Resistance of mitochondrial p53 to dominant inhibition
© Heyne et al; licensee BioMed Central Ltd. 2008
Received: 11 December 2007
Accepted: 12 June 2008
Published: 12 June 2008
Mutation of a tumor suppressor allele leaves the second as backup. Not necessarily so with p53. This homo-tetrameric transcription factor can become contaminated with mutant p53 through hetero-tetramerization. In addition, it can be out-competed by the binding to p53 DNA recognition motifs of transactivation-incompetent isoforms (ΔN and ΔTA-isoforms) of the p53/p63/p73 family of proteins. Countermeasures against such dominant-negative or dominant-inhibitory action might include the evolutionary gain of novel, transactivation-independent tumor suppressor functions by the wild-type monomer.
Here we have studied, mostly in human HCT116 colon adenocarcinoma cells with an intact p53 pathway, the effects of dominant-inhibitory p53 mutants and of Δex2/3p73, a tumor-associated ΔTA-competitor of wild-type p53, on the nuclear transactivation-dependent and extra-nuclear transactivation-independent functions of wild-type p53. We report that mutant p53 and Δex2/3p73, expressed from a single gene copy per cell, interfere with the stress-induced expression of p53-responsive genes but leave the extra-nuclear apoptosis by mitochondrial p53 largely unaffected, although both wild-type and mutant p53 associate with the mitochondria. In accord with these observations, we present evidence that in contrast to nuclear p53 the vast majority of mitochondrial p53, be it wild-type or mutant, is consisting of monomeric protein.
The extra-nuclear p53-dependent apoptosis may constitute a fail-safe mechanism against dominant inhibition.
Lesions that can contribute to cell transformation normally activate the homo-tetrameric transcription factor p53, primarily to stimulate genes whose products cause senescence or apoptosis . In addition, p53 can provoke apoptosis directly through its interaction with key factors of the apoptotic machinery at the mitochondria and in the cytoplasm [2, 3]. As a result, ideally the transformation process is ceased. Among the p53 target genes that can suppress cell proliferation, one of the most important is p21Waf/Cip1 (CDKN1A), whereas PUMA (p53 up-regulated modulator of apoptosis) constitutes a prime candidate for a p53-responsive master gene of transcription-dependent apoptosis, at least in some tissues [4, 5]. In contrast, transcription-independent apoptosis by p53, which might have evolved to ascertain faithful tumor suppression in the face of lesions that temporarily compromise transactivation , involves binding of p53 to Bcl-2 family proteins outside the nucleus. Remarkably, both the transactivation of genes and the interaction with apoptosis regulators are mediated through, and rely upon, the integrity of p53's core DNA binding domain [7, 8]. Tumor-derived mutant p53 proteins are thus usually bi-dysfunctional as they are predominantly mutated within this domain.
Stresses such as DNA-damage, oncogene expression, hypoxia, and reactive oxygen can trigger the translocation of approximately 2% of p53 to mitochondria  in many primary and some transformed cell types [6, 9–12]. Notably, this seems to occur fast and precede transcriptional effects in certain tissues [7, 9, 10, 13, 14]. Mitochondrial p53 is primarily present at the outer mitochondrial membrane where it may form, without the help of further factors, a permeabilizing, death-inducing complex [7, 12]. Alternatively, it may form complexes with the anti-apoptotic Bcl-2 and BclXL proteins [7, 8]. The affinities of these proteins for p53 are higher than for the pro-apoptotic BH123 proteins Bax and Bak; consequently, the latter are freed, form oligomers, and kill the cell. A further pathway may allow pro-apoptotic Bak to be released from the anti-apoptotic Mcl-1 or BclXL proteins upon their association with a distinct site of the p53 DNA binding domain, and then form oligomers and kill the cell . In addition to its mitochondrial action, p53 may bind to cytosolic BclXL and liberate Bax, and may then activate cytosolic monomeric Bax to form lethal oligomers by a mechanisms involving transient interaction of Bax with p53's polyproline-rich domain . Finally, p53 may act through a combined protein interaction and transactivation mechanism: The product of the p53 target gene PUMA resolves an inactive cytosolic p53/BclXL complex by binding to a distinct site on BclXL and allows the activation of Bax by free p53 . Which arm of the complex death program is primarily active almost certainly is cell type and context-dependent.
