Cloning of DNA from double minutes of Y1 mouse adrenocortical tumor cells: Evidence for gene amplification

We have isolated a metaphase chromosome fraction highly enriched in double minutes (dm) from a mouse adrenocortical tumor cell line (Y1-DM). We have cloned DNA from this dm-enriched fraction in the λ vector Charon 4A, and have characterized two randomly chosen recombinant bacteriophage clones from this dm DNA library. When ³²P-labeled DNA from each recombinant was hybridized to Southern blots of restriction endonuclease-digested DNA from different mouse cell lines, large differences were seen in the intensity of the resulting autoradiographic images, depending on the source of the genomic DNA. A very strong signal was obtained with DNA from the Y1-DM cells and with DNA from a related Y1 subline that lacks dm but contains a marker chromosome bearing a large homogeneously staining region (HSR). Hybridization to DNA from parental inbred mice and from two unrelated mouse cell lines produced a significantly weaker signal than that obtained with DNA from the Y1 cells, but the DNA fragments from these sources were of similar size. Based on results from filter hybridization analysis, we estimate that sequences homologous to the cloned fragments are approximately 100- to 200-fold more abundant in the genome of the Y1-DM cells than in the parental mouse cells. The data are consistent with the hypothesis that dm and HSRs in these cells contain amplified genes.     

Amplification of genome regions in the somatic cells of mammals resistant to colchicine. III. Localization of the amplified genes in minute chromatin bodies

Small chromatin bodies (SCB) were revealed in Djungarian hamster cells resistant to colchicine. They looked like single bodies or like clusters of small particles. SCB were localized both in nucleus and cytoplasm. Similar formations were earlier observed in oocytes of insects with amplified extrachromosomal rDNA genes. DNA in the SCB was able to replicate not only during the S phase but also during other phases of the cell cycle. The restriction analysis showed that in cells with SCB DNA amplified sequences were replicated autonomously too. These data indicate that SCB in colchicine-resistant cells contain amplified genes. Besides, SCB double-minute chromosomes (DMs) were observed in some resistant sublines. In one of them, DMs were the only karyotypic alteration. The relationship between SCB, chromosomal homogeneously staining regions (HSRs) and DMs was studied. Single SCB and DMs appeared at the early stage of the development of colchicine-resistance (the level of drug resistance is 16-22). Selection of variants 170-220-fold resistant to colchicine was usually accompanied by the decrease in the cell number with SCB and DMs and by the increase in the amount of cells containing the chromosomes with HSRs. During the further enhancement of drug resistance (700-750), some decrease in the number of cells with HSRs and the appearance of the great number of cells containing large groups of SCB were found. The loss of colchicine-resistance observed during cultivation in colchicine free medium was accompanied by the disappearance of HSRs, emergence of SCB and DMs and further elimination of SCB and DMs from cells. The quantity of autonomously replicating amplified DNA fragments after digestive by HindIII was increased with the enhancement of SCB number in cultures.     

Regular pattern of karyotypic alterations accompanying gene amplification in Djungarian hamster cells: study of colchicine,adriablastin, and methotrexate resistance

Chromosomal analysis of 26 Djungarian hamster cell lines obtained from 11 independent clones and possessing different levels of resistance to colchicine or adriablastin as a consequence of gene amplification revealed regular patterns in the karyotypic changes that accompanied the development of drug resistance. Usually the sequence of karyotypic changes was as follows: first an additional chromosome 4 appeared; then single unpaired small chromatin bodies (SCBs) arose; later in the middle part of the long arm of one of three chromosomes 4 long homogeneously staining regions (HSRs) and double minute chromosomes (DMs) were formed; and finally in the most resistant variants large clusters of SCBs appeared. The emergence of the clusters of the SCBs correlated well with the occurrence of autonomously replicating, amplified DNA sequences. In contrast to DNA of the HSRs the DNA of the SCBs could replicate outside the S-phase of the cell cycle. When kept in a non-selective medium, the cells gradually lost their resistance to colchicine: 1%–4% of the cells lost the capacity to form colonies in the selective medium independently of the pattern of location in them of amplified genes (in chromosomal HSRs, SCBs, or DMs). Loss of drug resistance was accompanied by disappearance of the chromosomal HSRs, SCBs, and DMs. Chromosomal analysis of the set of methotrexate-resistant Djungarian hamster cell lines indicated the following karyotypic evolution: first the additional material on the distal part of one of two chromosomes 3 appeared; then the light HSRs were formed on the distal part of one of two chromosomes 4; later clusters of SCBs and HSRs arose on the distal part of the short arm of chromosome 3. Probably the amplification of different genes is characterized by specific patterns of karyotypic alterations.     

