DNA-bound lipids of normal and tumor cells: retrospective and outlooks for functional genomics.

By very soft phenol method, the high-molecular-mass natural DNA complexes (10(8)-10(9) Da), which contain 1-3% specific lipids, were isolated from different eukaryotic and prokaryotic cells. Two pools of DNA-bound lipids were isolated: loosely bound (extracted with 35% ethanol) and tightly bound lipids (extracted after additional treatment DNAse I). The composition of these two lipid pools of different sources (rat thymus, liver, regenerating liver, loach sperm, pigeon erythrocytes, Zajdel ascites hepatoma, Ehrlich ascites carcinoma, sarcoma 37, Escherichia coli B, T2 phage) was studied. The DNA-bound lipid pools consist of neutral lipids (NL) and phospholipids (PL), moreover NL is always in a few fold more than PL. The composition of these lipid pools of eukaryotes distinguishes between themselves, mainly, by free cholesterol (minor fraction), cardiolipin (major fraction), and by phosphatidylcholine. Only the tightly bound lipid pool was present in T2 phage DNA. The dramatic redistribution effect between all fractions of NL pools (free and ester cholesterol, free fatty acids, diglycerides) was observed in DNA synthesis phase of cell cycle on the background of the unchanged composition of PL pools. Comparative analysis of DNA-bound lipid pools of normal and cancer cells was carried out. The DNA-bound lipid pools of transformed cells significantly differ from the same normal cells both by PL composition (cardiolipin) and by the presence of additional fractions (mono- and triglycerides) as well. The possible functions of DNA-bound lipid pools, especially of cardiolipin and cholesterol at the attachment of DNA loops to the nuclear matrix, DNA replicon organization, replication, and transcription are discussed.

Specific natural DNA-bound lipids in post-genome era. The lipid conception of chromatin organization.

Two pools of DNA-bound lipids were isolated from DNA supramolecular complex (SC-DNA): loosely bound (extracted with 35% ethanol) and tightly bound lipids (extracted after additional treatment DNase I). The compositions of the two lipid pools from different sources (rat thymus, liver, loach sperm, pigeon erythrocytes, Zajdel ascites hepatoma, Ehrlich ascites carcinoma, sarcoma 37, Escherichia coli B and T2 phage) were studied. The possible functions of DNA-bound lipids, especially of cardiolipin and cholesterol, at the attachment of DNA loops to the nuclear matrix, in DNA replicon organization, replication and transcription are discussed.

Chromatin-bound cardiolipin: the phospholipid of proliferation.

Chromatin-bound lipids, cardiolipin (CL), diglycerides, cholesterol, and cholesterol esters, together with nonhistone proteins, play a key role in structural and functional organization of the chromatin genome during various stages of evolution. There are two pools of chromatin lipids, namely loosely- and tightly-bound lipids. The entire chromatin cardiolipin is bound to DNA. The CL molecule has a common 'interphosphate' structural motive with DNA, i.e. DNA and CL phosphate moieties separated from each other with six chemical bonds and equidistant, which is important for CL functional role, the regulation of gene expression. The CL dominates in the DNA of the active genome but not in the DNA of the repressed genome. The amount of CL in the DNA from the repressed genome of pigeon erythrocytes (one CL molecule per 20 nucleosomes) is 20 times less than in the DNA from the active genome of rat thymus and liver and in the DNA of transformed cells. Cardiolipin provides A-form DNA in the complex with RNA-polymerase, which is necessary for transcription. The biological and structural function of cardiolipin can be realised only when unsaturated fatty acyl residues are present in its structure.

Structural and functional aspects of nuclear lipids in normal and tumor cells.

This review summarizes available data on the structural and functional role of neutral lipids and phospholipids in normal and tumor eukaryotic cells. The role of acidic phospholipids (cardiolipin, phosphatidylinositol, and phosphatidylglycerol) in regulation of activities of DNA- and RNA-polymerases, DNA-topoisomerases I and II, DNA-methylases, and replication initiation proteins (dnaA and T-antigen) is discussed. The role of sphingolipids is emphasized considering, on one hand, the involvement of sphingosines in signal transduction, chromatin association-dissociation, and regulation of DNA and RNA synthesis and protein kinase C and, on the other hand, participation of ceramides and dihydroceramides in apoptosis. The possible role of sphingomyelin, sphingosine, cardiolipin, and diglycerides in the contacts of DNA loops with nuclear matrix is analyzed. Lipid hormones indirectly influence supercoiled DNA conformation; the effect of hormones on metabolism of phospholipids and neutral lipids in chromatin and nuclear matrix is reviewed. Characteristics of lipid composition in chromatin and nuclear matrix of the tumor cells are discussed.

[The role of disulfide bridges of the residual protein in chromosomal DNA organization]. / Rol' disul'fidnykh mostikov ostatochnogo belka v organizatsii khromosomal'noi DNK.

