Diadenosine 5', 5"'-P1, P4-tetraphosphate: a pleiotropically acting alarmone?
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Adenine Nucleotides↗
Biomedical subjects
A Varshavsky
Publications and source records attributed to A Varshavsky.
Mitogenic hormones and tumor promoters greatly increase the incidence of colony-forming cells bearing amplified dihydrofolate reductase genes.
Previous work has shown that the presence of a phorbol ester tumor promoter, phorbol 12-myristate 13-acetate (PMA), during a single-step selection for methotrexate (MTX)-resistant mouse 3T6 cells results in an up to 100-fold increase in the incidence of MTX-resistant, colony-forming cells. MTX resistance of most of these cells is due to amplification of the gene for dihydrofolate reductase (DHFR), the target enzyme for MTX. We show here that other active, noncytotoxic phorbol ester tumor promoters, such as phorbol 12, 13-didecanoate and 20-phorbol 12,13-butyrate, at their optimal concentrations (approximately equal to 0.1 microM) are approximately equal to PMA in increasing the incidence of MTX-resistant 3T6 colonies. Mezerein, a potent second-stage tumor promoter, but a weak complete promoter, increases the incidence of MTX resistance up to 350-fold, the strongest effect for any of the agents so far tested. PMA analogs that are inactive as tumor promoters, such as phorbol or phorbol 12,13,20-triacetate, have no effect on the incidence of MTX-resistant 3T6 colonies. Anthralin, a nonphorbol tumor promoter, is approximately equal to 40% as active as PMA in the MTX selection assay. Remarkably, the hormones insulin, arginine vasopressin, and epidermal growth factor, all of which are mitogenic for 3T6 cells, also exert a strong PMA-like effect on the incidence of MTX-resistant 3T6 colonies under conditions of MTX selection. The effect of insulin at its optimal concentration (approximately equal to 1 microgram/ml) is approximately equal to 70% that of PMA. Although the effect of PMA on the incidence of MTX-resistant 3T6 colonies does not significantly depend on the initial density of seeded cells or volume of the medium added, the analogous effect of insulin is strongly influenced by these parameters. Mevalonic acid, arachidonic acid, thymidine, caffeine, and nicotine, all of which are known to influence patterns of DNA synthesis in mammalian cells, were tested at their highest noncytotoxic concentrations and failed to produce any significant effect on the incidence of MTX-resistant 3T6 colonies. We discuss possible mechanisms of hormone- and tumor promoter-facilitated gene amplification in mammalian cells, relationship of mitogenic hormones to tumor promoters, and also implications of our findings for the problem of drug resistance in cancer chemotherapy.
Animals↗
Loss of unstably amplified dihydrofolate reductase genes from mouse cells is greatly accelerated by hydroxyurea.
Previous work has shown that mammalian cells that carry unstably amplified genes for dihydrofolate reductase (DHFR) gradually lose the amplified DHFR genes when grown in the absence of the DHFR inhibitor methotrexate (MTX). Unstably amplified genes occur on small acentric chromosomes called double minutes (DMs) or even smaller chromatin fragments, in contrast to stably amplified genes, which reside in centromere-containing chromosomes. We have found that the rate of loss of the unstably amplified DHFR genes can be greatly oncreased by growing the cells in the presence of a nonlethal concentration of hydroxyurea. For example, in one MTX-resistant subline studied, approximately equal to 90% of the original DHFR gene dosage is lost in 25-30 cell doublings in the absence of MTX. The same degree of loss is achieved, however, in less than 4 doublings if cells are grown in the presence of 50 microM hydroxyurea. This new effect of hydroxyurea does not appear to be due to changes in plating efficiency or selective cytotoxicity. In particular, no increase in cell death occurs at 50 microM hydroxyurea, and cells continue to multiply, albeit 1/2 to 2/3 as fast as in the absence of hydroxyurea. The ability to selectively accelerate the loss of amplified genes from mammalian cells as shown in the present work may have important implications both for the problem of drug resistance in cancer chemotherapy and for curing mammalian cells of extrachromosomally maintained DNA genomes of pathogenic viruses.
