Form Of Multidrug Resistance Research Articles

Clustered base substitutions in CTP synthetase conferring drug resistance in Chinese hamster ovary cells.

Dominantly acting mutations that eliminate the allosteric regulation of CTP synthetase confer a form of multidrug resistance and a mutator phenotype to cultured Chinese hamster ovary cells. Mutations responsible for this phenotype have been identified in 23 independent strains selected for resistance to arabinosyl cytosine and 5-fluorouracil. All these mutations were due to base substitutions at seven sites within a highly conserved region of the ctps gene. This clustering should make it feasible to assess the role of such mutations in the development of drug resistance encountered in the treatment of malignant disease.

Radiation and heat sensitivity of human T-lineage acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) clones displaying multiple drug resistance (MDR)

The hyperthermia as well as radiation responses of multidrug resistant (CEM/VLB 100 with classical MDR and CEM/VM-1 with atypical MDR), methotrexate resistant (CEM/MTX) subclones of CCRF-CEM T-lineage ALL cell line were compared with those of a drug sensitive (CEM-1–3) subclone from the same parent cell line. Also analyzed were the hyperthermia as well as radiation responses of multidrug resistant (HL60/AR) and drug sensitive subclones of the HL60 AML cell line. Notably, the drug resistant subclones of CEM and HL60 were as sensitive to hyperthermia as were the drug sensitive subclones. Importantly, no thermotolerant plateau was observed in the hyperthermia survival curves of the drug resistant subclones, indicating that drug/multidrug resistance is not associated with a greater likelihood of thermal tolerance development during hyperthermia. Similarly, the drug resistant CEM and HL60 subclones were not more radiation resistant than the drug sensitive subclones. Thus, the classical or atypical forms of multidrug resistance or methotrexate resistance of the analyzed leukemic cell lines were not associated with radiation resistance. Furthermore, the radiation survival curves of the drug resistant subclones lacked a distinct initial shoulder and their n values were not greater than those of the drug sensitive subclones, suggesting that multidrug resistance is not associated with an increased ability to repair or accumulate sublethal radiation damage. Our findings provide evidence that there is no apparent association between drug/multidrug resistance and heat or radiation sensitivity of CEM T-lineage ALL or HL60 AML leukemia cells. The results of this study indicate that acquired resistance to methotrexate, vinblastine, vincristine, etoposide, actinomycin-D, adriamycin, or daunomycin, or pleiotropic multidrug resistance do not necessarily confer radiation resistance for human leukemic cells.

Do Anti-P-Glycoprotein Antibodies Have a Future in the Circumvention of Multidrug Resistance?

Discovering how to circumvent or overcome cancer drug resistance has become the Holy Grail of experimental chemotherapists and clinicians. It is clear that patients' tumors can become resistant to all the therapies they receive, and outwitting the tumor cells' resistance mechanisms has become an increasingly difficult challenge. In recent years, we have learned that the phenomenon of multidrug resistance, characterized by cross-resistance to many natural product anticancer drugs, decreases drug accumulation and retention and that Pglycoprotein (Pgp) expression (7) may be an important factor in some of the clinical failures of cancer chemotherapy. A number of tumors have been shown to express Pgp either de novo or after chemotherapy (23), and recent results suggest that Pgp expression may be an important indicator of therapeutic outcome (4). While non-Pgp forms of natural-product multidrug resistance have been described experimentally (5,6), they have not yet been shown in patients' tumors, principally because appropriate reagents that detect them are presently unavailable. Accordingly, efforts directed at circumventing multidrug resistance are focused on inhibition or modulation of Pgp function or expression and include screening and rational design of Pgp-inhibitory chemicals, as well as the use of modulator combinations, inhibitory antibodies, and antisense oligonucleotides (7,8). Most experimental and clinical efforts to circumvent multidrug resistance have used drugs such as verapamil, quinidine, amiodarone, and cyclosporine that likely block the efflux function of Pgp (8). This approach has not yet proven satisfactory in the clinical setting in part because of the limitation of available modulators and because of their toxic effects (9,10). Planned or in progress are clinical trials of compounds (11,12) that appear to have a far more favorable therapeutic index than some of the first generation modulators (such as verapamil) and may be effective in maximizing the chemotherapy of drug resistant disease associated with the expression of Pgp. Among the other possibilities to circumvent Pgp-associated multidrug resistance, several would seem to hold promise for the near future. Application of modulator combinations, which has provided some encouraging preliminary results in cell culture (13,14), has the advantage of maximum inhibition of Pgp without the dose-limiting toxic effects seen with single modulator treatment. This approach, however, may also suffer from lack of specificity and nonspecific toxicity. By contrast, the use of anti-Pgp antibodies to circumvent Pgp-mutlidrug resistance offers the prospect of specificity, since the antibodies should only target Pgp; toxicity would only be that associated with the administration of protein. It is obvious that only an antibody that recognizes surface determinants of Pgp will have a chance of modulating Pgp-multidrug resistance in vivo. Anti-Pgp antibodies have been administered in vivo alone, in combination with anticancer drugs, and coupled to a toxin. Tsuruo et al. developed two antibodies that recognize surfaceexposed epitopes of Pgp (15) and showed that one, MRK-16 monoclonal antibody, blocked the growth of multidrug resistant human tumor xenografts in athymic mice (76). In this issue of the Journal, Pearson et al. (17) have taken the experiment one step further: using a transplantable human colon carcinoma cell line infected with a retrovirus carrying a complementary DNA containing the MDR1 gene (also known as PGY1), the authors also showed that MRK-16 monoclonal antibody was able to overcome Pgp-multidrug resistance in vitro and, more importantly, in xenografts in vivo when used in combination with vincristine. Of interest, neither the antibody alone nor vincristine alone were able to enhance the survival of mice bearing the Pgpexpressing xenografts. A preliminary account of similar findings was reported last year by Rittman-Grauer et al. (18) using the other antibody that recognizes surface determinants of Pgp, HYB-241. By contrast, Fitzgerald et al. (79) coupled MRK-16 monoclonal antibody to ricin and showed that this immunotoxin was effective in selectively killing tumor cells that expressed

