ref-1) multifunctional DNA base excision repair gene during fetal development and in adult rat brain and testis

DNA Repair

ELSEVIER

Mutation Research 362 (1996) 237-248

Differential expression of the apurinic/apyrimidinic endonuclease (APE/ref-1) multifunctional DNA base excision repair gene during fetal development and in adult rat brain and testis Teresa M. Wilson a, Scott A. Rivkees a,b, Walter A. Deutsch c, Mark R. Kelley a,b,* a Department of Pediatrics, Herman B Wells Center for Pediatric Research. Indiana University School qfMedicine, 702 Barnhill D r Room 2664, Indianapolis, IN 46202, USA b Department o[`Biochemistrv and Molecular Biology, Indiana Universi~" School (>['Medicine, Indianapolis, IN 46202, USA c Department of Biochemistry, Louisiana State University, Baton Rouge, LA 70803, USA

Received 4 August 1995;revised 12 October 1995: accepted 13 October 1995

Abstract The multifunctional mammalian apurinic/apyrimidinic (AP) endonuclease is responsible for the repair of AP sites in DNA. In addition, this enzyme has been shown to function as a redox factor facilitating the DNA binding capability of Jun-Jun homodimers and Fos-Jun heterodimers by altering their redox state and to be involved in calcium mediated transcriptional repression of the parathyroid hormone gene. Previous studies examining the tissue specific distribution of the AP endonuclease (APE) transcript and protein by Northern analysis and enzymatic assays, respectively, have shown that this gene is expressed in all tissues at relatively similar levels. In the current study, adult and fetal rat tissue sections were examined for the expression of the APE transcript in specific subpopulations of cells and during development by in situ hybridization. In the adult brain, the APE transcript showed a widespread, but heterogeneous pattern of expression. Predominant levels of transcript were detected in the suprachiasmatic nuclei, the supraoptic and paraventricular nuclei, the hippocampus and the cerebellum. During fetal development, transcript was detected in all somatic sites examined with very high levels in the thymus, liver and developing brain. Examination of the adult testis indicated that the expression of the transcript varies with the stage of spermatogenesis with the highest levels being present over round spermatids. These results provide evidence that the APE gene is not homogeneously expressed, but rather is found in subpopulations of cells in the brain and testes and during development. Keywords: AP endonuclease;Base excision repair; DNA repair; In situ

1. Introduction

* Corresponding author. Tel.: (317) 274-2755: Fax: (317) 2748679; E-mail: [email protected].

The inherent chemical instability of DNA, the production of reactive oxygen species during normal cellular metabolism (Demple and Harrison, 1994),

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and the continuous exposure to environmental mutagens represent a potential threat to the genetic information of cells (Friedberg, 1985; Larson et al., 1985; Loeb and Preston, 1986; Saffhill et al., 1985). If unrepaired, damaged DNA can have deleterious biological consequences for the organism and result in cell transformation or death (Friedberg, 1985). Thus, to protect against DNA damage, organisms have evolved an elaborate array of DNA repair mechanisms such as nucleotide excision repair (NER), mismatch/recombination repair and DNA base excision repair (BER) (Barnes et al., 1993; Lindahl, 1992, 1994). DNA base excision repair involves two major classes of repair enzymes, namely DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases (Demple and Harrison, 1994; Doetsch and Cunningham, 1990; Lindahl, 1992; Sancar and Sancar, 1988). DNA glycosylases are enzymes that hydrolyze the N-glycosidic bond between the damaged base and the deoxyribose moiety, leaving behind an AP site on the DNA backbone (Demple and Harrison, 1994; Doetsch and Cunningham, 1990; Wallace, 1988). Cytotoxic lesions such as N3-methyladenine, N 7methylguanine, and N3-methylguanine, are all released by the N-methylpurine DNA glycosylase (MPG) enzyme (Mitra and Kaina, 1993; Sakumi and Sekiguchi, 1990; Sancar and Sancar, 1988). A recent report has also demonstrated MPG glycosylase activity directed towards 8-oxoguanine, a major deleterious lesion occurring during oxidative DNA damage (Bessho et al., 1993). AP sites produced by the action of N-glycosylases are acted upon by APEs, which can make an incision either 3' to the AP site (class I AP lyase) or 5' to the AP site (class II AP endonuclease) (Demple and Harrison, 1994: Doetsch and Cunningham, 1990). Furthermore, it is estimated that tens of thousands of AP sites are produced per cell per day, primarily by spontaneous base hydrolysis, and cellular metabolites reacting with oxygen radicals (Lindahl, 1993). Unrepaired AP sites bypassed during DNA replication result in mutations and genetic instability (Loeb and Preston, 1986). The major cellular enzymes initiating repair of AP sites ('class II' AP endonucleases) have been identified and characterized in bacteria, yeast, Drosophila

