Expression of a drug resistance gene in human neuroblastoma cell lines: modulation by retinoic acid-induced differentiation.

Expression of a multidrug resistance gene (mdr1) and its protein product, P-glycoprotein (Pgp), has been correlated with the onset of multidrug resistance in vitro in human cell lines selected for resistance to chemotherapeutic agents derived from natural products. Expression of this gene has also been observed in normal tissues and human tumors, including neuroblastoma. We therefore examined total RNA prepared from human neuroblastoma cell lines before and after differentiation with retinoic acid or sodium butyrate. An increase in the level of mdr1 mRNA was observed after retinoic acid treatment of four neuroblastoma cell lines, including the SK-N-SH cell line. Western blot (immunoblot) analysis demonstrated concomitant increases in Pgp. However, studies of 3H-vinblastine uptake failed to show a concomitant Pgp-mediated decrease in cytotoxic drug accumulation. To provide evidence that Pgp was localized on the cell surface, an immunotoxin conjugate directed against Pgp was added to cells before and after treatment with retinoic acid. Incorporation of [3H]leucine was decreased by the immunotoxin in the retinoic acid-treated cells compared with the undifferentiated cells. These results demonstrate that whereas expression of the mdr1 gene can be modulated by differentiating agents, increased levels of expression are not necessarily associated with increased cytotoxic drug accumulation.

Various methods of analysis of mdr-1/P-glycoprotein in human colon cancer cell lines.

BACKGROUND: Multidrug resistance (MDR) mediated by high levels of mdr-1 (also known as PGY1)/P-glycoprotein (Pgp) has been studied in tissue culture systems; however, most tumor samples which express mdr-1/Pgp have much lower levels. PURPOSE: We wanted to determine if levels seen clinically could be detected by commonly used methods and to determine if these levels conferred MDR reversible by Pgp antagonists. METHODS: We studied multi-drug-resistant cell lines and sublines with levels of mdr-1/Pgp expression comparable to those seen clinically. We evaluated the expression of mdr-1 RNA by Northern blot analysis, slot blot analysis, polymerase chain reaction (PCR) analysis, and in situ hybridization. We evaluated protein expression by immunofluorescence, immunohistochemistry, fluorescence-activated cell sorting, and immunoblotting analyses. Drug resistance and reversibility were determined by cell growth during continuous drug exposure. RESULTS: In most cases, the low level of mdr-1/Pgp present in these cell lines could be detected by each method, but the assays were at the limit of sensitivity for all methods except the PCR method. These low levels of mdr-1/Pgp are capable of conferring MDR, which can be antagonized by verapamil. CONCLUSIONS: Levels of mdr-1/Pgp similar to those found in clinical samples can be detected by each of these methods, but the PCR method was the most sensitive and most reliably quantitative. IMPLICATIONS: In vitro sensitization by the addition of verapamil in cell lines with these low levels of mdr-1/Pgp suggests that clinically detected levels may confer drug resistance in vivo.

Identification of mutations at DNA topoisomerase I responsible for camptothecin resistance.

A camptothecin-resistant cell line that exhibits more than 600-fold resistance to camptothecin, designated CPT(R)-2000, was established from mutagen-treated A2780 ovarian cancer cells. CPT(R)-2000 cells also exhibit 3-fold resistance to a DNA minor groove-binding ligand Ho33342, a different class of mammalian DNA topoisomerase I inhibitors. However, CPT(R)-2000 cells exhibit no cross-resistance toward drugs such as Adriamycin, amsacrine, vinblastine, and 4'-dimethyl-epipodophyllotoxin. The mRNA, protein levels, and enzyme-specific activity of DNA topoisomerase I are relatively the same in parental and CPT(R)-2000 cells. However, unlike the DNA topoisomerase I activity of parental cells, which can be inhibited by camptothecin, that of CPT(R)-2000 cells cannot. In addition, parental cells after camptothecin treatment results in a decrease in the level of DNA topoisomerase I, whereas CPT(R)-2000 cells are insensitive to camptothecin treatment. These results suggested that the mechanism of camptothecin resistance is most likely due to a DNA topoisomerase I structural mutation. This notion is supported by DNA sequencing results confirming that DNA topoisomerase I of CPT(R)-2000 is mutated at amino acid residues Gly717 to Val and Thr729 to Ile. We also used the yeast system to examine the mutation(s) responsible for camptothecin resistance. Our results show that each single amino acid change results in partial resistance, and the double mutation gives a synergetic effect on camptothecin resistance. Because both mutation sites are near the catalytic active center, this observation raises the possibility that camptothecin may act at the vicinity of the catalytic active site of the enzyme-camptothecin-DNA complex.

