Diagnosis and reversal of multidrug resistance in paediatric cancers.
Explore the source record for details and available documents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Biomedical subjects
V Ling
Publications and source records attributed to V Ling.
Ormaplatin resistance is associated with decreased accumulation of its platinum (II) analogue, dichloro(D,L-trans)1,2-diaminocyclohexaneplatinum (II).
Ormaplatin (also known as tetraplatin) is a platinum-containing analogue which has recently undergone clinical trials. Ormaplatin may undergo conversion to dichloro(D,L-trans)-1,2-diaminocyclohexaneplatinum(II) [P1Cl2(trans-dach)]. The cisplatin-resistant murine lymphoma cell lines E8 and E5, were found to be cross-resistant to ormaplatin and PtCl2(trans-dach). We found an inverse rank correlation between drug resistance and drug accumulation for PtCl2(trans-dach) similar to our previous findings with cisplatin; however, accumulation of ormaplatin in the resistant cells was increased. Ormaplatin cytotoxicity appears to result primarily from extracellular conversion to PtCl2(trans-dach), since ormaplatin cytotoxicity was decreased under conditions where extracellular conversion to PtCl2(trans-dach) was minimised. Co-incubation with different inhibitors of energy metabolism resulted in a 65-70% increase in PtCl2(trans-dach) accumulation in the parental cell line R1.1 and a 113-307% increase in the resistant cell line E5 which suggests that the decrease in accumulation in E5 may be at least partly energy dependent. We conclude from these findings that cross-resistance to ormaplatin is associated with an energy-dependent decreased accumulation of PtCl2(trans-dach) in these cisplatin-resistant cell lines.
Animals↗
Combining cyclosporin with chemotherapy controls intraocular retinoblastoma without requiring radiation.
Chemotherapy without radiation has not controlled most intraocular retinoblastoma, perhaps because of the common high expression of multidrug resistance P-glycoprotein that we found in retinoblastoma. Cyclosporin blocks P-glycoprotein-induced efflux of vincristine and teniposide in vitro, and possibly modulates responses to carboplatin. To avoid eye irradiation in bilateral retinoblastoma patients with RB1 germline mutations, which incurs a high second malignancy rate, we added cyclosporin A to a vincristine-teniposide-carboplatin protocol and consolidated chemotherapy responses with focal therapy. We scored patients requiring irradiation, enucleation, or focal ablation of central vision as failures. In 21 study patients, the overall relapse-free rate at a median follow-up of 3.3 years was 76%, with a rate of 92% for newly diagnosed and 50% for previously treated, relapsed retinoblastoma. Our results for the most unfavorable tumors with vitreous seeds (86% at 3.5 years) are better than published success rates of irradiation for similar tumors, or irradiation with the same chemotherapy without cyclosporin (45% at 2. 6 years). These results also exceeded our historic success rate with similar chemotherapy without cyclosporin, focal therapy, and/or radiation in 19 equivalently poor-risk patients (relapse-free rate 37% at a median follow-up of 5.6 years, P = 0.032), 16 of whom were previously untreated (relapse-free rate also 37%, P = 0.012). A better outcome occurred with higher cyclosporin blood levels and projected tissue exposure. Cyclosporin did not enhance the usual chemotoxicity. This clinical study suggests that cyclosporin improves the long-term response of retinoblastoma to chemotherapy, possibly by more than one mechanism.
Antineoplastic Agents↗
P-glycoprotein: its role in drug resistance.
Explore the source record for details and available documents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Involvement of cytoplasmic factors regulating the membrane orientation of P-glycoprotein sequences.
Chinese hamster pgpl P-glycoprotein (Pgp) is a membrane transport protein that causes multidrug resistance (MDR) by actively extruding a wide variety of cytotoxic agents out of cells. It may also function as a peptide transporter and as a chloride channel. Previously, we have shown that hamster pgpl Pgp is expressed in more than one topological form and that the generation of these structures is modulated by charged amino acids flanking the predicted transmembrane (TM) segments 3 and 4. Different topological structures of Pgp may be involved in different functions. In this study, we examined the role of cytoplasmic components in cell-free translation systems in modulating the topologies of Pgp. By using rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE) expression systems, we showed that WGE contains a soluble, heat-labile, high molecular weight fraction that regulates the membrane topology of truncated Pgp molecules. These results and our previous findings indicate that the membrane topology of a mammalian polytopic membrane protein may be regulated both by the amino acid sequence of the protein and by soluble cytoplasmic component(s). We speculate that Pgp expressed in various cell types may have different topological structures modulated by specific cytoplasmic factors.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Reconstitution of drug transport by purified P-glycoprotein.
