Resistance Of Human Tumor Cells Research Articles

Divergent Transcriptional Control of Multidrug Resistance Genes in Saccharomyces cerevisiae

Improper control of expression of ATP binding cassette transporter-encoding genes is an important contributor to acquisition of multidrug resistance in human tumor cells. In this study, we have analyzed the function of the promoter region of the Saccharomyces cerevisiae YOR1 gene, which encodes an ATP binding cassette transporter protein that is required for multidrug tolerance in S. cerevisiae. Deletion analysis of a YOR1-lacZ fusion gene defines three important transcriptional regulatory elements. Two of these elements serve to positively regulate expression of YOR1, and the third element is a negative regulatory site. One positive element corresponds to a Pdr1p/Pdr3p response element, a site required for transcriptional control by the homologous zinc finger transcription factors Pdr1p and Pdr3p in other promoters. The second positive element is located between nucleotides -535 and -299 and is referred to as UASYOR1 (where UAS is upstream activation sequence). Interestingly, function of UASYOR1 is inhibited by the downstream negative regulatory site. Promoter fusions constructed between UASYOR1 and the PDR5 promoter, another gene under Pdr1p/Pdr3p control, are active, whereas analogous promoter fusions constructed with the CYC1 promoter are not. This suggests the possibility that UASYOR1 has promoter-specific sequence requirements that are satisfied by another Pdr1p/Pdr3p-regulated gene but not by a heterologous promoter.

Abrogation of P53 function by transfection of HPV16 E6 gene does not enhance resistance of human tumour cells to ionizing radiation.

Suppression of wild-type p53 expression has been shown to enhance the radiation resistance of human diploid fibroblasts, but results concerning the role of p53 expression in the sensitivity of human tumour cells have been conflicting. In order to address this question, we transfected four human tumour cell lines with the human papilloma virus 16 E6 gene and compared the radiosensitivity of subclones expressing E6 with that of subclones transfected with the neo gene alone. E6 binds to wild-type p53 promoting its degradation and abrogating its function. Two of these cell lines, one derived from a squamous cell carcinoma and the other an osteogenic sarcoma, expressed wild-type p53. The other two cell lines were of similar origins and histologies but expressed mutant or no p53 (null). Insertion of E6 into the cell was accomplished by two techniques: (1) to-transfection of plasmid vectors containing neo and E6; (2) infection with a retroviral vector containing neo and E6. Multiple transfected subclones were examined for each cell line. Transfection with E6 and abrogation of p53 function had no significant influence on the radiosensitivity of any of the cell lines tested. In particular, there was no evidence that loss of wild-type p53 function increased the resistance of these human tumour cell lines to ionizing radiation.

Use of hydroxyurea to alter drug resistance of human tumor cells.

Tumor cell resistance to cancer chemotherapeutic agents is a well-recognized problem for clinicians. Efforts are being made to develop agents that are not affected by cross-resistance to other drugs, as observed with the mdr phenotype. Other efforts are focused on reversing drug resistance to enhance chemotherapeutic intervention. Gene amplification accounts for one mechanism through which tumor cells develop drug resistance. Since amplified genes may be unstable, the elimination of these genes is likely to be a promising new target for cancer chemotherapy. The use of HU at low concentrations either to reestablish tumor sensitivity to chemotherapeutic agents or to decrease tumorigenicity, accomplished by the reduction of oncogene copy number, continues to be investigated. Studies thus far all report similar effects of noncytotoxic concentrations of HU on unstably amplified genes (EC DNA elimination), regardless of what gene is harbored on the EC DNA. The next essential step in the evaluation of HU-induced EC DNA elimination is to study the phenomena in vivo. In spite of extensive tissue distribution, HU appears to have pharmacokinetic properties, due to its short half-life, that may limit investigators' ability to study its use in prototype animal tumor models such as the nude mouse. In contrast, HU's half-life in humans (3.5 to 4.5 hours) [122] is comparatively longer, and therefore clinical trials may prove less troublesome.

Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport.

The multidrug resistance-associated protein (MRP) is a 180- to 195-kDa glycoprotein associated with multidrug resistance of human tumor cells. MRP is mainly located in the plasma membrane and it confers resistance by exporting natural product drugs out of the cell. Here we demonstrate that overexpression of the MRP gene in human cancer cells increases the ATP-dependent glutathione S-conjugate carrier activity in plasma membrane vesicles isolated from these cells. The glutathione S-conjugate export carrier is known to mediate excretion of bivalent anionic conjugates from mammalian cells and is thought to play a role in the elimination of conjugated xenobiotics. Our results suggest that MRP can cause multidrug resistance by promoting the export of drug modification products from cells and they shed light on the reported link between drug resistance and cellular glutathione and glutathione S-transferase levels.

Effect of unmodified triple helix-forming oligodeoxyribonucleotide targeted to human multidrug-resistance gene mdr1 in MDR cancer cells

The human mdr1 gene encodes a transmembrane glycoprotein the over-expression of which is associated with development of multidrug resistance in human tumor cells. A negative modulation of human mdr1 has been attempted via a 27-mer unmodified triple helix-forming oligonucleotide, named 1D, targeted to a homopurine sequence in the coding region of the gene. By administering 10 μM of 1D we could find a significant reduction in MDR1 mRNA levels in the human drug-resistant cell line CEM-VLB 100. This effect appears to be specific and due to a transient block of RNA polymerase mediated by triple helix formation.

