Phosphorylation of the multidrug resistance associated glycoprotein

Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.     

糖基因在髓性白血病耐药性中特征性改变

目的通过研究糖基因在髓性白血病中的差异表达,明确这些糖基因与白血病耐药的相关性,从而为预测和诊断髓性白血病耐药性,寻求逆转药物提供新策略和靶点。方法采用real-time PCR技术筛选髓性白血病细胞及其耐药细胞株中差异表达的糖基因,筛选出两组细胞差异表达3倍以上的糖基因,初步探索糖基因在髓性白血病耐药性中的特征性改变;采用流式细胞仪分析髓性白血病耐药细胞株与多种FITC标记植物凝集素的结合能力,表征比较细胞膜表面糖链的特征。结果 12个糖基因在NB4和NB4/ADR细胞株中表达具有显著的差异;高表达的糖基因与FITC标记植物凝集素的结合能力增强。结论髓性白血病细胞及其耐药细胞株中糖基因、细胞膜表面糖链特征均有显著差异,这些特征性改变与白血病多药耐药具有相关性。     

Atypical multidrug resistance may be associated with catalytically active mutants of human DNA topoisomerase II alpha

In human cells, atypical drug resistance was previously identified with reduced catalytic activity or nuclear localization efficiency of DNA topoisomerase II alpha (TOP2 alpha). We have shown two etoposide resistant hTOP2 alpha mutants, K798L and K798P confer resistance to etoposide. In this work, we showed these mutants are also resistant against doxorubicin and mAMSA in vivo in the yeast strain ISE2, rad52, top2-4 at the non-permissive temperature. We purified these mutants to characterize the drug resistant mechanism. Purified recombinant proteins were 8- to 12-fold more resistant to etoposide and doxorubicin than wild type TOP2 alpha, and 2-fold more resistant to amsacrine, as measured by accumulation of cleavable DNA. These data show that K798L and K798P may be intrinsically resistant against these drugs in vitro and that this character may confer atypical multidrug resistant phenotype in vivo in yeast.     

Cytogenetically balanced translocations are associated with focal copy number alterations

Current cytogenetic methods (e.g., G-banding and multicolor chromosomal painting) allow detection of translocation events but lack the resolution to (a) locate the breakpoints precisely at the chromosome band level or (b) discriminate balanced translocations from translocations with copy number alterations not previously reported, or imperfectly balanced translocations. In this study, we demonstrate that cytogenetically balanced translocations are in fact frequently associated with segmental gain or loss of DNA. The recent development of a whole genome tiling path BAC array has enabled tiling resolution analysis of genomic segmental copy number status. Combining tiling resolution BAC array comparative genomic hybridization (array CGH) with G-Banding analysis and multicolor chromosomal painting approaches such as spectral karyotyping (SKY) facilitates high-resolution mapping of genomic alterations associated with imperfectly balanced translocations. Using a refined version of our CGH array we have deduced the copy number status throughout the genomes of three cytogenetically well-characterized prostate cancer cell lines (PC3, DU145, LNCaP) to determine whether translocations are associated with focal gains and losses of DNA. At 78 kb tiling resolution we identified the boundaries of 170, 80, and 34 known and novel copy number alterations (CNA) in these cell line genomes, respectively. Thirty-three of the 36 known translocations (92%, P < 0.001) in DU145 were associated with segmental CNA. Likewise, 80% (P < 0.001) of the known translocations showed association in LNCaP. Although many translocation breakpoints exhibit segmental alteration in PC3, the pattern of chromosomal rearrangements is too complex for use in comprehensive association with CNA boundaries. Our results reveal that imperfectly balanced translocations in tumor genomes are a phenomenon that occurs at frequencies much higher than previously demonstrated. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Mitochondrial NADH:ubiquinone oxidoreductase alterations are associated with endometriosis

Genetic alterations and aberrant expression of ‘mitochondrial membrane complex I’ (MMC-I) underlie several complex human disorders, but no reports are documented to date in endometriosis. Sequencing of mitochondrially encoded MMC-I subunits revealed 72 mutations of which 2 missense (G10398A; A13603A/G) mutations and 1 synonymous (T10400C) mutation showed higher prevalence in patients. In silico functional analysis predicted A13603A/G, a novel heteroplasmy as a ‘damaging variant’. Our results indicate higher endometriosis risk for haplotype ‘10398A/10400C/13603AG’ and haplogroup ‘N’. Immunohistochemical analysis revealed elevated MMC-I expression in eutopic endometria of patients compared to controls. In conclusion, MMC-I alterations may constitute an inheritable risk factor for endometriosis.     