P-glycoprotein confers multidrug resistance upon cells in which it is highly expressed, reducing the effectiveness of numerous cytotoxic drugs, including many of those used for chemotherapy of cancer. Although P-glycoprotein is widely believed to function as an ATP-dependent drug efflux pump, the unusually broad substrate specificity of P-glycoprotein has engendered the proposal of other, less direct mechanisms. None of the hypothetical mechanisms has been definitively tested, however, in a purified system where other cellular components and processes are absent. We have used a fluorescent substrate of P-glycoprotein, Hoechst 33342, to measure transport activity in real-time of highly purified P-glycoprotein in a reconstituted liposome system in which the P-glycoprotein has a uniformly inside-out orientation. Using this system, we demonstrated MgATP-dependent, chemosensitizer-inhibitable transport of Hoechst 33342. Transport was prevented by omission of Mg2+, by substitution of nonhydrolyzable adenylyl-beta,gamma-imidodiphosphate for ATP, by inhibition of the ATPase activity of P-glycoprotein with vanadate and N-ethylmaleimide, and by the chemosensitizers verapamil and amiodarone. Measurements of intraliposomal pH during Hoechst 33342 transport detected no large pH changes in P-glycoprotein-containing liposomes. These results are inconsistent with a mechanism in which P-glycoprotein affects drug accumulation by directly altering intracellular pH. The Hoechst 33342 transport assay results are consistent with mechanisms in which P-glycoprotein alone is sufficient to transport drugs out of the membrane bilayer.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Hemolysin transport in Escherichia coli. Point mutants in HlyB compensate for a deletion in the predicted amphiphilic helix region of the HlyA signal.
The alpha-hemolysin transporter of Escherichia coli, a member of the ATP-binding cassette transporter super-family, is responsible for secretion of the 107-kDa protein toxin HlyA across both membranes of the Gram-negative envelope in a single step. Secretion of HlyA is dependent on a signal sequence, which occupies the C-terminal 50-60 amino acids of HlyA. Previously, it was shown that point mutants in the transmembrane domain of the transporter HlyB could partially correct the transport defect caused by a deletion of the C-terminal 29 amino acids of HlyA. These suppressor mutations demonstrated a direct interaction between HlyA and HlyB. They also displayed suppressor effects on a broad spectrum of HlyA signal mutants. In the present study, we selected HlyB alleles that complemented an internal deletion of 29 amino acids in HlyA containing a predicted amphiphilic helix region immediately upstream from the previous deletion. This set of HlyB mutants identifies further sites in HlyB that modulate substrate specificity but display allele-specific effects on a range of HlyA signal mutants. The inability to isolate mutations with effects restricted to either half of the signal sequence suggests that the signal is not recognized in a modular fashion by the transporter but rather functions as an integrated whole. We also report the isolation of the first substrate specificity mutation, which lies within the ATP-binding domain of HlyB. This could support a model in which the region of the ATP-binding cassette between the two Walker consensus motifs involved in ATP binding interacts with either the substrate or the transmembrane domains.
Amino Acid Sequence↗
Structural analysis and comparison of the C-terminal transport signal domains of hemolysin A and leukotoxin A.
NMR spectroscopy was used to study the structure of the C-terminal signal sequences of the bacterial toxins, hemolysin A(HlyA) and leukotoxin A (LktA). The two signals share little sequence homology; however, both can direct toxin transport with equal efficiency. We report here that in a membrane mimetic environment both peptides form two short non-interacting alpha-helices separated by a short loop. This higher order structure may be a common feature of C-terminal signals and may be required for interaction with the membrane associated transporter complex.
Amino Acid Sequence↗
Identification of a sister gene to P-glycoprotein.
The P-glycoproteins (Pgps) are a small family of transport proteins associated with the multidrug resistance phenotype of cell lines selected for growth in cytotoxic drugs. Utilizing low stringency screening, we have identified a novel gene closely related to the Pgps expressed in the pig and other mammalian liver which we have called Sister of P-glycoprotein (spgp). Sequence of this gene shows it to be a member of the ATP-binding cassette family of transporters and the gene most closely related to Pgp identified to date. The function of spgp is not known, but it can be recognized by at least one Pgp mAb, C219. This cross-reactivity has implications for expression studies in tissues and tumors utilizing this and other Pgp antibodies.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Secretion and circular dichroism analysis of the C-terminal signal peptides of HlyA and LktA.
The secretion of the 107 kDa hemolysin A (HlyA) from Escherichia coli is mediated by membrane proteins hemolysin B (HlyB) and hemolysin D (HlyD). The signal for transport has been mapped to the C-terminal 60 amino acids of the HylA molecule. We have shown previously that the C-terminal 70 amino acids of leukotoxin (LktA) from Pasteurella hemolytica can substitute functionally for the HlyA signal sequence. This 70 amino acid peptide contains little primary sequence similarity to the HlyA signal sequence, and we have hypothesized that these signal sequences assume a similar higher-order structure which is recognized by the HlyB/D transporter. In the present study, we have expressed and purified small peptides containing the C-terminal 61 amino acids of HlyA and the C-terminal 70 amino acids of LktA. We show that these signal peptides are sufficient for secretion from E. coli in a HlyB/D dependent manner. Circular dichroism analyses show that both molecules exhibit common biophysical properties. In aqueous solution, they appear to be mainly unstructured, but in a membrane mimetic environment they assume a helical secondary structure. The conformational change observed for both peptides going from an aqueous to a membrane mimetic environment may be an important feature of these signal sequences necessary for their recognition and transport.