Gene amplification-associated cytogenetic aberrations and protein changes in vincristine-resistant Chinese hamster, mouse, and human cells

We carried out cytogenetic studies of four Chinese hamster, mouse, and human cell lines selected for high levels of resistance (500- to 4,000-fold) to vincristine (VCR) by a multistep selection procedure. All cells examined contained gene amplification-associated metaphase chromosome abnormalities, either homogeneously staining regions (HSRs), abnormally banding regions (ABRs), or double-minute chromosomes (DMs); control actinomycin D- and daunorubicin-resistant hamster lines did not exhibit this type of chromosomal abnormality. VCR-resistant Chinese hamster sublines exhibited both increased synthesis of the protein V19 (Mr 19,000; pl = 5.7) and increased concentrations of V19 polysomal mRNA. When VCR-resistant cells were grown in drug-free medium, level of resistance, synthesis of V19, and amount of V19 mRNA declined in parallel with mean length of the HSR or mean number of DMs per cell. Cross-resistance studies indicate that VCR-resistant cells have increased resistance both to antimitotic agents and to a wide variety of agents unrelated to VCR in chemical structure and/or mechanism of action. Our studies of tubulin synthesis in Chinese hamster cells indicate no overproduction of tubulin or presence of a mutant tubulin species. Comparison with antifolate-resistant Chinese hamster cells known to contain amplified dihydrofolate reductase genes localized to HSRs or ABRs strongly suggests that the HSRs, ABRs, or DMs of the Vinca alkaloid-resistant sublines likewise represent cytological manifestations of specifically amplified genes, possibly encoding V19, involved in development of resistance to VCR.     

Novel DNA sequences at chromosome 10q26 are amplified in human gastric carcinoma cell lines: molecular cloning by competitive DNA reassociation.

Molecular cloning of genomic sequences altered in cancer cells is believed to lead to the identification of new genes involved in the initiation and progression of the malignant phenotype. DNA amplification is a frequent molecular alteration in tumor cells, and is a mode of proto-oncogene activation. The cytologic manifestation of this phenomenon is the appearance of chromosomal homogeneously staining regions (HSRs) or double minute bodies (DMs). The gastric carcinoma cell line KATO III is characterized by a large HSR on chromosome 11. In-gel renaturation analysis confirmed the amplification of DNA sequences in this cell line, yet none of 42 proto-oncogenes that we tested is amplified in KATO III DNA. We employed the phenol-enhanced reassociation technique (PERT) to isolate 21 random DNA fragments from the amplified domain, and used 6 of them to further clone some 150 kb from that genomic region. While in situ hybridization performed with some of these sequences indicated that in KATO III they are indeed amplified within the HSR on chromosome 11, somatic cell hybrid analysis and in situ hybridization to normal lymphocyte chromosomes showed that they are derived from chromosome 10, band q26. The same sequences were found to be amplified in another gastric carcinoma cell line, SNU-16, which contains DMs, but were not amplified in other 70 cell lines representing a wide variety of human neoplasms. One of these sequences was highly expressed in both KATO III and SNU-16. Thus, the cloned sequences supply a starting point for identification of novel genes which might be involved in the pathogenesis of gastric cancers, and are located in a relatively unexplored domain of the human genome.     

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.     

Amplification of a plasmid bearing a mammalian replication initiation region in chromosomal and extrachromosomal contexts

Amplified genes in cancer cells reside on extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSRs). We used a plasmid bearing a mammalian replication initiation region to model gene amplification. Recombination junctions in the amplified region were comprehensively identified and sequenced. The junctions consisted of truncated direct repeats (type 1) or inverted repeats (type 2) with or without spacing. All of these junctions were frequently detected in HSRs, whereas there were few type 1 or a unique type 2 flanked by a short inverted repeat in DMs. The junction sequences suggested a model in which the inverted repeats were generated by sister chromatid fusion. We were consistently able to detect anaphase chromatin bridges connected by the plasmid repeat, which were severed in the middle during mitosis. De novo HSR generation was observed in live cells, and each HSR was lengthened more rapidly than expected from the classical breakage/fusion/bridge model. Importantly, we found massive DNA synthesis at the broken anaphase bridge during the G1 to S phase, which could explain the rapid lengthening of the HSR. This mechanism may not operate in acentric DMs, where most of the junctions are eliminated and only those junctions produced through stable intermediates remain.     

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.     

Isolation of recombinant bacteriophage containing male-specific mouse DNA

The mammalian Y chromosome is an isolated piece of genetic material that directs sexual determination and gametogenesis. Very little is understood about the mechanism whereby the Y chromosome carries out these functions. Also, there is a severe lack of genetic markers on this chromosome. In order to understand the structure and function of the Y chromosome at the level of its DNA sequences and to provide genetic markers, we are isolating clones of DNA whose sequences are found primarily in DNA from male mice. To this end, we have developed a procedure for the identification of such clones. Application of this screening procedure to a lambda library derived from mouse sperm DNA has yielded 12 distinct clones, part of whose sequences are present predominantly in male DNA. Besides this DNA, they also contain other sequences that are shared with female DNA. These clones are either derived from the Y chromosome or they represent autosomal sequences specifically amplified during male development.     

