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Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Patologia Cellulare e Molecolare, Universita’ degli Studi di Napoli "Federico II," 80131 Naples, Italy [M. F., G. M. P., M. T. B., S. B., M. D., M. S.]; Istituto Nazionale dei Tumori, Fondazione Giovanni Pascale, 80131 Naples, Italy [G. B., G. V.]; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032 [N. M., D. T.]; and Dipartimento di Medicina Sperimentale e Clinica, Universita’ degli Studi di Catanzaro, 88100 Catanzaro, Italy [A. F.]
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ABSTRACT |
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INTRODUCTION |
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Disruption of the HMGA2 gene results in a mouse pygmy phenotype and a decreased growth rate of embryonic fibroblasts (10) , which suggests that protein HMGA2 plays a role in cell proliferation. Indeed, HMGA proteins have been immunolocalized in vivo to the AT-rich G/Q and C bands of mammalian metaphase chromosomes. Therefore, they could be involved in the changes in chromosome structure that occur during the cell cycle. In addition, HMGA proteins are efficient substrates for cdc2 kinase phosphorylation that alters HMGA DNA-binding affinity in a cell cycle-dependent fashion (11, 12, 13) .
HMGA protein concentrations are high during embryogenesis (10 , 14) and low in normal adult tissues. Increased expression of HMGA proteins is associated with the transformation of rat thyroid cells (15, 16, 17) , and HMGA genes play a pivotal role in thyroid cell transformation (18) . In fact, thyroid cells infected by oncogenic retroviruses failed to exhibit phenotypic markers of neoplastic transformation when HMGA protein synthesis was specifically inhibited by antisense constructs. HMGA1 gene overexpression is a constant feature of human malignant neoplasias (19, 20, 21) , and blockage of HMGA1 expression in human thyroid anaplastic carcinoma cell lines results in apoptotic cell death (22) .
Rearrangements of the HMGA2 gene are frequent in human benign tumors of mesenchymal origin. They consist in the loss of the COOH-terminal tail and the fusion with ectopic sequences. Alterations of the HMGA1 gene also occur in several benign tumors, but they often result in gene overexpression rather than truncation (23, 24, 25, 26) . In fact, we recently described a case of thyroid microfollicular adenoma with an extragenic translocation in the HMGA1 locus that caused overexpression of the gene (27) .
Because HMGA proteins are necessary for the induction of the transformed phenotype in rat thyroid cells (18) , we investigated whether overexpression of the HMGA1b protein is sufficient to induce cellular transformation. To this purpose, we transfected the PC Cl 3 cell line (28) , a normal rat thyroid cell line expressing all of the typical markers of thyroid differentiation, with the HMGA1b cDNA under the transcriptional control of the cytomegalovirus promoter. Although the transfected cells entered the S phase earlier than normal cells, they stopped replicating at G2-M phase and underwent programmed cell death. The third AT-hook domain and the acetylation site K60 are required for HMGA1b-induced apoptosis. The HMGA1a isoform exerts similar biological effects. Taken together, these data demonstrate that HMGA1b alone does not transform normal rat thyroid cells and implicate HMGA1 proteins in the regulation of cell cycle and apoptosis.
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MATERIALS AND METHODS |
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A); pHA-YK60 contains the entire coding sequence (1–96) with a mutation on the second acetylation domain (K60E); pHA-YK54,K60 contains the entire coding sequence (aa 1–96) with mutations on both acetylation sites (K54A, K60E). pHA-I (aa 1–107) contains the entire coding sequence of the HMGA1a isoform.
Cell Culture and Transfection Experiments.
PC Cl 3 cells are described elsewhere (28)
. They were grown in Ham’s F-12 medium, Coon’s modification (Sigma Chemical Co.) supplemented with 5% calf serum (Life Technologies, Inc., Paisley, PA) in the presence of a mix containing six growth factors (6H: 10 nM thyrotropic hormone, 10 nM hydrocortisone, 100 nM insulin, 5 µg/ml transferrin, 5 nM somatostatin, and 20 µg/ml glycyl-histidyl-lysine). The calcium phosphate procedure was used for transfections (30)
. PC Cl 3 cells were transfected with the above-described plasmids or with the backbone vector. Stable transfectants PC/Y were selected for neomycin resistance in 400 µg/ml G418.
