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Human Molecular Genetics Pages 1355-1361  

A cellular model that recapitulates major pathogenic steps of Huntington's disease
Introduction
Results
   Formation of inclusions in cells expressing mutant huntingtin
   Processing of mutant full-length huntingtin
   Apoptosis and expression of mutant proteins
Discussion
Materials And Methods
   Plasmid construction
   Generation of inducible cell lines
   Culturing and differentiation of cell lines
   Western blot analysis
   Immunocytochemical studies
Acknowledgements
References

A cellular model that recapitulates major pathogenic steps of Huntington's disease

A cellular model that recapitulates major pathogenic steps of Huntington's disease

Astrid Lunkes and Jean-Louis Mandel*

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/Université Louis Pasteur, 1 rue Laurent Fries, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France

Received May 18, 1998; Revised and Accepted June 30, 1998

To gain insight into the pathogenic mechanisms of Huntington's disease (HD), we have developed a stable cellular model, using a neuroblastoma cell line in which the expression of full-length or truncated forms of wild-type and mutant huntingtin can be induced. While the wild-type forms have the expected cytoplasmic localization, the expression of mutant proteins leads to the formation of cytoplasmic and nuclear inclusions in a time- and polyglutamine length-dependent manner. The inclusions are ubiquitinated, appear more rapidly in cells expressing truncated forms of mutant huntingtin and are correlated with enhanced apoptosis. In lines expressing mutant full-length huntingtin, major characteristics present in Huntington's patients could be modelled. Selective processing of the mutant, but not the wild-type, full-length huntingtin was observed at late time points, with appearance of a breakdown product corresponding to a predicted caspase-3 cleavage product. A more truncated N-terminal fragment of huntingtin is also produced, that appears involved in building up cytoplasmic inclusions at early time points, and later on also nuclear inclusions. This fits with the finding that inclusions in the brain of HD patients are detected only using antibodies directed against epitopes very close to the polyglutamine stretch. This unique model should thus be useful to study the processing mechanism of mutant huntingtin, its role in the formation of intracellular aggregates and the effect of the latter on cellular physiology.

INTRODUCTION

Huntington's disease (HD) (1) and six other neurodegenerative diseases including spinal and bulbar muscular atrophy (SBMA) (2), dentatorubral-pallidoluysian atrophy (DRPLA) (3,4) and spinocerebellar ataxia (SCA) types 1, 2, 3 and 7 (5-10) are caused by expansion of a CAG repeat coding for a polyglutamine stretch in the respective proteins. A strong inverse correlation between age of onset of the disease and the length of the CAG repeat expansion is observed. Genetic arguments as well as data from mouse models have indicated that the expansion confers a gain of toxic property to the respective proteins (1,2,11,12). Recent studies in mouse models as well as in HD, SCA1, SCA3 and DRPLA patients have indicated that the pathogenesis of polyglutamine disorders correlates with the presence of ubiquitinated intranuclear inclusions in neurons affected in the respective diseases (13-17). At least in HD it seems that these aggregates represent short truncated derivatives of huntingtin protein as they are detected only by antibodies recognizing epitopes very close to the polyglutamine stretch (15). Several lines of evidence suggest that truncated mutant forms are more toxic than the mutant full-length proteins. This was the case in transgene mouse models for HD and SCA3, and in cellular models where inclusions and apoptosis were observed only by expressing truncated proteins encompassing the expanded polyglutamine stretch (16-23).

To date, in none of the models designed for HD disease could the in vivo situation be simulated by expression of full-length mutant protein. We have established a regulated system for the expression of huntingtin under the control of the reverse tetracycline-inducible transactivator (24) in the mouse-rat neuroblastoma-glioma cell line NG108-15. In this system, the addition of doxicycline to the culture medium induces huntingtin expression, thus preventing potential toxic effects of overproduced mutant huntingtin when growing the cells. Since the NG108-15 cells display neuronal-like properties after differentiation (25) they allow the study of expression of mutant huntingtin over much longer times (up to 18 days) than in transient transfection systems described previously (16,17, 20,22,23). Cells expressing full-length mutant huntingtin show formation of cytoplasmic and nuclear inclusions in a time- and polyglutamine length-dependent manner. These inclusions are ubiquitinated and contain only a short N-terminal part of mutant huntingtin, thus appearing similar to these observed in the brain of HD patients (15). This indicates that a processing step generating fragments with high aggregation potential operates in these cells.

