Nuclear transport as an ultimate step of multidrug resistance.

Adriamycin (ADM) incorporation into nuclei of whole multidrug resistant (MDR) CEM cells is lower than into sensitive ones (S), that is mostly thought to be the consequence of a decrease of drug related to the activity of the multidrug resistance plasma membrane protein P 170. Isolated nuclei of the lymphoblastic tumor cell line CEM, which structures were controlled by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy, where incubated with 10(-6) mole/l of ADM. Incorporation into DNA was quantified by spectrofluorimetry. It was lower and slower into MDR nuclei than into S ones. Different modulators of active transport influence drug transfer into S nuclei and had no effect in MDR nuclei. The nuclear transfer into S nuclei appeared divided into two components: one was decreased by WGA, increased by cytosolic factors and an other part was purely passive in an identical intensity to MDR nuclei. Resistance of MDR nuclei seemed indebt to a defect, in these cells, of factors that mediate and/or activate nuclear transport of drug.

Involvement of the 92-kDa gelatinase (matrix metalloproteinase-9) in the ceramide-mediated inhibition of human keratinocyte growth.

Triggering the ceramide pathway by exogenous treatment with neutral sphingomyelinase (Smase) inhibited human keratinocyte growth rate, while having no influence on cell apoptosis. Increasing the ceramide content of keratinocytes with Smase (100 U/ml) or C6-ceramide (1 microM) enhanced matrix metalloproteinase (MMP)-9 production. On the contrary, levels of MMP-2 secretion were unchanged. The inhibition of keratinocyte growth rate induced by ceramide could be annihilated by a peptide hydroxamate MMP inhibitor or an MMP-9 blocking antibody. In addition, inhibiting MMP-9 activity in control keratinocyte culture was found to stimulate keratinocyte proliferation. These data suggest a pivotal function of MMP-9 in the control of keratinocyte growth.

Enhancement of all-trans-retinoic acid efficiency in granulocytic differentiation of HL-60 cells by incorporation into low density lipoprotein.

All-trans-retinoic acid (ATRA) has been proven to lead to complete remission of acute promyelocytic leukemia by inducing differentiation into granulocytes except when an acquired resistance occurred. High levels of low density lipoprotein (LDL) receptor in cancer cells suggested the use of ATRA incorporated into LDL. 50% of HL-60 cell differentiation were obtained with 5 nmoles/l of ATRA-LDL compared to 150 moles/l of ATRA. Maximal differentiation (80%) was reached at 25 nmoles/l and 1,000 nmoles/l respectively. This higher efficiency suggests the involvement of LDL receptor in ATRA-LDL internalization and/or the protection of the drug, from eventual catabolism, by LDL particles.

[Detection of metastatic activity of the MCF-7 multidrug resistant cell line cultured in spheroids]. / Mise en évidence d'une activité métastasiante dans la lignée MCF-7 multidrogue résistante cultivée en sphéroïdes.

The occurrence of solid tumors spreading through the body is a major concern for the clinicians. Moreover, in numerous cases, metastases exhibit a multidrug resistant (MDR) pattern. This dual characteristic still remains supported by few biological explanations. The purpose of our study was to compare invasive properties of sensitive and MDR MCF-7 cells. Spheroids were chosen as experimental model since they exhibit a number of characteristics (i.e. tridimensional structure) close to the growth of an in vivo tumor. MDR spheroids formed more compact structures compared to sensitive ones. In every experiment, spheroids made from sensitive cells were more resistant to doxorubicin than the same cells grown as monolayers, a characteristic not observed with MDR cells. On an other hand, a form of multicellular resistance appeared in spheroids of sensitive cells, a fact which was not present in MDR spheroids. Incubation of MDR spheroids in Boyden's chambers put in evidence increased motility and invasive properties through Matrigel which were not present in sensitive MCF-7 cells. Zymograms of culture media and membrane extracts were performed in polyacrylamide gels. Two metalloproteases, progelatinases A et B were detected in culture media conditioned by monolayers and spheroids of both sensitive and resistant cells. In contrast, 2 unidentified serine proteases were detected only in media conditioned by spheroids of both cell types. An intense band of pro-MMP2 was present only in membrane extracts from MDR spheroids. Taken altogether, these results demonstrate that spheroids of MDR cells exhibit a number of properties which could lead to an increased ability to form metastases.

Sequential overexpression of LRP and MRP but not P-gp 170 in VP16-selected A549 adenocarcinoma cells.

Chemoresistance remains the major obstacle to successful therapy of lung cancer. In order to understand drug resistance mechanisms, the expression of three proteins involved in multidrug resistance (P-gp, MRP and LRP) was studied, using the non-small cell lung cancer (NSCLC) A549 cell line. In addition, 3 levels of resistance were obtained by continuous exposure of cells to etoposide (VP16), which led to a 22-fold increase of the resistance index. The wild-type A549 strongly expressed the LRP protein while MRP protein was found at a moderate level. Induction of resistance paralleled an increase of the expression of the mrp gene and a decrease of the lrp gene; the mdr1 gene was not expressed. Taken together, these results indicate that intrinsically resistant NSCLC cells exhibit a complex pattern of MDR proteins, still susceptible to evolve under treatment. Such a fact would have to be considered in clinical situations.

HL60 cells exhibit particular ultrastructural features during all-trans retinoic acid-induced apoptosis.

