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Biomedical subjects

V Ling

Publications and source records attributed to V Ling.

At least 127 records · Page 7Linked to original sources
PubMed1990

Identification of a membrane glycoprotein overexpressed in murine lymphoma sublines resistant to cis-diamminedichloroplatinum(II).

A series of murine thymic lymphoma cell sublines was selected in vitro for resistance to cis-diamminedichloroplatinum(II) (CDDP). The level of CDDP resistance correlated with reduced drug accumulation in these cells. A rabbit antiserum was raised against the plasma membrane of a CDDP-resistant subline and used in Western blot analyses. Increased expression of a surface antigen of approximately 200 kDa was observed and found to correlate with the degree of resistance. Further biochemical and immunological studies demonstrated that this is a plasma membrane glycoprotein. However, it is different from the multidrug resistance-associated P-glycoprotein with a molecular weight of about 170,000. We have called this unique CDDP resistance-associated membrane protein CPR-200.

Animals↗
PubMed1990

Overexpression of P-glycoprotein in mammalian tumor cell lines after fractionated X irradiation in vitro.

We observed that in vitro exposure of mammalian tumor cells to fractionated x irradiation results in the expression of drug resistance. The cause of this resistance was investigated in a series of Chinese hamster ovary cell lines that had survived exposure to multiple lethal doses of radiation. These cell lines had increased levels of P-glycoprotein (Pgp), the multidrug-resistance-associated membrane glycoprotein. Consistent with the classic multidrug resistance phenotype, they exhibited cross-resistance to multiple drugs, as well as sensitivity to reversal of vincristine resistance by verapamil. However, the cell lines showed no change in their sensitivity to x rays. Pgp overexpression occurred in these cells, despite a lack of Pgp gene amplification or of significant alteration in Pgp messenger RNA levels. Although the cause of increased Pgp levels is not yet known, these data suggest a biological basis for the clinical problem of drug resistance that can occur in previously irradiated tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Sex-dependent and independent expression of the P-glycoprotein isoforms in Chinese hamster.

P-glycoprotein (Pgp) is a small family of membrane proteins which belongs to a superfamily of energy-dependent membrane transport proteins identified in phylogenetically distant species, from bacteria to man. Among mammalian species, some of the Pgp isoforms can mediate multidrug resistance by acting as an energy-dependent drug efflux pump. However, the physiologic functions of the Pgp isoforms have not been defined. In this study we examined the expression of the three hamster Pgp isoforms in normal hamster tissues, by using isoform-specific monoclonal antibodies in a competitive immunohistochemical assay. We showed that each Pgp isoform is predominantly expressed in a small, distinct group of differentiated cells, where it is likely to function in specific secretory pathways. The expression of the Pgp isoforms appears to be tightly regulated and, at least in some cells, under complex hormonal control. Furthermore, there is a striking sex difference in Pgp content of the adrenal cortex. These findings are important for the ultimate understanding of the normal physiologic roles of the Pgp gene family members.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Detection of P-glycoprotein isoforms by gene-specific monoclonal antibodies.

P-glycoprotein is a highly conserved membrane protein shown to be overexpressed in many multidrug-resistant tumor cell lines. P-glycoprotein is encoded by a small gene family in mammalian cells. Class I and II isoforms cause multidrug resistance, whereas class III does not. In this report, we have characterized three P-glycoprotein-specific monoclonal antibodies (mAbs) by high-resolution epitope mapping with a series of hexapeptides. mAb C494 is gene specific, binding to a sequence present only in the class I isoform of hamster and human. The mAb C32 recognizes a sequence conserved in hamster class I and II isoforms but not in class III isoforms. In contrast, the mAb C219 recognizes a highly conserved amino acid sequence found in all P-glycoprotein isoforms characterized to date. These mAbs were used to reveal differential expression and specific localization of the three P-glycoprotein isoforms in hamster tissues by immunohistochemical staining and competition with epitope-specific peptides. Colonic epithelial cells expressed predominantly the class I isoform in a polarized manner, adrenal cortical cells expressed predominantly the class II isoform, whereas a small percentage of skeletal muscle fibers expressed the class III isoform of P-glycoprotein. These findings suggest that the P-glycoprotein isoforms have distinct physiological roles associated with specialized cell functions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Immunohistochemical detection of P-glycoprotein: prognostic correlation in soft tissue sarcoma of childhood.