The review of literature data and our investigation on the role of disulfide bridges of residual protein (RP) in structural organization of chromosomal DNA is presented. It was studied the action of several S-S cleaving agents (2-mercaptoethanol, dithiothreitol, NaBH4, glutathione reductase) on native DNA-RP complexes, isolating from the different eukaryotic and prokaryotic cells. It was shown, that the thiols result the fragmentation of DNA-RP complex in double-strand subunits of several size (5 x 10(5), (18-20) x 10(6), 70 x 10(6) Da) on dependence of the incubate condition (concentration of thiols, pH, time). It was observed, that specific S-S bonds (thiol-sensitivity at neutral or acid conditions, glutathione reductase-sensitivity) are present in DNA-RP complexes, which may control of different structural levels of DNA into chromosome (gen-transcription-replicon-domain). The possible quasisubunit structure of chromosomal DNA with participation of polypeptide S-S bonds and complementary "sticky" ends of subunits is discussed.

[DNA-binding lipids: composition and possible functions]. / DNK-sviazannye lipidy: sostav i vozmozhnye funktsii.

The experimental data concerning the composition of DNA-bound lipids of different eukaryotic and prokaryotic cells have been summarized. Using X-ray diffraction patterns, circular dichroism, microcalorimetry, electron microscopy, viscoelastometry and sedimentation methods, it has been proved that the lipids are important integral components of chromosomal DNA. It was shown that the DNA-bound lipids have a specific composition which differs from that of chromatin, nuclear membrane and matrix lipids. The composition of these lipids changes depending on the activity of the genome and the phase of the cell cycle as well as when DNA passes from a supercoiled into a relaxed state. The DNA of cancer cells has a specific composition of the lipid component. The lipids take part in the regulation of transcription. The DNA-bound lipids are hypersensitive target sites for ionizing radiation and anticancer agents. The role of this lipid class in structure-functional organization of chromosomal DNA is discussed.

[Thiol-induced fragmentation of chromosomal DNA]. / Tiol-indutsirovannaia fragmentatsiia khromosomal'noi DNK.

Supramolecular complexes of DNA (SC DNA) were isolated from loach sperm, loach erythrocytes and hen erythrocytes by the phenol method. By the use of UV-sedimentation on neutral 5-20% sucrose gradient, we studied the effect of 2-mercaptoethanol (ME), dithiothreitol (DTT) and NaBH4 on SC DNA at different pH and long-time incubation (5 and 10 days). It appeared that ME treatment at pH 4.4 fragmented SC DNA of three objects into subunits of size 5 x 10(5)D. Incubation with DTT at pH 8 in the presence of EDTA resulted in subunits of size 1-2 x 10(7)D. However, NaBH4 at pH 8 failed to induce fragmentation of SC DNA. It is shown that ME-induced at pH 4.4 fragmentation is accompanied by a decrease in hyperchromatic effect of subunits, indicating the presence of "sticky" ends. Thus, ME-induced fragmentation of SC DNA results from a "clayting" double-strand break, involving, on an average, 180 bp.

[DNA-bound lipids from eukaryotic (loach spermatozoa, pigeon erythrocytes) and prokaryotic (E. coli B, phage T2) cells]. / DNK-sviazannye lipidy kletok éukariot (sperma v'iuna, éritrotsity golubia) i prokariot (E. coli B, fag T2).

Using thin-layer chromatography, some specific DNA-bound neutral lipids and phospholipids of loach spermatozoa, pigeon erythrocytes, E. coli B and phage T2 cells were studied. These lipids are represented by loosely and firmly bound components. The content of neutral lipids in the above DNAs (per mg of DNA) is 10.6, 4.8, 7.81 and 1.43 micrograms, respectively; that of phospholipids is 4.31, 1.28, 1.14 and 0.54 micrograms, respectively. The eucaryotic DNA-bound lipids are highly deficient of free cholesterol, phosphatidylcholine, phosphatidylinositol and phosphatidylserine but are rich in cardiolipin, phosphatidylethanolamine, cholesterol esters, diglycerides and free fatty acids. The quantitative and qualitative composition of DNA-bound lipids of loach spermatozoa changes during the transition from the superhelical to the relaxed conformation of DNA. Procaryotic DNA-bound neutral lipids are also represented by the free cholesterol, diglyceride and free fatty acid fractions, whereas the DNA-bound phospholipids of procaryotes consist of only two fractions, i.e., cardiolipin and phosphatidylethanolamine. The role of DNA-bound lipids in the structural and functional organization of eucaryotic and procaryotic genomes is discussed.

[Changes induced by sarcolysine in vivo in the composition of DNA-bound lipids in sarcoma 37]. / Izmenenie sostava DNK-sviazannykh lipidov sarkomy 37, indutsirovannoe sarkolizinom in vivo.