Mutant p53 is present at the mitochondria regardless of apoptotic stimulus , suggesting that in contrast to wild-type (wt) p53, a translocation mechanism is active for the mutant proteins regardless of stress, or that the presence of mutant p53 in and by itself constitutes a death stimulus, as is the case with many other oncoproteins. Apart from exhibiting a wt p53-independent oncogenic 'gain-of-function', which at least in part seems to be mediated through inactivating interaction with other pro-apoptotic members of the p53 family (reviewed in ), studies of Li-Fraumeni individuals with an inherited mutated allele and of knock-in mice have clearly indicated that mutant proteins can act dominant-inhibitory, either through hetero-oligomerization with wt p53 expressed from the second allele or through the sequestration of limiting factors [18, 19]. Clearly, these interactions can compromise the transcriptional activity of wt p53.
Another potent mechanism of dominant inhibition seems to employ target gene promoter occupation by transactivation-incompetent (ΔN and ΔTA-) members of the p53 family. Interestingly, inhibition of transactivation by ΔN-p73 occupying p53 recognition motifs appears to play an important physiological role in the protection of developing sympathetic neurons from p53- and p63-provoked apoptosis (reviewed in ), whereas a similar mechanism based on aberrantly spliced p73 giving rise to the Δex2/3p73 isoform, can be active in human tumors (for instance, ). Here we began to examine to what extent dominant inhibition by the described mechanisms would affect the extra-nuclear apoptotic functions of p53.
The following studies were performed primarily in human HCT116 colon adenocarcinoma cells. HCT116 is a poorly differentiated, growth factor-insensitive cell line exhibiting microsatellite instability (MIN) caused by deficiency for the essential hMLH1 mismatch repair factor. Many forms of stress except aberrant oncogene expression can stabilize and activate the wt p53 tumor suppressor present in these cells and elicit the expected responses, including apoptosis and cell cycle arrest [22–24]. 5-fluorouracil (5FU), the mainstay chemotherapeutic for colon cancer, induces apoptosis in a p53-dependent manner in HCT116 cells, primarily through the interference of FdUMP with RNA metabolism . α-amanitin, which causes a global transcription inhibition through the initiation of RNA polymerase II degradation, can also provoke HCT116 cell apoptosis. This transactivation-independent cell death was shown to rely on p53 acting at the mitochondria .
When exponentially proliferating HCT116 cultures were treated with α-amanitin (10 μM) and analyzed by flow cytometry, the number of cells with a sub-2n DNA content indicative of apoptosis increased with time (see Additional file 1A). However, this increase was not as marked as the one observed by others with the same cell type . In contrast, 5FU (375 μM) induced a robust apoptosis under similar conditions. In agreement with previous reports [6, 23, 25], both agents provoked apoptosis in dependence of p53 as HCT116 p53-/- cells failed to respond in this manner (see Additional file 1A). Robust cell death also ensued when HCT116 cultures were simultaneously treated with 5FU and α-amanitin. As expected, inclusion of α-amanitin in the drug cocktail blocked the strong stimulation by p53 of the p21Waf/Cip1 gene that was normally observed in the presence of 5FU at both the transcript and protein levels (see Additional file 1B). Thus, in accord with earlier findings , this shows that 5FU plus α-amanitin can trigger a p53-mediated cell death in HCT116 cultures in the absence of Pol II-mediated transcriptional transactivation. This cell death was apoptotic as it was accompanied by cytochrome c release from the mitochondrial intermembrane space and by caspase 3 activation (see Additional file 1C).
Apoptosis by 5FU, α-amanitin, or both combined (FA hereafter) was preceded by an increased appearance of p53 in the mitochondrial fraction prepared by biochemical cell fractionation (see Additional file 2A). Remarkably and in agreement with previous reports [7, 9], HCT116 p53-/- cells retrovirally infected to express p53 conformational mutant 175H or DNA contact mutant 273H, had mutant p53 in their mitochondrial fraction regardless of stress (see Additional file 2B). To confirm the presence of mutant p53 at the mitochondria, immune electron microscopy was performed on the cell lines in the absence of stress, and the number of gold grains at the mitochondria was counted in a blinded study. Additional file 2C shows that the number of mitochondria-associated grains was significantly higher in the cell lines that express p53 when compared to the vector-only cell line (Chi-square test: P < 0.001 for 273H and P < 0.04 for 175H). As neither mutant can interact with the Bcl-2 family of proteins [7, 8] or transactivate genes , no enhanced apoptosis was apparent in these cell lines (data not shown).
