Cellular adaptation to drug exposure: evolution of the drug-resistant phenotype

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. For etoposide (VP-16), increased expression of MDR-1 or MRP and alterations in topoisomerase IIalpha have been shown to confer tolerance. To further understand resistance to VP-16, three sublines, designated MCF-7-VP17, ZR-75B-VP13, and MDA-MB-231-VP7, were initially isolated as single clones from parental cells by exposure to VP-16. Subsequently, a population of cells from each subline was exposed to 3-fold higher drug concentrations, allowing stable sublines to be established at higher extracellular drug concentrations. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolates. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 6-314-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased and VP-16 accumulation decreased. This adaptation allowed for partial restoration of topoisomerase II activity as a result of increased expression (MCF-7-VP17 and ZR-75B-VP13) or hyperphosphorylation (MDA-MB-231-VP7), with a resultant increase in growth rate. In MDA-MB-231-VP7 cells, hyperphosphorylation coincided with increased casein kinase II mRNA and protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase IIalpha protein levels secondary to an acquired 600-bp deletion in one topoisomerase IIalpha allele, which resulted in reduced protein levels. In all three sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from MRP overexpression helping to confer high levels of resistance.     

Correlations among Epworth Sleepiness Scale scores, multiple sleep latency tests and psychological symptoms

Mouse leukemic cell subline L1210/VCR exerts expressive multidrug resistance (MDR) that is mediated by P-glycoprotein. Cells originally adapted to vincristine are also extremely resistant to doxorubicin. Resistance to both vincristine and doxorubicin is connected with depression of drug uptake. While resistance of L1210 cells to vincristine could be reversed by verapamil as chemosensitizer, resistance of cells to doxorubicin was insensitive to verapamil. Action of verapamil (well-known inhibitor of PGP activity) on multidrug resistance was often used as evidence that MDR is mediated by PGP. From this point it may be possible that the resistance of L1210/VCR cells to vincristine is mediated by PGP and the resistance to doxorubicin is mediated by other PGP-independent system. Another and more probable explanation of different effect of verapamil on resistance of L1210/VCR cells to vincristine and doxorubicin may be deduced from the following fact: Using UV spectroscopy we found that doxorubicin dissolved in water buffered medium interacts effectively with verapamil. This interaction may be responsible for the decrease of concentration of both drugs in free effective form and consequently for higher survival of cells. In contrast to doxorubicin vincristine does not give any interaction with verapamil that is measurable by UV spectroscopy and resistance of L1210/VCR cells to vincristine may be fully reversed by verapamil.     

Reduced drug accumulation and multidrug resistance in human breast cancer cells without associated P-glycoprotein or MRP overexpression

MCF-7 human breast cancer cells selected in Adriamycin in the presence of verapamil developed a multidrug resistant phenotype, which was characterized by as much as 100,000-fold resistance to mitoxantrone, 667-fold resistance to daunorubicin, and 600-fold resistance to doxorubicin. Immunoblot and PCR analyses demonstrated no increase in MDR-1 or MRP expression in resistant cells, relative to parental cells. This phenotype is similar to one previously described in mitoxantrone-selected cells. The cells, designated MCF-7 AdVp, displayed a slower growth rate without alteration in topoisomerase II alpha level or activity. Increased efflux and reduced accumulation of daunomycin and rhodamine were observed when compared to parental cells. Depletion of ATP resulted in complete abrogation of efflux of both daunomycin and rhodamine. No apparent alterations in subcellular daunorubicin distribution were observed by confocal microscopy. No differences were noted in intracellular pH. Molecular cloning studies using DNA differential display identified increased expression of the alpha subunit of the amiloride-sensitive sodium channel in resistant cells. Quantitative PCR studies demonstrated an eightfold overexpression of the alpha subunit of the Na+ channel in the resistant subline. This channel may be linked to the mechanism of drug resistance in the AdVp cells. The results presented here support the hypothesis that a novel energy-dependent protein is responsible for the efflux in the AdVp cells. Further identification awaits molecular cloning studies.     

