Search PubMed⌕ Search

Biomedical subjects

V Ling

Publications and source records attributed to V Ling.

At least 163 records · Page 9Linked to original sources
PubMed1986

Overexpression and amplification of five genes in a multidrug-resistant Chinese hamster ovary cell line.

Multidrug-resistant cells are cross-resistant to a wide range of unrelated drugs, many of which are used in cancer chemotherapy. We constructed a cDNA library from RNA of the multidrug-resistant Chinese hamster ovary cell line CHRC5. By differential screening we isolated cDNAs derived from mRNAs that are overexpressed in this cell line. The cDNAs could be grouped in five classes on the basis of transcript lengths detected in RNA blots. We infer that each class codes for a separate protein. The corresponding genes are amplified 10 or 30 times in CHRC5 DNA, providing an explanation for the constitutive overexpression found in this cell line. Despite differential amplification, the genes may be linked in one large amplicon as indicated by the hybridization analysis of large fragments of CHRC5 DNA separated by pulsed field gradient gel electrophoresis. Therefore, some of these genes might be fortuitously coamplified and not contribute functionally to the resistant phenotype. It is also possible, however, that genes involved in drug resistance are clustered. One of our clones cross-hybridized with the recently described cDNA pCHP1 (J. R. Riordan, K. Deuchars, N. Kartner, N. Alon, J. Trent, and V. Ling, Nature [London] 316:817-819, 1985) encoding part of the 170-kilodalton P-glycoprotein, a protein which is frequently overproduced in multidrug-resistant cells. The nature of the four other genes is still unknown. Sequences of four of the five classes of cDNAs are conserved in mouse and human DNA.

Animals↗
PubMed1986

Multidrug resistance.

Multidrug resistance describes a complex phenotype whose predominant feature is resistance to a wide range of structurally unrelated cytotoxic compounds, many of which are anticancer agents. This phenotype occurs frequently in mammalian cell lines and transplantable tumours selected for resistance to a single drug. Reduced cellular accumulation of the drugs involved appears to account for the resistance. This may be a consequence of reduced drug influx, increased drug efflux, or both. A wide variety of biochemical changes have been identified in multidrug resistant cell lines, the most consistent of which is the increased expression of P-glycoprotein, a conserved, high molecular weight, plasma membrane glycoprotein. The level of P-glycoprotein expression correlates with the degree of drug resistance in a variety of different cell types. In a number of multidrug resistant cell lines, overexpression of P-glycoprotein results from gene amplification. While the function of P-glycoprotein is unknown, independent lines of evidence support the notion that P-glycoprotein is the causative molecule mediating the multidrug resistance phenotype. Significant levels of P-glycoprotein expression have been detected in some biopsy specimens from patients with ovarian and sarcoma tumours. These findings suggest that multidrug resistant tumour cells can occur in human malignancies. The presence of such cells may affect the outcome of chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1985

Quantitative genetic analysis of tumor progression.

Metastasis and resistance to chemotherapy are common features of progressed cancers. With respect to the latter phenotype, it is thought that during tumor growth drug-resistant cells arise spontaneously at rates characteristic of the genetic alterations involved. On application of chemotherapy, such variant tumor cells are more likely to survive, and they may eventually dominate, resulting in a non-responsive malignancy. Aspects of this model have been confirmed in a number of experimental systems and in patients. In contrast to our understanding of drug resistance, steps involved in the progression to metastatic spread of tumor cells are much less well-understood. In this review we describe methodologies of quantitative genetic analysis with reference to development of drug resistance. We then describe attempts by ourselves and others to use a similar approach to investigate metastatic properties. Based on these studies, we have proposed the quantitative 'dynamic heterogeneity' model of tumor metastasis, which is presented here. Using an 'experimental' metastasis assay and Luria-Delbruck fluctuation analysis, we determined that in murine KHT fibrosarcoma and B16 melanoma lines, 'metastatic' variants with a distinct phenotype are generated at high rates. These variants are relatively unstable resulting in a dynamic equilibrium between generation and loss of metastatic variants. The metastatic ability of such a tumor population is thus dependent on the frequency of a subpopulation of metastatic variants which are turning over rapidly. This dynamic heterogeneity model is able to quantitatively provide a unifying explanation for a wide range of observations concerning tumor heterogeneity and clonal instability. Genetic mechanisms involving rapid rates have been characterized in drug-resistant variants. We speculate that similar processes may be involved in different aspects of tumor progression such as those resulting in metastasis.

Animals↗
PubMed1985

Differential expression of three alpha-tubulin genes in Chinese hamster ovary cells.

