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Regulatable killing of eukaryotic cells by the prokaryotic proteins Kid and Kis - PubMed

  • ️Wed Jan 01 2003

Regulatable killing of eukaryotic cells by the prokaryotic proteins Kid and Kis

Guillermo de la Cueva-Méndez et al. EMBO J. 2003.

Abstract

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.

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Figures

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Fig. 1. Mechanism of action of protein prokaryotic killer systems and genetic organization of the parD killer system of plasmid R1. (A) The parD killer system of R1 is organized in a bicistronic operon that ensures that transcription of the antidote kis occurs before that of the toxin kid. After transcription, some bicistronic mRNAs are degraded by exonucleolytic processing from their 3′ end up to an inverted repeat that folds in a stem–loop structure (white arrowheads). This ensures an excess of monocistronic mRNAs that encode only for the antidote. Translation of kid starts from a Shine–Dalgarno sequence (indicated by asterisks) that overlaps with the 3′ end of the kis gene. Thus, kid translation can start only if the antidote has been translated previously. All these strategies ensure that, under normal circumstances, the antidote is always in excess over the toxin and they allow the formation of a non-toxic protein complex that interacts with an inverted repeat located in the parD promoter (black arrows) to repress its own transcription. (B) Toxicity of protein prokaryotic killer systems is neutralized in plasmid-containing cells by continuous synthesis of an unstable antidote (light grey spheres). Loss of the plasmid during bacterial division prevents further production of the antidote and its preferential degradation allows the toxin to exert its lethal effect on bacterial segregants (dark grey spheres).

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Fig. 2. Independent transcriptional control of kis and kid allows activation of the parD system in S.cerevisiae inhibiting cell proliferation conditionally. (A) Scheme depicting plasmid p303MKCKd. (B) Analysis of growth rates of budding yeast transformed with p303MKCKd or with pRS303 (control) in solid media supplemented with the indicated amounts (µM) of methionine and/or Cu2+. Samples were diluted in sterile water at the cell densities indicated and 10 µl of each dilution were used for each dot. Abbreviations: Met25, methionine repressible promoter; CUP1, copper inducible promoter; CYC1, CYC1 terminator.

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Fig. 3. Kid inhibits cell proliferation in X.laevis embryos; Kis neutralizes this effect. (A) Scheme depicting microinjection experiments of Xenopus embryos. One blastomere of a two-cell embryo was injected with either Kid, MBPKis, Kid plus MBPKis or buffer. Development was allowed to proceed until the control uninjected embryos reached stage 9 (late blastula). (B) Representative embryos from the experiment shown in (A) when buffer, MBPKis, Kid, or Kid plus Kis proteins were microinjected. Almost identical results were obtained with all the embryos injected with Kid (43 in total), MBPKis (15 in total) and Kid plus MBPKis (19 in total). (C) A section of one embryo microinjected into one blastomere with Kid protein. Embryos were fixed, paraffin-embedded, sectioned and stained for DNA. The uninjected half embryo developed normally whereas the Kid-injected half showed very few cells, most of which were anucleate. The yellow arrowheads indicate the only two nuclei present in the Kid-injected half embryo.

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Fig. 4. In the absence of its antidote Kis, toxin Kid kills human cells. (A) Plot showing that only 9 out of 154 HeLa cells (5.8%) survived 24 h after Kid microinjection, whereas 155 out of 168 HeLa cells (92.5%) did so after Kid plus MBPKis microinjection. (B) Plot showing that only 9 out of 72 SW480 cells (12.5%) survived 48 h after Kid microinjection, whilst 65 out of 65 (100%) of these cells did so when they were microinjected with Kid plus MBPKis.

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Fig. 5. Independent transcriptional control of kis and kid allows regulated inhibition of cell proliferation and cell death in HeLa cells. (A) Scheme depicting plasmids pNATHA1i and pNATHA2i, in which kid (if present) is constitutively expressed from a CMV promoter whilst kis expression is repressed in Tet Off cell lines by doxycycline addition to the culture media. (B) Time course comparing the relative growth of HeLa Tet Off (control), kis+ (pNATHA1i+) and kis+/kid+ (pNATHA2i+) stably transfected cells in the presence (Pr kis Off) and the absence (Pr kis On) of doxycycline. Each line represents a comparison of the cell proliferation rate in the cultures without doxycycline (Pr kis On) with the rate for cells grown in the presence of doxycycline (Pr kis Off). Control HeLa cells and pNATHA1i (kis+) cells grow identically with or without kis transcription. In contrast, relative growth decreases over time when kis transcription is repressed by doxycycline in pNATHA2i (kis+/kid+) cells. (C) Cell death rates for the experiment shown in (B). At least 250 cells were assessed at each time point.

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Fig. 6. Expression of kid without kis causes widespread and premature cell death by apoptosis in HeLa cells. (A) Low power fields of control HeLa Tet Off, kis+ (pNATHA1i+) and kis+/kid+ (pNATHA2i+) cell cultures grown in the absence (Pr kis On) and the presence (Pr kis Off) of doxycycline for 10 days and stained with propidium iodide (red) and FITC-linked Annexin-V (green). (B) Percentage of Annexin-V positive cells in the different cultures analysed in (A). (C) Magnified image of one kis+/kid+ (pNATHA2i+) cell grown in the presence of doxycycline (Pr kis Off) for 10 days and stained as in (A).

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Fig. 7. Regulated expression of Kid and Kis for selective killing of cancer cells. Scheme depicting a possible approach for anti-cancer gene therapy based on the transcriptional activation of the kis gene by wild-type p53 and reinforced by activation of the kid toxin gene by p53 bearing a common mutation. Blue spheres, Kis; red spheres, Kid.

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