RNA Isolation and Northern Blot Analysis.
Total RNA was extracted from the G418-selected cell clones with the RNAfast Isolation System (Molecular System, San Diego, CA). Northern blot hybridization was performed as described previously (31)
using Hybond-N membranes (Amersham Pharmacia BioTech Italia) according to the manufacturer’s instructions. All cDNA probes were radiolabeled with a random prime synthesis kit (Amersham Pharmacia BioTech Italia). Hybridization reactions were performed at 42°C with 2 x 106 cpm/ml of hybridization solution. After washes, filters were air-dried and exposed to autoradiographic film for 1–7 days. The entire HMGA1b cDNA was used as a molecular probe for the HMGA1 gene. A mouse glyceraldehyde-3-phosphate dehydrogenase cDNA probe was used to equalize RNA loading.
Protein Extraction, Western Blotting, and Antibodies.
Cells were washed once in cold PBS and lysed in a buffer containing 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.5), 1% (v/v) Triton X-100; 50 mM NaCl; 5 mM EGTA, 50 mM NaF; 20 mM Na PPi; 1 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride, and 0.2 mg/ml each of aprotinin and leupeptin. Lysates were clarified by centrifugation at 10,000 x g for 15 min, and the supernatant was stored at -70°C. Protein concentration was estimated by a modified Bradford assay (Bio-Rad). Total proteins were separated by SDS-PAGE and transferred to membranes (Immobilon-P Millipore Corp., Bedford, MA). Membranes were blocked with 5% nonfat milk proteins and incubated with the appropriate antibodies. Anti-HMGA1 are polyclonal antibodies raised against a synthetic peptide located in the NH2-terminal region of the HMGA1 proteins (32)
at a dilution of 1:1,000; antibodies against various cyclins, CDK, and CDKIs were from Santa Cruz Biotechnology (anti-cyclin D3 C-16, anti-CDK7, C-19, and anti-cyclin B1 GNS1), Oncogene Science (anti-cyclin A AB-2), PharMingen (anti-cyclin E HE12, anti-CDK2, and anti-CDK4), and Calbiochem (anti-cdc2 Ab-1). Anti 
-tubulin (Santa Cruz Biotechnology) was used to equalize the amount of proteins loaded. Bound antibodies were detected by the appropriate secondary antibodies and revealed with the Amersham enhanced chemiluminescence system.
Growth Rate and [3H]thymidine Incorporation.
The cells (105/dish) were plated in 60-mm plates and cultured in complete medium. They were counted every day for 10 consecutive days to extrapolate the growth curves. The doubling time was measured when the cells were in the logarithmic phase of growth.
To evaluate [3H]thymidine incorporation, the cells were plated as above and allowed to adhere for 12 h in serum-containing medium. They were then washed three times in PBS and left for 3 days in serum-free medium. After 3 days of starvation, the cells were allowed to grow in serum-containing medium and harvested after 0, 12, 24, 48, and 72 h of incubation with pulses of 4 µCi/ml of [3H]thymidine (Amersham Pharmacia BioTech Italia) added to each plate 3 h before each harvesting. The plates were then washed three times with PBS, and the cells were fixed in 5% trichloroacetic acid at 4°C for 20 min, washed three times in trichloroacetic acid and solubilized in 0.5 N NaOH (0.3 ml). This solution was neutralized with 0.5 N HCl (0.3 ml), and radioactivity was measured by liquid scintillation counting. Two plates for each time point were used to analyze growth rate and [3H]thymidine incorporation, and the experiments were performed in triplicate.
Flow-Cytometry.
