Mutagenesis, Vol. 18, No. 2, 151-158,
March 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Environmental factors affecting transcription of the human L1 retrotransposon. II. Stressors
Radiation Oncology Research Laboratory, University of California–San Francisco, 1855 Folsom Street, MCB 200, San Francisco, CA 94103, USA and 1 Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, 221 Burwood Highway, Burwood, 3125 Victoria, Australia
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Retrotransposons have clearly molded the structure of the human genome. The reverse transcriptase coded for by long interspersed nuclear elements (LINEs) accounts for 35% of the human genome, with 8–9 x 105 copies of the most common human LINE element, L1Hs. Retrotransposons cycle through an RNA intermediate with transcription as the rate limiting step. Because various retrotransposons have been demonstrated to be induced by environmental stimuli, we investigated the response of the L1Hs promoter to various agents. L1Hs promoter activity was analyzed by transfecting an L1Hs-expressing cell line with plasmids containing one of two L1Hs promoters fused to the LacZ reporter gene. L1Hs promoter activity was then monitored with a ß-galactosidase assay. Treatment with UV light and heat shock resulted in a small increase in ß-galactosidase activity from one promoter, while treatment with tetradecanoylphorbol 13-acetate resulted in small increases in ß-galactosidase activity from both promoters. No increase in ß-galactosidase activity was observed after exposure to X-rays or hydrogen peroxide.
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Introduction |
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LINEs are retrotransposons that lack long terminal repeats, encode retrotransposition enzymes and transpose through an RNA intermediate (Eickbush, 1992
). LINEs are present in many different organisms. The three families of human LINEs are 
6 kb in length and contain a 5'-untranslated promoter region (UTR), a 3'-UTR and remnants of a poly(A) tail. Human LINEs contain two open reading frames (ORFs), the first of which encodes a multimer-forming protein with nucleic acid chaperone activity that is crucial for retrotransposition (Moran et al., 1996; Hohjoh and Singer, 1997; Martin and Bushman, 2001). The second ORF encodes a protein with endonuclease and reverse transcriptase functions (Mathias et al., 1991; Fang et al., 1996). There is one human LINE family, L1Hs, that contains active, full-length elements, while the rest of the 868 000 copies (20% of the human genome) of human LINEs are defective due to truncations at the 5'-end (Lander et al., 2001).
LINE elements are a major force in shaping mammalian genomes. While the number of LINEs and SINEs is probably underestimated, it is considered that L1 activity is responsible for 33% of the human genome (Lander et al., 2001; Venter et al., 2001). In addition to insertional mutations, LINE elements can have a variety of other influences on the genome. The LINE reverse transcripase is thought to be responsible for the creation of pseudogenes (Jurka, 1997) and psuedogene-derived genes (Kleene et al., 1998), as well as the creation of new genes through exon shuffling (Moran et al., 1999; Esnault et al., 2000). It has also been observed that retrotransposon sequences have been utilized as coding sequences (Murnane and Morales, 1995) or regulatory sequences (Nigumann et al., 2002) for other genes. Lastly, it has been shown recently that LINE element insertion sites are associated with a variety of other chromosome rearrangements (Gilbert et al., 2002; Kazazian and Goodier, 2002; Symer et al., 2002). Taking into account the host of ways L1Hs activity has impacted on the human genome, a central questions is ‘under what circumstances do human LINE elements retrotranspose?’. Even though L1Hs transcription is the rate limiting step in retrotransposition (Mathias and Scott, 1993; Skowronski et al., 1988), little is known about the regulation of L1Hs transcription or the relationship between L1Hs transcription and retrotransposition. L1Hs contains an internal promoter that is 952 bp in length (Swergold, 1990) that is a binding site for a variety of cellular proteins (Minakami et al., 1992; Mathias and Scott, 1993; Yang et al., 1998), including transcription factors YY1 (Becker, 1995; Kurose et al., 1995) and a SRY-like protein (Tchenio et al., 2000).