Like the full-length mutants, 175ΔC and 273ΔC were present in the mitochondrial fraction of unstressed cells (Figure 1B; compare with Additional file 2B). In contrast, wt p53 translocated to the mitochondria in these mutant p53-transduced cells only after 5FU treatment (Figure 1B). 175ΔC and 273ΔC, like the full-length mutants but unlike mutant proteins with a deletion in the oligomerization domain, were competent for homo-oligomerization, as indicated by chemical crosslinking with Bis-maleimidohexane (BMH) that crosslinks sulfhydryl groups, and with glutaraldehyde (GLD) that crosslinks amino groups (Figure 1C). Interestingly, 175H and 175ΔC consistently showed a weaker propensity to homo-oligomerize in this cell type than 273H or 273ΔC, a tendency observed with both BMH and GLD crosslinkings (Figure 1C). This became also apparent in an assay of hetero-oligomerization when the mutants were employed to co-immunoprecipitate, with p53 antibody DO-1, a full-length p53 carrying a mutated DO-1 epitope (Figure 1D). Thus, 273H and 273ΔC seem to form stable oligomers more readily than 175H and 175ΔC in HCT116 cells. Combined, these data indicate that the presence of the mutants 175ΔC and 273ΔC at approximately two-fold increased levels compared to wild-type, does not interfere with the 5FU-induced mitochondrial translocation of wt p53.
To confirm the inhibitory effect of 273ΔC on the induced expression of p21Waf/Cip1, chromatin immunoprecipitation (ChIP) was performed. Consistent with an inefficient promoter occupation by the hetero-oligomers, the promoter sequence of p21Waf/Cip1 was less frequently precipitated with an anti-p53 antibody from cultures expressing 273ΔC than from cultures harbouring vector only. Again, no differences were observed between the 175ΔC and control cells (Figure 2C).
We next asked whether the observed effects of mutant 273ΔC on transactivation might translate into a phenotype. The seeding of 1,000 live cells of the respective cell lines onto 10 cm dishes and the maintenance of these cultures under stress-free conditions resulted in approximately equal numbers of colonies. However, when the cells were exposed to different sub-lethal concentrations of the DNA-damaging drug etoposide, only cells that expressed mutant p53 were able to grow colonies at a threshold concentration of the drug, and again, 175ΔC proved less dominant than 273ΔC in this assay (Figure 2D). Etoposide was chosen as the stressor because it induces primarily cell cycle arrest whereas 5FU induces primarily apoptosis in HCT116 cells [23, 28]. Altogether, the data suggest that p53 mutant 273ΔC expressed from a single gene copy and produced, on average, at approximately 1.5-times the level of endogenous wt p53, can act dominant-inhibitory on transactivation and the suppression of colony formation by wt p53 in stressed HCT116 cells. 175ΔC may be impaired in these assays owing to its lower propensity to oligomerize.
BMH crosslinks sulfhydryl groups while glutaraldehyde (GLD) crosslinks amino groups. To confirm the absence of detectable p53 oligomers in the mitochondrial fraction, a similar study was performed with GLD. As shown in Figure 5B, GLD was able to crosslink p53 in total cell protein samples but not in the mitochondrial fraction. To test whether this was limited to HCT116 cells, we next employed human MCF7 breast adenocarcinoma cells with an intact wt p53 pathway. 5FU induced mitochondrial translocation of p53 in MCF7 cells (Figure 5C). As in HCT116 cells, the mitochondrial fraction of MCF7 cells was devoid of oligomeric p53 upon BMH of GLD crosslinking while oligomers were readily detectable in the total cell protein sample (Figure 5D). Combined, these data suggest that the majority of mitochondrial p53, be it wild-type or mutant, is monomeric. We cannot exclude that mitochondrial p53 consists of unstable oligomers, or that a small fraction of p53 is present at the mitochondria as tetramers and is trapped in megacomplexes after crosslinking. In support of the conclusion that mitochondrial p53 is mostly monomeric, p53DD capable of forming immuno-precipitable hetero-oligomers with wt p53 in HCT116-p53DD cells, was not detectable at the mitochondria whereas wt p53 was (Figure 5E).
