The class A of basic helix-loop-helix (bHLH) proteins are ubiquitously expressed transcription factors playing a pivotal role in the regulation of cell growth and differentiation. We determined that enforced expression of all four different mammalian members of this family, E12, E47, E2-2, and HEB, suppresses the cell colony-forming efficiency of several cell lines. To gain insights into the mechanisms by which class A bHLH factors affect cell growth, we have investigated their role in the transcriptional regulation of cyclin-dependent kinase inhibitors. We found that p21CIP1/WAF1, p15INK4B, and p16INK4B promoter sequences contain E-boxes that render these genes competent for class A bHLH-mediated transcriptional activation and Id-mediated repression. The mechanism underlying the class A bHLH-mediated inhibition of cell growth does not involve an arrest of G1 progression in 293T cells. In fact, contrary to what has been found in 3T3 NIH fibroblasts,we found that enhanced expression of class A bHLH proteins led to a decreased proliferation rate by promoting cell death associated with the induction of apoptosis. These findings highlight the role of the class A bHLH proteins as general negative regulators of cell proliferation through a mechanism(s) that involves both enhancement of several cyclin-dependent kinase inhibitor genes expression and promotion of cell death.

The bHLH3family of transcription factors have been shown to play important roles in cell growth and differentiation (reviewed in Refs. 1and 2). There are two distinct classes of bHLH proteins. Ubiquitously expressed class A bHLH, often also referred as the “E”proteins, consist of E2-2 (3), HEB (4), and the differentially spliced products of the E2A gene, E12 and E47 (5). The class B proteins represent a large yet expanding group of transcription factors, the expression of which is developmentally regulated, such as MyoD (6), NeuroD(7), and Tal1 (8). These proteins contain an HLH domain, mediating homo- and heterodimerization, which consists of two amphipathic helixes separated by a loop, plus an adjacent DNA-binding region rich in basic amino acids (5). The bHLH dimers bind to a DNA consensus sequence known as the E-box (CANNTG). E-box sites are present in a wide variety of tissue-specific enhancers,driving their specific activity (9, 10, 11).

Another class of HLH proteins is defined by the Id genes,which share a highly homologous HLH domain but lack the basic DNA-binding region (12, 13, 14, 15, 16, 17, 18, 19). Thus, the Id proteins counteract the E protein-mediated gene regulation by forming with them inactive heterodimers, thereby preventing binding of bHLH factors to the E-box sites (20). Down-regulation of Idgene expression is necessary for differentiation to proceed in many cell lineages (12, 21, 22), and overexpression of Id proteins enhances cell proliferation of several cell lines (23, 24). However, there is an increasing amount of evidence in which this general rule is reverted: up-regulation of single Id proteins accompanies cell differentiation (25, 26), and it has been reported recently that ectopic expression of Id2 can induce apoptosis after growth factor withdrawal, at least in some cellular settings (27).

The function of class A bHLH factors has been investigated extensively through targeted mutation in mice. The generation of mice with E2A null mutations has revealed an essential role of this gene in lymphopoiesis, with B-cell development being completely blocked at the earliest identifiable stages (28), and with an increased susceptibility to develop T-cell lymphomas (29). Studies using cell cultures have shown that increasing the intracellular amount of E47 causes withdrawal from the cell cycle before G1-S transition in NIH 3T3 cells(23). Conversely, antisense-mediated down-regulation of Id proteins delays the reentry of these serum-starved cells into the cell cycle (30). Furthermore, it has been shown recently that ectopic expression of E47 or E12 proteins promotes the death of E2A-deficient lymphomas (31), and restoration of E2A activity in Jurkat leukemic T cells results in an inhibition of cell growth and induction of cell death associated with an increase in apoptotic cells (32). On the basis of these findings, we reasoned that class A bHLH factors might inhibit cell proliferation through the transcriptional activation of genes that can negatively regulate cell cycle progression. In has been suggested recently that the CDKI p21 could represent a potential effector of the mechanisms by which E2A negatively controls cell growth(33).