Increased expression of P-glycoprotein is associated with multidrug resistance (MDR) in many cell lines. Significant levels of P-glycoprotein have been detected in a number of human tumors. The purpose of this study was to determine whether P-glycoprotein expression correlates with both response to chemotherapy and prognosis in soft tissue sarcoma of childhood. In a retrospective study, biopsy samples from 30 cases of rhabdomyosarcoma (RMS) and undifferentiated sarcoma (US) treated at The Hospital for Sick Children in Toronto were analyzed using a semiquantitative immunohistochemical procedure. P-glycoprotein was detected in nine patients, four at diagnosis, and five at subsequent biopsy. All nine patients relapsed after a clinical response (complete [CR] 55%, partial [PR] 45%) to chemotherapy. Twenty of 21 patients with consistently P-glycoprotein-negative tumors received chemotherapy and they all responded clinically (CR 80%, PR 20%). Only one of these 20 patients has relapsed. The probability of relapse-free survival was significantly different (P less than .000000012) in chemotherapy-treated patients whose tumors contained detectable levels of P-glycoprotein (n = 9), compared with those whose tumors contained no detectable P-glycoprotein (n = 20). The overall probability of survival was also significantly different in these two groups (P less than .0000267). Both relapse-free and overall survivals remained statistically different in the two groups of patients when analyzed by the log-rank method, after adjustment for differences in stages and sites. The incidence of other adverse prognostic factors in the two groups, for example, younger and older ages, low pretreatment lymphocyte counts, large tumors, and unfavorable histology were not significantly different. Thus, detectable P-glycoprotein appears to be an important adverse prognostic factor in children with soft tissue sarcoma, and consistent absence of the protein is associated with a favorable prognosis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Identification of P-glycoprotein in renal brush border membranes.

A monoclonal antibody (C219) that recognizes the P-glycoprotein (Mr = 170,000) in plasma membranes of multidrug-resistant Chinese hamster ovary (CHO) cell lines was used to assay renal brush border membrane (BBM) and basolateral membrane (BLM) fractions for the presence of a cross-reactive polypeptide. The C219 antibody bound to a 155,000 dalton protein in immunoblots of rat BBM but not BLM proteins resolved by sodium dodecyl sulfate gel electrophoresis. The corresponding human kidney BBM and dog kidney BBM proteins had molecular weights of 170,000 and 160,000 respectively. The glycoprotein nature of the renal protein was shown by its sensitivity to N-glycanase treatment which reduced the apparent molecular weight of the dog protein to 120,000. In addition, dog P-glycoprotein could be bound to and eluted from immobilized wheat germ agglutinin. The molecular weight, antibody crossreactivity, glycosidase sensitivity and lectin binding show that this protein is a normal kidney analogue of the P-glycoprotein induced in multidrug resistant cell lines.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

P-glycoprotein expression in multidrug-resistant human ovarian carcinoma cell lines.

Multiple selections with either vinblastine or vincristine in the human ovarian carcinoma cell line SKOV3 resulted in variants with increasing degrees of multidrug resistance. SKOV3 derivatives that span a wide range in resistance (4- to 2000-fold) were obtained and analyzed for P-glycoprotein expression. In general, we observed a progressive increase in P-glycoprotein level (detected by Western blot) that paralleled the increase in multidrug resistance. However, a more detailed analysis of the P-glycoprotein mRNA and gene level indicated that the amount of P-glycoprotein expressed may be under complex control. At low levels of resistance, only an increase in P-glycoprotein mRNA and protein was observed. At intermediate to high levels of resistance P-glycoprotein gene amplification became evident. At the high level of resistance, an example was observed where only the amount of P-glycoprotein was increased without a concomitant increase in mRNA or gene copy. The mechanisms through which the content of P-glycoprotein in the plasma membrane is mediated are not understood; it is possible that the resistant variants identified here represent perturbations at different levels of regulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Drug resistance in KHT fibrosarcoma cell lines with different metastatic ability.