For the reasons detailed above, we had employed C-terminally truncated versions of mutant p53 and shown that these, although capable of dominance towards wt p53-mediated transactivation of genes, failed to interfere with the transcription-independent apoptosis by p53 at the mitchondria. To study the effects of full-length mutants, expression plasmids producing HA-tagged full-length 175H, 273H and also 248W as another common tumor-associated conformational mutant were generated. All mutants were expressed to approximately equal levels upon transfection into HCT116 p53-/- cells (see Additional file 3A). In a first study we determined the ratio of mutant vs. wt p53 expression plasmids that knocked down the transactivation by wt p53 of the endogenous p21 gene (see Additional file 3B). All mutants including 175H were able to efficiently block p21 activation, indicating that 175H, unlike its truncated variant 175ΔC, was exerting dominance efficiently. This ratio of vector/wt p53 or mutant/wt p53 plasmid was then used to transfect cultures and determine the levels of transcription-independent apoptosis by p53. The results are summarized in Additional file 3C. p53-negative cultures receiving vector-only produced background levels of apoptosis (6–8%). Cultures transfected with mutant p53 plasmids alone showed apoptosis at or even below background (not shown). In contrast, wt p53 expression alone entailed significant cell death (23%). Notably, over-expression of mutant p53 to levels sufficient to inhibit p21 transactivation nonetheless allowed significant transcription-independent apoptosis to occur (14–16%). The reduction of apoptosis in cells receiving mutant p53 plasmid in addition to wt p53 plasmid may reflect anti-apoptotic function(s) of over-produced mutant p53. Combined, we interpret these results to support that the transcription-independent mitochondrial apoptosis by p53 in HCT116 cells is relatively resistant to dominant inhibition by mutant p53.
p53 binds to its DNA recognition motif as a tetramer; monomers can not function as a transcription factor . Multimeric transcription factors, in contrast to monomeric, can be subject to dominant inhibition not only through i) the sequestration of limiting factors by an overproduced mutant, or ii) promoter-occupation by a transactivation-defective isoform, but also through iii) contamination of the multimer by mutated monomers. Although the latter two mechanisms have been documented to be active against p53 in vitro and in vivo (reviewed in [18, 37, 38], some major conflicting observations have for some time challenged the concept of dominant-negative action against p53. Doubtlessly, the observation that of the 159 annotated human tumors which had been studied for loss-of-heterozygosity by mid-2007, and which each expressed one out of 90 distinct mutated p53 alleles, approximately 60% have lost the wild-type allele (Olivier and Hainaut in ), makes a case against the global importance of dominance. Similarly negative was the observation that approximately 50% of the tumors that develop in Li-Fraumeni patients with one inherited mutated full-length p53, have lost their wild-type allele [40, 41]. On the other hand, there are several important observations in strong favour of dominance. For example, while only 50% of the Li-Fraumeni tumors with a mutated full-length p53 are wt p53-deficient, this is true for almost 100% of the Li-Fraumeni tumors with an inherited p53 allele mutated to produce no protein [40, 41].
Strong support for dominant inhibition comes also from studies on transgenic mice, although in this system dominance can be somewhat more difficult to assess, mostly for two reasons. First, the two p53 mutations introduced into the mouse germ line by knock-in in two central studies [42, 43], 172H and 270H (corresponding to human 175H and 273H, respectively), display additional, wt p53-independent oncogenic functions (gain-of-function) that may superimpose dominant-negative effects. Second, whereas human p53+/- cells almost always loose their wt allele in the course of transformation, p53+/- cells from mice often retain it  indicating that in mice, in contrast to humans, gene dose reduction can impair critical p53 functions to a degree as to mimic, with respect to tumor development, complete p53-deficiency. Nonetheless, careful studies of the efficacy of known wt p53 activities in primary fibroblasts from 172H/+ and 270H/+ mice, compared to cells from -/+ animals, have clearly demonstrated that the mutant proteins can act dominant-negative [42, 43]. Combined, the available data clearly indicate that hetero-tetramerization can incapacitate wt p53 as a transcription factor [1, 18, 37, 38]. The degree of inhibition by a mutant depends on the mutant-to-wt protein levels, the site of mutation, and the cell type [34, 35, 39].
Here we have presented evidence suggesting that the extra-nuclear, mitochondrial death pathway of p53 protein can be resistant to dominant inhibition because mitochondrial p53 is mostly monomeric. This raises important questions. For example, can monomeric p53 trigger mitochondrial apoptosis? Moll and colleagues have convincingly demonstrated that a mitochondrially targeted monomeric p53 was not only capable of provoking apoptosis but was indeed the most efficient cell death inducer of all tested p53 proteins . That group also showed that mitochondrially targeted p53 can induce apoptosis in cells expressing mutant p53 . So clearly, monomeric p53 at the mitochondria can trigger apoptosis.