and mammals (Demple and Harrison, 1994; Demple et al., 1991; Doetsch and Cunningham, 1990). These repair proteins hydrolyze the phosphodiester backbone immediately 5' to an AP site generating an abasic deoxyribose 5-phosphate which is released by a 5'-deoxyribophosphodiesterase (dRPase) or 5'-exonuclease followed by DNA synthesis and ligation. These enzymes have also been shown to contain repair activity for 3'-terminal oxidative lesions (Demple and Harrison, 1994; Demple et al., 1986; Henher et al., 1983; Ramotar et al., 1991). By hydrolyzing T-blocking fragments from oxidized DNA, these enzymes can produce 3'-hydroxyl nucleotide termini, permitting DNA repair synthesis. Recently, the human APE repair enzyme has been found to display multiple functions. Besides AP site DNA repair activity, APE potentiates the binding of Jun-Jun homodimers or Fos-Jun heterodimers to DNA by altering the redox state of these proteins (Walker et al., 1993; Xanthoudakis and Curran, 1992; Xanthoudakis et al., 1992, 1994; Yao et al., 1994). Hypoxic conditions in cell lines have been shown to increase APE mRNA levels (Walker et al., 1994; Yao et al., 1994). APE expression has recently been shown to be modulated in epithelium in a pig wound-healing model (Harrison et al., 1995). Finally, the APE enzyme may also be involved in transcription repression of the parathyroid hormone gene through altered extracellular calcium levels (Okazaki et al., 1994). Currently our knowledge of the sites of APE expression are limited. Northern analysis studies have shown that this gene is expressed in all tissues, albeit at relatively similar levels. Little, however, is known about whether this gene is differentially expressed in specific subpopulations of cells or differentially during development. To provide insights into the cellular sites of APE action, we have used in situ hybridization to examine the expression of APE gene expression in rat tissues. We now provide evidence that the APE gene is not homogeneously expressed, but rather is found in subpopulations of cells in the brain and testes. We also provide evidence for widespread APE gene expression during development, with some differential levels of specific tissue expression.

T.M. Wilson et al. / Mutation Research 362 (1996) 237-248

2. Materials and methods 2.1. Animals

Wistar-Harlan rats were supplied by Harlan Laboratories (Indianapolis, IN). For studies of adult animals (brain and testis studies), 400 g 60-90 dayold-male animals were used. For developmental studies, pregnant dams (gestation day 0 = day of sperm positively) were used. Animals were killed by decapitation while anesthetized. Specimens were rapidly dissected, immediately frozen in chilled 2-methylbutane ( - 20°C), and stored at -80°C. 2.2. Biochemisto,

APE assays were performed as has been previously described (Huq et al., 1995; Wilson III et al., 1994) and is briefly described below. PM2 DNA was partially depurinated by heating at 70°C at pH 5.2 for 15 min to produce roughly one AP site per DNA circle. Assay mixtures (50 /xl) contained 25 mM Tris-HC1, pH 7.5, 10 mM MgCI 2, 170 fmol of PM2 [3H]DNA molecules (27 cpm/fmol molecules), and a sample of protein. Incubations were for 10 rain at 37°C. The number of nicks introduced into closed circular PM2 DNA was determined by a nitrocellulose filter binding assay as described by Kuhnlein et al. (1978). 2.3. In situ hybridization