RESULTS

Formation of inclusions in cells expressing mutant huntingtin

We developed a cellular model for HD using the reverse tetracycline-inducible system (24). In a first transfection, the reverse tetracycline-inducible transactivator (rtTA) was stably integrated in NG108-15 cells. To study the fate of the full-length huntingtin versus truncated forms as well as the effect of polyglutamine length, double stable cell lines expressing the following proteins under the control of the tetracycline transactivator promoter were generated in a second stable transfection: full-length huntingtin (FL-hd) with 15, 73 and 116 repeats; truncated huntingtin (T-hd; 502 amino acids) with 15 and 73 repeats; and a very truncated huntingtin protein (VT-hd; 80 amino acids) with 15, 73 and 120 repeats (Fig. 1). All these proteins carry the flag-tag epitope at their N-terminus.


Figure 1. Features of the huntingtin-derived proteins. The full-length and truncated huntingtin cDNA constructs with different lengths of CAG repeats were flag-tagged at the N-terminus and cloned into the pUHD172-1neo vector. The hashed boxes correspond to polyglutamine repeats with of 15, 73 and 116. The localization of the epitopes (grey boxes) of the antibodies used and the predicted caspase-3 cleavage sites are indicated.

Huntingtin expression was assessed by immunofluorescence in cells following differentiation and doxicycline induction, using the anti-flag monoclonal antibody (mAb) or the huntingtin-specific antibodies 4C8 (monoclonal) and 567 (polyclonal) that recognize domains C-terminal to the polyglutamine (Fig. 1). Homogenous cytoplasmic staining was observed in all lines expressing the normal huntingtin constructs (as an example VT-hd15 is shown in Fig. 2). In contrast, distinct intracellular inclusions [cytoplasmic inclusions (CIs) or nuclear inclusions (NIs)] were seen in the mutant lines T-hd73 and FL-hd73, co-existing in most instances with homogenous cytoplasmic staining (Fig. 2). Both NIs and CIs also reacted with an anti-ubiquitin antibody (Fig. 2). Significantly, in line VT-hd73 expressing the shortest huntingtin protein, no homogenous cytoplasmic staining was observed. Instead, the protein appeared to aggregate as dense inclusions, either in the cytoplasm (CI) (not shown) or in the nucleus (NI) (Fig. 2). In a time course experiment, the frequency of CIs decreased from 49% (day 4) to 4% (day 16) in the latter line, whereas the frequency of NIs increased from 20% (day 4) to 84% (day 16) (Fig. 3a). Cells expressing the longer truncated construct T-hd73 showed a delayed and less efficient formation of dense NIs (up to 17%) compared with VT-hd73 (Fig. 3b).

In line FL-hd73, a large majority of cells showed only cytoplasmic staining up to day 14 when using the anti-flag mAb (Table 1). However, CIs were detected in 17-36% of cells, either co-existing with homogenous cytoplasmic staining, or without such staining. Two kinds of NIs were also detected. From day 4 to day 6, 2-3% of cells showed very large NIs (Fig. 2), that often appeared less dense than NIs observed in the VT-hd73 line (Fig. 2). The proportion of such cells did not change significantly from day 4 to day 18. Small dense NIs, resembling those observed in VT-hd73, appeared at day 16 in a small percentage of cells that lacked cytoplasmic staining (Table 1).

