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Molecular Endocrinology, doi:10.1210/me.2006-0122
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Molecular Endocrinology 20 (12): 3212-3227
Copyright © 2006 by The Endocrine Society

Involvement of the Pituitary-Specific Transcription Factor Pit-1 in Somatolactotrope Cell Growth and Death: An Approach Using Dominant-Negative Pit-1 Mutants

Isabelle Pellegrini, Cathy Roche, Marie-Helene Quentien, Mireille Ferrand, Ginette Gunz, Sylvie Thirion, Claude Bagnis, Alain Enjalbert and Jean-Louis Franc

Laboratoire Interactions Cellulaires Neuroendocriniennes (I.P., C.R., M.-H.Q, M.F., G.G., S.T., A.E., J.-L.F.), Centre National de la Recherche Scientifique Unité Mixte de Recherche 6544-Université de la Méditerranée, Marseille, France; and Département de Thérapie Cellulaire et Génique (C.B.), Etablissement Français du Sang Alpes Méditerranée, Marseille, France

Address all correspondence and requests for reprints to: Dr. Jean-Louis Franc, Laboratoire Interactions Cellulaires Neuroendocriniennes, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6544, Université de la Méditerranée, Institut Fédératif Jean Roche, Faculté de Médecine Nord, Boulevard P. Dramard, 13916 Marseille cedex 20, France. E-mail: franc.jl{at}jean-roche.univ-mrs.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The anterior pituitary-specific transcription factor Pit-1 was initially identified and cloned as a transactivator of the prolactin (PRL) and GH genes and later as a regulator of the TSHb gene. It was found to be a major developmental regulator, because natural Pit-1 gene mutations cause a dwarf phenotype in mice and cause combined pituitary hormone deficiency associated with pituitary hypoplasia in humans. To further investigate the growth-promoting effects of Pit-1, we used a strategy based on the use of dominant-negative Pit-1 mutants as an alternative means of inactivating endogenous Pit-1 functions. R271W, a Pit-1 mutant identified in one allele in patients with severe combined pituitary hormone deficiency, and Pit-1{Delta}1-123, a deletion mutant in which only the DNA binding domain of Pit-1 is conserved, were generated, and their ability to abolish the effects of the endogenous native Pit-1 in the differentiated proliferating somatolactotrope GH4C1 cell line was investigated. Enforced expression of the dominant-negative mutants in GH4C1 cells using recombinant lentiviral vectors decreased the levels of expression of known Pit-1 target genes such as PRL and GH, abolished the hormone release, and reduced cell viability by decreasing the growth rate and inducing apoptosis via a caspase-independent pathway. These results show for the first time that the growth-promoting effects of Pit-1 are at least partly due to the fact that this transcription factor prevents apoptotic cell death.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RECENT STUDIES IN which several homeodomain transcription factors expressed in a cell-specific manner were identified in the anterior pituitary gland have shown the existence of a transcriptional cascade orchestrating a developmental program that leads to the expression of five mature cell types (1). The anterior pituitary-specific transcription factor Pit-1 was initially identified and cloned as a transactivator of the prolactin (PRL) and GH genes, and was later found to be also a regulator of the TSH b-subunit (TSHb) gene (2). It was also found to be a major regulator of lactotrope, somatotrope, and thyrotrope development and differentiation, in view of the role played by naturally occurring alterations in the Pit-1 gene in the Snell (dw) and Jackson (dwJ) dwarf mouse strains. These mice carry recessive Pit-1 point (Snell) and null (Jackson) mutations and are characterized by low or undetectable levels of Pit-1 expression; the absence of PRL, GH and TSHb expression; and a marked lack of lactotrope, somatotrope and thyrotrope proliferation, resulting in hypoplasia of the gland (3). Previous findings suggested that Pit-1 might be involved in cell proliferation, because Pit-1 antisense oligonucleotides not only block GH and PRL transcription but also inhibit the proliferation of pituitary somatotrope (GC) and lactotrope 235-1 cell lines (4).

Although our present knowledge of how Pit-1 is involved in development was mostly based on rodent animal models, Pit-1 is now known to carry out similar functions in humans. Analysis of Pit-1 gene expression in normal and tumoral human pituitary tissues has shown that Pit-1 gene expression is restricted to cells and tumors belonging to the thyrotrope, somatotrope, and lactotrope lineages (5). Furthermore, various recessive and dominant inactivating mutations in the Pit-1 gene have been identified in patients with combined pituitary hormone deficiency (CPHD) associated with pituitary hypoplasia (6). Most mutations have been found to occur in the POU domain of Pit-1, which is important for its ability to dimerize and bind to DNA. However, although some of these mutants prevent Pit-1 activity by disrupting the DNA binding process, other mutations that do not affect DNA-binding still act as strong inhibitors via mechanisms that still remain to be elucidated (7).

To further investigate the growth-promoting effects of Pit-1, we developed a strategy based on the use of dominant-negative Pit-1 mutants as an alternative means of inactivating endogenous Pit-1 functions. R271W, a Pit-1 mutant identified in one allele in patients with severe CPHD in which a single Arg residue in the POU-homeodomain was replaced by Trp (8), and Pit-1{Delta}1-123, a deletion mutant in which the whole transactivation domain was lacking and only the DNA binding domain was conserved, were generated. The ability of the Pit-1 mutant proteins to abolish the activity of endogenous Pit-1 in the GH4C1 cell line was established. The GH- and PRL-producing pituitary tumor cell line GH4C1 provides a convenient model for testing this hypothesis. Despite their unlimited proliferative capacity, these cells express somatolactotrope differentiation markers and high levels of Pit-1. The results obtained here show that enforced expression of the Pit-1 dominant-negative mutants in GH4C1 cells induced using recombinant lentiviral vectors resulted in a marked decrease in the levels of expression of known Pit-1 target genes such as PRL and GH and a marked decrease in cell growth and viability due to the apoptosis induced by these mutants.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Functional Characterization of Pit-1 Mutants
Wild-type (WT) and mutant 35S-labeled Pit-1 proteins were generated in an in vitro transcription/translation (TNT) system and run on SDS-PAGE. As shown in Fig. 1AGo, two bands corresponding to apparent molecular sizes 31 and 33 kDa (depending on the methionine at which the translation was initiated) were detected in the case of WT Pit-1. A similar pattern was obtained with the mutant carrying the R271W point mutation, whereas a band corresponding to a molecular mass of approximately 18 kDa, consistent with a truncated protein, was observed with the Pit-1{Delta}1-123 N-terminal deletion mutant, in which only the DNA binding domain is conserved. Parallel TNT reactions were therefore run in the absence of 35S-Met, and aliquots of recombinant WT and mutant Pit-1 proteins were subsequently assayed to determine their DNA binding activities (Fig. 1BGo). Under our conditions, as previously reported (9), WT Pit-1 protein formed a single specific binding complex with the P1 element of the human PRL (hPRL) proximal promoter (lanes 3–4). Preincubation with an anti-Pit-1 monoclonal antibody (mAb) directed against the N-terminal domain of the protein resulted in a supershift in mobility (lane 5, open arrow). Under the same conditions, the R271W (lanes 6–7) and Pit-1{Delta}1-123 (lanes 9–10) mutant proteins were both found to bind to the P1 probe. Adding the Pit-1 Mab resulted in a supershift with the R271W (lane 8) but not the Pit-1{Delta}1-123 mutant (lane 11). A nonspecific band was observed with unprogrammed reticulocyte lysate.