The ability of environmental factors to stimulate transposable element activity was first proposed by McClintock (1984). McClintock’s ‘genomic shocks’ hypothesis proposes that environmental stimuli may mobilize transposable elements. This hypothesis is supported by evidence that retrotransposons from various organisms can be mobilized by a variety of environmental stresses. With regard to LTR retrotransposons, heat shock and the DNA-damaging agents UV light, ethylmethyl sulfonate, ionizing radiation and 4-nitroquinoline oxide have been shown to induce transcription and transposition of the yeast Ty LTR retrotransposon (Morawetz, 1987; Bradshaw and McEntee, 1989). Copia elements in Drosophila melanogaster have been shown to be transcriptionally responsive to heat, hydrogen peroxide and sodium azide (Strand and McDonald, 1985). The DNA-damaging agent mitomycin C was shown to induce genomic rearrangements involving the gypsy (mdg4) LTR retrotransposon in Drosophila (Georgeiv et al., 1990). In mammals, anoxia causes a 500-fold induction of the LTR retrotransposon-like VL30 elements in rat fibroblasts (Anderson et al., 1988). Many plant retrotransposons are also known to be activated by environmental stressors (Wessler, 1996). For example, the copy number of the plant LTR retrotransposon BARE-1 correlates with environmental conditions, suggesting stress-induced mobilization (Kalendar et al., 2000).
The transcription of several mouse, rat and human endogenous retroviruses are also inducible by a wide variety of stresses, including 5-azacytidine, cycloheximide, 12-O-tetradecanoylphorbol 13-acetate (TPA) and benzo[a]pyrene diol epoxide, diethylnitrosamine and 1,3-butadiene (Hsieh et al., 1987; Hsiao and Chang, 1999). The human immunodeficiency virus is responsive to a variety of environmental stressors such as UV, ionizing radiation and other DNA-damaging agents (Stein et al., 1989; Kumar et al., 1996).
Non-LTR retrotransposons may also be responsive to environmental stress. Zepp, a green algae LINE, is inducible by heat shock (Higashiyama et al., 1997). Interestingly, mouse LINE mRNA is increased when cultured cells are exposed to several treatments, such as serum starvation, 5-azacytidine and TPA (Tchenio et al., 1993). Mouse lymphoid precursor cells were shown to increase endogenous reverse transcriptase activity when exposed to several DNA-damaging agents (Rudin and Thompson, 2001). Recently, mouse LINEs in smooth muscle cells were shown to be transcriptionally induced by benzo[a]pyrene (Lu et al., 2000). Another type of retroelement, SINEs (short interspersed nuclear elements), also show responsiveness to environmental stimuli. Alu elements in humans (Rudin and Thompson, 2001), Bm1 elements in silkworm and B2 elements in Chinese hamster cells have been reported to be transcriptionally inducible by heat shock and other stresses (Fornace et al., 1989; Liu et al., 1995; Kimura et al., 1999; Li et al., 1999).
Although the influence of various environmental agents on retrotransposition has been studied extensively in some organisms, there are few data on how environmental factors affect retrotransposition in human cells. To study the influence of various factors on retrotransposition in human cells, clones of the human coriocarcinoma Jeg-3 cell line were established that contained stably integrated copies of plasmids containing one of two different L1Hs promoters fused to the LacZ reporter gene. Changes in the level of expreesion of the LacZ gene was then monitored with an assay for ß-galactosidase following exposure of these clones to UV, TPA, heat shock, X-rays and hydrogen peroxide.
