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1.
Int J Cancer ; 72(6): 1021-6, 1997 Sep 17.
Article in English | MEDLINE | ID: mdl-9378536

ABSTRACT

Multidrug resistance (MDR) in human cancer cells is multifactorial. Previously, we reported on the association between expression of P-glycoprotein (Pgp), the multidrug resistance-associated protein (MRP), and the lung resistance protein (LRP) with the MDR phenotype in the NCI panel of 60 human cancer cell lines used for in vitro anticancer drug screening. Eight cell lines from this panel, manifesting widely divergent levels of in vitro drug resistance were chosen to investigate the role of MRP and LRP expression at the molecular level. LRP mRNA levels, as determined by ribonuclease protection assay, varied significantly among the 8 cell lines, and correlated closely with in vitro drug resistance to both MDR and non-MDR related drugs. LRP mRNA expression was determined to be a stronger correlate of drug sensitivity than protein expression. In contrast, MRP mRNA levels were not significantly correlated with drug sensitivity. The rates of newly transcribed LRP or MRP mRNA did not correlate with mRNA levels, indicating that mRNA stability or other features of processing may be important in regulation of LRP and MRP mRNA levels. Using Southern blot analysis, LRP gene amplification was shown not to be associated with LRP overexpression. These data suggest that LRP expression may be an important determinant of the MDR phenotype in cell lines intrinsically resistant to cancer chemotherapeutic agents.


Subject(s)
Antineoplastic Agents/toxicity , Drug Resistance, Multiple/genetics , Glycoproteins/biosynthesis , Neoplasm Proteins/biosynthesis , Transcription, Genetic , Vault Ribonucleoprotein Particles , ATP-Binding Cassette Transporters/biosynthesis , Amsacrine/toxicity , Cell Nucleus , Cisplatin/toxicity , Colonic Neoplasms , Doxorubicin/toxicity , Female , Humans , Kidney Neoplasms , Leukemia , Lung Neoplasms , Melphalan/toxicity , Multidrug Resistance-Associated Proteins , Ovarian Neoplasms , Phenotype , RNA, Messenger/biosynthesis , Tumor Cells, Cultured
2.
Antimicrob Agents Chemother ; 41(7): 1521-30, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9210678

ABSTRACT

We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide.


Subject(s)
Anti-HIV Agents/isolation & purification , Bacterial Proteins , Carrier Proteins/isolation & purification , HIV Envelope Protein gp120/metabolism , Acquired Immunodeficiency Syndrome/transmission , Amino Acid Sequence , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Fusion , Cell Survival/drug effects , Enzyme-Linked Immunosorbent Assay , Humans , Molecular Sequence Data , Molecular Weight , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Titrimetry , Ultrafiltration
3.
J Natl Cancer Inst ; 89(11): 807-13, 1997 Jun 04.
Article in English | MEDLINE | ID: mdl-9182980

ABSTRACT

BACKGROUND AND PURPOSE: Cancer cells that express P-glycoprotein, multidrug resistance-associated protein (MRP), or lung resistance protein (LRP) have demonstrated resistance to a wide variety of chemotherapeutic drugs. Recently, we reported that human colon carcinoma cells that express all three proteins exhibit reduced P-glycoprotein gene expression and a loss of multidrug resistance after exposure to tumor necrosis factor-alpha, a hormone-like protein produced by cells of the immune system. In this study, we examined the effects of tumor necrosis factor-alpha on MRP and LRP gene expression in the same colon carcinoma cells. METHODS: HCT15 and HCT116 colon carcinoma cells were incubated with tumor necrosis factor-alpha at 100 U/mL for 2, 12, 24, 48, or 72 hours; alternatively, cells transfected with an expression vector containing a human tumor necrosis factor-alpha complementary DNA were studied. The effects of tumor necrosis factor-alpha on MRP and LRP messenger RNA expression were evaluated by means of reverse transcription and the polymerase chain reaction; effects on MRP and LRP protein expression were examined by use of specific monoclonal antibodies and flow cytometry. The flow cytometry data were analyzed by use of the two-sided, nonparametric Mann-Whitney rank sum test. RESULTS: Treatment with exogenous tumor necrosis factor-alpha reduced the level of LRP messenger RNA in both cell types in an apparently time-dependent fashion; in HCT15 cells, almost no LRP messenger RNA was detected after 48 hours of treatment. In contrast, the level of MRP messenger RNA was increased in HCT116 cells by such treatment, but the level in HCT15 cells was unchanged. Treatment with exogenous tumor necrosis factor-alpha induced changes in LRP and MRP protein expression in the two cell types that paralleled the changes found for messenger RNA. In transfected cells, the endogenous production of tumor necrosis factor-alpha reduced LRP gene expression (both messenger RNA and protein) and increased MRP gene expression (both messenger RNA and protein), regardless of cell type. CONCLUSION: In human colon carcinoma cells, tumor necrosis factor-alpha influences MRP and LRP gene expression in opposite ways. The findings for LRP gene expression parallel our earlier findings for P-glycoprotein expression in these cells. IMPLICATION: In developing strategies for overcoming multidrug resistance in tumor cells, the possibility that an agent can suppress one or more mechanisms of drug resistance and enhance others should be considered.


Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , Drug Resistance, Multiple/genetics , Neoplasm Proteins/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Tumor Necrosis Factor-alpha/physiology , Vault Ribonucleoprotein Particles , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/genetics , Clone Cells , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Gene Expression/drug effects , Humans , Multidrug Resistance-Associated Proteins , Neoplasm Proteins/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/metabolism , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolism
4.
Oncol Res ; 7(7-8): 363-9, 1995.
Article in English | MEDLINE | ID: mdl-8747599

ABSTRACT

Previous results from this laboratory indicated that EMT6 cells treated with inhibitors of the intracellular pH (pHi) regulatory mechanisms were resistant to cisplatin (Laurencot, C.M.; Kennedy, K.A. The effect of low pH on the cytotoxicity of cisplatin in EMT6 mouse mammary tumor cells. Oncol. Res. 7:371-380; 1995.) This inhibitor-induced cisplatin resistance was independent of pH. The purpose of the research presented here was to characterize further cisplatin resistance observed in cells cultured with the Na+/H+ antiport inhibitor 5-(N,N hexamethylene) amiloride (NHMA) and the HCO3-/Cl- exchanger inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). Using [195mPt]-DDP, the accumulation of cisplatin into EMT6 mouse mammary tumor cells treated with NHMA and SITS was not changed. Total DNA cross-link formation and DNA-interstrand cross-link formation, however, decreased in NHMA- and SITS-treated cells. Since cisplatin accumulation was unchanged in NHMA- and SITS-treated cells but the amount of DNA cross-links decreased, the intracellular activation of cisplatin appeared to be altered in the cisplatin-resistant cells. Because SITS interferes with chloride transport and chloride has been proposed to be involved in cisplatin activation, cisplatin toxicity in EMT6 cells was evaluated in chloride-deficient medium. EMT6 cells cultured in chloride-deficient medium were less sensitive to cisplatin than cells cultured in chloride-containing medium, but this sensitivity was not altered by NHMA and SITS. Furthermore, the resistance to cisplatin in cells treated with NHMA and SITS was similar in chloride-containing and chloride-deficient medium. These data suggest that the concentration of other ions, in addition to chloride, may be important for cisplatin toxicity.


Subject(s)
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/antagonists & inhibitors , Amiloride/analogs & derivatives , Cisplatin/pharmacology , Mammary Neoplasms, Experimental/metabolism , Amiloride/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cisplatin/metabolism , Cross-Linking Reagents/pharmacology , Drug Resistance, Neoplasm , Hydrogen-Ion Concentration , Mammary Neoplasms, Experimental/drug therapy , Mice , Tumor Cells, Cultured
5.
Oncol Res ; 7(7-8): 371-9, 1995.
Article in English | MEDLINE | ID: mdl-8747600

ABSTRACT

Pathophysiological factors exist within solid tumors that lead to a low pH environment. Therefore, pH-sensitive cancer chemotherapeutic agents may selectively target and kill tumor cells while sparing the normal tissue from toxicity. Using colony forming assays to assess cell survival, we found that EMT6 mouse mammary tumor cells were more sensitive to cisplatin cytotoxicity when cultured in pH 6.0 medium than in pH 7.2, 6.8, or 6.4 medium. The pH-dependent cytotoxicity of cisplatin resulted from an increase in cisplatin accumulation and an increase in the amount of DNA cross-links at pH 6.0 compared with pH 7.2. Because DNA is the cytotoxic target of cisplatin, intracellular pH (pHi) may be an important factor in determining the cytotoxicity of anticancer drugs at low extracellular pH (pHe). Therefore, manipulating the pHi of cells could be one method to enhance the effectiveness of the pH-sensitive chemotherapeutic agents. The pHi of EMT6 cells varied with pHe: at pHe 7.2, pHi was 7.54; at pHe 6.8, pHi was 7.29; at pHe 6.4, pHi was 7.02; and at pHe 6.0, pHi was 6.64. Using inhibitors to the ion transport mechanisms which regulate pHi, 5-N,N-hexamethylene amiloride (NHMA) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), the pHi and pHe of EMT6 cells were equilibrated. To evaluate the importance of pHi in determining drug toxicity, cell survival was determined for cells treated with cisplatin in the presence of NHMA and SITS. Cells cultured with NHMA and SITS were less sensitive to cisplatin. The cisplatin resistance obtained was independent of pH and could be attributed to the presence of SITS.


Subject(s)
Cisplatin/pharmacology , Hydrogen-Ion Concentration , Mammary Neoplasms, Experimental/metabolism , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/pharmacology , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Survival/drug effects , Cross-Linking Reagents/pharmacology , Drug Resistance, Neoplasm , Mammary Neoplasms, Experimental/drug therapy , Mice , Sodium Bicarbonate/pharmacology , Tumor Cells, Cultured , Tumor Stem Cell Assay
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