Microcell-mediated cotransfer of genes specifying methotrexate resistance, emetine sensitivity, and chromate sensitivity with Chinese hamster chromosome 2.

Many selectable mutants of somatic Chinese hamster cells have been described, but very few of the mutations have been mapped to specific chromosomes. We have utilized the microcell-mediated gene transfer technique to establish the location of three selectable genetic markers on chromosome 2 of Chinese hamster. Microcells were prepared from the methotrexate-resistant MtxRIII line of Flintoff et al. (Somatic Cell Genet. 2:245-261, 1976) and fused to wild-type CHO cells, and microcell hybrids (transferants) were selected in medium containing methotrexate. All transferants were karyotyped and found to contain a marker chromosome from the donor MtxRIII line. This marker chromosome, called 2p-, consisted of a chromosome 2 with a reduced short arm resulting from a reciprocal translocation between 2p and 5q. In experiments utilizing emetine-resistant (Emtr) or chromate-resistant (Chrr) recipient cells it was found that the emt+ and chr+ wild-type genes were cotransferred with the 2p- chromosomes. Karyotype analysis of several transferants with rearranged or broken 2p- markers allowed regional localization of the emt and chr loci to the proximal third of the long arm and localization of the gene or genes conferring methotrexate resistance to the short arm. These results confirm our earlier assignment of the emt and chr loci to chromosome 2 in Chinese hamster.

Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer.

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.

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.

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.

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.

Localization of a human reduced folate carrier protein in the mitochondrial as well as the cell membrane of leukemia cells.

IgG polyclonal antiserum was generated in New Zealand White rabbits immunized with a 16-mer peptide consisting of a specific amino acid sequence at residues corresponding to the sixth to seventh predicted transmembrane domain of the human reduced folate carrier (RFC). Using Western immunoblotting to examine the cytosolic and membrane fractions of the human CCRF-CEM T-cell lymphoblastic leukemia cell line, polyclonal antihuman RFC antiserum recognized two bands in the cytosolic fraction (approximately 60 kDa and approximately 70 kDa) on 10% polyacrylamide gels. In the membrane fraction, an approximately 60-kDa protein was identified. Comparative studies of a panel of human tumor cell lines including the HT1080 fibrosarcoma, 8805 malignant fibrous histiocytoma, and the MCF breast cancer cell lines revealed similar findings. Likewise, a recombinant approximately 60-kDa membrane protein was identified after expression of baculovirus-infected Sf9 insect cells containing cDNA of the human RFC. In the CEM-7A cell line, a variant of the CCRF-CEM cell line that overexpresses the RFC, 21-fold overexpression of the approximately 60-kDa membrane protein (RFC) was shown by Western analysis. To characterize further the cellular distribution of the human RFC, immunohistochemical analyses were performed in CCRF-CEM T-cell lymphoblastic leukemia cells. Predominantly membrane localization of the antibody reacting sites was detected; however, a cytoplasmic component was noted as well. By confocal microscopy and by immunogold electron microscopy, the cytoplasmic expression was found to be largely of mitochondrial origin. These findings were corroborated by Western immunoblotting of mitochondrial membrane isolates from the CCRF-CEM cell line, which demonstrate an approximately 60-kDa protein. The localization of the human RFC to the mitochondrial membrane is a novel finding, and it suggests a role for the mitochondrial membrane in the transport of folates.

Probing for the human coronavirus OC43 in multiple sclerosis.

Tissues acquired at autopsy from four patients with MS and biopsies from one patient with a probable diagnosis of MS were probed for the presence of OC43 RNA. This human coronavirus was not detected.

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.

No evidence for linkage to the type 1 or type 2 neurofibromatosis loci in Noonan syndrome families.