Chinese hamster ovary cells contain a complex family of ca. 16 unique alpha-tubulin sequences and a similar multiplicity of beta sequences. To examine which members of this multigene family are expressed, we constructed cDNA libraries from two Chinese hamster ovary cell lines according to the method of H. Okayama and P. Berg (Mol. Cell. Biol. 3:280-289, 1983). Each library consisted of 5.5 X 10(5) transformants and contained a high percentage of full-length tubulin clones. Three different alpha-tubulin genes were identified by sequence analysis of the 3' noncoding regions of these tubulin clones. The relative abundance of the transcripts corresponding to the three genes was estimated by gene-specific dot blotting of 96 cDNA alpha-tubulin clones and was found to be 71, 24, and 5%. There is little homology in the 3' noncoding sequences of these genes; however, a strong interspecies homology exists in this region for two of the Chinese hamster ovary genes with the two alpha-tubulin genes previously described in other systems. The third Chinese hamster ovary gene, with an expression frequency of 24%, is unique in that its 3' noncoding region is unlike that of the other mammalian alpha-tubulin genes. In addition, limited sequence data from the coding region of this gene indicates it codes for a unique alpha-tubulin protein.

Animals↗
PubMed1985

Cloning of 11 alpha-tubulin gene sequences from the genome of Chinese hamster ovary cells.

We have analyzed the complex tubulin gene family in clonal lines of Chinese hamster ovary cells. There are approximately 16 alpha-tubulin genes and a similar multiplicity of beta-tubulin genes. The alpha-tubulin genes are not closely linked to each other nor to the beta-tubulin genes. A genomic library has been constructed in the vector lambda charon 4A containing insert sizes of 13-20 kilobases. The library has been screened with both inter- and intra-species alpha-tubulin probes. Eleven alpha-tubulin clones with different restriction patterns have been isolated and characterized. At least seven of these clones contain the complete gene coding region. One clone appears to represent the transcribed alpha-tubulin gene II. The sequence of an intron from this gene is compared with that from an equivalent gene in the rat.

Animals↗
PubMed1985

Detection of P-glycoprotein in ovarian cancer: a molecular marker associated with multidrug resistance.

A multidrug resistance phenotype is frequently observed in animal and human cell lines selected for in vitro resistance to a single chemotherapeutic agent. Overexpression of a highly conserved cell-surface glycoprotein (P-glycoprotein) is consistently associated with this phenotype in these mutant lines. A monoclonal antibody against P-glycoprotein was used to examine tumor samples from five patients with advanced ovarian cancer for evidence of P-glycoprotein overexpression. High levels of P-glycoprotein were detected in samples from two patients suggesting that a multidrug resistance mutation may also occur in ovarian cancer. This finding has broad implications for the understanding of nonresponse to chemotherapy in a variety of human neoplasms, and may provide a rational explanation for failure of chemotherapy in treatment of advanced ovarian cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1985

Microscopic analysis of the mastoid bone in chronic serous otitis media.

The pathologic changes of the middle ear and mastoid bone mucosa in two pediatric patients with long standing chronic serous otitis media were studied by electron and light microscopy. The embryological development of the eustachian tube, middle ear cleft, and mastoid bone suggests a common physiological and anatomical continuity. The histological changes by light and electron microscopy in these patients demonstrates the metaplastic changes of the basal cell of the mucosa differentiating into mucous and keratin cells. These metaplastic changes are associated with the exudative process known as chronic serous otitis media.

Biopsy↗
PubMed1984

Dynamic heterogeneity: rapid generation of metastatic variants in mouse B16 melanoma cells.

The ability of clonally derived lines of B16F1 and B16F10 melanoma cells to form experimental metastases in C57BL mice after intravenous injection was examined. Luria- Delbruck fluctuation analysis was applied to the results obtained with parallel subclones grown to small population sizes before testing for metastatic ability. The analysis demonstrated that variant cells capable of forming experimental metastases were generated in B16F1 cell populations at an effective rate of about 1.3 X 10(-5) per cell per generation while in B16F10 cell populations the effective rate of production was about 5 X 10(-5) per cell per generation. These results are consistent with a dynamic heterogeneity model of tumor progression. They suggest that the majority of cells in both lines are effectively nonmetastatic and that the higher metastatic ability of the B16F10 population may be due in part to a higher rate of generation of metastatic variants.

Animals↗
PubMed1984

Dynamic heterogeneity and metastasis.

Parallel clonal populations grown to small defined sizes were used to quantitate rates of generation of metastatic cells. In murine KHT fibrosarcoma and B16 melanoma lines, metastatic cells are generated at effective rates of 10(-5) per cell per generation, or greater. These variant cells are unstable, and are apparently lost at very high rates. It thus appears that metastases could arise from unstable variants, and that rapid rates of change in some phenotypes may be an important feature of malignant progression. We have called such rapid variations dynamic heterogeneity. This may be a useful concept for further investigating aspects of tumor heterogeneity.

Animals↗
PubMed1984

Multidrug resistance.

Mutations resulting in a complex phenotype of cross-resistance and collateral-sensitivity are frequently observed in mammalian cell lines. A cell surface 170 000 dalton glycoprotein (P-glycoprotein) has been identified to be intimately associated with this multidrug resistance phenotype. We speculate that such mutations also occur in advanced cancers and play a major role in contributing to the non-response to combination chemotherapy. In this context, P-glycoprotein may be a useful molecular marker for diagnostic and therapeutic applications.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1984

Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells.

