Role of the high mobility group A* proteins in human lipomas

Monica Fedele1, Sabrina Battista1, Guidalberto Manfioletti2, Carlo Maria Croce3, Vincenzo Giancotti2 and Alfredo Fusco1,4,5

1 Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli, Via Pansini, 5, I-80131 Naples, Italy, Istituto Nazionale dei Tumori Fondazione Senatore Pascale, via M. Semmola, I-80131 Naples, Italy,
2 Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, University of Trieste, Italy,
3 Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PE 19107, USA and
4 Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia di Catanzaro, Università degli Studi di Catanzaro, via Tommaso Campanella 5, I-88100 Catanzaro, Italy


    Abstract
 Top
 Abstract
 Introduction
 HMGA genes in human...
 Conclusions
 References
 
The HMGA family is comprised of four proteins: HMGA1a, HMGA1b, HMGA1c and HMGA2. The first three proteins are products of the same gene, HMGA1, generated through an alternative splicing mechanism. The HMGA proteins are involved in the regulation of chromatin structure and HMGA DNA-binding sites have been identified in functional regions of many gene promoters. Rearrangements of the HMGA2 gene have been frequently detected in human benign tumors of mesenchymal origin including lipomas. 12q13-15 chromosomal translocations involving the HMGA2 gene locus, account for these rearrangements. The HMGA proteins have three AT-hook domains and an acidic C-terminal tail. The HMGA2 modifications consist in the loss of the C-terminal tail and fusion with ectopic sequences. A pivotal role of the HMGA2 rearrangements in the process of lipomagenesis is suggested by experiments showing that transgenic mice carrying a truncated HMGA2 gene showed a giant phenotype together with abdominal/pelvic lipomatosis. As HMGA2 null mice showed a great reduction in fat tissue, a positive role of the HMGA2 gene in adipocytic cell proliferation is proposed. More recently, similar alterations of the HMGA1 gene have been described. As the block of the HMGA1 protein synthesis induces an increase in growth rate of the pre-adipocytic cell line 3T3-L1, we suggest a negative role of the HMGA1 proteins in adipocytic cell growth and, therefore, we propose that adipocytic cell growth derives from the balance of the HMGA1 and HMGA2 protein functions.


    Introduction
 Top
 Abstract
 Introduction
 HMGA genes in human...
 Conclusions
 References
 
HMGA protein family**
HMGA1/Hmga1 and HMGA2/Hmga2 are two genes (human/murine, respectively) that encode four proteins named HMGA1a, HMGA1b, HMGA1c and HMGA2, being the first three proteins spliced forms of the HMGA1/Hmga1 gene.

The HMGA1a, HMGA1b and HMGA2 proteins (previously HMGI, HMGY and HMGI-C, respectively) are composed of 107, 96 and 108 amino acid residues, respectively (13). Each protein contains three basic domains, named AT-hooks and an acidic C-terminal region. The HMGA1a protein differs from HMGA1b in that it has an additional insertion of 11 amino acid residues between the first and the second AT-hook domains. The structure of HMGA2 protein is very similar to that of HMGA1a; however, the first 25 amino acid residues are totally different. Moreover, in HMGA2 there is a short peptide of 12 amino acid residues between the third AT-hook and the C-terminal acidic tail. These proteins are very well conserved during evolution, and only a few differences can be detected between the human and the murine HMGA sequences (410). A new HMGA1 isoform has been recently isolated and found to be a variant of HMGA1a. It has been designated HMGA1c and has a deletion of 67 nucleotides compared with the HMGA1 sequence. This deletion results in a frameshift so that the two proteins are identical in their first 65 amino acids and differ thereafter. HMGA1c encodes a protein of 172 amino acids and has a molecular weight of 26–27 kDa (11). Little is known about this form that, however, appears to be the only isoform present in normal human and mouse testis (Chieffi et al., manuscript in preparation).

The three AT-hook domains are responsible for binding to AT-rich sequences inside the minor groove of DNA (12,13) and are characterized by the amino acidic sequence BBXRGRPBB where B is a basic amino acid residue (K or R) and X is a glycine or proline residue (14). Structural analysis of the protein–oligonucleotides complex has shown that the conserved sequence RGR is important for this interaction. Although each AT-hook binds with a low specificity to DNA, a high affinity has been described when two or three AT-hook domains bind simultaneously to a single DNA molecule. The C-terminal region (15 amino acid residues) is highly acidic and contains several hydroxylic amino acids that are phosphorylated by casein kinase II. This region may be important in protein–protein interaction and in the negative regulation of cell growth, as it is lost in human benign tumors.

The genomic structure of the HMGA genes
The Hmga2 gene (see Figure 1Go) consists of five exons and spans for >200 kb with a large intronic sequence (>100 kb) separating the third from the fourth exon. The Hmga2 transcripts, among which the major one is of 4.1 kb, originate from multiple transcription initiation sites in the 5' flanking region and have a long 3' untranslated region. The first exon encodes the first 37 amino acid residues which includes the first AT-hook domain. The second and third AT-hook domains are encoded by the second and third exon, respectively. The fourth exon encodes the 12 amino acid residues that separate the third AT-hook domain from the C-terminal acidic domain, which is encoded by the last exon (7,15).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1. Schematic representation of the HMGA genomic organization. White and shaded boxes indicate non-coding and coding sequences, respectively.

 
The HMGA1 gene has the same exon–intron organization except it lacks the region corresponding to the fourth exon of HMGA2. The HMGA1 gene spans 10 kb; it is processed in a mRNA of 1.8 kb that encodes three different isoforms through an alternative splicing. At the 3' end of the HMGA1-specific mRNA there is a long untranslated region of 1.3 kb. The HMGA1 gene has eight exons and a number of different transcription start sites (5,16).

