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Cloning, characterization and expression of two Xenopus bcl-2-like cell-survival genes
Gene. 158 (1995) 171 179 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
171
GENE 08877
Cloning, characterization and expression of two Xenopus bcl-2-1ike cell-survival genes (Programmed cell death; apoptosis; amphibian metamorphosis; c-myc; oncogenes)
Jorge Cruz-Reyes* and Jamshed R. Tata Division of Developmental Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Received by J.K.C. Knowles: 16 December 1994; Accepted: 30 January 1995; Received at publishers: 23 February 1995
SUMMARY
We describe two cloned cDNAs, termed xR1 and x R l 1, isolated from a Xenopus laevis stage 28-30 embryonic head cDNA library. Comparison of amino acid (aa) sequences derived from nucleotide (nt) sequences of xR1 and xR11 cDNAs revealed substantial homology with bcl-2-related genes, especially with bcl-xL. In particular, there was a marked conservation of the BH1 and BH2 domains considered to be important for the anti-cell death and heterodimerisation properties of bcl-2. Constitutive expression of x R l l in cultured rat fibroblast (Rat-l) cells conferred a strong protection against cell death induced by the cytotoxic agents staurosporine and cycloheximide, by serum deprivation and specific deregulation of c-myc. Measurement of xR1 and xR11 mRNAs by RNase protection assay revealed similar widespread expression in Xenopus embryos and tadpoles. Except for an abrupt increase in the accumulation of xR1 and xRI1 mRNAs in brains of mid-metamorphic and post-metamorphic tadpoles and adults, there was insignificant modulation of their expression in tissues undergoing total regression (tail) or morphogenesis (limb) during natural or thyroid hormone-induced metamorphosis. These findings raise the possibility of continuing expression of cell survival genes in tissues undergoing total regression during post-embryonic development.
Programmed cell death (PCD) is now recognised to be an integral part of, and essential requirement for, many developmental processes (for reviews, see Ellis and Horvitz, 1986; Bowen and Bowen, 1990; Oppenheim, 1991; Ellis et al., 1991; Tomei and Cope, 1991; Lavin and Watters, 1993; Dexter e~: al., 1994). It is particularly important during post-embryonic development and is
associated with such diverse phenomena as neurogenesis, morphogenesis, remodelling of tissues, immune responses and tissue regression or organolysis. In the last decade a number of genes have been identified in different systems whose products are associated with PCD, often manifested as apoptosis, which is characterised by nuclear condensation and DNA fragmentation (Hengartner et al., 1992; Korsmeyer, 1992; Yuan and Horvitz, 1992; Yuan et al., 1993; White et al., 1994; Woronicz et al., 1994).
Correspondence to: Dr. J.R. Tata, Laboratory of Developmental Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Tel: (44-181) 959-3666 ext. 2106; Fax: (44-181) 913-8583; e-mail: [email protected] * Present address: Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2185, USA. Tel: (1-410) 955-7419.
central nervous system; cRNA, RNA complementary to cDNA; DMEM, Dulbecco's modified Eagle's medium; EF-lct, elongation factor-let; ER, estrogen receptor; FCS, fetal calf serum; kb, kilobase(s); nt, nucleotides; ORF, open reading frame; PCD, programmed cell death; SEM, standard error of the mean; Ss, staurosporin; T3, 3.3',5-triiodo-L-thyronine; TH, thyroid hormone(s); XR, Xenopus cellsurvival (anti-apoptosis) protein; XR, gene (DNA, RNA) encoding XR.
INTRODUCTION
Abbreviations: aa, amino acid(s}; bcl, B cell leukemia; bp, base pair(s); cDNA, DNA complementary to RNA; Chx, cycloheximide; CNS, SSDI 0378-1119(95)00159-X
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Fig. 1. Deduced aa sequence, based on cDNA nt sequences, of xR1, xRll and other Bcl-2-type proteins that modulate apoptosis. Areas of aa identity between the Xenopus sequences, human Bcl-2, Bcl-xL and Bax, C. elegans Ced-9, and viral bhrf-1 are indicated by stippling (Vaux et al., 1988; Boise et al., 1993; Oltvai et al., 1993; Hengartner, 1992; Henderson, 1993). The highly conserved BH1 and BH2 domains of Bcl-2 protein are indicated by open bars, and aa essential for suppression of apoptosis and heterodimerisation to Bax are marked by dots (Yin et al., 1994). The 63-aa region of human Bcl-xL deleted in human Bcl-xs is denoted by arrows. A predicted 19-aa hydrophobic domain and flanking charged residues that are present in all the sequences are indicated by a stippled bar (Chen-Levy and Cleary, 1990). The nt sequences of x R l l and xR1 mRNAs have been deposited with EMBL Nucleotide Sequence Database under the accession Nos. X82461 and X82462, respectively. Methods: ~.Zap-cDNA libraries prepared from tails of mid-metamorphic Xenopus tadpoles (stages 53-56), and embryonic head (stages 28-30), using a kit obtained from Stratagene, were screened with a human bcl-2 cDNA, kindly provided by Dr. G. Evan, Imperial Cancer Research Fund London, at low stringency. The filters were hybridised in 6xSSC ( l x S S C is 0.15M NaCI/0.015M Na 3.citrate, pH 7.6)/1 x Denhardt's solution/24mM Na.phosphate buffer (pH 6.5)/100 gg salmon sperm DNA per ml/0.5% SDS at 63°C overnight. The final wash conditions were 20 min at 42°C in 0.2 x SSC. cDNA inserts from two hybridising clones to bcl-2 cDNA, termed xR1 and xR11, were subcloned, xR1 was in vivo excised in pBluescript SK (+/ ), whereas xR11 was amplified by PCR using pBluescript primers with EcoRI and SacI linkers and subcloned into pBluescript KS ( + / - ) (Stratagene, La Jolla, CA, USA). Both xR1 and xR11 cDNAs were
These genes have been classified into two groups: (i) those whose products induce cell death and (ii) those that protect against cell death or confer survival. In mammalian cells it has been established that the bcl-2 gene plays an important role in cell survival (Korsmeyer, 1992; Korsmeyer et al., 1993); it is homologous, structurally and functionally to ced-9 gene of C. elegans, as demonstrated by the prevention of cell death by human bcl-2 introduced into ced-9 mutants of developing nematodes (Vaux et al., 1992). These findings, and a continuously growing family of bcl-2-related molecules that modulate apoptosis (e.g., bcl-x, bax, bhrf-1), argue for the existence of other bcl-2-1ike genes, each playing a unique role in maintaining development or regulating PCD (Boise et al., 1993; Oltvai et al., 1993; Henderson et al., 1993). Our interest in studying PCD stems from work on amphibian metamorphosis, which is a most dramatic example of post-embryonic development. The wideranging phenotypic changes produced in metamorphosis, such as limb morphogenesis, acquisition of new hepatic functions, remodelling of CNS and extensive tissue regression, resemble many associated with mammalian fetal and neonatal development. They are all obligatorily induced and regulated by thyroid hormones 3,3',5triiodo-L-thyronine and thyroxine (T3, T 4 ) , and are dependent on new protein synthesis (Tata, 1993). A striking feature of amphibian metamorphosis is the widespread cell death in regressing larval tissues, such as the tadpole tail, gills, and gut, which accompanies morphogenesis of limb buds and lungs and transformation of skin and the central nervous system (Tata, 1966; Lockshin, 1981; Yoshizato, 1989; Tata et al., 1991). It was considered that bcl-2-1ike cell survival genes may themselves be positively and negatively regulated in these contrasting postembryonic morphogenetic and regressive developmental processes. We, therefore, embarked upon the cloning and characterisation of two homologues of the bcl-2 family of genes in Xenopus laevis termed xR1 and xRll, examine their role in protection against PCD, and search for a
isolated from the embryonic head library and sequenced by a dideoxy termination method in plasmid DNA (US Biochemical, Amersham International, Amersham, UK). For transfection and subsequent functional analysis, a SacI-SalI xR11 insert was excised from pBluescript KS ( + / - ) , and subcloned into pSP64 plasmid (Promega, Madison, WI, USA), from which an EcoRI x R l l insert was isolated and subcloned into the EcoRI site of pBabe-puro (Morgenstern and Land, 1990). Orientation of the insert was determined by restriction enzyme mapping. A plasmid with the insert in the forward orientation was designated pBabe-puro-xR1 sense (S), while a plasmid with the insert in the reverse orientation was named pBabe-puro-xRll antisense (A). Sequence comparisons and peptide analyses were performed with the Genetics Computer Group (GCG, Madison, WI, USA) Programs for the GCG Package (1991): BLAST, MAP, GAP, PILEUP, GELSTART and other sequence assembling programmes.