Figure 1
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  		Fig. 1. Mutants R271W and Pit-1{Delta}1-123 Bind to Target DNA Elements

A, SDS-PAGE analysis of [35S]Met-radiolabeled WT and mutant R271W and Pit-1{Delta}1-123 resulting from in vitro TNT reactions. WT, R271W Pit-1, and Pit-1{Delta}1-123 proteins were expressed in similar levels. B, EMSA using the P1 Pit-1 element of the proximal hPRL promoter as a probe and 1 or 2 µl of WT or mutant R271W and Pit-1{Delta}1-123 proteins translated in the absence of [35S]Met. UP, Unprogrammed reticulocyte lysate. The arrows in bold print indicate the binding complexes involving WT, R271W, and Pit-1{Delta}1-123, and the clear arrow indicates the supershifted complex obtained after adding a Pit-1 mAb. NS, Nonspecific bands.

 
The transactivating properties of the Pit-1 mutant proteins were measured by performing transient transfection assays on CV-1 cells, which do not express the endogenous Pit-1 gene. Increasing amounts of an expression vector encoding WT or mutant Pit-1 were cotransfected together with a natural Pit-1-responsive luciferase reporter construct encoding 164 bp of the hPRL proximal promoter (hPRLluc). Transfecting low amounts of WT Pit-1 resulted in a sharp increase in reporter activation, reaching a maximum of 8-fold at the highest concentrations used (Fig. 2AGo). By comparison, the Pit-1{Delta}1-123 mutant, which lacks the major transactivating domain, only weakly activated (~2-fold) the hPRLluc reporter at all the doses tested. The R271W mutant showed an intermediate response (level of activation). Although low amounts of transfected R271W induced a lower hPRLluc activation than equivalent amounts of WT Pit-1, a similar level of activation to that induced by WT Pit-1 was induced at the maximum doses of R271W used (Fig. 2AGo).


Figure 2
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  		Fig. 2. Differential Transcriptional Activation of the hPRL-164luc Promoter by WT and Mutant Pit-1 in CV-1 Cells

A, Nonpituitary CV-1 cells were transfected by means of the calcium phosphate method, using reporter plasmid hPRL-164luc and varying amounts (0, 0.05, 0.1, 0.2, 0.3, and 0.6 µg) of CMV-pcDNA3 expression vectors encoding WT Pit-1, R271W, or Pit-1{Delta}1-123 cDNAs. A CMV-ß-galactosidase plasmid was used as the internal control to check the transfection efficiency. Cells were harvested after 48 h and assayed for luciferase. Results were normalized with respect to ß-galactosidase activity and are expressed as fold activation over the control value. Transfections were performed in triplicate under each of the conditions tested during a single experiment. Data are expressed as means ± SE of three independent experiments. B, CV-1 cells were cotransfected with the hPRL-164luc construct and either WT, R271W, or Pit-1{Delta}1-123 mutant alone or combined, as indicated. Two different amounts (0.1 or 0.3 µg) of expression vectors were tested. Transfections were performed in triplicate under each condition within a single experiment. Data are expressed as means ± SE of five independent experiments.

 
We then addressed the question of whether the mutant proteins might mainly inhibit WT Pit-1 activity. WT and mutant Pit-1 expression vectors were cotransfected into CV-1 cells in a 1:1 ratio, keeping the total expression vector DNA levels constant. The expression vectors were tested at two different concentrations (0.1 and 0.3 µg) (Fig. 2BGo). At limiting doses (0.1 µg), R271W did not reverse but rather additively enhanced the transcriptional effects of WT Pit-1, whereas Pit-1{Delta}1-123 had no significant effects. At higher concentrations (0.3 µg), the R271W mutant had no significant effects on the activity of WT Pit-1. Under these conditions, no additive effects are possible because the activation of the promoter is already maximum. By contrast, the Pit-1{Delta}1-123 mutant conspicuously decreased the WT Pit-1-induced hPRLluc transactivation process (P < 0.05).

The inhibitory activities of the Pit-1 mutants were next examined in the context of cells expressing the endogenous Pit-1 gene. For this purpose, the pituitary somatolactotrope GH4C1 cell line was used as a model. In these cells, which also express endogenous PRL and GH genes, the baseline activity of the PRL promoter construct was high, but this activity could still be significantly stimulated in a dose-dependent manner by overexpressing Pit-1 (Fig. 3AGo). Conversely, the expression of increasing amounts of Pit-1{Delta}1-123 mutant reduced the baseline activity of the hPRLluc construct in a dose-dependent manner (65% inhibition at the maximum doses tested). In the case of the R271W mutant, contrary to what occurred with CV-1 cells, cotransfection of the mutant into GH4C1 cells also resulted in a dose-dependent inhibition of the hPRLluc reporter activity, and the magnitude of this effect was similar to that observed with the Pit-1{Delta}1-123 mutant (Fig. 3AGo). Cotransfecting each of these two mutants with the hGHluc reporter construct resulted in a similar dose-dependent inhibition of the promoter gene activity (Fig. 3BGo). In the control experiments performed with a pTKLuc construct under the same conditions, no significant effects of the WT or mutant Pit-1 constructs were observed (Fig. 3CGo).


Figure 3
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  		Fig. 3. The R271W and the Pit-1{Delta}1-123 Mutants Inhibit Pituitary Promoter Baseline Activities in GH4C1 Cells

A, GH4C1 cells were transfected using a liposome-based transfection method with the hPRLluc or hGHluc constructs, and the indicated amounts (µg) of expression vectors encoding WT Pit-1, R271W, or Pit-1{Delta}1-123 mutants. WT Pit-1 induced dose-dependent promoter activation, whereas both the R271W and Pit-1{Delta}1-123 mutants reduced the baseline activities of the reporter constructs in a dose-dependent manner. B, The transfections in A were repeated with a luciferase reporter containing the hGH promoter. Again, the R271W and Pit-1{Delta}1-123 mutants specifically decreased the promoter activation in a dose-dependent manner. C, The transfections in A were repeated with a luciferase reporter containing the thymidine kinase promoter as a control. None of the WT or mutant constructs had any significant effects. Transfections were performed in triplicate under each condition within a single experiment. Data are expressed as means ± SE of three independent experiments.

 
Gene Transfer Efficiency of Lentiviral Vectors in GH4C1 Cells
To investigate the long-term effects of Pit-1 dominant-negative mutants on somatolactotrope cell differentiation and growth, recombinant viral vectors obtained from HIV (10, 11) were used to transfer genes into GH4C1 cells (Fig. 4Go). The transduction efficiency of lentiviral vectors in GH4C1 cells was initially assessed by flow cytometry using enhanced green fluorescent protein (EGFP) as a marker. GH4C1 cells were transduced with pGK (human phosphoglycerate kinase)-EGFP at multiplicities of infection (MOIs) ranging from 0.1–100 and were assayed by performing fluorescence activated cell sorting (FACS) analysis 72 h after the transduction. The percentage of EGFP-positive cells increased with the MOI, reaching 95–100% of EGFP-positive cells at an MOI of 5 (Fig. 4BGo). It is worth noting that, in this context, a particularly high mean fluorescence intensity value was obtained with the human phosphoglycerate kinase promoter. The percentage of EGFP-positive cells and that of mean fluorescence intensity both remained stable for at least 5 wk (data not shown). MOIs ranging from 2–10 were used in subsequent experiments with recombinant Pit-1 vectors. In these experiments, more than 90% of the cells were found to be transduced, as assessed from the EGFP marker expression levels. Considering that the translation of the EGFP is driven by the internal ribosome entry site (IRES) element and originates from a bicistronic mRNA containing the Pit-1 cDNA, the percentage of EGFP-positive cells observed by FACS analysis suggests that up to 90% of the cells express the lentivirally transduced Pit-1 gene.


