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TELOMERASE-DEPENDENT REACTIVATION OF DNA SYNTHESIS IN MACROPHAGES IMPLIES ALTERATION OF TELOMERES
Cell Biology International 2002, Vol. 26, No. 12, 1019–1027 doi:10.1006/cbir.2002.0961, available online at http://www.idealibrary.com on
TELOMERASE-DEPENDENT REACTIVATION OF DNA SYNTHESIS IN MACROPHAGES IMPLIES ALTERATION OF TELOMERES Y. E. YEGOROV1*, E. V. KAZIMIRCHUK1, S. M. TEREKHOV2, D. N. KARACHENTSEV3, E. A. SHIROKOVA1, A. L. KHANDAZHINSKAYA1, J. A. MESHCHERYAKOVA4, D. R. COREY5 and A. V. ZELENIN1 1
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov str., Moscow, 119991, Russia; 2Institute of Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia; 3 Moscow Physico-Technical Institute (PhysTech), Dolgoprudnyi, Moscow, Russia; 4Center of Bioengineering, Russian Academy of Sciences, Moscow, Russia; 5Department of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9041, U.S.A. Received 21 March 2002; accepted 28 August 2002
In previous work we demonstrated that various types of cultured cells with a limited life span could not reactivate DNA synthesis in the nuclei of mouse peritoneal macrophages in heterokaryons. We now investigate the role of telomerase in the process of the macrophage nucleus reactivation in heterokaryons with immortal telomerase-positive 3T3 Swiss mouse fibroblasts and human fibroblasts with introduced hTERT gene. We report that introduction of the hTERT gene into human diploid fibroblasts results in emergence of telomerase activity in these cells and the ability to induce the reactivation of DNA synthesis in the macrophage nuclei in heterokaryons. Inhibition of telomerase activity in heterokaryons by reverse transcriptase inhibitors (azidothymidine and guanosine polyphosphonate analogues) and by a 2 -O-methylRNA oligonucleotide anti-sense to the template region of telomerase RNA, block reactivation of DNA synthesis in macrophage nuclei without inhibiting DNA synthesis in the nuclei of fibroblasts. Our results suggest alterations (shortening or damage) in the macrophage telomere structure. As far as we know, heterokaryons with macrophages are the first cellular model for rapid investigation of the effects of telomerase inhibitors. 2002 Elsevier Science Ltd. All rights reserved.
K: telomerase; telomeres; telomerase inhibition; macrophages; heterokaryons; human fibroblasts; proliferation; differentiation.
INTRODUCTION The mechanisms that block cell proliferation at the stage of terminal differentiation and maintain differentiated cells in the non-dividing state are poorly studied. An important approach to the problem of cessation of proliferation is based on hybridization of somatic cells. Terminally differentiated nondividing cells can be fused with proliferating ones and their interaction in hybrids (heterokaryons) can be investigated. Heterokaryons are artificially constructed cells that contain nuclei of a dissimilar origin. *To whom correspondence should be addressed: Fax: (095) 135-1405; E-mail: [email protected] 1065–6995/02/$-see front matter
In our previous work, we investigated the regulation of DNA synthesis in heterokaryons formed by macrophages from normal mouse peritoneum and various proliferating cells. Altogether, we used 15 different cell strains and lines (Prudovsky et al., 1985; Prudovsky et al., 1989a,b; Zelenin and Prudovsky, 1989; Prudovsky et al., 1993). No reactivation of DNA synthesis in the macrophage nuclei was detected in heterokaryons with cells of limited proliferative life-span, although nuclei of actively proliferating partners synthesized DNA on their own. We did, however observe reactivation of DNA synthesis in the macrophage nuclei in heterokaryons with immortal cells: in up to 90% of heterokaryons that contained 2002 Elsevier Science Ltd. All rights reserved.
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the DNA-synthesizing 3T3 nucleus, the macrophage nuclei were also labeled within 24 h after fusion. In our early work (Prudovsky et al., 1989a,b) we looked for the relationship between the presence of active oncogenes and the ability of proliferating cells to induce reactivation of DNA synthesis in the macrophage nuclei in heterokaryons. After the discovery of telomerase we proposed that it could be responsible for this reactivation. We had previously developed efficient strategies for inhibiting telomerase (Yegorov et al., 1996; Yegorov et al., 1997a,b,c; Yegorov et al., 1999), and in this study we explore the effects of telomerase inhibition in heterokaryons with macrophages. We also introduce the gene of the telomerase catalytic subunit (hTERT) into human diploid fibroblasts and check their ability to induce reactivation of DNA synthesis in the macrophage nuclei in heterokaryons.
MATERIALS AND METHODS Cells All cells were maintained in the accordance with ‘UKCCCR Guidelines on the use of Cell Lines in Cancer Research’. 3T3 Swiss cells were obtained from ICRF (London) and grown in DMEM (Sigma) supplemented with 10% fetal calf serum (FCS) (HyClone, U.S.A.) and 40 U/ml of gentamycin. The human skin diploid fibroblasts (HDF) (strain 1608) were obtained from the collection of the Institute of Medical Genetics (Russian Academy of Medical Sciences, Moscow). Cells were grown in DMEM with 10% fetal calf serum (HyClone), 5% human umbilical cord serum (PanEco, Moscow), and 40 U/ml gentamycin. The cultures were grown at 37C with 5% CO2 in the atmosphere. The cells were replated using the trypsin-EDTA treatment as soon as they achieved the monolayer status. HDF, passed 20–40 population doublings, were used in the work. Resident (unstimulated) macrophages were obtained from peritoneal cavities of CBA mice. The peritoneal cavities of 2-month-old mice were washed with the phosphate-buffered saline from a syringe. The resultant cell suspension was put in penicillin vials containing cover slips (2106 cells per vial). After incubation for 1 h at 37C, nonadherent cells (predominantly lymphocytes) were washed out and the complete culture medium was added. After 24 h incubation cover slips were
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treated for 1 h with trypsin solution (0.25%) at 37C. This procedure allowed us to obtain preparations containing pure macrophages (unlike other cells, macrophages are trypsin-resistant). Within the following 24 h macrophages were fused with culture cells. Freedom from contaminants in the cell lines, used in the work, checked continuously by autoradiography.
Transfection of hTERT gene Plasmid p190 containing the complete hTERT gene under control of CMV promoter/enhancer in the pCI-neo vector (Promega) originated from the laboratory of Prof. R. Weinberg (Whitehead Institute of Biomedical Research, U.S.A.) was the courtesy of Prof. P. Donini (University of Rome, Italy). The plasmid contains the neomycin phosphotransferase gene responsible for the G-418 resistance. We electroporated the plasmid in human skin fibroblasts (strain 1608) with an Electro Cell Manipulator (BTX, U.S.A.). A single pulse (4 kV/cm, 100 s) in Ca-Mg-free PBS was used. Then cells were plated at a low density (104 cells per well) in 6-well plates (Costar). The medium used was DMEM supplemented with 10% FCS, 10% human umbilical cord serum, and 40 U/ml gentamycin. G-418 (400 g/ml) was added on the third day. From 10 to 14 days later, single G-418resistant clones were harvested. Then the telomerase activity was determined by a direct method as described previously (Yegorov et al., 1996; Yegorov et al., 1997a).
