Plasmid R1 inhibits growth of bacteria by synthesizing an inhibitor of cell proliferation, Kid, and a neutralizing antidote, Kis, which binds tightly to the toxin. Here we report that this toxin and antidote, which have evolved to function in bacteria, also function efficiently in a wide range of eukaryotes. Kid inhibits cell proliferation in yeast, Xenopus laevis and human cells, whilst Kis protects. Moreover, we show that Kid triggers apoptosis in human cells. These effects can be regulated in vivo by modulating the relative amounts of antidote and toxin using inducible eukaryotic promoters for independent transcriptional control of their genes. These findings allow highly regulatable, selective killing of eukaryotic cells, and could be applied to eliminate cancer cells or specific cell lineages in development.
Prokaryotic plasmids have developed genetic systems that increase their stable maintenance in bacterial hosts. One group of them, called killer systems, eliminate bacteria that have lost the plasmid during cell division (Jensen and Gerdes, 1995). ParD is a protein killer system of Gram‐negative plasmid R1, which is composed of two genes: kis (for killing supressor) and kid (for killing determinant) that encode the antidote (Kis; 10 kDa) and the toxin (Kid; 12 kDa), respectively. Both genes are organized in a bicistronic operon in which the toxin is located downstream of the antidote, and their expression is controlled in several ways: by coupled transcription; by post‐transcriptional processing of some of its bicistronic mRNAs to produce kis+/kid− and kis+/kid+ messengers; by overlapping translation of both genes; and by a very tight interaction between Kis and Kid to generate a non‐toxic complex that represses its own transcription (Bravo et al., 1987, 1988; Ruiz‐Echevarría et al., 1991, 1995). These controls avoid the synthesis of the toxic component if its antidote has not been translated previously and ensure a balanced production of the antidote relative to the toxin (Figure 1A). Under normal circumstances, both components of this killer system are synthesized at a basal level from their plasmid, allowing the bacterial host to survive. However, the stability of the antidote Kis is lower than that of the toxin Kid (Tsuchimoto et al., 1992). In bacteria that lose the plasmid and thus lack continuous synthesis of the unstable antidote, its more rapid degradation leads to an excess of non‐neutralized Kid protein, which is toxic to the host and inhibits its proliferation (Figure 1B).
Pathogenic bacteria have evolved many toxins to attack eukaryotic cells and many of these have had valuable practical applications (Fitzgerald, 1996; Culver, 1997). However, plasmid stability systems differ from these. They have evolved in bacteria to kill bacteria, but under the tight control of effective antidotes. If a similar system could be developed for eukaryotic cells, it would have many applications. For example, gene therapy approaches for selectively killing cancer cells depend on highly selective targeting or expression of toxins to cause maximum damage to cancer cells whilst minimizing damage to normal cells. A higher degree of selectivity could be achieved if the toxin is targeted to tumour cells and non‐tumour cells are protected from the action of the toxin by a specific antidote. In this work, we show that Kid inhibits cell proliferation in eukaryotes and kills human cells by apoptosis. Furthermore, we demonstrate that the antidote Kis overcomes the toxic effect of Kid in yeast, Xenopus laevis and human cells. We also establish that it is possible to regulate these effects in eukaryotes by means of independent transcriptional regulation of kis and kid, and we discuss strategies for exploiting these results for selectively killing tumour cells and for selective cell‐lineage knock‐outs in development.