Rapid recovery of a functional MDR phenotype caused by MRP after a transient exposure to MDR drugs in a revertant human lung cancer cell line

Prior studies have shown that, in some human tumour cells, increased expression of the multidrug resistance gene MDR1 can be induced in response to certain stress conditions such as a transient exposure to cytotoxic agents. Little is known about the possibility of increasing the expression of the recently cloned multidrug resistance-associated protein (MRP) in response to a transient exposure to cytotoxic drugs. In order to examine this possibility, we have used sensitive assays (RT-PCR, flow cytometry) and the sensitive large cell lung cancer cell line, COR-L23/P, and the revertant line (COR-L23/Rev), generated by growing the doxorubicin-selected, MRP-overexpressing resistant variant COR-L23/R without drug exposure for 24-28 weeks. COR-L23/Rev overexpresses MRP, but to a lesser extent than COR-L23/R. COR-L23/Rev rapidly recovered similar levels of MRP mRNA, protein expression, resistance and drug accumulation deficit as COR-L23/R after a 48-72 h exposure to cytotoxic concentrations of doxorubicin or vincristine but not cisplatin. The increase in MRP mRNA could only be detected 3 to 4 days after the transient exposure to drugs. However, when the parental line, COR-L23/P, was exposed to equitoxic doses of doxorubicin, vincristine or cisplatin, no increase in the levels of MRP mRNA could be observed at higher doses (5- to 10-fold the IC50) of doxorubicin or vincristine (but not of cisplatin), we detected a transient increase in the levels of MDR1 mRNA immediately after short-term exposure. In conclusion, we have shown that a human revertant lung cancer cell line (COR-L23/Rev) has the ability to recover quickly, similar levels of MRP expression and resistance as COR-L23/R after a transient exposure to the MDR-drugs doxorubicin and vincristine.     

Using the national cancer institute anticancer drug screen to assess the effect of MRP expression on drug sensitivity profiles

The MRP gene contributes to one form of multidrug resistance. To identify drugs interacting with MRP, we measured MRP mRNA expression by quantitative PCR in 60 cell lines of the National Cancer Institute Anticancer Drug Screen. Expression was detected in all cell lines (highest in lung carcinomas and central nervous system tumors) with a range of 14-fold. A mean graph of MRP mRNA levels was constructed to determine Pearson correlation coefficients (PCCs) with mean graphs of >40,000 compounds using the COMPARE analysis. Only 20 compounds had PCCs of >/=0.500. The PCCs for VP-16, doxorubicin, and vincristine were 0.008, 0.13, and 0.257, respectively. Initially, 36 compounds with PCCs of >/=0.428 were analyzed using two MRP-overexpressing cell lines; low levels of cross-resistance was demonstrated for 23 compounds (1.3-9.4-fold). Twenty-four compounds also were available for further studies. Using a fluorescence activated cell sorter assay to measure competition of calcein efflux from MRP-overexpressing cells, 10 compounds were found to increase calcein retention by >/=2-fold. Ten compounds also were able to reduce ATP-dependent [3H]LTC4 transport into vesicles from MRP-overexpressing cells. These results contrast with previous studies with MDR-1 in which high correlations were found and confirmed for a large number of compounds. Although other assays may be more revealing, in these unselected cell lines, MRP mRNA expression was a poor predictor of drug sensitivity. This raises the possibility that other factors, including conjugating enzymes, glutathione levels, or other transporters, confound the MRP effect.     

Coordinated action of glutathione S-transferases (GSTs) and multidrug resistance protein 1 (MRP1) in antineoplastic drug detoxification. Mechanism of GST A1-1- and MRP1-associated resistance to chlorambucil in MCF7 breast carcinoma cells

