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W F Flintoff

Publications and source records attributed to W F Flintoff.

At least 37 records · Page 2Linked to original sources
PubMed1988

The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure.

We have previously cloned and characterized two different dihydrofolate reductase amplicon types from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). The largest of these (the type I amplicon) is 273 kilobases (kb) in length. In the present study, we utilized clones from the type I amplicon as probes to analyze the size and variability of the amplified DNA sequences in five other independently isolated methotrexate-resistant Chinese hamster cell lines. Our data indicated that the predominant amplicon types in all but one of these cell lines are larger than the 273-kb type I sequence. In-gel renaturation experiments as well as hybridization analysis of large SfiI fragments separated by pulse-field gradient gel electrophoresis showed that two highly resistant cell lines (A3 and MK42) have amplified very homogeneous core sequences that are estimated to be at least 583 and 653 kb in length, respectively. Thus, the sizes of the major amplicon types can be different in different drug-resistant Chinese hamster cell lines. However, there appears to be less heterogeneity in size and sequence arrangement within a given methotrexate-resistant Chinese hamster cell line than has been reported for several other examples of DNA sequence amplification in mammalian systems.

Animals↗
PubMed1987

Rat glial C6 cells are defective in murine coronavirus internalization.

Rat C6 glial cells were resistant to infection by several strains of murine coronaviruses. The restriction was not at the adsorption stage, since virus adsorbed to the C6 cells in a similar manner to mouse L cells which supported a lytic infection. The virus could not be internalized by the C6 cells. However, if the virus was introduced into the C6 cells by polyethylene glycol fusion, viral replication occurred and progeny virions were released from the infected cells. These studies indicated that the C6 cells were restrictive to coronavirus replication by preventing the early penetration stage of the viral replicative cycle.

Adsorption↗
PubMed1984

Replication of murine coronaviruses in somatic cell hybrids between murine fibroblasts and rat schwannoma cells.

The replication of the murine coronaviruses MHV3 and JHM has been studied in somatic cell hybrids formed between murine fibroblast L2 cells which support lytic infections with both these agents, and rat RN2 Schwannoma cells which support the replication of JHM in a temperature-sensitive, persistent manner but are restrictive to the replication of MHV3. The results described in this report indicate that the totally permissive state is dominant over the persistent or restricted state since the hybrid cells permit the replication of both these viral agents in a lytic manner.

Animals↗
PubMed1984

Characterization and chromosomal location of endogenous mouse mammary tumor virus loci in GR, NFS, and DBA mice.

Endogenous mouse mammary tumor virus (MMTV) proviral copies were characterized in three genetically dissimilar mouse strains: GR, a high-tumor-incidence strain bred in Europe that carries an MMTV proviral copy associated with early mammary tumors; DBA, a high-tumor-incidence laboratory strain bred in the USA with an endogenous copy that is associated with MMTV antigen expression in the milk; and NFS, a recently inbred line of the low-tumor-incidence NIH Swiss mouse. MMTV proviral loci were studied using restriction endonuclease analysis and the Southern transfer procedure in genetic crosses and in somatic cell hybrids. By studying the segregation of MMTV-specific EcoRI, BamHI, and PstI fragments, the organization of these fragments into MMTV proviral loci was determined and it was shown that (1) many homologous proviral loci are present in these three mouse strains, (2) these MMTV proviruses differ in their pattern of internal restriction sites, and (3) the MMTV loci are distributed on multiple chromosomes including 1 and 7.

Animals↗
PubMed1984

In vivo and in vitro models of demyelinating disease X. A Schwannoma-L-2 somatic cell hybrid persistently yielding high titres of mouse hepatitis virus strain JHM.

Following infection of RN2 rat Schwannoma cells with unfiltered JHMV inocula, a cell line with an altered phenotype evolved, which was shown to be a somatic cell hybrid of RN2 and mouse L-2 cells. This cell line, EJ, persistently yields JHMV at titres greater than 10(6) pfu/ml and does not show the suppression of virus production at 39.5 degrees C that is characteristic of a persistently infected RN2 line. Intracellular viral nucleocapsids are demonstrated. Cloning of EJ hybrids yields cell lines that show a variety of responses to infection by JHMV or MHV3.