173 possible developmental and thyroid hormonal regulation of expression of these genes during early embryogenesis, metamorphosis of tadpoles and in fully developed adults.
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RESULTS AND DISCUSSION
(a) Cloning of xR1 and x R l l We used a human bcl-2 cDNA probe to clone Xenopus genes by low-stringency hybridisation of multiple cDNA libraries made from developing head and tail tissues. Two cDNA clones, thus isolated, were termed xR1 (2.0 kb) and xR11 (1.1 kb), which exhibited moderate and strong hybridisation to the bcl-2 cDNA probe, respectively. These two cDNAs were sequenced and Fig. 1 shows their deduced aa sequences, xR1 and xR11 displayed a high level of sequence identity with members of the bcl-2 gene family reported to modulate experimentally induced apoptosis, xR1 and xR11 both exhibit an overall 40% aa identity to bcl-2. They contain the BH1 and BH2 domains of Bcl-2 required for inhibition of apoptosis and heterodirnerisation with the cell death inducing Bcl-2-derived fragment bax (Yin et al., 1994) especially as regards aa known to be essential for these two processes (e.g., Gly 145'194and Trp 188"195of Bcl-2), and a C-terminal hydrophobic region that functions as a signal-anchor sequence responsible for the integral membrane position of Bcl-2 (Chen-Levy and Cleary, 1990). xRl 1 shares 57% aa identity with human Bcl-XL the large spliced form of Bcl-x (compared with 44% between Bcl-2 and Bcl-XL), and contains a 204-aa ORF. To determine if either of these Xenopu:~ cDNA clones give rise to an appropriate sized protein product, they were transcribed as RNA and subjected to in vitro translation. As seen in Fig. 2, only x R l l cDNA yielded a translation product of the approximate size predicted by the O R F (approx. 22.5 kDa). Consequently, it was decided to fully characterise only x R l l cDNA functionally by over-producing xR11 protein in tissue culiture cells by transfection. bcl-2-1ike
(b) x R l l is a negative regulator of apoptosis Because of the close aa sequence similarity between human bcl-xL and the O R F of xR11 (Fig. 1), and the transcription-translation of the cDNA to the protein of expected size, we sought to determine whether x R l l could serve a similar function as Bcl-XL and other Bcl-2like proteins in preventing apoptotic cell death (Boise et al., 1993; Williams and Smith, 1993). Towards this end, the rat fibroblast cell line Rat-1 was transfected with the x R l l cDNA inserted in both orientations into the EcoRI cloning site of the pBABE-puro expression plasmid (Morgenstern and Land, 1990). This approach has been successfully exploited to study the survival function of
<-22.5kDa
Fig. 2. In vitro translation of xRl and xRll mRNAs, xR1 and xRll mRNA were used for in vitro translation in the presence of [aSS]methionine. The translation products were resolved by SDSPAGE. Size of the resulting xR11 protein is indicated on the right in kDa. Translation of a constructcontainingluciferasecDNA was used as a control (C). Methods: pBluescript plasmids containing xR1 and xR11 cDNA inserts were linearised at the 3' multiple cloning site with EcoRI and transcribed with T3 and T7 RNA polymerases,respectively, for 1 h at 37°C. The resulting run-off transcripts were phenolchloroformextractedand ethanol precipitated. In vitro translation was then performed with a rabbit reticulocytelysate kit (Promega) in the presenceof [35S] methioninefor 1 h at 30°C.The lysate(5 ~tl)was added to SDS loading buffer and subjected to 0.1% SDS-15% PAGE. Gels were dried and exposed to X-ray (X-OMA) AR-film(Kodak). human Bcl-2 (Fanidi et al., 1992). High levels of expression of xR11 mRNA in transfected rat cells was demonstrated by RNase protection assay with an x R l l sensespecific riboprobe (data not shown). Puromycin-resistant cells were selected and then clonal populations were tested for resistance to experimentally induced apoptosis. xRll-transfected cells had similar growth kinetics to the parental cell line, as well as to the puromyein-transfected control cells (not shown). Puromycin-resistant clones were thus subjected to the cytotoxic agents Ss and Chx, serum-deprived culture conditions, and to c-myc deregulation. Apoptosis induced by all these procedures, has been previously shown in several cell lines, and can be blocked by Bcl-2 over expression (Bissonnette et al., 1992; Fanidi et al., 1992; Jacobson et al., 1993). We characterised apoptosis of Rat-1 fibroblasts both by propidium iodide fluorescence of DNA to visualise chromatin condensation and fragmentation, and DNA size separation by agarose gel electrophoresis. Rat-1 cells transfected with xR11 eDNA in the anti-sense orientation underwent rapid cell death following the addition of Ss or Chx as determined by nuclear condensation and fragmentation (Fig. 3), and chromatin cleavage at nucleosomal intervals
174
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Fig. 3. Protection against apoptosis by overexpressionofxRll in Rat-1 fibroblasts. Cells were cultured in 10% FCS and 1 ~tM Ss for 18-20 h to induce apoptosis. (A) Rat-1 clones expressing xR11 in the antisense orientation. Note a high proportion of cells exhibiting chromatin condensation and nuclear fragmentation (arrowed) indicative of apoptosis. (B) Constitutive expression of xR11, subcloned in the sense orientation, prevents Ss-induced apoptosis. Most cells show a regular diffuse chromatin staining pattern characteristic of viable cells. Fluorescence microscopic examination was performed under a 100 × magnification objective. Methods: Rat-1 cells, kindly provided by Dr. M. Jacobson, University College London, were cultured as previously described (Evan, 1992). The isolation of appropriately infected Rat-1 cell lines with the pBabe-puro retrovirus, directing constitutive expression, was also carried out as previously described (Morgenstern and Land, 1990). Cell lines expressingxRll in both orientations were isolated by selecting puromycin-resistant clones. Three independent transfected Rat-1 cell lines (for each xRll orientation) were studied. The results with one of each xR1/-expressing subclones, and control with no insert are shown; in similar independent experiments, the behaviour of the other subclones was indistinguishable, pBabe-puro-xRll cells were maintained in 5 ~tg/ml of puromycin (Sigma). Cells were assayed for constitutive expression of xR11 by RNase protection analysis.To assay for apoptosis induced by the cytotoxic drug Ss (Sigma, Poole, UK), cells were removed from culture dishes with trypsin, EDTA, washed once in DMEM containing 10% FCS, resuspended in DMEM and plated on poly-L-lysine-coated13-mm glass coverslips in 24-wellFalcon plates at a density of 10000 cells per well. Microscopic examination of nuclei was performed by fixing the cells with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4, and staining with propidium iodide (Sigma) as previously reported (Jacobson, 1993). Apoptotic and normal cells were scored by examination under a 40 × magnification objective in a fluorescence microscope.
(Fig. 4). I n contrast, Rat-1 cells transfected with sense x R l l c D N A d e m o n s t r a t e d significant resistance to cell death after 24 h of t r e a t m e n t (Figs. 3 a n d 4 show a representative analysis after 12 h). As regards xR1, transfection of Rat-1 cells with this c D N A did n o t exhibit the same strong p h e n o t y p e as with x R l l cDNA. The failure of transfected xR1 to protect these cells against d r u g - i n d u c e d apoptosis m a y result from the fact that, unlike x R l l , its c D N A is n o t fulllength, despite the retention of the 'anti-cell death' domains. The above procedure of i n d u c i n g cell death with Chx
Fig. 4. Size fractionation of DNA to indicate apoptosis in Rat-1 cells expressing xR11 subcloned in the sense (S) and antisense (A) orientations. Cells were treated with 1 ktM Ss or 10 ~tM Chx for 6 and 12 h to induce apoptosis. Cells expressing antisense xRll exhibit rapid breakdown of DNA to nucleosome-size fragments with either treatment, whereas the constitutive expression of sense xR11 protects Rat-1 cells from DNA fragmentation. Methods: To confirm that cell death was due to apoptosis, 106 cells transfected with xRll in either orientation and treated with 1 ~tM Ss or 10 ~tM Chx, were lysed, the DNA resolved by electrophoresis on a 1.2% agarose gel and stained with ethidium bromide, as previously described (Boise et al., 1993). All other procedures as in Fig. 3.