To examine the role of multidrug resistance protein 1 (MRP1) and glutathione S-transferases (GSTs) in cellular resistance to antineoplastic drugs, derivatives of MCF7 breast carcinoma cells were developed that express MRP1 in combination with one of three human cytosolic isozymes of GST. Expression of MRP1 alone confers resistance to several drugs representing the multidrug resistance phenotype, drugs including doxorubicin, vincristine, etoposide, and mitoxantrone. However, co-expression with MRP1 of any of the human GST isozymes A1-1, M1-1, or P1-1 failed to augment MRP1-associated resistance to these drugs. In contrast, combined expression of MRP1 and GST A1-1 conferred approximately 4-fold resistance to the anticancer drug chlorambucil. Expression of MRP1 alone failed to confer resistance to chlorambucil, showing that the observed protection from chlorambucil cytotoxicity was absolutely dependent upon GST A1-1 protein. Moreover, using inhibitors of GST (dicumarol) or MRP1 (sulfinpyrazone), it was shown that in MCF7 cells resistance to chlorambucil requires both intact MRP1-dependent efflux pump activity and, for full protection, GST A1-1 catalytic activity. These results are the first demonstration that GST A1-1 and MRP1 can act in synergy to protect cells from the cytotoxicity of a nitrogen mustard, chlorambucil.     

The responses of secondary endings of cat soleus muscle spindles to succinyl choline

In this report we examine biochemical and genetic alterations in DNA topoisomerase II (topoisomerase II) in K562 cells selected for resistance in the presence of etoposide (VP-16). Previously, we have demonstrated that the 30-fold VP-16-resistant K/VP.5 cell line exhibits decreased stability of drug-induced topoisomerase II/DNA covalent complexes, requires greater ATP concentrations to stimulate VP-16-induced topoisomerase II/DNA complex formation, and contains reduced mRNA and protein levels of the M(r) 170,000 isoform of topoisomerase II, compared with parental K562 cells. K/VP.5 cells grown in the absence of VP-16 for 2 years maintained resistance to VP-16, decreased levels of topoisomerase II, and attenuated ATP stimulation of VP-16-induced topoisomerase II/DNA binding, compared with K562 cells. Sequencing of cDNA coding for two consensus ATP binding sites and the active site tyrosine in the K/VP.5 topoisomerase II gene indicated that no mutations were present in these domains. In addition, single-strand conformational polymorphism analysis of restriction fragments encompassing the entire topoisomerase II cDNA revealed no evidence of mutations in the gene for this enzyme in K/VP.5 cells. Nuclear extracts from K562 (but not K/VP.5) cells contained a heat-labile factor that potentiated VP-16-induced topoisomerase II/DNA covalent complex formation in isolated nuclei from K/VP.5 cells. Immunoprecipitated topoisomerase II from K/VP.5 cells was 2.5-fold less phosphorylated, compared with enzyme from K562 cells. Collectively, our data suggest that acquired VP-16 resistance is mediated, at least in part, by altered levels or activity of a kinase that regulates topoisomerase II phosphorylation and hence drug-induced topoisomerase II/DNA covalent complex formation and stability.     

Expression of topoisomerase II, bcl-2, and p53 in three human brain tumor cell lines and their possible relationship to intrinsic resistance to etoposide

We characterized three human brain tumor cell lines (D54, HBT-20, and HBT-28) with respect to resistance to etoposide (VP-16), a topoisomerase II-reactive drug. All three cell lines were inherently resistant to VP-16 when compared to other human cell lines, with D54 showing the greatest resistance using colony formation assays. Resistance to VP-16 has been attributed to decreased drug uptake and changes in topoisomerase II; however, drug uptake and topoisomerase II protein levels (immunoblot) were no lower in D54 than in HBT-20 and HBT-28, cell lines relatively more sensitive to VP-16. More to the point, measurement of topoisomerase II-mediated DNA cleavage of cellular DNA after treatment with VP-16 showed that the topoisomerase II in these cells was active. These data indicate mechanisms other than those attributable to decreased drug uptake or altered topoisomerase II exist for clinical resistance to VP-16. VP-16-induced DNA cleavage has been associated with apoptosis in some cell lines; however, neither DNA laddering nor morphological changes characteristic of apoptosis were detected in these cell lines after treatment with VP-16. Bcl-2 and mutant p53 were present in these cells. Either of these conditions can prevent apoptosis and could explain a dissociation between the proximal mediator of VP-16-induced cytotoxicity (topoisomerase II-DNA complex formation) and cellular death.     

