[Logistics and European laws on allogeneic tissue transplantation]. / Logistik und europäische Gesetze für allogene Gewebetransplantate.

European rules stipulate quality and safety requirements on recruitment, testing, preserving, and distributing human tissues for application in patients in order to obtain a high level of health safety. In order to avoid transmissible diseases in tissue transplantation, specific requirements for all individual groups of tissues of human origin were defined. European Member States need to implement these directives in order to obtain a high level of health protection in human application. Precise instructions for tissue institutes regarding organization, management, documentation, and quality controls are required. Adverse effects have to be registered by these institutions. Costs need to be contained and therefore uniform administrative directives will engineer the modern techniques of communication. A uniform European code is needed for easy traceability of human tissues. The directive respects the basic human rights as laid down in the Charter of Fundamental Rights of the European Union.

Comparison of the functional characteristics of the nucleotide binding domains of multidrug resistance protein 1.

Multidrug Resistance Protein 1 (MRP1) transports diverse organic anionic conjugates and confers resistance to cytotoxic xenobiotics. The protein contains two nucleotide binding domains (NBDs) with features characteristic of members of the ATP-binding cassette superfamily and exhibits basal ATPase activity that can be stimulated by certain substrates. It is not known whether the two NBDs of MRP1 are functionally equivalent. To investigate this question, we have used a baculovirus dual expression vector encoding both halves of MRP1 to reconstitute an active transporter and have compared the ability of each NBD to be photoaffinity-labeled with 8-azido-[(32)P]ATP and to trap 8-azido-[(32)P]ADP in the presence of orthovanadate. We found that NBD1 was preferentially labeled with 8-azido-[(32)P]ATP, while trapping of 8-azido-[(32)P]ADP occurred predominantly at NBD2. Although trapping at NBD2 was dependent on co-expression of both halves of MRP1, binding of 8-azido-ATP by NBD1 remained detectable when the NH(2)-proximal half of MRP1 was expressed alone and when NBD1 was expressed as a soluble polypeptide. Mutation of the conserved Walker A lysine 684 or creation of an insertion mutation between Walker A and B motifs eliminated binding by NBD1 and all detectable trapping of 8-azido-ADP at NBD2. Both mutations decreased leukotriene C(4) (LTC(4)) transport by approximately 70%. Mutation of the NBD2 Walker A lysine 1333 eliminated trapping of 8-azido-ADP by NBD2 but, in contrast to the mutations in NBD1, essentially eliminated LTC(4) transport activity without affecting labeling of NBD1 with 8-azido-[(32)P]ATP.

Membrane topology of the multidrug resistance protein (MRP). A study of glycosylation-site mutants reveals an extracytosolic NH2 terminus.

Multidrug resistance protein, MRP, is a 190-kDa integral membrane phosphoglycoprotein that belongs to the ATP-binding cassette superfamily of transport proteins and is capable of conferring resistance to multiple chemotherapeutic agents. Previous studies have indicated that MRP consists of two membrane spanning domains (MSD) each followed by a nucleotide binding domain, plus an additional extremely hydrophobic NH2-terminal MSD. Computer-assisted hydropathy analyses and multiple sequence alignments suggest several topological models for MRP. To aid in determining the topology most likely to be correct, we have identified which of the 14 N-glycosylation sequons in this protein are utilized. Limited proteolysis of MRP-enriched membranes and deglycosylation of intact MRP and its tryptic fragments with PNGase F was carried out followed by immunoblotting with antibodies known to react with specific regions of MRP. The results obtained indicated that the sequon at Asn354 in the middle MSD is not utilized and suggested approximate sites of N-glycosylation. Subsequent site-directed mutagenesis studies established that Asn19 and Asn23 in the NH2-terminal MSD and Asn1006 in the COOH-terminal MSD are the only sites in MRP that are modified with N-linked oligosaccharides. N-Glycosylation of Asn19 and Asn23 provides the first direct experimental evidence that MRP has an extracytosolic NH2 terminus. This finding, together with those of previous studies, strongly suggests that the NH2-terminal MSD of MRP contains an odd number of transmembrane helices. These results may have important implications for the further understanding of the interaction of drugs with MRP.