Cell fusion Fusion in a monolayer was induced by polyethylene glycol (PEG) 1500 (Loba) as described previously (Prudovsky et al., 1985). Culture cells (1–3105) were seeded into vials containing cover slips with macrophages and incubated for 2 h. Immediately before fusion, the cover slips with cells were rinsed with warm DMEM and then consequently transferred into vials containing (1) 50% PEG solution in DMEM (pH 8.5), (2) 25% PEG solution, or (3) DMEM. The cover slips remained in each vial for 1–2 s and the whole procedure was repeated five times for each cover slip. The cover slips were then transferred into the complete culture medium. Media and PEG solutions were at 37C.
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Identification of heterokaryons and detection of DNA synthesis Culture cells were labeled before fusion with 3 H-thymidine (0.5–1 Ci/ml, 2.5 Ci/mmol). The labeling took 4 days for 3T3 Swiss cells and 7 days for human fibroblasts. Immediately after fusion, 14 C-thymidine (0.1 Ci/ml, 50.8 mCi/mmol) was added to the culture medium. 24 h after fusion cells were fixed with 96% ethanol (10 min). Then preparations were treated for 5 min with 5% trichloroacetic acid at 4C, washed with running tap water for 2 h, covered with the type M emulsion (Gosniikhimfotoproekt, Moscow), and developed after exposure for a week. Cells were stained with a specially prepared mixture of methylene and toluidine blue. In autoradiographs, the culture cell nuclei were heavily labeled with 3H-thymidine. About half of the area over the nuclei was covered with silver grains. If these nuclei had incorporated 14 C-thymidine, an additional relatively weak label appeared over the nuclei and near them. The macrophage nuclei with reactivated DNA synthesis contained only 14C-label. Microphotography was performed through an Olympus (Japan).
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(1) is complementary to the template region of telomerase RNA, (2) contains two mismatches (underlined). Preparations with heterokaryons were transfected with 3.5 l SuperFect (Qiagen, U.S.A.) and 1 M oligomer in 165 l of culture media (DMEM with 10% FCS and 40 U/ml of gentamycin) according to the manufacturers’ protocols. After 4 h incubation at 37C, the transfecting mixture was removed, and complete medium with 14 C-thymidine was added.
Chemicals 3 -azido-3 -deoxythymidine (AZT) was from Aldrich (U.S.A.). The synthesis of compound GF2G will be published elsewhere. The synthesis and properties of triphosphonate GP3 were published in (Shipitsyn et al., 1999).
Investigation of DNA synthesis in heterokaryons The 14C-labeled macrophage and culture cell nuclei in heterokaryons were counted. Only heterodikaryons, i.e., heterokaryons with one culture cell nucleus and one macrophage nucleus, were studied. The macrophage reactivation index was calculated as percent of 14C-labeled macrophage nuclei in heterokaryons with 14C-labeled culture cell nuclei. At least five preparations were investigated in each experiment (50–500 heterokaryons). The mean errors were determined with P=0.95. Each experiment was repeated at least three times and the results of all experiments always coincided. Introduction of 2 -O-meRNA oligomers into heterokaryons The heterokaryon preparations on cover slips were transfected with 2 -O-meRNA (Oligos Etc. Inc., U.S.A.) approximately within 3 h after fusion: 5 -CAG-T-T-A-G-G-G-T-TAG-3 (1) 5 -CAG-T-T-A-G-A-A-T-TAG-3 (2) All bases are 2 -O-methyl-RNA, —phosphorothioate linkages.
The idea to utilize dinucleotide tetraphosphonate analogues as polymerase inhibitors was suggested by A. A. Kraevsky. These compounds are ready for use by cellular polymerases, because they do not need to be recognized and be repeatedly phosphorylated by cellular kinases, they exhibit an increased stability (phosphonates) and can easily penetrate into cells (due to shielding of phosphorus charges by nucleoside bases). Compounds GF2G and GP3 are carbocyclic analogues of d4GTP (2 ,3 -dideoxy-2 ,3 didehydroguanosine 5 -triphosphate). They bear a modified triphosphonate residue instead of a triphosphate one, what results in an enhanced resistance to dephosphorylating enzymes. In particular, the half-lives of these analogues in the human blood serum are 3 h (transformation GF2G to GP3) and 7 h (transformation GP3 to monophosphonate) versus 20 min for dGTP (Shirokova et al., 2002). As compared with analogue GP3, compound GF2G is more lipophilic, which may stimulate its better penetration through cell membranes. Compounds GP3 and GF2G displayed the properties of terminating substrates of HIV reverse transcriptase in the cell-free system. The
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Fig. 1. DNA synthesis in heterokaryons of 3T3 Swiss cells with macrophages: a—no reactivation, b—reactivation of DNA synthesis in the macrophage nucleus. Nuclei of 3T3 Swiss cells are labeled with 3H and 14C, nuclei of macrophages are unlabeled (a) or labeled with 14C only (b).
50% inhibitory effect was achieved at the concentrations of 15–25 M (Shipitsyn et al., 1999). Compounds GF2G and GP3 in the concentration of 5 M significantly inhibited the telomerasecatalysed DNA synthesis in a cell-free system (data not shown).
RESULTS Effect of AZT addition on DNA synthesis in heterokaryons To investigate the ability of 3T3 Swiss cells to reactivate DNA synthesis in macrophage nuclei upon fusion and the ability of AZT to inhibit this process we performed first set of experiments with fusion of 3T3 Swiss cells and macrophages. Two examples of obtained heterokaryons are presented on the (Fig. 1). Double isotope technique allows us to exactly distinguish the derivation of each nucleus in heterokaryons. After throughout observation of the preparations we calculated four parameters:
(1) DNA synthesis in free macrophages (%), (2) macrophage reactivation index—percentage of heterokaryons containing DNA synthesizing macrophage nuclei in heterokaryons with DNA synthesizing 3T3 Swiss nuclei, (3) DNA synthesis in 3T3 Swiss cells nuclei in heterokaryons (%), (4) DNA synthesis in free 3T3 Swiss cells in the same preparations (%). Last two parameters we need to evaluate possible inhibition of DNA synthesis by AZT. Macrophage reactivation index is the measure of macrophage nuclei reactivation that is independent on the percentage of DNA synthesis in the nuclei of partner cells. The macrophage reactivation index varied from experiment to experiment from 27 to 83% (Fig. 2c). DNA synthesis in free macrophages in the same preparations did not exceed 0.2%. Thus we can conclude that 3T3 Swiss cells induce reactivation of DNA synthesis in macrophage nuclei in the heterokaryons. As AZT is reverse transcriptase inhibitor and can inhibit telomerase function in cells (Yegorov et al.,
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Fig. 2. The AZT effect on the DNA synthesis in free 3T3 Swiss cells and in heterokaryons between 3T3 Swiss cells and macrophages. a—DNA synthesis in free 3T3 Swiss cells in the preparations containing heterokaryons, b—DNA synthesis in the 3T3 Swiss cell nuclei in heterokaryons, c—the macrophage reactivation index (percentage of 14C-labeled macrophage nuclei in heterokaryons with 14C-labeled 3T3 Swiss nuclei). The results were obtained in eight independent experiments.