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Materials and methods |
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Plasmids P1LZ and pL1.2LZ
The plasmid P1LZ was obtained from Gary D.Swergold at the FDA (Swergold, 1990
) and was re-sequenced. The L1Hs promoter sequence in plasmid P1LZ was derived from a full-length cDNA and has been named ‘Pito’ (Figure 1). The plasmid pL1.2LZ is similar to P1LZ, except that it contains the L1Hs promoter named ‘Pita’, derived from pL1.2A, a plasmid constructed from a cloned active L1Hs element isolated from a L1Hs insertion into the factor VIII gene of a hemophilia patient (Dombroski et al., 1991). pL1.2A was obtained from Julie McMillan (NIH) and was not re-sequenced. Both promoters are transcriptionally active, yet only Pita is part of an actively retrotransposing element. Both promoters are fused to a LacZ reporter gene to monitor promoter activity using the Galactolight assay for detection of ß-glactosidase. The LacZ reporter gene was fused to the L1Hs 3'-UTR for transcription termination and poly(A) addition sequences (Figure 1). Sequence analysis of the Pito and Pita promoters demonstrated the presence of a variety of putative transcriptional response elements (Figure 1b). Both promoter regions possess androgen receptor-like binding sites (Morales et al., 2002), a collagenase-like AP-1 site (Schonthal et al., 1988; Stein et al., 1989) and heat shock elements [(A/G)GAAN] similar to those in the HSP40 promoter (Amin et al., 1988; Xiao and Lis, 1988; Hata and Ohtsuka, 1998).
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Cell culture and transfection
The human choriocarcinoma cell line Jeg-3 was obtained from the American Tissue Culture Collection (Camden, NJ). Jeg-3 was grown in minimum Eagle’s medium (Life Technologies) with 10% bovine calf serum (Hyclone). The Jeg-3 clones used in these experiments, W32, W31 and W8, were derived by transfection with the plasmid P1LZ or pL1.2LZ, respectively. Transfections were performed with 20 µg plasmid using the CaPO4 precipitation method. Selection for cells with integrated plasmid was performed by co-transfection with pSV2Neo (2 µg) and growth in selective medium containing G418 (400 µg/ml). Individual surviving colonies were confirmed as stable transfectants by long PCR (Stratagene) with primers to the L1Hs promoter and the LacZ gene (data not shown). Transient transfections were conducted with Lipofectin (Life Technologies) using the protocol provided by the manufacturer.
Treatment of cells
Cells were plated at 5 x 105 cells/60 mm dish, grown for 2 days and then exposed to the various agents. In several UV and TPA experiments these treatments were conducted on cells that were previously serum-starved for 24 h (indicated in the figure legends as ‘...in the absence of serum’). The procedure for treatment with UV used standard fluences and incubation periods (Fornace et al., 1988; Stein et al., 1989). After 1 day the medium was removed from the dishes and pooled and any excess was aspirated. The dishes were then irradiated with UV light at either 10 or 20 J/m2 and the same medium was replaced on the cells. The cells were then incubated at 37°C and lysates were made 1, 2 and 4 h after irradiation, the times previously shown to demonstrate standard induction of UV- and TPA-inducible genes (Fornace et al., 1988; Stein et al., 1989). The lysates were then stored at –72°C. The UVC light source was an in-house construction that generated 254 nm light at 1.3 J/s (kindly provided by James Cleaver, UCSF). TPA was dissolved in 100% ethanol at a concentration of 10 mg/ml. TPA was then diluted in medium with and without 10% serum to a concentration of 0, 50 or 100 ng/ml immediately before exposing the cells. Cells were grown for 2 days prior to addition of low serum medium (2% serum) for 18 h, after which TPA-containing medium was added to the dishes. Lysates were harvested after 1, 2 and 4 h incubations. For heat shock treatments the cells were placed in an incubator at either 37, 42 or 45°C for 20 min and then placed back at 37°C. Lysates were made 1, 2 and 4 h after removal from the higher temperatures, a common time of induction of heat shock proteins (Jacquier-Sarlin et al., 1995; Holmberg et al., 1997). The samples were then stored at –72°C prior to analysis. For X-rays cells was irradiated with 0, 250 and 500 cGy using a Phillips RT250 X-ray machine (250 kV). Lysates were collected 1, 2 and 4 h after irradiation and stored at –72°C. Hydrogen peroxide was dissolved in phosphate-buffered saline (PBS) buffer (30% w/w) immediately prior to use. The medium was aspirated off, the monolayer washed twice with PBS and the cells were chilled on ice for 10 min. Cold hydrogen peroxide at 10 or 100 µM was then added to the dishes. The cells with hydrogen peroxide were kept on ice for 20 min. At the end of this time cells were warmed by pouring on medium at 37°C and after 1, 2 and 4 h lysates were prepared and stored at –72°C.