APE gene expression was studied by in situ hybridization using cRNA probes. 3sS-labeled antisense and sense probes were generated from the coding region of the rat APE cDNA (Rivkees and Kelley, 1994; Wilson et al., 1994). Probes were labeled with [c~-3SS]thio-UTP (New England Nuclear, Boston, MA) using the Gemini System (Promega; Madison, WI) (Reppert et al., 1991; Rivkees et al., 1992; Rivkees and Kelley, 1994). In situ hybridization was performed similar to as described (Reppert et al., 1991; Rivkees et al., 1992; Rivkees and Kelley, 1994). Sections were first incubated in 4% paraformaldehyde (30 rain), 0.2 HC1 (30 rain), acetylated in 0.1 M triethanolamine containing 0.2% acetic anhydride (10 min), and dehydrated through alcohol. Sections were covered with 50 /xl of hy-

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bridization buffer containing 5 × 10 6 cpm of probe per ml. Glass coverslips were applied, and sections were incubated overnight at 55°C. The next day, coverslips were removed, and sections were washed in 2 X SSC (30 rain, 20°C), RNase A (5.0 mg/ml, 60 rain, 37°C), 2 X SSC (30 rain, 20°C), 0.1 X SSC (60 min, 60°C), and 0.1 X SSC (30 min, 20°C). Sections were then dehydrated in alcohol containing 0.3 M ammonium acetate and dried. Film autoradiographs were generated by exposing slides to Kodak SB5 film for 2 to 7 days. Emulsion autoradiographs were generated by dipping sections in Kodak NTB2 emulsion and exposed for 5-14 days. Sections were developed using Kodak 19 developer (4 rain), and then stained with 0.5% toluidine blue to facilitate identification of anatomical structures. Labeled brain structures were classified according to the atlas of Paxinos and Watson (1986). For developmental studies the atlas of Rugh (1964) was used. 2.4. PCR primers

Oligonucleotides used for the comparative RTPCR of the APE mRNA expression levels were as follows: 5' oligonucleotide; 5'-CTGGACTTACATGATGAATGCCCG-3' and 3' oligonucleotide; 5'GAAGAGATAACGCACTGGTCTCCT-3'. These oligonucleotides correspond to the region of the rat APE gene at 748 bp 3' of the ATG and 47 bp 3' of the TGA, respectively. 2.5. RNA isolation, reuerse transcription, polymerase chain reaction, Southern blotting and DNA hybridization

Total RNA was extracted from the tissues according to the procedure of Chomcyznski and Sacchi (1987) and as previously described (Kelley et al., 1993; Wilson et al., 1995). Briefly, the cells were homogenized in guanidinium thiocyanate, followed by acid phenol-chloroform extraction, precipitation with isopropanol, reextraction with guanidinium thiocyanate, and isopropanol precipitation. This procedure resulted in RNA isolation without genomic DNA contamination. The methodology for semi-quantitative or comparative reverse transcription-polymerase chain reac-

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tion (cRT-PCR) has been described in detail in our previous publications (Kelley et al., 1993; Wilson et al., 1995). A brief description follows: Three micrograms of total RNA were added to a reaction tube containing 1 x reverse transcription buffer, dNTPs, 10 U RNasin (Promega, RNase inhibitor), 100 pmol oligo(dT) primers (Bethesda Research Laboratories, Gaithersburg, MD), and RNase-free deionized distilled water to a final volume of 19/xl. This mixture was heated for 10 rain at 65°C and quenched on ice, and 200 U Moloney murine leukemia virus RNase H reverse transcriptase (Superscript, Bethesda Research Laboratories) in 1 /xl are added for a total reaction volume of 20 ml. This mixture was incubated at room temperature for 10 rain and then at 42°C for 1 h. The reaction was terminated by heating at 95°C for 5 rain and quenching on ice. RNase-H (2 U) was then added to the reaction and incubated for 20 rain at 37°C. Five microliters of the reverse transcription reaction from each sample were diluted into a final volume of 100 /xl in 1 X PCR buffer and 2 U Tfl thermostable polymerase (Epicentre, Madison, WI). The polymerase amplification was carried out using a MJ Research programmable heating/cooling dry block for 25 cycles of amplification (94°C, 30 s; 60 C, 1 rain; 72 C, 2 rain), followed by 10 rain at 72°C. A reaction was performed as described above, but without the addition of reverse transcriptase as a test for the presence of contaminating genomic DNA in the RNA samples. The oligonucleotide primers spanned at least one intron for genomic contamination controls. Following amplification, 10 /~1 of the PCR products were electrophoresed on 2% agarose gels containing 0.5 m g / m l ethidium bromide in Tris borateEDTA buffer. After photography of the gels using UV light, they were prepared for transfer and blotted onto nitrocellulose. The nitrocellulose was prehybridized in 5 x Denhardt's solution, 250 m g / m l denatured salmon sperm DNA, 1% ( w / v ) sodium dodecyl sulfate (SDS), 0.1% tetrasodium pyrophosphate, and 50% deionized formamide for 12 h at 42 C. Hybridization was performed overnight under stringent conditions at 42°C in the same solution as that used for prehybridization, but included in the mixture was a [ c~-32P]dCTP random primed APE or histone H3.3 cDNA. The blots were washed under