Animals↗
Cellular and SV40 chromatin: replication, segregation, ubiquitination, nuclease-hypersensitive sites, HMG-containing nucleosomes, and heterochromatin-specific protein.
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Animals↗
Acquisition and loss of amplified genes: dramatic effects of hormones, tumor promoters and cytotoxic drugs.
We discuss our recent findings in three related areas of the gene amplification field. 1) We have found that tumor-promoting phorbol esters, nonphorbol tumor promoters, and most significantly, mitogenic hormones, such as insulin, vasopressin, and epidermal growth factor (EGF), greatly increase the incidence of methotrexate (MTX) resistance in 3T6 cells under condition of MTX selection. Most of these MTX-resistant cells bear amplified dihydrofolate reductase (DHFR) genes. 2) We have discovered that when mouse cells bearing unstably amplified DHFR genes are grown in the presence of nonlethal concentrations of hydroxyurea (HU), the rate of loss of the DHFR genes from these cells is greatly increased. 3) We have developed a new method for detection and mapping of homologous, repeated and amplified DNA sequences, and have used this method to detect and clone amplified DNA fragments in mammalian cells resistant simultaneously to a number of different drugs.
Animals↗
On the chromatin structure of the amplified, transcriptionally active gene for dihydrofolate reductase in mouse cells.
The method for two-dimensional hybridization mapping of nucleosomes (Levinger, L., Barsoum, J., and Varshavsky, A. (1981) J. Mol. Biol. 146, 287-304) was used to analyze chromatin structure of the gene for dihydrofolate reductase (DHF reductase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase (EC 1.5.1.3)) in L5178Y-R mouse cells. The DHF reductase gene in these cells is amplified about 350-fold as a result of selection for resistance to methotrexate. Dramatic overproduction of DHF reductase mRNA in L5178Y-R cells suggests that most of the DHF reductase genes in these cells are transcribed. We report that all major mononucleosomal species resolvable by two-dimensional fractionation are detected by both DHF reductase- and satellite DNA-specific hybridization probes. Although the DHF reductase and satellite hybridization patterns differ somewhat from each other and from the total mononucleosomal pattern, their overall similarity is very high. In particular, no large differences in the abundance of mononucleosomes containing high mobility group non-histone proteins (HMG) 14 and 17 are seen between the DHF reductase and satellite chromatin regions under a wide variety of conditions for chromatin isolation, digestion, and fractionation. Possible interpretations of the apparent lack of selectivity of HMG-chromatin interactions in this system are discussed. We also found that the amplified DHF reductase genes possess a wide range of nucleosomal repeat lengths close to that in the bulk chromatin. In contrast, the range of nucleosomal repeat lengths in the satellite chromatin is much narrower than in both DHF reductase and bulk chromatin.
Animals↗
Selective arrangement of ubiquitinated and D1 protein-containing nucleosomes within the Drosophila genome.
We have a new approach, two-dimensional hybridization mapping of nucleosomes, to compare the structures of mononucleosomes from different regions of the Drosophila melanogaster genome. Approximately one in two nucleosomes of the transcribed copia and heat-shock 70 (hsp 70) genes in nonshocked cultured cells contains ubiquitin-H2A (uH2A) semihistone, a covalent conjugate of histone H2A and a small protein, ubiquitin. In striking contrast, less than one in 25 nucleosomes of tandemly repeated, nontranscribed 1.688 satellite DNA contains uH2A, suggesting that most of the nucleosomal uH2A is located in transcribed genes. Approximately 25% of all nucleosomes are ubiquitinated in nonsynchronized cultured Drosophila cells. The hsp 70 genes in nonshocked cells occur in nucleosomes, are greatly enriched in uH2A and are not digested preferentially by staphylococcal nuclease. In contrast, the same genes in chromatin from heat-shocked cells are highly sensitive to staphylococcal nuclease and no longer possess nucleosomal organization recognizable with this probe. Histone ubiquitination in transcribed nucleosomes may prevent formation of higher order chromosomal structures by modifying nucleosome-nucleosome interactions. The observed loss of nucleosomal organization in very actively transcribed genes, such as the hsp 70 genes in shocked cells, may be related to the recent finding that ubiquitin conjugates are substrates for the cytoplasmic ATP-dependent proteolytic system. We have also found that 1.688 satellite mononucleotomes contain a specific approximately 50,000 dalton nonhistone protein, D1, in addition to being extremely under-ubiquitinated. D1 may be involved in formation of the highly compact structure of satellite heterochromatin.