In this study, we have determined that enforced expression of all four class A bHLH factors (E12, E47, E2-2, and HEB) suppresses the cell colony-forming efficiency in several cell lines, suggesting that all of the members of class A bHLH act as general negative regulators of cell proliferation. We have then investigated the role of different members of the class A bHLH family in the transcriptional regulation of several CDKIs. We have found that p21CIP1/WAF1, p15INK4B, and p16INK4B promoter sequences contain E-boxes, which represent specific target sites accessible for transcriptional activation mediated by E2A factors. In contrast with previous results(23) suggesting that the E2A gene products could block G1 progression when overexpressed in NIH 3T3 cells, we found that in 293T cells the mechanism underlying the class A bHLH-mediated inhibition of cell growth does not involve an arrest of G1 progression. Rather, we have found that enhanced expression of the class A bHLH protein promotes cell death associated with the induction of apoptosis.

Plasmids.

The CMV-based expression vectors for E12, E47, E2–2, HEB, and Id4 were described previously (34, 35). Expression vectors for Id1,Id2, and Id3 were generous gifts from Dr. G. Peters (Imperial Cancer Research Fund, London, United Kingdom) and were described elsewhere (36, 37). The CMV-based expression vector for bcl-2 was kindly provided by Antonio Giordano (Jefferson Medical College, Philadelphia, PA). To construct the p15-Luciferase reporter, a PCR reaction was performed on genomic DNA from HeLa cells to amplify a DNA fragment corresponding to residues −113 to +303 with respect to the transcriptional start site (residues 12–406 of the p15 promoter sequence deposited in GenBank, no. S75756). The PCR product was cloned into the SmaI and HindIII sites of pGL3 basic(Promega) to obtain p15L-Luc and into pBluescript KS (Stratagene) to obtain p15KS; this plasmid DNA was cut with SacI, and the insert was cloned into the SacI site of pGL3 to obtain p15S-Luc. To construct the p16-Luc, a PCR reaction was performed on genomic DNA from HeLa cells to amplify a DNA product corresponding to residues −832 to −63 of the promoter sequences defined by Hara et al. (Ref. 38; residues 348-1139 on the sequence deposited as GenBank no. X94154). The resulting amplification product was cloned into the SacI and HindIII sites of pGL3. The p21-luciferase reporter contains the residues from−2329 to +11 of the p21 promoter (residues 2257 to 4595 of the sequence deposited in GenBank, no. U24170) subcloned from p21WWP into the HindIII site of pGL3 basic. To construct the p57-luciferase reporter, a pBluescript clone of a p57KIP2 genomic fragment corresponding to residues 1 to 4895 (GenBank D64137) was provided by Dr. Y. Nakamura (Institute of Medical Science, The University of Tokyo, Tokyo, Japan), and the promoter region from −983 to +258 was cloned into the KpnI and MluI sites of pGL2 (Promega).

For mutant constructs, the p21-S luciferase reporter was constructed by inserting the SmaI-HindIII (nucleotides −61 to+11) fragment into pGL3. Point mutations were introduced into the E-box sequences upon PCR reactions with oligonucleotide pairs in which one internal primer is centered on the mutated E-box and the other anneals on external sequences. The p21-S/E mutant was obtained by cutting the p21-S/E2 with PvuII and HindIII, followed by blunting of the overhang generated by HindIII, gel purification of the larger fragment, and ligation. The p15-S/E was obtained by cutting p15-S with EcoRI and PvuII,followed by fill-in of the 3′ overhang generated by EcoRI and self ligation. The p16/E2,p16/E1, and p16E1/2were constructed by PCR mutagenesis as described below. Details of the primer sequences and PCR parameters are available upon request. All of the mutations were verified by sequencing with the T7 dideoxy sequencing kit (Amersham Pharmacia Biotech).

Cell Lines and Transient Transfections.

U2OS and SaOS-2 osteosarcoma cells, HeLa papillary carcinoma cells,293T human kidney carcinoma cells, ΦNX cells, and NIH3T3 murine fibroblasts were grown at 37°C in DMEM supplemented with 10%FCS (Life Technologies). Subconfluent cell cultures were transfected with Lipofectin (Life Technologies) for HeLa cells and Lipofectamine(Life Technologies) for 293T cells, following the supplied instructions. Cells were seeded at 40% confluence on six-well plates;16 h later, DNA lipid complexes were prepared by mixing up to 4μg of DNA (0.05 μg of pRL-CMV, 0.4 μg of reporter, and up to 3.5μg of effector plasmid DNAs, respectively) and 5 μl of lipofectin or 10 μl of Lipofectamine in a 200-μl total DMEM volume. All transfections included a reference sample with pGL3 basic. Forty-eight h later, cells were harvested, and luciferase activity was determined with the Dual Luciferase Reporter Assay (Promega) in a TD20 luminometer(Turner designs), following the manufacturer’s instructions. The luciferase activity was calculated by subtracting the intrinsic activity as measured by samples corresponding to the pGL3 basic and then normalized to transfection efficiency as measured by the activity deriving from pRL-CMV (Promega), an expression vector for the Renilla luciformis luciferase.