The actin-based motility of the facultative intracellular pathogen Listeria monocytogenes

Drug resistance is a major obstacle to successful cancer chemotherapy. P-glycoprotein, which transports various antitumor agents outside the resistant tumor cells, plays a key role in multidrug resistance. We found that MRK-16, a monoclonal antibody against P-glycoprotein, and cyclosporine, synergistically enhanced the antitumor effects of vincristine and adriamycin in multidrug-resistant K562/ADM cells. On the other hand, the combined use of MRK-16 with verapamil or FK-506 did not show such synergistic effects. Drug accumulation studies revealed that MRK-16 remarkably increased the accumulation of cyclosporine, but not verapamil, in K562/ADM cells. This increased accumulation of cyclosporine by MRK-16 in K562/ADM cells directly resulted in the enhanced accumulation of vincristine and adriamycin in the cells. The synergistic effect of MRK-16 and cyclosporine was further confirmed by isobologram analysis in three different highly multidrug-resistant tumor cells. Moreover, while MRK-16 alone did not enhance the sensitivity of the KB-8-5 cells moderately resistant to vincristine, it increased two-fold the reversing effect of cyclosporine at 1 microM, an achievable blood concentration. Since MRK-16 alone showed therapeutic effects against multidrug-resistant tumors, the combined use of MRK-16, cyclosporine and antitumor agents would provide therapeutic benefits for the treatment of resistant tumors.     

Functional analysis of the nucleotide binding domains of the multidrug resistance protein (MRP)

HeLa cells were transfected with full-length multidrug resistance protein (MRP) cDNA and with MRP cDNAs that had been mutated at certain nucleotide binding domains. Stable transfectants were isolated and those producing equivalent amounts of P190 were tested in cytotoxicity assays using a variety of chemotherapeutic agents. The results demonstrate that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to adriamycin, vincristine, or etoposide (VP-16). Single-site mutations of lysine in these same motifs reduce IC50 values but less than that observed with the deletion mutants. Additional studies have demonstrated an increase in drug accumulation and reduction in drug efflux in NBD deletion and single-site mutants. The results of this study therefore identify two lysines of the NBD A- and C-motifs that are critical for MRP-mediated multidrug resistance. The results also provide definitive evidence that resistance occurring as a result of MRP overexpression is related to enhanced levels of an ATP-dependent efflux pump.     

Influence of catheter materials and tissue composition on low dose rate iridium-192 seed implant dosimetry

Drug sensitivity was studied for the tubulin inhibitors taxol, taxotere, rhizoxin and for doxorubucin and cisplatin, in human lung and breast cancer cell lines, including drug-selected cell lines, overexpressing the membrane transporter P-glycoprotein (Pgp) or the multidrug resistance protein (MRP). All tubulin-inhibiting agents were more potent than doxorubicin and cisplatin in all cell lines. In the drug resistance-selected cell lines (doxorubicin or mitoxantrone resistant) there was cross-resistance between the tubulin inhibitors and the selecting agent; however, MRP overexpressing cells were relatively less resistant to taxanes than the Pgp overexpressing cells. Polymerization of microtubules after exposure to taxol was observed in drug sensitive cell lines, but not in resistant cell lines, even at high taxol concentrations and after long exposure times. In the Pgp overexpressing cell lines, steady accumulation of 14C-taxol was defective and could be reverted by verapamil. MRP overexpressing cells did not have a significant accumulation defect of taxol, compared to the parental cell lines, and verapamil did not have any effect. These data confirm that the Pgp overexpression is an important mechanism of resistance to taxanes and rhizoxin in human lung and breast tumor cells. However, the presence of mechanisms other than transport defects may play an important role in non-Pgp expressing cells, and these may include an altered function of tubulins.     

Mechanisms of resistance of human small cell lung cancer lines selected in VP-16 and cisplatin