Animals↗
Protein D1 preferentially binds A + T-rich DNA in vitro and is a component of Drosophila melanogaster nucleosomes containing A + T-rich satellite DNA.
Our previous work [Levinger, L. & Varshavsky, A. (1982) Cell 28, 375-385] has shown that D1, a 50-kilodalton chromosomal protein of Drosophila melanogaster, is specifically associated with isolated nucleosomes that contain a complex A + T-rich satellite DNA with buoyant density of 1.688 g/ml. We show here that D1 is also a component of nucleosomes containing a simple-sequence, pure A + T satellite DNA, buoyant density 1.672 g/ml. Furthermore, using a modification of a protein blotting technique in which proteins are not exposed to dodecyl sulfate denaturation, we have found that D1 preferentially binds to A + T-rich double-stranded DNA in vitro, and it is apparently the only abundant nuclear protein in cultured D. melanogaster cells that possesses this property. Synthetic poly[d(A-T)].poly[d(A-T)] and poly(dA).poly(dT) duplexes effectively compete in vitro with A + T-rich D. melanogaster satellite DNAs for binding to D1, whereas total Escherichia coli DNA is an extremely poor competitor. These findings strongly suggest that D1 is a specific component of A + T-rich, tandemly repeated, heterochromatic regions, which constitute up to 15-20% of the total D. melanogaster genome. Possible functions of D1 protein include compaction of A + T-rich heterochromatin and participation in microtubule-centromere interactions in mitosis. In addition, D1 may prevent nonspecific binding to A + T-rich satellite DNA of other nuclear proteins that have a preference for AT-DNA, such as RNA polymerase or regulatory proteins, and may also participate in the higher-order chromatin organization outside tandemly repetitive regions by binding to nonrandomly positioned stretches of A + T-rich DNA.
Adenine Nucleotides↗
New way to isolate simian virus 40 nucleoprotein complexes from infected cells: use of a thiol-specific reagent.
A new method for the isolation of simian virus 40 nucleoprotein complexes from nuclei of lytically infected cells is described. The method is based on the addition of a thiol-specific reagent, 5'5'-dithiobis(2-nitrobenzoic acid), to lysis and extraction buffers. By inhibiting an uncoating activity during simian virus 40 extraction, 5'5'-dithiobis (2-nitrobenzoic acid) allows the use of efficient extraction buffers, such as one containing Triton X-100 and EDTA, for the isolation of native simian virus 40 minichromosomes and virion-type structures. Use of the method is illustrated by following encapsidation of simian virus 40 minichromosomes in a pulse-chase experiment. Since 5'5'-dithiobis (2-nitrobenzoic acid) is an inhibitor of many different enzymes, the 5',5'-dithiobis (2-nitrobenzoic acid) extraction technique may be useful for the isolation of not only papovaviruses but also other viruses and possibly cellular chromatin.
Animals↗
Two-dimensional hybridization mapping of nucleosomes. comparison of DNA and protein patterns.
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Animals↗
Phorbol ester dramatically increases incidence of methotrexate-resistant mouse cells: possible mechanisms and relevance to tumor promotion.