Methods to detect P-glycoprotein-associated multidrug resistance in patients' tumors: consensus recommendations.

Multidrug resistance (MDR), especially that associated with overexpression of MDR1 and its product, P-glycoprotein (Pgp), is thought to play a role in the outcome of therapy for some human tumors; however, a consensus conclusion has been difficult to reach, owing to the variable results published by different laboratories. Many factors appear to influence the detection of Pgp in clinical specimens, including its low and heterogeneous expression; conflicting definitions of detection end points; differences in methods of sample preparation, fixation, and analysis; use of immunological reagents with variable Pgp specificity and avidity and with different recognition epitopes; use of secondary reagents and chromogens; and differences in clinical end points. Also, mechanisms other than Pgp overexpression may contribute to clinical MDR. The combined effect of these factors is clearly important, especially among tumors with low expression of Pgp. Thus, a workshop was organized in Memphis, Tennessee, to promote the standardization of approaches to MDR1 and Pgp detection in clinical specimens. The 15 North American and European institutions that agreed to participate conducted three preworkshop trials with well-characterized MDR myeloma and carcinoma cell lines that expressed increasing amounts of Pgp. The intent was to establish standard materials and methods for a fourth trial, assays of Pgp and MDR1 in clinical specimens. The general conclusions emerging from these efforts led to a number of recommendations for future studies: (a) although detection of Pgp and MDR1 is at present likely to be more reliable in leukemias and lymphomas than in solid tumors, accurate measurement of low levels of Pgp expression under most conditions remains an elusive goal; (b) tissue-specific controls, antibody controls, and standardized MDR cell lines are essential for calibrating any detection method and for subsequent analyses of clinical samples; (c) use of two or more vendor-standardized anti-Pgp antibody reagents that recognize different epitopes improves the reliability of immunological detection of Pgp; (d) sample fixation and antigen preservation must be carefully controlled; (e) multiparameter analysis is useful in clinical assays of MDR1/Pgp expression; (f) immunostaining data are best reported as staining intensity and the percentage of positive cells; and (g) arbitrary minimal cutoff points for analysis compromise the reliability of conclusions. The recommendations made by workshop participants should enhance the quality of research on the role of Pgp in clinical MDR development and provide a paradigm for investigations of other drug resistance-associated proteins.

Intrinsic drug resistance in human kidney cancer is associated with expression of a human multidrug-resistance gene.

The cloning of the cDNA for the mdr1 gene, whose expression is associated with the development of multidrug-resistance in cultured cells, has made it possible to explore the mechanism of multidrug resistance in human tumors. We have found that normal human kidney, six of eight adenocarcinomas of the kidney, and four cell lines derived from kidney adenocarcinomas express high levels of mdr1 mRNA. Two criteria suggest that primary multidrug resistance in human adenocarcinomas of the kidney results, at least in part, from expression of the mdr1 gene: (1) mdr1 mRNA levels are elevated in four unselected kidney adenocarcinoma cell lines that show a multidrug-resistant phenotype; and (2) multidrug resistance in these kidney cancer cell lines is reversed by verapamil and quinidine, agents known to reverse mdr1-associated drug resistance in cell lines selected for multidrug resistance in vitro. These results suggest that appropriate pharmacological intervention to reverse multidrug resistance might make adenocarcinomas of the kidney more sensitive to chemotherapy with agents such as Adriamycin (Adria Laboratories, Columbus, OH) and the vinca alkaloids.

Expression of the multidrug resistance, MDR1, gene in neuroblastomas.

Metastatic neuroblastoma is a childhood malignancy that is frequently responsive to chemotherapy with doxorubicin, vincristine, and teniposide (VM26), among other drugs, but in the majority of treated patients, the tumor recurs during or after chemotherapy. In this work, we have examined the hypothesis that the development of resistance to chemotherapy in neuroblastoma might be related to the expression of the human MDR1 gene, which encodes a multidrug transporter that functions as an energy-dependent drug efflux pump. RNA samples from 49 neuroblastomas were analyzed, including 31 from untreated and 18 from treated patients. MDR1 RNA was detectable in the majority of treated and untreated tumors using a sensitive, semiquantitative slot blot assay. Of the samples from treated patients, five of 18 were found to have high MDR1 RNA levels, whereas only three of 31 from untreated patients had high MDR1 levels, a statistically significant difference (P less than .01). These results show that high levels of MDR1 RNA are often associated with resistance to chemotherapy in neuroblastoma and suggest that they may contribute to this resistance. Many of the neuroblastoma samples were also evaluated for N-myc amplification but there was no correlation between N-myc copy number and the level of MDR1 mRNA expression.