Expression of Kid inhibits yeast cell growth and co‐expression of Kis rescues
The first purpose of this work was to test whether the components of the parD system of plasmid R1 could function in eukaryotes. As mentioned above, this killer system is kept silent in Escherichia coli by means of a complex genetic and molecular regulatory circuit, based mainly on the bicistronic nature of the parD operon. Although some bicistronic operons exist in eukaryotes (McBratney et al., 1993; Cornelis et al., 2000), it would be technically difficult to use them effectively for this purpose. Therefore we decided to study the effect of Kis and Kid in Saccharomyces cerevisiae using independent transcriptional control, rather than the native bicistronic nature of the parD operon. Budding yeast was transformed with the integrative plasmid p303MKCKd, in which kis expression is repressed in the presence of methionine and kid expression is activated in the presence of Cu2+ (Figure 2A). Yeast growth was severely inhibited in this transformant in the presence of methionine and Cu2+, but not in their absence or in the presence of Cu2+ only (Figure 2B). These results indicate that expression of Kid inhibits cell proliferation in S.cerevisiae and that co‐expression of its antidote, Kis, protects against inhibition. Importantly, they also indicate that antidote alone has no apparent side effects on yeast cell viability.
Microinjected Kid inhibits cell proliferation in frog embryos and kills human cells; Kis protects
Next, we injected purified proteins to see whether Kid inhibits cell proliferation in X.laevis embryos and whether Kis protects from that effect. Two‐cell embryos of X.laevis were microinjected near the animal pole of one of the blastomeres with Kid protein, or an active fusion of the Kis protein (maltose‐binding protein fused to Kis, MBPKis), or both proteins or buffer alone. The effects of these injections on subsequent cell divisions were followed with time (Figure 3A). Kid‐injected blastomeres failed to develop normally unlike the non‐injected half of the embryo. On the other hand, blastomeres injected with MBPKis, MBPKis and Kid, or buffer alone progressed normally in all cases until at least mid‐blastula (Figure 3B). Nuclear staining of sections of the different embryos showed that cells injected with Kid underwent a limited number of divisions (Figure 3C).
These results encouraged us to test the effects of Kis and Kid microinjection in human cells. Thus, we performed similar experiments using HeLa and SW480 cells. Microinjection of Kid into these cells dramatically decreased their survival and eventually led to the death of all Kid‐injected cells. This effect was completely abolished when Kis was pre‐incubated with Kid before injection (Figure 4) and absent when only buffer was injected instead (data not shown).
Kid‐mediated toxicity and Kis‐mediated survival can be regulated in human cells
The results so far demonstrate that the consequences of Kid toxicity are similar in both eukaryotes and prokaryotes. Kid inhibits cell proliferation whilst Kis neutralizes the toxicity of Kid. They also demonstrate that, in yeast, it is possible to substitute the complex prokaryotic regulatory circuits that regulate parD in E.coli by separate control of transcription of the antidote and the toxin. We asked whether independent transcriptional control of kis and kid would regulate cell killing or cell survival in human cells. For that purpose two plasmids, pNATHA1i and pNATHA2i, were constructed. Their design was based on previous observations of relative transcription from a constitutive cytomegalovirus (CMV) early promoter compared with a tetracycline repressible promoter in the presence or the absence of tetracycline (or its analogue doxycycline) in HeLa Tet Off cells (Yin et al., 1996) (Figure 5A).
HeLa Tet Off cell line was stably transfected with plasmids kis+ (pNATHA1i+) and kis+/kid+ (pNATHA2i+) and clones were selected in the absence of doxycycline so that the promoter for the antidote kis is On (Pr kis On). Figure 5B shows that conditions that switch Kis synthesis Off (Pr kis Off) inhibited proliferation only in cells that contain kid (pNATHA2i+). Furthermore, as expected from the results shown in Figure 4, this inhibition of proliferation was associated with widespread cell death when Kid is expressed without Kis beyond three days (Figure 5C and see below) and with total cell death after 15 days (data not shown). These results demonstrate that, as in the case of S.cerevisiae, inhibition of cell proliferation can be modulated in human cells by independent transcriptional control of kis and kid. Importantly, and as seen previously in the case of yeast and X.laevis, expression of kis alone has no apparent phenotypic effects in HeLa cells.