1996; Yegorov et al., 1997a,b,c; Yegorov et al., 1999) we used AZT addition to heterokaryons to see possible inhibition of reactivation of DNA synthesis in macrophage nuclei. Indeed, AZT inhibited the reactivation of DNA synthesis in the macrophage nuclei in heterokaryons (Fig. 2c). Inhibition was observed in all experiments in the whole range of AZT concentrations used (from 5 to 30 M). In contrast to the macrophage nuclei, the addition of AZT to the culture medium in concentrations up to 30 M had no significant effect on DNA synthesis both in free 3T3 Swiss cells (Fig. 2a) and in 3T3 Swiss nuclei within heterokaryons (Fig. 2b). It means that observed inhibition of reactivation of macrophage nuclei can not be explained by direct influence of AZT.
Transfection of human skin diploid fibroblasts (strain 1608) with the hTERT gene allowed us to obtain few single clones in the selective medium. The telomerase activity was detected in all clones studied, whereas no telomerase activity could be detected in the original cells. All the clones exhibit different levels of telomerase activity, different morphology and growth rate. We chose one of the clones (clone 7) for experiments with heterokaryons. The clone 7 cells resemble young human diploid fibroblasts. By the present time they passed over 160 population doublings without alteration of cell morphology and growth rate. In the experiments we used clone 7 cells, passed 30–80 PD. Heterokaryons with telomerased HDF
Creation of telomerase-positive human fibroblasts As we knew that human diploid fibroblasts have no ability to reactivate DNA synthesis in macrophage nuclei in heterokaryons (Zelenin and Prudovsky, 1989) we decided to introduce hTERT gene into them and to check their ability in experiments with heterokaryons.
To check directly whether telomerase participates in the process of the DNA synthesis reactivation in the macrophage nuclei in heterokaryons we investigated heterokaryons of macrophages with original HDF (strain 1608) and with telomerasetransfected HDF (clone 7). There was no reactivation of DNA synthesis in macrophage
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Table 1. DNA synthesis in heterokaryons of macrophages with telomerased (clone 7) and nontelomerased human fibroblasts* Macrophage partner cells Telomerased fibroblasts Non-telomerased fibroblasts
Percentage of labeled free fibroblasts
Percentage of labeled fibroblast nuclei in heterokaryons
Index of macrophage reactivation in heterokaryons (%)
76.216.9 54.318.7
72.924.6 80.96.7
70.812.5 1.23.7
*The results were obtained in four independent experiments.
Table 2. Effect of antisense oligonucleotides to the template region of telomerase RNA on DNA synthesis in heterokaryons of macrophages and immortal human fibroblasts (clone 7)* Introduced oligonucleotides
Antisense Mismatch
Percentage of labeled free fibroblasts
Percentage of labeled fibroblast nuclei in heterokaryons
Index of macrophage reactivation in heterokaryons (%)
782.8 752.9
71.15.1 70.16.2
546.7 697.4
*The results were obtained in three independent experiments.
nuclei in heterokaryons with HDF (with only one exception) (Table 1). Index of reactivation of macrophage nuclei in heterokaryons with the telomerase-positive HDF reached 70% (Table 1). Effect of the telomerase RNA antisense oligonucleotides on DNA synthesis in heterokaryons In this series of experiments heterokaryons were treated with highly specific telomerase inhibitors— 2 -O-methyl RNA oligonucleotides complementary to the template region of human telomerase RNA. These oligonucleotides specifically inhibited reactivation of DNA synthesis in the macrophage nuclei in heterokaryons with clone 7 cells only and did not influence DNA synthesis in free clone 7 cells or in clone 7 nuclei within heterokaryons (Table 2). The mismatch oligonucleotides had no effect (compare Table 1 (first row) and Table 2 (second row)). Effect of modified guanosine analogues on DNA synthesis in heterokaryons We evaluated the ability of dinucleotide analogue GF2G and the corresponding nucleotide triphosphonate GP3 to block the telomerase function at the cellular level, namely, in heterokaryons. Like AZT, these compounds were shown to be effective
inhibitors of DNA synthesis catalysed by the HIV reverse transcriptase in cell-free systems, but in contrast to AZT, they were essentially inactive (in concentrations 3–10 M) in the HIV-infected cell cultures (Shipitsyn et al., 1999). Our experiments showed that both GF2G and GP3 in low concentrations (1–5 M) had no significant effect on DNA synthesis in heterokaryons (data not shown), but in high concentrations (100 M) inhibited the reactivation of DNA synthesis in the macrophage nuclei in heterokaryons (Table 3) and did not affect DNA synthesis both in 3T3 Swiss cells and 3T3 Swiss cell nuclei in heterokaryons. The inhibition efficacy was practically the same for GF2G and GP3.
DISCUSSION Heterokaryons are a good model for investigation of DNA synthesis regulation because they contain several internal controls of the inhibitor action. We can control the inhibitory effect on free cells (both partners) in the same preparations; we can control the influence of all experimental procedures by comparing DNA synthesis in heterokaryons and in free cells. It follows from our results that the reverse transcriptase inhibitor AZT influences only DNA synthesis in the macrophage nuclei within
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Table 3. DNA synthesis in heterokaryons of macrophages with immortal 3T3 Swiss mouse fibroblasts in the presence of reverse transcriptase inhibitors* Inhibitors
GF2G (100 M) GP3 (100 M) Without inhibitors
Percentage of labeled free fibroblasts
Percentage of labeled fibroblast nuclei in heterokaryons
Index of macrophage reactivation in heterokaryons (%)
834.3 824.4 844.1
575.0 614.6 644.5
296.1 255.3 514.6
*The results were obtained in four independent experiments.