The standard error of the mean for each time point/treatment is indicated as an error bar. The percent increase in the expression of ß-galactosidase in the treated samples relative to the appropriate control is presented in each figure. The values that are marked in the figures (dark rectangles) are significantly different from the untreated controls at that time point, with P values of 
0.05 as determined by ANOVA and the ANOVA post hoc tests of Tukey–Kramer (Kramer, 1956; Keselman and Rogan, 1978) and Dunnett (1964). Each of the statistical tests utilized the same sets of data and were conducted using Statview 5.01 for the Macintosh (www.statview.com). The ANOVA test was used to determine whether the means of a set of variables were equal to each other. The Tukey–Kramer and Dunnett’s tests made multiple comparisons to determine if the means of a set of variables were significantly different from each other. The number of experiments are indicated in the figure legends.
The Galactolight assay
The Galactolight assay (Tropix) was used to determine the level of ß-galactosidase activity from the L1Hs promoters. The Galactolight assay was conducted as recommended by the manufacturer (Tropix) with the following alterations. The cells were washed with Saline A (137 mM NaCl, 5.36 mM KCl, 0.55 mM dextrose, 0.45 mM NaHCO3) instead of PBS and 50 µl of DNase I (1 µg/ml) was added to the lysis buffer to make the pellet more compact. The lysates were centrifuged at 14 000 g for 10 min and the supernatant was centrifuged again at 14 000 g for 2 min. In order to inactivate endogenous ß-galactosidase activity, the lysates were heat inactivated at 48°C for 50 min (Jain and Magrath, 1991). The protein concentrations of the final supernatants were determined using the standard Bradford assay (Bio-Rad) and luminescence was measured using a Monolight 2000 (Analytical Luminescence) luminometer.
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Results |
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P1LZ and pL1.2LZ clones
Cell lines isolated from choriocarcinomas, embryonal carcinomas, breast carcinomas and a medulloblastoma are some of the only ones known to express L1Hs. The choriocarcinoma cell line utilized in these studies, Jeg-3, was previously demonstrated to express L1Hs (Leibold et al., 1990
). To monitor L1Hs promoter activity, Jeg-3 was transfected with the P1LZ and pL1.2LZ plasmids (Figure 1a
) and individual clones were selected and screened for an increase in ß-galactosidase activity. Initially, clones W31 and W32 had levels of ß-galactosidase activity that were increased 6- and 20-fold, respectively, over that found in the parental Jeg-3 cell line (Table I). In comparison, the level of ß-galactosidase activity in a cell clone stably transfected with the lacZ gene on a fos promoter, previously found to result in high levels of ß-galactosidase activity (Schilling et al., 1991), was increased 83-fold over that found in Jeg-3. Heat inactivation was used to eliminate endogenous ß-galactosidase activity (Jain and Magrath, 1991), which resulted in a 7- to 12-fold reduction in ß-galactosidase activity in the parental Jeg-3 cell line (Table I). Following heat inactivation, clones W8, W31 and W32 had levels of ß-galactosidase activity that were 3-, 9- and 59-fold higher, respectively, than that found in Jeg-3 (Table I). While the level of ß-galactosidase activity of the W32 clone decreased over time due to promoter silencing (data not shown), the data presented were derived from multiple experiments over various time points and normalized as a percentage of control. Long PCR with primers to the L1Hs promoter and LacZ gene was used to confirm that these clones contained the stably integrated and unrearranged plasmid, as demonstrated by the presence of the 4 kb fragment found in the original plasmids (data not shown). Using these criteria, the Jeg-3 clones W32 and W8 contained the intact, stably transfected P1LZ and pL1.2LZ plasmids, respectively, and were used for evaluating the effect of environmental stimuli on L1Hs promoter activity.
