Differential sensitivity of double minute chromosomes to hydroxyurea treatment in cultured methotrexate-resistant mouse cells

Treating mammalian cells with continuous sub-lethal doses of Hydroxyurea (HU) causes the loss of double minute chromosomes (DMs) containing amplified oncogenes in culture. Recently, we have shown that treating glioblastoma multiforme cells in culture with low doses of HU causes the loss of DMs containing epidermal growth factor receptor genes. Loss of amplified EGFR genes was accompanied by cessation of growth, and greatly decreased tumorigenicity. To further study HU-induced elimination of DMs we have now followed the fate of dihydrofolate reductase gene (DHFR) amplifying DMs in methotrexate-resistant mouse cells during simultaneous treatment with both MTX and HU. We report that in the presence of both HU and MTX, the amplified genes decreased to 25% of starting levels in the first week of treatment, but that ultimately the cells become resistant to HU and reamplify the DHFR gene. We also report that some DHFR amplifying DMs are much more sensitive to HU than others. This study demonstrates that HU does not simply increase the rate of passive loss of DMs.     

Differential sensitivity of double minute chromosomes to hydroxyurea treatment in cultured methotrexate-resistant mouse cells.

Treating mammalian cells with continuous sub-lethal doses of Hydroxyurea (HU) causes the loss of double minute chromosomes (DMs) containing amplified oncogenes in culture. Recently, we have shown that treating glioblastoma multiforme cells in culture with low doses of HU causes the loss of DMs containing epidermal growth factor receptor genes. Loss of amplified EGFR genes was accompanied by cessation of growth, and greatly decreased tumorigenicity. To further study HU-induced elimination of DMs we have now followed the fate of dihydrofolate reductase gene (DHFR) amplifying DMs in methotrexate-resistant mouse cells during simultaneous treatment with both MTX and HU. We report that in the presence of both HU and MTX, the amplified genes decreased to 25% of starting levels in the first week of treatment, but that ultimately the cells become resistant to HU and reamplify the DHFR gene. We also report that some DHFR amplifying DMs are much more sensitive to HU than others. This study demonstrates that HU does not simply increase the rate of passive loss of DMs.     

Autonomously replicating episomes contain mdr1 genes in a multidrug-resistant human cell line.

Gene amplification in human tumor cells is frequently mediated by extrachromosomal elements (e.g., double minute chromosomes [DMs]). Recent experiments have shown that DMs can be formed from smaller, submicroscopic circular precursors referred to as episomes (S. M. Carroll, M. L. DeRose, P. Gaudray, C. M. Moore, D. R. Needham-Vandevanter, D. D. Von Hoff and G. M. Wahl, Mol. Biol. 8:1525-1533, 1988). To investigate whether episomes are generally involved as intermediates in gene amplification, we determined whether they mediate the amplification of the mdr1 gene, which when overexpressed engenders cross resistance to multiple lipophilic drugs. A variety of methods including electrophoresis of undigested DNAs in high-voltage gradients, NotI digestion, and production of double-strand breaks by gamma irradiation were used to distinguish between mdr1 sequences amplified on submicroscopic circular molecules and those amplified within DMs or chromosomal DNA. The gamma-irradiation procedure provides a new method for detecting and determining the size of circular molecules from 50 kilobases (kb) to greater than 1,000 kb. These methods revealed that some of the amplified mdr1 genes in vinblastine-resistant KB-V1 cells are contained in supercoiled circular molecules of approximately 600 and approximately 750 kb. Analysis of the replication of these molecules by a Meselson-Stahl density shift experiment demonstrated that they replicate approximately once in a cell cycle. The data lend further support to a model for gene amplification in which DMs are generally formed from smaller, autonomously replicating precursors.     

CpG island mapping of a mouse double-minute chromosome.

The development of double-minute chromosomes (DMs) and subsequent gene amplification are important genomic alterations resulting in increased oncogene expression in a variety of tumors. The molecular mechanisms mediating the development of these acentric extrachromosomal elements have not been completely defined. To elucidate the mechanisms involved in DM formation, we have developed strategies to map amplified circular DM DNA. In this study, we present a long-range restriction map of a 980-kb DM. A cell line cloned from mouse EMT-6 cells was developed by stepwise selection for resistance to methotrexate. This cloned cell line contains multiple copies of the 980-kb DM carrying the dihydrofolate reductase (DHFR) gene. A long-range restriction map was developed in which a hypomethylated CpG-rich region near the DHFR gene served as a landmark. This strategy was combined with plasmid-like analysis of ethidium bromide-stained pulsed-field gels and indicated that a single copy of the DHFR gene was located near a hypomethylated region containing SsII and NotI sites. At least 490 kb of this DM appears to be composed of unrearranged chromosomal DNA.     

Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.

This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.     

Murine erythroleukemia cell variants: isolation of cells that have amplified the dihydrofolate reductase gene and retained the ability to be induced to differentiate

A series of murine erythroleukemia cell (MELC) variants was generated by selection for the ability to grow in increasing concentrations of the folate antagonist methotrexate (MTX). Growth of the parental MELC strain DS-19 was completely inhibited by 0.1 microM MTX. We isolated cells able to grow in 5, 40, 200, 400, and 800 microM MTX. Growth rates and yields were essentially the same in the presence or absence of the selective dose of MTX for all variants. MTX resistance was not the result of a transport defect. Dihydrofolate reductase (DHFR) from our variants and DS-19 was inhibited to the same extent by MTX. Variants had increased dihydrofolate reductase activities. The specific activity of DHFR was proportional to the selective concentration of MTX employed to isolate a given variant. DNA dot blotting established that the cloned variant (MR400-3) had a 160-fold increase in DHFR gene copy number relative to the parental strain (DS-19). Hybridization studies performed in situ established the presence of amplified DHFR genes on the chromosomes of the MTX-resistant but not the MTX-sensitive (parental) cells. Quantitation of DHFR mRNA by cytoplasmic dot blotting established that the amplified DHFR gene expression was proportional to gene copy number. Thus, MTX resistance was due to amplification of the DHFR gene. The variants retained the ability to be induced to differentiate in response to dimethyl sulfoxide and hexamethylenebis(acetamide) as evaluated by the criteria of globin mRNA accumulation, hemoglobin accumulation, cell volume decreases, and terminal cell division.(ABSTRACT TRUNCATED AT 250 WORDS)     

Double-minute chromosomes as megabase cloning vehicles.

Radiation-reduced chromosomes provide valuable reagents for cloning and mapping genes, but they require multiple rounds of x-ray deletion mutagenesis to excise unwanted chromosomal DNA while maintaining physical attachment of the desired DNA to functional host centromere and telomere sequences. This requirement for chromosomal rearrangements can result in undesirable x-ray induced chromosome chimeras where multiple non-contiguous chromosomal fragments are fused. We have developed a cloning system for maintaining large donor subchromosomal fragments of mammalian DNA in the megabase size range as acentric chromosome fragments (double-minutes) in cultured mouse cells. This strategy relies on randomly inserted selectable markers for donor fragment maintenance. As a test case, we have cloned random segments of Chinese hamster ovary (CHO) chromosomal DNA in mouse EMT-6 cells. This was done by cotransfecting plasmids pZIPNeo and pSV2dhfr into DHFR-CHO cells followed by isolation of a Neo + DHFR + CHO donor colony and radiation-fusion-hybridization (RFH) to EMT-6 cells. We then selected for initial resistance to G418 and then to increasing levels of methotrexate (MTX). Southern analysis of pulsed-field gel electrophoresis of rare-cutting restriction endonuclease digestions of DNA from five RFH isolates indicated that all five contain at least 600 kb of unrearranged CHO DNA. In situ hybridization with the plasmids pZIPNeo and pSV2dhfr to metaphase chromosomes of MTX-resistant hybrid EMT-6 lines indicated that these markers reside on double-minute chromosomes.     

Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification

Initiation of DNA synthesis occurs with high frequency at oriß, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oriß/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oriß/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oriß/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.     

Much more than you expected: The non-DHFR-mediated effects of methotrexate

BackgroundFor decades, methotrexate (MTX; amethopterin) has been known as an antifolate inhibitor of dihydrofolate reductase (DHFR), and it is widely used for the treatment of various malignancies and autoimmune diseases. Although the inclusion of MTX in various therapeutic regimens is based on its ability to inhibit DHFR and consequently to suppress the synthesis of pyrimidine and purine precursors, recent studies have shown that MTX is also able to target other intracellular pathways that are independent of folate metabolism.Scope of reviewThe main aim of this review is to summarize the most important, up-to-date findings of studies regarding the non-DHFR-mediated mechanisms of MTX action.Major conclusionsThe effectiveness of MTX is undoubtedly caused by its capability to affect various intracellular pathways at many levels. Although the most important therapeutic mechanism of MTX is strongly based on the inhibition of DHFR, many other effects of this compound have been described and new studies bring new insights into the pharmacology of MTX every year.General significanceIdentification of these new targets for MTX is especially important for a better understanding of MTX action in new protocols of combination therapy.     