Ultrastructural features induced by 1 micromol/l all-trans retinoic acid (ATRA) treatment of HL60 cells were observed with transmission electron microscopy. Whilst some cells were unaffected, most of the others underwent granulocytic differentiation, starting point for apoptosis and then, possibly, secondary necrosis. First steps of apoptosis led to nuclear fragmentation into dense bodies. Then three different ways were observed: i) cells shrank and dense bodies were expelled, mostly associated with a fine lamellae of cytoplasm, ii) a secondary necrosis involved the release of apoptotic bodies without cytoplasm or iii) cells showed a dual or bipolar structure, not previously described.

Cofactors in in vitro induction of apoptosis in HL60 cells by all-trans retinoic acid (ATRA).

The aim of this study was to determine the culture conditions that could modulate the induction of apoptosis by all-trans retinoic acid (ATRA). Cell viability was evaluated by trypan blue test, differentiation by nitro blue tetrazolium test, and apoptosis by morphological analysis. ATRA induced apoptosis in HL60 cells only when more than 100,000 cells/mL were seeded, while differentiation was induced regardless of the seeded cell concentration. Reduction in the concentration of foetal calf serum or glutamine in the medium led to a weak increase in apoptosis. In contrast, a dramatic enhancement of apoptosis occurred when the culture medium was supplemented with glucose or when the culture pH was decreased. These characteristics were independent of the mechanism of action of ATRA, but the action of glucose could be of significance in diabetic patients. An exchange of supernatants after 3 days of culture showed that supernatants from control cultures seeded at high cell density were better apoptosis inducers than supernatants from cultures treated with ATRA, but seeded at low cell density. Factor(s) in this supernatant which induced apoptosis was (were) removed by ultrafiltration. In conclusion, our results showed that ATRA alone cannot induce apoptosis, but can do so in conjunction with cofactors. The depletion of some components of the medium and the appearance of secreted macromolecule(s) could be cofactor(s) in the induction of apoptosis.

Ultrastructural changes related to multidrug resistance in CEM cells: role of cytoplasmic vesicles in drug exclusion.

The multidrug resistance phenotype is found to be frequently associated with the overexpression of proteins which lead to a decrease of drug accumulation within human tumor cells. A 170 kDa membrane glycoprotein which is related to the overexpression of the mdr1 gene is inserted in the plasma membrane and pumps the cytotoxic drugs out of the cells. The aim of this work was to study the morphological modifications of resistant CEM/VLB 100 cells relative to their parental drug-sensitive ones and the detection of the ultrastructural localization of P-glycoprotein at the cytoplasmic level. Using a scanning electron microscope, CEM resistant cells showed wide smooth protrusions while CEM sensitive cells showed microvilli and fine folds. With transmission electron microscopy, an enhanced secretory system was observed in CEM resistant cells: both electron transparent and electron opaque vesicles were associated with the Golgi system, revealed by wheat germ agglutinin-colloidal gold labelling. These vesicles were the binding site of C 219 and MRK 16 antimembrane glycoprotein antibodies, and some of them were determined to belong to the lysosomal system after PTA staining. These vesicles may be an additional way to decrease the cellular uptake of drugs in multidrug resistant cells. Moreover, some nuclear and nucleolar modifications were also observed. These observations show that MDR has wide morphological features which concern several organelles.

Monitoring of bacterial growth and structural analysis as probed by FT-IR spectroscopy.

Fourier-transform infrared spectroscopy was used to explore structural changes in bacteria under different incubation conditions. In particular, differences between Bradyrhizobium japonicum (BRJ) grown in liquid and on solid media were investigated, as well as the rearrangement of BRJ after transfer from one medium to the other. The FT-IR absorption bands located between 1200 and 900 cm-1 region, vary in spectral shape and intensity when BRJ were suspended in solution medium or plated on solid medium. In agreement with the electronic micrograph data, these spectroscopic changes are due to the changes involving the bacterial wall (peptidoglycan) when BRJ are plated in agar medium. By means of this FT-IR ultrastructural study of Bradyrhizobium japonicum bacteria, it has been possible to follow and to evaluate the rate of the molecular change in bacteria without any destructive interference. This indicates that FT-IR spectroscopy can prove to be a valuable technique in the monitoring of metabolic events in bacterial cells relevant to agriculture as well as environmental and health sciences.

[The multidrug resistance phenotype: some morphological and biological characteristics except efflux pump]. / Le phénotype de résistance multidrogue: quelques caractéristiques morphologiques et biologiques autres que la pompe à efflux.

Recent data from the literature together with personal results strongly suggest that multidrug resistance phenotype is overwhelming the sole expression of P170 glycoprotein efflux pump. Morphological alterations have been put in evidence in MDR cells after transmission and scanning electron microscopy. They include presence of osmiophilic vesicles and modifications of nuclear and nucleolar chromatin. Biological characteristics include the hypersecretory pattern of lysosomal enzymes from MDR cells. Such a fact could be more or less related to the increased occurrence of mdr1 RNA in metastasis, especially in breast cancers, compared to primary tumors. If the P170-mediated efflux is one of the key mechanism of MDR, a decreased influx of anticancer drugs cannot be excluded. Liposomes, for instance made of cardiolipin, are thus able to increase the intracellular drug uptake of vinblastine without any action upon efflux mechanism.