Figure 4
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  		Fig. 4. Diagram Showing the Proviral Structure of the Lentiviral Vectors and the High Transduction Efficiency of the Recombinant Lentivirus in GH4C1 Cells

A, Lentiviral vectors expressing the Pit-1 gene and the R271W and Pit-1{Delta}1-123 mutants were obtained from pRRLTpgkEGFP sin18, an HIV-1-based self-inactivating vector in which the transgene is placed under the control of the human phosphoglycerate kinase promoter. PGK, Human phosphoglycerate kinase promoter; LTR, long terminal repeat; rre, rev response element; {Delta}gag, residual silent gag coding sequence; {Delta}U3, deletion in the U3 sequence of the LTR that features self-inactivating vectors; I, IRES. Not drawn to scale. B, GH4C1 cells were seeded onto six-well plates and transduced with RRLT-pGK-EGFP-WPRE at an MOI of 5. Fluorescence image of cells expressing the EGFP transgene 72 h after transduction. At an MOI of 5, the percentage of the cells expressing EGFP reached 95%.

 
The effects of increasing viral inputs on the levels of expression of the WT and mutant Pit-1 transgenes were then determined by performing Northern blot analysis. In agreement with the flow cytometry data, the levels of WT and mutant Pit-1 transgene expression increased with increasing MOIs. The blot presented in Fig. 5AGo, corresponding to an MOI of 5, shows that high levels of transgenes were present in comparison with the levels of endogenous Pit-1 gene expressed. Besides the 2.4-kb band corresponding to the endogenous Pit-1 gene transcripts, a band with a size of approximately 3 kb, consistent with the predicted Pit-1-ires-EGFP and the R271W-ires-EGFP transcripts, was specifically detected in cells transduced with the pGK-Pit-1-ires-EGFP and the pGK-R271W-ires-EGFP vectors. The mRNA transcribed from the pGK-{Delta}1-123-ires-EGFP vector migrated to roughly the same position as the endogenous Pit-1 mRNA, yielding a superimposed signal [cf. the signal obtained in mock-infected cells (CT) or pGK-EGFP-infected cells used as controls, which is also shown in Fig. 5Go for the sake of comparison].


Figure 5
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  		Fig. 5. Gene Transfer Efficiency of Lentiviral Vectors

A, Northern blot analysis of WT and mutant Pit-1 transgene expression performed 10 d after infecting the cells with pGK-EGFP, pGK-Pit-1-ires-EGFP, pGK-R271W-ires-EGFP, and pGK-Pit-1{Delta}1-123-ires-EGFP lentiviral vectors (MOI = 5) or without lentilviral vectors (mock infected cells, CT). Besides the 2.4-kb band corresponding to the expression of the endogenous Pit-1 gene detected in all the samples, a 3-kb band corresponding to the bicistronic mRNA containing the Pit-1 and EGFP cDNAs was specifically detected in cells transduced with WT and R271W Pit-1 vectors. The mRNA transcribed from the pGK-{Delta}1-123-ires-EGFP vector is superimposed on the endogenous Pit-1 mRNA. B, Time course of transgene mRNA accumulation in GH4C1 cells transduced with WT Pit-1 lentiviral vector. Replicate wells of GH4C1 cells were transduced with EGFP or Pit-1 lentiviral vectors. ARN was prepared on the day of infection (d 0), and on postinfection d 3, 7, and 10 and was analyzed with a cDNA probe corresponding to the POU domain of Pit-1. Rehybridization of the blots with an 18S probe shown in the lower panel indicates that gene transfer with lentiviral vectors had no cytotoxic effects per se.

 
Finally, the accumulation with time of transgenes delivered by the lentiviral vector was determined. The results presented in Fig. 5BGo for the Pit-1 WT form at an MOI of 10 indicate that maximum levels of expression of the transgene were reached on d 7. On d 10, the rates of expression of endogenous and Pit-1-ires-EGFP seemed to increase, but this was due to cell multiplication, as shown by the increase in the 18S RNA signal. In addition, the fact that the 18S RNA levels did not differ between pGK-EGFP-, pGK-Pit-1-ires-EGFP-, and mock-infected cells shows that gene transfer with lentiviral vectors had no cytotoxic effects per se on the transduced GH4C1 cells (Fig. 5BGo).

Effects of Dominant-Negative Pit-1 on Endogenous PRL and GH Gene Expression and Hormone Secretion
We next assessed the impact of WT and mutant Pit-1 gene delivery on the levels of endogenous PRL and GH gene expression. GH4C1 cells were infected with WT and mutant Pit-1 vectors at an MOI of 10, and RNA was prepared 10 d later. Northern blot analysis of PRL and GH gene expression (Fig. 6AGo) followed by quantification against the 18S band (Fig. 6BGo) showed that overexpressing WT Pit-1 resulted in a significant increase in the PRL as well as the GH mRNA levels (295% and 145% of the control value, respectively). By contrast, both PRL and GH mRNA levels were significantly lower in cells transduced with the R271W and Pit-1{Delta}1-123 mutants (Fig. 6BGo). As shown in Fig. 6Go, C and D, these effects were both dose and time dependent. Lastly, a decrease in the intensity of the 18S RNA band, suggestive of lower cellular material, was observed in wells corresponding to 10 d of Pit-1 mutant expression (Fig. 6AGo and see below).


Figure 6
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  		Fig. 6. Expression of the Endogenous PRL and GH Genes Increased in GH4C1 Cells Transduced with WT Pit-1 and Decreased in GH4C1 Cells Transduced with the R271W and Pit-1{Delta}1-123 Mutants

A, Northern blot analysis of PRL and GH mRNA levels performed 10 d after infection with GFP, WT Pit-1, R271W, or Pit-1{Delta}1-123 mutant at a MOI of 10. The blot was successively hybridized with PRL, GH, and 18S cDNA probes as described in Materials and Methods. B, Quantification of the blots presented in A. The PRL and GH mRNA levels were quantified against the intensity of the 18S band. Values given for each construct are means ± SD of three replicate wells. C, Quantitative analysis of PRL and GH mRNA levels in GH4C1 cells transduced with increasing MOIs of GFP, WT Pit-1, R271W, or Pit-1{Delta}1-123 lentiviral vectors. Results from one representative experiment of three are shown. Each value is that obtained on a single well. D, Time course of the effects of WT and mutant Pit-1 expression on PRL and GH mRNA levels in GH4C1 cells transduced with increasing MOIs of GFP, WT Pit-1, R271W or Pit-1{Delta}1-123 lentiviral vectors. Results from one representative experiment of three are shown. Each value corresponds to a single well.