high stringency conditions in 1 X SSC and 0.5% SDS at room temperature for 15 rain and then three times in 0.2 X SSC-0.5% SDS at 65°C for 20 rain/wash. The washed filters were blotted dry and exposed to X-ray film at - 7 0 ° C with enhancing screens (Dupont/New England Nuclear, Boston, MA). Autoradiographs were scanned using a MicrotekII scanner and the data quantified using SigmaScan software (Jandel Scientific). Oligonucleotides to the Histone H3.3 gene were used as an internal control for reverse transcription and amplification variance. H3.3 has been extensively used and does not change its expression during the cell cycle (Kelley et al., 1993; Wilson et al., 1995).

2.6. Ouerexpression of glutathione S-transferase: APE fusion proteins fi~r biochemisto, analysis For overexpression of glutathione S-transferase: rat and human APE fusion protein (GST-APE), bacterial cultures (10 ml) containing the rat or human GST-APE fusion constructs were grown overnight at 37°C in LB plus 100 ng//xl ampicillin, and as described previously for other DNA repair genes in our laboratory (Wilson Ill et al., 1994). Overnight cultures were diluted I to 10 in fresh, pre-warmed (37°C) LB medium supplemented with ampicillin and grown for 1 h at 37°C with shaking. Expression of the GST-APE fusion proteins was induced by adding isopropyl-/3-D-thio-galactoside (IPTG) to a final concentration of 0.1 mM and growing the cells for an additional 3.5 h at 37°C. Cells were harvested by centrifugation at 1000 × g for 10 rain and washed once with PBS, pH 7.4. Packed cells were resuspended in 3 ml of PBS and lysed by mild sonication (two 20-s bursts) on ice. Both the rat and human APE fusion proteins were totally soluble. Cellular debris was pelleted by centrifugation at 8500 X g for 10 rain at 4°C and the supernatant collected containing the soluble rat and human APE fusion proteins. The soluble fraction of each protein had Triton X- 100 added to a final concentration of 1% and loaded onto a glutathione Sepharose 4B column, pre-equilibrated with PBS. Binding of the GST-APE proteins was carried out for 3 b on a nutator at 4°C. The column was subsequently washed with 20 column volumes of PBS containing 1% Triton X-100. Following the

T.M. Wilson et al./ Mutation Research 362 (1996) 237-248 wash, the fusion proteins were eluted with 50 mM Tris, pH 7.5, containing 10 mM glutathione, fractions collected and analyzed by SDS-polyacrylamide gel electrophoresis. The column purified proteins were used for biochemical analysis and antibody production.