a n d Ss implies that apoptosis m a y be induced by multiple pathways associated with the toxicity of these substances. A n a d d i t i o n a l a p p r o a c h was therefore a d o p t e d to obviate this difficulty in studying the cell survival function of x R l l . Towards this end, we transfected Rat-1/c-myc-ER cells, which have been transformed with a functional estrogen receptor construct ( E v a n et al., 1992) with p B A B E - p u r o directing constitutive expression of x R l l , a n d then observed the effect of xR11 expression on apoptosis i n d u c e d by serum depletion a n d de n o v o synthesis of c-myc. I n these cells the c-myc p r o m o t e r is u n d e r the control of ER, so that the c-myc gene, which is inactive in the absence of estrogen, can be selectively activated by the simple a d d i t i o n of the h o r m o n e . For this purpose, Rat- 1/c-myc-ER a n d Rat- 1/c-mycER/xRl I cells were first arrested in phase Go by serum depletion, after which the progression of apoptosis was followed as a function of time in the presence or absence of de n o v o synthesised c-myc, c-myc was i n d u c e d by a d d i t i o n of estradiol-17~ to
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29 Time of culture (h) Fig. 5. Protection of Rat-1 fi~roblasts by xRll against apoptosis induced by serum deprivation and activation of c-myc. Rat-1 cells expressing c-myc fused to the human estrogen receptor (c-myc/ER), were infected with the retrovirus pEabe-puro alone or expressing xR11 in sense (S) or antisense (A) orientation. The cells were growth-arrested in culture in 0.05% FCS, while control cultures were performed in 10% FCS. Activation of c-myc in Rat-1/c-myc/xRll cells was by addition of 2 laM estradiol-17 13(E2) to the medium after 29 h of culture, as indicated by the arrow. The cells did not reach confluence. Cells were fixed, stained with propidium iodide, and apoptotic nuclei were scored with a fluorescence microscope under a 40 x magnification objective (see Fig. 3). The results represent the means + SEM of triplicate cultures. At leasl 100 cells were counted per culture. Methods: Rat-1/c-myc-ER cells, kindly provided by Dr. G. Evan, Imperial Cancer Research Fund, London, were cultured as previously described (Evan, 1992). Myc-ER cells were maintained in phenol red-free DMEM supplemented with 10% charcoal-dextran stripped FCS and 1 mg Geneticin/ml. To assay for c-myc-induced apoptosis, Rat-1/c-myc-ER cells, expressing xR11 or not, were growth-arrested in 0.05% FCS for 29 h before activating c-myc by the addition of E 2 to the medium at a final concentration of 2 ~tM. All other procedures were as for Fig. 3.
fibroblast cultures after 29 h of serum-depletion. As shown in Fig. 5, the expression of xR11 effectively abolished apoptosis induced both by serum-deprivation and c-myc induction. The percentage of dying cells was determined daily by propidium iodide staining of DNA to visualize condensed and fragmented nuclei, as shown in Fig. 3. Thus, xR11 is an efficient repressor of apoptosis induced in the absence of protein synthesis, and by c-myc activation.
(c) xR1 and xR11 are expressed in most tissues throughout development We next investigated the expression patterns for both xR11 and xR1 during natural development of Xenopus tadpoles up to adult stages, as well as following T 3induced metamorphosis. Because of the difficulty of detecting xR11 and xR1 transcripts by Northern blotting, due to their low concentrations in most tissues and developmental stages, the more sensitive RNase protection
assay was adopted for this purpose. We first estimated the relative concentrations of xR1 and x R l l transcripts in total RNA from whole Xenopus embryos and early tadpoles at different developmental stages. As seen in Fig. 6, both mRNAs were detected from stage-12 embryos to stage-50 tadpoles. A small increase in the steady-state levels can be clearly observed for the x R l l transcript, after correction for the EF-lct mRNA assayed simultaneously in the same samples of total RNA, as development progressed. Northern blot and densitometric analysis showed that the concentration of EF-I~ mRNA was very similar as compared to skeletal muscle actin mRNA and rRNA in the animals at the above stages (data not shown). For later developmental stages until completion of natural metamorphosis and in adult Xenopus, the levels of xR1 and xR11 mRNA were measured in individual tissues. In the tadpole tail, which undergoes total regression, the concentrations of both xR1 and xRI1 m R N A increased slightly from premetamorphosis (stages 51 to 54) to metamorphic climax at stage 61-62 (Fig. 7A). Of all the other tadpole tissues examined during natural metamorphosis, only the brain showed a substantial developmental regulation of xR1 and xR11. A severalfold enrichment of xR1 and xR11 mRNAs in the brain of mid-metamorphic and post-metamorphic tadpoles (stages 58-64), relative to RNA from the whole head of tadpoles until the onset of metamorphosis (stages 51-57), can be observed in Fig. 7B. This enrichment of xR1 and x R l l mRNAs, seen in metamorphosing tadpole brain, was also reflected in its relatively high concentration in adult Xenopus brain, as compared with other tissues (Fig. 7C). Interestingly, the relative levels of these transcripts in the head at all stages were very similar. Also, in brain of tadpoles at all stages and adult Xenopus the mRNA levels were similar and remained high (Fig. 7B). In data not shown, xR1 and x R l l mRNA concentrations were also found to be very similar in growing hind limbs of stages 56-60 tadpoles. To determine whether metamorphosis accelerated by the administration of exogenous thyroid hormone could affect the levels of xR1 and xR11 mRNAs, total RNA was prepared from different regions of the tadpoles. It is known that by 3 days after hormone addition, the expression of several genes associated with metamorphosis, is up- or down-regulated (Buckbinder and Brown, 1992; Shi and Brown, 1993; Wang and Brown, 1993; Tata, 1993). When RNA samples from head, middle region, tail (stages 53-57), and limbs (stages 56-60) sections were analysed by RNase protection assays, no obvious T3-inducible change was observed in the concentration of xR1 and x R l l mRNAs (data not shown). Thus, xR1 and xR11 genes are widely transcribed in embryonic and post-
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Fig. 6. Expression of xR1 and xR11 mRNAs in whole Xenopus embryos and pre-metamorphic tadpoles. Relative levels of specific transcripts were determined by RNase protection assay after hybridising 15 ~tg of total RNA from stage 12 50 embryos and tadpoles to 0.5 × 106 cpm of gel-purified 3zp-labelled antisense xR1 or xRl 1 cRNAs. To control the RNA loading, antisense EF-lct RNA was used. Long and short autoradiograph exposures of xR1 and x R l l , and EF-I~, respectively, are shown together in each figure. Methods: Adult Xenopus laevis were purchased from Blade Biologicals (Cowden, UK) and maintained at 22°C. Tissues from adult Xenopus were obtained from female animals. Embryos and tadpoles were obtained as previously described (Kawahara et al., 1991) and staged according to Nieuwkoop and Faber (1967). The treatment of tadpoles with T 3 (2× 10 -9 M), was for different periods of time with a daily change of water and hormone. Total RNA from individual adult Xenopus tissues (pooled from 5 animals), batches of whole early embryos, or from the head, middle (containing liver, kidney, intestine and pancreas) and tail regions of tadpoles, at different stages (pooled from 10-20 animals) before and during metamorphosis, was prepared by the LiCl-urea procedure (Baker and Tata, 1990). The presence and relative amounts of xR1 and xR11 were established by RNase protection assay as described by Krieg and Melton (1987). xR1 antisense RNA was transcribed with T7 polymerase from a 5'-end fragment (NotI-HinclI) subcloned into pBluescript, and linearised with BamHI to give an approx. 300-nt RNase-protected fragment. xR11 antisense RNA was transcribed with T3 RNA polymerase from a 5'-end fragment (SacI-HindIII) subcloned into pBluescript, and linearised with EcoRV to give an approx. 260-nt protected fragment. Both probes represent largely the 5' untranslated mRNA sequences. As probe for loading control in gel electrophoresis antisense RNA was prepared from Xenopus EF-lct mRNA. To ensure that the RNase protection assay was quantitative, EF-lct mRNA was subjected to the assay in the same sample at the same time in the range of 5 to 30 ~tg of total RNA from tissues of Xenopus tadpoles undergoing metamorphosis, followed by densitometric analysis of the autoradiograms to establish linearity of RNA concentration. EF-I ct cRNA was synthesized with 10 gCi of 3000 Ci/mmol [a2p] UTP, and 3 x 104 cpm of gel-purified probe was used per RNase protection reaction. EF-I ct mRNA was present at very similar overall concentrations from stage-30 Xenopus tadpoles onwards, as seen by Northern blotting and densitometric analysis, and was thus considered to be a reliable loading control for determining significant changes in the xR1 and xR11 mRNA levels. All RNase protection assays were performed with 10-15 gg of total RNA, at least twice, the RNA resolved by 6% PAGE and the autoradiograms analysed by densitometry. In some cases the RNA samples from a particular developmental stage or tissue were obtained from different batches of animals.