BACKGROUND: The combination of VP-16 and cisplatin is one of the most active regimens available for the treatment of small cell lung cancer (SCLC), however, most tumors eventually become resistant to these drugs. METHODS: To investigate the problem of resistance to VP-16 and cisplatin in patients with SCLC, we established two resistant sublines from the drug sensitive human SCLC line, NCI-H209, by in vitro selection in VP-16 and cisplatin. RESULTS: The VP-16-selected cell line, H209/VP, was more than 100-fold resistant to VP-16, and displayed cross-resistance to VM-26 and other topoisomerase II interactive drugs, but not to vinca alkaloids. There was no difference in accumulation of VP-16 in H209/VP compared with its parent cell line. The level of topoisomerase II-alpha was reduced to 8% of that in the parent cell line, and there was an altered form of this enzyme with a molecular weight of 160 kilodaltons (kDa), in addition to the normal 170 kDa protein. The cisplatin-selected cell line, H209/CP, was 11.5-fold resistant to cisplatin, with only a low level of cross-resistance to other platinum compounds including carboplatin, tetraplatin, iproplatin, and lobaplatin. This line was highly cross-resistant to vinca alkaloids, but not to anthracyclines or epipodophyllotoxins. The H209/CP cell line was not resistant to cadium chloride, suggesting that alterations in metallothionein are unlikely to be a cause of resistance. Although glutathione (GSH) levels were increased nearly 2-fold in H209/CP, there was no difference in levels of the GSH-related enzymes glutathione-S-transferase, glutathione peroxidase, and glutathione reductase, compared with the parent line. The H209/CP line had a 1.4-fold elevation of topoisomerase II-alpha. The accumulation of cisplatin was reduced in this cell line, and there were fewer DNA-interstrand cross links formed in the presence of cisplatin in H209/CP, compared with the parent line. Neither H209/VP nor H209/CP expressed MDR1, the gene for P-glycoprotein. The MRP gene was expressed at a slightly higher level in the H209/VP cell line, but there was no significant increase in expression of this gene in the H209/CP cell line. CONCLUSIONS: The resistance of the H209/VP cell line is associated with an alteration of topoisomerase II-alpha, whereas the resistance in the H209/CP line is associated with reduced drug accumulation.     

Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C

We selected a human colon carcinoma cell line in increasing concentrations of mitoxantrone to obtain a resistant subline, S1-M1-3.2, with the following characteristics: profound resistance to mitoxantrone; significant cross-resistance to doxorubicin, bisantrene, and topotecan; and very low levels of resistance to Taxol, vinblastine, colchicine, and camptothecin. This multidrug resistance (MDR) phenotype, which was not reversed by verapamil or another potent P-glycoprotein (Pgp) inhibitor, CL 329,753, was dependent, in part, upon an energy-dependent drug efflux mechanism. Pgp and the multidrug resistance protein (MRP) were not elevated in the resistant cells relative to the drug-sensitive parent, suggesting that resistance was mediated by a novel pathway of drug transport. A cell-based screen with S1-M1-3.2 cells was used to identify agents capable of circumventing this non-Pgp, non-MRP MDR. One of the active agents identified was a mycotoxin, fumitremorgin C. This molecule was extremely effective in reversing resistance to mitoxantrone, doxorubicin, and topotecan in multidrug-selected cell lines showing this novel phenotype. Reversal of resistance was associated with an increase in drug accumulation. The compound did not reverse drug resistance in cells with elevated expression of Pgp or MRP. We suggest that fumitremorgin C is a highly selective chemosensitizing agent for the resistance pathway we have identified and can be used as a specific pharmacological probe to distinguish between the diverse resistance mechanisms that occur in the MDR cell.     

Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione

The M(r) 190,000 multidrug resistance protein (MRP) confers resistance to a broad spectrum of natural product drugs. However, it has not been possible to demonstrate that MRP can actively transport unmodified forms of these compounds, although the protein has been shown to transport structurally diverse glutathione (GSH)- and glucuronide-conjugated molecules. Previously, we showed that ATP-dependent uptake of vincristine by MRP-enriched, inside-out membrane vesicles could be stimulated by physiological concentrations of GSH (Loe et al., J. Biol. Chem., 271: 9675-9682, 1996). We have now established that the ATP/GSH dependent vincristine uptake is both proportional to the level of MRP in the membrane vesicles and can be inhibited by monoclonal antibodies shown previously to inhibit transport of established MRP substrates, such as leukotriene C4. We also show that short-chain alkyl derivatives of GSH can stimulate drug uptake, which suggests that vincristine transport does not necessarily involve a change in redox state or glutathionylation of the protein. Furthermore, vincristine uptake is accompanied by ATP- and drug-dependent accumulation of GSH, which can also be stimulated to a lesser extent by vinblastine but not daunorubicin or doxorubicin. Although GSH or vincristine alone are very poor inhibitors of MRP-mediated transport of leukotriene C4, together they act as relatively potent competitive inhibitors. Overall, our data demonstrate that MRP can actively cotransport GSH and unmodified vincristine and that these compounds probably interact, either with the leukotriene C4 binding site(s) on the protein or with a mutually exclusive site.     