Location of a protease-hypersensitive region in the multidrug resistance protein (MRP) by mapping of the epitope of MRP-specific monoclonal antibody QCRL-1.

Multidrug resistance protein (MRP) is a Mr 190,000 integral membrane phosphoglycoprotein which has been shown by transfection studies to confer multidrug resistance. We have previously raised and characterized a panel of MRP-specific monoclonal antibodies (MAbs) which detect distinct epitopes in the MRP molecule (D. R. Hipfner et A, Cancer Res., 54. 5788-5792, 1994), and, in the present study, we have identified the epitope of one of these, MAb QCRL-1. Immunoblot analysis of MRP fragments generated by digestion with formic acid or trypsin suggested that the MAb QCRL-1 epitope was located in the region connecting the two halves of MRP. Subsequent analyses of a series of truncated bacterial glutathione S-transferase fusion proteins containing segments of human MRP further localized the MAb QCRL-1 epitope to a region encompassing amino acids 903-956. Similar experiments with an analogous segment of murine MRP demonstrated that MAb QCRL-1 was highly specific for the human protein. The reactivity of MAb QCRL-1 with a series of overlapping hexapeptides and heptapeptides within this region identified the human MRP-specific heptapeptide SSYSGDI (corresponding to amino acids 918-924) as the epitope, and this peptide was shown to specifically inhibit MAb QCRL-1 binding to MRP. The results of these studies confirm that this epitope has a cytoplasmic location consistent with the topology of MRP predicted from hydrophobicity analyses. These experiments also revealed the presence of a number of protease-sensitive sites on either side of the MAb QCRL-1 epitope in the cytoplasmic domain connecting the two halves of MRP. Future epitope-mapping studies with other MRP-specific MAbs win provide additional insights into the topology of MRP, and may help to identify functionally important regions of this protein. Moreover, definition of the epitope recognized by MAb QCRL-1 as well as the other MAbs will facilitate the use of these reagents for immunohistological studies of MRP expression in drug-resistant tumors.

Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione-dependent vincristine transport.

The 190-kDa multidrug resistance protein (MRP) has recently been associated with the transport of cysteinyl leukotrienes and several glutathione (GSH) S-conjugates. In the present study, we have examined the transport of leukotriene C4 (LTC4) in membrane vesicles from MRP-transfected HeLa cells (T14), as well as drug-selected H69AR lung cancer cells which express high levels of MRP. V(max) and K(m) values for LTC4 transport by membrane vesicles from T14 cells were 529 +/- 176 pmol mg(-1) min(-1) and 105 +/- 31 nM, respectively. At 50 nM LTC4, the K(m) (ATP) was 70 micron. Transport in T14 vesicles was osmotically-sensitive and was supported by various nucleoside triphosphates but not by non- or slowly-hydrolyzable ATP analogs. LTC4 transport rates in membrane vesicles derived from H69AR cells and their parental and revertant variants were consistent with their relative levels of MRP expression. A 190-kDa protein in T14 membrane vesicles was photolabeled by [3H]LTC4 and immunoprecipitation with MRP-specific monoclonal antibodies (mAbs) confirmed that this protein was MRP. LTC4 transport was inhibited by an MRP-specific mAb (QCRL-3) directed against an intracellular conformational epitope of MRP, but not by a mAb (QCRL-1) which recognizes a linear epitope. Photolabeling with [3H]LTC4 was also inhibitable by mAb QCRL-3 but not mAb QCRL-1. GSH did not inhibit LTC4 transport. However, the ability of alkylated GSH derivatives to inhibit transport increased markedly with the length of the alkyl group. S-Decylglutathione was a potent competitive inhibitor of [3H]LTC4 transport (K(i(app)) 116 nM), suggesting that the two compounds bind to the same, or closely related, site(s) on MRP. Chemotherapeutic agents including colchicine, doxorubicin, and daunorubicin were poor inhibitors of [3H]LTC4 transport. Taxol, VP-16, vincristine, and vinblastine were also poor inhibitors of LTC4 transport but inhibition by these compounds was enhanced by GSH. Uptake of [3H]vincristine into T14 membrane vesicles in the absence of GSH was low and not dependent on ATP. However, in the presence of GSH, ATP-dependent vincristine transport was observed. Levels of transport increased with concentrations of GSH up to 5 mM. The identification of an MRP-specific mAb that inhibits LTC4 transport and prevents photolabeling of MRP by LTC4, provides conclusive evidence of the ability of MRP to transport cysteinyl leukotrienes. Our studies also demonstrate that MRP is capable of mediating ATP-dependent transport of vincristine and that transport is GSH-dependent.