A very different pattern was observed in line FL-hd116. Even at early time points (days 4/6), only 35% of cells presented a homogenous cytoplasmic staining, and this decreased after day 10 (Table 1). From day 4 to day 10, the majority of cells showed CIs (Fig. 4a). Their proportion decreased at later time points, where ~50% and more expressing cells showed small dense NIs lacking cytoplasmic staining (Fig. 4a). While the more C-terminal 4C8 mAb detected the homogenous cytoplasmic staining, none of the compact NIs could be detected with this antibody. This result was confirmed by the absence of co-localization using the antibody combination anti-Flag/566 (Fig. 4b), suggesting that these NIs are composed either of a fragment cleaved N-terminal to the 4C8 epitope, or that the 4C8 epitope is masked in the aggregates. Interestingly, the 4C8 mAb and the C-terminal 567 polyclonal antibody detected the large, non-compact NIs (Fig. 2, shown for FL-hd73), which were also seen in T-hd73 cells (up to 7%) and in cells expressing the respective non-mutant proteins (up to 2%). Their presence suggests that the whole huntingtin transgene protein can be imported in the nucleus. However, nuclear import appears to be more efficient for the truncated mutant form.

Table 1. Time course study of the expression pattern in cells expressing mutant full-length huntingtin
Days induction cyto only (%) cyto + CI (%) CI only (%) NI only (%) n
Phenotype of cells expressing FL-hd73
4/6 81 13 4 0 47
8/10 79 11 10 0 61
12/14 80 10 8 0 163
16/18 53 28 8 3 152
Phenotype of cells expressing FL-hd116
4/6 35 22 36 7 55
8/10 40 20 33 4 95
12/14 9 12 25 47 127
16/18 7 4 18 67 333
Immunofluorescence analysis was performed using the N-terminal anti-flag mAb. cyto, cytoplasmic; CI, cytoplasmic inclusion; NI, nuclear inclusion; n, total number of expressing cells. Large NIs co-existing with cytoplasmic staining were found in a small percentage of cells, and therefore are not listed separately in the table.

Processing of mutant full-length huntingtin


Figure 2. Confocal microscopy analysis of NG108-15 cells expressing various huntingtin transgenes. Differentiated and doxicycline-induced NG108-15 cells encoding VT-hd15 (as an example for wild-type transgene) and mutant T-hd73 and FL-hd73 transgenes were investigated using the N-terminal anti-flag mAb, the more C-terminal huntingtin-specific mAbs 4C8 and 567, and an anti-ubiquitin antibody. An even cytoplasmic distribution is shown for line VT-hd15 (the dot is not within a cell and therefore unspecific), whereas a cell expressing VT-hd with 73 polyglutamines displays two nuclear inclusions without any cytoplasmic staining. A cell expressing T-hd73 shows both nuclear and cytoplasmic inclusions (also detected by the anti-ubiquitin antibody in a co-localization experiment), and an even cytoplasmic staining. Here, the anti-ubiquitin antibody was also positive in a nucleus where no inclusions were detected with the anti-flag mAb. An example of the rare large inclusions is shown for a cell expressing FL-hd73, detected by the 4C8 and the distal 567 antibodies in a co-localization experiment (the polyclonal 567 antibody stains processes even in cells that do not express the transgene).

Figure 3. Formation of nuclear inclusions is a time-dependent process. (a) Fate of the VT-hd73 transgene protein. The frequency of cells bearing CIs decreased over time, whereas the NIs increased. (b) Comparison of FL-hd73 and T-hd73 lines by immunofluorescence using the anti-flag mAb. Both, CIs and especially NIs are more frequent in T-hd73 than in FL-hd73. Bars represent the standard error.

Figure 4. Formation of inclusions from a line expressing full-length huntingtin (FL-hd116). (a) Cells expressing full-length huntingtin with 116 polyglutamines were differentiated, doxicycline induced for 16 days and subjected to confocal immunofluorescence analysis using the anti-flag mAb. The transgene protein was identified as cytoplasmic (left) or nuclear (right) inclusions without cytoplasmic staining. (b) Co-localization of the transgene using the N-terminal anti-flag mAb and the more C-terminal huntingtin-specific polyclonal antibody 566 (performing conventional fluorescence microscopy). The anti-flag mAb detected the nuclear inclusions, while the 566 polyclonal antibody was negative for these. The 566 antibody detected a homogenous cytoplasmic staining that may correspond to endogenous and transgene huntingtin.