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L B S
Fig. 7. Relative concentrations of xR1 and x R l l mRNAs in individual tissues of Xenopus tadpoles at different stages of metamorphosis and adult Xenopus, determined by RNase protection assay. (A) 20 ~tg of total tail RNA of stage 51-61 tadpoles. (B) 15 ~tg of total RNA from heads of tadpoles until the onset of metamorphosis (stages 51-57) and brains of mid-metamorphosing, post-metamorphosing and adult animals (B). The staging is indicated in vertical numbers. (C) 20 ktg of total RNA from adult Xenopus kidney (K), heart H), liver (L), brain (B), spleen (S) and skeletal muscle (M). All other details as in Fig. 6.
embryonic development, while their more differential expression was detected in late-metamorphosis and in adult animals. (d) Discussion (I) There is considerable evidence, from different experimental approaches, that some members of the bcl-2 gene family in vertebrates, invertebrates and viruses encode cell survival factors (Hengartner et al., 1992; Korsmeyer, 1992; Vaux et al., 1992; Boise et al., 1993; Oltvai et al., 1993; Henderson et al., 1993; Allsop et al., 1993; Merino et al., 1994; see Dexter et al., 1994). Our
177 sequence data (Fig. 1) indicate that xR1 and x R l l are the first members of the amphibian bcl-2-1ike genes to be identified. The conservation in xR1 and xRll of aa residues within the BH1 and BH2 domains of bcl-2, that are essential for the cell survival function and heterodimerization with bax (Yin et al., 1994) gave the first indication of their potential role. In our studies, xRI1 expressed constitutively in rat fibroblasts protected these cells against apoptosis induced experimentally by cytotoxic agents (Ss and Chx), withdrawal of serum from the culture medium and deregulation of c-myc expression. The anti-apoptotic function of xRll was manifested as prevention of nuclear fragmentation and generation of nucleosome-length DNA ladder (Figs. 3-5). Thus, xRll is both structurally and functionally similar to Bcl-2 in suppressing cell death in 1:he absence of new protein synthesis (Jacobson et al., 1994). Furthermore, xRll may play a role during Xenopus development by acting cooperatively with c-myc and growth factors (Fanidi et al., 1992). (2) The failure to obt~Jn an xR1 translation product (Fig. 2) leaves in doubt its role in programmed cell death. Although xR1 did not exhibit as high a sequence homology with Bcl-XL as xRll (57%), it resembles more the negative regulators (Bcl-2 and Bcl-XL) than the positive effectors (Bax and Bcl-xs) of cell death. Furthermore, xR1 shares the two highly con~erved BH 1 and BH2 functional domains of Bcl-2 (Oltvai et al., 1993; Yin et al., 1994). This suggests that, like xR11, xR1 may also have a role in modulating cell death. (3) Studies on bcl-2 in man and mouse showed that this gene is more widely expressed in embryos than in adults (LeBrun et al., 1993; Merry et al., 1994). Our RNase protection analysis indicates that both xR1 and xRl I are widely expressed in all embryonic, larval and adult stages of development (Figs. 6 and 7). Of particular interest is the strong and selective accumulation of these mRNAs in the brain of mid-metamorphic and postmetamorphic tadpoles and adults compared to other tissues (Fig. 7A,B). It is worth considering the fact that at the moment we do not know if the variations in the steady-state levels of these mRNAs is due to transcriptional or post-transcriptional regulation, or both. Only studies carried out at the cellular level could help in resolving this issue. If it is accepted that xR1 and xRll play a role in preventing cell death, then one would expect a significant decrease or increase in their mRNA concentration in tissues undergoing extensive regression and growth, respectively. Therefore, the continuous expression of xR1 and x R l l mRNAs in the tadpole tail and limbs during natural and T3-induced metamorphosis (Fig. 7), was at first sight surprising and inexplicable. However, in the developing mouse brain, where up to
80% of all cells are lost during its maturation, there is no overall decrease in bel-2 expression (Oppenheim, 1991; Merry et al., 1994). It may well turn out that the expression of cell survival genes is not significantly developmentally modulated, but that cell death is brought about by enhanced expression or activation of positive effectors. It has also been suggested that developing tissues, whatever their ultimate fate, require a high level of expression of apoptosis-repressor genes, or that bcl-2-1ike genes may play a role beyond simply protecting them from developmental cell death (Allsop et al., 1993; Korsmeyer et al., 1993; Merry et al., 1994; Jacobson et al., 1994). Interestingly, the overexpression of c-myc, which is able to activate cell death can also, in the same cell and under specific circumstances, stimulate cell proliferation (Evan et al., 1992) while Ich-2 encodes both positive and negative regulators of PCD (Wang et al., 1994). At the same time, the multiplicity of such genes implies some degree of redundancy of their products. Such redundancy or, as yet unknown, additional functions could explain the continuing expression of xR1 and xR11 during natural or T3-induced metamorphosis in the tadpole tail and limbs. (4) Our results also raise some important issues for future studies. First, the present findings in heterologous cell transfections have to be extended to the expression in vivo of xR1 and xR11 (or other bcl-2-1ike genes) during natural and thyroid hormone-induced metamorphosis. The specific inhibition of metamorphosis by prolactin (Tata, 1993) offers an additional approach to investigating the role of cell survival factors. Preliminary investigations in our laboratory have confirmed the feasibility of following the progression of cell death in the intact tail, during natural and T3-induced programmed cell death, in vivo and in organ culture, by both morphological and biochemical criteria of apoptosis. Another important approach to be taken in extending our work on metamorphosis, in view of the relative constancy of expression of xR1 and xR11, is to focus on the expression of positive and early inducers of cell death, such as ced-3 or interleukin 1B converting enzyme (ICE), Ich-2, Nedd-2, bcl-xs and nur-77 (Yuan et al., 1993; Boise et al., 1993; Oitvai et al., 1993; Wang et al., 1993; Woronicz et al., 1994; Kumar et al., 1994). We have now initiated work along both these lines.
ACKNOWLEDGEMENTS We are most grateful to Dr. Gerard Evan, Imperial Cancer Research Fund, London, and Dr. Michael Jacobson, University College London, for providing us with Rat-1 and Rat-1/c-myc-ER cells and human bcl-2 cDNA, and for careful reading of the manuscript. We
178 would like to thank Mrs. Betty Bennett for her participation in some aspects of the experimental work and Dr. Abdallah Fanidi for his help on transfection with retroviruses. We also thank Mrs Ena Heather for preparation of the manuscript.
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Hockenbery, D.M., Oltvai, Z.N., Yin, X.-M., Milliman, C.L. and Korsmeyer, S.J.: Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75 (1993) 241-251. Jacobson, M.D., Burne, J.F., King, M.P., Miyashita, T., Reed, J.C. and Raft, M.C.: Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature 361 (1993) 365-368. Jacobson, M.D., Burne, J.F. and Raft, M.C.: Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J. 13 (1994) 1899-1910. Kawahara, A., Baker, B. and Tata, J.R.: Developmental and regional expression of thyroid hormone receptor genes during Xenopus metamorphosis. Development 112 (1991) 933-943. Korsmeyer, S.J.: Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood 80 (1992) 870 886. Korsmeyer, S.J., Shutter, J.R., Veis, D.J., Merry, D.E. and Oltvai, Z.N.: Bcl-2/Bax: a rheostat that regulates an anti-oxidant pathway and cell. Semin. Cancer Biol. 4 (1993) 327 332. Kumar, S., Kinoshita, M., Noda, M., Copeland, N.G. and Jenkins, N.A.: Induction of apoptosis by the mouse Nedd-2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-113-converting enzyme. Genes Dev. 8 (1994) 1613-1626. Lavin, M. and Watters, D.: Programmed Cell Death. The Cellular and Molecular Biology of Apoptosis. Harwood, Chur, 1993. LeBrun, D.P., Warnke, R.A. and Cleary, M.L.: Expression of bcl-2 in fetal tissues suggests a role in morphogenesis. Am. J. Pathol. 142 (1993) 743-753. Lockshin, R.A.: Cell death in metamorphosis. In: Bowen, I.D. and Lockshin, R.A. (Eds.) Cell Death in Biology and Pathology. Chapman and Hall, London, 1981, pp. 79 121. Merino, R., Ding, L., Veis, D.J., Korsmeyer, S.J. and Nunez, G.: Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes. EMBO J. 13 (1994) 683-691. Merry, D.E., Veis, D.J., Hickey, W.F. and Korsmeyer, S.J.: bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS. Development 120 (1994) 301-311. Morgenstern, J.P. and Land, H.: Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18 (1990) 3587-3596. Nieuwkoop, P.O. and Faber, J.: Normal Table of Xenopus laevis (Daudin), 2nd ed. North Holland, Amsterdam, 1967. Oltvai, Z.O., Milliman, C.L. and Korsmeyer, S.J.: Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell 74 (1993) 609-619. Oppenheim, R.W.: Cell death during development of the nervous system. Annu. Rev. Neurosci. 14 (1991) 453-501. Shi, Y.°B. and Brown, D.D.: The earliest changes in gene expression in tadpole intestine induced by thyroid hormone. J. Biol. Chem. 268 (1993) 20312 20317. Tata, J.R.: Requirement for RNA and protein synthesis for induced regression of the tadpole tail in organ culture. Dev. Biol. 13 (1966) 77-94. Tata, J.R.: Gene expression during metamorphosis: an ideal model for post-embryonic development. BioEssays 15 (1993) 239-248. Tata, J.R., Kawahara, A. and Baker, B.S.: Prolactin inhibits both thyroid hormone-induced morphogenesis and cell death in cultured amphibian larval tissues. Dev. Biol. 146 (1991) 72-80. Tomei, L.D. and Cope, F.O.: (Eds) Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1991. Vaux, D.L., Weissman, I.L. and Kim, S.K.: Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science 258 (1992) 1955 1957. Veis, D.J., Sorenson, C.M., Shutter, J.R. and Korsmeyer, S.J.: Bcl-2
179 deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75 (1993) 229-240. Wang, Z. and Brown, D.D.: The thyroid hormone-induced gene expression program for amphibiarL tail resorption. Proc. Natl. Acad. Sci. USA 88 (1993) 11505-11509. Wang, L., Miura, M., Bergeron, L., Zhu, H. and Yuan, J.: Ich-2, an Ice~ ced-3-related gene, encodes both positive and negative regulators of programmed cell death. Cell 78 (1994) 739-750. While, K., Grether, M.E., Abrams, J.M., Young, L., Farrell, K. and Steller, H.: Genetic control c f programmed cell death in Drosophila. Science 264 (1994) 677-683. Williams, G.T. and Smith, C.A.: Molecular regulation of apoptosis: genetic controls on cell death. Cell 74 (1993) 777 779. Woronicz, J.D., Cainan, B., Ngo, V. and Winoto, A.: Requirement for the orphan steroid receptor Nur 77 in apoptosis of T-cell hybridomas. Nature 367 (1994) 277 281.
