Regulation of CDPK isoforms during tuber development

CDPK activities present during tuber development were analysed. A high CDPK activity was detected in the soluble fraction of early stolons and a lower one was detected in soluble and particulate fractions of induced stolons. The early and late CDPK activities displayed diverse specificity for in vitro substrates and different subcellular distribution. Western blot analysis revealed two CDPKs of 55 and 60 kDa that follow a precise spatial and temporal profile of expression. The 55 kDa protein was only detected in early-elongating stolons and the 60 kDa one was induced upon stolon swelling, correlating with early and late CDPK activities. A new member of the potato CDPK family, StCDPK3, was identified from a stolon cDNA library. Gene specific RT-PCR demonstrated that this gene is only expressed in early stolons, while the previously identified StCDPK1 is expressed upon stolon swelling. This expression profile suggests that StCDPK3 could correspond to the 55 kDa isoform while StCDPK1 could encode the 60 kDa isoform present in swelling stolons. StCDPK1 has myristoylation and palmitoylation consensus possibly involved in its dual intracellular localization. Transient expression studies with wild-type and mutated forms of StCDPK1 fused to GFP were used to show that subcellular localization of this isoform is controlled by myristoylation and palmitoylation. Altogether, our data suggest that sequential activation of StCDPK3 and StCDPK1 and the subcellular localisation of StCDPK1 might be critical regulatory steps of calcium signalling during potato tuber development.     

P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance

We isolated a full-length MDR1 cDNA from human adrenal where P-glycoprotein is expressed at high level. The deduced amino acid sequence shows two amino acid differences from the sequence of P-glycoprotein obtained from colchicine-selected multidrug resistant cultured cells. The amino acid substitution Gly----Val at codon 185 in P-glycoprotein from colchicine resistant cells occurred during selection of cells in colchicine. As previously reported, cells transfected with the MDR1 cDNA carrying Val185 acquire increased resistance to colchicine compared to other drugs. The other amino acid substitution Ser----Ala at codon 893 probably reflects genetic polymorphism. The MDR1 gene, the major member of the P-glycoprotein gene family expressed in human adrenal, is sufficient to confer multidrug-resistance on culture cells.     

StGA2ox1 is induced prior to stolon swelling and controls GA levels during potato tuber development

The formation and growth of a potato ( Solanum tuberosum ) tuber is a complex process regulated by different environmental signals and plant hormones. In particular, the action of gibberellins (GAs) has been implicated in different aspects of potato tuber formation. Here we report on the isolation and functional analysis of a potato GA 2-oxidase gene ( StGA2ox1 ) and its role in tuber formation. StGA2ox1 is upregulated during the early stages of potato tuber development prior to visible swelling and is predominantly expressed in the subapical region of the stolon and growing tuber. 35S-over-expression transformants exhibit a dwarf phenotype, reduced stolon growth and earlier in vitro tuberization. Transgenic plants with reduced expression levels of StGA2ox1 showed normal plant growth, an altered stolon swelling phenotype and delayed in vitro tuberization. Tubers of the StGA2ox1 suppression clones contain increased levels of GA₂₀, indicating altered GA metabolism. We propose a role for StGA2ox1 in early tuber initiation by modifying GA levels in the subapical stolon region at the onset of tuberization, thereby facilitating normal tuber development and growth.     

Localization of the multidrug resistance-associated 170 kDa P-glycoprotein gene to mouse chromosome 5 and to homogeneously staining regions in multidrug-resistant mouse cells by in situ hybridization

This report describes the localization of the 170 kDa P-glycoprotein gene(s) to mouse chromosome 5, subbands A2 or A3. Overproduction of P-glycoprotein is associated with multidrug resistance (MDR). MDR cell lines derived from the SEWA mouse tumor carry multiple copies of the P-glycoprotein gene. These were found to reside in homogeneously staining regions, situated in different locations in different sublines.     

Identification of the Multidrug Resistance-Related Membrane Glycoprotein as an Acceptor for Cyclosporine

Abstract

The immunosuppressive agent cyclosporine A (CSA) has been shown to reverse multidrug resistance (MDR) in malignant cells. In the present study, a ³H-cyclosporine diazirine analogue (³H-PL-CS) was used to photolabel viable MDR cells. The 170 kDa membrane P-glycoprotein, which functions as a drug efflux pump, was strongly labeled. The binding of ³H-cyclosporine diazirine analogue to P-glycoprotein was competable by excess cyclosporine A and by the nonimmunosuppressive cyclosporine H. These results suggest that cyclosporine reverses the MDR phenotype by binding directly to P-glycoprotein and that this binding is not dependent on the immunosuppressive potential of the cyclosporine derivative. The identification of P-glycoprotein as a cyclosporine binding protein has obvious implications for cancer chemotherapy.     