Animals↗
PubMed1984

Moderate-level gene amplification in methotrexate-resistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene.

In previous studies, we have described several classes of methotrexate-resistant Chinese hamster ovary cell lines. Although the RI class is resistant because of an altered target enzyme, dihydrofolate reductase, the RIII class derived from RI cells is somewhat more resistant because of a moderate amplification of the altered dhfr structural gene (Flintoff et al., Mol. Cell. Biol. 2:275-285, 1982). In one RIII line, a translocation between the short arm (p) of chromosome 2 and the long arm (q) of chromosome 5 was observed, and the amplified RIII gene complex was mapped to the p arm of the 2p-marker chromosome derived from the translocation (Worton et al., Mol. Cell. Biol. 1:330-335, 1981). We tested the hypothesis that chromosomal translocation is a general feature of RIII cells and that such translocation involves a site at or near the dhfr structural gene. Thus, we examined four independently derived RIII-type mutants and found that each had a moderate amplification of the dhfr gene sequences, and karyotype analysis revealed that each carried a translocation involving the 2p arm at or near band 2p25. That this chromosomal rearrangement involves a site near the dhfr locus was demonstrated by mapping the altered but unamplified structural gene coding for the RI phenotype to the short arm of an unaltered chromosome 2. This suggests that a highly specific rearrangement involving an exchange at or near the site of the unamplified gene is a necessary prerequisite for the amplification process. A model for gene amplification involving chromosomal rearrangements and sister chromatid exchange is described.

Animals↗
PubMed1983

Transport of methotrexate in Chinese hamster ovary cells: a mutant defective in methotrexate uptake and cell binding.

The uptake of several folate compounds has been investigated in wild-type and one class of methotrexate-resistant Chinese hamster ovary cells. The wild-type cells can take up methotrexate, folic acid, and 5-methyl tetrahydrofolate. The uptake of methotrexate is characterized by a KT of 1.9 +/- 0.2 microM and a Vmax of 1 +/- 0.8 pmol/min/mg protein, is competitively inhibited by folic acid and 5-methyl tetrahydrofolate, and is sensitive to p-hydroxymercuriphenyl sulfonic acid. The resistant cells are unable to take up methotrexate, folic acid, and 5-methyl tetrahydrofolate. In addition, the resistant cells are unable to bind methotrexate specifically, whereas the wild-type cells bind the drug with an apparent KD of 2 +/- 0.4 microM and a Vmax of 1.3 +/- 0.3 pmol/mg protein. These data indicate that the resistant cells are resistant because of an inability to take up the drug resulting from a defective membrane-binding component. The data also suggest that both methotrexate and folic acid are transported by the same system in Chinese hamster ovary cells.

Animals↗
PubMed1982

Overproduction of dihydrofolate reductase and gene amplification in methotrexate-resistant Chinese hamster ovary cells.

Stable isolates of Chinese hamster ovary cells that are highly resistant to methotrexate have been selected in a multistep selection process. Quantitative immunoprecipitations have indicated that these isolates synthesize dihydrofolate reductase at an elevated rate over its synthesis in sensitive cells. Restriction enzyme and Southern blot analyses with a murine reductase cDNA probe indicate that the highly resistant isolates contain amplifications of the dihydrofolate reductase gene number. Depending upon the parenteral line used to select these resistant cells, they overproduce either a wild-type enzyme or a structurally altered enzyme. Karyotype analysis shows that some of these isolates contain chromosomes with homogeneously staining regions whereas others do not contain such chromosomes.

Animals↗
PubMed1980

Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate.