Proteome alterations in gamma-irradiated human T-lymphocyte leukemia cells

Analyses of the protein expression profiles of irradiated cells may be beneficial for identification of new biomolecules of radiation-induced cell damage. Therefore, in this study we exploited the proteomic approach to identify proteins whose expression is significantly altered in gamma-irradiated human T-lymphocyte leukemia cells. MOLT-4 cells were irradiated with 7.5 Gy and the cell lysates were collected at different times after irradiation (2, 5 and 12 h). The proteins were separated by two-dimensional electrophoresis and quantified using an image evaluation system. Proteins exhibiting significant radiation-induced alterations in abundance were identified by peptide mass fingerprinting. We identified 14 proteins that were either up- or down-regulated. Cellular levels of four of the proteins (Rho GDP dissociation inhibitor 1 and 2, Ran binding protein 1, serine/threonine protein kinase PAK2) were further analyzed by two-dimensional immunoblotting to confirm the data obtained from proteome analysis. All identified proteins were classified according to their cellular function, including their participation in biochemical and signaling pathways. Taken together, our results suggest the feasibility of the proteome method for monitoring of cellular radiation responses.     

Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells

Leukemia stem cells are known to exhibit multidrug resistance by expression of ATP-binding cassette (ABC) transporters which constitute transmembrane proteins capable of exporting a wide variety of chemotherapeutic drugs from the cytosol. We show here that human promyeloblastic leukemia KG-1a cells exposed to the histone deacetylase inhibitor phenylbutyrate resemble many characteristics of leukemia stem cells, including expression of functional ABC transporters such as P-glycoprotein, BCRP and MRP8. Consequently, KG-1a cells display resistance to the induction of apoptosis by various chemotherapeutic drugs. Resistance to apoptosis induction by chemotherapeutic drugs can be reversed by cyclosporine A, which effectively inhibits the activity of P-glycoprotein and BCRP, thus demonstrating ABC transporter-mediated drug resistance in KG-1a cells. However, KG-1a are highly sensitive to apoptosis induction by salinomycin, a polyether ionophore antibiotic that has recently been shown to kill human breast cancer stem cell-like cells and to induce apoptosis in human cancer cells displaying multiple mechanisms of drug and apoptosis resistance. Whereas KG-1a cells can be adapted to proliferate in the presence of apoptosis-inducing concentrations of bortezomib and doxorubicin, salinomycin does not permit long-term adaptation of the cells to apoptosis-inducing concentrations. Thus, salinomycin should be regarded as a novel and effective agent for the elimination of leukemia stem cells and other tumor cells exhibiting ABC transporter-mediated multidrug resistance.     

Thiolactomycin resistance in Escherichia coli is associated with the multidrug resistance efflux pump encoded by emrAB.

Thiolactomycin (TLM) and cerulenin are antibiotics that block Escherichia coli growth by inhibiting fatty acid biosynthesis at the beta-ketoacyl-acyl carrier protein synthase I step. Both TLM and cerulenin trigger the accumulation of intracellular malonyl-coenzyme A coincident with growth inhibition, and the overexpression of synthase I protein confers resistance to both antibiotics. Strain CDM5 was derived as a TLM-resistant mutant but remained sensitive to cerulenin. TLM neither induced malonyl-coenzyme A accumulation nor blocked fatty acid production in vivo; however, the fatty acid synthase activity in extracts from strain CDM5 was sensitive to TLM inhibition. The TLM resistance gene in strain CDM5 was mapped to 57.5 min of the chromosome and was an allele of the emrB gene. Disruption of the emrB gene converted strain CDM5 to a TLM-sensitive strain, and the overexpression of the emrAB operon conferred TLM resistance to sensitive strains. Thus, activation of the emr efflux pump is the mechanism for TLM resistance in strain CDM5.     

Chromosomal instability,tolerance of mitotic errors and multidrug resistance are promoted by tetraploidization in human cells

Up to 80% of human cancers, in particular solid tumors, contain cells with abnormal chromosomal numbers, or aneuploidy, which is often linked with marked chromosomal instability. Whereas in some tumors the aneuploidy occurs by missegregation of one or a few chromosomes, aneuploidy can also arise during proliferation of inherently unstable tetraploid cells generated by whole genome doubling from diploid cells. Recent findings from cancer genome sequencing projects suggest that nearly 40% of tumors underwent whole genome doubling at some point of tumorigenesis, yet its contribution to cancer phenotypes and benefits for malignant growth remain unclear. Here, we investigated the consequences of a whole genome doubling in both cancerous and non-transformed p53 positive human cells. SNP array analysis and multicolor karyotyping revealed that induced whole-genome doubling led to variable aneuploidy. We found that chromosomal instability (CIN) is a frequent, but not a default outcome of whole genome doubling. The CIN phenotypes were accompanied by increased tolerance to mitotic errors that was mediated by suppression of the p53 signaling. Additionally, the expression of pro-apoptotic factors, such as iASPP and cIAP2, was downregulated. Furthermore, we found that whole genome doubling promotes resistance to a broad spectrum of chemotherapeutic drugs and stimulates anchorage-independent growth even in non-transformed p53-positive human cells. Taken together, whole genome doubling provides multifaceted benefits for malignant growth. Our findings provide new insight why genome-doubling promotes tumorigenesis and correlates with poor survival in cancer.     