Structure and expression of the human MDR (P-glycoprotein) gene family.

The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragments of MDR1 cDNA and by cloning and sequencing of genomic fragments. We have found no evidence for any other cross-hybridizing MDR genes. The sequence of two exons of the MDR2 gene was determined from genomic clones. Hybridization with single-exon probes showed that the human MDR1 gene is closely related to two genes in mouse and hamster DNA, whereas MDR2 corresponds to one rodent gene. The human MDR locus was mapped by field-inversion gel electrophoresis, and both MDR genes were found to be linked within 330 kilobases. The expression patterns of the human MDR genes were examined by enzymatic amplification of cDNA. In multidrug-resistant cell lines, increased expression of MDR1 mRNA was paralleled by a smaller increase in the levels of MDR2 mRNA. In normal human tissues, MDR2 was coexpressed with MDR1 in the liver, kidney, adrenal gland, and spleen. MDR1 expression was also detected in colon, lung, stomach, esophagus, muscle, breast, and bladder.     

Identification of residues in the first cytoplasmic loop of P-glycoprotein involved in the function of chimeric human MDR1-MDR2 transporters.

The human MDR1 gene encodes the multidrug transporter (P-glycoprotein), a multidrug efflux pump. The highly homologous MDR2 gene product does not appear to be a functional multidrug pump. We have constructed a chimeric protein in which the first intracytoplasmic loop and the third and fourth transmembrane domains of the MDR1 protein were replaced by the analogous region of MDR2. Substitution of the MDR2 sequences encompassing amino acid residues 140 to 229 resulted in 17 amino acid changes, 10 in the intracytoplasmic loop (amino acids 141-188) and 7 in the transmembrane regions. This chimeric protein was expressed on the surface of NIH 3T3 cells where it bound [3H]azidopine but did not confer drug resistance. When only 4 residues, 165, 166, 168, and 169, were changed back to MDR1 amino acids, a functional drug transporter was recovered. When residues 165, 166, 168, and 169 from MDR2 were substituted into a functional MDR1 cDNA, the resulting construction was not able to confer drug resistance. These results indicate that the major functional differences between MDR1 and MDR2 in this region of P-glycoprotein reside in a small segment of the first intracytoplasmic loop. We also independently analyzed the effect of replacing Asn183 of MDR1 with Ser which occurs in MDR2. Substitution of Ser at position 183 in combination with Val at position 185 in P-glycoprotein resulted in a relative increase in resistance to actinomycin D, vinblastine, and doxorubicin in transfected NIH 3T3 cells. These results emphasize the importance of the first intracytoplasmic loop in P-glycoprotein in determining function and relative drug specificity of the transporter.     

Polymorphism of the human <Emphasis Type="Italic">MDR1</Emphasis> gene in Siberian and Central Asian populations

The multidrug resistance gene MDR1 (ABCB1) codes for a P-glycoprotein that acts as an ATP-dependent transporter and is involved in removing drugs, xenobiotics, and peptides from the cell. MDR1 is expressed in the brain, kidneys, liver, and gastrointestinal tract. The P-glycoprotein is thought to play a role in individual resistance to xenobiotics and infections. Several polymorphisms of MDR1 are associated with the level of its expression and resistance to various neurodegenerative and gastrointestinal diseases. The allele and haplotype frequencies, genetic differentiation, and linkage disequilibrium for five MDR1 single nucleotide polymorphisms (3435C/T, 2677G/T/A, 1236C/T, +139C/T, and ?1G/A) were studied in the Russian, Tuvinian, and northern and southern Kyrgyz populations. Significant genetic differences were observed between Russians and northern Kyrgyz and between Tuvinians and northern Kyrgyz. The linkage disequilibrium pattern was characterized by high population specificity.     