I have tested the hypothesis that at least some of the known tumor promoters may act by facilitating gene amplification. A series of single-step selections for resistance to methotrexate, a specific inhibitor of dihydrofolate reductase (DHFR), was carried out with 3T6 cells in the presence and in the absence of a potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Incidence of methotrexate-resistant, colony-forming 3T6 cells is increased up to 100 fold if selection is carried out in the presence of TPA. For a major portion of this new TPA effect to be observed, it is sufficient to add TPA simultaneously with methotrexate. The effect of TPA on the incidence of methotrexate resistance is detectable at less than 20 nM TPA and is maximal at about 200 nM TPA. Phorbol (a nonpromoting analog of TPA), thymidine and dimethylsulfoxide each fail to produce any TPA-like effect in this system. DHFR gene copy numbers per cell in clones resistant to 100, 200 and 300 nM methotrexate are approximately 3, 10 and 16 times higher, respectively, than the DHFR gene copy number in the parental 3T6 cells. These numbers do not depend on the presence or absence of TPA during methotrexate selection.
Animals↗
Arrest of segregation leads to accumulation of highly intertwined catenated dimers: dissection of the final stages of SV40 DNA replication.
When SV40-infected cells are placed into hypertonic medium, newly synthesized DNA accumulates as form C catenated dimers. These molecules consist of two supercoiled monomer circles of SV40 DNA interlocked by one or more topological inter-twinings and are seen as transiently labeled inter-mediates during normal replication. Form C catenated dimers represent pure segregation intermediates, replicative DNA structures in which DNA synthesis is complete but which still require topological separation of the two daughter circles. Hypertonic shock seems to block selectively a type II topoisomerase activity involved in disentangling the two circles. This is reflected in the fact that form C catenated dimers that accumulate during the block are highly intertwined with catenation linkage numbers up to C(L) = 20. While initiation of replication is also inhibited by hypertonic treatment, ongoing SV40 DNA synthesis is not affected, and replication is free to proceed from the earliest cairns structure through to form C catenated dimers. The block to segregation is rapidly and completely released by shifting the cells back to normal medium. A much slower recovery of DNA segregation takes place on prolonged incubation in hypertonic medium, perhaps because of some cellular homeostatic mechanism. The results of this work lead to a detailed view of the final stages of SV40 DNA replication.
Chromosomes↗
On the possibility of metabolic control of replicon "misfiring": relationship to emergence of malignant phenotypes in mammalian cell lineages.
Constraints of a multireplicon chromosomal organization and of the necessity to maintain constant gene dosages demand that each origin of replication in a eukaryotic cell "fire" (initiate replication) only once per cell cycle. The central idea of this work is that a low probability of an extra ("illegitimate") round of DNA replication (called below "replicon misfiring") within any given chromosomal domain could be increased by certain substances of either intra- or extracellular origin. The term " "firone" is proposed for such a substance. It is shown that existence of firones could greatly speed up evolution of cellular systems under selection pressure, a developing tumor being one example of such a system. Experimentally testable predictions of the firone hypothesis are discussed.
Animals↗
Heat-shock proteins of Drosophila are associated with nuclease-resistant, high-salt-resistant nuclear structures.
Proteins produced in cultured Drosophila cells during the heat-shock response (HSPs) were recently shown by autoradiography to be confined in large measure to the cell nucleus. We report here that nuclear HSPs are not associated with nucleosomes solubilizes by treatment with staphylococcal nuclease at low ionic strength nor are HSPs released by extraction with high salt, which solubilized most of the remaining histones and DNA. Possible functions of nuclear HSPs are discussed.
Animals↗
Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers.