Colony Formation Assays.

Subconfluent (70%) cell cultures were transfected by calcium phosphate method with 9 μg of the CMV-based expression vector and 1 μg of pRC-CMV (Invitrogen), conferring the resistance to G418. Eight h later,the cells were split at dilutions of 1:6 into 10-cm plates, and after incubation overnight, G418 was added at a concentration of 0.5 or 1 mg/ml for SaOS and U2OS or HeLa and C33A cell lines, respectively. After 10–15 days of selection, the plates were washed with PBS, fixed with 10% methanol and 10% acetic acid, and stained with cresylviolet in 10% ethanol. The colonies were scored by visual inspection and counted.

Flow Cytometry Analysis.

Transfections into ΦNX (39) cells were performed with the calcium-phosphate/chloroquine method as described(40). Briefly, cells were plated in six multiwell plates at a density of 200,000 cells/well 2 days before transfection, then transfected with 5 μg of the indicated expression vector and the medium was changed 8 h after transfection. The cell cycle was analyzed 48 and 72 h after transfection. The DNA profile of the fixed cells was evaluated by FACScan (FACS-Vantage; Becton Dickinson,Omaha, CA) flow cytometry, according to Lamm et al.(40). The transfection efficiency was measured using 1μg of a GFP-expressing plasmid in parallel experiments.

Transient Transfection Analysis.

Subconfluent cultures of 293T cells were transfected with Lipofectamine, typically using 4 μg of CMV-based vector as described in the text. In the experiments using two different expression constructs 2 μg of Bcl2 and 2 μg of bHLH vectors were used. For BrdUrd incorporation assays, 24 h after transfection cells were trypsinized and seeded on coverslips. After an additional 12 h of incubation, cell samples were pulse-labeled for 1 h with BrdUrd and processed with the BrdU Detection Kit I (Roche) according to the instructions supplied. Nuclei were stained with diaminophenylindole(Sigma). Coverslips were mounted in glycerol-PBS, visualized under a Polyvar microscope, and photographed. To evaluate the extent of apoptosis in 293T transfected cells, 24 h after transfection cells were trypsinized and seeded on coverslips. After an additional 12 h of incubation, cell samples were analyzed by the TUNEL assay using the In Situ Cell Death Detection kit, POD (Roche). Coverslips were mounted, visualized, and photographed. The number of viable cells after transfections was determined by trypan blue dye exclusion. For quantitative determination of dead cells, 36 h after transfection cells were trypsinized and stained with 0.4% trypan blue in PBS at room temperature.

All Four Members of Class A bHLH Proteins Negatively Affect Cell Proliferation.

It was reported that the E2A gene products, E47 and E12,suppress cell growth in cell colony formation assay (23). We set out to examine whether the remaining class A proteins shared the growth-inhibitory properties of E2A gene products in this type of assay. Colony formation assays were carried out using different cell lines (i.e., HeLa, U2OS, and SaOS-2). CMV-based expression vectors for E12, E47, E2-2, and HEB were cotransfected in each cell lines along with a selectable neoR marker. After 10–14 days of selection with G418, the number of cell colonies was determined. As reported in Fig. 1 (and data not shown), we found that for each of the class A bHLH proteins tested, we observed a reduction of the number of cell colonies with respect to the control transfection in which CMV-based empty vector was used. Although our results are meant to be qualitative rather than quantitative, we constantly observed that cell colony reduction was less marked when HEB was overexpressed. Results similar to those presented in Fig. 1 were also observed using NIH 3T3 and 293T cell lines (data not shown).