ATP-dependent 17 beta-estradiol 17-(beta-D-glucuronide) transport by multidrug resistance protein (MRP). Inhibition by cholestatic steroids.

In addition to its ability to confer resistance to a range of natural product type chemotherapeutic agents, multidrug resistance protein (MRP) has been shown to transport the cysteinyl leukotriene, LTC4, and several other glutathione (GSH) S-conjugates. We now demonstrate that its range of potential physiological substrates also includes cholestatic glucuronidated steroids. ATP dependent, osmotically sensitive transport of the naturally occurring conjugated estrogen, 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), was readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells (Vmax 1.4 nmol mg-1 min-1, K(m) 2.5 micron). The involvement of MRP was confirmed by demonstrating that transport was completely inhibited by a monoclonal antibody specific for an intracellular conformational epitope of the protein. MRP-mediated transport of LTC4, was competitively inhibited by E(2)17 beta G (K(i(app)) 22 micron), despite the lack of structural similarity between these two substrates. Competitive inhibition of [3H]E(2)17 beta G transport was also observed with a number of other cholestatic conjugated steroids. All of these compounds prevented photolabeling of MRP with [3H]LTC4, demonstrating that the cholestatic steroid and leukotriene conjugates compete either for the same or possibly overlapping sites on the protein. Consistent with the presence of overlapping but non-identical sites, studies using chemotherapeutic drugs to inhibit MRP-mediated E(2)17 beta G transport indicated that daunorubicin had the highest relative potency of the drugs tested, whereas it was the least potent inhibitor of LTC4 transport. Non-cholestatic steroids glucuronidated at the 3 position of the steroid nucleus, such as 17 beta-estradiol 3-(beta-D-glucuronide), did not compete for transport of E(2)17 beta G by MRP, nor did they inhibit photolabeling of the protein with [3H]LTC4. These data identify MRP as a potential transporter of cholestatic conjugated estrogens and demonstrate site-specific requirements for glucuronidation of the steroid nucleus.

Characterization of the M(r) 190,000 multidrug resistance protein (MRP) in drug-selected and transfected human tumor cell.

Overexpression of multidrug resistance-associated protein (MRP) has been detected in resistant cell lines derived from a variety of tumor types. The deduced amino acid sequence of MRP suggests that it is a member of the ATP-binding cassette transmembrane transporter superfamily that may be glycosylated and/or phosphorylated [S. P. C. Cole et al., Science Washington, DC), 258: 1650-1654, 1992]. Recently, transfection of HeLa cells with MRP expression vectors has demonstrated that the protein is capable of increasing resistance to natural product drugs such as anthracyclines, Vinca alkaloids, and epipodophyllotoxins (C. E. Grant et al., Cancer Res., 54: 357-361, 1994). Although the resistance phenotype of the transfectants is similar to that of the human small cell lung cancer cell line, H69AR, from which MRP was originally cloned, the transfectants differ in their drug accumulation characteristics, relative resistance to certain drugs, and MRP mRNA:protein ratio. Such differences have also been observed among drug-selected cell lines that overexpress MRP, and the underlying causes of these variable phenotypes are presently not known. We have utilized polyclonal anti-MRP-peptide antibodies to compare MRP post-translational modification, stability, processing, and subcellular distribution in the HeLa transfectants and in the drug-selected H69AR cells. These studies establish that MRP in both the transfected and selected cells is an ATP-binding, integral membrane glycophosphoprotein with an apparent molecular weight of 190,000. No obvious differences were detected in the extent or type of glycosylation or the kinetics of processing and turnover of the protein that might contribute to the different characteristics of the transfected and drug-selected cells. Analyses of the subcellular distribution of MRP by isopyknic density gradient centrifugation revealed that approximately 80% of MRP in the HeLa transfectants was associated with a low density plasma membrane fraction while the comparable fraction in the drug-selected H69AR cells contained only approximately 50% of the protein. The remaining MRP and plasma membrane markers were codistributed in higher density fractions consistent with the presence of MRP in endocytotic vesicles. The relatively high proportion of MRP associated with these fractions in H69AR cells may contribute to the lack of an observable accumulation defect in these cells when compared with the transfectants.