If the p53-mediated mitochondrial apoptosis is resistant to dominant inhibition, why are the 40% of tumors with a mutant p53 allele that have retained the wt allele (dominant inhibition active) not dying through this pathway? Several aspects may be relevant here. First, such tumors may have evolved to impair this pathway through other means, as for instance the overproduction of Bcl2/BclXL or down-regulation of Bax/Bak. Second, even in cells expressing mutant p53, the translocation of wt p53 to the mitochondria required a stress signal (see Figure 1B). Therefore, (some) p53-dependent apoptosis may well occur in these tumors despite of the inhibition of p53's nuclear transactivator function by the mutant, but this may need stress, as for example under chemotherapy, to become apparent. The HCT116 cells used in this study had to be exposed to genotoxic stress for p53 translocation to occur; the oncogenic deregulation present in these cells was insufficient to act as p53 stressor, probably primarily because HCT116 cells fail to express the oncogenic stress signal mediator p14ARF . Third, Chipuk and colleagues have documented that cell types exist in which efficient mitochondrial apoptosis by p53 is dependent upon the transactivation of the p53 target gene PUMA. PUMA resolves an inactive cytosolic p53/BclXL complex and allows the activation of pro-apoptotic Bax by free cytoplasmic p53 . Thus, if tumors that co-express wt and dominant-negative mutant p53 belong to this cell type, extra-nuclear apoptosis induction by p53 might be impaired despite of the resistance of mitochondrial p53 to dominant inhibition by hetero-tetramerization. Forth, some of these PUMA-sensitive tumors may have developed from cell types that are able to mobilize the p53-relatives p63 or p73 for the transactivation of PUMA. In such tumors, it will make a difference whether the expressed mutant p53 is of class I (wild-type conformation) or class II (conformation-altered) type. Class II but not class I mutants can bind and inhibit p63 and p73, which is considered to be one mechanism of 'gain-of-function' . Consequently, tumors of this type with the genotype (wt p53/class II mutant), but not (wt p53/class I mutant) tumors, may exhibit a gain-of-function phenotype with respect to the impairment of mitochondrial apoptosis. Importantly, these inferences can be subject to experimental testing. In sum, the resistance of mitochondrial monomeric p53 to dominant inhibition by hetero-tetramerization should increase the selection pressure on a stressed tumor with a mutated p53 allele to loose the mitochondrial apoptosis function.
Contamination of the wt p53 tetramer with mutant p53 through hetero-tetramerization is a remarkably inefficient way to inactivate p53 [34, 35] whereas dominant inhibition through transactivation-defective (ΔN or ΔTA) isoforms of, for instance, p53 or p73, is very efficient ([21, 35], see also Figure 6). The latter mechanism can involve the formation of hetero-tetramers between wt p53 and ΔN-p53, where one truncated protein per tetramer is sufficient to incapacitate it for transactivation , and can involve competitive promoter-occupation by ΔN-p53 or ΔN-p73 [21, 35]. The extraordinary efficiency of this inhibitory mechanism may be one reason for the fact that it has gained important roles in several physiological processes. For example, ΔN-p73 is a very efficient and perhaps the primary inhibitor of p63-mediated apoptosis during neuronal development, and of p53-mediated apoptosis in response to adult neuron injury and neurodegeneration . With this efficacy of inhibition, it is no surprise that ΔTA-isoforms of p73 have also been found in tumor cells. One might thus speculate that the ΔTA-p73-resistant apoptotic function of mitochondrial p53 has evolved, at least in some cell types, as part of a fail-safe mechanism against the powerful dominant-negative effects of these molecules.
Monomeric p53 not only escapes dominant inhibition through hetero-tetramerization but also ubiquitylation through the E3/E4 ubiquitin ligase MDM2, as MDM2 binds exclusively to tetrameric p53 . On the other hand, (multi-)mono-ubiquitylation of p53 by MDM2, as opposed to poly-ubiquitylation associated with p53 degradation, can promote mitochondrial p53 translocation , and in accord with this, the apoptosis- and tetramerization-defective p53 mutant 337C  occurring in Li-Fraumeni patients fails to translocate to the mitochondria upon stress (our unpublished observation). Thus, the recent finding that cytoplasmic p53 – mono-ubiquitylated by cytoplasmic MDM2 – travels to the mitochondria without shuttling MDM2 , might suggest that monomerization of p53 occurs in stressed cells somewhere between this modification and p53's final destination at the mitochondria.