3. Results 3. I. Biochemical activit3, o f the rat APE We have previously published the cloning and coding region of the rat APE (Wilson et al., 1994). The data presented in Table 1 demonstrate the specific activity for the rat APE on AP containing DNA. In direct comparisons of recombinant rat and human APE activity, both had similar activity on AP DNA and no activity on non-AP containing DNA. These proteins were assayed as glutathione S-transferase (GST) fusion proteins with the GST portion neither hindering the activity nor adding significantly to the specific activity of the rat and human APEs (Table 1; Huq et al., 1995). A more detailed analysis of the rat AP endonuclease activities and kinetics is beyond

Table 1 Rat APE repair activity Addition

H3.3

Nicks introduced/DNA molecule

GST-rAPE GST-hAPE GST

0.43 0.61

AP

NT

0.0 l < 0.01 0.11

0.09

Reactions (50 ~1) contained 25 mM Tris-HCl, pH 7.5. 10 mM MgC12, 170 fmol of PM2 [3HIDNA in which the depurinated DNA substrate containedroughly one AP site per DNA circle and 50 ng of protein, GST-rAPE is the rat APE (Huq et al., 1995) and GST-hAPEis the human APE (Demple et al., 1991) produced as glutathione Stransferase fusion proteins in E. coli.

the focus of this discussion and will be published elsewhere (Huq et al., 1995). 3,2. Regional expression o f APE mRNA To identify the sites of APE expression, multiple tissue comparative reverse transcription-polymerase chain reaction (RT-PCR) analysis for the rat APE gene was performed (Fig. 1). After normalizing APE

::

rAPEN

241

...

-

-- 231 bp

--

-- 213 bp

Fig. 1. Comparative RT-PCR (cRT-PCR) of the steady-state mRNA levels in various adult rat tissues for the APE gene. Total RNA was isolated and subjectedto RT-PCR(25 cycles) using the Histone H3.3 gene as an internalcontrol for reverse transcriptionand amplification variance. The blot was transferred and probed with the rat cDNA for the APE gene or a Histone H3.3 specific DNA fragment.

T.M. Wilson et al. / Mutation Research 362 (1996) 237-248

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APE

Fig. 2. In situ hybridization analysis during rat fetal development. The developmental expression pattern of APE mRNA was examined at gestation days (GD) 8, 14 and 17, from left to right respectively. PL, placenta; S, spinal cord; H, heart; LV, liver; T, telencephalon; M, mesencephalon; SM, submandibular gland; TH, thymus; I, intestine; K, kidney; L, lung; BF, brown fat. In all experiments, the sense RNA probe gave essentially no background (see Fig. 5 for example of lack of background signal with a sense APE cRNA probe).

m R N A levels for the internal control (historic H3.3), it was determined the A P E gene is expressed at relatively the same levels in all tissues e x a m i n e d and similar results are obtained using Northern blot analysis (data not shown), This has also been o b s e r v e d in human tissues as well (data not shown). H o w e v e r , as shown below, this type of analysis does not reveal the true nature of A P E tissue specific expression and leads to erroneous and misleading conclusions concerning the lack o f differential expression o f A P E

m R N A in various cells, in particular, repair, in general.

and D N A

3.3. In situ analysis o f A P E gene expression during rat fetal development To ascertain whether the A P E gene was differentially expressed in any tissues, we e x a m i n e d A P E g e n e expression during rat fetal d e v e l o p m e n t to determine if A P E expression varied d e v e l o p m e n t a l l y .

APE Fig. 3. In situ hybridization of APE mRNA on adult, male rat sagittal and coronal brain tissue sections. APE mRNA expression was examined using sagittal or coronal tissue sections at 100 #m intervals spanning the entire brains of rats (n = 3 or more per orientation, although only one section per orientation is shown). Areas of higher APE expression are; pyriform cortex (PC), hippocampus (H), cerebellum (CB), suprachiasmatic nuclei (SC) and supraoptic nuclei (SO). Only the antisense probes are shown as the sense probes showed no background level of hybridization (see Fig. 5). T, thalamus: LM, lateral mammillary body; SN, substantia nigra; PN, pontine nucleus.

TM. Wilson et a l . / Mutation Rese~wch 362 (1996) 237-248

At all ages examined, which spanned periods of organogenesis and neurogenesis, a high level APE mRNA expression was present in all somatic sites examined (Fig. 2). At gestation day (GD) 8, the fetus was examined within the uterus. A moderate hybridization signal was also present over the embryo. At GD 14, the somatic pattern of labeling was widespread and a strong hybridization signal was present over all organs that could be identified (Fig. 2). Although APE mRNA was expressed at high levels in all structures examined, when relative by-

243

bridization signals were compared among different organs, APE mRNA levels were actually the greatest in the liver, thymus and brain. At GD 17, the pattern of hybridization was similar to that observed at GD 14.