heterokaryons and does not interfere with DNA synthesis in the nuclei of proliferating cells. The ability of AZT to inihibit telomerase is well known (Yegorov et al., 1996; Yegorov et al., 1997a,b,c; Yegorov et al., 1999; Strahl and Blackburn, 1994; Strahl and Blackburn, 1996; Melana et al., 1998; Gomez et al., 1995; Gomez et al., 1998; Murakami et al., 1999), suggesting that AZT may inhibit reactivation of the macrophage nuclei via blocking the telomerase function. These results stimulated our studies of the influence of the telomerase introduction into heterokaryons via gene transfection. To introduce telomerase activity we transfected HDF with hTERT gene. It is well known that such transfection of HDF leads to emergence of telomerase activity, stabilization of telomeres, and dramatic increase in proliferative potential (practically immortalization) (Bodnar et al., 1998; Vaziri and Benchimol, 1998; Nakayama et al., 1998). In our experiments, HDF passed over 160 population doublings after transfection with hTERT. Control experiments did not reveal reactivation of DNA synthesis in the macrophage nuclei upon fusion with HDF. The experiments on heterokaryons with the hTERT-transfected HDF showed that the macrophage reactivation index exceeded 70%. This is similar to the results obtained after fusion with 3T3 Swiss cells (Fig. 2c). Thus, a different approach (telomerase addition) once more demonstrated the necessity of telomerase functioning for the DNA synthesis reactivation in the macrophage nuclei in heterokaryons. To support the hypothesis that telomerase inhibition blocks reactivation of macrophage function, we performed another set of experiments with the most specific anti-telomerase compounds—antisense oligonucleotides. Previous work (Pitts and Corey, 1998) had shown the 2 -O-methyl-RNA oligomers to be potent and sequence-selective inhibitors of human telomerase. We observed
inhibition of DNA synthesis in the macrophage nuclei in heterokaryons without any effect on DNA synthesis in clone 7 cells, both free and within heterokaryons (Table 2). In these experiments we used the SuperFect transfection reagent. This reagent gives the possibility to work in the presence of serum. It decreases cytotoxicity and influences DNA synthesis. These experiments once more confirmed our conclusion that the telomerase blocking inhibits the process of reactivation of DNA synthesis in the macrophage nuclei in heterokaryons. The data obtained in this work suggest that telomerase is involved in the DNA synthesis reactivation in the macrophage nuclei in heterokaryons with proliferating cells. (i) Only proliferating cells which possess telomerase activity induce this reactivation, (ii) telomerase blockade abolishes the reactivation. This conclusion is consistent with the previous data that only immortal cells have such ability. Many types of mortal cells (telomerase-negative) in our hands were not able to reactivate DNA synthesis in the macrophage nuclei in heterokaryons: mouse embryo fibroblasts, rat embryo fibroblasts, HDF, rat chondrocytes, and proliferating precursors of macrophages (Prudovsky et al., 1985; Prudovsky et al., 1989a,b; Zelenin and Prudovsky, 1989). At the same time, a number of immortal cells (telomerase-positive) have such an ability: 3T3 Swiss, NIH 3T3, SV3T3, HeLa, C3H10T1/2, and the p53-transformed rat chondrocytes (Prudovsky et al., 1985; Prudovsky et al., 1989a,b; Zelenin and Prudovsky, 1989). As telomerase works mainly on telomeres, it is reasonable to conclude that telomeres of mouse peritoneal macrophages in heterokaryons are altered (have an altered conformation). Macrophages in heterokaryons have one exclusive property. In contrast to many types of nondividing cells (Prudovsky et al., 1993; Rabinovich and Norwood, 1980; Stein and Yanishevsky, 1981; Ringertz et al., 1985; Laeng et al., 1985; Prudovsky
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et al., 1990), they have no inhibitory effect on DNA synthesis in heterokaryons. The absence of such an effect of macrophages on DNA synthesis in the benign cell nuclei in heterokaryons (this work (Prudovsky et al., 1985; Prudovsky et al., 1989a,b)) means that they do not contain any diffusible intracellular negative regulator of cell proliferation. The absence of inhibitors of proliferation and, simultaneously, maintenance of nondividing state in macrophages led us in 1989 to the supposition that ‘some changes in the macrophage genome prevent replication and that the elimination, or neutralization of these changes by products of immortalizing oncogenes takes place in heterokaryons’ (Zelenin and Prudovsky, 1989; Prudovsky et al., 1993). The results described in this work confirm the hypothesis. As far as we know, heterokaryons with macrophages are the first system that is suitable for investigation of telomerase inhibitors at the cellular level. This technique can be used with low amounts of inhibitors (sufficient for few milliliters of culture medium) and the answer can be obtained during few days (especially with a luminescent technique). An alternative approach—growing cell cultures in the presence of telomerase inhibitors and the telomere measurement—is much more time- and inhibitor-consuming. When we became convinced of the telomerasedependent macrophage reactivation, we decided to check some inhibitors of the DNA synthesis catalysed by the HIV reverse transcriptase on heterokaryons. We chose the guanine-derived compounds, since guanosine residues comprise 50% of the telomeric sequence and, in theory, guanine analogues may be the most effective inhibitors. Low concentrations (1–5 M) of these compounds had no significant effect on DNA synthesis in heterokaryons, which agrees with the results obtained in the HIV-infected cell cultures (Shipitsyn et al., 1999). Both compounds in high concentrations (100 M) had a similar inhibitory effect on the macrophage nuclei reactivation in heterokaryons. GF2G is suspected to be not sufficiently stable in the cell environment, and its effect is mediated by GP3 that emerged as a result of GF2G hydrolysis. Thus, the only investigations of new compounds on heterokaryons showed that both compounds are active at the cellular level. There are many questions concerning changes in the macrophage telomeres. To answer them, some additional experiments directed to investigation of their structure should be done. We suppose that the telomere damage may not require significant telomere shortening. In 1997 we proposed the loop
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structure for telomeres (Yegorov et al., 1997a) which can explain the appearance of ‘bad’ (damaged) telomeres with long telomeric sequences by alteration of DNA-telomere protein relationships. Relatively rapid damage to telomeres may make cells more sensitive to existing chemotherapeutic agents and make clinical use of anti-telomerase agents more practical.
ACKNOWLEDGEMENTS The authors thank their many colleagues, particularly M. Eldarov, S. S. Akimov, A. K. Akhmalisheva who contributed to the experiments and/or discussions of this work. This work was supported by the Russian Foundation for Basic Research (projects 96-1597640, 96-04-49086, 99-04-48073), the Russian State Program for Support of Scientific Schools (project 00-15-97833), the Russian Human Genome Program, the ISCC grant (project 1244), and the United States National Institutes of Health (CA-85363). REFERENCES B AG, O M, F M, H SE, C C-P, M GB, H CB, S JW, L S, W WE, 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349–352. G DE, K A, O OA, 1995. Preferential incorporation of 3 -azido-2 ,3 -dideoxythymidine (AZT) in telomeric sequences of CHO cells. Inter J Oncology 7: 1057–1060. G DE, T AM, O OA, 1998. Irreversible telomere shortening by 3 -azido-2 ,3 -dideoxythymidine (AZT) treatment. Biochem Biophys Res Commun 246: 107–110. L H, H DT, S R, 1985. Proliferative quiescence of normal mast cells resembles that of coldsensitive mutant mastocytoma cells. Dominant expression of the quiescent state in heterokaryons. Exp Cell Res 158: 170–176. M SM, H JF, P BJ, 1998. Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by 3 -azido-3 deoxythymidine. Clin Cancer Res 4: 693–696. M J, N N, S K, O K, 1999. Inhibition of telomerase activity and cell proliferation by a reverse transcriptase inhibitor in gynaecological cancer cell lines. Eur J Cancer 35: 1027–1034. N J, T H, T E, S M, I K, N H, N T, I T, I F, 1998. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nature Genet 8: 65–68. P AE, C DR, 1998. Inhibition of human telomerase by 2 -O-methyl-RNA. Proc Natl Acad Sci USA 95: 11549–11554.