Evaluation of the reversal of multidrug resistance by MDR1 ribonucleic acid interference in a human colon cancer model using a Renilla luciferase reporter gene and coelenterazine

The reversal effect of multidrug resistance (MDR1) gene expression by adenoviral vector-mediated MDR1 ribonucleic acid interference was assessed in a human colon cancer animal model using bioluminescent imaging with Renilla luciferase (Rluc) gene and coelenterazine, a substrate for Rluc or MDR1 gene expression. A fluorescent microscopic examination demonstrated an increased green fluorescent protein signal in Ad-shMDR1- (recombinant adenovirus that coexpressed MDR1 small hairpin ribonucleic acid [shRNA] and green fluorescent protein) infected HCT-15/Rluc cells in a virus dose-dependent manner. Concurrently, with an increasing administered virus dose (0, 15, 30, 60, and 120 multiplicity of infection), Rluc activity was significantly increased in Ad-shMDR1-infected HCT-15/Rluc cells in a virus dose-dependent manner. In vivo bioluminescent imaging showed about 7.5-fold higher signal intensity in Ad-shMDR1-infected tumors than in control tumors (p < .05). Immunohistologic analysis demonstrated marked reduction of P-glycoprotein expression in infected tumor but not in control tumor. In conclusion, the reversal of MDR1 gene expression by MDR1 shRNA was successfully evaluated by bioluminescence imaging with Rluc activity using an in vivo animal model with a multidrug resistance cancer xenograft.     

马铃薯块茎发育机理及其基因表达

马铃薯(Solanum tuberosum L.)块茎是有块茎马铃薯植物的地下变态器官,它由匍匐茎顶端膨大形成。对于马铃薯块茎形成的生理机制已有许多研究,这些研究表明,块茎发生受许多因素的影响,总体来讲短日照、较低的温度以及离体条件下培养基较高的蔗糖浓度等有利于块茎形成,同时,块茎形成过程中内源激素亦发生一系列变化。然而,对于块茎形成中相关基因表达,进而调控块茎形成的系统研究目前还较滞后。已有研究显示,块茎形成与膨大涉及到一系列基因的表达与关闭,同时它也与淀粉合成和块茎储藏蛋白基因的表达有关。综述了这一领域现有的研究进展。     

马铃薯块茎发育机理及其基因表达

马铃薯（Solanum tuberosum L.)块茎是有块茎马铃薯植物的地下变态器官,它由匍匐茎顶端膨大形成,对于马铃薯块茎形成的生理机制已有许多研究,这些研究表明,块茎发生受许多因素的影响,总体来讲短日 照,较低的温度以及离体条件下培养基较高的蔗糖浓度等有利于块茎形成,同时,块茎形成过程中内源激素亦发生一系列变化,然而,对于块茎形成中相关基因表达,进而调控块茎形成的系统研究目前还较滞后,已有研究显示,块茎形成与膨大涉及到一系列基因的表达与关闭,同时它也与淀粉合成和块茎储藏蛋白基因的表达有关,综述了这一领域现有的研究进展。     

MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping

The human MDR3 gene is a member of the multidrug resistance (MDR) gene family. The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine. The MDR1 P-glycoprotein related transports cytotoxic drugs. Its overexpression can make cells resistant to a variety of drugs. Attempts to show that MDR3 P-glycoprotein can cause MDR have been unsuccessful thus far. Here, we report an increased directional transport of several MDR1 P-glycoprotein substrates, such as digoxin, paclitaxel, and vinblastine, through polarized monolayers of MDR3-transfected cells. Transport of other good MDR1 P-glycoprotein substrates, including cyclosporin A and dexamethasone, was not detectably increased. MDR3 P-glycoprotein-dependent transport of a short-chain phosphatidylcholine analog and drugs was inhibited by several MDR reversal agents and other drugs, indicating an interaction between these compounds and MDR3 P-gp. Insect cell membranes from Sf9 cells overexpressing MDR3 showed specific MgATP binding and a vanadate-dependent, N-ethylmaleimide-sensitive nucleotide trapping activity, visualized by covalent binding with [alpha-(32)P]8-azido-ATP. Nucleotide trapping was (nearly) abolished by paclitaxel, vinblastine, and the MDR reversal agents verapamil, cyclosporin A, and PSC 833. We conclude that MDR3 P-glycoprotein can bind and transport a subset of MDR1 P-glycoprotein substrates. The rate of MDR3 P-glycoprotein-mediated transport is low for most drugs, explaining why this protein is not detectably involved in multidrug resistance. It remains possible, however, that drug binding to MDR3 P-glycoprotein could adversely affect phospholipid or toxin secretion under conditions of stress (e.g. in pregnant heterozygotes with one MDR3 null allele).     

Expression of a human multidrug resistance cDNA (MDR1) in the bone marrow of transgenic mice: resistance to daunomycin-induced leukopenia.