To study the processing of the huntingtin protein, we performed western blotting analysis of cells expressing mutant full-length huntingtin. Breakdown products of ~90 kDa for FL-hd73 (not shown) and ~98 kDa for FL-hd116 were detected from day 10 onwards by both the anti-flag mAb (not shown) and the 4C8 mAb (Fig. 5). The size of these fragments corresponds well with that of the predicted N-terminal caspase-3 cleavage product of huntingin (26), which includes the 4C8 epitope. Although endogenous huntingtin is produced at levels similar to mutant huntingtin (as detected by the 4C8 mAb), the corresponding caspase-3 fragment was not observed in these lines, consistent with the hypothesis that glutamine length is a determining factor for caspase-3 cleavage of full-length huntingtin (26). However, while inclusions were already detected at high frequency at days 4/6 (Table 1), the potential caspase-3 fragment appeared rather late (day 10). This suggests that this fragment is not involved in the initial formation of inclusions. It might, however, accelerate aggregation at later time points, as in the T-hd73 line expressing a truncated form of mutant huntingtin (502 amino acids), that is very close in size to the predicted caspase-3 fragment (510 amino acids), a much higher percentage of inclusions was observed than in cells expressing FL-hd73 (Fig. 3b). Interestingly, the 4C8 mAb detected CIs in up to 11% of cells expressing T-hd73, whereas it did not recognize the NIs at all (data not shown). This result further supports the idea that an N-terminal fragment shorter than the proposed caspase-3 cleavage product is liberated from huntingtin. However, we could not observe in our western blot analysis the shorter fragment predicted from the immunofluorescence studies of inclusions.

Apoptosis and expression of mutant proteins


Figure 5. Cleavage of the mutant full-length huntingtin protein. Cells expressing FL-hd116 were differentiated and doxicycline-induced over a period of 18 days, and samples were taken at the indicated time points for western blotting. Using the huntintin-specific mAb 4C8, a breakdown product corresponding to the size of the putative caspase-3 fragment was detected. No cleavage product was detected for the endogenous huntingtin (also detected by the 4C8 mAb), which is expressed at about the same level as the mutant transgene protein.

To address the question of whether the mutant proteins affect the viability of the cells, apoptosis was assessed by counting apoptotic nuclei in huntingtin-expressing cells versus non-huntingtin-expressing cells. In lines FL-hd73 and T-hd73, apoptosis in expressing cells increased with time from ~12 to 30% between days 6 and 16 (Fig. 6), whereas it stayed at basal level in non-expressing cells grown within the same culture dish (9 ± 2%) and in cells expressing constructs with 15 CAG repeats (11 ± 2%) (not shown). Strikingly, in cells expressing the shortest version of huntingtin (VT-hd73), 30% of the cells were apoptotic by day 6, a time point where apoptotic counts did not surpass basal levels in FL-hd73- and T-hd73-expressing cells. After 12 days of culture, cell lines expressing the intermediate form of huntingtin (T-hd73) also displayed apoptotic levels above background, whereas no detectable increase was observed in cell lines expressing full-length huntingtin until day 16 of culture. The observation that the cell death rate did not surpass the 30% level is most probably due to the detachment of dead cells during culturing. Thus, in these three cell lines, earlier apoptosis was correlated with expression of shorter mutant huntingtin, that also displayed a higher potential to form inclusions. However, there might also be other cellular factors that modulate the extent of apoptosis, as one cell line expressing the VT form with 120 glutamines showed a high accumulation of NIs, but no increased cell death (data not shown).


Figure 6. Cell death susceptibility of each of the lines expressing mutant constructs. Apoptosis was assessed over a period of 16 days by counting apoptotic bodies in the respective huntingtin-expressing cells versus non-huntingtin-expressing cells. For line FL-hd73, an increase in apoptosis compared with non-expressing cells grown within the same culture dish was observed at day 16. Line T-hd73 showed an increased apoptosis at day 12, and VT-hd73 cells already at day 6, compared with non-expressing cells. The arrow indicates the cell death frequency of cells expressing huntingtin with 15 glutamines.

