Gene amplification and tumor progression.
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Animals↗
Biomedical subjects
V Ling
Publications and source records attributed to V Ling.
Mechanism of multidrug resistance.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Electrophoretic analysis of P-glycoproteins produced by mouse J774.2 and Chinese hamster ovary multidrug-resistant cells.
P-glycoprotein (P-gp) plays a fundamental role in multidrug resistance. The quantity of P-gp relates to the degree of drug resistance. A comparison was made between P-gps in mouse and hamster cell lines in both Laemmli and modified Fairbanks gel systems. Both proteins are derived from precursors of similar size that undergo differential N-linked glycosylation. The electrophoretic mobility and the amount of P-gp are remarkably dependent on the conditions of analysis. Notably, boiling P-gp before Laemmli gel electrophoresis decreases its mobility by an amount that is equivalent to approximately equal to 15 kDa and results in an apparent diminution in the amount of protein. The latter effect can give a false impression concerning the quantity of P-gp in cells.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Reduced cyclosporin accumulation in multidrug-resistant cells.
Cyclosporin accumulation was reduced by 50% or more in multidrug- resistant CHRC5 CHO cells with high levels of P-glycoprotein expression compared to drug sensitive AuxB1 CHO cells. This difference could be overcome by verapamil which is known to interact with P-glycoprotein and reverse multidrug resistance. The difference in cyclosporin accumulation between sensitive and resistant cells decreased with increasing cyclosporin concentrations suggesting that cyclosporine itself regulated its own accumulation through interaction with P-glycoprotein. Indeed, cyclosporin also reversed differences in vinblastine accumulation between resistant and sensitive cell lines. Since P-glycoprotein is highly expressed in the kidney which is also a target for cyclosporin toxicity, the effects of verapamil on cyclosporin accumulation were studied in two renal cell lines, rat mesangial cells and LLCPK1, cells. Verapamil increased cyclosporin accumulation by approximately 70%. These results suggest that cellular cyclosporine accumulation is regulated at least in part by its interaction with P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Altered plasma membrane ultrastructure in multidrug-resistant cells.
Multidrug resistance is mediated by P-glycoprotein, an integral plasma membrane component which is thought to function as a drug export pump. This model can explain drug resistance, but fails to account for the broader pleiotropy of the multidrug resistance phenotype. We report here a freeze-fracture study revealing increases in the densities of protoplasmic face intramembrane particles in multidrug-resistant Chinese hamster ovary (CHO) and human leukemic cells. The intramembrane particle density in a CHO cell revertant which had lost the characteristics of the multidrug resistance phenotype was indistinguishable from that of the drug-sensitive parental cell line. This demonstration of a global multidrug resistance-linked change in plasma membrane architecture may have significant implications for understanding the variety of concurrent membrane-related changes which are not easily explained by the current model for multidrug resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Resistance of multidrug-resistant lines to natural killer-like cell-mediated cytotoxicity.
Multidrug resistance (MDR) refers to a complex phenotype that describes a number of features characterized primarily by resistance to a wide range of structurally unrelated drugs. In this paper we investigated the relationship between drug resistance and resistance to NK-mediated cytotoxicity. Studies with two independently selected multidrug-resistant cell lines indicated that increased drug resistance was associated with both an increased resistance to NK-mediated cytotoxicity and increased levels of membrane P-glycoprotein expression. This resistance to cytotoxicity appears to result partly from an alteration in the membrane structure of the target cells inasmuch as there was a reduction in effector:target cell recognition. Resistance to NK-mediated cytotoxicity should be included with the numerous pleiotropic changes associated with the multidrug resistance phenotype.
Cell Line↗
A sensitive method for immunocytochemical detection of P-glycoprotein in multidrug-resistant human ovarian carcinoma cell lines.
P-glycoprotein, a molecular weight 170 kilodalton membrane component can be accurately detected in a series of human ovarian carcinoma cells with increasing degrees of multidrug resistance by using a modified immunoperoxidase "sandwich" method. Drug-resistant derivatives were selected from a drug-sensitive parent ovarian carcinoma cell line, SKOV3, by continuous exposure to increasing concentrations of the cytotoxic drug vincristine. These cells had corresponding overexpression of P-glycoprotein demonstrable at both protein and mRNA levels. Monoclonal antibodies against P-glycoprotein localized staining for P-glycoprotein to the plasma membrane and the Golgi region in individual drug-resistant cells, in proportion to their P-glycoprotein expression. P-glycoprotein was not demonstrable in drug-sensitive SKOV3 cells by either immunoblotting or immunocytochemical staining methods. The immunocytochemical staining method allowed detection of P-glycoprotein in the least drug-resistant cell line with as low as 8-fold relative resistance to vincristine. This method is as sensitive as Northern blot, and more sensitive than standard Western blot in detection of P-glycoprotein. We conclude that this highly sensitive immunocytochemical staining method for P-glycoprotein can be suitable for determination of P-glycoprotein expression in biopsy samples of tumors, and it can be a powerful diagnostic and prognostic tool in the study of the natural history of drug resistance. This may have important applications in the clinical management of cancer chemotherapy.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Generation of drug-resistant variants in metastatic B16 mouse melanoma cell lines.