PC Cl 3 and PC/Y cells were analyzed for DNA content as described previously (33)
. Cells were collected and washed in PBS. DNA was stained with propidium iodide (50 µg/ml) and analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) that was interfaced with a Hewlett-Packard computer (Palo Alto, CA). Cell cycle data were analyzed with the CELL-FIT program (Becton Dickinson).
Immunoprecipitation Analysis and Kinase Assay.
PC and PC/Y cells, starved or treated for 12, 24, and 36 h with complete medium, were lysed in NP40 lysis buffer [50 mM HEPES (pH 7); 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.5 mM Na3VO4, and 1 mM DTT supplemented with Na4P2O7, aprotinin, leupeptin, pepstatin, and benzamidine]. Four hundred µg of extract were incubated with 1 µg of anti-cdc2 Ab-1 (Calbiochem) rabbit polyclonal antiserum or with 1 µg of anti-Cyclin B1 GNS1 (Santa Cruz Biotechnology) mouse monoclonal antibodies for 1 h at 4°C. A/G Sepharose protein (Santa Cruz Biotechnology) was then added, and lysates were incubated for 15 h at 4°C. Immunocomplexes were collected by centrifugation at 10,000 rpm for 1 min at 4°C and washed four times in NP40 lysis buffer. After two additional washes in 10 mM Tris-HCl (pH 6.8), 20 mM MgCl2, one-tenth of the immunoprecipitates was used for kinase assay and nine-tenths were resolved on polyacrylamide gels and probed with anti-cyclin B or anti-cdc-2 antibodies. For in vitro kinase assay, immunoprecipitates were resuspended in cdc-2 kinase assay buffer (Upstate Biotechnology) for 20 min in the presence of 2 µg of histone H1 (Upstate Biotechnology) as substrate and 0.5 µCi of [-32P] ATP (3,000 Ci/mmol; Amersham Pharmacia Biotech) in a final volume of 25 µl. Samples were incubated for 20 min at 30°C, and the reaction was stopped by adding 25 µl of 2 x Laemmli loading dye. Phosphorylated histone H1 was visualized by autoradiography and quantified with the GS-525 Molecular Imager (Bio-Rad) interfaced with an Hewlett-Packard computer.
Cell Viability, DNA Laddering, and TUNEL Assay.
To assess cell viability, nonadherent and adherent cells were collected, and aliquots were mixed with an equal volume of 0.4% trypan blue (Life Technologies, Inc.). The percentage of cells that picked up the dye (dead cells) was determined. The data are representative of at least three independent culture dishes per time point.
For the DNA laddering assay, adherent and nonadherent cells from a 100-mm Petri dish were collected, washed in PBS, and resuspended gently in 0.4 ml of lysis buffer [0.5% Triton X-100, 5 mM Tris-HCl (pH 7.4), 20 mM EDTA]. After a 20-min incubation on ice, the samples were centrifuged at 12,000 rpm for 30 min. The supernatants were extracted with phenol-chloroform and precipitated in ethanol. The pellets were washed in 70% ethanol and resuspended in TE buffer [10 mM Tris (pH 8)-1 mM EDTA] containing 0.4 µg/µl RNaseA. Extracted nucleic acids were treated with RNaseA at 37°C for 30 min and then analyzed on 1.2% agarose gels stained with ethidium bromide.
The TUNEL assay was performed with the In Situ Cell Death Detection Kit (Boehringer, Mannheim) following the manufacturer’s instructions. Briefly, the air-dried cells were fixed with a freshly prepared paraformaldehyde solution [4% in PBS (pH 7.4)] for 30 min at room temperature. The slides were rinsed with PBS and incubated in permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) for 2 min on ice. Then the slides were rinsed twice with PBS, and 50 µl of TUNEL reaction mixture was added. After 30 min incubation at 37°C, substrate solution was added. After 10 min incubation at room temperature the slides were mounted and analyzed under the light microscope.
Inhibition of Cell Death.
Cells (4 x 105 ) were plated in six multiwells and allowed to grow for 4 days before a treatment with 10 and 100 µM of z-VAD-fmk, z-DEVD-cho, Ac-YVAD-cho, and z-FA-cho (Calbiochem) for 72 h. Death was assessed by measuring the percentage of stained cells with trypan blue as described above.