Bacterial Proteins↗
Topological determinants of internal transmembrane segments in P-glycoprotein sequences.
P-glycoprotein (Pgp) is a polytopic membrane protein responsible for multidrug resistance in cancer cells. Previously, we have used a coupled cell-free translation/translocation system to investigate the membrane orientation of Pgp sequences and have made the unexpected observation that predicted transmembrane (TM) segments from both the NH2-terminal and COOH-terminal halves inserted in microsomal membranes in two different orientations (Zhang, J.-T., Duthie, M., and Ling, V. (1993) J. Biol. Chem. 268, 15101-15110). How these topological forms of Pgp are regulated is not known. In the present study, we have used site-directed mutagenesis to investigate if the amino acids surrounding the internal TM segments of Pgp may affect their orientation. We discovered that the charged amino acids flanking TM4 are important in determining the membrane orientation of the NH2-terminal half molecule of Pgp. This is a novel observation demonstrating the existence of internal topogenic sequences in a mammalian polytopic membrane protein. These findings thus suggest A) that the topological structure of a mammalian polytopic membrane protein does not integrate into the membrane simply by following the lead of the first inserted TM segment but that internal TMs may have independent topogenic information and B) that the TM segments in a multi-spanning membrane protein may be more dynamic than have been previously anticipated, i.e. mutations in the amino acids surrounding internal TMs could drastically change the overall topology of the molecule.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
P-glycoprotein stability is affected by serum deprivation and high cell density in multidrug-resistant cells.
The control of P-glycoprotein (Pgp) expression in multidrug-resistant cells (MDR) is complex and may be regulated at different levels. We have investigated Pgp stability in four different human and hamster MDR cell lines. Using a pulse-chase procedure we show that Pgp half-life is between 14 and 17 h in all these cell lines when they are growing exponentially. However, in the presence of a low level of serum, Pgp half-life is increased four to sixfold. A similar effect is observed when the cell cultures are maintained in high cell density. The increased Pgp stability appears to be differently regulated as serum deprivation results in a general enhanced degradation of total cytoplasmic and membrane proteins. Moreover, the observed serum effect suggests the involvement of growth factors in the control of Pgp stability. These findings suggest that protein stability may be an important factor in the regulation of Pgp expression.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Using purified P-glycoprotein to understand multidrug resistance.
Since P-glycoprotein was discovered almost 20 years ago, its causative role in multidrug resistance has been established, but central problems of its biochemistry have not been definitively resolved. Recently, major advances have been made in P-glycoprotein biochemistry with the use of purified and reconstituted P-glycoprotein, as well as membranes from nonmammalian cells containing heterologously expressed P-glycoprotein. In this review we describe recent findings using these systems which are elucidating the molecular mechanism of P-glycoprotein-mediated drug transport.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Multidrug drug resistance in pediatric sarcomas.
PURPOSE: Although P-glycoprotein overexpression is an important cause of multidrug resistance in vitro, whether this resistance mechanism is equally applicable to the clinic has still to be fully established. This review has examined the immunohistochemical and molecular biologic tools used to evaluate soft tissue sarcomas of children and adults, in order to determine whether P-glycoprotein can limit the efficacy of chemotherapy. DESIGN: Because soft tissue sarcomas are successfully treated by cytotoxic substrates of P-glycoprotein in many, but not all children, these tumors may be useful models for determining whether this drug efflux transporter is a clinically relevant cause of chemoresistance. RESULTS: Certain studies of acute myelogenous leukemia, lymphoma, and myeloma in adults and rhabdomyosarcoma, neuroblastoma, and acute lymphoblastic leukemia in children have provided the best current evidence for a strong corelation between the expression of P-glycoprotein and outcome of chemotherapy. Based on these observations, several clinical trials have been initiated to determine whether pharmacologic chemosensitization improves chemotherapy responses and cure rates in P-glycoprotein-expressing malignancies. Thus, early identification of lower levels of P-glycoprotein in tumors that still might respond to chemosensitizer-modulated chemotherapy may be highly pertinent to conducting more informative multidrug resistance reversal trials. CONCLUSION: The question of whether the multidrug resistance P-glycoprotein is a clinically relevant cause of chemoresistance may ultimately be answered by the successful prevention of chemotherapy failure by chemosensitizers that specifically reverse this drug efflux mechanism.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Multidrug resistance in pediatric malignancies.