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S GH, Y RM, 1981. Quiescent human diploid cells can inhibit entry into S-phase in replicative nuclei in heterokaryons. Proc Natl Acad Sci USA 78: 3025–3029. S C, B EH, 1994. The effect of nucleoside analogs of telomerase and telomeres in Tetrahymena. Nucl Acids Res 22: 93–900. S C, B EH, 1996. Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortal human cell lines. Mol Cell Biol 16: 53–56. V H, B S, 1998. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol 8: 279–282. Y YE, C DN, A SS, B NL, K AA, Z AV, 1996. Reverse transcriptase inhibitors suppress telomerase function and induce senescence-like processes in cultured mouse fibroblasts. FEBS Letters 389: 115–118. Y YE, C DN, A SS, A AK, S YB, S DB, S IV, Y IN, Z AV, 1997a. Blockade of Telomerase Function by Nucleoside Analogs. Biochemistry (Moscow) 62: 1296–1305 (http://puma.protein.bio.msu.su/biokhimiya/). Y YE, A AK, S YB, S DB, C DN, Z AV, K AA, 1997b. Azidothymidine, blocking telomerase functioning, shortens telomeric repeats in transformed human cells. Russian J Genet 33: 1238–1240. Y YE, C DN, A SS, B’ NL, K AA, Z AV, 1997c. Effect of reverse transcriptase inhibitors on telomerase function in immortal mouse fibroblasts. Mol Biol (Moscow) 31: 108–113. Y YE, A SS, A AK, S IV, S YB, K AA, Z AV, 1999. Blockade of telomerase function in various cells. Anti-Cancer Drug Design 14: 305–316. Z AV, P IA, 1989. Regulation of DNA synthesis investigated in heterokaryons of dividing and nondividing cells. Inter Rev Cytology 117: 179–214.
TELOMERASE-DEPENDENT REACTIVATION OF DNA SYNTHESIS IN MACROPHAGES IMPLIES ALTERATION OF TELOMERES Y. E. YEGOROV1*, E. V. KAZIMIRCHUK1, S. M. TEREKHOV2, D. N. KARACHENTSEV3, E. A. SHIROKOVA1, A. L. KHANDAZHINSKAYA1, J. A. MESHCHERYAKOVA4, D. R. COREY5 and A. V. ZELENIN1 1
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov str., Moscow, 119991, Russia; 2Institute of Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia; 3 Moscow Physico-Technical Institute (PhysTech), Dolgoprudnyi, Moscow, Russia; 4Center of Bioengineering, Russian Academy of Sciences, Moscow, Russia; 5Department of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9041, U.S.A. Received 21 March 2002; accepted 28 August 2002
In previous work we demonstrated that various types of cultured cells with a limited life span could not reactivate DNA synthesis in the nuclei of mouse peritoneal macrophages in heterokaryons. We now investigate the role of telomerase in the process of the macrophage nucleus reactivation in heterokaryons with immortal telomerase-positive 3T3 Swiss mouse fibroblasts and human fibroblasts with introduced hTERT gene. We report that introduction of the hTERT gene into human diploid fibroblasts results in emergence of telomerase activity in these cells and the ability to induce the reactivation of DNA synthesis in the macrophage nuclei in heterokaryons. Inhibition of telomerase activity in heterokaryons by reverse transcriptase inhibitors (azidothymidine and guanosine polyphosphonate analogues) and by a 2 -O-methylRNA oligonucleotide anti-sense to the template region of telomerase RNA, block reactivation of DNA synthesis in macrophage nuclei without inhibiting DNA synthesis in the nuclei of fibroblasts. Our results suggest alterations (shortening or damage) in the macrophage telomere structure. As far as we know, heterokaryons with macrophages are the first cellular model for rapid investigation of the effects of telomerase inhibitors. 2002 Elsevier Science Ltd. All rights reserved.
K: telomerase; telomeres; telomerase inhibition; macrophages; heterokaryons; human fibroblasts; proliferation; differentiation.
INTRODUCTION The mechanisms that block cell proliferation at the stage of terminal differentiation and maintain differentiated cells in the non-dividing state are poorly studied. An important approach to the problem of cessation of proliferation is based on hybridization of somatic cells. Terminally differentiated nondividing cells can be fused with proliferating ones and their interaction in hybrids (heterokaryons) can be investigated. Heterokaryons are artificially constructed cells that contain nuclei of a dissimilar origin. *To whom correspondence should be addressed: Fax: (095) 135-1405; E-mail: [email protected] 1065–6995/02/$-see front matter
In our previous work, we investigated the regulation of DNA synthesis in heterokaryons formed by macrophages from normal mouse peritoneum and various proliferating cells. Altogether, we used 15 different cell strains and lines (Prudovsky et al., 1985; Prudovsky et al., 1989a,b; Zelenin and Prudovsky, 1989; Prudovsky et al., 1993). No reactivation of DNA synthesis in the macrophage nuclei was detected in heterokaryons with cells of limited proliferative life-span, although nuclei of actively proliferating partners synthesized DNA on their own. We did, however observe reactivation of DNA synthesis in the macrophage nuclei in heterokaryons with immortal cells: in up to 90% of heterokaryons that contained 2002 Elsevier Science Ltd. All rights reserved.
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the DNA-synthesizing 3T3 nucleus, the macrophage nuclei were also labeled within 24 h after fusion. In our early work (Prudovsky et al., 1989a,b) we looked for the relationship between the presence of active oncogenes and the ability of proliferating cells to induce reactivation of DNA synthesis in the macrophage nuclei in heterokaryons. After the discovery of telomerase we proposed that it could be responsible for this reactivation. We had previously developed efficient strategies for inhibiting telomerase (Yegorov et al., 1996; Yegorov et al., 1997a,b,c; Yegorov et al., 1999), and in this study we explore the effects of telomerase inhibition in heterokaryons with macrophages. We also introduce the gene of the telomerase catalytic subunit (hTERT) into human diploid fibroblasts and check their ability to induce reactivation of DNA synthesis in the macrophage nuclei in heterokaryons.
MATERIALS AND METHODS Cells All cells were maintained in the accordance with ‘UKCCCR Guidelines on the use of Cell Lines in Cancer Research’. 3T3 Swiss cells were obtained from ICRF (London) and grown in DMEM (Sigma) supplemented with 10% fetal calf serum (FCS) (HyClone, U.S.A.) and 40 U/ml of gentamycin. The human skin diploid fibroblasts (HDF) (strain 1608) were obtained from the collection of the Institute of Medical Genetics (Russian Academy of Medical Sciences, Moscow). Cells were grown in DMEM with 10% fetal calf serum (HyClone), 5% human umbilical cord serum (PanEco, Moscow), and 40 U/ml gentamycin. The cultures were grown at 37C with 5% CO2 in the atmosphere. The cells were replated using the trypsin-EDTA treatment as soon as they achieved the monolayer status. HDF, passed 20–40 population doublings, were used in the work. Resident (unstimulated) macrophages were obtained from peritoneal cavities of CBA mice. The peritoneal cavities of 2-month-old mice were washed with the phosphate-buffered saline from a syringe. The resultant cell suspension was put in penicillin vials containing cover slips (2106 cells per vial). After incubation for 1 h at 37C, nonadherent cells (predominantly lymphocytes) were washed out and the complete culture medium was added. After 24 h incubation cover slips were
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treated for 1 h with trypsin solution (0.25%) at 37C. This procedure allowed us to obtain preparations containing pure macrophages (unlike other cells, macrophages are trypsin-resistant). Within the following 24 h macrophages were fused with culture cells. Freedom from contaminants in the cell lines, used in the work, checked continuously by autoradiography.