For each class A protein tested, we found that reduction of colony formation was independent of the pRB or p53 status, because it was observed in cells that are either positive for their function (U2OS) or negative (SaOS-2). These findings indicate that all four members of class A bHLH proteins induce reduction of cell colony formation, and such inhibitory function could be easily detected with all of the cell lines tested, thereby identifying the whole class A bHLH proteins as negative regulators of cell proliferation.

Transactivation of the p21, p16, and p15 Promoters by Class A bHLH Factors.

It has been shown previously that the E47 protein enhances transcription of the CDKI p21CIP1 promoter when overexpressed in HeLa cells, and it has been suggested that activation of p21 may be relevant to the inhibitory function of E47(33). Because all four members of class A bHLH factors display a similar negative regulatory activity on cell proliferation,we reasoned that homo/heterodimers of different bHLH factors such as HEB, E2-2, and E12 might also be able to activate the p21 promoter in transient transfection assays. In addition, we also sought to test whether bHLH-mediated activation was restricted to the p21 promoter or whether other CDKIs, such as p15INK4B, p16INK4and p57KIP2, may also be transcriptionally induced by the class A bHLH proteins. To test these hypotheses, we used luciferase reporter constructs driven by the p21, p15, p16, and p57 promoter sequences: p21-Luc, p15-Luc p16-Luc, and p57-Luc, respectively. Transient transfection experiments were carried out in the presence of CMV-based expression vectors encoding the four different members of the class A bHLH family: CMV-E47, CMV-E2–2, CMV-E12, and CMV-HEB,respectively.

In line with previously published results, stimulation of p21-Luc occurred in cotransfections with the E47 protein. Moreover, we found the p15-Luc and p16-Luc promoter activities were also enhanced by coexpression of E47. In addition, a modest but reproducible activation of the p15, 16, and p21 promoter activities was observed when the other class A HLH factors, i.e., E2–2, E12, and HEB, were overexpressed. In sharp contrast, the activity of the p57-Luc promoter was unaffected in cotransfections with any of the class A proteins tested. Similar bHLH-mediated activation of the luciferase promoter constructs was also observed using the HeLa cell line (data not shown). The activity p21-Luc promoter construct was enhanced, albeit at a different extent, by the presence of each of the bHLH factors tested in HeLa cells (Fig. 2 A). These findings suggest that overexpression of each member of the class A bHLH factor family results in the activation of different CDKI promoter constructs. Thus, at least three members of the family of CDKIs, i.e., p21, p15, and p16, are subjected to a positive transcriptional control by the class A bHLH transcription factors, with the E47 protein being the strongest transcriptional stimulator of this particular group of CDKIs.

To further evaluate the E47-mediated transactivation of the CDKI promoters, we sought to determine whether ectopic expression of Id proteins, which are known to suppress the bHLH binding to DNA by forming heterodimers with them, could have a significant effect on the E47-mediated activation of the CDKI promoter constructs. CMV-based expression vectors for Id1, Id2, Id3, and Id4 were cotransfected along with each CDKI luciferase-driven promoter constructs in the presence of CMV-E47 expression. The results reported in Fig. 2,B are relative to the Id4 cotransfections; however, we observed similar results for any of the Id family members (data not shown). As shown in Fig. 2,B, we determined that coexpression of the Id4 protein suppresses the stimulatory effect mediated by E47. Moreover, as shown in Fig. 2 B, Lanes 3, 7, and 11, we found that overexpression of the Id4 protein not only blocks the bHLH-mediated activation but also represses the basal activity of p16, p15, and p21 promoter.

Taken together, the data presented in Fig. 2 suggest that the promoters of several CDKI genes, i.e.,p21, p15, and p16, are competent for regulation by all members of the class A bHLH factors, and such regulation is subjected to a negative control exerted by the dnHLH Id proteins.

E-Box-mediated Activation of p21, p15, and p16 by Class A bHLH Factors.

It has been shown previously that E47 protein-stimulated p21 expression is strictly dependent upon the presence of E-box consensus sequences located within the p21 promoter region (33). We identified putative E-box sites in the proximal promoter regions of p15INK4B and p16INK4A. To assess the contributory role of the E-box sites in bHLH-mediated activation of these promoters, we carried out site-directed mutagenesis of the E-box sites present in p21, p15, and p16 promoters, respectively.