The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump.

The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an expression vector containing MRP cDNA. MRP-overexpressing SW-1573 cells are resistant to doxorubicin, daunorubicin, vincristine, VP-16, colchicine, and rhodamine 123, but not to 4'-(9-acridinylamino)methanesulfon-m-anisidide or taxol. The intracellular accumulation of drug (daunorubicin, vincristine, and VP-16) is decreased and the efflux of drug (daunorubicin) is increased in the transfectant. The decreased accumulation of daunorubicin is abolished by permeabilization of the plasma membrane with digitonin, showing that MRP can lower the intracellular daunorubicin level against a concentration gradient. Anti-MRP antisera predominantly stain the plasma membrane of MRP-overexpressing cells. We conclude that MRP is a plasma membrane drug-efflux pump.

Characterization of transport-mediated methotrexate resistance in human tumor cells with antibodies to the membrane carrier for methotrexate and tetrahydrofolate cofactors

An earlier report (Matherly, L. H., Czajkowski, C. A., and Angeles, S. M. (1991) Cancer Res. 51, 3420-3426) described a K562 human erythroleukemia line (K562.4CF), characterized by an elevated uptake capacity for methotrexate (MTX) and 5-formyltetrahydrofolate, and the identification of a highly glycosylated membrane transporter (GP-MTX) by radioaffinity labeling with N-hydroxysuccinimide [3H] methotrexate. In the present study, radioaffinity-labeled GP-MTX from K562.4CF cells was isolated by Ricinus communis agglutinin I-agarose affinity chromatography, coupled with gel filtration and preparative electrophoresis. Antiserum to the purified, radio-labeled protein was raised in a rabbit and screened by immunoblot analysis of K562.4CF plasma membrane proteins or purified GP-MTX. The antiserum detected a broad GP-MTX band centered at 92 kDa on 7.5% gels. On 4-10% gels, the apparent molecular mass for GP-MTX shifted to 99 kDa. Antiserum specificity was established by quantitatively converting the immunoreactive glycoprotein in plasma membrane homogenates to its N- and O-deglycosylated forms with N- and O-glycanases, respectively. Whereas the methotrexate uptake capacity of K562.4CF cells was elevated 6.1-fold over parental cells, the GP-MTX content on immunoblots was increased approximately 3-fold. For two methotrexate-resistant K562 lines (33- and 70-fold), decreased drug uptake (28 and 18% of parental levels) closely correlated with their reduced GP-MTX contents. A GP-MTX isoform was also detected on immunoblots of membrane proteins from CCRF-CEM human lymphoblastic leukemia cells. With a transport-impaired CCRF-CEM line (13% of wild type uptake), an aberrant electrophoretic migration for GP-MTX was observed, establishing the presence of structural modifications in the transport protein. These structural differences were independent of carrier glycosylation since they were detected following the glycosidase treatments. These findings implicate important roles for distinct carrier-specific alterations in the expression of diminished drug transport in methotrexate-resistant human tumor cells.

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.

Faster repair of DNA double-strand breaks in radioresistant human tumor cells

To investigate the molecular basis of radiation resistance in human tumor cells, the induction and repair of radiation-induced DNA single- and double-strand breaks was determined by DNA elution analysis in two normal human cell lines and 12 early-passage human tumor cell lines of varying radiosensitivities. The radiosensitivities (D 0) of the cell lines ranged from 1 to 2.9 Gy. Inherent cellular radiosensitivity was found to directly correlate with the rate at which the DNA double-strand breaks were repaired. Radioresistant cell lines repaired approximately 90% of their radiation-induced DNA double-strand breaks within 1 hr of irradiation while more radiosensitive cell lines required 2–4 hr to repair the same fraction of damage. Radioresistant cell lines also had lower initial DNA double-strand break frequencies. DNA single-strand break induction and repair was not found to be an important factor in the radiation response of human tumor and normal cell lines. Therefore, the rate at which DNA double strand breaks are repaired is a critical factor underlying radioresistance in human tumor cell lines.

Resistance of human tumor cells in vitro to oxidative cytolysis.

Nine human cell types, six of them malignant, displayed a marked resistance to lysis by hydrogen peroxide (LD50, 2-20 mM). Of the reactive oxygen intermediates generated extracellularly, only H2O2 lysed all the cell types. OH was lytic to one of four, OI- to one of one, and O-2 to none of four cell types tested. Resistance to oxidative lysis did not correlate with specific activity of catalase, glutathione (GSH) peroxidase, other peroxidases, or glutathione disulfide reductase, or with specific content of GSH. Resistance to H2O2 seemed to occur via mechanisms distinct from those responsible for cellular consumption of H2O2. Consumption was inhibitable by azide and was probably due to catalase in each cell type. In contrast, resistance to oxidative lysis occurred via distinct routes in different cells. One cell type used the GSH redox cycle as the primary defense against H2O2, like murine tumors previously studied. Other cells seemed to utilize catalase as the major defense against H2O2. Nonetheless, with both catalase and the GSH redox cycle inhibited, all the human cells tested exhibited an inherent resistance to oxidative lysis, that is, resistance independent of detectable degradation of H2O2.