 
Given the low levels of PRL and GH gene expression observed in GH4C1 cells transduced with Pit-1 mutants forms, it was predicted that the hormonal release into the culture media would also be relatively low. The data obtained by measuring the rates of PRL release on d 1, 3, 6, and 9 after transduction with the various vectors are shown in the case of a representative experiment in Fig. 7Go. The amount of PRL secreted per 24 h per 105 cells in control wells was stable over this period of time. By comparison, a significant increase in PRL secretion was observed in culture wells corresponding to WT Pit-1 overexpression, whereas a concomitant decrease in PRL secretion was observed in cells transduced with either of the two Pit-1 mutants. These data support the idea that the repression of PRL gene transcription by dominant-negative Pit-1 mutants ultimately results in a decrease in the level of hormone synthesis and secretion.


Figure 7
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  		Fig. 7. Time Course of PRL Secretion by GH4C1 Cells Transduced with WT and Mutant Pit-1

The release of PRL was measured on d 1, 3, 6, and 9 after the transduction of GH4C1 cells with the various lentiviral vectors. The number of viable cells was estimated in the same well using the Cell Titer Glo assay (see Materials and Methods). Results are expressed as ng/24 h/105 cells. Values are means ± SE of those obtained on three culture wells.

 
Effects of Dominant-Negative Pit-1 on GH4C1 Cell Growth
To investigate whether the disrupted Pit-1 signaling processes resulting from dominant-negative mutant expression might affect cell growth, GH4C1 cell growth was assayed at 3-d intervals during a 9-d period after infecting the cells with WT or mutant Pit-1. Direct cell counts (data not shown) and indirect estimates of the number of viable cells based on a luminescent cell viability assay (Fig. 8AGo) yielded similar results. As shown in Fig. 8AGo, the growth pattern of cells overexpressing WT Pit-1 was similar to that of control and EGFP-transduced cells. By contrast, the number of viable cells present in wells infected with R271W or Pit-1{Delta}1-123 mutants increased as in the control wells during the first 3 d after infection but stopped increasing and even decreased thereafter, so that by d 8–10 the number of viable cells had dropped below the initial values measured in the control wells (P < 0.05). This decrease appears to have been at least partly due to the cell loss that became evident by postinfection d 6–7, when a large number of floating dead cells were observed (data not shown). This decrease in cell viability accounts for the decrease in the 18S RNA signal that was previously observed 10 d after cells expressing Pit-1 mutants were infected (Fig. 6AGo). By d 7, 10 times more cells were present in control cells and cells infected with EGFP and Pit-1 than in cells infected with R271W and {Delta}1-123 mutants.


Figure 8
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  		Fig. 8. R271W and Pit-1{Delta}1-123 Mutant Expression Decreases Somatolactotrope Cell Viability

A, Growth curves of GH4C1 cells after transduction with pGK-EGFP, pGK-Pit-1-ires-EGFP, pGK-R271W-ires-EGFP, and pGK-Pit-1{Delta}1-123-ires-EGFP vectors. Cell viability was monitored using the Cell Titer Glo assay. Values are means ± SD of three replicates obtained at each postinfection time. (*, P < 0.05 compared with initial values recorded in control wells.) B, Cell viability measurements were performed 6 d after transduction of {alpha}T3 mouse gonadotroph cells, human fibroblasts, and rat somatolactotrope SMtTW cells transduced with the various lentiviral vectors. Values are means ± SD of those obtained on three replicate wells. (*, P < 0.05 compared with control values.)

 
To assess the cell specificity of these Pit-1 mutants’ effects on cell growth, lentiviral transduction of Pit-1 mutants was subsequently performed into another cell type expressing the endogenous Pit-1 gene (rat pituitary somatolactotrope SMtTW tumoral cells; see Ref. 12) and, for the sake of comparison, into cells not expressing the endogenous Pit-1 gene [mouse {alpha}T3 pituitary gonadotrope cells (13) and human primary fibroblasts]. In these three cell types, preliminary flow cytometry analysis of the EGFP expression levels indicated that a plateau of infected cells was reached at an MOI of 10, when 55–80% of the cells expressed the transgene (data not shown). SMtTW, {alpha}T3, and human fibroblasts were subsequently transduced with the WT and mutant Pit-1 vectors, and cell viability measurements were performed 6 d after the transduction step. As shown in Fig. 8BGo, transduction of somatolactotrope nondividing SmtTW cells with Pit-1 mutants resulted in a low but nevertheless significant 25–29% decrease in cell viability in comparison with cells transduced with the EGFP- or WT-Pit-1 vectors (Fig. 8BGo). By contrast, in similar experiments performed on {alpha}T3 gonadotrope cells and human fibroblasts, the levels of Pit-1 mutant expression were not found to have any effect on cell viability (Fig. 8BGo). The inhibitory effects of Pit-1 dominant-negative mutants on cell growth therefore seem to be specific to somatolactotrope pituitary cells.

To determine the mechanism underlying the inhibitory effects of Pit-1 dominant-negative mutants on GH4C1 cell growth, cells transduced with the various vectors mentioned above were examined using flow cytometry methods after undergoing DNA staining with propidium iodide (PI). The histograms of the flow cytometry data obtained on postinfection d 6 show that expression of either R271W or Pit-1{Delta}1-123 mutants markedly decreased the number of cells present in the G2/M phases in comparison with noninfected and EGFP-infected cells, without affecting the percentages of cells in the G1/S phases (Fig. 9AGo). Furthermore, the concomitant increase observed in the subdiploid sub-G1 population representing cell death suggests that the expression of Pit-1 dominant-negative mutants induced apoptosis. No differences were observed between the cell cycles of cells overexpressing Pit-1 and those of mock- or EGFP-infected cells. These data are summarized in Table 1Go, which gives the results of three independent experiments.


Figure 9
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  		Fig. 9. Cell Cycle Distribution Analysis and Hoechst Fluorescent Staining of Apoptotic and Mitotic-Positive GH4C1 Cells Transduced with pGK-EGFP, pGK-Pit-1-ires-EGFP, pGK-R271W-ires-EGFP, and pGK-Pit-1{Delta}1-123-ires-EGFP

A, Six days after the transduction step, cells were incubated with PI, and the PI-DNA content of GH4C1 cells was evaluated using a FACS sorter. M1, G1/S phases; M2, G2/M phases; M3, sub-G1 populations. A clear-cut increase in the subdiploid sub-G1 population representing cell death was observed in cells transduced with the dominant-negative mutants. B, Glass slides with infected GH4C1 cells were fixed with 4% formaldeyde and stained with Hoechst 33342. Arrows indicate cells with nuclear condensation. C, Cell division (mitotic events) was quantified. The number of events per field was expressed as the mean value obtained on 50 or 100 fields. White bar, EGFP; hatched bar, R271W; black bar, {Delta}1-123. D, Cells with a nuclear condensation were quantified. The number of events per field was expressed as the mean value obtained on 50 or 100 fields. *, P < 0.05; **, P < 0.01. White bar, EGFP; hatched bar, R271W; black bar, {Delta}1-123.

 

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  		Table 1. FACS Cell Cycle Analysis of GH4C1 Cells Expressing WT or Mutant Pit-1

 
To confirm these data, GH4C1 cells were stained with Hoechst 3332 dye 4 or 6 d after being infected or not with dominant-negative mutants. Apoptotic events were detected by nuclear condensation and DNA fragmentation, and cell division was reflected in mitotic events. A larger number of apoptotic events (Fig. 9Go, B and D) and a smaller number of mitotic events (Fig. 9Go, B and C) were found to occur in cells transduced with these two mutants.