3.4. Distribution and differential expression of APE mRNA in adult rat brain Following up on the striking observation that APE mRNA was highly expressed in specific regions of the developing rat brain, we next examined the

Fig. 4. Emulsionsof the in situ pattern of APE gene expression in adult male rat brains. Labeling was seen in several cortical layers in emulsion autoradiographs(A and B). Detailed analysis of the hippocampus is presented in C and D and in the cerebellum in E and 1=. Py, pyramidal cells or neurons:DG, dentate gyrus; H, dentate hilus; CAI, CA2 and CA3 are regions of Ammon's born: Pc or P, Purkinjecells: M, molecularlayer; G or GR, granule layer; W. white matter: CN. deep cerebellarnuclei.

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TM. Wilson et aL /Mutation Research 362 (1996) 237 248

regional expression of brain APE gene expression by in situ hybridization. Examination of the distribution of APE mRNA in the brain revealed a widespread, but heterogeneous pattern of distribution. APE mRNA was expressed at moderate levels throughout the entire brain; however, it was expressed at very high levels in several distinct brain regions (Figs. 3 and 4). APE mRNA was expressed at higher levels in the hippocampus, suprachiasmatic nuclei (SCN), the site of a circadian clock, and in the supraoptic nuclei (SON) and paraventricular nuclei (PVN); nuclei which regulate water level balance. High levels of APE expression were also found in the pyriform cortex (Figs. 3 and 4). Emulsion autoradiographs revealed that cortex labeling was heaviest over the pyramidal cells in layers III and V. Lower levels of labeling were also seen over cells in the other cortex layers (Fig. 4). APE mRNA expression was also detected at high levels in the hippocampal formation (Figs. 3 and 4). Emulsion autoradiographs revealed heavy labeling over granule cells in the dentate gyrus (Fig. 4). In the dentate hilus, labeling was present over pyramidal neurons and not over interneurons (Fig. 4). In Ammon's horn, heavy labeling was seen over pyramidal cells in the CAI, CA2 and CA3 regions. Specific, but low level labeling of interneurons could be detected in the stratum oriens, lacunosum-moleculare, and radiatum (Fig. 4). In the cerebellum (Figs. 3 and 4), Purkinje cells were the most heavily labeled cell type. In the molecular layer, there also was low level labeling of cells, while in the granule layer, there was a moderate hybridization signal. The deep cerebellar nuclei also expressed high levels of APE mRNA (Fig. 4). In the basal ganglia, which contains different subpopulations of neurons, moderate labeling of all neurons was observed. The higher labeling seen in the specific brain regions is not just due to cell density. As seen in the cortical region (Fig. 4), there is heavier labeling over the different cells in the various layers, yet the cell density is not dramatically different between these layers. Also, as seer in Fig. 4F, the Purkinje cells have a much greater number of grains than neighboring cells in the molecular or granular regions, again demonstrating increased expression of APE mRNA and not just a cell density artifact.

3.5. Differential expression of APE in adult rat testis Because of the differential expression of the APE gene in post-mitotic tissue, we were interested in the expression pattern of APE in a tissue undergoing vigorous cellular differentiation and cell-cycling. The testis are ideal for these in vivo studies since they fit this criteria and understanding DNA repair gene expression in developing germ cells has important ramifications concerning which DNA repair systems are active in maintaining germ line DNA integrity. Emulsion autoradiograpbs generated from in situ hybridization studies revealed that APE mRNA expression varied with the stage of spermatogenesis (Fig. 5). APE mRNA was present at modest levels over spermatogonia and spermatocytes. However, the highest level of APE mRNA expression was detected over round spermatids, which are post-meiotic cells. Sense probes on these, and the previously shown developmental and brain sections, revealed no nonspecific hybridization (Fig. 5).