Increased expression of sorcin is associated with multidrug resistance in leukemia cells via up-regulation of MDR1 expression through cAMP response element-binding protein

Sorcin, a 22 kDa Ca²⁺ binding protein, was first identified in a vincristine-resistant Chinese hamster lung cell line, and was later demonstrated to be involved in the development of multidrug-resistance (MDR) phenotypes in a variety of human cancer cell lines. However, the exact role of sorcin in MDR cells is yet to be fully elucidated. Here we explored the role of sorcin in the development of MDR in leukemia cells, and revealed that the expression level of sorcin was directly correlated to the expression of MDR1/P-glycoprotein (P-gp). In addition, it was shown that sorcin induced the expression of MDR1/P-gp through a cAMP response element (CRE) between −716 and −709 bp of the mdr1/p-gp gene. Furthermore, overexpression of sorcin increased the phosphorylation of CREB1 and the binding of CREB1 to the CRE sequence of mdr1/p-gp promoter, and induced the expression of MDR1/P-gp. These findings suggested that sorcin induces MDR1/P-gp expression markedly through activation of the CREB pathway and is associated with the MDR phenotype. The new findings may be helpful for understanding the mechanisms of MDR in human cancer cells, prompting its further investigation as a molecular target to overcome MDR.     

Reduced drug accumulation and multidrug resistance in human breast cancer cells without associated P-glycoprotein or MRP overexpression

MCF-7 human breast cancer cells selected in Adriamycin in the presence of verapamil developed a multidrug resistant phenotype, which was characterized by as much as 100,000-fold resistance to mitoxantrone, 667-fold resistance to daunorubicin, and 600-fold resistance to doxorubicin. Immunoblot and PCR analyses demonstrated no increase in MDR-1 or MRP expression in resistant cells, relative to parental cells. This phenotype is similar to one previously described in mitoxantrone-selected cells. The cells, designated MCF-7 AdVp, displayed a slower growth rate without alteration in topoisomerase IIα level or activity. Increased efflux and reduced accumulation of daunomycin and rhodamine were observed when compared to parental cells. Depletion of ATP resulted in complete abrogation of efflux of both daunomycin and rhodamine. No apparent alterations in subcellular daunorubicin distribution were observed by confocal microscopy. No differences were noted in intracellular pH. Molecular cloning studies using DNA differential display identified increased expression of the alpha subunit of the amiloride-sensitive sodium channel in resistant cells. Quantitative PCR studies demonstrated an eightfold overexpression of the alpha subunit of the Na+ channel in the resistant subline. This channel may be linked to the mechanism of drug resistance in the AdVp cells. The results presented here support the hypothesis that a novel energy-dependent protein is responsible for the efflux in the AdVp cells. Further identification awaits molecular cloning studies. J. Cell. Biochem. 65:513–526. © 1997 Wiley-Liss Inc.     

Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural and functional characteristics, this bacterial protein is classified as a member of the P-glycoprotein cluster of the ABC transporter superfamily. LmrA can even substitute for P-glycoprotein in human lung fibroblast cells, suggesting that this type of transporter is conserved from bacteria to man. The functional similarity between bacterial LmrA and human P-glycoprotein is further exemplified by their currently known spectrum of substrates, consisting mainly of hydrophobic cationic compounds. In addition, LmrA was found to confer resistance to eight classes of broad-spectrum antibiotics, and homologs of LmrA have been found in pathogenic bacteria, supporting the clinical and academic value of studying this bacterial protein. Current studies are focused on unraveling the mechanism by which ABC multidrug transporters, such as LmrA, couple the hydrolysis of ATP to the translocation of drugs across the membrane. Recent evidence indicates that LmrA mediates drug transport by an alternating two-site transport mechanism.     

