Mechanisms o f Ageing and Development, 51 (1990) 133--138 Elsevier Scientific Publishers Ireland Ltd.
133
ABSENCE OF REVERSE TRANSCRIPTASE ACTIVITY IN HUMAN DIPLOID FIBROBLASTS
ARUN SRIVASTAVA*, ROBERT J. SHMOOKLER REIS b and SAMUEL GOLDSTEIN** Departments o f Medicine and Biochemistry and Molecular Biology, University o f Arkansas f o r Medical Sciences and Geriatric Research Education and Clinical Center, John L. McClellan Memorial Veterans Hospital, Little Rock, Arkansas 72205 (U.S.A.) (Received January 23rd, 1989) (Revision received April 18th, 1989)
SUMMARY
The reverse flow of genetic information from RNA transcripts to DNA has been postulated to be the mechanism for the generation of pseudogenes, dispersed repetitious DNA families, and small extrachromosomal circular DNAs in eukaryotic cells. We were unable to detect reverse transcriptase-like activity in several strains of cultured human cells, with or without induction by a variety of chemical and physical agents known to induce latent retroviruses.
Key words: Reverse transcriptase; Genetic flux; Human fibroblasts; Chromosomal breakage syndromes INTRODUCTION
Reverse transcriptase catalyzes the copying of RNA into DNA and thus effects the reverse flow of genetic information [1--3]. While this activity is encoded in the genome of retroviruses and is readily assayable in eukaryotic cells infected with these agents, its presence in normal cells has hitherto only been inferred, as the mechanism responsible for several forms of genetic flux in eukaryotic genomes. These include the generation of a variety of dispersed repetitive sequences [4--6] and pseudogenes [7,8] many of which bear evidence of RNA processing [9,10], and also the production of small extrachromosomal circular DNAs which are believed to be intermediates in reverse transcription [11,12]. Although normal mammalian cells have *Present address: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, U.S.A. **To whom all correspondence should be addressed. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
134
generally not been shown to contain reverse transcriptase activity, there have been occasional reports that "reverse transcriptase-like" activity can be detected in normal human and non-human primate placentas and amniotic fluid [13--16]. Because of the far-reaching significance of this mechanism in determining the structure, function and evolution of eukaryotic genomes, we have analyzed several strains of cultured human fibroblasts, from one of which we have cloned extrachromosomal circular DNA molecules [17], for the presence of low levels of reverse transcriptase activity. We report here that reverse transcriptase activity was undetectable in two normal strains of human diploid fibroblasts, at early and late passage, and other strains derived from subjects with genetically-determined disorders of chromosomal breakage, even after treatment by a variety of chemical and physical agents known to damage DNA a n d / o r alter its expression, and previously shown to be effective in eliciting active viral replication in latent infections [18,19]. MATERIALS AND M E T H O D S
Cultured fibroblasts H u m a n fibroblast strains included A2 from skin of a normal 11-year-old male [20], MRC5 from fetal lung, and mutant fibroblasts derived from subjects with xeroderma pigmentosum and ataxia telangiectasia obtained from the Genetic Mutant Repository (Institute for Medical Research, Camden, N J). All cells were propagated in Eagle's minimal essential medium supplemented with 15°70 fetal bovine serum [20]. Fibroblast strains MRC5 and A2 were propagated to replicative senescence (late passage) as described [17]. Reverse transcriptase assays All enzyme assays were carried out in a 100/al reaction mix containing 50 mM Tris--HCl, pH 8.0, 1 mM dithiothreitol, 100/ag/ml bovine serum albumin, l /ag of template-primer, 25 laM of all four deoxynucleoside triphosphates, 5 /aCi of 3Hlabeled d T T P or d G T P , 50 mM KC1, 5 mM MgCI 2 or 1 mM MnCl 2 as indicated, and medium (up to 80 gl) or various sub-cellular fractions (approximately 10/ag protein), at 37 °C for 60 min. The reactions were terminated with the addition of 10o70 trichloroacetic acid and the acid-precipitable radioactivity was measured [21]. The final concentration of NP-40 was 0.05 o70. Medium was obtained after incubation with cell monolayers for 24--96 h (with and without 15°70 fetal bovine serum). Sub-cellular fractions were prepared by standard methods [22] and protein concentrations measured by a modified Lowry procedure [23]. Chemical and physical treatments Duplicate cultures were treated with indicated amounts of chemicals and incubated at 37°C for 24 h. Cultures were re-fed and incubated without the chemicals for an additional 24 h. Medium samples were then removed for analysis.