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The influence of UV
The effect of UV irradiation on L1Hs promoter activity was examined for a number of reasons. First, different kinds of retroelements have been shown to be inducible by UV radiation (Stein et al., 1989; Servomaa and Rytomaa, 1990
; Faure et al., 1996). Second, LINE expression was shown to be effected by UV irradiation (Deragon et al., 1990; Servomaa and Rytomaa, 1990). Third, a sequence search of the L1Hs promoter in the plasmid revealed a sequence identical to the UV-phorbol ester response element (URE) of the collagenase (+ strand, TGAGTCA) and GADD153 (– strand, TGACTCA) genes (Holbrook and Fornace, 1991). The URE is present in other L1Hs (Hohjoh et al., 1990; Holmes et al., 1994), suggesting that it may play an important role in retrotransposition and that UV irradiation might affect L1Hs transcription (Angel et al., 1987). A sequence comparison of the two L1Hs promoters (Pito and Pita) revealed notable differences (Figure 1b), including many single base pair polymorphisms and insertions and three non-homologous regions of 11–20 bp. One notable difference is that the collagenase AP-1 site, which is identical to the AP-1 consensus sequence in promoter Pita, is different by a single base in promoter Pito.
After UVC irradiation, clone W32, stably transfected with the P1LZ plasmid, demonstrated no increase in ß-galactosidase activity at either 10 or 20 J/m2, with or without serum (Figure 2). In fact, at the higher fluences of UVC and at longer times after irradiation, ß-galactosidase activity decreased in clone W32. Higher wavelength UVB also failed to increase ß-galactosidase activity in clone W32 (data not shown). Another P1LZ clone, W31, also did not demonstrate a statistically significant increase in ß-galactosidase activity when irradiated with UVC under similar conditions (data not shown). In contrast, in clone W8, containing the pL1.2LZ plasmid, a statistically significant increase in ß-galactosidase activity was seen at 1, 2 and 4 h after treatment with 20 J/m2 UVC (Figure 3). Jeg-3 cells transiently transfected with the pL1.2LZ plasmid also showed similar small inductions after UVC irradiation at 20 J/m2 (data not shown). In contrast, the untransfected parental Jeg-3 cell line showed a 50% decrease in endogenous ß-galactosidase activity when irradiated under similar conditions (data not shown).
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The influence of TPA
Because TPA is a potent tumor-promoting agent that can stimulate the expression of many genes containing UREs in their promoters (Buscher et al., 1988
), we assessed the responsiveness of the L1Hs promoter to TPA. In clone W32, a small but significant increase in ß-galactosidase activity was observed 1–4 h after exposure to 100 ng/ml TPA (Figure 4). W32 treated with 50 ng/ml TPA failed to produce an increase in ß-galactosidase activity up to 2 h after incubation in TPA, but did show a significant increase after 4 h. Like clone W32, clone W8 also showed a small but significant increase in ß-galactosidase activity with TPA, but unlike W32 showed an increase 1 and 2 h after treatment with 50 ng/ml (Figure 4). As previously reported (Morales et al., 2002), both clones W32 and W8 showed an increase in ß-galactosidase activity after treatement with medium containing serum (Figure 4), however, no additional increase in ß-galactosidase was seen after treatment with serum in combination with TPA.
