Factors contributing to the resistance to apoptosis induced by topoisomerase I inhibitors in vincristine resistant cells.

The activity of numerous antineoplasic drugs is correlated with their capacity to induce the apoptotic process. In this study, apoptosis induced by the topoisomerase I (Topo I) inhibitors camptothecin (CPT) and the CPT-11 active metabolite SN-38 was evaluated on HL-60 cells and their multidrug resistant variant HL-60-Vincristine cells. Both CPT and SN-38 induced high levels of apoptosis in sensitive cells but very low levels in MDR cells. The role of the different genes and proteins usually implicated in the drug resistance phenomenon was studied. The Pgp independence of the two drugs was suggested by the lack of modulation of anti-Topo I effects with verapamil. Moreover CPT and SN-38 induced a strong decrease of mdr1 mRNA in MDR treated cells. MRP mRNA expression was very low in drug sensitive and resistant cells and decreased during treatments in both cell lines. However, MRP protein was not detected in control and MDR cells suggesting that this pump was probably not implicated in this resistance phenomenon. Topo I and BCL-2 proteins displayed a higher expression in MDR cells but only Topo I proteins decreased during treatments in the two cell lines. These data suggest that in addition to the classical multidrug resistance phenotype, dysregulation of proteins associated with DNA replication and apoptotic process could contribute to acquired resistance to a large panel of drugs, including those which are not considered as substrates for Pgp.

A method for simultaneous quantification of monoclonal antibody Ki-67 and DNA content by flow cytometry. Application to breast carcinomas.

OBJECTIVE: To seek a method assessing Ki-67 immunostaining and DNA content by flow cytometry simultaneously. STUDY DESIGN: The murine monoclonal antibody Ki-67 (Ki) identifies a nuclear protein complex expressed by all nonquiescent tumor cells. Since the antigen detected by Ki is quite labile in most fixation and embedding protocols, a new method for simultaneous quantification of nuclear Ki immunofluorescence and DNA content by flow cytometry was developed. Unfixed, solid tumor cells are permeabilized only with saponin to preserve Ki antigen. The percentage of Ki-positive cell subpopulations calculated by subtraction of the related aspecific fixation histogram gives optimal results more rapidly than by cytogram analysis. Application to breast carcinoma shows the feasibility of the method. RESULTS: Significant correlations between Ki staining and the S-phase fraction were observed. Mean Ki labelling rates of aneuploid tumors were significantly higher than those of the diploid tumors, and significant differences between histologic types were found. CONCLUSION: This technique can be considered a fast, sensitive and optimal method to evaluate the proliferative activity of breast carcinomas and possibly of other solid tumors in a department of pathology.

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.

Multicentric evaluation of the MDR phenotype in leukemia. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations. To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available. Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996. The same batches of MRK16, 4E3 and UIC2 were used. Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested. Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry. The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR. Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results). For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another. No correlations could be found between techniques. Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation. The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed. In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry. Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100. For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100. Whatever the technique used, concordant results were obtained for K562/HHT300. The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers. The group does not recommend this technique for circulating tumoral cells.

[In vivo evaluation of potential doubling time (Tpot) of tumors by flow cytometry: practical difficulties and possible solutions]. / Evaluation du temps potentiel de doublement des tumeurs in vivo (Tpot) par cytométrie en flux: difficultés pratiques et solutions possibles.