135
Membranes were prepared 96 h post-treatment from treated cells and untreated controis. Duplicate cultures (,,02 x 106 cells) of strain A2 and complementation groups A and F (extremely UV-sensitive) of xeroderma pigmentosum [24] were rinsed with PBS and irradiated through air at room temperature with a germicidal UV source (0.4--2 j/m2/s) at various doses (see Table I), calibrated with a UV-flux meter (Model J-225, Ultraviolet Products, Inc., San Gabriel, CA). Warm medium was added immediately following irradiation and cultures were incubated at 37°C. Medium and membranes were assayed as above. Normal and ataxia telangiectasia cells (1 x 10~) sensitive to ionizing radiation [25] were suspended in growth medium and irradiated (178 rads/min) at 0°C with a J.L. Shepherd model 143 ~37Cs irradiator (1.33 meV) at various doses. Cells were re-plated in 100-mm dishes in fresh medium, incubated at 37 °C and assayed as described above. RESULTS AND DISCUSSION
We assayed directly for reverse transcriptase activity, since this method is as sensitive as radioimmunoassay or antigen capture assay [26] and is more general since it avoids the requirement inherent in immunologic procedures that specific antibodies will cross-react with existing retroviral poi enzymes. We performed standard assays for reverse transcriptase using a variety of synthetic template-primers as well as activated DNA [21], with the growth medium and plasma membranes o f all strains, since these compartments have been found to contain reverse transcriptase activity after cells are infected with retroviruses [16]. The results are summarized in Table I. All experiments were performed in triplicate, using MgC12 as divalent cation; those assays involving untreated A2 and MRC5 normal fibroblasts (uppermost panel in Table I), were also repeated for each strain, in triplicate, with MnCI 2 as the divalent cation. Purified reverse transcriptase from avian myeloblastosis virus, used as a positive control, catalyzed the incorporation of 99--126 pmol o f radioactive precursor into acid-precipitable nucleic acids with all template-primers used. Poly(rC)-oligo(dG)~0 was the most efficient template-primer, consistent with published reports [27,28], and was therefore used in subsequent assays with human fibroblasts. Activated DNA was not acceptable as a templateprimer, since nuclear and mitochondrial fractions of A2 fibroblasts supported the incorporation o f 18 and 6 pmol o f nucleoside monophosphates, respectively, only when using this substrate (not shown). Such incorporation was presumably due to endogenous DNA polymerase activity of nuclei and mitochondria. However, using the ribo-homopolymers which are specific for reverse transcriptase, no activity was detectable with either Mg 2÷or Mn 2÷cations, in any fractions from any of the strains, whether derived from normal persons and examined at early and late passage, or following treatment with chemical reagents known to cause general DNA demethylation (5-azacytidine), or to induce mutagenesis (BrdU), or otherwise induce expression o f repressed genes (db-cAMP). The same was true when assays were per-
136
TABLE I ASSAYS OF REVERSE TRANSCRIPTASE Fibroblast strain
Treatment
IN H U M A N D I P L O I D F I B R O B L A S T S Template-primer
p m o l [~H]Deoxynucleoside monophosphates incorporated ° Medium
Membrane AMV-RT~'
133
ABSENCE OF REVERSE TRANSCRIPTASE ACTIVITY IN HUMAN DIPLOID FIBROBLASTS
ARUN SRIVASTAVA*, ROBERT J. SHMOOKLER REIS b and SAMUEL GOLDSTEIN** Departments o f Medicine and Biochemistry and Molecular Biology, University o f Arkansas f o r Medical Sciences and Geriatric Research Education and Clinical Center, John L. McClellan Memorial Veterans Hospital, Little Rock, Arkansas 72205 (U.S.A.) (Received January 23rd, 1989) (Revision received April 18th, 1989)
SUMMARY
The reverse flow of genetic information from RNA transcripts to DNA has been postulated to be the mechanism for the generation of pseudogenes, dispersed repetitious DNA families, and small extrachromosomal circular DNAs in eukaryotic cells. We were unable to detect reverse transcriptase-like activity in several strains of cultured human cells, with or without induction by a variety of chemical and physical agents known to induce latent retroviruses.
