Multidrug resistance in a human leukemic cell line selected for resistance to trimetrexate

Trimetrexate (TMQ) is a lipophilic antifolate shown to have antitumor activity in humans. TMQ-resistant sublines of the MOLT-3 human acute lymphoblastic leukemia cell line were developed and were designated as MOLT-3/TMQ200, MOLT-3/TMQ800, and MOLT-3/TMQ2500 based on degrees of resistance to TMQ. The TMQ resistance was accompanied by 5- to 7-fold increases in dihydrofolate reductase activity and markedly reduced cellular TMQ accumulation. Methotrexate accumulation was not impaired in TMQ-resistant cells. TMQ retention (efflux) was unchanged in these TMQ-resistant cells. Verapamil enhanced the TMQ accumulation in the resistant cells to the level seen in the parent cells but had no effects on the TMQ retention. These sublines were cross-resistant not only to methotrexate but also to vincristine, doxorubicin, daunorubicin, and mitoxantrone. There was no cross-resistance to bleomycin or cisplatin. Resistance to vincristine, doxorubicin, daunorubicin, and mitoxantrone was reversed by verapamil. TMQ resistance was only minimally reversed by verapamil and methotrexate resistance not affected at all. Both cellular accumulation and retention of vincristine and daunorubicin in the TMQ-resistant cells were markedly decreased. Verapamil enhanced both accumulation and retention of the drug. Plasma membrane fractions of the TMQ-resistant cells analyzed by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue revealed the presence of a distinct band with a molecular weight of 170,000. Immunoblot analysis with 125I-labeled monoclonal antibody raised against P-glycoprotein of multidrug-resistant Chinese hamster ovary cells (C219) cross-reacted with the Mr 170,000 protein of the TMQ-resistant cells. These results show that the TMQ-resistant cells displayed not only decreased TMQ uptake and increased dihydrofolate reductase but also characteristics associated with a classical multidrug-resistant phenotype. Multidrug resistance includes lipophilic antifolate.     

Multidrug resistance in a human small cell lung cancer cell line selected in adriamycin

A multidrug resistant variant (H69AR) of the human small cell lung cancer cell line NCI-H69 was obtained by culturing these cells in gradually increasing doses of Adriamycin up to 0.8 microM after a total of 14 months. H69AR expresses the multidrug resistant phenotype because it is cross-resistant to anthracycline analogues including daunomycin, epirubicin, menogaril, and mitoxantrone as well as to acivicin, etoposide, gramicidin D, colchicine, and the Vinca alkaloids, vincristine and vinblastine. H69AR is also similar to other multidrug resistant cell lines in that it displays little or no cross-resistance to bleomycin, 5-fluorouracil, and carboplatin. It has a slight collateral sensitivity to 1-dehydrotestosterone and lidocaine. H69AR has increased cell-cell adhesiveness compared to H69, but a similar growth rate in vitro and tumorigenicity in nude mice. When cultured in the absence of Adriamycin, there is a 40% decrease in resistance by 35 days of culture, compared to cells in continuous culture in drug, but no further decrease in resistance up to 181 days. Monoclonal antibodies to P-glycoprotein have no detectable reactivity with H69AR cells as determined by enzyme-linked immunosorbent assay and immunoblotting techniques. Thus, unlike most multidrug resistant cell lines, H69AR does not appear to express enhanced levels of P-glycoprotein. H69AR will provide a useful model for the study of multidrug resistance in human small cell lung cancer.     

Multidrug Resistance Phenotype in the RMS-GR Human Rhabdomyosarcoma Cell Line Obtained after Polychemotherapy

Classical cytotoxic treatment of rhabdomyosarcoma (RMS), the most common soft tissue malignancy in children, is often accompanied by significant morbidity and poor response. Chemotherapy may induce multidrug resistance (MDR) associated with the expression of P-glycoprotein, a drug efflux pump which modifies the sensitivity of tumoral cells to drugs. To analyze MDR in RMS we used the RMS-GR cell line, obtained from an embryonal rhabdomyosarcoma treated in vivo with polychemotherapy. The RMS-GR cells showed cross-resistance to vincristine, doxorubicin and actinomycin D, the drugs of choice in the conventional treatment of RMS. Polymerase chain reaction (PCR) analysis showed that these RMS cells overexpressed mdr1 /P-glycoprotein. The pattern of resistance and the level of P-glycoprotein expression were similar to those found in the resistant RMS TE.32.7.DAC cell line obtained in vitro . Southern blot analysis showed that mdr1 overexpression was not due to amplification of the gene. Our results showed that the in vivo treatment of embryonal RMS may induce an MDR phenotype mediated by mdr1 /P-glycoprotein in RMS cells.     