Genetic instability is recognized as an important aspect of the development of tumor heterogeneity and malignancy. In a previous study [Hill et al. Science (Wash. DC), 244:998-1001, 1984], we demonstrated that metastatic variants are generated at a more rapid rate in the highly metastatic B16F10 mouse melanoma cell line than in the less metastatic B16F1 cell line. The metastatic variants were phenotypically unstable, being generated and lost at high rates; consequently, we proposed a dynamic heterogeneity model of tumor metastasis which describes these properties quantitatively. As an extension of this work, we have examined the ability of these two melanoma cell lines to generate variants resistant to the drugs methotrexate and N-(phosphonacetyl)-L-aspartate. We observed that the highly metastatic B16F10 cell line generated variants resistant to a given concentration of methotrexate or N-(phosphonacetyl)-L-aspartate at higher rates than the B16F1 cell line. We conclude that B16F10 cells are genetically less stable than B16F1 cells and since resistance to methotrexate and N-(phosphonacetyl)-L-asparate usually results from gene amplification that B16F10 cells possess increased ability to amplify DNA. This higher rate of generation of drug-resistant variants corresponds to the higher rate of generation of metastatic variants we observed previously and suggests that a gene amplification mechanism may be involved in the generation of a metastic phenotype in B16 melanoma cells.
Animals↗
Chromosomal location of human P-glycoprotein gene sequences.
Nonresponse to chemotherapy may result from the acquisition of multidrug resistance by malignant cells. Overexpression of the 170,000 dalton cell surface P-glycoprotein is associated with this phenotype and this appears to result from amplification of a multigene family coding for this protein. A cDNA encoding a conserved portion of P-glycoprotein has been cloned from hamster cells, and this was used in the present study to localize human P-glycoprotein gene sequences to chromosome 7q36.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Frequencies of independent and simultaneous selection of Chinese hamster cells for methotrexate and doxorubicin (adriamycin) resistance.
We have determined the frequency with which Chinese hamster cells become resistant to either methotrexate or doxorubicin (former generic name, adriamycin) alone or to the two drugs simultaneously. We find that the frequency of acquisition of simultaneous resistance is 10-100 times higher than that predicted from the frequency of each resistance selected independently. In approximately 50% of cloned resistant variants, resistance is the result of amplification of the dihydrofolate reductase gene (methotrexate) and/or of the multiple-drug-resistance P-glycoprotein gene (doxorubicin). Prior exposure of cells to hypoxia markedly enhances these resistance frequencies. Our results indicate that the simultaneous emergence of resistance to these two cancer chemotherapeutic agents are not independent events, and we interpret them to constitute two consequences of the same basic process occurring at a high frequency.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Multidrug resistance and P-glycoprotein expression.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Simultaneous expression of two P-glycoprotein genes in drug-sensitive Chinese hamster ovary cells.
Overexpression of P-glycoprotein is characteristic of multidrug-resistant cells. We analyzed four P-glycoprotein transcripts that are simultaneously expressed in a drug-sensitive Chinese hamster ovary cell line. We concluded that these transcripts are encoded by two distinct members of a P-glycoprotein multigene family, each of which has two alternative polyadenylation sites. A comparison of the two hamster sequences with the single reported human and mouse P-glycoprotein cDNA sequences demonstrates that P-glycoprotein is a highly conserved protein, that the hamster multigene family is undergoing concerted evolution, and that differences between gene family members are maintained across species. These conserved differences suggest that there may be functional differences between P-glycoprotein molecules.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Expression of hamster P-glycoprotein and multidrug resistance in DNA-mediated transformants of mouse LTA cells.
The overexpression of a plasma membrane glycoprotein, P-glycoprotein, is strongly correlated with the expression of multidrug resistance. This phenotype (frequently observed in cell lines selected for resistance to a single drug) is characterized by cross resistance to many drugs, some of which are used in cancer chemotherapy. In the present study we showed that DNA-mediated transformants of mouse LTA cells with DNA from multidrug-resistant hamster cells acquired the multidrug resistance phenotype, that the transformants contained hamster P-glycoprotein DNA sequences, that these sequences were amplified whereas the recipient mouse P-glycoprotein sequences remained at wild-type levels, and that the overexpressed P-glycoprotein in these cells was of hamster origin. Furthermore, we showed that the hamster P-glycoprotein sequences were transfected independently of a group of genes that were originally coamplified and linked within a 1-megabase-pair region in the donor hamster genome. These data indicate that the high expression of P-glycoprotein is the only alteration required to mediate multidrug resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
P-glycoprotein in human sarcoma: evidence for multidrug resistance.