Key words: Reverse transcriptase; Genetic flux; Human fibroblasts; Chromosomal breakage syndromes INTRODUCTION
Reverse transcriptase catalyzes the copying of RNA into DNA and thus effects the reverse flow of genetic information [1--3]. While this activity is encoded in the genome of retroviruses and is readily assayable in eukaryotic cells infected with these agents, its presence in normal cells has hitherto only been inferred, as the mechanism responsible for several forms of genetic flux in eukaryotic genomes. These include the generation of a variety of dispersed repetitive sequences [4--6] and pseudogenes [7,8] many of which bear evidence of RNA processing [9,10], and also the production of small extrachromosomal circular DNAs which are believed to be intermediates in reverse transcription [11,12]. Although normal mammalian cells have *Present address: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, U.S.A. **To whom all correspondence should be addressed. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
134
generally not been shown to contain reverse transcriptase activity, there have been occasional reports that "reverse transcriptase-like" activity can be detected in normal human and non-human primate placentas and amniotic fluid [13--16]. Because of the far-reaching significance of this mechanism in determining the structure, function and evolution of eukaryotic genomes, we have analyzed several strains of cultured human fibroblasts, from one of which we have cloned extrachromosomal circular DNA molecules [17], for the presence of low levels of reverse transcriptase activity. We report here that reverse transcriptase activity was undetectable in two normal strains of human diploid fibroblasts, at early and late passage, and other strains derived from subjects with genetically-determined disorders of chromosomal breakage, even after treatment by a variety of chemical and physical agents known to damage DNA a n d / o r alter its expression, and previously shown to be effective in eliciting active viral replication in latent infections [18,19]. MATERIALS AND M E T H O D S
Cultured fibroblasts H u m a n fibroblast strains included A2 from skin of a normal 11-year-old male [20], MRC5 from fetal lung, and mutant fibroblasts derived from subjects with xeroderma pigmentosum and ataxia telangiectasia obtained from the Genetic Mutant Repository (Institute for Medical Research, Camden, N J). All cells were propagated in Eagle's minimal essential medium supplemented with 15°70 fetal bovine serum [20]. Fibroblast strains MRC5 and A2 were propagated to replicative senescence (late passage) as described [17]. Reverse transcriptase assays All enzyme assays were carried out in a 100/al reaction mix containing 50 mM Tris--HCl, pH 8.0, 1 mM dithiothreitol, 100/ag/ml bovine serum albumin, l /ag of template-primer, 25 laM of all four deoxynucleoside triphosphates, 5 /aCi of 3Hlabeled d T T P or d G T P , 50 mM KC1, 5 mM MgCI 2 or 1 mM MnCl 2 as indicated, and medium (up to 80 gl) or various sub-cellular fractions (approximately 10/ag protein), at 37 °C for 60 min. The reactions were terminated with the addition of 10o70 trichloroacetic acid and the acid-precipitable radioactivity was measured [21]. The final concentration of NP-40 was 0.05 o70. Medium was obtained after incubation with cell monolayers for 24--96 h (with and without 15°70 fetal bovine serum). Sub-cellular fractions were prepared by standard methods [22] and protein concentrations measured by a modified Lowry procedure [23]. Chemical and physical treatments Duplicate cultures were treated with indicated amounts of chemicals and incubated at 37°C for 24 h. Cultures were re-fed and incubated without the chemicals for an additional 24 h. Medium samples were then removed for analysis.
135
Membranes were prepared 96 h post-treatment from treated cells and untreated controis. Duplicate cultures (,,02 x 106 cells) of strain A2 and complementation groups A and F (extremely UV-sensitive) of xeroderma pigmentosum [24] were rinsed with PBS and irradiated through air at room temperature with a germicidal UV source (0.4--2 j/m2/s) at various doses (see Table I), calibrated with a UV-flux meter (Model J-225, Ultraviolet Products, Inc., San Gabriel, CA). Warm medium was added immediately following irradiation and cultures were incubated at 37°C. Medium and membranes were assayed as above. Normal and ataxia telangiectasia cells (1 x 10~) sensitive to ionizing radiation [25] were suspended in growth medium and irradiated (178 rads/min) at 0°C with a J.L. Shepherd model 143 ~37Cs irradiator (1.33 meV) at various doses. Cells were re-plated in 100-mm dishes in fresh medium, incubated at 37 °C and assayed as described above. RESULTS AND DISCUSSION
We assayed directly for reverse transcriptase activity, since this method is as sensitive as radioimmunoassay or antigen capture assay [26] and is more general since it avoids the requirement inherent in immunologic procedures that specific antibodies will cross-react with existing retroviral poi enzymes. We performed standard assays for reverse transcriptase using a variety of synthetic template-primers as well as activated DNA [21], with the growth medium and plasma membranes o f all strains, since these compartments have been found to contain reverse transcriptase activity after cells are infected with retroviruses [16]. The results are summarized in Table I. All experiments were performed in triplicate, using MgC12 as divalent cation; those assays involving untreated A2 and MRC5 normal fibroblasts (uppermost panel in Table I), were also repeated for each strain, in triplicate, with MnCI 2 as the divalent cation. Purified reverse transcriptase from avian myeloblastosis virus, used as a positive control, catalyzed the incorporation of 99--126 pmol o f radioactive precursor into acid-precipitable nucleic acids with all template-primers used. Poly(rC)-oligo(dG)~0 was the most efficient template-primer, consistent with published reports [27,28], and was therefore used in subsequent assays with human fibroblasts. Activated DNA was not acceptable as a templateprimer, since nuclear and mitochondrial fractions of A2 fibroblasts supported the incorporation o f 18 and 6 pmol o f nucleoside monophosphates, respectively, only when using this substrate (not shown). Such incorporation was presumably due to endogenous DNA polymerase activity of nuclei and mitochondria. However, using the ribo-homopolymers which are specific for reverse transcriptase, no activity was detectable with either Mg 2÷or Mn 2÷cations, in any fractions from any of the strains, whether derived from normal persons and examined at early and late passage, or following treatment with chemical reagents known to cause general DNA demethylation (5-azacytidine), or to induce mutagenesis (BrdU), or otherwise induce expression o f repressed genes (db-cAMP). The same was true when assays were per-
136
TABLE I ASSAYS OF REVERSE TRANSCRIPTASE Fibroblast strain
Treatment
IN H U M A N D I P L O I D F I B R O B L A S T S Template-primer
p m o l [~H]Deoxynucleoside monophosphates incorporated ° Medium
Membrane AMV-RT~'