Transfection of hTERT gene Plasmid p190 containing the complete hTERT gene under control of CMV promoter/enhancer in the pCI-neo vector (Promega) originated from the laboratory of Prof. R. Weinberg (Whitehead Institute of Biomedical Research, U.S.A.) was the courtesy of Prof. P. Donini (University of Rome, Italy). The plasmid contains the neomycin phosphotransferase gene responsible for the G-418 resistance. We electroporated the plasmid in human skin fibroblasts (strain 1608) with an Electro Cell Manipulator (BTX, U.S.A.). A single pulse (4 kV/cm, 100 s) in Ca-Mg-free PBS was used. Then cells were plated at a low density (104 cells per well) in 6-well plates (Costar). The medium used was DMEM supplemented with 10% FCS, 10% human umbilical cord serum, and 40 U/ml gentamycin. G-418 (400 g/ml) was added on the third day. From 10 to 14 days later, single G-418resistant clones were harvested. Then the telomerase activity was determined by a direct method as described previously (Yegorov et al., 1996; Yegorov et al., 1997a).
Cell fusion Fusion in a monolayer was induced by polyethylene glycol (PEG) 1500 (Loba) as described previously (Prudovsky et al., 1985). Culture cells (1–3105) were seeded into vials containing cover slips with macrophages and incubated for 2 h. Immediately before fusion, the cover slips with cells were rinsed with warm DMEM and then consequently transferred into vials containing (1) 50% PEG solution in DMEM (pH 8.5), (2) 25% PEG solution, or (3) DMEM. The cover slips remained in each vial for 1–2 s and the whole procedure was repeated five times for each cover slip. The cover slips were then transferred into the complete culture medium. Media and PEG solutions were at 37C.
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Identification of heterokaryons and detection of DNA synthesis Culture cells were labeled before fusion with 3 H-thymidine (0.5–1 Ci/ml, 2.5 Ci/mmol). The labeling took 4 days for 3T3 Swiss cells and 7 days for human fibroblasts. Immediately after fusion, 14 C-thymidine (0.1 Ci/ml, 50.8 mCi/mmol) was added to the culture medium. 24 h after fusion cells were fixed with 96% ethanol (10 min). Then preparations were treated for 5 min with 5% trichloroacetic acid at 4C, washed with running tap water for 2 h, covered with the type M emulsion (Gosniikhimfotoproekt, Moscow), and developed after exposure for a week. Cells were stained with a specially prepared mixture of methylene and toluidine blue. In autoradiographs, the culture cell nuclei were heavily labeled with 3H-thymidine. About half of the area over the nuclei was covered with silver grains. If these nuclei had incorporated 14 C-thymidine, an additional relatively weak label appeared over the nuclei and near them. The macrophage nuclei with reactivated DNA synthesis contained only 14C-label. Microphotography was performed through an Olympus (Japan).
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(1) is complementary to the template region of telomerase RNA, (2) contains two mismatches (underlined). Preparations with heterokaryons were transfected with 3.5 l SuperFect (Qiagen, U.S.A.) and 1 M oligomer in 165 l of culture media (DMEM with 10% FCS and 40 U/ml of gentamycin) according to the manufacturers’ protocols. After 4 h incubation at 37C, the transfecting mixture was removed, and complete medium with 14 C-thymidine was added.
Chemicals 3 -azido-3 -deoxythymidine (AZT) was from Aldrich (U.S.A.). The synthesis of compound GF2G will be published elsewhere. The synthesis and properties of triphosphonate GP3 were published in (Shipitsyn et al., 1999).
Investigation of DNA synthesis in heterokaryons The 14C-labeled macrophage and culture cell nuclei in heterokaryons were counted. Only heterodikaryons, i.e., heterokaryons with one culture cell nucleus and one macrophage nucleus, were studied. The macrophage reactivation index was calculated as percent of 14C-labeled macrophage nuclei in heterokaryons with 14C-labeled culture cell nuclei. At least five preparations were investigated in each experiment (50–500 heterokaryons). The mean errors were determined with P=0.95. Each experiment was repeated at least three times and the results of all experiments always coincided. Introduction of 2 -O-meRNA oligomers into heterokaryons The heterokaryon preparations on cover slips were transfected with 2 -O-meRNA (Oligos Etc. Inc., U.S.A.) approximately within 3 h after fusion: 5 -CAG-T-T-A-G-G-G-T-TAG-3 (1) 5 -CAG-T-T-A-G-A-A-T-TAG-3 (2) All bases are 2 -O-methyl-RNA, —phosphorothioate linkages.
The idea to utilize dinucleotide tetraphosphonate analogues as polymerase inhibitors was suggested by A. A. Kraevsky. These compounds are ready for use by cellular polymerases, because they do not need to be recognized and be repeatedly phosphorylated by cellular kinases, they exhibit an increased stability (phosphonates) and can easily penetrate into cells (due to shielding of phosphorus charges by nucleoside bases). Compounds GF2G and GP3 are carbocyclic analogues of d4GTP (2 ,3 -dideoxy-2 ,3 didehydroguanosine 5 -triphosphate). They bear a modified triphosphonate residue instead of a triphosphate one, what results in an enhanced resistance to dephosphorylating enzymes. In particular, the half-lives of these analogues in the human blood serum are 3 h (transformation GF2G to GP3) and 7 h (transformation GP3 to monophosphonate) versus 20 min for dGTP (Shirokova et al., 2002). As compared with analogue GP3, compound GF2G is more lipophilic, which may stimulate its better penetration through cell membranes. Compounds GP3 and GF2G displayed the properties of terminating substrates of HIV reverse transcriptase in the cell-free system. The
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Fig. 1. DNA synthesis in heterokaryons of 3T3 Swiss cells with macrophages: a—no reactivation, b—reactivation of DNA synthesis in the macrophage nucleus. Nuclei of 3T3 Swiss cells are labeled with 3H and 14C, nuclei of macrophages are unlabeled (a) or labeled with 14C only (b).
50% inhibitory effect was achieved at the concentrations of 15–25 M (Shipitsyn et al., 1999). Compounds GF2G and GP3 in the concentration of 5 M significantly inhibited the telomerasecatalysed DNA synthesis in a cell-free system (data not shown).
RESULTS Effect of AZT addition on DNA synthesis in heterokaryons To investigate the ability of 3T3 Swiss cells to reactivate DNA synthesis in macrophage nuclei upon fusion and the ability of AZT to inhibit this process we performed first set of experiments with fusion of 3T3 Swiss cells and macrophages. Two examples of obtained heterokaryons are presented on the (Fig. 1). Double isotope technique allows us to exactly distinguish the derivation of each nucleus in heterokaryons. After throughout observation of the preparations we calculated four parameters:
(1) DNA synthesis in free macrophages (%), (2) macrophage reactivation index—percentage of heterokaryons containing DNA synthesizing macrophage nuclei in heterokaryons with DNA synthesizing 3T3 Swiss nuclei, (3) DNA synthesis in 3T3 Swiss cells nuclei in heterokaryons (%), (4) DNA synthesis in free 3T3 Swiss cells in the same preparations (%). Last two parameters we need to evaluate possible inhibition of DNA synthesis by AZT. Macrophage reactivation index is the measure of macrophage nuclei reactivation that is independent on the percentage of DNA synthesis in the nuclei of partner cells. The macrophage reactivation index varied from experiment to experiment from 27 to 83% (Fig. 2c). DNA synthesis in free macrophages in the same preparations did not exceed 0.2%. Thus we can conclude that 3T3 Swiss cells induce reactivation of DNA synthesis in macrophage nuclei in the heterokaryons. As AZT is reverse transcriptase inhibitor and can inhibit telomerase function in cells (Yegorov et al.,
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Fig. 2. The AZT effect on the DNA synthesis in free 3T3 Swiss cells and in heterokaryons between 3T3 Swiss cells and macrophages. a—DNA synthesis in free 3T3 Swiss cells in the preparations containing heterokaryons, b—DNA synthesis in the 3T3 Swiss cell nuclei in heterokaryons, c—the macrophage reactivation index (percentage of 14C-labeled macrophage nuclei in heterokaryons with 14C-labeled 3T3 Swiss nuclei). The results were obtained in eight independent experiments.