DISCUSSION

We have established inducible cell lines that are able to model major pathogenic features of HD. In cell lines expressing full-length mutant huntingtin, we observed CIs and NIs that share two major properties with those observed in patients. They contain a short fragment of the mutant protein and they are ubiquitinated. This implies that a processing mechanism operates in these cells to generate a fragment with high aggregation potential. Furthermore, expression of the mutant protein resulted in increased apoptosis.

The formation of inclusions, and especially of NIs, is much more efficient when the mutant protein carries 116 instead of 73 glutamines. This reflects the preferential observation of NIs in the brain of HD patients with early age of onset (and large expansions) (15). The dense NIs are detected in immunofluorescence analysis by the anti-flag mAb, but not by the 4C8 mAb (recognizing an epitope ~400 amino acids distal to the polyglutamine stretch), suggesting that an N-terminal cleavage event is involved in their formation. This is in agreement with the finding that inclusions in the brain of HD patients are only detected with N-terminal antibodies close to the polyglutamine stretch (15). We failed to detect shorter fragments on western blot, which might be due to their high aggregation potential. Indeed, even for lines expressing the VT form of huntingtin with 73 or 120 polyglutamine repeats, the corresponding band on western blot was very weak, although the inclusions were easily visible by immunofluorescence. As also observed by others, expression of a very truncated mutant fragment results in a more rapid formation of NIs (16,17,20,22,23). In fact, most of the very truncated form aggregates as early as 4 days after induction of expression, either in the cytoplasm or in the nucleus, with no homogenous cytoplasmic staining. The timing of appearance of inclusions suggests that the CIs may be precursors of the NIs. Import of such inclusions might be linked to anomalies of the nuclear membrane such as the indentations of the nuclear membrane observed in a mouse model for HD (13). However, in the latter model, indentations of the nuclear membrane occurred after the appearance of NIs.

The cellular models presented up to now for polyglutamine expansion diseases were based on transient transfections (16,17,20,22,23). In those short duration assays, the formation of CIs was observed only in cells expressing truncated mutant forms (20,22,23), or at low level in cells expressing a full-length huntingtin with 128 glutamines, while NIs were only detected in cells expressing a very short form of mutant huntingtin (exon-1 with 128 CAG repeats) (20). We have chosen cells that express neuronal properties and that sustain long-term expression of the transgene, and one or both of these features may allow the production of truncated precursors of inclusions. The protease activity responsible for the processing of mutant huntingtin is likely to play an important role in the pathogenesis of HD, and our cellular model is a tool to study and identify these activities.

Ubiquitination of inclusions has been observed in several diseases unrelated to polyglutamine disorders (27,28), thus reflecting attempts of the cell to remove misfolded or aggregated proteins. Among the polyglutamine disorders, ubiquitination has been observed in brains of HD, SCA3 and DRPLA patients and in mouse models (13-17), but it had only once been reported for cellular systems (22). Investigation of our cell lines at early time points should allow the analysis of timing of ubiquitination. Since huntingtin has been found to interact with a ubiquitin-conjugating enzyme (29), the proteasomal pathway may thus be implicated in the processing of huntingtin to a short truncated N-terminal fragment, which then aggregates. We could observe inclusions even after 12 days of shutting down the expression of the mutant huntingtin transgene (data not shown), indicating that the cell machinery is not capable of removing the aggregates.

The dense NIs were not stained by the 1C2 mAb that is specific for expanded polyglutamines (30), whereas this antibody detected the large non-compact NIs. Most probably, the pathological epitope is masked in the dense aggregates, possibly due to interaction of polyglutamine tracts with each other to form [beta]-pleated sheets (31). This is consistent with the lack of staining of inclusions by 1C2 in transgenic HD mice (13), its very weak reaction with NIs seen in the brain of SCA3 patients (16) and with its failure to detect aggregates of the mutant androgen receptor (21).