Mutant p53 can compromise transactivation by wild-type p53 through hetero-tetramerization, and p53/p63/p73 isoforms lacking a transactivation domain through competitive promoter-occupation as well as hetero-tetramerization. p53 has evolved to command a second, transcription-independent pathway of apoptosis induction at the mitochondria. This second pathway – although probably less efficient than full apoptosis by p53, provoked by both pathways in concert, – is resistant against dominant inhibition in HCT116 cells. Moreover, mitochondrial p53 – in contrast to nuclear – is mostly monomeric. Thus, the extra-nuclear p53-dependent apoptosis may constitute a fail-safe mechanism against dominant inhibition.
Plasmids, chemicals, and antibodies
pLRNL-175ΔC, pLRNL-175ΔO, pLRNL-273ΔC, pLRNL-273ΔO pLRNL-175H and pLRNL-273H have been described elsewhere . Full-length mutant p53 genes 175H, 273H and 248W were cloned into expression plasmid pCMV-pA. Plasmid pLXSNp53DD was kindly provided by Moshe Oren, Rehovot, Israel. Plasmid p73Δ2/3 β-pcDNA-3.1 was kindly provided by Thorsten Stiewe, Würzburg, Germany. The p73Δ2/3 β insert was cut out with BamHI and subcloned into pLRNL. The drugs α-amanitin, 5-fluorouracil (5-FU) and etoposide were from Sigma (St Louis, USA), as were propidium iodide for DNA content analysis, glutaraldehyde and crystal violet for fixation and colony staining, the β-actin monoclonal antibody, and the peroxidase-conjugated secondary anti-mouse and anti-rabbit antibodies. The crosslinker BMH was from Pierce (Rockford, USA). G418 and the transfection reagent Nanofectin I were purchased from PAA (Pasching, Austria). The p53 monoclonal antibodies DO-1 and PAb421, and puromycin were purchased from Calbiochem (San Diego, USA); the p53 polyclonal antibody CM-1 was from Biocare Medical (Pike Lane, USA); the p73Ab-4 antibody cocktail (clones ER-13 + ER-15 +GC-15) was purchased from Lab Vision Corporation (Fremont, USA); the monoclonal cytochrome c antibody (clone 7H8.2C12) was from BD Biosciences (Franklin Lakes, USA); the cytochrome c oxidase subunit IV monoclonal antibody (10G8) was from Molecular Probes (Eugene, USA) and the anti-HA antibody 12CA5 was from Sigma (St. Louis, USA). The monoclonal caspase-3 antibody (8G10) was purchased from Cell Signaling Technology (Boston, USA). The monoclonal p21 antibody was from BD Pharmingen (Franklin Lakes, USA). The polyclonal Bax antibody (sc-493) was acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Protein G Sepharose (4 Fast Flow) was from GE Healthcare (Uppsala, Sweden). The Annexin V:PE apoptosis detection kit was purchased from BD Biosciences (Franklin Lakes, USA).
Cell culture, viruses, and transfection
293GP retrovirus producer cells, MCF-7, and H1299 cells were cultured in DMEM, and HCT116 cells and derivatives in McCoy's 5A medium, all supplemented with 10% FCS and grown in a humidified 7% CO2 atmosphere at 37°C. Retroviruses were produced in the 293GP producer line after a 1:1 transfection with a vector coding for the stomatitis virus G protein and either empty retroviral vector or vector expressing one of the p53 mutants, p53DD, or Δex2/3p73. The viruses were harvested 48 h after transfection. The supernatant was filtered through a 0.45 μM filter (Renner, Germany). Virus stocks were titered on 293 cells and frozen in aliquots at -80°C. For infection, cultures were incubated with virus stocks at a multiplicity of infection (MOI) of approximately 0.1 colony-forming units (cfu)/cell to assure transfer of only one transgene per infected cell. Infection was carried out in the presence of 4 μg/ml polybrene (Sigma) for 4 h, and virus-infected cells were selected in 400 μg/ml G418, beginning at 48 h after infection, for approximately 7 days. Exponentially growing cultures that had been out of selection for at least 2 days were drug-treated as outlined in the main text and figure legends. As transfection reagent, Nanofectin I from PAA (Pasching, Austria) was employed according to the manufacturer's recommendation.
Flow cytometry analysis of DNA content and apoptosis
Twenty-four h prior to drug treatment, cells were seeded in six-well dishes to approximately 30% confluency. At the indicated time points, the cells on the dishes were harvested by trypsinization, washed in PBS, resuspended in 200 μl of 0.9% NaCl, squeezed through a 23.5-gauge needle into 1.8 ml of methanol, and fixed overnight at -20°C. Cells were resuspended in PBS supplemented with RNase A (25 μg/ml) at approximately 106 cells per ml, and were stained with PI (25 μg/ml) for >1 h at 4°C. DNA fluorescence was measured with a Becton Dickinson FACSCanto (Bedford, USA) and the data were analyzed with BD FACSDiva software from Becton. FACS detection of Annexin V:PE at apoptotic cells was done according to the protocol supplied with the Annexin V:PE apoptosis detection kit from BD Biosciences (Franklin Lakes, USA).