Implantation of recombinant rat myocytes into adult skeletal muscle: a potential gene therapy.

The ability of skeletal muscle to regenerate provides an excellent therapeutic entry, via genetic engineering, for correcting diseases of skeletal muscle and other tissues. We have used a retrovirus to transfer the cDNA for the human multidrug transporter, encoded by the MDR1 gene, into the genomes of the rat muscle cell line L6 and into primary rat myocytes. The MDR1 gene confers drug resistance to cells, and thus serves as a selectable marker in vitro. In cultured cells, the retroviral promoter-driven human MDR1 cDNA was shown to be stable in the presence or absence of drug selection or muscle cell fusion. MDR1 mRNA was synthesized, as shown by RNA blot analysis and in situ hybridization. The protein product was localized to the plasma membrane of transduced myocytes and myotubes by immunofluorescence. As a model for skeletal muscle gene therapy, transduced L6 myocytes were implanted into the tibialis anterior muscle of Wistar rats. The retroviral sequences of the human MDR1 gene and its mRNA were present in the muscles of Wistar rats 5 days, but not 12 days, after implantation, possibly because of immunorejection. On the other hand, the human MDR1 cDNA was stable in the tibialis anterior muscle of nude mice, which are incapable of immunorejection, at least 4 weeks after implantation of myocytes. Immunosuppression of Wistar rats with cyclosporine A delayed immunorejection of recombinant myocytes, and MDR1 cDNA and mRNA was detected 3-4 weeks after implantation. In situ hybridization revealed that injected recombinant myocytes remain in discrete foci in adult rodent skeletal muscle and express MDR1 mRNA for at least 30 days in nude mice and cyclosporine-treated rats.

Mitotane enhances cytotoxicity of chemotherapy in cell lines expressing a multidrug resistance gene (mdr-1/P-glycoprotein) which is also expressed by adrenocortical carcinomas.

P-Glycoprotein (Pgp), product of the mdr-1 gene, is a 130- to 180-kDa plasma membrane phosphoglycoprotein which mediates multidrug resistance in cell culture by increasing efflux of the natural product chemotherapeutic agents. High levels of expression of mdr-1/Pgp are found in both the normal adrenal and adrenocortical cancers. By RNA in situ hybridization the expression in adrenocortical cancer is shown to be widely distributed. The present study demonstrates that decreased drug accumulation mediated by mdr-1/Pgp can be overcome by clinically achievable concentrations of mitotane (o,p'-DDD). The increase in drug accumulation with the addition of mitotane is due at least in part to a decrease in drug efflux and results in an increase in cytotoxicity when agents of the natural product class are used. This effect is observed in cells with a broad range of mdr-1/Pgp expression, including levels comparable to those found in most adrenocortical cancers. Similar increases in drug accumulation can be demonstrated in an unselected adrenocortical cancer cell line that expresses mdr-1/Pgp. The finding that multidrug resistance mediated by mdr-1/Pgp can be reversed by mitotane provides a rational basis for exploring the use of mitotane in combination with natural product chemotherapeutic agents in adrenocortical cancer.

Initial pharmacokinetics and bioavailability of PSC 833, a P-glycoprotein antagonist.

Resistant cancer cells have been shown to overexpress a 170-kd membrane glycoprotein called P-glycoprotein. P-glycoprotein, a product of the multidrug resistance 1 gene, functions as an energy-dependent efflux pump that decreases intracellular drug concentrations. A variety of nonchemotherapeutic agents have been shown to inhibit P-glycoprotein-dependent drug efflux including cyclosporin. PSC 833 is a nonimmunosuppressive derivative of cyclosporin D with the ability to reverse multidrug resistance because of P-glycoprotein overexpression in vitro. As part of early clinical development of PSC 833, the authors investigated the bioavailability of an oral formulation of PSC 833. PSC 833 (3 mg/kg) was administered as a 2-hour intravenous infusion on day 1 of the treatment cycle. Serial blood samples for the determination of PSC 833 whole blood concentrations were obtained after both the intravenous and oral doses. On day 5 of the study, patients received a single oral dose (9 mg/kg) of PSC 833. A total of 14 patients were treated. The intravenous data were best described by a two-compartment open model. The oral data also were described using a two-compartment model, with oral absorption incorporating a lag time to account for possible delays in absorption. There was large intra- and interpatient variability in the pharmacokinetics of PSC 833 in these patients. The absolute bioavailability of PSC 833 was 34% but ranged from 3% to 58% of the administered dose. The clearance (CI) of PSC 833, in general, was consistent between the two dose forms administered. The pharmacokinetic behavior of PSC 833 appears to be similar to that of cyclosporine.