1996; Yegorov et al., 1997a,b,c; Yegorov et al., 1999) we used AZT addition to heterokaryons to see possible inhibition of reactivation of DNA synthesis in macrophage nuclei. Indeed, AZT inhibited the reactivation of DNA synthesis in the macrophage nuclei in heterokaryons (Fig. 2c). Inhibition was observed in all experiments in the whole range of AZT concentrations used (from 5 to 30 M). In contrast to the macrophage nuclei, the addition of AZT to the culture medium in concentrations up to 30 M had no significant effect on DNA synthesis both in free 3T3 Swiss cells (Fig. 2a) and in 3T3 Swiss nuclei within heterokaryons (Fig. 2b). It means that observed inhibition of reactivation of macrophage nuclei can not be explained by direct influence of AZT.
Transfection of human skin diploid fibroblasts (strain 1608) with the hTERT gene allowed us to obtain few single clones in the selective medium. The telomerase activity was detected in all clones studied, whereas no telomerase activity could be detected in the original cells. All the clones exhibit different levels of telomerase activity, different morphology and growth rate. We chose one of the clones (clone 7) for experiments with heterokaryons. The clone 7 cells resemble young human diploid fibroblasts. By the present time they passed over 160 population doublings without alteration of cell morphology and growth rate. In the experiments we used clone 7 cells, passed 30–80 PD. Heterokaryons with telomerased HDF
Creation of telomerase-positive human fibroblasts As we knew that human diploid fibroblasts have no ability to reactivate DNA synthesis in macrophage nuclei in heterokaryons (Zelenin and Prudovsky, 1989) we decided to introduce hTERT gene into them and to check their ability in experiments with heterokaryons.
To check directly whether telomerase participates in the process of the DNA synthesis reactivation in the macrophage nuclei in heterokaryons we investigated heterokaryons of macrophages with original HDF (strain 1608) and with telomerasetransfected HDF (clone 7). There was no reactivation of DNA synthesis in macrophage
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Table 1. DNA synthesis in heterokaryons of macrophages with telomerased (clone 7) and nontelomerased human fibroblasts* Macrophage partner cells Telomerased fibroblasts Non-telomerased fibroblasts
Percentage of labeled free fibroblasts
Percentage of labeled fibroblast nuclei in heterokaryons
Index of macrophage reactivation in heterokaryons (%)
76.216.9 54.318.7
72.924.6 80.96.7
70.812.5 1.23.7
*The results were obtained in four independent experiments.
Table 2. Effect of antisense oligonucleotides to the template region of telomerase RNA on DNA synthesis in heterokaryons of macrophages and immortal human fibroblasts (clone 7)* Introduced oligonucleotides
Antisense Mismatch
Percentage of labeled free fibroblasts
Percentage of labeled fibroblast nuclei in heterokaryons
Index of macrophage reactivation in heterokaryons (%)
782.8 752.9
71.15.1 70.16.2
546.7 697.4
*The results were obtained in three independent experiments.
nuclei in heterokaryons with HDF (with only one exception) (Table 1). Index of reactivation of macrophage nuclei in heterokaryons with the telomerase-positive HDF reached 70% (Table 1). Effect of the telomerase RNA antisense oligonucleotides on DNA synthesis in heterokaryons In this series of experiments heterokaryons were treated with highly specific telomerase inhibitors— 2 -O-methyl RNA oligonucleotides complementary to the template region of human telomerase RNA. These oligonucleotides specifically inhibited reactivation of DNA synthesis in the macrophage nuclei in heterokaryons with clone 7 cells only and did not influence DNA synthesis in free clone 7 cells or in clone 7 nuclei within heterokaryons (Table 2). The mismatch oligonucleotides had no effect (compare Table 1 (first row) and Table 2 (second row)). Effect of modified guanosine analogues on DNA synthesis in heterokaryons We evaluated the ability of dinucleotide analogue GF2G and the corresponding nucleotide triphosphonate GP3 to block the telomerase function at the cellular level, namely, in heterokaryons. Like AZT, these compounds were shown to be effective
inhibitors of DNA synthesis catalysed by the HIV reverse transcriptase in cell-free systems, but in contrast to AZT, they were essentially inactive (in concentrations 3–10 M) in the HIV-infected cell cultures (Shipitsyn et al., 1999). Our experiments showed that both GF2G and GP3 in low concentrations (1–5 M) had no significant effect on DNA synthesis in heterokaryons (data not shown), but in high concentrations (100 M) inhibited the reactivation of DNA synthesis in the macrophage nuclei in heterokaryons (Table 3) and did not affect DNA synthesis both in 3T3 Swiss cells and 3T3 Swiss cell nuclei in heterokaryons. The inhibition efficacy was practically the same for GF2G and GP3.
DISCUSSION Heterokaryons are a good model for investigation of DNA synthesis regulation because they contain several internal controls of the inhibitor action. We can control the inhibitory effect on free cells (both partners) in the same preparations; we can control the influence of all experimental procedures by comparing DNA synthesis in heterokaryons and in free cells. It follows from our results that the reverse transcriptase inhibitor AZT influences only DNA synthesis in the macrophage nuclei within
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Table 3. DNA synthesis in heterokaryons of macrophages with immortal 3T3 Swiss mouse fibroblasts in the presence of reverse transcriptase inhibitors* Inhibitors
GF2G (100 M) GP3 (100 M) Without inhibitors
Percentage of labeled free fibroblasts
Percentage of labeled fibroblast nuclei in heterokaryons
Index of macrophage reactivation in heterokaryons (%)
834.3 824.4 844.1
575.0 614.6 644.5
296.1 255.3 514.6
*The results were obtained in four independent experiments.