Newly synthesized dihydropyridine derivatives as modulators of P-glycoprotein-mediated multidrug resistance

Newly synthesized 1,4-dihydropyridine derivatives possessing alkyl chains at the 4-position screened whether they could overcome P-glycoprotein-mediated multidrug resistance in cultured cancer cells and also leukemia-bearing animals. Of these derivatives, some could overcome drug resistance to doxorubicin and vincristine in multidrug resistant human cancer cell lines. Combined administration of vincristine and some of the derivatives significantly increased the life span of P-glycoprotein overexpressing multidrug-resistant P388 leukemia-bearing mice. The calcium antagonistic activities, an undesirable effects, were weaker than that of verapamil. These results suggested that the introduction of alkyl groups at the 4-position were effective for both overcoming multidrug resistance and reducing the calcium antagonistic activity.     

Retrovirus-mediated gene transfer of the multidrug resistance-associated protein (MRP) cDNA protects cells from chemotherapeutic agents

Transduction of hematopoietic progenitors with a multidrug resistance gene like mdr-1 or mrp aims to protect bone marrow from toxicity of chemotherapeutic agents. The interest in the use of mrp as an alternative to mdr-1 gene transfer for bone marrow protection lies in its different modulation. Indeed, classical P-gp reversal agents, tested in the clinic to decrease mdr-1 tumor resistance, have little or no effect on MRP function. This would allow, in the same patient, the use of reversal agents to decrease P-gp tumor resistance without reversing bone marrow protection of the transduced hematopoietic cells provided by multidrug resistance-associated protein (MRP). As a first step, we have constructed and tested two different mrp-containing vectors with either the Harvey retroviral long terminal repeat (LTR) or PGK as promoters and generated ecotropic producer cells. We have shown by Southern blot analysis that retroviral supernatant from these producer cells can efficiently transmit the mrp gene to target cells. Mrp expression could be detected by fluorescence-activated cell sorting (FACS) analysis in the producer cells. The transduced cells have increased resistance to doxorubicin, vincristine, and etoposide. Furthermore, chemoprotection of the transduced cells was increased after selection with chemotherapeutic agents in the presence of glutathione, a co-factor for MRP function. These data indicate that mrp retroviral vectors may be useful for chemoprotection and selection.     

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resistance is conferred by the full-length enzyme carrying a mutated active site (Y783F) or a truncation at aa 1044. The level of phosphorylation of topoisomerase II was previously shown to correlate with drug resistance in cultured cells, hence we tested mutants in the major casein kinase II acceptor sites in the C-terminal domain of yeast topoisomerase II for changes in drug sensitivity. Neither ectopic expression of the C-terminal domain alone nor phosphoacceptor site mutants significantly alter the host cell's sensitivity to etoposide.     

Respiratory function tests in the newborn and the infant

AIM: To study the mechanisms of the resistance to harringtonine (Har) in the HL60 cells. METHODS: Growth inhibition, karyotype analysis, flow cytometry, Western blotting and polymerase chain reaction. RESULTS: The Har-resistant HL60 cell line, named HR20, showed cross resistance to homoharringtonine, doxorubicin, daunorubicin, vincristine, and colchicine. The growth doubling time and the cell numbers in G1 phase were increased. The accumulation of cellular daunorubicin in the resistant cells was obviously reduced, but distinctly increased by tetrandrine and verapamil. The numbers of telocentromeric chromosome increased and the chromosomal aberration more occured in the resistant cells. The resistant cells overexpressed multidrug resistant mdr-1 gene and P-glycoprotein 150 kDa. CONCLUSION: The Har-resistant HL60 cell strain belonged to a multidrug resistance strain, overexpressing mdr-1 gene and P-glycoprotein.