Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Multidrug resistance (MDR) is frequently found in haematologic malignancies. It has been shown that MDR is often related to the expression of a membrane glycoprotein (P-170) which actively pumps many hydrophobic agents out of the cells. Previous electron microscopic investigations revealed morphological differences between P-388 resistant and P-388 sensible cell membranes, but the modulation of the membrane morphology seems to be related to the tumor-cell environment. In order to establish if morphological differences exist between sensitive and resistant cells, both sensitive and resistant strains from three different cell lines were studied by scanning electron microscopy: human leukaemia CEM and vinblastine resistant cells (CEM/VBL100), human breast cancer MCF-7 and mice leukaemia P-388 with the doxorubicin resistant strains (MCF-7/DX and P-388/DX, respectively). The surface of the membranes of the sensitive cells was regular, unlike the resistant ones which proved to be irregular, endowed with long villus-like processes or numerous folds and ruffles. The addition of albumin to the culture medium induced a shift from the resistant to the sensitive phenotype, thus suggesting that the P-388/DX morphology may be linked to the concentration of protein in the culture medium. Exposure to DX, verapamil (VRP) or monoclonal antibody against P-170 (mAb-57) did not modify the surface of the resistant strains, demonstrating that surface irregularities are probably not linked to P-glycoprotein function. Blasts from four P-170 positive leukaemic patients were also analyzed: an irregular shape was always found.
Cell Mol Biol (Noisy-le-grand) 1993 Jul
PMID:Scanning electron microscopy of multidrug resistant cells in haematological and mammary malignancies. 810 68

Inside-out membrane vesicles prepared from multidrug resistant human leukemic cells (CEM/VBL1000), but not from sensitive cells, transported [3H]-labelled vinblastine (VBL) in an ATP-dependent manner, reaching a plateau level by 15 min. The transport occurred with an apparent Km of 60 +/- 20nM. Verapamil (10 microM), and taxol (IC50 = 1 microM) prevented VBL uptake and evoked VBL diffusion from vesicles when added after VBL uptake had reached steady state. The channel forming agent alamethicin prevented net uptake of VBL and addition of alamethicin to the vesicles after the steady-state had been reached resulted in the rapid efflux of [3H]VBL. Very low concentrations of Triton X-100 (0.01 % v/v) also prevented net uptake of VBL, whilst addition of Triton X-100 and making the medium hypo-osmotic after the steady state had been reached caused the [3H]VBL to rapidly diffuse out of the vesicles. These observations indicate that VBL is actively transported into the lumen of inside-out vesicles from multidrug resistant leukaemia cells.
Biochem Mol Biol Int 1993 Jul
PMID:Vinblastine transport by membrane vesicles from human multidrug-resistant CCRF-CEM leukaemia cells: inhibition by taxol and membrane permeabilising agents. 810 21

Oxygenated derivatives of cholesterol inhibit cholesterol synthesis, prevent lymphoid cell growth, and evoke cell death. We have employed a novel selection method to isolate M10 cells, a line of oxysterol-resistant cells, from the sensitive clone CEM C7. Concentrations of the potent sterol 25-hydroxycholesterol that occupy the oxysterol binding protein cause cell death in CEM C7, but not in M10 cells. Both cell lines have similar amounts of the oxysterol binding protein with similar affinities for oxysterol. However, in neither line are the levels of oxysterol binding protein mRNA affected by 1 microM 25-hydroxycholesterol. Furthermore, both cells express the cellular nucleic acid binding protein (CNBP), a 7 zinc finger, DNA-binding protein of unknown function, regulated by oxysterols. The levels of CNBP mRNA are significantly reduced by 25-hydroxycholesterol in the sensitive CEM C7 cells, in which the dose response and time course are consistent with occupancy of the oxysterol binding protein by oxysterol and with subsequent cell kill. However, in the resistant M10 cells, CNBP mRNA levels are unaffected by these concentrations of the 25-hydroxycholesterol. Our results suggest a role for CNBP in oxysterol-induced regulation of cell viability and growth.
J Steroid Biochem Mol Biol 1994 Mar
PMID:Oxysterol sensitive and resistant lymphoid cells: correlation with regulation of cellular nucleic acid binding protein mRNA. 814 9