Previous reports [Flintoff, W. F., Davidson, S. V., & Siminovitch, L. (1976) Somatic Cell Genet. 2, 245--261; Gupta, R. S., Flintoff, W. F., & Siminovitch, L. (1977) Can. J. Biochem. 55, 445--452] described a series of Chinese hamster ovary cells that were resistant to the cytotoxic action of methotrexate and contained a dihydrofolate reductase that was less sensitive to inhibition by the drug than wild-type enzyme. In this study, binding of labeled methotrexate to the reductase--NADPH complex and separation of free and bound drug by filtration through Sephadex G--25 have been used to demonstrate that clonal isolates of these resistant cells contain a dihydrofolate reductase varying between 2.5- and 6-fold lower in affinity for the drug than the wild-type enzyme. The apparent dissociation constant for the wild-type enzyme is 0.5 x 10(-9) M. Using two-dimensional polyacrylamide gel electrophoresis, 11 independently selected resistant isolates have been shown to contain a reductase with a similar overall net charge as the wild-type enzyme. Reductase purified from either wild-type or resistant cells contains two components after isoelectric focusing in polyacrylamide gels. The major component represents about 90% of the total protein and has a pI of about 8.0. The minor component representing about 10% of the reductase protein has a pI between 7.2 and 7.6.

Animals↗
PubMed1980

Selection of wild-type revertants from methotrexate-resistant cells containing an altered dihydrofolate reductase.

A selection system for wild-type revertants from methotrexate-resistant Chinese hamster ovary cells is described. In the absence of exogenous thymidine, cells use the folate metabolic pathway to generate thymidine 5'-monophosphate from deoxyuridine 5'-monophosphate. Thus, in the presence of methotrexate, the incorporation of labeled deoxyuridine into phenotypic wild-type cells is inhibited whereas resistant cells that are cycling incorporate sufficient radioactivity to be killed. Using several suicide cycles, wild-type revertants have been isolated from methotrexate-resistant cells containing a structurally altered dihydrofolate reductase. These revertants possess a wild-type sensitivity to the cytotoxicity of the drug and contain a reductase with similar properties as wild-type enzyme.

Animals↗
PubMed1978

Isolation of mutant mammalian cells altered in polyamine transport.

Chinese hamster ovary and rat myoblast cells resistant to the toxic action of methylglyoxal bis guanylhydrazone (MGBG), an antimitotic agent and inhibitor of polyamine synthesis, have been isolated by single step selection. Mutagenesis with ethyl methanesulfonate increases the recovery of the variants at least 30-fold. Intracellular accumulation of MGBG is greatly reduced in resistant cells. This property is accompanied by a 99% decrease in the uptake of all three naturally occurring polyamines. Both the resistant phenotype and the defect in polyamine transport behave recessively in somatic cell hybrids.

Biological Transport↗
PubMed1977

Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells.

We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levls of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W.F., Davidson, S.V., and Siminovitch, L. (1976) Somatic Cell Genet.2,245-261). By selecting for increased resistance form these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In the report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.

Cell Line↗
PubMed1976

Isolation and partial characterization of three methotrexate-resistant phenotypes from Chinese hamster ovary cells.

Three mechanisms for resistance to methotrexate (Mtx) have been identified in Chinese hamster ovary (CHO) cells selected from resistance to this drug. First-step selections produce cells with either an apparent structural alteration in the enzyme dihydrofolate reductase (class I), or a decreased permeability to the drug (class II). Mutagenesis with ethyl methanesulfonate increases the proportion of Mtx-resistant cells 5-10-fold. Second-step selections to higher resistance using class I resistant cells as parents results in cells with an increased activity of the reductase enzyme (class III) with no apparent further qualitative alterations in the enzyme. All three classes of resistant cells retain their Mtx-resistant phenotype when cultured under nonselectivve conditions.

Cell Line↗
PubMed1976

Genetic characterization of methotrexate-resistant chinese hamster ovary cells.

In a previous report, we described the selection and partial characterization of three methotrexate (Mtx)-resistant Chinese hamster ovary cells (CHO) (1). Class I cells contained an apparent structural alteration in dihydrofolate reductase. Class II cells had an alteration affecting the permeability of the drug. Class III cells, selected from Class I cells, had an increased activity of the altered enzyme. In the work described here, it has been shown that the spontaneous mutation rate to Class I resistance is in the order of 2 X 10-9 mutations per locus per generation and that in single-step mutagenized selections the number of resistant colonies of Class I and II are about equal. Class I and Class III resistance is expressed codominantly in somatic cell hybrids, whereas the Class II resistant marker is a recessive trait.

Animals↗