A genetic system comprised of mammalian cell mutants which demonstrate concomitant resistance to a number of unrelated drugs has been described previously. The resistance is due to reduced cell membrane permeability and is correlated with the presence of large amounts of a plasma membrane glycoprotein termed P-glycoprotein. This system could represent a model for multiple drug resistance which develops in cancer patients treated with chemotherapeutic drugs. We demonstrate here that the multiple drug resistance phenotype can be transferred to mouse cells with DNA from a drug-resistant mutant and then amplified quantitatively by culture in media containing increasing concentrations of drug. The amount of P-glycoprotein was correlated directly with the degree of drug resistance in the transformants and amplified transformants. In addition, the drug resistance and expression of P-glycoprotein of the transformants were unstable and associated quantitatively with the number of double minute chromosomes. We suggest that the gene for multiple drug resistance and P-glycoprotein is contained in these extrachromosomal particles and is amplified by increases in double minute chromosome number. The potential use of this system for manipulation of mammalian genes in general is discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1984

Rapid phenotype variation in cells derived from lung metastases of KHT fibrosarcoma.

We have established previously that intravenously derived metastatic variants are generated in KHT fibrosarcoma cells at an effective rate of 10(-5)/cell/generation. To study the properties of these variants further, we examined several lines of KHT fibrosarcoma cells obtained from experimental lung metastases. When tested using an experimental metastasis assay, some of the lines were highly metastatic, relative to parental lines, but these highly metastatic phenotypes were often rapidly lost as the lines were grown in vitro, and both decreases and increases in metastatic ability were observed. In another set of experiments, lines obtained by 10 serial selections of experimental lung metastases without intervening in vitro growth between passages were also analyzed. Again, while highly metastatic phenotypes were observed in some instances, they did not persist beyond 1 or 2 in vivo passages, and the series as a whole failed to reveal a persistent increase in ability to form experimental metastases. We conclude from these experiments that although metastatic variants are generated at high rates in KHT cell lines, the phenotype is lost at even higher rates, and metastatic variants represent only a small proportion of the tumor cell population. Thus, it appears that in this system rapid phenotypic variation may play an important role in the metastatic process.

Animals↗
PubMed1984

Selection for experimental metastatic ability of heterologous tumor cells in the chick embryo after DNA-mediated transfer.

The chick embryo is an immune-deficient host able to support growth of a wide variety of transformed cells. Since growth of normal cells is not observed, this system appears to be generally useful for investigating malignant properties of different cells. Recently, we developed a sensitive assay to quantitate and select for rodent cells able to survive and grow in embryonic chick organs following i.v. injection (Cancer Res., 42: 4018-4025, 1982). We envisage this assay as a model system for studying aspects of the metastatic process. We have used DNAs from murine and human melanoma cell lines (which grow well in chick embryos after i.v. injection) to transfect murine LTA cells (which do not grow in chicks after i.v. injection). From the transfected LTA cells, we were able to isolate clones which grow well in the chick after i.v. injection. Such clones were not observed in untransfected LTA cells or with LTA cells transfected with LTA DNA. These experiments clearly demonstrate the feasibility of using the chick embryo as a host system to study genes involved in growth control alteration of the sort seen in malignant transformation.

Animals↗
PubMed1983

Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines.

The plasma membranes of hamster, mouse, and human tumor cell lines that display multiple resistance to drugs were examined by gel electrophoresis and immunoblotting. In every case, increased expression of a 170,000-dalton surface antigen was found to be correlated with multidrug resistance. This membrane component is of identical molecular size and shares some immunogenic homology with the previously characterized P-glycoprotein of colchicine-resistant Chinese hamster ovary cells. This finding may have application to cancer therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1983

Daunorubicin-resistant Chinese hamster ovary cells expressing multidrug resistance and a cell-surface P-glycoprotein.

Independent lines of Chinese hamster ovary cells resistant to the antineoplastic drug, daunorubicin, were obtained by clonal isolation in increasing drug concentrations. A single daunorubicin-resistant phenotype typified by reduced cellular drug accumulation was observed. These mutants displayed a complex phenotype of resistance to a variety of unrelated drugs. Such properties are similar to those of membrane-altered colchicine-resistant lines (V. Ling and L.H. Thompson, J. Cell. Physiol., 83: 103-116, 1974). Analysis of the plasma membrane components of the daunorubicin-resistant clones by gel electrophoresis revealed a prominent cell surface glycoprotein with a molecular weight of about 170,000. This component was immunologically cross-reactive with the cell surface P-glycoprotein of about the same molecular weight, previously identified in colchicine-resistant cells. Thus, it appears that the mechanism of resistance characterized by P-glycoprotein expression could be the basis of many drug-resistant phenotypes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