Cellular and pharmacologic aspects of drug resistance in acute myeloid leukemia

Modern treatments and supportive care regimens for acute myeloid leukemia have produced some cures of what was once a uniformly fatal disease. To enhance the cure rate, intense efforts are now being put forth to understand the mechanisms of resistance to chemotherapeutic regimens that actually arise in clinical cases of acute myeloid leukemia, and to develop ways and means to circumvent such resistance. This review focuses on such efforts involving daunorubicin and cytarabine, the two most effective agents currently available for treatment of the disease. Most current published studies of daunorubicin have focused on detecting the classic form of multidrug resistance. For cytarabine, pharmacologically directed treatment approaches and enhancement of cytarabine leukemic cell kill by increasing the distribution of cells in S-phase and manipulating cellular cytarabine transport are currently under investigation.

Transgenic mice that express the human multidrug-resistance gene in bone marrow enable a rapid identification of agents that reverse drug resistance.

The development of preclinical models for the rapid testing of agents that circumvent multidrug resistance in cancer is a high priority of research on drug resistance. A common form of multidrug resistance in human cancer results from expression of the MDR1 gene, which encodes a Mr 170,000 glycoprotein that functions as a plasma membrane energy-dependent multidrug efflux pump. We have engineered transgenic mice that express this multidrug transporter in their bone marrow and demonstrated that these animals are resistant to leukopenia by a panel of anticancer drugs including anthracyclines, vinca alkaloids, etoposide, taxol, and actinomycin D. Differential leukocyte counts indicate that both neutrophils and lymphocytes are protected. Drugs such as cisplatin, methotrexate, and 5-fluorouracil, which are not handled by the multidrug transporter, produce bone marrow suppression in both normal and transgenic mice. The resistance conferred by the MDR1 gene can be circumvented in a dose-dependent manner by simultaneous administration of agents previously shown to be inhibitors of the multidrug transporter in vitro, including verapamil isomers, quinidine, and quinine. Verapamil and quinine, both at levels suitable for human trials that produced only partial sensitization of the MDR1-transgenic mice, were fully sensitizing when used in combination. We conclude that MDR1-transgenic mice provide a rapid and reliable system to determine the bioactivity of agents that reverse multidrug resistance in animals.