Transfection of a human topoisomerase II alpha gene into etoposide-resistant human breast tumor cells sensitizes the cells to etoposide.

The etoposide-resistant human breast cancer cell line MDA-VP was derived from MDA-parent cells by sequential selection in increasing concentrations of etoposide. MDA-VP cells express a lower amount of topoisomerase II alpha mRNA than the MDA-parent does, have mutations in topoisomerase II alpha (topo II) cDNA, and show cross-resistance to doxorubicin and amsacrine. We investigated whether transfer of a normal human topoisomerase II alpha (H-topo II) gene into MDA-VP cells could overcome their resistance to etoposide. H-topo II in a mammalian expression vector containing a glucocorticoid-inducible mouse mammary tumor virus (MMTV) promoter (pMAMneo) was transfected into MDA-VP cells (MDA-VP-hTOP2MAM). These H-topo II-transfected cells showed increased H-topo II mRNA expression and protein levels compared with MDA-VP parental cells or with MDA-VP cells transfected with the control pMAM vector (MDA-VP-MAM). Following cell exposure to dexamethasone, DNA-protein cleavable complex formation and cytotoxicity induced by etoposide, doxorubicin, and amsacrine were increased in the MDA-VP-hTOP2MAM cells compared with MDA-VP-MAM cells. However, these changes were short-lived, and by 24 h, cytotoxicity, cleavable DNA-protein complex formation, and H-topo II protein levels returned to baseline values. These results indicate that sensitivity of MDA-VP cells correlated with changes in cellular H-topo II. The gene transfer of a normal H-topo II gene can sensitize MDA-VP cells to the actions of multiple antineoplastic agents that target topo II.

Respiratory cryptosporidiosis in a patient with malignant lymphoma. Report of a case and review of the literature.

Respiratory cryptosporidiosis is a rare complication of intestinal infection by cryptosporidia, with only six cases reported (to our knowledge) since its first description in 1983. We report the first case of respiratory cryptosporidiosis recognized at the National Institutes of Health, Bethesda, Md. An antemortem diagnosis was made based on recognition of acid-fast cryptosporidia in an induced sputum specimen obtained from a 64-year-old woman with malignant lymphoma and an associated profound immunodeficiency. Autopsy confirmed the presence of cryptosporidia along the apical aspect of the respiratory epithelium lining the trachea, bronchi, and bronchioles. Cryptosporidia were also identified in the duodenum and gallbladder. Immunohistochemical staining of the paraffin-embedded autopsy lung sections using a monoclonal antibody verified the diagnosis of cryptosporidiosis. Review of our case and the literature suggests that respiratory cryptosporidiosis is characterized by a chronic tracheitis, bronchitis, and bronchiolitis but generally does not cause severe pulmonary dysfunction.

Multidrug resistance due to P-glycoprotein.

P-glycoprotein is a cell membrane transport protein in various human tissues that acts as an efflux pump, apparently to protect normal cells from environmental toxins. It is also a mechanism used by some cancer cells to resist chemotherapy. The gene responsible for P-glycoprotein is partially defined. Agents that inhibit the protein in tumors may make chemotherapy more effective and less toxic.

Effects of acetylation and guanidination on alkaline conformations of chymotrypsin.

Guanidination leads to stabilization of several globular proteins, including bovine chymotrypsinogen, as determined by hydrogen isotope exchange (P. Cupo, W. El-Deiry, P.L. Whitney, and W.M. Awad, Jr. (1980) J. Biol. Chem. 255, 10828-10833). The present study examined the binding of proflavin to guanidinated, acetylated, and native chymotrypsins in order to compare conformational flexibilities. The order of decreasing alkaline stabilities of the catalytically active conformations of the different delta-chymotrypsin forms was guanidinated, native, and acetylated proteins; delta-chymotrypsin showed greater stability than alpha-chymotrypsin. In each case removal of calcium reduced the amount of the catalytically active conformation. The alkaline pH dependence for the decrease of the catalytically active conformation of guanidinated alpha-chymotrypsin could not be attributed to the titration of a single group, indicating that the alpha-amino group of isoleucine-16 is not the sole feature regulating the conformational transition for this derivative. At neutral pH values delta-chymotrypsin exists completely in an active conformation and the percentage of alpha-chymotrypsin in this form is only slightly lower. These differences from earlier results are possibly due to differences in buffers, calcium ion concentrations, and ionic strength. The rate of inactivation of guanidinated delta-chymotrypsin with methyl acetimidate was much lower than the corresponding rate for the native enzyme. This suggests that guanidination increases enzyme stability which in turn leads to a reduced accessibility of the alpha-amino group of isoleucine-16.