Effects of Dominant-Negative Pit-1 Mutants on Various Apoptotic Signaling Pathways
The release of cytochrome C from the mitochondrial intermembrane space, which is one of the key events in the apoptotic process, can occur in both caspase dependent and independent programmed cell death (PCD) (14). To investigate whether mitochondrial apoptosis may occur in cells transduced with dominant-negative mutants, the release of cytochrome C from mitochondria was analyzed by performing flow cytometry 4 and 6 d after cell infection. Plasma membrane-permeabilized GH4C1 cells were stained with a fluorescent dye-labeled antibody to cytochrome C and the cells were subjected to flow cytometry. No differences were observed between the results obtained with EGFP-infected cells and the control values (Fig. 10AGo), whereas the R271W mutant induced cytochrome C release rates of 3.2 and 5.7% 4 and 6 d after the infection step, respectively, which indicates that cytochrome C may be involved in this PCD process. This experiment was performed three times, and similar effects were consistently observed.


Figure 10
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  		Fig. 10. Detection of Cytochrome C Release, Determination of Caspase Activity, and Effects of Caspase Activity on Cell Growth

GH4C1 cells were infected or not with pGK-EGFP or pGK-R271W-ires-EGFP. A, Four or 6 d after the infection step, cytochrome C translocation into the cytoplasm was assessed by selectively permeabilizing the plasma membrane and then performing immunocytochemistry and flow cytometry. The loss of cytochrome C from mitochondria is indicated by a shift in the fluorescence to the left (from M2 to M1). This experiment was performed three times, and this figure gives the results of one representative experiment. B, To determine caspase activity, 100,000 GH4C1 cells were infected with pGK-EGFP or pGK-R271W-ires-EGFP vector in a 24-well plate. Caspase activities were determined on postinfection d 2, 5, and 7 using the Caspase Glo 3/7 Assay and Caspase Glo 9 Assay (Promega) as described by the manufacturer. The 100% activity level corresponds to the activity of the EGFP- infected cells. C and D, GH4C1 cells were infected or not ({circ}, bullet) with pGK-EGFP ({triangleup}, {blacktriangleup}), or pGK-R271W-ires-EGFP ({square}, {blacksquare}). Sixteen hours after being infected, cells were incubated with or without ({circ}, {triangleup}, {square}) 50 µM Z-VAD-CMK (C, bullet, {blacktriangleup}, {blacksquare}) or 50 µM Ac-LEMD-CMK (D, bullet, {blacktriangleup}, {blacksquare}) and cell viability was monitored using the Cell Titer Glo assay.

 
It is well known that cytochrome C released from mitochondria induces a series of biochemical reactions resulting in caspase activation, which play an essential role in PCD (15, 16). To determine whether the cytochrome C release may have induced an increase in caspase 3 and 9 activities, these latter were measured in EGFP-, and Pit-1-R271W infected cells on postinfection d 2, 5, and 7. As shown in Fig. 10BGo, no increase in the caspase 3 and 9 activities was found to occur in the mutant infected cells as compared with the control EGFP-infected cells.

To confirm this result, a cell-permeable general caspase inhibitor, Z-VAD-CMK, and a cell-permeable caspase 9 inhibitor, Ac-LEMD-CMK, were used to abolish the caspase activities in infected cells, and the cell growth was assayed for a period of 7 d. The growth of GH4C1 cells infected with R271W mutant was not affected by these inhibitors (Fig. 9Go, C and D). It is also worth noting that a specific caspase 3 inhibitor, Z-DEVD-CMK, gave the same results. The latter data confirm that caspases are not involved in the GH4C1 cell death induced by Pit-1-R271W.

To determine whether even low rates of cytochrome C release can induce cell death, experiments were performed using staurosporine, a drug known to enhance caspase 3 activity in GH4C1 cells (17). The results obtained showed that small amounts of cytochrome C released greatly increased caspase 3 and 9 activities, resulting in cell death (see the supplemental data published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Cyclosporine A, an inhibitor of pore transition permeability, partially blocked the cytochrome C release, caspase activities, and cell death. All in all, these results show that even a small increase in cytochrome C release can have significant effects and lead to cell death.

Several caspase-independent pathways have been described (18), involving various factors, including L-DNAse II, which originates from the leukocyte elastase inhibitor (LEI). The latter is transformed by endoproteolytic cleavage into L-DNAse II, a molecule bearing endonuclease activity (18). The accompanying change of molecular weight can be observed by performing SDS-PAGE and Western blotting procedures using antibodies directed against LEI and L-DNAse II. No differences in the level of expression of LEI and DNAse II were observed after infecting the cells with Pit-{Delta}1-123 and Pit-1-R271W in comparison with the control values and those obtained with EGFP-infected cells (data not shown). L-DNAse II is therefore not involved in this PCD process.

Another factor can be implicated in the caspase independent pathway: the apoptosis-inducing factor (AIF). This latter can be released from the mitochondria during some PCD processes and is subsequently involved in DNA fragmentation and the subsequent chromosomal condensation. The possibility that AIF might be translocated from the mitochondria to the nucleus was investigated using confocal microcopy 4 d after cell infection. Positive control cells were obtained by treating cells with peroxide. Under these conditions, punctate nuclear AIF staining was observed (Fig. 11Go). Contrary to what occurred in the case of these control cells, no nuclear translocation of AIF was observed in EGFP-, Pit-1-R271W-, or Pit-1-{Delta}1-123-infected cells.


Figure 11
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  		Fig. 11. Detection of AIF Delocalization

GH4C1 cells were infected with pGK-EGFP-, pGK-R271W ires EGFP, or pGK-1-123 ires EGFP or treated with 100 µM H2O2 to serve as positive control cells. Four days after infection or 4 h after adding H2O2, the cells were fixed and the AIF subcellular localization was determined under confocal microscopy. Arrow points indicate diffuse distribution of the AIF serving as the positive control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Pit-1 has been identified as a pituitary-specific signal integrator that directs both positive and negative regulatory stimuli to genes in somatolactotrope cells. This signal integration appears to be regulated by the binding of Pit-1 in the form of either a monomer or a dimer to a variety of cis-elements, as well as by functional interactions with other regulatory factors such as the estrogen receptor, the thyroid hormone receptor, Oct-1, GATA-2, Plim, Pitx1/2, Ets-1, or CCAAT/enhancer binding protein {alpha} (1, 19), and with cofactors (20). Consistent with the deletion of the whole transactivation domain (21), it was observed in the present study that the Pit-1{Delta}1-123 mutant was still able to bind to DNA but was markedly deficient in its capacity of transactivate target genes. Furthermore, this mutant acts in a dominant-negative manner, repressing Pit-1-induced prolactin promoter activation in both heterologous CV-1 cells and somatolactotrope GH4C1 cells. Because the Pit-1{Delta}1-123 protein does not appear to have any intrinsic repressive activity, the dominant-negative effects are likely to be due to the formation of nonfunctional complexes between WT Pit-1 and the Pit-1{Delta}1-123 mutant proteins and/or Pit-1{Delta}1-123 homodimers. This hypothesis is supported by previous studies indicating that the POU domain and Pit-1 can form a heterodimer on DNA (21) and by the recent report that the POU domain alone was unable to recruit its coactivator, the cAMP response element binding protein-binding protein, in the transcriptional binding complex (22).