In eukaryotic cells oxysterols inhibit cholesterol biosynthesis and cell growth. A potent oxysterol, 25-hydroxycholesterol, was used to investigate the biological effects of oxysterols on three clonal lines of either glucocorticoid-sensitive or -resistant CEM cells, human leukemic T-lymphocytes. In addition, the glucocorticoid sensitivity of an oxysterol-resistant CEM cell line was tested. Oxysterols blocked growth and caused the lysis of cells regardless of their glucocorticoid response. All cells studied herein possessed an oxysterol binding protein with high affinity for 25-hydroxycholesterol. For all clones grown in serum-free medium, the half-maximal cytolytic concentration of 25-hydroxycholesterol (20-40 nM) correlated with its affinity (Kd = approximately 31 nM) for this oxysterol binding protein. Both cholesterol and mevalonate reversed 25-hydroxycholesterol cytotoxicity; 3-6 microM cholesterol or 0.1 mM mevalonate decreased 60 nM 25-hydroxycholesterol cytotoxicity by 50%. This cholesterol or mevalonate reversal appeared possible even after several days of 60 nM oxysterol treatment. The protective effect of cholesterol could be overcome by increasing 25-hydroxycholesterol concentrations. Cholesterol and mevalonate did not prevent glucocorticoid-mediated lymphocytolysis. Furthermore, the oxysterol-resistant line was sensitive to dexamethasone lysis. These data support the hypothesis that oxysterols and glucocorticoids act independently to block the growth of human leukemic lymphoblasts.
J Steroid Biochem Mol Biol 1993 Oct
PMID:Oxysterol-induced cell death in human leukemic T-cells correlates with oxysterol binding protein occupancy and is independent of glucocorticoid-induced apoptosis. 821 73

It has been reported that thymidylate synthase (TS) is a component of a multienzyme complex associated with DNA replication based on observations that enzyme activity was decreased when cells were treated with various DNA synthesis inhibitors (Plucinski, T. M., Fager, R. S., and Reddy, G. P. V. (1990) Mol. Pharmacol. 38, 114-120). The veracity of the TS assay (known as the tritium release assay) employed in these experiments may be compromised, however, because it requires the S phase-specific enzyme thymidine kinase (TK) to phosphorylate the substrate [5-3H]dUrd. In our study, this problem was further illustrated as the phosphorylated products of [14C]dCyd and [6-3H]dUrd were simultaneously quantitated to determine the activities of TS, TK, and dCyd kinase in intact CCRF-CEM cells. TS and dCyd kinase were unaffected by aphidicolin, hydroxyurea, and 1-beta-D-arabinofuranosylcytosine, whereas TK was strongly inhibited by these agents. Elevation of the cellular dTTP pool that accompanied drug treatment was not the primary mechanism affecting TK activity because incubation of cells with dCyd elevated the dTTP pool to similar levels, but did not inhibit TK to the same extent as did the drugs. Furthermore, after cells were washed from aphidicolin, [6-3H]dCyd incorporation, which primarily labels dTMP in DNA, proceeded at a linear rate, whereas a lag period of 15 min was observed before [3H]dThd was incorporated at a linear rate. These results suggest that TK activity is affected by more than one mechanism in intact cells. Because the activities of dCyd kinase and dCMP deaminase do not fluctuate as much as that of TK in response to changes in DNA synthesis activity and cell cycle, dCyd incorporation appears to be a more reliable assay of TS in intact cells than does dUrd incorporation. Our findings also imply that [3H]dThd incorporation assays may overestimate inhibition of DNA synthesis.
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PMID:Regulation of thymidine kinase and thymidylate synthase in intact human lymphoblast CCRF-CEM cells. 822 47