Multidrug resistance and rhabdomyosarcoma (Review)

Classical cytotoxic treatment of rhabdomyosarcoma (RMS) is often accompanied by significant morbidity and poor response. This cytotoxic therapy may induce a multidrug resistance (MDR) phenotype in RMS which is associated with decreased effectiveness of chemotherapy. The majority of MDR molecules belong to a family of ABC (ATP binding cassette) transporters. Studies of drug resistance in RMS suggest that there are various mechanisms acting simultaneously, which might explain the low percentage of long-term survival in this malignancy. Moreover, although cells exposed to cytotoxic agents increase expression of muscle differentiation markers indicating myogenic differentiation, multidrug resistance may be a major obstacle in differentiation therapy for RMS. This review briefly discusses the current knowledge of resistance in RMS and emphasizes the importance of understanding the different aspects of MDR status in these patients.     

Multidrug (pleiotropic) resistance in doxorubicin-selected variants of the human sarcoma cell line MES-SA

The emergence of drug-resistant tumor cells is a major limiting factor in cancer chemotherapy. There is little information about the nature of such resistant variants among human cancer cell populations. Doxorubicin (DOX)-resistant sublines of the human sarcoma cell line MES-SA were selected by continuous in vitro exposure to DOX. Stepwise increases in DOX concentration produced variants which were 25- and 100-fold resistant to DOX. These sublines displayed marked cross-resistance to daunorubicin, dactinomycin, mitoxantrone, colchicine, vincristine, vinblastine, and etoposide and moderate resistance to mitomycin C and melphalan. Cross-resistance was not observed, however, to methotrexate, 5-fluorouracil, bleomycin, carmustine, or cisplatin. DOX resistance in these cell lines appeared to be stable despite long periods of growth in drug-free medium. Two additional marker chromosomes were identified in the 100-fold resistant variant, which indicated clonal selection during drug exposure, but no double minute chromosomes or homogeneously staining regions were noted. Doxorubicin accumulation in the DOX-resistant cells was reduced by approximately 50% compared to that of the sensitive MES-SA cells, as a result of enhanced efflux of DOX from the resistant cells. There was no evidence of appreciable DOX metabolism by either the sensitive or resistant cells. These studies demonstrate marked DOX resistance and multidrug resistance arising in a human sarcoma line during exposure to DOX. The pleiotropic nature of this resistance is similar to that described in other models. Decreased drug accumulation due to enhanced drug efflux is identified as a major mechanism of resistance in these cells, although other factors may also be involved.     

Therapeutic differentiation in a human rhabdomyosarcoma cell line selected for resistance to actinomycin D

Classical cytotoxic treatment of rhabdomyosarcoma (RMS) is accompanied often by significant morbidity and poor response. The use of cytotoxic agents may induce a multidrug resistance phenotype, which plays an important role in the sensitivity of tumoral cells to drugs. Using actinomycin D, a drug of choice in the treatment of RMS, we induced resistance in the TE.32.7 human RMS cell line. The TE.32.7-DAC-resistant cell line exhibited cross-resistance to vincristine and doxorubicin and showed mdr1/P-glycoprotein over-expression, suggesting that this mechanism was involved in the reduction in intracellular drug concentration and may be responsible for the failure of treatment of RMS with classical cycles of cytotoxics. Furthermore, this resistant cell line showed increased expression of the muscle differentiation markers desmin and α-actinin and ultrastructural changes which clearly indicated myogenic differentiation. Our findings suggest that, although this tumor is probably arrested along the normal myogenic pathway to maturation, induction of cell differentiation with anti-neoplastic drugs may be an alternative therapeutic approach. However, the failure of TE.32.7-DAC cells to completely re-enter the program of myogenic differentiation supports the hypothesis that multidrug resistance is a major obstacle in differentiation therapy for RMS. Int. J. Cancer 75:379–383, 1998. © 1998 Wiley-Liss, Inc.     