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The influence of heat shock, X-rays and hydrogen peroxide
Heat shock, an important non-genotoxic cellular stress, was evaluated for its ability to induce L1Hs transcription for two reasons. First, heat shock is known to produce an increase in transcription and transposition of other LTR and non-LTR retroelements (Bradshaw and McEntee, 1989
; Kimura et al., 1999; Li et al., 1999; Rudin and Thompson, 2001). Second, we observed that the L1Hs promoter contains sequences similar to the putative heat shock responsive element (A/G)GAAN found in the HSP40 promoter (Amin et al., 1988; Xiao and Lis, 1988; Hata and Ohtsuka, 1998). Heating clone W32 to 42 and 45°C resulted in a small but significant increase in the levels of ß-galactosidase activity after 2 and 4 h (Figure 5). In contrast, although X-rays and hydrogen peroxide are reported to induce some genes (Holbrook and Fornace, 1991; Herrlich et al., 1992, 1997) and rat LINEs (Servomaa and Rytomaa, 1990), treatment of clone W32 with X-rays or hydrogen peroxide did not result in an increase in ß-galactosidase activity (Figure 6).
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Discussion |
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The results presented here suggest that the expression of two different L1Hs promoters can be slightly increased by environmental factors. Because the transcriptional activity of retrotransposons is thought to be the rate limiting step of retrotransposition (Scowronski et al., 1988
; Mathias and Scott, 1993), this observation suggests that stressors may influence retrotransposition in human cells. While it cannot be ruled out that the relatively small increases in ß-galactosidase activity that we have observed are due to changes in the turnover rate of the mRNA or protein or changes in translation, relatively small increases in RNA expression in response to environmental stimuli are not unexpected. In view of the mutagenic properties of retrotransposition, it is likely that there would be a strong evolutionary selection against high levels of expression of retrotransposons. In addition, relatively small increases in expression of retrotransposons can have conspicuous effects on retrotransposition frequency. In the absence of exogenous reverse transcriptase, the normally undetectable levels of LINE element gene products in HeLa cells were still able to result in a detectable LINE insertion frequency (Moran et al., 1996). Therefore, in view of the dramatic events associated with retrotransposon insertions, small changes in transcription could have a major impact on genomic instability. Consistent with this possibility, the influence of small changes in transcription on retrotransposition can be seen in Drosophila I–R hybrid dysgenesis or the transient elevation of I element retrotransposition in the germline of SF female progeny resulting from crosses of inducer and reactive strains. The SF females are sterile, due to high frequencies of I element-mediated insertions and rearrangements (Lim and Simmons, 1994).de La Roche Saint Andre et al.(1998) investigated whether I element transcription correlated with reactivity and retrotransposition frequency. They found that the amount of a full-length I element mRNA in SF females issued from reactive mothers is only 5-fold above the amount of I element mRNA from non-dysgenic progeny. These results suggest that the highest known in vivo LINE retrotransposition frequency is mediated by only a 5-fold increase in transcription over background.
The environmental stimuli examined in our study included both genotoxic and non-genotoxic stressors. Of the two L1Hs promoters tested, only one was responsive to genotoxic stressors. The W8 clone showed a small increase in ß-galactosidase activity in response to UVC irradiation in the absence of serum (Figure 3). However, all fluences and wavelengths of UV radiation failed to elicit an increase in ß-galactosidase activity in clone W32. In fact, at 4 h both doses of UVC repressed ß-galactosidase activity compared with controls with and without serum. These results suggest variability in the response of different L1Hs promoters to specific types of genotoxic stressors. The Pita promoter in clone W8 contains a sequence identical to the UV-responsive collagenase AP-1 element, while this sequence in the Pito promoter in clones W31 and W32 differs by 1 bp from the consensus sequence for this element (Stein et al., 1992). Various other sequence differences between these L1Hs promoters may also contribute to the differences in their responsiveness to genotoxic stressors. Alternatively, differences in the site of integration could be responsible for the difference in the response of clone W8 compared to clones W31 and W32, since the integration site of a transgene can affect its transcriptional status. However, this alternative seems less likely, since transient transfections performed with the pL1.2LZ plasmid gave results with UVC that were similar to those obtained with clone W8. Regardless, in view of the large number of integration sites of L1Hs in the human genome, the response of the L1Hs promoter at any location is relevant.