Non-P-glycoprotein-mediated multidrug-resistant human KB cells selected in medium containing adriamycin, cepharanthine, and mezerein.

Human epidermoid KB cell lines resistant to high levels of adriamycin, C-A90, C-A120, C-A500, and C-A1000, were isolated in selection medium containing increasing concentrations of adriamycin, 1 microgram/ml of cepharanthine, a multidrug-resistance (MDR) reversing agent, and 100 nM of mezerein, a protein kinase C activating agent. One of the adriamycin-resistant KB cell lines, C-A500, was cross-resistant to drugs that typify the classical multidrug resistance phenotype, such as vincristine, actinomycin D, VP-16, and colchicine. The accumulation of adriamycin and vincristine was decreased in C-A500 cells and the efflux of adriamycin from C-A500 was enhanced compared with parental KB-3-1 cells. These adriamycin-resistant KB cells did not contain detectable levels of P-glycoprotein or overexpress MDR1. Multidrug-resistance-associated protein (MRP) and MRP mRNA were expressed in the adriamycin-resistant KB cells, C-A120, C-A500, and C-A1000, but not in parental KB-3-1 and revertant C-AR cells. The MRP gene was amplified in all the MDR cells that overexpressed MRP mRNA. DNA topoisomerase II levels were markedly decreased in C-A500 and C-A1000 cells but only slightly decreased in C-A120 cells. These results indicate that MRP overexpressed in the resistant cells may be responsible for the reduced accumulation of adriamycin and vincristine and that both the increased expression of MRP and decreased levels of topoisomerase II underlie the drug resistance in C-A120, C-A500, and C-A1000 cell lines.

Deletion and transposon mutagenesis and sequence analysis of the pRO1600 OriR region found in the broad-host-range plasmids of the pQF series.

The nucleotide sequence of the replicative origin (OriR) region of the small cryptic broad-host-range plasmid, pRO1600, which forms the basis of a number of useful cloning vectors has been determined. In addition it has been subjected to Tn5 mutagenesis, deletion analysis, and subcloning in order to define the regions essential for replication in Pseudomonas aeruginosa. The sequence (1894 bp) contains a fragment derived from transposon Tn1. The OriR region is structurally related to other replication (Rep) protein-dependent origins in that it has an A-T-rich region upstream of four 17-bp direct repeats (iterons) which presumably function in initiator protein binding. The sequence also contains a DNA-A-binding site and an open reading frame which could encode a basic (pI 10.6) 25,343-Da Rep protein with homology to RepA from the Neisseria gonorrhoeae beta-lactamase plasmid pFA3. The possible evolutionary origin of this plasmid in P. aeruginosa (RP1) is discussed.

Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs.

Amplification of the gene encoding multidrug resistance-associated protein (MRP) and overexpression of its cognate mRNA have been detected in multidrug-resistant cell lines derived from several different tumor types. To establish whether or not the increase in MRP is responsible for drug resistance in these cell lines, we have transfected HeLa cells with MRP expression vectors. The transfectants display an increase in resistance to doxorubicin that is proportional to the levels of a M(r) 190,000, integral membrane protein recognized by anti-MRP antibodies. The transfectants are also resistant to vincristine and VP-16 but not to cisplatin. The results demonstrate that MRP overexpression confers a multidrug resistance phenotype similar to that formerly associated exclusively with elevated levels of P-glycoprotein.