Overexpression of an immunologically conserved, cell-surface glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines in vitro. A preliminary survey of specimens from 12 solid tumor types in our laboratories indicates significant overexpression of P-glycoprotein in some sarcomas. When tested by immunoblotting with monoclonal antibodies directed against P-glycoprotein; tumors from six of 25 sarcoma patients displayed elevated levels of P-glycoprotein. Three of the sarcoma patients exhibiting P-glycoprotein had not previously been exposed to chemotherapy, implying that overexpression of this marker and possible concomitant multidrug resistance may not depend only on selection during prior drug treatments. The P-glycoprotein overexpression in the sarcoma specimens is evidence for the presence of multidrug resistant cells in these tumors; thus, our data suggest that this mode of resistance may have clinical significance in sarcoma patients.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Dynamic heterogeneity: characterization of two cell lines derived from experimental lung metastases of mouse KHT fibrosarcoma.
We have recently demonstrated that KHT fibrosarcoma variant cells that form experimental metastases are generated by a stochastic process at high effective rates (approximately 10(-5)/cell/generation). The metastatic variant cells are unstable and are rapidly lost with an effective rate of reversion of approximately 10(-1)/cell/generation. In this study, we have examined the dynamics of generation of metastatic variant cells for two lines of KHT cells derived from individual experimental lung metastases in a single step selection. When grown in vitro, the KHT35-L1 cell population maintained a highly metastatic phenotype whereas the KHT3-L1c cell population reverted to a poorly metastatic phenotype. Our results indicate that there is an approximately 10-fold higher effective rate of generation of metastatic variant cells for KHT35-L1 cells relative to KHT3-L1c cells. Thus, a higher effective rate of generation of metastatic variants accompanies a stably expressed metastatic ability in fibrosarcoma cells and, as we have previously reported, in melanoma cells.
Animals↗
The mdr1 gene, responsible for multidrug-resistance, codes for P-glycoprotein.
The development of simultaneous resistance to multiple drugs in cultured cells occurs after selection for resistance to single agents. This multidrug-resistance phenotype is thought to mimic multidrug-resistance in human tumors treated with chemotherapy. Both the expression of a membrane protein, termed P170 or P-glycoprotein, and the expression of a cloned DNA fragment, termed mdr1, have been shown independently to be associated with multidrug-resistance in cultured cells. In this work, we show that human KB carcinoma cells which express the mdr1 gene also express P-glycoprotein, and that cDNAs encoding P-glycoprotein cross-hybridize with mdr1 cDNAs. Thus, the mdr1 gene codes for P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
The structure of HSAG-1, a middle repetitive genetic element which elicits a leukemia-related cellular surface antigen.
HSAG-1 is a cloned member of a heterogeneous middle repetitive family of genetic elements which is capable of eliciting a leukemia-related surface antigen detected with a monoclonal antibody after DNA transformation of mouse cells. HSAG-1 was originally isolated from a Chinese hamster-human leukemia hybrid cell gene library both by sib-selection for antigen producing activity and by hybridization with labelled human genomic human DNA. We show here that the human labelled site is at the right hand end of the insert, while the antigen-eliciting portion is included in a 1450 bp fragment at the left hand end of the insert. We also present the complete nucleotide sequence of the 3369 bp insert. The sequence contains 12 elements which bear a significant resemblance to accepted consensus sequences for Alu repetitive elements. The right hand end contains adjacent elements with close sequence similarity to portions of the human and hamster type I and type II Alu consensus sequences. All of the other Alu-related elements have diverged relative to the Alu consensus sequences by additions, long deletions and substitutions. The left hand portion of the insert which has the antigen-producing activity contains four of these diverged elements representing a relatively high proportion (26%) of the nucleotide sequence. The sequence is thus consistent with our previous observations of a repetitive family with biological function.
Animals↗
Complete sequence of three alpha-tubulin cDNAs in Chinese hamster ovary cells: each encodes a distinct alpha-tubulin isoprotein.
The genome of Chinese hamster ovary (CHO) cells contains a complex family of approximately 16 alpha-tubulin genes, many of which may be pseudogenes. We present here the complete cDNA sequences of three expressed alpha-tubulin genes; one of these genes has been identified only in CHO cells. The noncoding regions of these three CHO alpha-tubulin genes differed significantly, but their coding regions were highly conserved. Nevertheless, we observed differences in the predicted amino acid sequences for the three genes. A comparison of the CHO alpha-tubulin sequences with all of the sequences available for mammals allowed assignment of the alpha-tubulin genes to three classes. The proteins encoded by the members of two of these classes showed no class-specific amino acids among the mammalian species examined. The gene belonging to the third class encoded an isoprotein which was clearly distinct, and members of this class may play a unique role in vivo. Sequencing of the three alpha-tubulin genes was also undertaken in CMR795, a colcemid-resistant clonal CHO cell line which has previously been shown to have structural and functional alterations in its tubulin proteins. We found differences in the tubulin nucleotide sequence compared with the parental line; however, no differences in the alpha-tubulin proteins encoded in the two cell lines were observed.
Amino Acid Sequence↗