We constructed a p21 minimal promoter reported bearing sequences from−61 to +11 with respect to the transcription start site, and we determined that this p21 minimal promoter was transactivated by E47 in a manner comparable with that of the parental p21 promoter construct(compare E47-mediated activation in Fig. 2,A and in Fig. 3,A). The p21 sequences spanning from −61 to +11 contain two putative E-box consensus elements, both located downstream of the TATA box. To determine the relative contribution of these two elements in the E47-mediated activation, we constructed appropriate p21 promoter constructs bearing mutations on either E-box elements and tested the ability of E47 to regulate the p21 mutant promoters. As reported in Fig. 3 A, we found that mutations of each single E-box resulted in a weaker E47 transactivation effect, and mutations of both E-boxes abolished transactivation. Thus, it appears that E47 transactivation of p21 promoter activity is mediated by the presence of at least two E-box elements present downstream the TATA box, and each of them contribute in an additive manner to the E47 activity.

A similar strategy was used to determine the cis-acting elements present in the p15 promoter responsible for the E47-mediated activation. We found that the p15 promoter sequences bearing residues spanning from −113 to +71 with respect to the Inr element was activated by E47 as much as larger promoter sequences (compare E47 activation shown in Fig. 2,A and 3,B), and this promoter construct contains a single putative E-box located at position−33. As shown in Fig. 3 B, E47 transactivation was completely abrogated when an E-box deletion mutant was used.

Finally, we determined the functional role of the E-box sites in the p16 promoter sequences. A computer-assisted search of the p16 promoter sequences indicated the presence of two putative E-box sites at position −316 and −50 relative to the most upstream start sites reported previously (38). To determine the relative contribution of these two elements in the E47-mediated activation, we introduced site-specific mutations in either E-box elements and tested the ability of E47 to regulated the p16 mutant promoters. As reported in Fig. 3 C, we found that single mutations of each single E-box resulted in a weaker E47 transactivation effect, and mutations of both E-boxes abolished E47 transactivation. Thus, as seen previously in the case of the p21CIP1 promoter, both the E-boxes found in this promoter are responsible for its transcriptional stimulation by E47.

Our findings demonstrated that the p21, p16, and p15 promoter sequences contain E-box sequences, which represent specific target sites accessible for transcriptional activation by class A bHLH factors. In addition, our data strongly suggest that bHLH-mediated activation is strictly dependent upon the presence of the E-box sequences.

Ectopic Expression of Class A bHLH Promotes Cell Death Associated with Apoptosis.

The phenomenon of cell colony reduction described in Fig. 1 can be interpreted in at least two ways: (a) ectopic expression of class A proteins might either lead to slower cell growth by inhibiting a transition between two phases of the cell cycle; and (b)or increase the number of dying cells as a result of cellular toxicity or induction of apoptosis. To gain insight into the mechanism(s)responsible for the cell cycle arrest, we performed transfection experiments using the ΦNX cells. This cell line is a 293T-derived highly transfectable amphotropic packaging cell line (39). Control transfections using a GFP expression vector followed by flow cytometric analysis of the transfected cells indicated that 90–95% of transfected cells were scored positive for GFP expression (data not shown). At different times (48 and 72 h) after transfection, the cells were harvested, and the cell cycle profiles were determined by flow cytometry on a FACSort. The relative amounts of cells with a DNA content of less than 2N were determined accordingly to the procedure reported previously (40). Two type of controls were used in these experiments, the empty CMV vector and a p21-expressing vector. As expected, overexpression of p21CIP1 gene product induced a G1 arrest (Table 1). Conversely, class A transfected-cells displayed an increase in the fraction of cells in S phase with a substantial increase of cells with a subdipolid DNA content. Significantly, the accumulation of cells in S phase and the percentage of presumptive apoptotic cell populations in these experiments were largely coincident. By contrast, no significant increase in apoptosis above the level of control-transfected cells was observed in cells transfected with the p21 expression vector. These finding were somewhat unexpected, because it was shown previously that overexpression of E47 induces a G1 arrest in NIH3T3 cells (23). In contrast, the flow cytometry data collected at different times after transfections clearly indicate that overexpression of any class A bHLH protein induced a strong increase of cells with subdiploid DNA content, an indicative characteristic of the apoptotic DNA fragmentation.