Kid triggers premature cell death by apoptosis in HeLa cells
Figures 4 and 5C show that excess Kid promotes widespread death in HeLa cells. This observation raised the interesting question of whether the lethal effect of Kid in these cells was due to activation of apoptosis. This pathway, which exists in human cells (Evan and Littlewood, 1998), is absent from S.cerevisiae (Shaham et al., 1998) and the first stages of Xenopus embryonic development (Hensey and Gautier, 1997). To address this question, samples studied in Figure 5 were stained with propidium iodide and fluorescein‐linked Annexin‐V, an early marker for apoptosis, and analysed by confocal microscopy. After 10 days of growth, HeLa Tet Off cell line and kis+ (pNATHA1i+) stable transfectant cells showed a similar small percentage of apoptotic cells in both the absence or presence of doxycycline. In contrast, the percentage of Annexin‐V positive kis+/kid+ (pNATHA2i+) cells increased almost four‐fold upon addition of doxycycline to the growth medium for 10 days (Figure 6A and B). A similar analysis upon longer exposures to doxycycline was not possible as, consistently, all pNATHA2i (kis+/kid+) cells were dead beyond day 10. A magnified view of one of these cells further confirmed apoptosis by morphological criteria (Figure 6C). These results clearly show that widespread cell death observed upon prolonged exposure to Kid in HeLa cells is due to activation of the apoptotic program.
The parD system of plasmid R1 has evolved to provide a finely balanced mechanism for regulated bacterial cell killing. This system has evolved to ensure the selfish maintenance of the R1 plasmid (Bravo et al., 1987, 1988; Ruiz‐Echevarría et al., 1991, 1995; Tsuchimoto et al., 1992). When the plasmid is present, the antidote exceeds the toxin and the cell survives. If the plasmid is lost during cell division, the antidote decays and the residual toxin kills the plasmid‐free daughter cell; thus, the possession of the plasmid can be likened to holding a hand grenade with the pin pulled; drop it and it kills you. Here we report the surprising finding that the protein components of this prokaryotic system can be used to manipulate cell proliferation or cell survival in a wide range of eukaryotic organisms. Kid inhibits their proliferation and equally important, Kis antagonizes this inhibition. To achieve these effects, it was necessary to place kis and kid under control of eukaryotic promoters. We show in this paper that independent control of both genes using eukaryotic promoters still allows regulatable arrest of cell proliferation and, in some cases, cell death.
Regulated expression of Kid and Kis for selective killing of cancer cells and for targeted cell ablation
Gene therapy approaches for selectively killing cancer cells depend on highly selective targeting or expression of toxins to cause maximum damage to cancer cells, whilst minimizing damage to normal cells. Prokaryotic toxins have been used for this purpose previously (Fitzgerald, 1996), but Kid has the advantage that it can be antagonized by a specific protein antidote in eukaryotic organisms. Unlike simple toxins (Culver, 1997), which require highly specific gene delivery or expression in cancer cells (Zullo et al., 1998), the approach suggested by this work offers the opportunity to both regulate the expression and antagonize the effect of Kid by activating the expression of Kis in non‐tumour cells. Transcriptional regulators known to be inactivated in many human cancers, such as p53 (Hainaut and Hollstein, 2000), could be used to induce kis expression. If so, combining this approach with efficient delivery vehicles might allow a regulated and selective strategy for gene therapy of cancer (Figure 7). Our results show that Kid‐induced cell death does not depend on the presence of functional p53, as SW480 cells, which express a mutant P53 protein, die 48 h after injection of Kid (Figure 4B). The recent determination of the structure of Kid (Hargreaves et al., 2002) opens the possibility of designing small drugs that mimic its effects on human cells.
We have shown that Kid inhibits cell proliferation not only in somatic cells, but also in embryonic cells. Thus, an additional use for this approach to regulated inhibition of cell proliferation is the opportunity to perform highly regulated knockouts of cell lineages during development. Targeted ablation has been successfully used in developmental studies (Booth et al., 2000; Lee et al., 2000). Once again, tissue‐specific promoters and conditional promoters could be used to tune the relative expression of kis and kid to allow selected cell knockouts at specific stages of development. This approach could have value in studies of development as well as in studies of differentiation, organogenesis or degenerative disorders.