A linkage analysis has been performed on 6 two-generation families with classical Noonan syndrome to determine whether the syndrome is linked to neurofibromatosis type 1 on chromosome 17q or to neurofibromatosis type 2 on chromosome 22q. A significantly negative location score was obtained between 10 cM centromeric to and 15 cM telomeric from the neurofibromatosis type 1 locus. A significantly negative lod score was obtained with a marker mapping within the region where neurofibromatosis type 2 is thought to be located. These data indicate that Noonan syndrome is not tightly linked to either neurofibromatosis type 1 or type 2.

Exclusion of allelism of Noonan syndrome and neurofibromatosis-type 1 in a large family with Noonan syndrome-neurofibromatosis association.

A large four-generation family with Noonan syndrome (NS) and neurofibromatosis-type 1 (NF1) was studied for clinical association between the two diseases and for linkage analysis with polymorphic DNA markers of the NF1 region in 17q11.2. Nonrandom segregation between NS and NF1 phenotypes was observed. Neurofibromatosis was tightly linked to NF1 markers, whereas Noonan syndrome was found not be allelic to NF1. These results suggest that two mutations at two independent but closely linked loci are the cause of neurofibromatosis-Noonan syndrome (NF-NS) association in this family.

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.

The selection of wild-type revertants from methotrexate permeability mutants.

In a previous report, we described the selection and partial characterization of three distinct classes of methotrexate (Mtx)-resistant Chinese hamster ovary cells (CHO) (1). Class I cells contained a structural alteration in dihydrofolate reductase. Class II cells showed a 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, the sensitivity of these lines to the diaminopyrimidines has been investigated. Class I cells are as sensitive, class II cells are 5- to 10-fold more sensitive, and class III cells are 10- to 30-fold more resistant than wild-type cells. The increased sensitivity of the class II cells provided an opportunity to select for revertants of these mutants and such phentotypic wild-type revertant cells have been selected using one diaminopyrimidine, pyrimethamine. Such cells have drug sensitivities and permeability characteristics similar to wild-type cells. A second class has been identified which has wild-type drug sensitivities to the diaminopyrimidines but Mtx class II resistance to Mtx, and drug permeabilities characteristic of Mtx-resistant class II cells.

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.

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.

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.

Structural organization of the human reduced folate carrier gene: evidence for 5' heterogeneity in lymphoblast mRNA.

The reduced folate carrier (rfc1) gene encodes a protein that is involved in the intracellular accumulation of folates. Point mutations in this gene and alterations resulting in the down regulation of its message are major factors involved in the resistance to antifolate chemotherapeutic compounds. As a framework for understanding the significance of such changes in relation to gene expression and function, in this report we describe the organization of the rfc gene from human lymphoblasts. The gene contains 5 exons (2 to 6) coding for protein. At least four 5' exons, used in a mutually exclusive manner in the production of the rfc message from lymphoblast cells, are spliced to exon 2, which contains the translational start site. "Semi-quantitative" PCR indicates that exon 1 is preferentially used. The major transcriptional start site has been mapped by RACE and RNase protection to a region 109 to 135 base pairs 5' to the start of exon 1. The 5' region of the gene has no TATA box-like sequence but contains several consensus binding sites for transcriptional factors such as SP-1, MZF1, CREB, AP-1, ETS, GATA-1 and GATA-2. The overall organization of the human gene is similar to that of the hamster and mouse genes.

Isolation of a human cDNA that complements a mutant hamster cell defective in methotrexate uptake.

A clone has been isolated from a human lymphoblastic cDNA expression library that complements a mutant Chinese hamster cell defective in the uptake of the folate analogue methotrexate. When transfected with this clone the mutant cells regain the ability to transport the drug and, as a consequence, become sensitive to its cytotoxic action. The clone is 2863 base pairs long and has an open reading frame of 1770 base pairs that codes for a putative protein of 64 kDa. The putative protein has 51 and 50% identity at the amino acid level with the mouse and hamster functions, respectively, involved in the transport of reduced folates. Together these three proteins share 47% identity and have similar predicted structural features. The data are consistent with this human clone encoding either the reduced folate transporter or an auxiliary function that interacts with this transporter.