Kinetic parameters of tumor growth yield predictive values and allow an optimisation of the treatment schedule, especially for fractionation in radiotherapy. Among those parameters, the labelling index (LI) and the potential doubling time (Tpot) may be measured by flow cytometry, a semi-quantitative analysis, after in vivo administration of iododeoxyuridine (IdUrd) to humans. This Begg's recommended methodology needs the selection of thresholds and gates whose boundaries are arbitrary. They can be positioned on a more objective basis using a negative control (aspecific fixation). Moreover a previous identification of the different cell populations of the tumour samples according to the method of Vindelov allows a better determination of these cell populations processing IdUrd-DNA staining. This procedure was used with 11 tumour biopsies including mainly head and neck cancers. This method displayed results similar to the literature concerning LI and Tpot determinations as well as shortened Tpot when the patients recurred. One sample has no labelling at all. A small fraction, likewise up to 10% of cells exhibiting on S DNA content were not labelled by IdUrd. These cells leave the S phase or progress too slowly in order to display IdUrd uptake. Intra-tumoral hypoxia is a possible explanation of these findings. DNA ploidy and the percentage of cells in S phase could be worth while to precise the relationship between DNA index and tumour kinetic. The measurement of Tpot could also be used in other cancers and for optimisation of dose of chemotherapy.

Culture conditions modulate the effects of aclacinomycin A on growth, differentiation and apoptosis of HL60 cells.

The continuous incubation for several days of HL60 cells, in exponential growth, with aclacinomycin A (ACM) induces growth inhibition, necrosis, differentiation and apoptosis. Differentiation and apoptosis were assessed by optical microscopy (OM) and flow cytometry (FCM). ACM displayed dose-dependent effects, except for the differentiation induction, which was biphasic. Differentiation and apoptosis could also be induced after a 1 h ACM exposure only. The poor reproducibility of apoptosis induction led us to study the culture conditions described in the literature (without renewing the medium) where control cells are not growing exponentially during the 5 day incubation period. During kinetic studies with different ACM concentrations, the differentiation was detected earlier by FCM than by OM, while it was not the case for apoptosis. This induction appeared more reproducible when non optimal conditions of culture were used.

[Effects of aclarubicin on growth, differentiation and apoptosis of tumor cells in vitro]. / Effets de l'aclarubicine sur la croissance, la différenciation et l'apoptose de cellules tumorales in vitro.

Aclarubicine induces various effects after several days of incubation with human leukemic cells HL60: cell growth inhibition, inductions of differentiation, necrosis and apoptosis. Several methods of detection of differentiated and apoptotic cells were studied. The methods utilizing optical microscopy were used as references. Flow cytometry (FCM) appeared to have a higher sensitivity, but it yielded somewhat different results. Aclarubicine and doxorubicin have dose-dependent effects, except for the differentiation induction by aclarubicine, which had a maximum effect and then decreased; this probably means that the inductions of differentiation and apoptosis could be independent. Different kinetic studies indicated that differentiation increased quickly after 24 to 48 hours and reached a plateau toward 72 hours, where approximately 80% of the cells were differentiated. Necrosis increased at the same time, but less, with a 1 hour incubation with 75 nmol/l of aclarubicine. Apoptosis appeared in an irregular and non-reproducible manner. The kinetic study also indicated a certain independence between the differentiation and apoptosis inductions.

Effect of aclacinomycin-doxorubicin association on differentiation and growth of human erythroleukemic K562 cells.

Anthracycline antitumor drugs such as doxorubicin (DOX) and aclacinomycin (ACM) represent potent candidates for the induction of differentiation of leukemic cells. Human multipotent K562 cells were induced by DOX and ACM to differentiate towards the erythroid lineage. After 3 days of culture, DOX-induced differentiation was dose-related whereas ACM did not require total cell growth arrest to induce its optimum effect, indicating that both drugs act differently on the coupling of growth and differentiation. Simultaneous exposure to ACM and DOX and sequential exposure to ACM (30 min) first, followed immediately by DOX did not improve erythroid differentiation. However, it led to either a synergistic or a subadditive inhibition of cell growth. In contrast, DOX (30 min) first, followed by ACM, produced in a narrow range of concentrations (DOX 1000 nM/ACM 1.85 nM, 3.75 nM or 7.5 nM), a synergistic induction of differentiation. Thus, DOX 1000 nM/ACM 3. 75 nM resulted in 81% of differentiated cells compared to 63% for ACM 15 nM and 43% for DOX 30 nM when these were used alone (at their concentration inducing optimum differentiation). In conclusion, these data emphasize the importance of schedules for the combination of chemotherapeutic drugs acting as differentiation inducers.