Materials and methods
p303MKCKd was constructed in two steps. First, kis was amplified by PCR and cloned into the SmaI site of p424Met25 (Mumberg et al., 1994). The PvuI fragment of this plasmid containing kis was exchanged by the equivalent fragment of pRS303 (Sikorski and Hieter, 1989) to generate p303MK. Second, kid was subcloned from pCIKid (see below) into pSAL1 (Mascorro‐Gallardo et al., 1996) by exchanging their small ScaI–XhoI fragments. A blunt‐ended BamHI fragment from this plasmid was subcloned in blunt‐ended AatII‐digested p303MK to generate p303MKCKd. For pMALKis (MBPKis overproducer), kis flanked by an EcoRI site at 3′ was obtained by PCR using the oligonucleotides 5′‐ATGCATACCACCCGACTG‐3′ and 5′‐TCGGAATTCAGATTTCCTCCTG‐3′. This PCR product was cloned into XmnI and EcoRI sites of pMAL‐c2 (BioLabs). For pGCHisKisKid (Kid overproducer), kiskid DNA flanked by NdeI and BamHI was obtained by PCR using the oligonucleotides 5′‐GGAATTCCATATGCATACCACCCGACT‐3′ and 5′‐CGGGATCCTCAAGTCAGAATAGTGGACAGG‐3′. This PCR product was cloned into NdeI and BamHI sites of pET15b (Invitrogen) before subcloning its NcoI–BamHI small fragment into the same sites of pRG80‐recA‐Nhis (Giraldo et al., 1998). For pNATHA plasmids, kis was flanked by EcoRI and XbaI by PCR using the oligonucleotides 5′‐CGGAATTCATGCATACTACCACCCGACTG‐3′ and 5′‐CTCTAGATCAGATTTCCTCCTGACC‐3′. This PCR product was cloned into pTRE (Clontech) digested with EcoRI and XbaI. For pNATHA2i plasmid, kid flanked by XhoI and EcoRI sites was amplified by PCR using the oligonucleotides 5′‐CCGCTCGAGATGGAAAGAGGGGAAATCT‐3′ and 5′‐CGGAATTCTCAAGTCAGAATAGTGGACAGG‐3′. This PCR product was cloned in pCI‐neo (Promega) digested with XhoI and EcoRI. Neomycin resistance was deleted from pCI‐neoKid (and from parental pCI‐neo) by BstXI and SmaI digestion and religation. Resultant plasmids lacking the neomycin resistance gene were digested with BglII and BamHI. The CMV promoter‐containing fragments resulting from these digestions were blunt ended and subcloned into a blunt‐ended HindIII pTREKis to obtain pNATHA1i and pNATHA2i plasmids. Tail‐to‐tail orientations between CMV‐ and tetracycline‐dependent transcriptional units were chosen for pNATHA1i and pNATHA2i.
Yeast growth determination
Several fresh colonies of W303a strain (MATa, ade2‐1, trp1‐1, can1‐100, leu2‐3, 112, his3‐11, ura3, psi+) transformed with pRS303 (control) or p303MKCKd were diluted in sterile water to 108 cells/ml. Ten microlitres of this and four serial 1/10 dilutions were placed on SD plates without histidine, supplemented or not with 500 μM methionine and/or 200 μM CuSO4, as indicated in Figure 2, and grown at 23°C for 48 h.