heterokaryons and does not interfere with DNA synthesis in the nuclei of proliferating cells. The ability of AZT to inihibit telomerase is well known (Yegorov et al., 1996; Yegorov et al., 1997a,b,c; Yegorov et al., 1999; Strahl and Blackburn, 1994; Strahl and Blackburn, 1996; Melana et al., 1998; Gomez et al., 1995; Gomez et al., 1998; Murakami et al., 1999), suggesting that AZT may inhibit reactivation of the macrophage nuclei via blocking the telomerase function. These results stimulated our studies of the influence of the telomerase introduction into heterokaryons via gene transfection. To introduce telomerase activity we transfected HDF with hTERT gene. It is well known that such transfection of HDF leads to emergence of telomerase activity, stabilization of telomeres, and dramatic increase in proliferative potential (practically immortalization) (Bodnar et al., 1998; Vaziri and Benchimol, 1998; Nakayama et al., 1998). In our experiments, HDF passed over 160 population doublings after transfection with hTERT. Control experiments did not reveal reactivation of DNA synthesis in the macrophage nuclei upon fusion with HDF. The experiments on heterokaryons with the hTERT-transfected HDF showed that the macrophage reactivation index exceeded 70%. This is similar to the results obtained after fusion with 3T3 Swiss cells (Fig. 2c). Thus, a different approach (telomerase addition) once more demonstrated the necessity of telomerase functioning for the DNA synthesis reactivation in the macrophage nuclei in heterokaryons. To support the hypothesis that telomerase inhibition blocks reactivation of macrophage function, we performed another set of experiments with the most specific anti-telomerase compounds—antisense oligonucleotides. Previous work (Pitts and Corey, 1998) had shown the 2 -O-methyl-RNA oligomers to be potent and sequence-selective inhibitors of human telomerase. We observed
inhibition of DNA synthesis in the macrophage nuclei in heterokaryons without any effect on DNA synthesis in clone 7 cells, both free and within heterokaryons (Table 2). In these experiments we used the SuperFect transfection reagent. This reagent gives the possibility to work in the presence of serum. It decreases cytotoxicity and influences DNA synthesis. These experiments once more confirmed our conclusion that the telomerase blocking inhibits the process of reactivation of DNA synthesis in the macrophage nuclei in heterokaryons. The data obtained in this work suggest that telomerase is involved in the DNA synthesis reactivation in the macrophage nuclei in heterokaryons with proliferating cells. (i) Only proliferating cells which possess telomerase activity induce this reactivation, (ii) telomerase blockade abolishes the reactivation. This conclusion is consistent with the previous data that only immortal cells have such ability. Many types of mortal cells (telomerase-negative) in our hands were not able to reactivate DNA synthesis in the macrophage nuclei in heterokaryons: mouse embryo fibroblasts, rat embryo fibroblasts, HDF, rat chondrocytes, and proliferating precursors of macrophages (Prudovsky et al., 1985; Prudovsky et al., 1989a,b; Zelenin and Prudovsky, 1989). At the same time, a number of immortal cells (telomerase-positive) have such an ability: 3T3 Swiss, NIH 3T3, SV3T3, HeLa, C3H10T1/2, and the p53-transformed rat chondrocytes (Prudovsky et al., 1985; Prudovsky et al., 1989a,b; Zelenin and Prudovsky, 1989). As telomerase works mainly on telomeres, it is reasonable to conclude that telomeres of mouse peritoneal macrophages in heterokaryons are altered (have an altered conformation). Macrophages in heterokaryons have one exclusive property. In contrast to many types of nondividing cells (Prudovsky et al., 1993; Rabinovich and Norwood, 1980; Stein and Yanishevsky, 1981; Ringertz et al., 1985; Laeng et al., 1985; Prudovsky
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et al., 1990), they have no inhibitory effect on DNA synthesis in heterokaryons. The absence of such an effect of macrophages on DNA synthesis in the benign cell nuclei in heterokaryons (this work (Prudovsky et al., 1985; Prudovsky et al., 1989a,b)) means that they do not contain any diffusible intracellular negative regulator of cell proliferation. The absence of inhibitors of proliferation and, simultaneously, maintenance of nondividing state in macrophages led us in 1989 to the supposition that ‘some changes in the macrophage genome prevent replication and that the elimination, or neutralization of these changes by products of immortalizing oncogenes takes place in heterokaryons’ (Zelenin and Prudovsky, 1989; Prudovsky et al., 1993). The results described in this work confirm the hypothesis. As far as we know, heterokaryons with macrophages are the first system that is suitable for investigation of telomerase inhibitors at the cellular level. This technique can be used with low amounts of inhibitors (sufficient for few milliliters of culture medium) and the answer can be obtained during few days (especially with a luminescent technique). An alternative approach—growing cell cultures in the presence of telomerase inhibitors and the telomere measurement—is much more time- and inhibitor-consuming. When we became convinced of the telomerasedependent macrophage reactivation, we decided to check some inhibitors of the DNA synthesis catalysed by the HIV reverse transcriptase on heterokaryons. We chose the guanine-derived compounds, since guanosine residues comprise 50% of the telomeric sequence and, in theory, guanine analogues may be the most effective inhibitors. Low concentrations (1–5 M) of these compounds had no significant effect on DNA synthesis in heterokaryons, which agrees with the results obtained in the HIV-infected cell cultures (Shipitsyn et al., 1999). Both compounds in high concentrations (100 M) had a similar inhibitory effect on the macrophage nuclei reactivation in heterokaryons. GF2G is suspected to be not sufficiently stable in the cell environment, and its effect is mediated by GP3 that emerged as a result of GF2G hydrolysis. Thus, the only investigations of new compounds on heterokaryons showed that both compounds are active at the cellular level. There are many questions concerning changes in the macrophage telomeres. To answer them, some additional experiments directed to investigation of their structure should be done. We suppose that the telomere damage may not require significant telomere shortening. In 1997 we proposed the loop
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structure for telomeres (Yegorov et al., 1997a) which can explain the appearance of ‘bad’ (damaged) telomeres with long telomeric sequences by alteration of DNA-telomere protein relationships. Relatively rapid damage to telomeres may make cells more sensitive to existing chemotherapeutic agents and make clinical use of anti-telomerase agents more practical.
ACKNOWLEDGEMENTS The authors thank their many colleagues, particularly M. Eldarov, S. S. Akimov, A. K. Akhmalisheva who contributed to the experiments and/or discussions of this work. This work was supported by the Russian Foundation for Basic Research (projects 96-1597640, 96-04-49086, 99-04-48073), the Russian State Program for Support of Scientific Schools (project 00-15-97833), the Russian Human Genome Program, the ISCC grant (project 1244), and the United States National Institutes of Health (CA-85363). REFERENCES B AG, O M, F M, H SE, C C-P, M GB, H CB, S JW, L S, W WE, 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349–352. G DE, K A, O OA, 1995. Preferential incorporation of 3 -azido-2 ,3 -dideoxythymidine (AZT) in telomeric sequences of CHO cells. Inter J Oncology 7: 1057–1060. G DE, T AM, O OA, 1998. Irreversible telomere shortening by 3 -azido-2 ,3 -dideoxythymidine (AZT) treatment. Biochem Biophys Res Commun 246: 107–110. L H, H DT, S R, 1985. Proliferative quiescence of normal mast cells resembles that of coldsensitive mutant mastocytoma cells. Dominant expression of the quiescent state in heterokaryons. Exp Cell Res 158: 170–176. M SM, H JF, P BJ, 1998. Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by 3 -azido-3 deoxythymidine. Clin Cancer Res 4: 693–696. M J, N N, S K, O K, 1999. Inhibition of telomerase activity and cell proliferation by a reverse transcriptase inhibitor in gynaecological cancer cell lines. Eur J Cancer 35: 1027–1034. N J, T H, T E, S M, I K, N H, N T, I T, I F, 1998. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nature Genet 8: 65–68. P AE, C DR, 1998. Inhibition of human telomerase by 2 -O-methyl-RNA. Proc Natl Acad Sci USA 95: 11549–11554.
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