4. Discussion

The major responsibility of normal cells in their maintenance of genetic stability is most likely not through the much studied nucleotide excision repair (NER) pathway, but through the recombination/mismatch repair systems (Modrich, 1994) and through the DNA base excision repair (BER) pathway (Lindahl, 1994). This hypothesis is supported by the notion that the major insults to DNA are not bulky lesions, but minor alterations in the DNA due to replicative events, or via oxidative metabolism, spontaneous loss of bases through hydrolysis (apurinic/apyrimidinic sites), endogenous methylation (S-adensylmethione), and environmental agents, such as cancer chemotherapeutic agents, that can alter DNA structure or base sequence (Barnes et al., 1993; Doll and Peto, 1981; Friedberg, 1985; Lindahl, 1992, 1993, 1994). Therefore, the major challenge to normal cells, on a day to day basis, involves the removal of damaged or inappropriate bases and resynthesis of DNA using the undamaged complementary strand as a template. This is primarily accomplished using the DNA BER pathway which includes the APE gene as a major constituent in the

T.M. Wilson et al. / Mutation Research 362 (1996) 237-248

Fig. 5. In situ hybridization for APE mRNA on adult male testis. In three separate studies using testes from different animals, prominent hybridization signals were seen for APE mRNA in developing round spermatids. SG, spermatogonia; SC, sperrnatocytes; RS, round sperrnatids: ES, elongating spermatids; L, leydig cells. The top panel is a dark-field exposure of emulsions of a cross-section through the testis tubules. The middle panel represents a magnified view of the intense pattern of APE expression in the round spermatids. The bottom panel is an exposure of a similar section using an APE sense RNA probe showing the lack of any background signal. Not all tubules contain round spermatids as the tubules develop in an asynchronous pattern. Similar results with sense probes were observed in all tissue sections examined.

245

repair o f base lesions (Friedberg, 1985; Mitra and Kaina, 1993). Currently, the predominant theory stipulates that D N A repair genes are 'housekeeping' in nature and are not interesting from a differential e x p r e s s i o n / g e n e regulation point of view. However, although the APE gene was expressed in all tissues examined with some varying degree o f expression, the subtissue distribution of expression was dramatically different. Examination of the distribution of APE m R N A in the brain revealed a widespread, but heterogeneous pattern of distribution. Of these regions, most of which are either the more metabolically active brain cells or are sites of excitatory amino acid (EAA) action, several sites were of particular interest. In agreement with our previous observations, APE m R N A was highly expressed in the suprachiasmatic nuclei, which is the site of a circadian pacemaker (Moore et al., 1989; Rivkees and Kelley, 1994). APE m R N A levels were also very high in the supraoptic and paraventricular nuclei, which are involved in the regulation of water balance (Sben et al., 1992; Rivkees and Kelley, 1994). High levels of APE m R N A expression were detected in the hippocampal formation. The hippocampus is a metabolically active tissue that is a major site of excitatory amino acid (EAA) action (Coltman and Monaghan, 1986). The hippocampus is susceptible to EAA-induced cell damage, which triggers oxidative damage of D N A (Coltman and Monaghan, 1986). Because A P E is involved in oxidative D N A damage repair, the high level of APE expression in these cells may be an evolved protective mechanism Ibr this type of cellular damage. Furthermore, our results showed that highest levels of APE m R N A were present in granule cells of the dentate gyrus and pyramidal cells of A m m o n ' s horn, which are cells that are also major sites of E A A action (Coltman and Monaghan, 1986). Thus, high level APE gene expression may also protect neurons in these regions. W e were also intrigued by the observation that there was low level APE m R N A expression in the dentate hilus. The hilus is a major site of hippocampal degeneration in seizure disorders and E A A toxicity models (Swanson, 1995). In future studies it will be interesting to determine if low levels of APE in this region contributes to the vulnerability of cells in the hilus to exocitotoxic damage.