To confirm and extend our findings on the effects of overexpression of class A proteins on cell cycle progression, we examined 293T class A-transfected cells by: (a) trypan blue dye exclusion for evidence of cell death; (b) TUNEL assay for apoptosis; and(c) BrdUrd incorporation for evidence of the fraction of cells in S phase. Control transfection using a GFP expression vector demonstrated that ∼60% of the transfected 293T cells were scored positive for GFP expression (data not shown). As presented in Fig. 4,A, we observed a significant increase of cell death in the class A-transfected 293T cells relative to control cells. Next, we examined for the extent of apoptosis by the TUNEL assay, and it was evident that class A-transfected cells exhibited significantly elevated apoptosis when compared with vector-only transfectants (Fig. 4,B). Finally, BrdUrd incorporation assays revealed the accumulation of a significant fraction of transfected cells in S phase(Fig. 4 C). Significantly, the S phase and presumptive apoptotic cell populations in these experiments were largely coincident, a phenomenon that has been reported previously for E2F-1-,cyclin D1-, and Id3-induced apoptosis (41, 42, 43).

To further analyze the mechanisms of class A-induced cell growth arrest, we evaluated how the presence of cell survival Bcl2gene product affected the ability of E2A factors to induce apoptosis in transfected cells. 293T cells were transfected with class A expression vectors in the presence of Bcl2, and the relative amounts of apoptotic cells were scored by TUNEL assay. Consistent with the role of Bcl2 in cell survival, we found that cotransfection of Bcl2 effectively rescued cells from E2A-induced apoptosis (Fig. 4, D and E).

We have shown that enforced expression of each member of the class A bHLH transcription factors, E47, E12, E2–2, and HEB, suppresses the colony-forming efficiency of a variety of cell lines. These findings highlight the role of the class A bHLH proteins as negative regulators of cell growth. The precise mechanisms through which the different class A bHLH proteins function in cell proliferation are presently unknown. It is conceivable that up-regulation of dedicate cell cycle inhibitory genes can represent the physiological targets of bHLH function; however, the in vivo models of loss of E2A function have shown that in some cell contexts, E2A might induce apoptosis (32). Using transient transfection assays, we observed that at least three members of the family of CDKIs, i.e., p21, p15, and p16, are subjected to a positive transcriptional control by different members of the class A bHLH transcription factors, with the E47 protein being the strongest activator. A straightforward mechanism for such up-regulation would be that bHLH factors can bind to the cognate E-box sites present in the promoter sequences of the p21, p15, and p16 and directly activate transcription. Indeed, we found that each CDKI promoter contains putative E-box sites, and most importantly, E47-mediated activation of these promoters is exquisitely dependent upon the presence of the cognate E-box target sites. Moreover, we determined that E47-mediated regulation is subjected to a negative control by the Id proteins.

The ability of bHLH proteins such as E47 to activate expression of CDKI raises the possibility that these genes may represent a potential target of the mechanisms by which E2A negatively controls cell growth. It has been shown previously that enforced expression of E47 protein results in a withdrawal from cell cycle before G1-S transition in NIHT3 cells; however, in this study, the extent of cell death and apoptosis was not measured(23). It has been reported previously that endogenous p21 transcription is only partially influenced by ectopic expression of E47 protein (33, 44). Accordingly with these reports, we found that overexpression of E47 protein did not result in a significant increase of the endogenous CDKI mRNAs, as determined by Northern blot analysis (data not shown). We found that, although overexpression of p21 induces a G1 arrest, enforced expression of bHLH proteins promotes cell death associated with apoptosis, rather than a G1 arrest. Our data suggest that, at least in our experimental system, the cell growth inhibition exerted by class A proteins might only partially involve up-regulation of endogenous CDKI.

Our findings showing that enforced expression of bHLH proteins increases cell death associated with induction of apoptosis is fully compatible with two recent reports showing the ability of E2A proteins to induce apoptosis. It has been shown that ectopic expression of E2A promotes the death of E2A-deficient lymphomas because of the activation of a programmed cell death pathway involving a loss of mitochondrial transmembrane potential (31). In an another study,restoration of E2A activity in Jurkat leukemic T cells resulted in an induction of cell death associated with an increase of apoptosis (32). These studies suggested that loss of E2A might represent a critical step in tumor development. Our findings further support the proposed hypothesis that E2A gene products can act as tumor suppressors.