Control of P-glycoprotein activity by membrane cholesterol amounts and their relation to multidrug resistance in human CEM leukemia cells

P-glycoprotein (P-gp) is the most well-known ATP-binding cassette (ABC) transporter involved in unidirectional substrate translocation across the membrane lipid bilayer, thereby causing the typical multidrug resistance (MDR) phenotype expressed in many cancers. We observed that in human CEM acute lymphoblastic leukemia cells expressing various degrees of chemoresistance and where P-gp was the sole MDR-related ABC transporter detected, the amount of esterified cholesterol increased linearly with the level of resistance to vinblastine while the amounts of total and free cholesterol increased in a nonlinear way. Membrane cholesterol controlled the ATPase activity of P-gp in a linear manner, whereas the P-gp-induced daunomycin efflux decreased nonlinearly with the depletion of membrane cholesterol. All these elements suggest that cholesterol controls both the ATPase and the drug efflux activities of P-gp. In addition, in CEM cell lines that expressed increasing levels of elevated chemoresistance, the amount of P-gp increases to a plateau value of 40% of the total membrane proteins and remained unvaried while the amount of membrane cholesterol increased with the elevation of the MDR level, strongly suggesting that cholesterol may be directly involved in the typical MDR phenotype. Finally, we showed that the decreased daunomycin efflux by P-gp due to the partial depletion of membrane cholesterol was responsible for the efficient chemosensitization of resistant CEM cells, which could be totally reversed after cholesterol repletion.     

Phosphorylation-dependent structural alterations in the small hsp30 chaperone are associated with cellular recovery

Small heat shock proteins (hsps) act as molecular chaperones by preventing the thermal aggregation and unfolding of cellular protein; however, the manner by which cells regulate chaperone activity remains unclear. In the present study, we examined the role of phosphorylation on the chaperone function of the Xenopus small hsp30. Both heat stress and sodium arsenite treatment in A6 cells resulted in a rapid activation of p38alpha and MAPKAPK-2. Surprisingly, the association of MAPKAPK-2 with hsp30 and its subsequent phosphorylation were more prevalent during recovery after heat stress. Treatment of A6 cells with SB203580, an inhibitor of the p38 MAP kinase pathway, resulted in a loss of hsp30 phosphorylation. Phosphorylation resulted in the formation of smaller multimeric hsp30 complexes and resulted in a significant loss of secondary structure. Consequently the phosphorylation-induced structural changes severely compromised the ability of hsp30 to prevent the heat-induced aggregation of citrate synthase and luciferase in vitro. We confirmed that the loss of chaperone activity was coincident with an attenuated binding of phosphorylated hsp30 with target proteins. Our data suggest that phosphorylation may be necessary to regulate the post-heat stress molecular chaperone activity of hsp30.     

Reduced biliverdin reductase-A levels are associated with early alterations of insulin signaling in obesity

Biliverdin reductase-A (BVR-A) is a serine/threonine/tyrosine kinase involved in the regulation of insulin signaling. In vitro studies have demonstrated that BVR-A is a substrate of the insulin receptor and regulates IRS1 by avoiding its aberrant activation, and in animal model of obesity the loss of hepatic BVR-A has been associated with glucose/insulin alterations and fatty liver disease. However, no studies exist in humans. Here, we evaluated BVR-A expression levels and activation in peripheral blood mononuclear cells (PBMC) from obese subjects and matched lean controls and we investigated the related molecular alterations of the insulin along with clinical correlates. We showed that BVR-A levels are significantly reduced in obese subjects and associated with a hyper-activation of the IR/IRS1/Akt/GSK-3β/AS160/GLUT4 pathway. Low BVR-A levels also associate with the presence of obesity, metabolic syndrome, NASH and visceral adipose tissue inflammation. These data suggest that the reduction of BVR-A may be responsible for early alterations of the insulin signaling pathway in obesity and in this context may represent a novel molecular target to be investigated for the comprehension of the process of insulin resistance development in obesity.     

Epigenetic loss of CDH1 correlates with multidrug resistance in human hepatocellular carcinoma cells

Promoter CpG hypermethylation of tumor suppressor genes is an essential step in cancer progression but little is known about its effect on cancer multidrug resistance. In this study, we showed that CDH1 promoter was hypermethylated in drug resistance of a doxorubicin-induced multidrug resistant hepatocellular carcinoma cell line R-HepG2. Transfection of CDH1 cDNA into R-HepG2 cells led to increased amount of doxorubicin uptake, decreased cell viability, decreased P-glycoprotein expression and increased apoptotic population of cells exposed to doxorubicin. Proto-oncogene tyrosine-protein kinase FYN was over-expressed in R-HepG2 cells which displayed a negative correlation with the expression of CDH1. FYN was knocked down in R-HepG2 cells, leading to less drug resistance by increased cell viability, increased doxorubicin uptake and attenuated P-glycoprotein expression. Our findings identified epigenetic silencing of CDH1 in cancer cells might be a new molecular event of multidrug resistance.