Xu, Q., Baker, B.S. and Tata, J.R.: Developmental and hormonal regulation of the Xenopus liver-type arginase gene. Eur. J. Biochem. 211 (1993) 891-898. Yin, X.-M., Oltvai, Z.N. and Korsmeyer, S.J.: BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 369 (1994) 321-323. Yoshizato, K.: Biochemistry and cell biology of amphibian metamorphosis with a special emphasis on the mechanism of removal of larval organs. Int. Rev. Cytol. 119 (1989), 97 149. Yuan, J., Shaham, S., Ledoux, S., Ellis, H.M. and Horvitz, H.R.: The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-ll3-converting enzyme. Cell 75 (1993) 641 652. Yuan, J. and Horvitz, H.R.: The Caenorhabditis elegans cell death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed celldeath. Development 116(1992)309 320.
171
GENE 08877
Cloning, characterization and expression of two Xenopus bcl-2-1ike cell-survival genes (Programmed cell death; apoptosis; amphibian metamorphosis; c-myc; oncogenes)
Jorge Cruz-Reyes* and Jamshed R. Tata Division of Developmental Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Received by J.K.C. Knowles: 16 December 1994; Accepted: 30 January 1995; Received at publishers: 23 February 1995
SUMMARY
We describe two cloned cDNAs, termed xR1 and x R l 1, isolated from a Xenopus laevis stage 28-30 embryonic head cDNA library. Comparison of amino acid (aa) sequences derived from nucleotide (nt) sequences of xR1 and xR11 cDNAs revealed substantial homology with bcl-2-related genes, especially with bcl-xL. In particular, there was a marked conservation of the BH1 and BH2 domains considered to be important for the anti-cell death and heterodimerisation properties of bcl-2. Constitutive expression of x R l l in cultured rat fibroblast (Rat-l) cells conferred a strong protection against cell death induced by the cytotoxic agents staurosporine and cycloheximide, by serum deprivation and specific deregulation of c-myc. Measurement of xR1 and xR11 mRNAs by RNase protection assay revealed similar widespread expression in Xenopus embryos and tadpoles. Except for an abrupt increase in the accumulation of xR1 and xRI1 mRNAs in brains of mid-metamorphic and post-metamorphic tadpoles and adults, there was insignificant modulation of their expression in tissues undergoing total regression (tail) or morphogenesis (limb) during natural or thyroid hormone-induced metamorphosis. These findings raise the possibility of continuing expression of cell survival genes in tissues undergoing total regression during post-embryonic development.
Programmed cell death (PCD) is now recognised to be an integral part of, and essential requirement for, many developmental processes (for reviews, see Ellis and Horvitz, 1986; Bowen and Bowen, 1990; Oppenheim, 1991; Ellis et al., 1991; Tomei and Cope, 1991; Lavin and Watters, 1993; Dexter e~: al., 1994). It is particularly important during post-embryonic development and is
associated with such diverse phenomena as neurogenesis, morphogenesis, remodelling of tissues, immune responses and tissue regression or organolysis. In the last decade a number of genes have been identified in different systems whose products are associated with PCD, often manifested as apoptosis, which is characterised by nuclear condensation and DNA fragmentation (Hengartner et al., 1992; Korsmeyer, 1992; Yuan and Horvitz, 1992; Yuan et al., 1993; White et al., 1994; Woronicz et al., 1994).
Correspondence to: Dr. J.R. Tata, Laboratory of Developmental Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Tel: (44-181) 959-3666 ext. 2106; Fax: (44-181) 913-8583; e-mail: [email protected] * Present address: Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2185, USA. Tel: (1-410) 955-7419.
central nervous system; cRNA, RNA complementary to cDNA; DMEM, Dulbecco's modified Eagle's medium; EF-lct, elongation factor-let; ER, estrogen receptor; FCS, fetal calf serum; kb, kilobase(s); nt, nucleotides; ORF, open reading frame; PCD, programmed cell death; SEM, standard error of the mean; Ss, staurosporin; T3, 3.3',5-triiodo-L-thyronine; TH, thyroid hormone(s); XR, Xenopus cellsurvival (anti-apoptosis) protein; XR, gene (DNA, RNA) encoding XR.
INTRODUCTION
Abbreviations: aa, amino acid(s}; bcl, B cell leukemia; bp, base pair(s); cDNA, DNA complementary to RNA; Chx, cycloheximide; CNS, SSDI 0378-1119(95)00159-X
172
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Fig. 1. Deduced aa sequence, based on cDNA nt sequences, of xR1, xRll and other Bcl-2-type proteins that modulate apoptosis. Areas of aa identity between the Xenopus sequences, human Bcl-2, Bcl-xL and Bax, C. elegans Ced-9, and viral bhrf-1 are indicated by stippling (Vaux et al., 1988; Boise et al., 1993; Oltvai et al., 1993; Hengartner, 1992; Henderson, 1993). The highly conserved BH1 and BH2 domains of Bcl-2 protein are indicated by open bars, and aa essential for suppression of apoptosis and heterodimerisation to Bax are marked by dots (Yin et al., 1994). The 63-aa region of human Bcl-xL deleted in human Bcl-xs is denoted by arrows. A predicted 19-aa hydrophobic domain and flanking charged residues that are present in all the sequences are indicated by a stippled bar (Chen-Levy and Cleary, 1990). The nt sequences of x R l l and xR1 mRNAs have been deposited with EMBL Nucleotide Sequence Database under the accession Nos. X82461 and X82462, respectively. Methods: ~.Zap-cDNA libraries prepared from tails of mid-metamorphic Xenopus tadpoles (stages 53-56), and embryonic head (stages 28-30), using a kit obtained from Stratagene, were screened with a human bcl-2 cDNA, kindly provided by Dr. G. Evan, Imperial Cancer Research Fund London, at low stringency. The filters were hybridised in 6xSSC ( l x S S C is 0.15M NaCI/0.015M Na 3.citrate, pH 7.6)/1 x Denhardt's solution/24mM Na.phosphate buffer (pH 6.5)/100 gg salmon sperm DNA per ml/0.5% SDS at 63°C overnight. The final wash conditions were 20 min at 42°C in 0.2 x SSC. cDNA inserts from two hybridising clones to bcl-2 cDNA, termed xR1 and xR11, were subcloned, xR1 was in vivo excised in pBluescript SK (+/ ), whereas xR11 was amplified by PCR using pBluescript primers with EcoRI and SacI linkers and subcloned into pBluescript KS ( + / - ) (Stratagene, La Jolla, CA, USA). Both xR1 and xR11 cDNAs were
These genes have been classified into two groups: (i) those whose products induce cell death and (ii) those that protect against cell death or confer survival. In mammalian cells it has been established that the bcl-2 gene plays an important role in cell survival (Korsmeyer, 1992; Korsmeyer et al., 1993); it is homologous, structurally and functionally to ced-9 gene of C. elegans, as demonstrated by the prevention of cell death by human bcl-2 introduced into ced-9 mutants of developing nematodes (Vaux et al., 1992). These findings, and a continuously growing family of bcl-2-related molecules that modulate apoptosis (e.g., bcl-x, bax, bhrf-1), argue for the existence of other bcl-2-1ike genes, each playing a unique role in maintaining development or regulating PCD (Boise et al., 1993; Oltvai et al., 1993; Henderson et al., 1993). Our interest in studying PCD stems from work on amphibian metamorphosis, which is a most dramatic example of post-embryonic development. The wideranging phenotypic changes produced in metamorphosis, such as limb morphogenesis, acquisition of new hepatic functions, remodelling of CNS and extensive tissue regression, resemble many associated with mammalian fetal and neonatal development. They are all obligatorily induced and regulated by thyroid hormones 3,3',5triiodo-L-thyronine and thyroxine (T3, T 4 ) , and are dependent on new protein synthesis (Tata, 1993). A striking feature of amphibian metamorphosis is the widespread cell death in regressing larval tissues, such as the tadpole tail, gills, and gut, which accompanies morphogenesis of limb buds and lungs and transformation of skin and the central nervous system (Tata, 1966; Lockshin, 1981; Yoshizato, 1989; Tata et al., 1991). It was considered that bcl-2-1ike cell survival genes may themselves be positively and negatively regulated in these contrasting postembryonic morphogenetic and regressive developmental processes. We, therefore, embarked upon the cloning and characterisation of two homologues of the bcl-2 family of genes in Xenopus laevis termed xR1 and xRll, examine their role in protection against PCD, and search for a
isolated from the embryonic head library and sequenced by a dideoxy termination method in plasmid DNA (US Biochemical, Amersham International, Amersham, UK). For transfection and subsequent functional analysis, a SacI-SalI xR11 insert was excised from pBluescript KS ( + / - ) , and subcloned into pSP64 plasmid (Promega, Madison, WI, USA), from which an EcoRI x R l l insert was isolated and subcloned into the EcoRI site of pBabe-puro (Morgenstern and Land, 1990). Orientation of the insert was determined by restriction enzyme mapping. A plasmid with the insert in the forward orientation was designated pBabe-puro-xR1 sense (S), while a plasmid with the insert in the reverse orientation was named pBabe-puro-xRll antisense (A). Sequence comparisons and peptide analyses were performed with the Genetics Computer Group (GCG, Madison, WI, USA) Programs for the GCG Package (1991): BLAST, MAP, GAP, PILEUP, GELSTART and other sequence assembling programmes.