Colony formation assay
For colony formation assays, 103 live cells (counted with a FACSCanto) were seeded onto 10-cm dishes and grown for 24 h. The cultures were then incubated in the presence of etoposide for 10 days. Colonies were washed with PBS, fixed with 1.25% glutaraldehyde for 20 min, washed again and stained with 1% crystal violet in PBS for 1 h at room temperature.
Preparation of subcellular fractions and immunoblot analysis
Mitochondria protein fractions were prepared and tested essentially as described before . In brief, mitochondria were isolated with the Mitochondria Isolation Kit for Mammalian Cells (Pierce, Rockford, USA), following the supplier's protocol. The centrifugation step at 700 g was repeated three times. Mitochondria were lysed in an SDS-lysis buffer heated to 100°C, containing 100 mM Tris-HCl (pH 6.8), 100 mM DTT, 4% SDS, and 20% glycerol. 15 μg of mitochondrial protein were subjected to 8 or 13% SDS-PAGE and analyzed by Western blotting. For immunoblot analysis, cells were lysed in the SDS-lysis buffer heated to 100°C. Samples containing 15 or 30 μg of total cellular protein were subjected to 8, 10, or 13% SDS-PAGE and transferred to a PVDF membrane (Immobilon-P; Millipore, Bedford, USA). Signals were detected upon overnight incubation of the membranes with one of the indicated antibodies, followed by a final incubation with a peroxidase-conjugated secondary anti-mouse (1:2000) or anti-rabbit (1:2000) antibody and Pierce ECL Western Blotting Substrate (Rockford, USA), performed as specified by the supplier.
Cytochrome c release
Cells were treated with the indicated drugs, and nuclei and mitochondria were again separated from the cytosolic fraction with the Mitochdria Isolation Kit for Mammalian Cells from Pierce (three centrifugation steps at 700 g, one centrifugation step at 3000 g, one centrifugation step at 12000 g). The cytosolic fraction was then concentrated using Microcon® Centrifugal Filter Devices YM-10 (Millipore, USA) for 10,000 nominal molecular weight limit, performed as specified by the supplier. Concentrated samples were mixed with SDS-lysis buffer, heated to 100°C, and analyzed by Western blotting.
Cells were seeded in 10 cm-dishes and treated 24 h later with 5-FU. One day after drug-treatment medium was removed and solution D (236.4 g guanidinium thiocyanate; Sigma, USA, in 293 ml water, 17.6 ml 0.75 M sodium citrate pH 7.0, and 26.4 ml 10% sarcosyl, 0.72% 2-mercaptoethanol) was added. Cells were scraped off and 0.1 ml of 2 M sodium-acetate pH 4, 1 ml of water-saturated phenol (Roth, Germany), and 0.2 ml of chloroform-isoamylalcohol (49:1) were added, mixed, and cooled on ice for 15 min. After centrifugation (10,000 g, 4°C, 20 min) the aqueous phase was collected and precipitated with isopropanol overnight. After a further centrifugation (10,000 g, 20 min, 4°C), RNA was redissolved in solution D and precipitated with isopropanol at -20°C for 1 h. The pellet was washed in 70% ethanol and dissolved in DEPC-water. The RNA was digested with RNase-free DNase I (Roche, Germany) for 60 min at 37°C, and 4 μg was used for the first-strand cDNA synthesis with SuperScript™III (Invitrogen, USA) as specified by the manufacturer. Semiquantitative RT-PCR analysis was performed with HotStarTaq (Qiagen, Germany), using the primers: p21 (for: ggcggcagaccagcatgacagatt; rev: atgaagccggcccacccaacctc; TA: 64°C), bak (for: taggcgctggggagactgataact; rev: aggcttggaggcttctgacacg; TA: 65°C), bax (for: ccccgagaggtctttttccgagtg; rev: gaaaaatgcccatgtcccccaatc; TA: 65°C), and gapdh (for: tggtatcgtggaaggactcatgac; rev: agtccagtgagcttcccgttcagc; TA: 64°C).