Functional analyses of CPHD-inducing Pit-1 mutations have shed new light on the structure-function relationships of the Pit-1 protein (6). Some of these mutations, such as the recessive nonsense mutation R172X that causes deletion of a protein segment required for DNA binding (23), can be understood in terms of their negative effects on DNA binding. Other mutations that do not affect the DNA binding activity are more puzzling and rather favor the idea that interactions between the mutant protein and other nuclear factors may be involved (24). The most frequent CPHD-inducing mutation is R271W, which occurs in both a familial context and in sporadic cases and is inherited as a dominant trait (8). It occurs near the carboxy-terminal end of the homeodomain DNA recognition helix and therefore does not disrupt the DNA binding processes (8). Interestingly, crystallographic and structural analysis of the DNA/POU domain complex showed that Arg271 is involved in the formation of the dimer interface, because it forms an intermolecular hydrogen bond with a glutamine residue located on the POU-specific domain of the 2-fold-related monomer (25). The R271W mutation can therefore be viewed in terms of how it affects the ability of Pit-1 to dimerize on DNA. The weak increase in PRL promoter activation we observed in CV-1 cells when limiting amounts of transfected R271W were used is consistent with the idea that the process of dimerization on the PRL-1P element is altered (24, 25) and that the cooperativity in PRL promoter transactivation is subsequently impaired.

Because CPHD patients carrying the R271W mutation have only one mutant allele, it has been predicted that R271W is likely to have dominant-negative effects. In the initial paper by Radovick et al. (8), in which the first heterozygous R271W mutation in a patient with a marked CPHD phenotype was identified, the authors described the dominant-negative effects of the mutation on WT Pit-1 activation of target genes. However, recent studies performed under practically the same experimental conditions did not confirm these results (26). Under our conditions, R271W transfected into heterologous CV1 cells did not inhibit the activity of the WT protein and even enhanced this activity when low amounts of expression vectors were present. Similar results were obtained in Hela cells (Pellegrini, I., and J. L. Franc, unpublished results). This finding seems to simply suggest that the residual activity associated with R271W bound to DNA forming a monomer may have enhanced the transcriptional effects of WT Pit-1 bound to DNA forming a homodimer in an additive manner. By contrast, R271W was found to suppress Pit-1-activated PRL promoter activity when transiently transfected into GH4C1 cells and drastically decreased the levels of expression of the endogenous PRL and GH genes delivered by lentiviral vectors. All in all, these results suggest that the dominant-negative effects of R271W depend on conditions that are only present in the environment of somatolactotroph cells and therefore involve cell-specific interactions.

The ability of Pit-1 to participate in both cell-type-specific restriction and the activation of transcription is consistent with the hypothesis that Pit-1, like nuclear receptors, can be alternatively associated with either corepressors or coactivators (27). More specifically, the distinct configurations adopted by Pit-1 on specific DNA elements suffice to transform Pit-1 from a transactivator into a repressor through alternative associations with a particular class of cofactors (20). Likewise, the ability of Pit-1 to bind to specific DNA elements in the form of either a monomer or a dimer has been found to determine the domains it uses to synergize with other factors and to subsequently activate target genes in a cell-specific manner (28). Therefore, one possible explanation for the apparent cell specificity of R271W dominant-negative effects would be that R271W may interact with and inhibit the function of a limiting transcription factor specifically targeting the somatolactotrope lineage. Alternatively, it is possible that the disruption of the Pit-1 dimerization interface resulting from the mutation of Arg271 may prevent the formation of other protein-protein contacts. In line with this hypothesis, cooperative activation of PRL gene transcription by Pit-1 and CCAAT/ enhancer binding protein {alpha} was recently found to be correlated with changes in the subnuclear localization of these factors in living pituitary cells (29).

Cell growth is known to result from the combined activities of signaling pathways controlling the balance between cell proliferation and arrest and cell death and survival. For example, the infection of lactotrope GH4 cells with adenoviral particles containing dominant-negative forms of the estrogen receptor resulted in antiproliferative effects in vitro and in potent antitumorigenic effects when cells were transplanted into nude mice (30). Recently, Caporali et al. (31) established that the involvement of E2 in rat pituitary lactotrope cell growth is mediated by two distinct signaling pathways regulating the stimulation of cell cycle progression as well as preventing apoptotic cell death. Homeobox transcription factors have been thought to be crucial proteins regulating a variety of developmental and differentiation processes, but direct transcriptional regulation of cell cycle control genes by homeodomain factors has also been reported to occur. A few examples of this process have been described in the anterior pituitary gland. It has been suggested, for instance, that the sine oculus factors Six6 expressed in the pituitary primordium may repress genes encoding cell cycle inhibitors, thus promoting precursor cell proliferation in the developing pituitary gland (32). Pitx2 has also been found to play some crucial roles in the control of discrete proliferative events at early stages of development, by activating transcription processes in a subset of G1 growth control genes, such as cyclin D1 and D2, and c-myc (33, 34).

The inactivation of endogenous Pit-1 functions in GH4C1 cells resulting from the expression of dominant-negative mutants was found here to significantly reduce the growth rates and to induce cell death. On similar lines, dominant-negative Pit-1 mutants decreased the cell viability when these mutants were expressed in rat somatolactotrope nondividing SMtTW cells but not in Pit-1-defective cell types such as {alpha}T3 mouse gonadotrope cells or human fibroblasts, which suggests that this process is restricted to cells expressing the endogenous Pit-1 gene. These results, together with the finding that the cell growth rates were not increased in GH4C1 cells overexpressing the WT Pit-1 form, also indirectly suggest that Pit-1 may act as a survival factor in somatolactotrope cells. Moreover, cell cycle data showed that the sub-G1 population indicative of apoptosis observed in GH4C1 cells expressing dominant-negative Pit-1 mutants was associated with a decrease in the proportion of cells present in the G2/M fraction, which suggests that Pit-1 may also regulate cell cycle progression.

Apoptosis or PCD has been shown in many cases to be due to proteases called caspases (15, 16). In this pathway, apoptotic stimuli trigger cytochrome C release from the mitochondria, which results after a series of biochemical reactions in caspase activation (35). The results of the present study show that, in Pit-1-R271W infected cells, cytochrome C can be released into the cytoplasm in a small percentage of cells. It is difficult to determine whether this process occurs in all cells at different times, because it is not possible so far to obtain a dynamic picture of this event, and cell death may not be a synchronous process. On the other hand, it is not possible to determine whether mitochondrial permeabilization was necessary for this PCD process to occur, because mitochondrial pore permeability inhibitors such as cyclosporine A could not be used for a long time on this cell line because this drug induces cell death. However, our results clearly show that the dominant-negative mutants did not induce cell death via a caspase-dependent pathway because no increase was detected in these activities in the GH4C1 cells expressing the dominant-negative mutant R271W, and because inhibition of caspase activity did not protect these cells from death. It is well known that, in some cases, cells treated with an apoptotic inducer can also initiate a suicide program that does not respond to caspase activation (36). Other lethal factors involved in apoptosis such as AIF, Endo G, and L-DNAse II may be released upon mitochondrial outer membrane permeabilization or activated after proteolytic cleavage in the cytosol (18, 36). In the case of GH4C1 cells, it was previously reported that EGF induces death in a caspase-independent manner (17). The present results show that neither AIF nor DNAse II are involved in the PCD induced by dominant-negative forms of Pit-1. All in all, these data indicate that, in addition to activating the hormone genes, Pit-1 might trigger the gene activation programs required for cell survival and/or cell cycle progression. Unraveling whether cell death and cell cycle progression may be independently regulated by Pit-1 via separate mechanisms, or whether the execution of the apoptotic programs induced by suppressing Pit-1 functions may be due to G1 arrest, will require further investigations. Although the genes known to be directly regulated by Pit-1 are mainly those defining the somatolactotrope phenotype (PRL, GH, and Pit-1 itself), Pit-1 has also been found to directly control regulatory genes, including receptors involved in growth control (37), as well as the c-fos gene, an early marker of cell cycle initiation (38). In this context, studies focusing on selected Pit-1 mutants may provide important clues that will help to decipher the pattern of target gene expression involved in somatolactotrope cell proliferation and survival.