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In the cerebellum, high levels of APE mRNA were observed in Purkinje cells. These cells are very metabolically active and are innervated by excitatory projections (Coltman and Monaghan, 1986). The deep cerebellar nuclei, which also receive excitatory projections (Coltman and Monaghan, 1986), also expressed high levels of APE mRNA supporting the concept that APE is expressed at sites of EAA action or in metabolically active brain cells. Pyramidal cells of the cerebral cortex that receive excitatory projections also expressed relatively high levels of APE mRNA. Furthermore, DNA polymerase /3, the enzyme responsible for BER-related DNA synthesis, has been localized in the adult rat brain to the cortex, cerebellum (particularly the Purkinje cells) and hippocampus, similar regions as demonstrated here for the APE (Zucconi et al., 1992; Kelley and Rivkees, unpublished data). Besides a predisposition to cancer, individuals afflicted with a DNA-repair disorder display a bewildering array of clinical symptoms, including immunodeficiencies, neurological problems, skeletal abnormalities and altered growth. AP sites in DNA have been shown to hinder transcription and, if not acted upon by an APE, leads to altered transcripts (Zhou and Doetsch, 1993). An accumulation of changed transcripts and, therefore, altered protein products may result, over time, in neuronal degradation as has been observed in numerous DNA repair associated diseases and may be involved in aging processes (Friedberg, 1985; Bradley et al., 1989; Boerrigter et al., 1992; Boerrigter and Vijg, 1993). Thus, the role of DNA BER may be extremely important in postmitotic cells, particularly those that are transcriptionally active or at a higher risk due to increased oxidative metabolism. During development, which spanned periods of organogenesis and neurogenesis, high level APE mRNA expression was present in all somatic sites examined. The presence of widespread and high level APE expression during development is expected to help protect the embryo and fetus against potential DNA replication errors during active periods of meiosis and mitosis. Since we did not compare pre- and post-natal APE mRNA expression, we do not know if expression of this gene is greater during organogenesis compared to later stages. Furthermore, at the gestational times we studied, APE

mRNA levels were very high in the liver and thymus, along with the developing brain. At these times, the liver has a much larger role as an hematopoietic organ, as opposed to in adults when the liver is more involved in detoxification. The expression of the DNA BER pathway is currently being investigated in developing and purified populations of hematopoietic cells. Further detailed analyses of the differential expression of the BER pathway during development may contribute to understanding these observed the various clinical phenotypes observed. When APE mRNA expression was examined in the testis, it was found to vary with the stage of spermatogenesis. APE mRNA expression was present at modest levels over spermatogonia and spermatocytes. However, highest levels of APE mRNA expression were detected over round spermatids. These are post-meiotic ceils which mature into spermatozoa via a process that involves nuclear condensation (Clermont, 1972). Thus, although APE mRNA was expressed throughout germ cell development, its expression was greatest after completion of meiosis during spermatozoa maturation. High level APE expression at this stage would thus favor correction of DNA errors before mature sperm are formed. The results presented in this report represent an initial investigation of the relationship of DNA BER and cell specificity of expression. This type of analysis allows us to begin to understand the regulation and functional relationship of this gene product to the various cell types. For example, why is the level of expression of the APE gene so strikingly high in specific cells in the brain such as the SCN, PVN, SON, pyriform cortex, hippocampus and cerebellum? What is its function during fetal development and what controls the level of expression? Why do round spermatids in developing germ ceils express a much higher level of APE mRNA compared to the other stages of germ cell development? Furthermore, and maybe most importantly, the differential expression of the rat APE gene in specific regions of rat brain, testis and particular tissues during development is in marked contrast to the expression of the NER gene ERCC3, which has been shown to be ubiquitously expressed at the same levels in mouse brain during development (Hubank and Mayne, 1994). We hope to use various rat and human neuronal and glioma cell lines, along with in vivo

T.M. Wilson et al. / Mutation Research 362 (1996) 237-248

experimentation to further elucidate the regulation and functional significance of APE gene expression, and other BER genes, in specific sub-tissue locations.

Acknowledgements We would like to thank the Wells Center oligonucleotide synthesis facility which is supported by the Riley Memorial Association for synthesis of the various oligonucleotides used in this report. MRK and WAD are supported by NIH grant RR09884 and a grant from the Council for Tobacco Institute 3739. SAR is supported by the American Heart Association and NIH NS32624.

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