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RESULTS |
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for the expression of the transfected gene. Northern blot analysis showed that PC/Y cells express a specific HMGA1 transcript (Fig. 1A)
. Conversely, no HMGA1 expression was found in the untransfected (PC Cl 3) or in the backbone vector-transfected PC Cl 3 cells (PC/V; panel A). The PC Cl 3 cells transformed with the myeloproliferative sarcoma virus (PC MPSV; 28
) expressed the endogenous HMGA1-specific transcript of about 1.8 kb. Western blot analysis with anti-HMGA1 antibody (32)
showed an immunoreactive protein in the PC/Y and PC MPSV cells, but not in the parental PC Cl 3 or PC/V cells (Fig. 1B)
. We selected six PC/Y and two PC/V cell clones for additional analyses. All of the clones showed the same biological behavior. Therefore, the data shown here refer to one or two representative clones.
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. They retained a differentiated phenotype and were not tumorigenic (data not shown).
Growth of PC/Y Cells.
The growth potential of the PC/Y cells was evaluated from growth rate and [3H]thymidine incorporation. As shown in Fig. 2A
, the growth rate of PC/Y cells was reduced compared with PC/V cells. In fact, the doubling time of PC/Y cells (25 ± 3h) was significantly higher than that of PC/V cells (18 ± 3h), suggesting that HMGA1b may inhibit thyroid cell growth. These differences were significant (P < 0.01).
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. However, the rate of thymidine incorporation of PC/Y cells remained stable, and started to decrease at 48 h. Conversely, the rate of [3H]thymidine incorporation of PC/V cells continued to increase steadily (Fig. 2B)
. These results demonstrate that PC/Y cells enter the cell cycle from serum deprivation-induced G0 more rapidly than parental PC Cl 3 cells. However, the finding that, at later times, the [3H]thymidine incorporation rate of PC/Y cells is not maintained at levels comparable with those observed in PC/V cells and that the growth rate of PC/Y cells is lower versus parental cells, suggests that the constitutive expression of HMGA1b may interfere with the correct completion of the cell cycle, at least in thyroid cells.
Cell Cycle Profile of PC/Y Cells.
To verify the results of the [3H]thymidine incorporation assay, the cell cycle distribution of PC Cl 3, PC/V, and PC/Y cells was analyzed. When PC/V cells are starved of serum and hormones for 3 days, they become quiescent (G0), and can be stimulated to enter the S-phase by 24 h of serum and 6H administration. To determine whether PC/Y cells had a reduced G1 phase and synthesized DNA earlier than PC/V cells, PC/V and PC/Y cells were starved as described, stimulated to reenter the cycle with serum and 6H, and analyzed by flow cytometry at 0, 12, 24, and 36 h after treatment. The percentage of PC/Y cells in the S-phase compartment increased significantly 12 h after treatment, reaching a peak at 24 h (Fig. 3B)
. Conversely, most PC Cl 3 and PC/V cells were still in G0/G1 phase 12 h after treatment (Fig. 3A)
but entered S-phase 24 h after treatment (Fig. 3B)
. Furthermore, as shown in Fig. 3C
, the fraction of PC/Y cells in the G2-M compartment 24 h posttreatment was increased by about 2-fold with respect to PC/V cells (22.9% versus 12.3%, respectively). The difference between PC/V and PC/Y cells in the G2-M compartment was reduced 36 h after treatment. Overall, there were two types of cell cycle profile alterations in PC/Y cells compared with PC/V cells: early entry into S-phase and accumulation of cells in G2-M phase after 24–36 h of hormone and serum treatment.