173 possible developmental and thyroid hormonal regulation of expression of these genes during early embryogenesis, metamorphosis of tadpoles and in fully developed adults.
C
xR1 x R l l
M
RESULTS AND DISCUSSION
(a) Cloning of xR1 and x R l l We used a human bcl-2 cDNA probe to clone Xenopus genes by low-stringency hybridisation of multiple cDNA libraries made from developing head and tail tissues. Two cDNA clones, thus isolated, were termed xR1 (2.0 kb) and xR11 (1.1 kb), which exhibited moderate and strong hybridisation to the bcl-2 cDNA probe, respectively. These two cDNAs were sequenced and Fig. 1 shows their deduced aa sequences, xR1 and xR11 displayed a high level of sequence identity with members of the bcl-2 gene family reported to modulate experimentally induced apoptosis, xR1 and xR11 both exhibit an overall 40% aa identity to bcl-2. They contain the BH1 and BH2 domains of Bcl-2 required for inhibition of apoptosis and heterodirnerisation with the cell death inducing Bcl-2-derived fragment bax (Yin et al., 1994) especially as regards aa known to be essential for these two processes (e.g., Gly 145'194and Trp 188"195of Bcl-2), and a C-terminal hydrophobic region that functions as a signal-anchor sequence responsible for the integral membrane position of Bcl-2 (Chen-Levy and Cleary, 1990). xRl 1 shares 57% aa identity with human Bcl-XL the large spliced form of Bcl-x (compared with 44% between Bcl-2 and Bcl-XL), and contains a 204-aa ORF. To determine if either of these Xenopu:~ cDNA clones give rise to an appropriate sized protein product, they were transcribed as RNA and subjected to in vitro translation. As seen in Fig. 2, only x R l l cDNA yielded a translation product of the approximate size predicted by the O R F (approx. 22.5 kDa). Consequently, it was decided to fully characterise only x R l l cDNA functionally by over-producing xR11 protein in tissue culiture cells by transfection. bcl-2-1ike
(b) x R l l is a negative regulator of apoptosis Because of the close aa sequence similarity between human bcl-xL and the O R F of xR11 (Fig. 1), and the transcription-translation of the cDNA to the protein of expected size, we sought to determine whether x R l l could serve a similar function as Bcl-XL and other Bcl-2like proteins in preventing apoptotic cell death (Boise et al., 1993; Williams and Smith, 1993). Towards this end, the rat fibroblast cell line Rat-1 was transfected with the x R l l cDNA inserted in both orientations into the EcoRI cloning site of the pBABE-puro expression plasmid (Morgenstern and Land, 1990). This approach has been successfully exploited to study the survival function of
<-22.5kDa
Fig. 2. In vitro translation of xRl and xRll mRNAs, xR1 and xRll mRNA were used for in vitro translation in the presence of [aSS]methionine. The translation products were resolved by SDSPAGE. Size of the resulting xR11 protein is indicated on the right in kDa. Translation of a constructcontainingluciferasecDNA was used as a control (C). Methods: pBluescript plasmids containing xR1 and xR11 cDNA inserts were linearised at the 3' multiple cloning site with EcoRI and transcribed with T3 and T7 RNA polymerases,respectively, for 1 h at 37°C. The resulting run-off transcripts were phenolchloroformextractedand ethanol precipitated. In vitro translation was then performed with a rabbit reticulocytelysate kit (Promega) in the presenceof [35S] methioninefor 1 h at 30°C.The lysate(5 ~tl)was added to SDS loading buffer and subjected to 0.1% SDS-15% PAGE. Gels were dried and exposed to X-ray (X-OMA) AR-film(Kodak). human Bcl-2 (Fanidi et al., 1992). High levels of expression of xR11 mRNA in transfected rat cells was demonstrated by RNase protection assay with an x R l l sensespecific riboprobe (data not shown). Puromycin-resistant cells were selected and then clonal populations were tested for resistance to experimentally induced apoptosis. xRll-transfected cells had similar growth kinetics to the parental cell line, as well as to the puromyein-transfected control cells (not shown). Puromycin-resistant clones were thus subjected to the cytotoxic agents Ss and Chx, serum-deprived culture conditions, and to c-myc deregulation. Apoptosis induced by all these procedures, has been previously shown in several cell lines, and can be blocked by Bcl-2 over expression (Bissonnette et al., 1992; Fanidi et al., 1992; Jacobson et al., 1993). We characterised apoptosis of Rat-1 fibroblasts both by propidium iodide fluorescence of DNA to visualise chromatin condensation and fragmentation, and DNA size separation by agarose gel electrophoresis. Rat-1 cells transfected with xR11 eDNA in the anti-sense orientation underwent rapid cell death following the addition of Ss or Chx as determined by nuclear condensation and fragmentation (Fig. 3), and chromatin cleavage at nucleosomal intervals
174
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Fig. 3. Protection against apoptosis by overexpressionofxRll in Rat-1 fibroblasts. Cells were cultured in 10% FCS and 1 ~tM Ss for 18-20 h to induce apoptosis. (A) Rat-1 clones expressing xR11 in the antisense orientation. Note a high proportion of cells exhibiting chromatin condensation and nuclear fragmentation (arrowed) indicative of apoptosis. (B) Constitutive expression of xR11, subcloned in the sense orientation, prevents Ss-induced apoptosis. Most cells show a regular diffuse chromatin staining pattern characteristic of viable cells. Fluorescence microscopic examination was performed under a 100 × magnification objective. Methods: Rat-1 cells, kindly provided by Dr. M. Jacobson, University College London, were cultured as previously described (Evan, 1992). The isolation of appropriately infected Rat-1 cell lines with the pBabe-puro retrovirus, directing constitutive expression, was also carried out as previously described (Morgenstern and Land, 1990). Cell lines expressingxRll in both orientations were isolated by selecting puromycin-resistant clones. Three independent transfected Rat-1 cell lines (for each xRll orientation) were studied. The results with one of each xR1/-expressing subclones, and control with no insert are shown; in similar independent experiments, the behaviour of the other subclones was indistinguishable, pBabe-puro-xRll cells were maintained in 5 ~tg/ml of puromycin (Sigma). Cells were assayed for constitutive expression of xR11 by RNase protection analysis.To assay for apoptosis induced by the cytotoxic drug Ss (Sigma, Poole, UK), cells were removed from culture dishes with trypsin, EDTA, washed once in DMEM containing 10% FCS, resuspended in DMEM and plated on poly-L-lysine-coated13-mm glass coverslips in 24-wellFalcon plates at a density of 10000 cells per well. Microscopic examination of nuclei was performed by fixing the cells with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4, and staining with propidium iodide (Sigma) as previously reported (Jacobson, 1993). Apoptotic and normal cells were scored by examination under a 40 × magnification objective in a fluorescence microscope.