The human multidrug resistance gene (MDR1) encodes a drug efflux pump glycoprotein (P-glycoprotein) responsible for resistance to multiple cytotoxic drugs. A plasmid carrying a human MDR1 cDNA under the control of a chicken beta-actin promoter was used to generate transgenic mice in which the transgene was mainly expressed in bone marrow and spleen. Immunofluorescence localization studies showed that P-glycoprotein was present on bone marrow cells. Furthermore, leukocyte counts of the transgenic mice treated with daunomycin did not fall, indicating that their bone marrow was resistant to the cytotoxic effect of the drug. Since bone marrow suppression is a major limitation to chemotherapy, these transgenic mice should serve as a model to determine whether higher doses of drugs can cure previously unresponsive cancers.     

Role of the highly structured 5'-end region of MDR1 mRNA in P-glycoprotein expression

Overexpression of P-glycoprotein, encoded by the MDR1 (multidrug resistance 1) gene, is often responsible for multidrug resistance in acute myeloid leukaemia. We have shown previously that MDR1 (P-glycoprotein) mRNA levels in K562 leukaemic cells exposed to cytotoxic drugs are up-regulated but P-glycoprotein expression is translationally blocked. In the present study we show that cytotoxic drugs down-regulate the Akt signalling pathway, leading to hypophosphorylation of the translational repressor 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] and decreased eIF4E availability. The 5'-end of MDR1 mRNA adopts a highly-structured fold. Fusion of this structured 5'-region upstream of a reporter gene impeded its efficient translation, specifically under cytotoxic stress, by reducing its competitive ability for the translational machinery. The effect of cytotoxic stress could be mimicked in vivo by blocking the phosphorylation of 4E-BP by mTOR (mammalian target of rapamycin) using rapamycin or eIF4E siRNA (small interfering RNA), and relieved by overexpression of either eIF4E or constitutively-active Akt. Upon drug exposure MDR1 mRNA was up-regulated, apparently stochastically, in a small proportion of cells. Only in these cells could MDR1 mRNA compete successfully for the reduced amounts of eIF4E and translate P-glycoprotein. Consequent drug efflux and restoration of eIF4E availability results in a feed-forward relief from stress-induced translational repression and to the acquisition of drug resistance.     

The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression.

The cystic fibrosis gene product, CFTR, and the multidrug resistance P-glycoprotein (encoded by the MDR1 gene) are structurally related proteins and both are associated with epithelial chloride channel activities. We have compared their cell-specific expression in the rat by in situ hybridization. In all tissues examined the two genes were found to have complementary patterns of expression, demonstrating exquisite regulation in both cell-specific and temporal fashions. Additionally, a switch in expression from one gene to the other was observed in certain tissues. For example, expression in the intestine switches from CFTR to MDR1 as the cells migrate across the crypt-villus boundary. A switch from CFTR to MDR1 expression was also observed in the uterine epithelium upon pregnancy. These data suggest that CFTR and P-glycoprotein serve analogous roles in epithelial cells and provide additional evidence that P-glycoprotein has a physiological role in regulating epithelial cell volume. The patterns of expression suggest that the regulation of these two genes is coordinately controlled.     

Developmental changes in carbohydrate content and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum)

Tuberising stolon tips of potato ( Solanum tuberosum L. cv. Record) accumulate starch and sucrose but the hexose content, particularly fructose, declines rapidly. Similar changes occur in the region 2 cm behind the swelling apex but the decline in glucose is far more pronounced than in the developing tuber. Tuberisation is characterised by an apparent switch from an invertase-dominated sucrolytic system (both acid and alkaline invertases [EC 3.2.1.26] are present) to one dominated by sucrose synthase (EC 2.4.1.13). Sucrose synthase and fructokinase (EC 2.7.1.4) activities were, at a maximum, ca 10- and 5-fold higher, respectively in the swelling stolon tip compared with the non-tuberising region. At the highest starch contents attained, the starch level in the young developing tuber was approximately double that in the adjacent non-tuberising stolon region. Immunoblots revealed that developmental changes in sucrose synthase. fructokinase and alkaline invertase polypeptides corresponded with enzyme activities. Antibodies raised against the N-terminal amino acid sequence of a soluble invertase purified from mature tubers did not detect significant quantities of a polypeptide in stolons and young, developing tubers. Antibodies raised against an in vitro expression product of an apoplastic invertase cloned from a leaf cDNA library detected a polypeptide in developing tubers but not in mature ones. However, expression of the protein did not correlate well with acid invertase activity during early tuber formation.     

Calmodulin gene family in potato: developmental and touch-induced expression of the mRNA encoding a novel isoform

Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca²⁺-binding area. The expression patterns of different genes were studied by northern analysis using the 3-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the -glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.