Another interesting feature of our cell system is cleavage of mutant full-length huntingtin to a fragment corresponding in size to the predicted N-terminal caspase-3 cleavage product (26). Importantly, endogenous wild-type huntingtin appears unaffected by the cleavage. It was shown previously that in a biochemical assay, mutant huntingtin is more sensitive to caspase-3 than the corresponding normal form (26). Here, we can extend this conclusion to our more relevant cellular system. The rather late appearance of this cleavage (compared with the formation of inclusions) suggests that it may be linked to the induction of an apoptotic pathway by the presence of inclusions. However, this breakdown fragment may accelerate further the formation of inclusions, since cells expressing truncated huntingtin with 73 polyglutamines (having approximately the size of the caspase-3 fragment) generate them at a higher level than cells expressing full-length huntingtin with the same expanded repeat.

While we observed apoptosis linked to expression of mutant huntingtin, the correlation with the presence and level of inclusions was not striking. In other cells expressing expanded polyglutamine proteins, the effect on apoptosis was modest (16,21) or had even to be induced by further stimuli (20,22). This would indicate that NIs or CIs do not cause apoptosis directly. In fact, in animal models, neuronal dysfunction occurs well before cell death (13,32). Since the NG108-15 cells can express some neuronal properties, it will be interesting to see whether these are affected by the presence of mutant huntingtin. For SCA1, it has indeed been suggested that neuronal dysfunction might be provoked by sequestration of neuron-specific factors such as the Purkinje cell-specific protein LANP (33) in inclusions.

The model presented here provides a tool to elucidate the mechanisms leading to cleavage of full-length huntingtin, its role in the formation of intracellular aggregates and the effects of the latter on cellular physiology. It will be especially interesting to see whether in this system protease or transglutaminase inhibition may interfere with the formation of inclusions and to investigate their consequences on cell function and survival.

MATERIALS AND METHODS

Plasmid construction

An adaptor containing a flag-tag, a Kozak consensus sequence and a multiple cloning site was inserted in pUHD10.3 vector (24,34). NcoI-SacII fragments with 15 and 73 CAG repeats of an earlier described plasmid (35) were cloned in the modified vector pUHD10.3 vector to generate the VT-hd constructs. A VT-hd construct with 120 CAG repeats was obtained by spontaneous expansion from the initial 73 CAGs. T-hd and FL-hd constructs were obtained by cloning of a SacII-BstYI fragment (position bp 240-1506) and a SacII-XhoI fragment (position bp 240-9586), respectively, in the VT-hd constructs.

Generation of inducible cell lines

Founder cell lines were generated by transfecting the mouse-rat neuroblastoma-glioma hybrid cell line NG108-15 (in a 100 mm dish) with 20 µg of pUHD172-1neo (24), a plasmid containing a tetracycline-inducible transactivator together with the neo cassette, using the calcium-phosphate precipitation method. Individual G418-resistant colonies were isolated using 0.5 mg G418/ml and characterized by transient transfection with the [beta]-galactosidase reporter plasmid pUHC 16-3 (24,34). [beta]-Galactosidase activity in the presence or absence of doxicycline (a tetracycline analogue) was measured as described (36). The clone with the highest inducibility and tightest regulation was chosen.

The founder clone was co-transfected with 10 µg of each huntingtin construct and 1 µg of pPGK-hyg plasmid, carrying the hygromycin resistance gene under the control of the PGK promoter (37). Hygromycin-resistant colonies were isolated using 250 µg/ml hygromycin B, induced by 1 µg/ml doxicycline for 30 h and screened for transgene expression by western blot analysis using antibodies against the flag epitope-tag or the huntingtin-specific 4C8 mAb (Fig. 1). Tightly regulated and the highest expressing clones were selected for further study.

Culturing and differentiation of cell lines

NG108-15 clones were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS), antibiotics, 0.5 mg/ml G418 and 250 µg/ml hygromycin B. For differentiation and doxicycline induction, cells were split and allowed to attach overnight. Medium was then removed and replaced by differentiation medium composed of DMEM, 1% FCS, antibiotics, 10 µM forskolin and 100 µM IBMX (isobutylmethylxanthine) together with 1 µg/ml doxicycline. During the time course experiments, the medium was changed every 3 days.