Inducer-responsive expression of the cloned human interferon beta 1 gene introduced into cultured mouse cells.

A hybrid plasmid was constructed from pSV2-Ecogpt (Mulligan and Berg, 1981) and the cloned 1.8 kilobase chromosomal DNA segment which contains the human interferon (HIFN-beta 1) gene and its flanking sequences. Cultured mouse FM3A cells were transfected by the hybrid plasmid and several Ecogpt+ clones were selected. Southern blot analysis of the DNA from these mouse cell clones showed that two of them contained a few copies of the 1.8 kilobase human DNA. Synthesis of mRNA characteristic of the HIFN-beta 1 mRNA increased as measured by RNA blot analysis when those clones were treated with Newcastle disease virus or with poly(I):poly(C). In addition, the induced mouse cell clones gave rise to the synthesis of human IFN-beta 1. These results suggest the presence of a unique nucleotide sequence in the 1.8 kilobase DNA which is sufficient for the inducer-responsive expression of the human IFN-beta 1 gene.     

Molecular structure and evolution of double-minute chromosomes in methotrexate-resistant cultured mouse cells.

To determine whether microscopically visible double-minute chromosomes (DMs) are derived from submicroscopic precursors, we monitored the amplification of the dihydrofolate reductase (DHFR) gene in 10 independent isolates of methotrexate (MTX)-resistant mouse cells. At every other doubling in MTX concentration, the cells were examined both microscopically, to detect the presence of microscopically visible DMs, and by pulsed-field gel electrophoresis and hybridization to a DHFR-specific probe, to detect submicroscopic DMs. One of the cloned MTX-resistant isolates was examined in detail and was shown to originally contain amplified DHFR genes on circular DMs measuring 1 and 3 Mb in size; additionally, metaphase chromosome preparations from this cloned isolate were examined and were shown to contain microscopically visible DMs too large to enter a pulsed-field gel. During stepwise selection for increasing levels of MTX, the smaller DMs (not microscopically visible) were shown to be preferentially amplified, whereas the larger (microscopically visible) ones decreased in relative numbers. Rare-cutting NotI digestion patterns of total genomic DNA that includes the DMs containing the DHFR gene suggest that the DMs increase in copy number without any further significant rearrangements. We saw no evidence from any of the 10 isolates to suggest that microscopically visible DMs are formed from smaller submicroscopic precursors.     

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.     

Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification.

We analyzed the amplification of the CAD gene in independently isolated N-(phosphonacetyl)-L-aspartate-resistant clones derived from single parental clones in two mouse cell lines. We report for the first time that the CAD gene is amplified unstably in mouse cells, that the degree of instability varies greatly between clones, and that minute chromosomes and highly unstable chromosomelike structures contain the amplified sequences. These data are most consistent with the idea that the amplified unit in each clone consists of different flanking DNA and that such differences engender amplified sequences with unequal stability. We also introduced the mouse chromosome containing the CAD gene into hamster cells by microcell-mediated chromosome transfer to determine whether the propensity for unstable extrachromosomal amplification of the mouse CAD gene would prevail in the hamster cell nuclear environment. We report that the mouse CAD gene was amplified stably in expanded chromosomal regions in each of seven hybrids that were analyzed. This observation is consistent with the idea that the nuclear environment influences whether mutants containing intra- or extrachromosomally amplified sequences will be isolated.     

Novel cytogenetic expression of gene amplification in actinomycin D-resistant somatic cell hybrids: transfer of resistance by centric chromatin bodies

SEWATC13 mouse cells, resistant to 0.1 g/ml of actinomycin D (AMD), were fused to AMD-sensitive cells of the Chinese hamster ovary cell line (CHO). Twentytwo hybrid clones were isolated and put into serial culture in the selective medium. Unexpectedly, identifiable mouse chromosomes were found only in one of the hybrids. All the others had only hamster chromosomes and, in addition, numerous chromatin bodies (CBs), mostly small and irregularly shaped, but also larger, more chromosome-like ones. The CBs were distinctly C-band positive and a mouse satellite probe hybridized strongly to them. The AMD resistance of the murine parental cells had previously been attributed to gene amplification in two large homogeneously staining regions (HSR-AMD1 and 2). They were not observed in the hybrid cells but had supposedly reappeared in the guise of the CBs. It was established by Southern DNA blot analysis that amplified DNA sequences, localized to the HSRAMD1 and 2 of the SEWA parent were present in multiple copies in the hybrids. It was also established by in situ hybridization that they were located in the CBs. Unlike double minutes (DMs) the CBs were all centric.