Fig. 1.

Class A bHLH proteins inhibit the proliferation of several human cell lines. The histogram shows the average of four independent experiments for each bHLH protein. Cells were transfected by calcium phosphate with 9 μg of the CMV-based expression vector and 1 μg of pRC-CMV conferring the resistance to G418. After 10–15 days of selection, the plates were stained, and the colonies were scored by visual inspection and counted. The data are representative of several experiments; SD, thin lines at the top of the columns.

Fig. 1.

Class A bHLH proteins inhibit the proliferation of several human cell lines. The histogram shows the average of four independent experiments for each bHLH protein. Cells were transfected by calcium phosphate with 9 μg of the CMV-based expression vector and 1 μg of pRC-CMV conferring the resistance to G418. After 10–15 days of selection, the plates were stained, and the colonies were scored by visual inspection and counted. The data are representative of several experiments; SD, thin lines at the top of the columns.

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Fig. 2.

A, transcriptional activation of CDKI promoters by class A bHLH proteins. The p15, p16, p21, and p57 promoter sequences cloned upstream of the luciferase gene were transfected into 293T cells in the presence of the indicated CMV-based expression vectors for each bHLH protein as indicated. Two μg of reporter and effector plasmid DNA were used together with 0.1 μg of pRL-CMV for normalization of the transfections efficiencies. The values presented were normalized with the internal control as described in the text and are representative of three experiments. SD, thin linesat the top of the columns. B, p15-Luc, p16-Luc, and p21-Luc plasmid reporters (2μg) were transiently cotransfected into 293T cells along with CMV-based expression vectors (2 μg) encoding the E47 and Id4 proteins or with the empty CMV vector, as indicated. The values presented were normalized with the internal control as described in the text and are representative of four independent experiments. SD, thin lines at the top of the columns.

Fig. 2.

A, transcriptional activation of CDKI promoters by class A bHLH proteins. The p15, p16, p21, and p57 promoter sequences cloned upstream of the luciferase gene were transfected into 293T cells in the presence of the indicated CMV-based expression vectors for each bHLH protein as indicated. Two μg of reporter and effector plasmid DNA were used together with 0.1 μg of pRL-CMV for normalization of the transfections efficiencies. The values presented were normalized with the internal control as described in the text and are representative of three experiments. SD, thin linesat the top of the columns. B, p15-Luc, p16-Luc, and p21-Luc plasmid reporters (2μg) were transiently cotransfected into 293T cells along with CMV-based expression vectors (2 μg) encoding the E47 and Id4 proteins or with the empty CMV vector, as indicated. The values presented were normalized with the internal control as described in the text and are representative of four independent experiments. SD, thin lines at the top of the columns.

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Fig. 3.

The E-boxes within the promoter sequences mediate transactivation by the E47 bHLH protein. Left, schematic representation of the parental and mutated promoter constructs for p21(A), p15 (B), and p16 (C). In each case, the luciferase reporters (2 μg) were transiently cotransfected into 293T cells, along with 2 μg of CMV-E47 expression vector (▪) or empty CMV vector (□). The values presented were normalized with the internal control as described in the text and are representative of at least three independent experiments. SD, thin lines at the top of the columns.

Fig. 3.

The E-boxes within the promoter sequences mediate transactivation by the E47 bHLH protein. Left, schematic representation of the parental and mutated promoter constructs for p21(A), p15 (B), and p16 (C). In each case, the luciferase reporters (2 μg) were transiently cotransfected into 293T cells, along with 2 μg of CMV-E47 expression vector (▪) or empty CMV vector (□). The values presented were normalized with the internal control as described in the text and are representative of at least three independent experiments. SD, thin lines at the top of the columns.

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Fig. 4.

Effect of the overexpression of class A bHLH proteins on BrdUrd incorporation and induction of cell death and apoptosis. Subconfluent 293T cells were transfected with the empty CMV vector(column −) or with each of class A bHLH vectors as indicated. In A, cells were pulse-labeled with BrdUrd incorporation as reported in the text. In B, cell death was measured by trypan blue dye exclusion, and in C, the induction of apoptosis was determine by TUNEL assays. D,effect of the apoptosis-regulatory gene Bcl2 on E47- and E12-induced apoptosis. 293T cells were cotransfected with the indicated bHLH expression vectors (2 μg each) in the presence of CMV-empty vector (columns −) or with the Bcl2 vector as indicated. E, appearance of apoptotic cells revealed by TUNEL assay. At least 300–400 cells were evaluated in each experiment,and the data reported are the means of several independent experiments.