CdG, the carbocyclic analog of 2'-deoxyguanosine, is active against herpes, hepatitis B, and human cytomegaloviruses. We have studied the interaction of the tritiated enantiomers of CdG with the herpes simplex virus type 1-specific thymidine kinase (HSV-1 TK) and have examined their metabolism in uninfected and HSV-1-infected cells. D- and L-CdG were equally effective competitive inhibitors of the phosphorylation of thymidine (dThd) by the partially purified HSV-1 TK (Ki values were 2.1 and 3.4 microM, respectively) and were also equal as substrates (Km values were 17 and 26 microM, respectively, and Vmax values of the enantiomers were equal and about 50% greater than the Vmax for dThd). The partially purified enzyme preparation, which contained cellular nucleotide kinase activities (pyruvate kinase also was present in the assay medium), converted D-CdG almost exclusively to the triphosphate and L-CdG almost exclusively to the monophosphate. Similarly, in virus-infected cells the D-enantiomer was converted predominantly to the triphosphate and the L-enantiomer predominantly to the monophosphate. In uninfected cells the results were qualitatively similar. In CEM cells deoxycytidine (dCyd) kinase (EC 2.7.1.74) seemed to be the enzyme principally responsible for the phosphorylation of both enantiomers, as shown by competition studies. Thus, both the HSV-1 TK and cellular dCyd kinase (of CEM cells) showed no selectivity for the enantiomers of CdG. This lack of enantiomeric specificity has obvious implications for the design of inhibitors of both viral proliferation and cellular metabolism.
Mol Pharmacol 1993 Dec
PMID:Phosphorylation of the enantiomers of the carbocyclic analog of 2'-deoxyguanosine in cells infected with herpes simplex virus type 1 and in uninfected cells. Lack of enantiomeric selectivity with the viral thymidine kinase. 826 63

CCRF-CEM-C7 is a well characterized human leukemic clonal cell line which is lysed by dexamethasone (dex). Originating from the wild-type CEM-C7 cells are two dex-resistant clones which are not lysed by 1 microM dex and have functionally defective glucocorticoid receptors (GR). They are receptorless ICR27TK.3 and activation-labile 4R4 cells. ICR27TK.3 and 4R4 cells have distinct cellular phenotypes, as indicated by dissimilar numbers of dex-binding sites despite similar levels of GR mRNA and immunochemically detectable GR. We have now investigated the molecular defects in the GR of ICR27TK.3 and 4R4 cells by determining the nucleotide sequence of their GR. Our results support the biochemical evidence previously reported by others for the presence of both a normal (GR+) and a mutant (GR*) allele in CEM-C7 cells. We clearly show that the wild-type CEM-C7 cells express two alleles of GR, the normal GR+ and the abnormal GR*, which has a Leu753--> Phe753 mutation. We demonstrate that both ICR27TK.3 and 4R4 cells contain only the abnormal GR* and that the normal GR+ gene is deleted in both of these GR defective clones. Our results further show that the GR* is basically an activation-labile receptor and has diminished functional capability in a transfection assay measuring GR-driven transcription. Thus, these two phenotypically different cell lines express similar amounts of an identical GR* containing a single point mutation at amino acid 753. A single point mutation in the steroid-binding domain of the GR, therefore, may behave differently, depending on the cellular milieu in which it is expressed.
Mol Endocrinol 1993 May
PMID:Identification of the activation-labile gene: a single point mutation in the human glucocorticoid receptor presents as two distinct receptor phenotypes. 831 49

We have developed versatile synthetic routes that afford metal-free macrocycles containing different functionalities in their framework. Novel oxaziridine and amide containing macrocycles were synthesized, and the metal complexes of the latter were also prepared. A series of theophilline and thymidine side-arm containing podands as well as macrocycles were obtained employing the same methodology. The primary anti-viral tests of these synthetic compounds for anti-HIV-1 activity was carried out using the XTT-based cytopathicity assay (CEM-SS cells) with AZT as positive control. It was found that the nature of the macrocyclic headgroups affected the anti-HIV-1 activity. Heteroatom containing macrocyclic headgroups displayed activity in the micromolar range. Metal complexation did not enhance the activity and side-arm substitution resulted in inactive compounds. Cell viability determined in both Jurkat and CEM-SS cells was strongly dependent on the structure of the macrocyclic framework. The oxaziridine moieties in the macrocycle were highly toxic to CEM-SS and less toxic to Jurkat cell lines, while amide containing macrocycles were toxic to neither.
Cell Mol Biol (Noisy-le-grand) 1995
PMID:Synthesis, activity and toxicity of novel macrocyclic ligands against HIV-1 in Jurkat and CEM-SS cell lines. 857 52