The effect of UV on ß-galactosidase activity in clone W8 is consistent with the reported effect of UV irradiation on various retroelements including mammalian LINEs. The LTR of the Drosophila retrotransposon 1731, controlling the activity of the CAT reporter gene, is up-regulated by UVB irradiation (Faure et al., 1996). The human immunodeficiency virus HIV-1 has shown increases in transcription following irradiation with 20 J/m2 UVC (Stein et al., 1989a,b; Kumar et al., 1996). In terms of mammalian LINEs, Servomaa and Rytomaa (1990) observed an increase in L1 mRNA after UV irradiation of rat chloroleukemia cells. In human cells, Deragon et al. (1990) detected a 2- to 3-fold increase in L1Hs reverse transcriptase activity 60 min after irradiation of Ntera2D1 cells with 2 x 10–2 J/m2 UV of unknown wavelength.
Unlike UV, TPA caused an increase in ß-galactosidase activity in both clone W32 and clone W8 (Figure 4). The data presented here suggest that TPA can produce a 40–50% increase in the activity of the L1Hs promoter. The lack of additivity in the effect of TPA and serum on the level of ß-galactosidase activity suggests that TPA and serum act through the same pathways. Previous studies have reported that many mammalian retroviruses are also inducible by TPA (Hellman and Hellman, 1981; Hsieh and Weinstein, 1990; Larsson et al., 1996). Similarly, non-LTR retrotransposons have also been shown to be inducible by TPA, as shown by a 5-fold increase in the frequency of pseudogene formation mediated by the mouse LINE L1Md RT (Tchenio et al., 1993). This variability may be due to differences between the promoters in mouse and human LINEs or the fact that the relationship between reporter gene expression and the retrotransposition rate may not be directly proportional. As such, the studies in mice showed that the TPA-induced LINE mRNA and ORF I levels do not correlate directly with TPA-induced retrotransposition frequency (Tchenio et al., 1993).
Clone W32 showed a small increase in ß-galactosidase activity following heat shock (Figure 5). The effect of heat shock on L1Hs has a precedent with LTR retrotransposons, such as Copia and 1731 in Drosophila (Ziarczyk and Best-Belpomme, 1991), DIRS 1 and TDD-1 in Dictyostelium discoideum (Cappello et al., 1984; Rosen et al., 1983; Zuker et al., 1984) and Ty in Saccharomyces cerevisiae (Kawakami et al., 1992). Zepp, a non-LTR retrotransposon found in the green alga Chlorella vulgaris, appears to increase its transcription 2- and 5-fold when the cells are grown for 5 min at 30 and 37°C (Higashiyama et al., 1997). Some mammalian SINEs also appear to be inducible by heat shock or agents that mimic heat shock. Heat shock transiently increases the abundance of Alu RNA in human cells (Liu et al., 1995; Li et al., 1999). Heat shock also transiently induces B1, B2 (Fornace et al., 1989) and C element polymerase III RNAs in mouse, hamster and rabbit cells (Liu et al., 1995). Deragon et al.(1990) detected a 23% increase in L1Hs RT activity 60 min after heat shocking Ntera2D1 cells.
Clone W32 did not show an increase in ß-galactosidase activity after treatment with X-rays or hydrogen peroxide (Figure 6). This result contrasts with previous studies with murine retrotransposons. The activity of rat LINEs was induced by 3 Gy X-rays (Servomaa and Rytomaa, 1990) and murine endogenous reverse transcriptase activity was elevated following exposure to 20 Gy 
-radiation (Rudin and Thompson, 2001). The reason for these differences between human and murine retrotransposons is unclear.
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Acknowledgments |
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This work was supported by grant number RO1 CA69044 from the National Cancer Institute, NIH.
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Notes |
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2 To whom correspondence should be addressed: Tel: +1 415 476 9083; Fax: +1 415 476 9069; Email: murnane{at}rorl.ucsf.edu
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References |
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Received on May 13, 2002; revised on October 23, 2002; accepted on November 1, 2002.