We have shown previously that tumor cell variants able to form experimental metastases can be generated stochastically in mouse cell lines at high rates and that, in B16 melanoma cell lines, resistance to drugs is associated with metastatic ability. Recently, we reported on the ability of two lines of KHT fibrosarcoma cells derived from individual experimental lung metastases to generate metastatic variants. The KHT 35LI cell line maintained a relatively stable high metastatic ability while the parallel line KHT 3LIc possessed a high metastatic ability when first cultured from the lung but rapidly lost this ability and became a line with a metastatic ability similar to the KHT parental line. Subsequent rate analysis indicated that the rate of generation of metastatic variants was higher for KHT 35LI cells compared to KHT 3LIc or KHT parental cells. Here we examined the ability of these two fibrosarcoma cell lines to generate variants resistant to the drugs methotrexate (MTX) and N-(phosphonacetyl)-L-aspartate (PALA). We observed that the highly metastatic KHT 35LI cell line is more resistant to these two drugs than the KHT 3LIc or the parental KHT cell lines. Cloning studies indicated that the likely reason for this increased resistance is that the KHT 35LI cell line generates variants resistant to a given concentration of MTX or PALA at higher rates than the KHT 3LIc or KHT parental cell lines. Since resistance to MTX and PALA usually arises from gene amplification, the results suggest that KHT 35LI cells possess increased ability to amplify their DNA. The association between the rates of generation of both drug-resistant and metastatic variants supports the hypothesis that the mechanism of gene amplification may be involved in the generation of both phenotypes.

Animals↗
PubMed1989

Multidrug resistance in cancer.

Chemotherapy often fails because a tumor develops resistance to an array of different drugs. A single glycoprotein turns out to be responsible: it proliferates in some cells and pumps out the drugs. Now that the protein pump has been identified it may be possible to interfere with its action or to make it the target for drugs that destroy the cancer cell.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

P-glycoprotein: multidrug-resistance and a superfamily of membrane-associated transport proteins.

The study of multidrug resistance (MDR) in tumor cell lines has led to the discovery of the plasma membrane P-glycoprotein (Pgp) molecule. This protein functions as an energy-dependent pump for the efflux of diverse anticancer drugs from MDR cells. It now appears that Pgp-mediated MDR tumor cells do occur in human cancers, and that they are likely to play a role in the ultimate response of patients to chemotherapy. Chemosensitizers, compounds able to reverse the MDR phenotype, have been identified and offer the exciting possibility of improving efficacy for some nonresponsive malignancies. Surprisingly, Pgp-like molecules can be found in evolutionarily distant species among both eukaryotes and prokaryotes. As a group, these proteins form a superfamily of ATP-dependent transport proteins. This finding has broad implications and provides new insights into how living organisms use this fundamental transport system to regulate the trafficking of diverse molecules across biological membranes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Cloning of cDNA Sequences Encoding the Calcium-Binding Protein, Calmodulin, from Barley (Hordeum vulgare L.).

Full- and partial-length cDNAs encoding calmodulin mRNA have been cloned and sequenced from barley (Hordeum vulgare L.). Barley leaf mRNA, size-fractionated in sucrose density gradients, was used to synthesize double-stranded cDNA. The cDNA was cloned in lambdagt10 and screened with a synthetic, 14-nucleotide oligonucleotide probe, which was designed using the predicted coding sequences of the carboxy termini of spinach and wheat calmodulin proteins. The primary structure of barley calmodulin, predicted from DNA sequencing experiments, consists of 148 amino acids and differs from that of wheat calmodulin in only three positions. In two of the three positions, the amino acid changes are conservative, while the third change consists of an apparent deletion/insertion. The overall nucleotide sequence similarity between the amino acid coding regions of barley and vertebrate calmodulin mRNAs is approximately 77%. However, a region encoding 11 amino acids of the second Ca(2+)-binding domain is very highly conserved at the nucleotide level compared with the rest of the coding sequences (94% sequence identity between barley and chicken calmodulin mRNAs). Genomic Southern blots reveal that barley calmodulin is encoded by a single copy gene. This gene is expressed as a single size class of mRNA in all tissues of 7-day-old barley seedlings. In addition, these analyses indicate that a barley calmodulin cDNA coding region subclone is suitable as a probe for isolating calmodulin genes from other plants.

Journal Article↗
PubMed1989

Identification of members of the P-glycoprotein multigene family.

Overproduction of P-glycoprotein is intimately associated with multidrug resistance. This protein appears to be encoded by a multigene family. Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype. Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family. Using a highly conserved exon probe, we found that the hamster P-glycoprotein gene family consists of three genes. We also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys. The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains. We propose that the hamster P-glycoprotein gene family arose from gene duplication. The hamster pgp1 and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene. We speculate that the hamster pgp1 and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Identification of the multidrug resistance-related P-glycoprotein as a cyclosporine binding protein.

The immunosuppressive agent cyclosporine A has been shown to reverse multidrug resistance (MDR) in malignant cells. In the present study, a 3H-cyclosporine diazirine analogue was used to photolabel viable MDR Chinese hamster ovary 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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