Differential modulation of P-glycoprotein transport by protein kinase inhibition.

Previous studies of P-glycoprotein have demonstrated that its function can be modulated by phosphorylation. In the present study, inhibition of protein kinase C with calphostin C or stauroporine or prolonged treatment with the phorbol ester TPA decreased phosphorylation of P-glycoprotein, and impaired transport of vinblastine. Calphostin C also inhibited transport of actinomycin D, vincristine, rhodamine, and azidopine in SW620 Ad300 multidrug-resistant human colon carcinoma cells. Photoaffinity labeling of P-glycoprotein with azidopine was decreased by calphostin C, suggesting that dephosphorylation alters the affinity of P-glycoprotein for its substrates. Impaired transport of rhodamine in normal T lymphocytes treated with staurosporine demonstrates that modulation of P-glycoprotein function is not limited to cells selected for drug resistance in vitro. Transport of P-glycoprotein antagonists in SW620 Ad300 cells was also affected by calphostin C. Cyclosporin A transport decreased, while verapamil transport increased. Cyclosporin A in calphostin C-treated cells resulted in additive P-glycoprotein antagonism, while no additive effect could be demonstrated with verapamil, suggesting that the increase in verapamil transport makes it a poorer P-glycoprotein antagonist. These studies suggest that transport by P-glycoprotein is a dynamic process which can be modulated by phosphorylation, and that antagonists may block P-glycoprotein differently in different phosphorylation states.

Amplification of DNA sequences in human multidrug-resistant KB carcinoma cells.

Four KB carcinoma cell lines selected independently for resistance to either colchicine, adriamycin, or vinblastine were studied. All cell lines showed high levels of resistance to the selecting drug and cross-resistance to the other drugs and to actinomycin D. Double-minute chromosomes could be identified on chromosomal spreads of these multidrug-resistant KB cell lines. Amplification of specific DNA sequences was demonstrated by using the technique of in-gel renaturation. All the cell lines share common amplified sequences. There are also amplified sequences that are specific for each cell line. A revertant cell line that has reacquired drug sensitivity has lost its amplified sequences. Specific probes obtained by cloning amplified sequences from the cell line selected in vinblastine recognize amplified sequences in all the resistant lines. The presence of common amplified sequences in these cell lines is strong evidence for the importance of these regions in multiple drug resistance.

Effect of glycerol on protein acetylation by acetic anhydride.

We investigated the basis for the previously unexplained stabilization of proteins by glycerol during reaction with acetic anhydride [S. Siegel and W. M. Award, Jr. (1973) J. Biol. Chem. 248, 3233-3240]. Model studies showed that glycerol competes successfully for acetylation against protein hydroxyl groups. In contrast, amino groups are much more potent nucleophiles and their acetylation is not apparently affected. Since alpha-amino and phenolic pKa's did not change significantly in increasing glycerol concentrations, these findings are ascribed to glycerol's lower pKa value as compared to water, leading to the decreased acetylation of tyrosine, threonine, and serine hydroxyl groups in Pronase guanidine-stable chymoelastase. An additional mechanism is important and predominates in the protection against inactivation of bovine delta-chymotrypsin during acetylation and is explained by the recently described basis for protein stabilization in glycerol [K. Gekko and S. N. Timasheff (1981) Biochemistry 20, 4667-4676; 4677-4686]. Those studies demonstrated that glycerol increased the hydrophobicity of nonpolar residues, augmenting their tendency to be removed from protein surfaces. Therefore, the stabilization afforded by glycerol for chymotrypsin is attributed in part to a favoring of the native folded state which forces the side chains of isoleucine-16 and valine-17 to be buried, increasing the apparent pKa of the alpha-amino group of isoleucine-16 as it forms the charge pair with the beta-carboxyl group of aspartate-194. This conclusion was supported by stopped-flow analyses of the interaction of delta-chymotrypsin with proflavin in increasing concentrations of glycerol.