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To investigate the effects exerted by HMG-Y on PC Cl 3 cells, we determined the expression of cyclins, CDK, and CDK-inhibitor proteins in PC/V and PC/Y cells. Cells were starved of 6H and serum as described, stimulated to reenter the cycle after 3 days, and then lysed at 6, 12, 18, 24, and 36 h. Subsequently, the expression of cyclins, CDK and CDK inhibitor proteins was detected by Western blot. Cyclin D3 was the only D-type cyclin detected in PC Cl 3 cells. Cyclin D3 was not expressed in starved PC/V cells, but it was induced after 6 h of stimulation, with a peak at 18–24 h (Fig. 4)
. Conversely, cyclin D3 expression was elevated in PC/Y cells even when the cells were deprived of growth factors and hormones. Furthermore, cyclin D3 induction in PC/Y cells occurred earlier and peaked at 12 h of treatment. Similarly, the basal level of cyclin E protein was increased in starved PC/Y cells and increased even more after 6 h of treatment, i.e., 12 h earlier than in PC/V cells. This pattern of G1 cyclin expression coincides with the finding that PC/Y cells enter the cell cycle 12 h earlier than do PC/V cells. CDK2 expression was increased after 18–24 h of treatment in PC/V cells and after 12 h in PC/Y cells. Conversely, CDK4/6 and CDK7 expression was not altered in quiescent or proliferating PC/V or PC/Y cells. These findings coincide with the observation that PC/Y cells failed to arrest growth completely in G1 upon starvation, and that they enter S-phase 6–12 h earlier than do PC/V cells.
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cdc2 Activity in PC/Y Cells.
A major difference between PC/V and PC/Y cells occurred in the expression of the G2-M associated proteins, cdc-2 and cyclin B. Because activation of cyclin B/cdc-2 complexes regulates G2-M transition, and its inactivation is a prerequisite for correct completion of the M phase, we compared the activity of cyclin B/cdc-2 complexes in PC/Y cells versus PC/V cells. To this aim, PC/V and PC/Y cells were starved for 72 h and then cultured with growth factors and serum for 24 or 36 h. The lysates obtained were analyzed for cyclin B- and cdc-2-associated kinase activity. cdc-2 activity was very low in starved PC/V cells, but was increased 24 h after treatment (Fig. 5
, top, Lanes 1–3). This is consistent with the FACScan result that PC/V cells start to enter G2-M at 24 h. Differently, the activity of cyclin B/cdc-2 complexes appeared in PC/Y cells at 24 h (like in the PC/V cells), but was increased further at 36 h (Fig. 5
, top, Lanes 4–6). Similar results emerged from the analysis of cyclin B-associated activity (Fig. 5
, bottom). These data suggest that in PC Cl 3 cells, HMGI-Y protein may interfere with the activity of cyclin B/cdc-2 complexes.
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. To investigate the mechanisms of PC/Y cell death, we performed three apoptotic assays: DNA laddering, flow-cytometric analysis, and TUNEL assay. As shown in Fig. 6B
, the PC/Y cells underwent massive DNA fragmentation after 7 days in culture. Conversely, no DNA laddering was observed in PC/V cells.
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. Similarly, PC/Y but not PC/V cells stained positive at the TUNEL assay (data not shown). These results demonstrate that PC/Y cells undergo apoptosis.
Mechanisms Underlying HMGA1b-induced Apoptosis.
Initially, to gain insights into the signaling pathway implicated in HMGA1b-mediated apoptosis, we monitored activation of caspase-3, an interleukin 1ß converting enzyme-like cysteine protease that is central to many apoptosis systems (38, 39, 40, 41)
. Total cell extracts were blotted with an antibody raised against PARP, a substrate for active caspase-3 (39
, 41) , which recognizes the full-length Mr 112,000 PARP and the Mr 85,000 cleaved product observed after apoptotic stimuli (42)
. The cleavage of PARP in PC/Y cells (Fig. 7A)
suggests that the caspase-3 pathway is involved in induction of HMGA1b-dependent apoptosis. In contrast, no PARP cleavage was observed in PC/V cells.