Human immunodeficiency virus type 1 (HIV-1)-infected CEM cells were treated (as single agents or in combination) with (minus)-2', 3'-dideoxy-3'-thiacytidine (3TC) and the following HIV-1-specific non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs): 2', 5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5'-(4'-amino-1',2'-oxathi ole)-2',2'-dioxide derivative of 3-methylthymidine (TSAO-m3T), the thiocarboxanilides UC10 and UC42, bis(heteroaryl)piperazine (BHAP) derivative U90152, and the 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivative 5-isopropyl-1-ethoxymethyl-6-benzyluracil (MKC-442). When used individually, the compounds led to the emergence of HIV-1 strains containing the following mutations in the RT: Glu138 to lysine for TSAO-m3T, Met184 to valine for 3TC, Lys103 to threonine/asparagine for the thiocarboxanilides, and Tyr181 to cysteine for BHAP and MKC-442. When 3TC was combined with TSAO-m3T, UC10, UC42, BHAP, or MKC-442, breakthrough of virus was markedly delayed or even suppressed. For these drug combinations, the concentrations of the individual drugs could be lowered by > or = 25-50-fold to suppress virus breakthrough compared with the individual use of the compounds. The concomitant presence of the Lys138 and Ile/Val184 mutations was found in the RT of the mutant viruses that emerged with combination therapy of the lowest concentrations of 3TC with either the lowest concentrations of TSAO-m3T or UC10 (approximately 0.5-3-fold the EC50 value). These virus strains retained high sensitivity to other NNRTIs such as BHAP or HEPT. The virus mutants that arose in the presence of combinations of the lowest concentrations of 3TC with either BHAP or HEPT predominantly contained the Cys181 mutation in the RT. In one case, the Ile181 mutation was found. The latter mutations, particularly the Ile181 mutation, resulted in markedly decreased sensitivity to the NNRTIs but not to 3'-azido-2', 3'-dideoxythymidine or 3TC.
Mol Pharmacol 1996 May
PMID:Marked inhibitory activity of non-nucleoside reverse transcriptase inhibitors against human immunodeficiency virus type 1 when combined with (-)2',3'-dideoxy-3'-thiacytidine. 862 38

In humans infected with the HIV-1 virus there may be a disproportionate severity of signs and symptoms of illness compared to the fraction of CD4+ infected T-lymphoid cells. In part, this may be due to altered intercellular signalling systems and intracellular signal transduction. Glucocorticoids are well known for their effects on the vitality and function of lymphoid cells. Patients with HIV infections often show elevated circulating levels of cortisol, suggesting some misfunction in the regulatory systems that maintain the levels of this critical hormone. At the cellular level, it is known that both acute HIV infection and glucocorticoids can cause apoptotic cell death in thymic lymphocytes. However, chronically HIV-infected cells appear to be resistant to glucocorticoid-evoked cell death. Glucocorticoid receptor-ligand binding studies on patients' cells have shown reduced affinity between the receptor binding sites and test steroids. In vitro, chronically HIV-infected cells of the lymphoid CEM line displayed resistance to glucocorticoid-induced apoptosis. These cells showed reduced numbers of binding sites with little alteration in apparent affinity between ligand and receptor. Thus it appears that there may often be malfunction of the normal glucocorticoid response in HIV-infected cells probably due to altered interactions between the glucocorticoid receptor and its hormone. Such alterations may have clinical consequences, including the possibility of a relatively longer life span of infected CD4+ T-lymphocytes, as well as systemic effects of chronically elevated cortisol level.
J Steroid Biochem Mol Biol 1996 Mar
PMID:Lymphoid cell resistance to glucocorticoids in HIV infection. 863 61


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