Fig. 4.

Effect of the overexpression of class A bHLH proteins on BrdUrd incorporation and induction of cell death and apoptosis. Subconfluent 293T cells were transfected with the empty CMV vector(column −) or with each of class A bHLH vectors as indicated. In A, cells were pulse-labeled with BrdUrd incorporation as reported in the text. In B, cell death was measured by trypan blue dye exclusion, and in C, the induction of apoptosis was determine by TUNEL assays. D,effect of the apoptosis-regulatory gene Bcl2 on E47- and E12-induced apoptosis. 293T cells were cotransfected with the indicated bHLH expression vectors (2 μg each) in the presence of CMV-empty vector (columns −) or with the Bcl2 vector as indicated. E, appearance of apoptotic cells revealed by TUNEL assay. At least 300–400 cells were evaluated in each experiment,and the data reported are the means of several independent experiments.

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1

Supported by grants from Associazione Italiana per la Ricerca sul Cancro, Istituto Superiore Sanitá, and Ministero dell’Universitá e Ricerca Scientifica e Tecnologica(to L. L.). The work performed by A. P. was in partial fulfillment of his doctoral thesis.

3

The abbreviations used are: bHLH, basic helix-loop-helix; CDKI, cyclin-dependent kinase inhibitor; CMV,cytomegalovirus; GFP, green fluorescent protein; BrdUrd,5-bromo-2′-deoxyuridine; TUNEL, terminal deoxynucleotidyltransferase-mediated nick end labeling; FACS,fluorescence-activated cell sorter.

Table 1

FACSort analysis of transiently transfected cellsa

Effector48 h72 h
G1SG2-MSub-2NG1SG2-MSub-2N
CMV 55 ± 2 27 ± 3 15 ± 1 3 ± 1 50 ± 2 32 ± 3 15 ± 2 3 ± 2 
p21 70 ± 2 15 ± 2 10 ± 2 5 ± 1 75 ± 2 10 ± 2 9 ± 2 6 ± 2 
E47 33 ± 2 36 ± 2 21 ± 3 10 ± 3 36 ± 4 38 ± 4 12 ± 3 14 ± 2 
E2-2 41 ± 2 36 ± 3 18 ± 3 5 ± 1 39 ± 2 39 ± 2 12 ± 3 10 ± 2 
E12 32 ± 2 36 ± 4 20 ± 3 12 ± 3 37 ± 2 39 ± 1 10 ± 4 14 ± 3 
HEB 38 ± 1 33 ± 3 15 ± 3 14 ± 3 38 ± 3 42 ± 2 9 ± 2 11 ± 3 
Effector48 h72 h
G1SG2-MSub-2NG1SG2-MSub-2N
CMV 55 ± 2 27 ± 3 15 ± 1 3 ± 1 50 ± 2 32 ± 3 15 ± 2 3 ± 2 
p21 70 ± 2 15 ± 2 10 ± 2 5 ± 1 75 ± 2 10 ± 2 9 ± 2 6 ± 2 
E47 33 ± 2 36 ± 2 21 ± 3 10 ± 3 36 ± 4 38 ± 4 12 ± 3 14 ± 2 
E2-2 41 ± 2 36 ± 3 18 ± 3 5 ± 1 39 ± 2 39 ± 2 12 ± 3 10 ± 2 
E12 32 ± 2 36 ± 4 20 ± 3 12 ± 3 37 ± 2 39 ± 1 10 ± 4 14 ± 3 
HEB 38 ± 1 33 ± 3 15 ± 3 14 ± 3 38 ± 3 42 ± 2 9 ± 2 11 ± 3 
a

The values reported are the means of four independent experiments.

We thank Gordon Peters, Antonio Giordano, and Yoishi Nakamura for the generous gift of reagents and P. P. Pelicci for helpful discussion. We thank S. Ronzoni in helping with the FACS analysis, S. Trapani for help in the TUNEL assay, and R. Terracciano for technical help.

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