In conclusion, the results of the present study show that a Pit-1 heterozygous CPHD point mutant and an N-terminal truncation mutant serve as dominant negatives in the context of somatolactotroph cells. In particular, lentiviral delivery of these mutants to the GH4C1 somatolactotroph cell line abolishes not only cell differentiation but also cell growth by inducing apoptosis. To our knowledge, this is the first time the growth-promoting effects of Pit-1 have been found to be at least partly mediated by processes preventing apoptotic cell death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Constructs and Mutagenesis
The cytomegalovirus (CMV)-driven eukaryotic expression vector for human Pit-1 (pcDNA3hPit-1) and the hPRL-164luc and GHluc reporter plasmids used here were described previously (39). The R271W mutation in the pcDNA3hPit-1 construct was generated by PCR using the QuikChange Mutagenesis kit (Stratagene, La Jolla, CA) and the following commercially synthesized oligonucleotides (Life Technologies, Inc., Rockville, MD), where the mutation is indicated in bold print: R271W, 5'-GGCAGAGAGAAAAATGGGTGAAAACAAGTC-3'. A second construct, Pit-1{Delta}1-123, corresponding to a Pit-1 fragment encoding only the POU domain, was generated by PCR, with a primer encompassing the methionine at codon 124 upstream of the POU domain, and a primer located downstream of the stop codon. Plasmid DNA was purified using the Qiafilter Plasmid Maxi Kit (QIAGEN, Valencia, CA) and all mutations were confirmed by DNA sequencing (ABI Prism BigDye terminator cycle sequencing ready reaction kit; Applied Biosystems, Foster City, CA). To generate lentiviral vectors (Fig. 4Go), the NotI-SpeI fragment encompassing the Pit-1 or R271W mutant cDNA in pcDNA3Pit-1 or the NotI-BamHI fragment containing the Pit-1{Delta}1-123 mutant in pCRscript were inserted instead of the EGFP gene into pRRLTpgkEGFSin18, a previously described HIV-1-derived lentiviral vector construct (40).

Cell Culture and Transfection
GH4C1 somatolactotrope pituitary cells were grown in Ham’s F10 medium supplemented with 15% horse serum and 2.5% fetal calf serum (FCS). Primary cultures of SMtTW rat somatolactotrope cells obtained from spontaneous transplantable tumors (41), a gift from Pr. J. Trouillas (University of Lyon I, Lyon, France), were maintained in culture in DMEM ITSG (Invitrogen, Carlsbad, CA) supplemented with 2% FCS. African green monkey kidney fibroblast-like CV-1 cells, Hela cells, {alpha}T3 mouse pituitary cells derived from T antigen-immortalized gonadotrope cells (13) and human fibroblasts obtained from long-term primary cultures of human pituitary adenomas were grown in DMEM supplemented with 10% FCS and were used as control when required.

GH4C1 cells were transfected into triplicate wells by lipofection in serum-free medium using Transfast transfection kit (Promega, Madison, WI) in line with the manufacturer’s instructions. Briefly, cells were plated at 2 x 105 cells/well into 12-well plates 24 h before their transfection and were transfected with 1.5 µg of DNA [0.3 µg of reporter plasmid, 0.1–1 µg of effector plasmid(s), and 0.2 µg of CMV-ß-galactosidase plasmid as the internal control to check the transfection efficiency]. Cells were incubated with the DNA/liposome complexes for 1 h and were then supplemented with 1.5 ml complete medium. CV-1 cells were transfected using the calcium phosphate method with the MBS mammalian transfection kit (Stratagene) in line with the manufacturer’s instructions. Briefly, cells were plated at 1 x 105 cells/well in six-well plates 24 h before being transfected. Transfections were carried out in triplicate wells using 3 µg of reporter plasmid, 0.1–1 µg of effector plasmid(s), and 0.3 µg of CMV-ß-galactosidase. In all the transfection processes, total DNA was kept constant and nonspecific effects of viral promoters were controlled by using appropriate empty vectors.

Luciferase and ß-Galactosidase Assays
GH4C1 and CV-1 cells were harvested 48 h after transfection and lysed in 200 µl Reporter Lysis Buffer (Promega). After three sequential freeze-thaw cycles, cell debris were pelleted by centrifugation at 10,000 x g for 2 min at 4 C and 20-µl aliquots of the supernatant were used for subsequent luciferase (luciferase assay system; Promega) and ß-galactosidase assays. For each assay, the total luciferase activity normalized against ß-galactosidase activity was taken to be 1, and results were expressed as fold activation over the control value. The data presented are means ± SE of three to five independent experiments using different plasmid preparations of each construct.

Generation of Lentiviral Vectors and Transduction
Lentiviral vector particles were produced in 293T cells using a polyethylenimine (Aldrich, Milwaukee, WI)-mediated transfection procedure. 293T cells were transfected with 1) the packaging plasmid pCMV{Delta}R8.91 expressing HIV-1 gag, pol, tat and rev proteins; 2) the envelope plasmid pMD-G expressing the vesicular stomatitis virus envelope G glycoprotein (VSV-G); and 3) the lentiviral vector construct containing the transgene of interest (Fig. 4Go). Conditioned medium was harvested 48 and 72 h after transfection, cleared of debris by low-speed centrifugation, filtered through 0.22-µm membrane, and concentrated by centrifugation at 10,000 x g for 1 h at 4 C in the presence of polyethylene glycol (molecular weight, 8000). The viral pellets were then resuspended in 1/100 vol of PBS. Viral stocks were stored in aliquots at –80 C and the titers were determined by transducing HelaT cells in a limiting dilution assay. No replication competent virus was detected in the concentrated lentiviral stocks. For the transduction experiments, GH4C1 cell cultures plated in 24- or 12-well plates (5 or 10 x 104 cells/well) were supplemented with lentiviral particles containing medium in the presence of 2 µg/ml polybrene at the MOI indicated. After a 16-h incubation period, the cells were washed and grown in regular medium for further analysis.

TNT and EMSA
The TNT T7 Coupled Reticulocyte Lysate System (Promega) was used for TNT. Reactions were carried out in a total volume of 50 µl with reticulocyte lysate, 1 µg plasmid DNA, 1 mM amino acid mixture, RNasin (Invitrogen; 40 U/ml), and T7 RNA polymerase, in the presence or absence of 35S-Met (NEN, Boston, MA; 10 mCi/ml). 35S-Met-radiolabeled translation products were separated by SDS-PAGE and exposed to autoradiographic film.