We have identified a new class of SV40 replicative intermediates which consists of a least 20 discrete DNA species. All members of this class are catenated dimers, two circular molecules of SV40 duplex DNA linked topologically by one or more intertwining events. Most of these molecules are linked by several intertwining events, and the range of linkage states observed runs from L = 1 to L = 10. A catenated dimer with a given linkage state is assigned to one of three distinct families (A, B or C) depending on the open or covalently closed nature of its two circular components: in form A catenated dimers, both circles are nicked or gapped; in form B, one of the circles is supercoiled; and in form C, both circles are supercoiled. Members of all three of these families are found in SV40 chromatin pulse-labeled with 3H-thymidine, and together they comprise 10-20% of the total replicative form SV40 DNA, appearing as a discrete series of electrophoretically resolved bands superimposed upon a continuous smear of growing cairns structures. The distribution of linkage states varies between the families, A being the most intertwined and C the least intertwined. Upon a chase with cold thymidine, label is lost rapidly from all these catenated DNA species. We suggest that the sequence A leads to B leads to C leads to mature monomeric supercoiled SV40 DNA represents the final stages of SV40 replication, and that a special enzyme activity exists in vivo to uncatenate the SV40 daughter chromosomes.
DNA Replication↗
High-resolution fractionation of nucleosomes: minor particles, "whiskers," and separation of mononucleosomes containing and lacking A24 semihistone.
Staphyloccal nuclease digests of HeLa chromatin fractionated on low ionic strength nucleoprotein gels have been further analyzed by second-dimension DNA and protein gel electrophoresis. In vivo radioactive labeling of chromatin components and use of longer gels allowed a higher sensitivity and resolution than has been previously reported for this approach. A number of nonhistone protein spots and about 20 DNA spots can be detected in the mononucleosomal region of the second-dimension gel. In particular, there are three DNA spots identical in DNA size that correspond to three discrete kinds of core mononucleosomes resolved on the first-dimension nucleoprotein gel. Analysis of protein composition shows that the most rapidly migrating particle contains all four core histones but no A24 semihistone (A24 is a covalent conjugate of histone H2A and a specific nonhistone protein, ubiquitin), whereas the other two core mononucleosomes contain A24 semihistone. Thus, one can now quantitatively separate the A24-lacking core mononucleosomes from those containing A24, making it possible to directly address the question of whether A24 is associated with nucleosomes containing a specific subset of DNA sequences. Additional features of two-dimensional nucleoprotein-DNA patterns are "whiskers," which run slower than core mononucleosomes in the nucleoprotein dimension and both faster and slower than core-length DNA in the DNA dimension. In more extensive digests, "secondary whiskers" are observed, which run faster than core mononucleosomes in both dimensions and appear to coincide with previously described subnucleosomal particles SN7 and SN8 [Bakayev, V., Bakayeva, T. & Varshavsky, A. (1977) Cell 11, 619-629]. Possible mechanisms of whisker formation are discussed.
Base Sequence↗
Staphylococcal nuclease makes a single non-random cut in the simian virus 40 viral minichromosome.
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Chromosome Mapping↗
Amplification of specific DNA sequences correlates with multi-drug resistance in Chinese hamster cells.
Mammalian cells selected for resistance to certain cytotoxic drugs frequently develop cross-resistance to a broad spectrum of other drugs unrelated in structure to the original selective agent. This phenomenon constitutes a major problem in cancer chemotherapy. Multi-drug resistance arises from decreased intracellular drug accumulation, apparently due to an alteration of the plasma membrane. The observation of double minute chromosomes or homogeneously staining regions in some of the multi-drug-resistant cell lines suggests that gene amplification underlies this phenomenon. We have used the technique of DNA renaturation in agarose gels to detect, compare and clone amplified DNA sequences in Adriamycin- and colchicine-resistant sublines of Chinese hamster cells. We show that both Adriamycin- and colchicine-resistant cells contain amplified DNA fragments, some of which are amplified in both of these independently derived cell lines. Furthermore, loss of the multi-drug resistance phenotype on growth in the absence of drugs correlates with the loss of amplified DNA. These results strongly suggest that the DNA sequences which are amplified in common in multi-drug-resistant cell lines include the gene(s) responsible for a common mechanism of multi-drug resistance in these cells. We have cloned one of the commonly amplified DNA fragments and show that the degree of amplification of this fragment in the cells correlates with the degree of their drug resistance.
Animals↗