Immune electron microscopy
Whole cells were fixed in 4% formaldehyde/0.05% glutaraldehyde, dissolved in 0.1 M cacodylat buffer (pH 7.4) at RT, and stored overnight at 4°C. Pellets were resuspended in 2% low-melting point agarose at 40°C and solidified on ice. Whole cells were fixed to the agarose gel with the formaldehyde/glutaraldehyde fixative (see above). After washing the gel with 0.1 M phosphate buffer pH 7.2, small blocks (maximum 2 × 2 × 2 mm3) were cut out and dehydrated by the processive-lowering-of-temperature-method, using the following ethanol series and temperatures: 30%, 0°C; 50%, -20°C; 70, 90, 100% at -35°C; for 1 h each. Dehydrated gel blocks were infiltrated and embedded with the acrylate resin Lowicryl K4M (Polysciences, Eppelheim, Germany) at -35°C. The resin was UV-polymerized for 1 day at -35°C, 1 day at 0°C, and 1 day at RT. Ultrathin sections (70–80 nm) were placed on droplets (30 μl) of the following: glycine (50 mM in PBS); blocking solution; anti-p53-antibody DO-1 or IgG control diluted in blocking solution; blocking solution; goat anti-mouse antibody coupled to 10 nM colloidal gold (Aurion, Netherlands); blocking solution; PBS; 2.5% glutaraldehyde in 0.1 M phosphate buffer; PBS; and water. The blocking solution contained 0.5% cold water fish gelatine, 0.5% BSA, 0.01% Tween-20 (all from Sigma), dissolved in PBS. The incubations with the antibodies were carried out overnight at 4°C in a wet chamber. Finally, the sections were dried and stained with uranyl acetate and methylcellulose. All intact mitochondria detected at 68,000× magnification in randomly chosen fields were analysed with morphometric software (Analysis, SIS, Germany).
Chromatin immunoprecipitation and protein co-immunoprecipitation
Cells were seeded and 24 h later treated with 5-FU. The ChIP analyses were performed with the Chromatin Immunoprecipitation Assay Kit from Upstate (Lake Placid, USA) according to the manufacturer's recommendation, with the following modifications. 2.5 μg p53-antibody (DO-1) or irrelevant antibody, linked to Protein G sepharose 4 Fast Flow™, were used for immunoprecipitation. For PCR, 2 μl out of 50 μl of DNA extractions were employed. The primer sequences and PCR conditions used to amplify the corresponding promoter fragments were as follows: p21 (for: acctttcaccattcccctac; rev: gcccaaggacaaaatagcca; TA: 56°C); U6 (for: ggcctatttcccatgattcc; rev: atttgcgtgtcatccttgc; TA: 56°C). For protein co-immunoprecipitation, HCT116 cultures at a density of approx. 50% were transiently transfected by Nanofectin I (PAA, Austria) with the different expression plasmids, and 24 h later the truncated p53 protein with the intact DO-1 epitope was immunoprecipitated with antibody DO-1, following our standard IP protocol . The full-length p53 with a defective DO-1 epitope that was co-precipitated along with the truncated p53 could be detected in a Western blot with the polyclonal p53 antibody CM-1.
Total protein extracts were prepared by lysing the cells in a buffer containing 10 mM Tris pH 7.6, 140 mM NaCl, 0.5% Nonidet P-40 (NP-40), and proteinase inhibitor cocktail (Sigma, USA). After incubation for 30 min at 4°C the samples were centrifuged at 13.000 rpm for another 30 min at 4°C. For BMH crosslinking, whole cell lysate was incubated with 0.2 mM BMH (bis-maleimidohexane; Pierce) according to the provided protocol. After an incubation of 1 h at RT, monomers and multimers were separated on SDS-polyacrylamide gels and detected with p53 antibody DO-1 or Bax antibody sc-493. Mitochondria prepared with the Mitochondria Isolation Kit for Mammalian Cells (Pierce), were resuspended in the BMH conjugation-buffer and incubated with BMH (final concentration: 1 mM) at 37°C for 1 h. For crosslinking with glutaraldehyde (GLD), whole cell lysates were incubated with 0.0025% GLD for 15 min at RT, as described before . Isolated mitochondria were resuspended in PBS and incubated with 0.0025% GLD for 15 min at 37°C. Finally, monomers and multimers were again separated on SDS-polyacrylamide gels and detected with the respective antibodies.
We thank Gabi Kiefer, Institute of Anatomy, for excellent technical assistance with electron microscopy. This work was supported by German Research Foundation (DFG) Grant RO 1201/10-1 to KR.
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