Altered topoisomerase II alpha in a drug-resistant small cell lung cancer cell line selected in VP-16.

The H209/V6 cell line was derived from the H209 small cell lung cancer cell line by selection in etoposide (VP-16). Cytogenetic analysis indicates that the sensitive and resistant cell lines share 20 marker chromosomes and thus are clearly related. However, the H209/V6 cell line has four additional structurally altered chromosomes and a 2 N-modal chromosome number, while the H209 cell line is hypotetraploid (4 N-). H209/V6 cells are cross-resistant to some drugs that interact with topoisomerase II but not mitoxantrone. H209/V6 cells are also not cross-resistant to vincristine, trimetrexate, or cisplatin. The rates of VP-16 efflux are the same in the resistant and sensitive cell lines, which is consistent with the observation that P-glycoprotein mRNA is not detectable in either cell line. Fewer VP-16-induced DNA-protein complexes are observed in H209/V6 cells, and immunoblot analysis shows that levels of topoisomerase II alpha are reduced in H209/V6 cells compared to the sensitive H209 cells. Furthermore, the topoisomerase II alpha-related protein in H209/V6 cells has an increased electrophoretic mobility, with an apparent M(r) of 160,000. The levels of the topoisomerase II alpha 6.1-kilobase mRNA in H209/V6 cells are reduced > 10-fold. In addition, a second topoisomerase II alpha-related mRNA of approximately 4.8 kilobases is observed in H209/V6 cells but not in H209 cells. The quantity and electrophoretic mobility of the M(r) 180,000 topoisomerase II beta protein and its 6.1-kilobase mRNA are the same in the sensitive and resistant cell lines. The topoisomerase II strand-passing activity in H209/V6 nuclear extracts is reduced about 2-fold, but this activity is not more resistant to inhibition by VP-16 than the activity in H209 cells. However, band depletion immunoblot experiments show that the topoisomerase II alpha-related M(r) 160,000 protein in H209/V6 cells is not bound to DNA in the presence of concentrations of VP-16 that deplete the M(r) 170,000 topoisomerase II alpha in H209 cells and the M(r) 180,000 topoisomerase II beta in both the resistant and sensitive cells. We conclude that quantitative and qualitative alterations in topoisomerase II alpha have occurred in H209/V6 cells and are likely to contribute to its resistance phenotype.

Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line.

The doxorubicin-selected lung cancer cell line H69AR is resistant to many chemotherapeutic agents. However, like most tumor samples from individuals with this disease, it does not overexpress P-glycoprotein, a transmembrane transport protein that is dependent on adenosine triphosphate (ATP) and is associated with multidrug resistance. Complementary DNA (cDNA) clones corresponding to messenger RNAs (mRNAs) overexpressed in H69AR cells were isolated. One cDNA hybridized to an mRNA of 7.8 to 8.2 kilobases that was 100- to 200-fold more expressed in H69AR cells relative to drug-sensitive parental H69 cells. Overexpression was associated with amplification of the cognate gene located on chromosome 16 at band p13.1. Reversion to drug sensitivity was associated with loss of gene amplification and a marked decrease in mRNA expression. The mRNA encodes a member of the ATP-binding cassette transmembrane transporter superfamily.

The synthesis and immobilisation of cartilage-specific proteoglycan by human chondrocytes in different concentrations of agarose.

Chondrocytes were cultured in agarose gels of different concentrations. In this in vitro model these cells synthesize tissue-specific proteoglycans. The rate of proteoglycan synthesis was not dependent on the concentration of the surrounding gel. The immobilisation of these macromolecules in monomeric and in aggregated form were studied. 0.5% to 1.0% of agarose failed to retain important amounts of proteoglycan. Proteoglycan monomers and even aggregates diffused to the incubation medium. 2.0% and 4.0% of agarose immobilised the bulk of the aggregates and approximately 50% of the monomeric proteoglycans. Low-molecular proteoglycan species or break-down products freely moved out of the gel. The reproducibility of the variables concerning proteoglycan metabolism was very good.