Variable content of double minute chromosomes is not correlated with degree of phenotype instability in methotrexate-resistant human cell lines.

Several variants resistant to 1.8 x 10(-4) M DL-methotrexate (MTX) have been isolated from the human cell lines HeLa BU25 and VA2-B by exposing them to progressively increasing concentrations of the drug. A striking variability of phenotype and chromosome constitution was observed among the different variants. All resistant cell lines exhibited a greatly increased dihydrofolic acid reductase (DHFR) activity and DHFR content; however, the DHFR activity levels varied considerably among the variants, ranging between about 35 and 275 times the parental level. In the absence of selective pressure, the increased DHFR activity was unstable, and in all cell lines but one was completely lost over a period ranging in different variants between 25 and 200 days. The MTX-resistant cells lines showed anomalies in their chromosome constitution, which involved the occurrence of a duplicated set of chromosomes in most cells of some of the variants and the presence of double minute chromosomes in all cell lines. An analysis of the correlation of loss of double minute chromosomes and loss of DHFR activity in the absence of MTX has given results consistent with the idea that the double-minute chromosomes contain amplified DHFR genes. However, the most significant finding is that, in contrast to what has been reported in the mouse system, the recognizable double-minute chromosomes varied greatly in number in different variants without any relationship to either the level of DHFR activity or the degree of instability of MTX resistance in the absence of selective pressure. These and other observations point to the occurrence in the human MTX-resistant variants of another set of DHFR genes, representing a varied proportion of the total, which is associated with the regular chromosomes, and which may be unstable in the absence of selective pressure.     

Stable formation of mutated p53 multimers in a Chinese hamster cell line causes defective p53 nuclear localization and abrogates its residual function

We have previously described a methotrexate-resistant cell line (MTX M) characterized by amplified dihydrofolate reductase (DHFR) genes, cytoplasmic p53 localization, and p53 stable tetramers. To investigate the p53 functionality in MTX M, the effect of chemical/physical agents was studied. In MTX M cells, DNA damage did not induce p53 or mdm-2 protein, while in the parental V79 cells, a residual p53 activity was found. cDNA sequencing showed that V79 and MTX M cells share the same mutations, indicating that the complete loss of p53 function in MTX M cells was due to cytoplasmic sequestration of a mutated p53 with residual activity. In Chinese hamster, both p53 and DHFR genes map on short arm of chromosome 2 suggesting that p53 itself might be amplified. However, fluorescence in situ hybridization with a hamster p53 probe showed only a single signal. Thus, the presence of p53 stable tetramers in MTX M cells, although correlated with DNA amplification, could not be the consequence of either p53 or DHFR gene amplification. Expression of a C-terminal human p53 peptide does not induce p53 nuclear accumulation, indicating that the cytoplasmic localization is due to a mechanism different from that already described in cancer cell lines. Treatments with Sodium Butyrate induced beta-tubulin polymerization, but did not apparently organize a normal microtubule network, which is shown to be important for the p53 localization. Our data indicated that in MTX M cells, p53 is sequestered in the cytoplasm by a novel mechanism that abrogates p53 residual function.     

Coregulation of dihydrofolate reductase and thymidylate synthase in overproducer cell lines of wild carrot

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities are associated with a 285,000 molecular weight enzyme complex in carrot (Daucus carota L.). Selection for methotrexate (MTX) resistance by stepwise increase of the concentration of MTX results in a high frequency adaptation to MTX with little or no significant increase in DHFR activity. However, when as a second step following MTX selection a specific inhibitor of TS, 5-fluoro-2-deoxyuridine was used, DHFR overproducer lines were obtained. The overproduction phenotype of the lines was almost completely lost after 8 weeks of growth in the absence of selection pressure. Although DHFR and TS are independent gene products, their activities increase in proportion (~20-fold) in the overproducer lines. This strongly suggests that DHFR and TS are not only functionally and physically linked in the same enzyme complex, but also are coregulated. These cell lines resemble the MTX-induced DHFR overproducer amplified cell lines of mammalian origin in their mode of selection, high frequency of appearance, elevated enzyme activity, and increased specific mRNA levels.     