Characteristics of four Friend leukemia cell sublines resistant to adriamycin.

Resistance of Friend murine erythroleukemia cells was induced or selected by continuous stepwise exposure to adriamycin (ADM). The resistance index (R.I.) varied with different "mdr type" drugs, even when the compounds were closely related as ADM and daunorubicin (DNR). The cell uptake of anthracycline, evaluated by flow cytometry (FCM), was better correlated with the R.I. than the HPLC assessment. The quantitative cytology showed nuclear changes (size and color distribution); all the modifications were in part dependent on the degree of resistance. This study showed that the graded resistant sublines differed in their resistant phenotypes apart from their different degree of resistance.

Evaluation of a method for detection of cells with reduced drug retention in solid tumours.

A method for detection of cells with reduced drug retention was evaluated in solid tumours. After a 1 h incubation with daunorubicin (DNR), the right angle scatter (RAS), forward angle scatter (FAS), and specific fluorescence (Fluo) were measured in sensitive and resistant cells; only Fluo was related qualitatively, but not quantitatively, to resistance. Various incubation conditions were examined. When the pH of the incubation medium increased, the DNR retention increased in sensitive and resistant cells. In contrast, when the cell concentration increased, the DNR retention decreased. Using sensitive and resistant cell lines, a proportion of resistant cells lower than 10% can be detected in a mixture. To analyse cells from solid tumours, the cells were dissociated by repeated fine needle aspirations. Tumours from 22 patients have been processed with this technique; 8 samples were classified as S (sensitive); 2 as R (resistant); and 12 as I (intermediate). Further experiments were run to study and improve the method. Another method of detection of dead cells was tested. The intra-assay variability of the technique was found to be less than 10%. When the study was performed with different fragments of the same tumour, the variation, corresponding to the tumour heterogeneity, rose to 21 to 36%. The inter-assay reproducibility was too bad, so a variant of this technique has been adapted, using verapamil or cyclosporin A, which is able to block DNR efflux; this new method allows tumour cells to be used as their own controls.

[Induction of quiescence by differentiating agents]. / Induction de quiescence par des agents différenciants.

The growth fraction of cancer cells, estimated by the monoclonal antibody Ki-67 labelling, and DNA content were determined simultaneously en K562 human leukemic cells by flow cytometry. Adriamycin, aclacinomycin A and fagaronine induced differentiation, as assessed by benzidine staining and glycophorin A expression. These drugs decreases the fraction of Ki-67 positive cells, Ki-67 negative cells displayed a G1, but also a G2 and a S DNA content in different proportions, indicating that induction of quiescent cells by differentiating agents is not a uniform process and is worthy of interest.

Alterations of growth fraction and DNA content in K562 cells by differentiating agents.

The growth fraction, estimated by the monoclonal antibody Ki-67 labeling, and DNA content, assessed by ethidium bromide staining, were determined simultaneously in K562 leukemic cells by flow cytometry. A multiparametric analysis enabled the fraction of the cell population with G1, S, and G2 + M contents in Ki-67-positive and Ki-67-negative cells to be evaluated. Butyric acid (BUT) was used as positive control. The fraction of Ki-positive cells decreased with the BUT concentration, while the proportion of cells with G1 DNA content increased only in the Ki-negative cells. Adriamycin, aclacinomycin A, and fagaronine induced differentiation, as assessed by benzidine staining and glycophorin A expression. These drugs decreased the fraction of Ki-positive cells by more than 50% for both anthracyclines and by 25% for fagaronine. Following treatment, Ki-negative cells displayed a G1, but also a G2 and a S DNA content in different proportions, indicating that induction of quiescent cells by differentiating agents is not a uniform process and is worthy of interest.