The two DNA fractions were isolated from sarcoma 37 by the use of the phenol method: supramolecular complex of DNA (SC DNA, 60%) and "phenol" nuclear matrix DNA (PNM DNA, 40%). The lipids in SC DNA represented of light and tightly bound components, the latter was similar to the lipid composition of PNM DNA. SC DNA contains 20 micrograms of neutral lipids (NL) and 6.5 micrograms of phospholipids (PL), while PNM DNA contains 9.8 micrograms of NL and 3.5 micrograms of PL per mg DNA. SC DNA-bound lipids of sarcoma 37 are deficient in free cholesterol (FC, 13%), but rich in cholesterol esters (CE, 39%) and free fatty acids (FFA, 23%); very rich in cardiolipin (CL, 43%) and phosphatidylethanolamine (PE, 28%), but deficient in phosphatidylcholine (PC, 12%). The tumor contains triglycerides (TG) that is absent in DNA of the normal cells. The injection of sarcolysine (10 micrograms/kg) markedly increased (1.5-3 times) the content of all LN and PL fractions in SC DNA, which was accompanied by both the accumulation of FC, TG, PC and the reduction of the remaining lipid fractions in PNM DNA. It is supposed, that DNA-bound lipids may be the target for the action of sarcolysine.

[Changes in the composition of DNA-bound nonhistone protein in the thymus and liver of gamma-irradiated rats]. / Izmenenie sostava DNK-sviazannogo negistonovogo belka v timuse i pecheni gamma-obluchennykh krys.

A supramolecular DNA complex (SC DNA) and DNA of a phenol nuclear matrix (PNM DNA) were extracted, by the phenol method, from rat thymus and liver 15 min following 10 Gy gamma-irradiation. The method of electrophoresis in polyacrylamide gel was used to study a composition of nonhistone proteins firmly bound to these DNA fractions. Irradiation was shown to induce the occurrence of new proteins and redistribution of proteins between SC DNA and PNN DNA of rat organs.

[DNA-bound lipids of the cells of Zajdela ascites hepatoma and of Ehrlich ascites cancer]. / DNK-sviazannye lipidy kletok astsitnoi gepatomy Zaidela i astsitnogo raka Erlikha.

DNA-bound neutral lipids (NL) and phospholipids (PL) were isolated and characterized from the Zajdel ascites hepatoma (ZAH) and Ehrlich ascites carcinoma (EAC) cells. The lipids are represented by light- and tightly bound components. It was shown, that the tumour DNA contained minor amount of NL (25, 17 micrograms and 16.87 micrograms per mg DNA, respectively) and of PL (4.54 micrograms and 5.36 micrograms per mg DNA, respectively, for ZAH and EAC). The composition of the tumour DNA-bound lipids was shown to differ from that of DNA-bound lipids of liver and thymus of intact rats by the next parameters: NL/PL ratio is much more than one; increased content of FC; equal values of the three basic ratios--CE/FC, NL/PL, cholesterol/PL, presence of mono- and triglycerides.

[The action of cortifen and corticosterone on the thymocyte nucleoid of mice in vivo and in vitro]. / Deistvie kortifena i kortikosterona na nukleoid timotsitov myshei in vivo i in vitro.

The effect of therapeutic doses of a hormonal cytostatic cortifen and corticosterone on supramolecular DNA structures of mice thymocyte nucleoid was studied. Capillary elastoviscometry showed that in vivo damage to supramolecular DNA complex structure there was a difference already two hours after injection of these agents. After 24 hours the damaging effects on nucleoid were markedly higher. The role of the hormone and alkylating group of cortifen in the cytotoxic action of antitumor agents is discussed.

[Composition of DNA-bound lipids in the regenerating rat liver]. / Sostav DNK-sviazannykh lipidov v regeneriruiushchei pecheni krys.

Using thin-layer chromatography, the qualitative and quantitative composition of specific DNA-bound neutral lipids (NL) and phospholipids (PL) of regenerating rat liver 22 hours (S-phase) and 28 hours (G2-phase) after hepatectomy was studied. These lipids are represented by light and tightly bound components. The intact liver DNA contains minor amounts of NL and PL (15.02 micrograms and 5.82 micrograms per mg of DNA, respectively). The composition of DNA-bound lipids in rat liver differs markedly from that of nuclear membrane and chromatin total lipids. The former are strongly deficient in free cholesterol (FC), but are rich in cholesterol esters (CE), very rich in cardiolipin (CL) and deficient in phosphatidylcholine. The basic parameters of DNA-bound lipids of rat liver (NL/PL, CE/FC and cholesterol/PL) are more than unity and depend on the cell cycle. It was shown that in the S-phase the content of DNA-bound NL and PL increases 1.5-fold, in the G2-phase the NL content shows a still greater increase--2.3-fold, while that of DNA-bound PL decreases to normal values. The basic changes of the DNA-bound lipids in regenerating rat liver are due to FC, CE and CL, which determine the tissue specificity of these lipids.