Use of chromosome microdissection,the polymerase chain reaction,and dot blot hybridization to analyze double minute chromosomes

The potential usefulness of chromosome microdissertion, the polymerase chain reaction (PCR), and dot blot hybridization as a quick screening method for determining the genetic composition of double minute chromosomes (DMs) was evaluated. DMs or abrnomally banding regions (ABRs) were microdissected from from multidrug-resistant hamster cell lines and amplified with PCR using primers specific for the hamster multidrug-resistance (MDR) gene, pgp 1. The microdissected-PCR-amplified products were shown to (a) hybridize to a ³²P-labeled pCHP1 probe for the hamster MDR gene by using dot blot or Southern blot analysis and also (b) hybridize back to the chromosome region from which they were originally dissected by using fluorescent in situ hybridization. Microdissected/PCR-amplified DMs were also shown to hybridize to ABRs. When microdissected DMs and ABRs were amplified using hamster specific Alu primers, the resulting material was shown to hybridize with probes for hamster MDR and Alu. These results suggest that the DMs contained in these MDR hamster cell lines contain Alu-like sequences and the chromosome microdissection-PCR-hybridization approach might be used as a quick screening method for identifying genes amplified in DMs and ABRs in cell lines and human tumor samples.     

Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells.

Genomic DNA in higher eucaryotic cells is organized into a series of loops, each of which may be affixed at its base to the nuclear matrix via a specific matrix attachment region (MAR). In this report, we describe the distribution of MARs within the amplified dihydrofolate reductase (DHFR) domain (amplicon) in the methotrexate-resistant CHO cell line CHOC 400. In one experimental protocol, matrix-attached and loop DNA fractions were prepared from matrix-halo structures by restriction digestion and were analyzed for the distribution of amplicon sequences between the two fractions. A second, in vitro method involved the specific binding to the matrix of cloned DNA fragments from the amplicon. Both methods of analysis detected a MAR in the replication initiation locus that we have previously defined in the DHFR amplicon, as well as in the 5'-flanking region of the DHFR gene. The first of these methods also suggests the presence of a MAR in a region mapping approximately 120 kilobases upstream from the DHFR gene. Each of these MARs was detected regardless of whether the matrix-halo structures were prepared by the high-salt or the lithium 3,5-diiodosalicylate extraction protocols, arguing against their artifactual association with the proteinaceous scaffolding of the nucleus during isolation procedures. However, the in vitro binding assay did not detect the MAR located 120 kilobases upstream from the DHFR gene but did detect specific matrix attachment of a sequence near the junction between amplicons. The results of these experiments suggest that (i) MARs can occur next to different functional elements in the genome, with the result that a DNA loop formed between two MARs can be smaller than a replicon; and (ii) different methods of analysis detect a somewhat different spectrum of matrix-attached DNA fragments.     

Expression of amplified cDNA and genomic sequences encoding human interleukin 2 in Chinese hamster ovary cells

Expression of human interleukin 2 (IL-2) at high levels has been achieved in Chinese hamster ovary (CHO) cells by amplification of transfected sequences. Plasmids containing the human IL-2 cDNA or genomic DNA and mouse dihydrofolate reductase (DHFR) cDNA were transfected into DHFR-negative CHO cells. Transformants expressing DHFR were selected in media lacking nucleosides, and cells which amplified both DHFR and IL-2 genes were obtained by exposure to increasing methotrexate (MTX) concentrations. These cell lines constitutively expressed elevated levels of IL-2 at a concentration of 2 mg/liter. These cell lines continued to produce IL-2 stably through at least 1 month, even in the absence of MTX.     

Localization of dihydrofolate reductase amplified genes in Chinese hamster cells resistant to methotrexate

New sublines of BFFR1 and BFFR3 cells were obtained as a result of prolonged cultivation of Chinese hamster cells of Blld-ii-FAF 28 line (clone 431) in the presence of increasing concentrations of methotrexate (MTX). The lines obtained were resistant to 200 and 300 mcM of MTX, respectively. Amplification of the gene for dihydrofolate reductase (DHFR), similar to normal DHFR gene in restriction patterns, was proved by blot-hybridization of the resistant cells' DNA with 32P-labeled plasmid DHFR-26. Correlation is shown between the extent of gene amplification and resistance of the cell lines. In situ hybridization of the metaphase chromosomes of resistant cells with 3H-DHFR-26 results in preferential binding of the label with the regions of marker chromosomes 2 and 5, containing long, so called differential staining regions which are known to be the places of localization of amplified genes.