EMSAs were carried out with Pit-1 recombinant proteins and a 32P-labeled double-stranded oligonucleotide containing the P1 Pit-1 element of the hPRL promoter (5'-AATGCCTGAATCATTATATTCATGAAGATATC-3'). One hundred nanograms of annealed double-stranded DNA was 5'-end labeled with T4 polynucleotide kinase (Invitrogen). In vitro-translated proteins were incubated on ice for 15 min in a 20-µl reaction of 1x binding buffer containing 1 µg of poly(deoxyinosinic-deoxycytidylic) acid sodium salt) and 20,000 cpm of the radiolabeled probe. The bound proteins were separated from the free probe on 8% polyacrylamide gel in 0.5% Tris-borate EDTA by SDS-PAGE at 180 V for 3 h at 4 C before being exposed to autoradiographic film. A supershift was induced in the complexes by a Pit-1 mAb directed against the N-terminal domain of the protein (Transduction Laboratories, Inc., Lexington, KY).

Northern Blot Analysis
Total RNA was purified from GH4C1 cell culture wells using the RNeasy kit (QIAGEN), run on a 1% agarose/formaldehyde gel, and transferred to nylon membrane. Prehybridization was performed at 42 C in 50% formamide, 6x sodium saline citrate, 5x Denhardt’s solution, 0.5% sodium dodecyl sulfate, and 100 µg/ml denatured salmon sperm DNA. The blots were successively hybridized in the same buffer for 16 h at 42 C with several deoxycytidine triphosphate {alpha}32P-labeled cDNA probes (2 x 106 cpm/ml). DNA probes were cDNA fragments generated by RT-PCR specific for PRL (amino acids 4–199), GH (amino acids 19–203), and Pit-1 POU domain (amino acids 124–291). Blots were washed under stringent conditions, placed on a Phosphor screen (Molecular Imager; Bio-Rad, Hercules, CA), stripped, and finally rehybridized with an 18S-specific cDNA probe to normalize the results to allow for variations in sample concentration and loading.

Cell Viability
The effects of WT and dominant-negative Pit-1 forms on cell growth were assessed with a luminescent cell viability assay, using the manufacturer’s protocol (Cell Titer Glo; Promega Corp.). Cell viability was assayed in replicate wells at 3-d intervals during a 9-d period after infecting a constant fraction of the cell culture with lentiviral vectors. Each sample was assayed in duplicate.

PRL RIA
PRL secretion into the culture medium was determined using standard RIA techniques with the material and protocols kindly supplied by the National Institute of Diabetes and Digestive and Kidney Diseases rat pituitary hormone distribution program. Inter- and intraassay variations amounted to less than 10%. One hundred microliters of each sample were assayed in duplicate. Results are expressed as nanograms per day per 105 cells.

Hoechst Fluorescent Staining of Apoptotic and Mitotic-Positive Cells
Glass slides with infected GH4C1 cells were fixed with 4% formaldeyde and counterstained with Hoechst 33342 (1 mg/ml) for 5 min at room temperature. Cells were washed with PBS mounted with fluorSave Reagent (Calbiochem, France), and apoptotic and mitotic-positive cells were then viewed and scored manually with a Leica/Leitz (Darmstadt, Germany) DMRB microscope using a PL fluotar 100x objective.

Flow Cytometry Analysis
Transduction efficiency with recombinant EGFP lentiviral vector was evaluated 3 d after infection by FACS. Cells were treated with trypsin and resuspended in 500 µl of medium containing 50 µg/ml PI (BD Pharmingen, San Diego, CA) and run on a FACScalibur flow cytometer (Becton Dickinson Inc., San Jose, CA). Data were analyzed with the CellQuest program (Becton Dickinson Inc.). For cell cycle distribution analysis, GH4C1 cells transduced with lentiviral vectors were harvested by trypsin treatment 6 d after infection, fixed in 70% ethanol for 30 min, and treated with Rnase A (1 mg/ml) for 30 min at room temperature. DNA was stained with 50 µg/ml of PI for 30 min at 4 C and protected from light before relative DNA content determination by FACS. DNA cell distribution histograms of were analyzed using the CellQuest Pro software program. Ten thousand events were acquired for each analysis.

Analysis of Cytochrome C Release
Cytochrome C release was analyzed using flow cytometric methods as previously described (42, 43). The anti-cytochrome C mAb clone 7H8.2C12 (BD Pharmingen) was conjugated to Alexa fluor 467 using the Zenon labeling kit (Molecular Probes, Eugene, OR).

Immunostaining of AIF
Immunostaining of AIF was carried out as described previously (17). Cells were permeabilized (0.1% Triton X-100 for 45 min) and then incubated for 40 h at 4 C with mAb anti-AIF (E-1; Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1:100 in PBS, 0.5% BSA. The immunostaining was revealed using an Alexa Fluor 555 goat antimouse IgG antibody (Molecular Probes) diluted in PBS containing 10% normal goat serum (Jackson Immunoresearch, Suffolk, UK; and Immunotech, Marseille, France). Confocal image acquisition was performed on a Leica (Deerfield, IL) TCS SP2 laser scanning microscope, and image editing was performed using Adobe Photoshop.

Stastitical Analysis
The values presented here are means ± SEM. Statistical significance was determined by performing Mann-Whitney nonparametric tests. Differences were taken to be statistically significant at a probability level of P < 0.05.


   ACKNOWLEDGMENTS
 
We acknowledge the assistance of Dr. Charles Prevot and Dr. S. Chapel for the FACS analysis and that of Dr. Jean-Paul Herman for his critical reading of this manuscript. We also thank Dr. Pamela Mellon for the {alpha}T3 cell line, Dr. Jaqueline Trouillas for SMtTW tumors, Dr. Alicia Torriglia for L-DNAse II antibodies, and the National Hormone and Pituitary Program and Dr. A. F. Parlow for generously providing antisera to pituitary prolactin.


   FOOTNOTES
 
This work was supported by La Ligue contre le Cancer (2001), the Association pour la Recherche sur le Cancer (Grant no. 5146), and Centre National de la Recherche Scientifique (programme Puces à ADN 2000–2002). M.-H.Q. was supported by a postdoctoral fellowship from Fondation pour la Recherche Médicale.

Current address for I.P.: Institut National de la Santé et de la Recherche Médicale Unité 379, Institut Paoli-Calmette, 232, Bd de sainte-Marguerite, 13273 Marseille cedex 09, France.

Disclosure statement: The authors have nothing to disclose.

First Published Online August 10, 2006

Abbreviations: AIF, Apoptosis-inducing factor; CMV, cytomegalovirus; CPHD, combined pituitary hormone deficiency; EGFP, enhanced green fluorescent protein; FACS, fluorescence activated cell sorting; FCS, fetal calf serum; hPRL, human PRL: IRES, internal ribosome entry site; LEI, leukocyte elastase inhibitor; mAb, monoclonal antibody; MOI, multiplicity of infection; PCD, programmed cell death; pGK, human phosphoglycerate kinase; PI, propidium iodide; PRL, prolactin; TNT, in vitro transcription/translation; TSHb, TSH b-subunit; WT, wild type.

Received for publication March 14, 2006. Accepted for publication August 3, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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