Cell death was evaluated using the Hoechst dye 33258 which colours the nuclei of cells. Nuclei that were fragmented or condensed were scored as apoptotic. Apoptotic nuclei were counted in cells expressing the transgene and in non-expressing cells. The standard error was calculated as [radic]p(1 - p)/n, where p is the proportion of apoptotic cells and n is the total number of cells counted.

Western blot analysis

Whole cell extracts were obtained by homogenization in 50 mM Tris-HCl, pH 8.0, 10% (v/v) glycerol, 5 mM EDTA, 150 mM KCl, 1 mM phenylmethylsulfonyl fluoride (PMSF) and a cocktail of protease inhibitors, followed by a 10 min incubation on ice and sonication with five pulses. Protein concentration was determined using the the Bradford test. The extracts were heat denatured by 5 min boiling in Laemmli loading buffer. A 20 µg aliquot of total protein extract was analysed on 6% SDS-polyacrylamide gels. To look for the presence of breakdown products, 10 or 15% SDS-polyacrylamide gels were used. Proteins were transferred to nitrocellulose membranes, blocked with 3% non-fat dry milk and incubated with different primary antibodies, anti-flag monoclonal antibody 2B11 (diluted 1:1000) and 4C8 (35) (ascites fluid diluted 1:2000) for 1 h at room temperature. The secondary antibody (goat anti-mouse immunoglobulins) was coupled to peroxidase and detected using the Supersignal Substrate western blotting kit (Pierce, IL). Stripping and reprobing were performed as described elsewhere (35).

Immunocytochemical studies

Cells were washed with 1× phosphate-buffered saline (PBS), fixed with methanol/acetone (1:1) for 1 min at -20°C, air-dried for 10 min and rehydrated using 1× PBS. Cells were then incubated with the primary antibody: anti-flag M2 mAb (Kodak), diluted 1:4000; 4C8 mAb, ascites fluid diluted 1:2000; 1C2 mAb (30), supernatant diluted 1:50; polyclonal antibodies 566 and 567 diluted 1:200 [corresponding to the mAbs 4C8 and 2E8 (35)]; or polyclonal anti-ubiquitin (Dako) diluted 1:100 for 1 h at room temperature, washed with 1× PBS, followed by incubation with Cy3 (Jackson Laboratories) or Oregon green-conjugated anti-mouse IgG (Molecular Probes) and/or with tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit IgG (Jackson Laboratories). The cells were counterstained with Hoechst 33258 and observed by fluorescence microscopy. Confocal images were colourized and merged using the Adobe-Photoshop software program.

Immunocytochemical studies demonstrated that although the cell lines are clonal, expression of the transgene was observed in a time-dependent manner, in up to 30-50% of all cells. Similar results have been observed before, and may be due to differential uptake of doxicycline (38).

The standard error was calculated as [radic]p(1 - p)/n, where p is the proportion of cells having NIs or CIs and n is the total number of expressing cells counted.

ACKNOWLEDGEMENTS

We would like to thank D. Devys, Y. Trottier, G. Yvert for providing original constructs and antibodies, and carefully reading the manuscript, C. Weber for excellent technical assistance, F. Saudou for suggesting the NG108-15 line, and G. Imbert for useful material. We thank N. Messaddeq and J.-L. Vonesch for confocal microscopy [supported by the French MESR (95.V.0015)] and Y. Lutz for providing the monoclonal anti-flag antibody 2B11. This work was supported by funds of INSERM, CNRS, HUS and EEC (contract BMH4-CT96-0244). A.L. is supported by a fellowship of the DFG (Deutsche Forschungsgemeinschaft).

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*To whom correspondence should be addressed. Tel: +33 3 88 65 34 12; Fax: +33 3 88 65 32 46; Email: mandeljl@igbmc.u-strasbg.fr

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