PURPOSE: Increased expression of P-glycoprotein is an important cause of multidrug resistance in tumor cell lines in vitro. Whether this mechanism is equally relevant as a cause of clinical chemoresistance has not been established and is currently being investigated. This review has examined the immunohistochemical and molecular biologic tools suitable for assessing P-glycoprotein expression in patient samples and methodologic issues important for evaluating the results of clinical studies. Current evidence that supports a role for P-glycoprotein in limiting the efficacy of cancer chemotherapy has been reviewed. DESIGN: Malignancies that have been successfully treated by chemotherapeutic substrates of P-glycoprotein, in which a proportion of patients still fail therapy, may be the most useful models for determining whether this drug efflux transporter is a clinically relevant cause of chemoresistance. RESULTS: Studies of acute myelogenous leukemia, lymphoma, and myeloma in adults have so far provided the best evidence for a relevant role for P-glycoprotein as a cause of clinical multidrug resistance. A similar strong association has been observed between the expression of P-glycoprotein and outcome of treatment in certain malignancies in children, such as neuroblastoma, rhabdomyosarcoma, and acute lymphoblastic leukemia. Some apparent controversies related to this issue of clinical relevance may be explained by the differences in P-glycoprotein detection techniques, methodology, and experimental designs used in different studies. Because several clinical trials have already been initiated to determine whether pharmacologic chemosensitization improves the outcome of chemotherapy in certain malignancies, the successful verification of multidrug resistance limiting the cure rates of these tumors becomes a more critical issue, and identification of those patients with lower levels of P-glycoprotein expression early in the course of their disease, when they are most likely to benefit from multidrug resistance reversal, has assumed an even greater relevance. CONCLUSION: The clinical relevance of the multidrug resistance P-glycoprotein may ultimately be confirmed by the successful prevention of chemotherapy failure by chemosensitizers that specifically reverse this drug efflux mechanism.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Overexpression of the class II P-glycoprotein gene in primary rat hepatocyte culture: evidence for increased mRNA stability.
The overexpression of P-glycoprotein (Pgp) appears to be responsible for multidrug resistance in some human cancers. The molecular basis of this overexpression is not understood. We have used primary monolayer cultures of adult rat hepatocytes as a model system to study the regulation of Pgp gene expression (Lee et al., J. Cell. Physiol., 157: 392-402, 1993). We observed a dramatic and specific overexpression of class II Pgp as a function of the time in culture. This isoform of Pgp, which is expressed at a very low level in normal liver, has also been shown to be predominantly overexpressed in several models of rat liver carcinogenesis. In the present study, we have used nuclear run-on assays and mRNA decay studies to investigate the mechanism for the overexpression of class II Pgp in cultured hepatocytes. We conclude that an increased mRNA stability is the major factor involved in the increased expression of class II Pgp. Studies using various drugs indicate that the integrity of the cytoskeleton is important for the maintenance of high expression of class II Pgp. Disruption of the cytoskeleton in cultured hepatocytes with cytochalasin D did not affect the transcriptional activity of the class II Pgp gene but rapidly destabilized its mRNA. This raises the possibility that an association between class II Pgp mRNA and cytoskeletal elements may underlie the mechanism that regulates class II Pgp mRNA stability. These findings have important implications for our understanding of the overexpression of class II Pgp during liver carcinogenesis.
Actins↗
Differences between accumulation of 99mTc-MIBI and 201Tl-thallous chloride in tumour cells: role of P-glycoprotein.
Both 99mTc-MIBI and 201Tl have been used for tumour imaging. It has recently been reported that 99mTc-MIBI is a substrate for P-glycoprotein (Pgp), a membrane pump which mediates multidrug resistance. We have evaluated the role of Pgp in the cellular accumulation of 201Tl by using sensitive and resistant strains of Chinese hamster ovary (CHO) fibroblasts (AuxB1 and CHRC5, respectively) grown in suspension culture. 201Tl accumulation was the same in sensitive and resistant cells, whereas 99mTc-MIBI accumulation was much lower in resistant cells than in sensitive ones. Down-modulation of Pgp with 100 microM verapamil did not alter cellular accumulation of 201Tl while it significantly increased 99mTc-MIBI accumulation in both types of cell. Similarly, 10 microM verapamil did not affect the rate of washout of 201Tl from preloaded cells, while 99mTc-MIBI washout was greatly reduced in the presence of verapamil. These results suggest that 201Tl will accumulate in both sensitive and resistant tumour cells, whereas 99mTc-MIBI will be extruded from resistant cells and therefore may be less useful for tumour detection when the tumour cells express high Pgp levels.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
P-glycoprotein and tumor progression.
Explore the source record for details and available documents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