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, z-DEVD-cho prevented HMGA1b-induced apoptosis in a dose-dependent manner, a weak preventive effect was obtained with z-VAD-fmk, and there was no effect with either Ac-YVAD-cho or z-FA-cho. These results indicate that the apoptotic effect associated with the overexpression of the HMGA1b protein in PC Cl 3 cells is caspase-dependent and is mediated by caspase-3. Subsequently, we analyzed the expression of several genes known to be involved in apoptosis. No differences in Bcl-2, Bax, Bcl-X, and c-myc gene expression were observed between PC/V and PC/Y cells (data not shown).
Mapping of the HMGA1b Region Responsible for Induction of Apoptosis.
To map the HMGA1b regions required for the induction of apoptotic cell death in PC Cl 3 cells, we used a series of COOH-terminal deletions of the gene (29
, 45)
. These HMGA1b derivatives were transiently transfected on the PC Cl 3 cells, and an apoptotic assay (TUNEL assay) was performed 3 days after. Fig. 8
shows that deletion of the COOH-terminal tail (pHA-Y/T) did not significantly impair HMGA1b-induced apoptosis in PC Cl 3 cells. Conversely, deletion of the last AT-hook domain (pHA-Y1–63) and of the last AT-hook domain plus the region between the middle and the last basic domain (pHA-Y1–53), and also replacement of the region spanning aa 54–63 with an oligonucleotide coding for different aa (pHA-YmutA), drastically reduced the apoptotic effect. This region carries two acetylation sites: lysine 54, which is preferentially acetylated by CBP, and lysine 60, which is preferentially acetylated by P/CAF (29)
. To determine whether the acetylation sites are important in the apoptotic process, we examined the apoptotic ability of constructs with specific mutations in lysines 54 and 60. The single mutant K54A (pHA-YK54) did not impair apoptosis. Conversely, both the single mutant K60E (pHA-YK60) and the double mutant K54A-K60E (pHA-YK54,K60) inhibited the apoptotic effect. These findings suggest that the third AT-hook domain and/or the P/CAF mediated acetylation of the HMGA1b protein are involved in the induction of apoptosis in PC Cl 3 cells. Finally, we transiently overexpressed in PC Cl 3 the long HMGA1a isoform, which has an extra stretch of 11 aa between the first and second AT-hook domains. As shown in Fig. 8A
, HMGA1a exerts similar effects on the apoptotic phenotype, suggesting that the mechanisms underlying cell cycle deregulation and apoptosis are the same for both HMGA1 isoforms. A Western blot showing the protein levels of the various transfected genes and mutants is represented in Fig. 8B
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DISCUSSION |
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To assess whether the HMGA1 gene exerts a transforming effect, we transfected the HMGA1b cDNA under the transcriptional control of the cytomegalovirus promoter in normal rat thyroid cells. Surprisingly, HMGA1b overexpression did not induce thyroid cell transformation; instead, it deregulated cell growth and induced apoptosis. [3H]thymidine incorporation and flow-cytometric analysis demonstrated that HMGA1b-transfected cells (PC/Y) enter the S-phase compartment significantly earlier than untransfected cells, but they have an abnormally extended G2-M phase. Expression analysis of cyclins and CDK confirmed these data: the cyclins and CDK involved in the G1-S transition were expressed earlier, and the expression of the cyclinB1/cdc2 complex involved in exit from G2-M was more prolonged in PC/Y cells than in vector-transfected cells (PC/V). Moreover, cdc-2- and cyclin B-associated kinase activities peaked 12 h later in PC/Y cells compared with PC/V cells. Because inactivation of cyclin B/cdc-2 is necessary for completion of the M phase, these results suggest that PC/Y cells exit mitosis later than parental PC Cl 3 cells.