(Fig. 4). I n contrast, Rat-1 cells transfected with sense x R l l c D N A d e m o n s t r a t e d significant resistance to cell death after 24 h of t r e a t m e n t (Figs. 3 a n d 4 show a representative analysis after 12 h). As regards xR1, transfection of Rat-1 cells with this c D N A did n o t exhibit the same strong p h e n o t y p e as with x R l l cDNA. The failure of transfected xR1 to protect these cells against d r u g - i n d u c e d apoptosis m a y result from the fact that, unlike x R l l , its c D N A is n o t fulllength, despite the retention of the 'anti-cell death' domains. The above procedure of i n d u c i n g cell death with Chx
Fig. 4. Size fractionation of DNA to indicate apoptosis in Rat-1 cells expressing xR11 subcloned in the sense (S) and antisense (A) orientations. Cells were treated with 1 ktM Ss or 10 ~tM Chx for 6 and 12 h to induce apoptosis. Cells expressing antisense xRll exhibit rapid breakdown of DNA to nucleosome-size fragments with either treatment, whereas the constitutive expression of sense xR11 protects Rat-1 cells from DNA fragmentation. Methods: To confirm that cell death was due to apoptosis, 106 cells transfected with xRll in either orientation and treated with 1 ~tM Ss or 10 ~tM Chx, were lysed, the DNA resolved by electrophoresis on a 1.2% agarose gel and stained with ethidium bromide, as previously described (Boise et al., 1993). All other procedures as in Fig. 3.
a n d Ss implies that apoptosis m a y be induced by multiple pathways associated with the toxicity of these substances. A n a d d i t i o n a l a p p r o a c h was therefore a d o p t e d to obviate this difficulty in studying the cell survival function of x R l l . Towards this end, we transfected Rat-1/c-myc-ER cells, which have been transformed with a functional estrogen receptor construct ( E v a n et al., 1992) with p B A B E - p u r o directing constitutive expression of x R l l , a n d then observed the effect of xR11 expression on apoptosis i n d u c e d by serum depletion a n d de n o v o synthesis of c-myc. I n these cells the c-myc p r o m o t e r is u n d e r the control of ER, so that the c-myc gene, which is inactive in the absence of estrogen, can be selectively activated by the simple a d d i t i o n of the h o r m o n e . For this purpose, Rat- 1/c-myc-ER a n d Rat- 1/c-mycER/xRl I cells were first arrested in phase Go by serum depletion, after which the progression of apoptosis was followed as a function of time in the presence or absence of de n o v o synthesised c-myc, c-myc was i n d u c e d by a d d i t i o n of estradiol-17~ to
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29 Time of culture (h) Fig. 5. Protection of Rat-1 fi~roblasts by xRll against apoptosis induced by serum deprivation and activation of c-myc. Rat-1 cells expressing c-myc fused to the human estrogen receptor (c-myc/ER), were infected with the retrovirus pEabe-puro alone or expressing xR11 in sense (S) or antisense (A) orientation. The cells were growth-arrested in culture in 0.05% FCS, while control cultures were performed in 10% FCS. Activation of c-myc in Rat-1/c-myc/xRll cells was by addition of 2 laM estradiol-17 13(E2) to the medium after 29 h of culture, as indicated by the arrow. The cells did not reach confluence. Cells were fixed, stained with propidium iodide, and apoptotic nuclei were scored with a fluorescence microscope under a 40 x magnification objective (see Fig. 3). The results represent the means + SEM of triplicate cultures. At leasl 100 cells were counted per culture. Methods: Rat-1/c-myc-ER cells, kindly provided by Dr. G. Evan, Imperial Cancer Research Fund, London, were cultured as previously described (Evan, 1992). Myc-ER cells were maintained in phenol red-free DMEM supplemented with 10% charcoal-dextran stripped FCS and 1 mg Geneticin/ml. To assay for c-myc-induced apoptosis, Rat-1/c-myc-ER cells, expressing xR11 or not, were growth-arrested in 0.05% FCS for 29 h before activating c-myc by the addition of E 2 to the medium at a final concentration of 2 ~tM. All other procedures were as for Fig. 3.
fibroblast cultures after 29 h of serum-depletion. As shown in Fig. 5, the expression of xR11 effectively abolished apoptosis induced both by serum-deprivation and c-myc induction. The percentage of dying cells was determined daily by propidium iodide staining of DNA to visualize condensed and fragmented nuclei, as shown in Fig. 3. Thus, xR11 is an efficient repressor of apoptosis induced in the absence of protein synthesis, and by c-myc activation.
(c) xR1 and xR11 are expressed in most tissues throughout development We next investigated the expression patterns for both xR11 and xR1 during natural development of Xenopus tadpoles up to adult stages, as well as following T 3induced metamorphosis. Because of the difficulty of detecting xR11 and xR1 transcripts by Northern blotting, due to their low concentrations in most tissues and developmental stages, the more sensitive RNase protection
assay was adopted for this purpose. We first estimated the relative concentrations of xR1 and x R l l transcripts in total RNA from whole Xenopus embryos and early tadpoles at different developmental stages. As seen in Fig. 6, both mRNAs were detected from stage-12 embryos to stage-50 tadpoles. A small increase in the steady-state levels can be clearly observed for the x R l l transcript, after correction for the EF-lct mRNA assayed simultaneously in the same samples of total RNA, as development progressed. Northern blot and densitometric analysis showed that the concentration of EF-I~ mRNA was very similar as compared to skeletal muscle actin mRNA and rRNA in the animals at the above stages (data not shown). For later developmental stages until completion of natural metamorphosis and in adult Xenopus, the levels of xR1 and xR11 mRNA were measured in individual tissues. In the tadpole tail, which undergoes total regression, the concentrations of both xR1 and xRI1 m R N A increased slightly from premetamorphosis (stages 51 to 54) to metamorphic climax at stage 61-62 (Fig. 7A). Of all the other tadpole tissues examined during natural metamorphosis, only the brain showed a substantial developmental regulation of xR1 and xR11. A severalfold enrichment of xR1 and xR11 mRNAs in the brain of mid-metamorphic and post-metamorphic tadpoles (stages 58-64), relative to RNA from the whole head of tadpoles until the onset of metamorphosis (stages 51-57), can be observed in Fig. 7B. This enrichment of xR1 and x R l l mRNAs, seen in metamorphosing tadpole brain, was also reflected in its relatively high concentration in adult Xenopus brain, as compared with other tissues (Fig. 7C). Interestingly, the relative levels of these transcripts in the head at all stages were very similar. Also, in brain of tadpoles at all stages and adult Xenopus the mRNA levels were similar and remained high (Fig. 7B). In data not shown, xR1 and x R l l mRNA concentrations were also found to be very similar in growing hind limbs of stages 56-60 tadpoles. To determine whether metamorphosis accelerated by the administration of exogenous thyroid hormone could affect the levels of xR1 and xR11 mRNAs, total RNA was prepared from different regions of the tadpoles. It is known that by 3 days after hormone addition, the expression of several genes associated with metamorphosis, is up- or down-regulated (Buckbinder and Brown, 1992; Shi and Brown, 1993; Wang and Brown, 1993; Tata, 1993). When RNA samples from head, middle region, tail (stages 53-57), and limbs (stages 56-60) sections were analysed by RNase protection assays, no obvious T3-inducible change was observed in the concentration of xR1 and x R l l mRNAs (data not shown). Thus, xR1 and xR11 genes are widely transcribed in embryonic and post-
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Fig. 6. Expression of xR1 and xR11 mRNAs in whole Xenopus embryos and pre-metamorphic tadpoles. Relative levels of specific transcripts were determined by RNase protection assay after hybridising 15 ~tg of total RNA from stage 12 50 embryos and tadpoles to 0.5 × 106 cpm of gel-purified 3zp-labelled antisense xR1 or xRl 1 cRNAs. To control the RNA loading, antisense EF-lct RNA was used. Long and short autoradiograph exposures of xR1 and x R l l , and EF-I~, respectively, are shown together in each figure. Methods: Adult Xenopus laevis were purchased from Blade Biologicals (Cowden, UK) and maintained at 22°C. Tissues from adult Xenopus were obtained from female animals. Embryos and tadpoles were obtained as previously described (Kawahara et al., 1991) and staged according to Nieuwkoop and Faber (1967). The treatment of tadpoles with T 3 (2× 10 -9 M), was for different periods of time with a daily change of water and hormone. Total RNA from individual adult Xenopus tissues (pooled from 5 animals), batches of whole early embryos, or from the head, middle (containing liver, kidney, intestine and pancreas) and tail regions of tadpoles, at different stages (pooled from 10-20 animals) before and during metamorphosis, was prepared by the LiCl-urea procedure (Baker and Tata, 1990). The presence and relative amounts of xR1 and xR11 were established by RNase protection assay as described by Krieg and Melton (1987). xR1 antisense RNA was transcribed with T7 polymerase from a 5'-end fragment (NotI-HinclI) subcloned into pBluescript, and linearised with BamHI to give an approx. 300-nt RNase-protected fragment. xR11 antisense RNA was transcribed with T3 RNA polymerase from a 5'-end fragment (SacI-HindIII) subcloned into pBluescript, and linearised with EcoRV to give an approx. 260-nt protected fragment. Both probes represent largely the 5' untranslated mRNA sequences. As probe for loading control in gel electrophoresis antisense RNA was prepared from Xenopus EF-lct mRNA. To ensure that the RNase protection assay was quantitative, EF-lct mRNA was subjected to the assay in the same sample at the same time in the range of 5 to 30 ~tg of total RNA from tissues of Xenopus tadpoles undergoing metamorphosis, followed by densitometric analysis of the autoradiograms to establish linearity of RNA concentration. EF-I ct cRNA was synthesized with 10 gCi of 3000 Ci/mmol [a2p] UTP, and 3 x 104 cpm of gel-purified probe was used per RNase protection reaction. EF-I ct mRNA was present at very similar overall concentrations from stage-30 Xenopus tadpoles onwards, as seen by Northern blotting and densitometric analysis, and was thus considered to be a reliable loading control for determining significant changes in the xR1 and xR11 mRNA levels. All RNase protection assays were performed with 10-15 gg of total RNA, at least twice, the RNA resolved by 6% PAGE and the autoradiograms analysed by densitometry. In some cases the RNA samples from a particular developmental stage or tissue were obtained from different batches of animals.