HL-60/VCR多药耐药细胞株耐药机制的研究

背景与目的：白血病细胞对化疗药物的耐药是白血病治疗失败的主要原因,多药耐药细胞株为白血病多药耐药机制和逆转多药耐药性的研究提供了良好的模型。为探讨药物诱导产生多药耐药机制,我们对HL-60/VCR细胞的耐药机制进行了研究。方法：应用流式细胞术和一组抗体,对药物敏感细胞株HL-60和多药耐药细胞株HL-60/VCR细胞的耐药相关蛋白P-gp、MRP、LRP、BCRP、GST-π,以及凋亡调节蛋白bcl-2、bax、bcl-x、bad的表达进行分析。结果：在HL-60/VCR细胞株中,耐药相关蛋白P-gp、MRP、BCRP、GST-π分别是其在HL-60细胞株中的18.62、1.19、1.50、1.32倍,而LRP无变化。凋亡抑制蛋白bcl-2、bcl-x分别是其在HL-60细胞株中的2.48、1.25倍,凋亡调节蛋白bad是HL-60细胞株中的1.08倍;而凋亡诱导蛋白bax反而降低,是HL-60细胞株中的0.88倍。结论：多种机制参与HL-60/VCR的多药耐药,涉及耐药蛋白P-gp、MRP、BCRP和GST-π的表达增强,而且凋亡调节蛋白bcl-2、bax、bcl-x、bad均可能参与其耐药机制的形成。     

In vitro prevention of the emergence of multidrug resistance in a pediatric rhabdomyosarcoma cell line.

We have established preclinical models for the development of drug resistance to vincristine (a major drug used in the treatment of pediatric rhabdomyosarcoma) using cell lines. The RD cell line has a mutant P53 phenotype and does not have detectable P-glycoprotein (P-gp) or multidrug resistance-related protein (MRP) despite expressing low levels of mdr-1 mRNA, which encodes P-gp and mrp1 mRNA. Resistant variants of RD were derived by exposure to increasing concentrations of vincristine. This was repeated on six occasions, resulting in three cell lines which could tolerate 64 x the IC(50) concentration. Six independent agents were tested for their ability to prevent the development of resistance in this model. Despite at least 10 attempts, resistance did not develop in the presence of the multidrug resistance (MDR) modulators PSC833, VX710, and XR9576. This strongly suggests that these agents may delay or even prevent the development of resistance to vincristine. This was also confirmed in a second rhabdomyosarcoma cell line, Rh30. In contrast, the agents indomethacin (MRP1 modulator), CGP41251 (protein kinase C inhibitor), and dexrazoxane (putative MDR prevention agent) did not affect the development of resistance in the RD model. Characterization of the resistant cell lines indicated the presence of increased mdr-1 and P-gp expression, which resulted in resistance to the agents doxorubicin, etoposide, and vincristine but not cisplatin. The resistance could be modulated using PSC833 or VX710, confirming that functional P-gp is present. No apparent differences were seen between the resistant cell lines derived in the absence and presence of the various agents. These experiments strongly suggest that the development of MDR may be preventable using modulators of MDR and merit clinical studies to test this hypothesis.     

Multidrug resistance in human cancer

Resistance to cytotoxic chemotherapy continues to be a major obstacle to more effective treatment of human cancers. A particular problem in clinical cancer chemotherapy is the phenomenon of simultaneous resistance of cancers to a variety of unrelated cytotoxic agents. Such resistance to multiple drugs is observed much more often than resistance to individual compounds. A similar experimental phenomenon has been termed multidrug resistance or MDR. Much has been learned in recent years about molecular mechanisms which can lead to MDR in cancer cells and a number of studies has been performed to evaluate the clinical relevance of such mechanisms. In particular, P-glycoprotein-associated MDR (MDR1) has received a lot of attention. This review will discuss (i) some principal aspects of drug resistance in cancer with particular emphasis on MDR1; (ii) available data on drug resistance mechanisms in brain tumors; and (iii) our current knowledge on the putative role of P-glycoprotein in the blood-brain barrier.     