Despite the early entry into S-phase, but in agreement with the prolonged G2-M phase, PC/Y cells grow more slowly than either PC/V or PC Cl3 cells. We found that apoptotic cell death, assessed by three techniques, largely accounts for the reduced cell growth rate of PC/Y cells. In addition, we demonstrate that HMGA1b-induced apoptosis is associated with PARP cleavage and inhibited by the cysteine protease inhibitor z-DEVD-cho in a dose-dependent manner, but not inhibited or partially inhibited by Ac-YVAD-cho and z-VAD-fmk, respectively. The caspase inhibitor z-VAD-fmk does not inhibit caspase 9, whereas it is very effective in inhibiting caspase 3 activity (44) . Conversely, the z-YVAD-cho inhibitor is virtually specific in suppressing caspase 1 activity. Consequently, it appears that the caspase 3 rather than the caspase 1 pathway is involved in the programmed cell death caused by HMGA1b gene overexpression.
Using deletion mutants, we mapped the regions required for the apoptotic effect in the third DNA-binding domain and in a region spanning from aa 54 to aa 63. The latest region carries two acetylation sites: lysine 54, which is preferentially acetylated by CBP, and lysine 60, which is preferentially acetylated by P/CAF (29) . Lysine 60 is involved in the induction of apoptosis because mutation of this lysine impaired HMGA1b-induced apoptosis. In the context of the human ß-IFN enhanceosome, acetylation of HMGA1 by CBP, but not by P/CAF, leads to enhanceosome destabilization and disassembly, turning off IFNß gene expression (29) . It is intriguing to speculate that acetylation of HMGA1b may modulate the protein’s apoptotic potential, because acetylation of HMGA1b lysine 60 by P/CAF, similar to acetylation of HMGA1b lysine 54 by CBP on the ß-IFN promoter, might affect its DNA-binding activity on other promoters or its association with other transcriptional factors involved in apoptosis. Moreover, we show that the HMGA1a isoform has a similar apoptotic effect, suggesting that it plays the same role as HMGA1b in cell cycle deregulation and apoptosis in normal rat thyroid cells.
We show that HMGA1 overexpression exerts an opposite effect on the growth of neoplastic versus normal cells: it is required for neoplastic transformation, whereas it induces G2-M delay and apoptosis in normal cells. This suggests that the cell context plays an important role in determining whether HMGA overexpression causes cell proliferation or cell death. In the presence of such genetic alterations as active oncogene expression or inactivation of tumor suppressor genes, HMGA1b overexpression may protect neoplastic cells from apoptosis while exerting the opposite effect on normal cells. Similar results are reported for other genes, e.g., myc (48) . In serum-deprived cells, myc overexpression induces apoptosis, but myc-expressing p53-/-, casp9-/-, and Apaf-1-/- cells were resistant to apoptosis after growth factor depletion (44) .
Apoptosis is thought to be a default pathway in cells receiving conflicting cell proliferation signals (9 , 48) . Within this framework, we speculate that the proliferative input of HMGA1b overexpression, which allows cells to enter S-phase rapidly, and growth inhibitory signals from normal cells, which prevent completion of the G2-M phase, can cause apoptosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Associazione Italiana Ricerca sul Cancro, Consiglio Nazionale delle Ricerche, Ministero dell’Università e Ricerca Scientifica e Tecnologica, and fellowships from Federazione Italiana Ricerca Cancro (to G. M. P. and G. B.). 
2 To whom requests for reprints should be addressed, at Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita’ degli Studi di Napoli "Federico II," via Pansini 5, Italy. Phone: 39-081-7463056; Fax: 39-081-7463037 or 39-081-7701016; E-mail: afusco@napoli.com or alfusco{at}unina.it
3 The abbreviations used are: HMG, high mobility group; HA, hemagglutinin; aa, amino acid; 6H, six hormones; z-VAD-fmk, carbozenzoxy-Val-Ala-Asp-fluoromethyl ketone; Ac-YVAD-cho, carbobenzoxy-Tyr-Val-Ala-Asp-7-amino-4-trifluoromethylcoumarin; z-DEVD-cho, carbobenzoxy-Asp-Glu-Val-Asp-fluoromethyl ketone; z-FA-cho, carbobenzoxy-Phe-Ala-fluoromethyl ketone; CDK, cyclin-dependent kinase; PARP, poly(ADP-ribose) polymerase; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.
Received 7/10/00. Accepted 3/26/01.
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| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