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Fig. 7. Relative concentrations of xR1 and x R l l mRNAs in individual tissues of Xenopus tadpoles at different stages of metamorphosis and adult Xenopus, determined by RNase protection assay. (A) 20 ~tg of total tail RNA of stage 51-61 tadpoles. (B) 15 ~tg of total RNA from heads of tadpoles until the onset of metamorphosis (stages 51-57) and brains of mid-metamorphosing, post-metamorphosing and adult animals (B). The staging is indicated in vertical numbers. (C) 20 ktg of total RNA from adult Xenopus kidney (K), heart H), liver (L), brain (B), spleen (S) and skeletal muscle (M). All other details as in Fig. 6.
embryonic development, while their more differential expression was detected in late-metamorphosis and in adult animals. (d) Discussion (I) There is considerable evidence, from different experimental approaches, that some members of the bcl-2 gene family in vertebrates, invertebrates and viruses encode cell survival factors (Hengartner et al., 1992; Korsmeyer, 1992; Vaux et al., 1992; Boise et al., 1993; Oltvai et al., 1993; Henderson et al., 1993; Allsop et al., 1993; Merino et al., 1994; see Dexter et al., 1994). Our
177 sequence data (Fig. 1) indicate that xR1 and x R l l are the first members of the amphibian bcl-2-1ike genes to be identified. The conservation in xR1 and xRll of aa residues within the BH1 and BH2 domains of bcl-2, that are essential for the cell survival function and heterodimerization with bax (Yin et al., 1994) gave the first indication of their potential role. In our studies, xRI1 expressed constitutively in rat fibroblasts protected these cells against apoptosis induced experimentally by cytotoxic agents (Ss and Chx), withdrawal of serum from the culture medium and deregulation of c-myc expression. The anti-apoptotic function of xRll was manifested as prevention of nuclear fragmentation and generation of nucleosome-length DNA ladder (Figs. 3-5). Thus, xRll is both structurally and functionally similar to Bcl-2 in suppressing cell death in 1:he absence of new protein synthesis (Jacobson et al., 1994). Furthermore, xRll may play a role during Xenopus development by acting cooperatively with c-myc and growth factors (Fanidi et al., 1992). (2) The failure to obt~Jn an xR1 translation product (Fig. 2) leaves in doubt its role in programmed cell death. Although xR1 did not exhibit as high a sequence homology with Bcl-XL as xRll (57%), it resembles more the negative regulators (Bcl-2 and Bcl-XL) than the positive effectors (Bax and Bcl-xs) of cell death. Furthermore, xR1 shares the two highly con~erved BH 1 and BH2 functional domains of Bcl-2 (Oltvai et al., 1993; Yin et al., 1994). This suggests that, like xR11, xR1 may also have a role in modulating cell death. (3) Studies on bcl-2 in man and mouse showed that this gene is more widely expressed in embryos than in adults (LeBrun et al., 1993; Merry et al., 1994). Our RNase protection analysis indicates that both xR1 and xRl I are widely expressed in all embryonic, larval and adult stages of development (Figs. 6 and 7). Of particular interest is the strong and selective accumulation of these mRNAs in the brain of mid-metamorphic and postmetamorphic tadpoles and adults compared to other tissues (Fig. 7A,B). It is worth considering the fact that at the moment we do not know if the variations in the steady-state levels of these mRNAs is due to transcriptional or post-transcriptional regulation, or both. Only studies carried out at the cellular level could help in resolving this issue. If it is accepted that xR1 and xRll play a role in preventing cell death, then one would expect a significant decrease or increase in their mRNA concentration in tissues undergoing extensive regression and growth, respectively. Therefore, the continuous expression of xR1 and x R l l mRNAs in the tadpole tail and limbs during natural and T3-induced metamorphosis (Fig. 7), was at first sight surprising and inexplicable. However, in the developing mouse brain, where up to
80% of all cells are lost during its maturation, there is no overall decrease in bel-2 expression (Oppenheim, 1991; Merry et al., 1994). It may well turn out that the expression of cell survival genes is not significantly developmentally modulated, but that cell death is brought about by enhanced expression or activation of positive effectors. It has also been suggested that developing tissues, whatever their ultimate fate, require a high level of expression of apoptosis-repressor genes, or that bcl-2-1ike genes may play a role beyond simply protecting them from developmental cell death (Allsop et al., 1993; Korsmeyer et al., 1993; Merry et al., 1994; Jacobson et al., 1994). Interestingly, the overexpression of c-myc, which is able to activate cell death can also, in the same cell and under specific circumstances, stimulate cell proliferation (Evan et al., 1992) while Ich-2 encodes both positive and negative regulators of PCD (Wang et al., 1994). At the same time, the multiplicity of such genes implies some degree of redundancy of their products. Such redundancy or, as yet unknown, additional functions could explain the continuing expression of xR1 and xR11 during natural or T3-induced metamorphosis in the tadpole tail and limbs. (4) Our results also raise some important issues for future studies. First, the present findings in heterologous cell transfections have to be extended to the expression in vivo of xR1 and xR11 (or other bcl-2-1ike genes) during natural and thyroid hormone-induced metamorphosis. The specific inhibition of metamorphosis by prolactin (Tata, 1993) offers an additional approach to investigating the role of cell survival factors. Preliminary investigations in our laboratory have confirmed the feasibility of following the progression of cell death in the intact tail, during natural and T3-induced programmed cell death, in vivo and in organ culture, by both morphological and biochemical criteria of apoptosis. Another important approach to be taken in extending our work on metamorphosis, in view of the relative constancy of expression of xR1 and xR11, is to focus on the expression of positive and early inducers of cell death, such as ced-3 or interleukin 1B converting enzyme (ICE), Ich-2, Nedd-2, bcl-xs and nur-77 (Yuan et al., 1993; Boise et al., 1993; Oitvai et al., 1993; Wang et al., 1993; Woronicz et al., 1994; Kumar et al., 1994). We have now initiated work along both these lines.
ACKNOWLEDGEMENTS We are most grateful to Dr. Gerard Evan, Imperial Cancer Research Fund, London, and Dr. Michael Jacobson, University College London, for providing us with Rat-1 and Rat-1/c-myc-ER cells and human bcl-2 cDNA, and for careful reading of the manuscript. We
178 would like to thank Mrs. Betty Bennett for her participation in some aspects of the experimental work and Dr. Abdallah Fanidi for his help on transfection with retroviruses. We also thank Mrs Ena Heather for preparation of the manuscript.
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