Isolation of tumor cell growth-inhibiting factors from a human rhabdomyosarcoma cell line

Two types of growth-modulating factors were derived from the serum-free conditioned media of a human rhabdomyosarcoma cell line, A673. One type, Mr 18,000 to 22,000, competes for binding to epidermal growth factor receptors and enhances the growth of normal and tumor cells in soft agar. It has all of the biological properties ascribed to transforming growth factor type alpha (TGF alpha). A673 cells also produce factors which inhibit the growth of human tumor cells in soft agar and in monolayer cultures. These tumor cell growth-inhibiting factors (TIFs) are acid- and heat-stable peptides. The major TIF activities have molecular weights in the ranges of greater than 28,000, 18,000 to 22,000, 10,000 to 16,000, and 5,000 to 10,000 and do not possess the antiviral activity associated with interferon. Partially purified preparations of TIF-1 (Mr 10,000 to 16,000) inhibit the growth of all human tumor cell lines tested and stimulate the growth of normal human fibroblasts and epithelial cells in monolayer cultures. The growth of human lung carcinoma A549 cells in soft agar, which was enhanced by treatment with TGF alpha from A673-conditioned media, was inhibited by treatment with TIF-1 derived from the same media. The ratio of the two types of tumor cell-derived, growth-modulating factors (TIFs and TGF alpha), which are antagonistic in their biological effects, may determine the capacity of tumor cells for anchorage-independent growth.     

Two distinct tumor cell growth-inhibiting factors from a human rhabdomyosarcoma cell line

Tumor cell growth-inhibiting factors (TIFs) have been shown to inhibit the growth of tumor cell lines in culture. TIF-1, the first TIF to be described, is a low-molecular-weight, acid- and heat-stable polypeptide with no antiviral activity. A second class of TIFs (TIF-2) has now been isolated from the conditioned media of a human rhabdomyosarcoma cell line and partially purified by polyacrylamide gel filtration, cation exchange, and reverse-phase high-pressure liquid chromatography. Partially purified preparations of TIF-2 inhibit the growth of a variety of human tumor cells in soft agar and monolayer cultures and are mitogenic for normal human and mouse cells. TIF-2 has no antiviral activity. The growth-inhibitory effects of TIF-2 are reversible when the affected cells are no longer exposed to the factor. Although both TIF-1 and TIF-2 are obtained from the same source, they can be distinguished by their molecular weight, heat lability, elution pattern from reverse-phase high-pressure liquid chromatography, and their effect on the growth of mink lung epithelial cells. The growth of a human tumor cell variant, selected for resistance to growth inhibition by TIF-1, is inhibited by TIF-2. TIFs may therefore be a family of related polypeptides which selectively inhibit the growth of tumor cells.     

Radiation resistance in a doxorubicin-resistant human fibrosarcoma cell line.

In clinical practice, cancers refractory to chemotherapy can appear relatively radioresistant. Recent work in multidrug-resistant cell lines has yielded conflicting results concerning the relationship between drug resistance and radiation resistance. The current study examines the radiation response of a human fibrosarcoma (HT1080) and a doxorubicin-resistant subline (HT1080/DR4). Using soft-agar colony formation after graded doses of x-rays as an endpoint, HT1080/DR4 had an increased D0 (D0 = 2.1 Gy) and a broader initial shoulder (n = 2.7, Dq = 2.1 Gy) than the parental HT1080 line (D0 = 0.7, Gy, n = 1.2, Dq = 0.3 Gy), suggesting that HT1080/DR4 has an increased capacity to repair radiation-induced DNA damage. This possibility was tested by comparing the cell lines' ability to accumulate sublethal damage. In split-dose recovery experiments, HT1080/DR4 demonstrated increased ability to repair sublethal radiation damage following fractionated irradiation, compared with the HT1080 parental line. The mechanism for this radiation resistance is not clear, but a variety of cellular alterations seen in drug-resistant cell lines are discussed with reference to areas of further study.     

A human B-cell lymphoma line with a de novo multidrug resistance phenotype.

A human diffuse large cell lymphoma line (WSU-DLCL) expressing multidrug resistance (MDR) was established from a patient with primary chemotherapy-resistant disease. This cell line has the same phenotypic features as malignant cells from the patient. The established cell line has features of a mature B-cell neoplasm with no evidence for commitment to other lineages. WSU-DLCL grows in suspension forming relatively large clumps of cells with a doubling time of 20 hours. By light microscopic examination, the cells are very large with primitive lymphoid features, have a large amount of cytoplasm containing numerous vacuoles and an irregular outline. Immunophenotypic characterization by monoclonal antibodies and flow cytometric analysis showed a monoclonal IgM kappa B-cell phenotype with high expression of the multidrug-resistant P-glycoprotein compared with either normal peripheral blood lymphocytes or cells of the REH cell line. The cells were negative for T-cell and myeloid/monocyte antigens as well as Epstein-Barr virus nuclear antigen (EBNA). In addition, the cell line expressed high levels of MDR RNA. DNA histogram generated by flow cytometry indicated a DNA index of 1.83. Cytogenetic analysis confirmed hypertriploidy and showed complex chromosomal abnormalities including 14q+. This cell line should be a valuable tool to study the role of the MDR gene in the primary resistance of lymphomas to chemotherapy and to facilitate therapeutic investigations.     