MBPKis was expressed in DH5α and purified by affinity chromatography through an amylose resin (BioLabs) in 20 mM HEPES pH 7.5, 100 mM KCl, 1 mM DTT and 10% ethyleneglycol, following manufacturer's instructions. Kid was expressed in TG1 upon induction of the culture with Nalidixic acid (25 μg/ml; Merck). The soluble fraction was precipitated with 60% ammonium sulphate and resuspended in 20 mM Tris–HCl pH 7.5, 500 mM KCl and dialysed against the same buffer to eliminate the ammonium sulphate. The dialysed fraction was loaded in Fast‐Flow chelating Sepharose column (Pharmacia) activated with Ni2+. A gradient of 0–6 M guanidinium chloride (GnCl) in 20 mM Tris–HCl pH 7.5 was applied to the column until elution of denatured Kid. Kid was diluted in 20 mM HEPES pH 7.5, 6 M GnCl, 150 mM KCl, 20 mM β‐mercaptoethanol, 0.2 mM EDTA and 1.2% CHAPS and refolded by dialysis against 150 mM KCl, 20 mM HEPES pH 8.0, 10 mM β‐mercaptoethanol, 0.1 mM DTT and 10% ethyleneglycol. Soluble protein was concentrated in 3K Centricon tubes.
For embryo microinjections, 160 and 720 ng of Kid and MBPKis, respectively, dialysed in 20 mM Tris–HCl pH 8.0 and 50 mM KCl were mixed with each other or with dialysis buffer in 4 μl and incubated on ice for 10 min. Fifty nanolitres of each mix were microinjected into one of the cells of dejellied two‐cell embryos of X.laevis. Embryos were incubated in 4% ficoll 400 in MBS buffer at 18°C until mid‐blastula was reached in the non‐injected controls. Embryos where photographed, fixed and embedded in paraffin wax for sectioning as described previously (Butler et al., 2001). Sections were mounted and stained with Hoechst 33258 or with propidium iodide and analysed by microscopy. For HeLa and SW480 microinjections, proliferating cells were asynchronously grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and cultured in small chambers. Medium was replaced with warmed CO2‐independent medium (Gibco/BRL) for microinjection. MBPKis and Kid were diluted in water to 2.7 and 2.3 pmol/μl, respectively, and supplemented with 1 mg/ml Texas Red Dextran (70 000 MW; Molecular Probes) and injected into cells. For the Kis and Kid sample, proteins were pre‐mixed at those concentrations prior to injection. After injection, cells were returned to normal DMEM for continuing growth. Images were collected on a Bio‐Rad 1024 confocal microscope for determination of survival rates.
HeLa Tet Off cell line (Clontech) was co‐transfected with pNATHA plasmids and pTKHyg (Clontech) by the lipofectamine method (Gibco). Stable clones were selected following manufacturers' recommendations.
Calculation of cell growth and cell death rates for HeLa cells
For cell proliferation and cell death experiments, control HeLa Tet Off, kis+ (pNATHA1i+) and kis+/kid+ (pNATHA2i+) cells were each cultured in six plates at equally low density and in selective medium. Doxycycline (0.1 μg/ml) was added to five of the plates of each sample 10, 7, 5, 3 or 1 days before the end of the experiment. Dead and mitotic cells were added back to the plates when medium was changed. All cells were resuspended in PBS, and stained with Trypan Blue to count total and dead cells. At least 250 cells for each sample were counted in a haemocytometer.
Cells were plated at identical cell densities in the absence or the presence of 0.1 μg/ml doxycycline and grown for 10 days on coverslips. Cells were stained with fluorescein‐linked Annexin‐V (green; Clontech) as recommended by the manufacturer, and then fixed and DNA stained with propidium iodide (red) before analysis.
We thank J.O.Mascorro‐Gallardo and R.Giraldo for plasmids and K.Butler for technical assistance. We are also indebted to B.Pimentel, D.Santamaría, J.Pérez‐Martín, V.de Lorenzo and L.Ko Ferrigno for critical reading of the manuscript and helpful discussions, and to L.Sánchez‐Palazón, L.Howard, N.de la Cueva and A.Zaldivar for encouraging support. G.C.M. was supported by the Spanish Ministry of Education and Science (BIO92‐CO2‐O2) and by Cancer Reseach UK. This work was supported by Cancer Research UK, the Louis Jeantet Foundation and the Comisión Interministerial de Ciencia y Tecnología (BIO94‐0707).
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