 3but) or BuONa. Cytospin slide preparations and flow cytometric studies showed that HL-60 cells treated with 1 mM MAG = 3but or BuONa exhibited a reduction in cell volume and condensation of nuclear structure characteristic of apoptosis, associated with monocytic differentiation. Time course studies demonstrated that DNA fragmentation, as determined by agarose gel electrophoresis, was detected 4 hours after incubation with the drugs, while morphologic signs appeared at day 3. Apoptotic cells increased with culture time and reached a maximum at day 6 of 20 +/- 5% with BuONa, 25 +/- 5% with MAG = 3but, and only 5 +/- 2% in controls. These findings suggest that these drugs may exert their actions, at least in part, through induction of apoptosis.

Induction of high-affinity paf receptor expression during T cell activation.

Activated human T cells via the CD2 or the CD3 pathways exhibited a higher capacity than resting T lymphocytes to incorporate and metabolize [3H]pafacether (paf) at 37 degrees C. Resting T lymphocytes lacked specific binding capacity for paf, yet high-affinity paf receptors (paf-R) were induced on CD3- or CD2-dependent activation. This up-regulation in the number of paf-R became apparent by day 1 of culture, reached a maximum of about 25,000 sites cell by days 4 to 6 and subsequently declined. Interestingly, human recombinant interleukin-2 in a dose-dependent manner prevented the decrease of high-affinity paf-R expression on T cells. By contrast, the receptor affinity was constant throughout the culture period. Thus, paf-R at different stages of T cell activation were indistinguishable with respect to receptor-ligand interaction, and differed only in their number. Together, these data demonstrate that after activation human T cells develop membrane high-affinity paf-binding sites. They also suggest for the first time that expression of the paf-R are coupled to T cell activation and/or differentiation.