Synthetic androgens suppress the transformed phenotype in the human prostate carcinoma cell line LNCaP.

Experiments have been designed to investigate hormonal effects on the human prostatic carcinoma cell line LNCaP in the presence of complete foetal calf serum. At physiological concentrations (3.3 x 10(-9)M), several derivatives of 17 alpha-methyl-testosterone led to a significant reduction of cell proliferation, inhibition of colony formation in soft agar, change of morphology, induction of a prostate specific mRNA and down-regulation of c-myc RNA. Two different antiandrogens, hydroxyflutamide and cyproterone acetate, were capable of reversing the effects exerted by the synthetic androgens on growth properties. The proliferation rate of control cells devoid of androgen receptor was not inhibited by synthetic androgens. Our results indicate that the cellular androgen response mechanism of LNCaP cells is intact and that synthetic androgens elicit androgen receptor mediated suppression of the transformed phenotype. Rare cases of remission of prostatic cancer on androgen treatment have been reported. LNCaP cells may be a model of an uncommon class of prostatic cancer which responds favourably to androgen treatment.     

12-O-tetradecanoylphorbol-13-acetate-induced differentiation of a human rhabdomyosarcoma cell line

The effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on proliferation and differentiation of the human embryonal rhabdomyosarcoma cell line RD was investigated. The proliferation of RD cells is drastically and reversibly inhibited by 100 nM TPA. The effect is evident after 24 h of treatment and is maximal after 50-70 h. The reduction of proliferation in treated cells is followed by increased expression of differentiative characters such as a large increase in muscle myosin expression and in the binding of 125I-alpha-bungarotoxin. Moreover TPA induces the appearance of myotube-like structures, which contain bundles of thick and thin myofilaments along with Z bodies. The described effects are not observed if the TPA-containing medium is replaced daily, thus suggesting that these effects might be related to substances secreted by treated cells. The phosphorylation of three proteins is significantly stimulated by TPA within minutes of its administration to RD cells. Although with a different pattern, the stimulation of protein phosphorylation is still clearly detectable after 6 days of incubation with TPA. These results on human rhabdomyosarcoma cells are, to our knowledge, the first evidence for a growth-inhibiting and a differentiative effect of TPA on a solid tumor of mesodermal origin.     

塞来昔布逆转MCF-7/Adr细胞多药耐药及其机制探讨

目的观察环氧合酶-2（COX-2）抑制剂塞来昔布对肿瘤多药耐药（MDR）的逆转作用,并探讨其初步作用机制。方法在乳腺癌耐药细胞株MCF-7/Adr中,应用MTT方法检测塞来昔布对多柔比星（阿霉素,DOX）的耐药逆转倍数;RT-PCR及Western blot方法检测基因及蛋白的变化;流式细胞术（FCM）检测细胞膜P-gp的表达、功能、细胞周期和凋亡。结果塞来昔布作用24小时后,MCF-7/Adr细胞对DOX的敏感性明显增加,浓度为10μmol/L及20μmol/L时,耐药逆转倍数分别为1.82和3.80,呈现剂量依赖性。COX-2、mdr1、c-Jun、YB-1、survivin等在基因和蛋白水平明显下调（P〈0.01）,NF-κB无明显差异（P〉0.05）;20μmol/L作用24小时,P-gp的功能受抑制;G0＋G1期细胞增多,S期细胞减少（P〈0.05）,凋亡率明显增高（P〈0.05）。结论选择性环氧合酶-2抑制剂塞来昔布可有效逆转MCF-7/Adr细胞多药耐药,在一定的浓度范围内呈现剂量依赖性,初步机制可能涉及干预转录因子的表达而下调mdr1,阻滞细胞周期及增加细胞凋亡。