Mechanisms of multidrug resistance in human tumor cells. The roles of P-glycoprotein, DNA topoisomerase II, and other factors

Multidrug resistance (MDR) associated with overexpression of P-glycoprotein (Pgp) is a well-described experimental phenomenon that appears to have clinical correlates. However, recent descriptions of non-P-glycoprotein forms of MDR have complicated efforts to detect and circumvent MDR in the tumors of patients. One major form of natural product MDR appears to be due to alterations in the amount or activity of DNA topoisomerase II. Compared to Pgp-MDR cells, cells expressing this form of MDR (atMDR) do not overexpress the mdrl gene or its product, Pgp, are unaltered in drug accumulation and retention, are unaffected by such ‘modulators’ of Pgp-MDR as verapamil, and express this phenotype recessively. Recently, other MDR cell lines have been described with some characteristics of Pgp-MDR (decreased drug accumulation and retention, increased drug cytotoxicity by modulators of MDR), but not others (no expression of the mdrl gene or Pgp). Whether any non-Pgp forms of MDR occur in patients' tumors remains to be determined.

Decreased nuclear matrix DNA topoisomerase II in human leukemia cells resistant to VM-26 and m-AMSA

CEM leukemia cells selected for resistance to VM-26 (CEM/VM-1) are cross-resistant to various other DNA topoisomerase II inhibitors but not to Vinca alkaloids. Since DNA topoisomerase II is a major protein of the nuclear matrix, we asked if alterations in nuclear matrix topoisomerase II might be important in this form of multidrug resistance. Pretreatment of drug-sensitive CEM cells for 2 h with either 5 microM VM-26 or 3 microM m-AMSA reduced the specific activity of newly replicated DNA on the nuclear matrix by 75 and 50%, respectively, relative to that of the bulk DNA. However, neither VM-26 nor m-AMSA affected the relative specific activity of nascent DNA isolated from the nuclear matrices of drug-resistant CEM/VM-1 cells. The decatenating and unknotting activities of DNA topoisomerase II were 6- and 7-fold lower, respectively, in the nuclear matrix preparations from the CEM/VM-1 cells compared to parental CEM cells. Western blot analysis revealed that the amount of immunoreactive topoisomerase II in the nuclear matrices of the CEM/VM-1 cells was decreased 3.2-fold relative to that in CEM cells, but there was no significant difference in the amount of enzyme present in the nonmatrix (1.5 M salt soluble) fractions of nuclei from these cell lines. Increasing the NaCl concentration used in the matrix isolation procedure from 0.2 to 1.8 M resulted in a progressive decrease in the specific activity of topoisomerase II in matrices of CEM/VM-1 but not CEM cells, which suggested that the association of the enzyme with the matrix is altered in the resistant cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple Patterns of Resistance of Human Leukemia Cell Sublines to Amsacrine Analogues

The antileukemia drug amsacrine and seven analogues were tested for in vitro activity against five multidrug-resistant human leukemia cell sublines (two derived from each of two Jurkat parent lines and one from the K562 line) and the corresponding parent lines (Jurkat, K562, and P388 leukemia). Resistant cell lines were derived by exposure to either amsacrine or doxorubicin. The resistance factor was calculated as the ratio of the mean IC50 value for the resistant cell line to that for the parent line. IC50 was defined as the concentration of drug inhibiting cell growth to 50% of that in control (drug-free) cultures. Patterns of cross-resistance were visualized by plotting the deviations of resistance factors from the mean resistance factor, on a logarithmic scale. Considerable variations in resistance factors were noted for each cell subline as the amsacrine substituents were altered. Four main patterns were evident: a transport-related multidrug-resistance pattern (three sublines), a pattern similar to that for a murine P388 leukemia subline resistant to amsacrine, and two patterns not observed previously. Since some of the sublines tested showed evidence of altered topoisomerase II activity, it appears that changes in the resistance pattern in these lines may reflect changes in the stability of the ternary complexes formed by the drug, the altered enzyme, and the DNA. The resistance factor was reduced from more than 100-fold to about 13-fold in three of the sublines tested and from eightfold to twofold in two others. This finding suggests that appropriate drug design may overcome several forms of multidrug resistance in human leukemia and other types of cancer.

A monoclonal antibody-Pseudomonas toxin conjugate that specifically kills multidrug-resistant cells.

One form of multidrug resistance is due to the expression of a 170-kDa energy-dependent drug efflux pump called P-glycoprotein in the plasma membranes of human cancer cells. We have prepared conjugates of Pseudomonas toxin with the anti-P-glycoprotein monoclonal antibody MRK-16. These anti-P-glycoprotein-toxin conjugates specifically kill multidrug-resistant human KB cells. Similar conjugates could be useful in cancer therapy to reduce or eliminate multidrug-resistant tumor populations in tumors intrinsically resistant to chemotherapy or in populations that become resistant during combination chemotherapy.