Post-translational modifications of the HMGA proteins
Phosphorylation.
In rapidly growing mammalian cells, the HMGA1 proteins are among the most extensively phosphorylated chromosomal proteins (17). They are substrates of various protein kinases, including protein kinase C (PKC), cdc2 and casein kinase II. Two major (Ser43 and Ser63) and four minor PKC phosphorylation sites have been identified (18,19). These sites are distinct from those phosphorylated by cdc2 kinase, which phosphorylates Thr52 and Thr77 (20,21), and by casein kinase II, which phosphorylates two or three serine residues at the C-terminus of the protein (22,23). The two cdc2 kinase phosphorylation sites are adjacent to the N-terminus of two of the three AT-hooks. Moreover, one of the major PKC phosphorylation sites, Ser63, is adjacent to the C-terminus of the second AT-hook, whereas Ser43 is located within the region spanning between the first and the second AT-hook. Phosphorylation of HMGA1a by both PKC or cdc-2 kinases resulted in a strong reduction of the DNA-binding affinity. These phosphorylations occur in a cell cycle-dependent manner, at the beginning of S phase and during the G2/M transition, suggesting that phosphorylation of HMGA1a plays a critical role in cell cycle progression. Very recently, a link between apoptosis induced in leukaemic cells and the degree of phosphorylation of HMGA1a protein has been described. At the early stages of the apoptotic process, the HMGA1a protein is hyper-phosphorylated. Subsequently, when the apoptotic bodies are formed, the HMGA1 protein becomes almost completely de-phosphorylated (23).

Acetylation.
In addition to undergoing phosphorylation, HMGA1a and HMGA1b proteins are also regulated by acetylation. The transcriptional coactivators CBP/p300 (CREB-binding protein) and P/CAF (CBP-associated cofactor) acetylate HMGA1a at distinct lysine residues, causing distinct effects on transcription (24). Specifically, CBP preferentially acetylates Lys65 whereas P/CAF preferentially acetylates Lys71. In the context of the human ß-interferon gene expression, acetylation of HMGA1a by both CBP and P/CAF is required for the enhanceosome activation, whereas only CBP acetyltransferase activity is required for enhanceosome destabilization and post-induction turn-off (24).

Other modifications.
Some other post-translational modifications have also been reported for HMGA proteins (25,26). In fact, both ADP-ribosylation and methylation have been reported, but a clear relationship between these modifications and biological events, in which they are involved, has not been reported yet.

HMGA1 proteins in the transcriptional complexes and chromatin
HMGA proteins are involved in the regulation of chromatin structure and function (18,25,27,39). HMGA1a DNA-binding sites have been identified in functional regions of many gene promoters (2830,32,33,3538,40,41). This protein has been demonstrated to bind to other transcription factors (2841), such as NF-{kappa}B, Elf-1 and Tst-1/Oct-6, through protein– protein interaction in highly conserved regions, i.e. Ets domain of Elf-1 and POU domain of Tst-1/Oct-6. As the HMGA1 proteins form complexes on specific promoters that interact with the basal transcription machinery, they are considered able to induce transcription.

One of the best characterized examples of synergistic interactions between transcription factors is provided by the virus-inducible enhancer of the interferon-ß (IFN-ß) gene (3540). Transcripts of IFN-ß mRNA are not detected in uninfected cells, but after virus infection the gene is activated to very high levels and then undergoes a rapid post-induction turn-off. Detailed analysis of the IFN-ß gene upstream sequence has revealed a highly compact and complex organization of cis-acting regulatory elements (PRDI through PRDIV). PRDII, PRDIV and PRDIII are recognized by NF-{kappa}B, ATF-2/c-Jun heterodimer, and several members of the IRF family, respectively. HMGA1a plays a key role in the activation of this gene by functioning as the essential architectural component for the assembly and stability of the IFN-ß gene enhanceosome (3638). Binding of HMGA1a to the enhancer alters the structure of the DNA, allowing cooperative recruitment of the IFN-ß gene activators that, together with HMGA1a, assemble into a remarkably stable higher order nucleoprotein complex termed the IFN-ß enhanceosome (37,39,40). Thus, the assembly and function of the IFN-ß enhanceosome requires a complex network of protein–DNA and protein–protein interactions orchestrated by the HMGA1a protein.

Involvement of HMGA1a protein has been reported not only at the promoter region of specific genes, but also at more global nuclear structures related to higher order chromatin bound to the nuclear matrix and forming distinct nucleoproteic loops (4245). Such structures have been called MARs (Matrix Attachment Regions) or SARs (Scaffold Attached Regions) and it has been shown that they contain, together with specialized AT-rich DNA sequences with high unwinding aptitude called BURs (Base-unpairing Regions), a variety of proteins including histone H1, topoisomerase II, SAF-A and SAF-B (Scaffold Attachment Factor A and B), lamin B1, p53, SATB1, nucleolin, p114 and HMGA1a/b proteins (4449). HMGA1a/b proteins specifically bind to BURs (48), although binding should regard DNA regions different from those to which histone H1 is bound. In fact, it has been reported that HMGA1a displaces histone H1 from chromatin and nuclease sensitive chromatin releases HMGA1a, HMGA1b and HMGA2 proteins but not histone H1 (49). At the same time, in an immunocytochemical study it has been demonstrated that topoisomerase II and HMGA1a/b proteins co-localize in the interphase nucleus of HeLa cells (50).

The mutually exclusive localization of histone H1 and HMGA1a protein could account for a different involvement of these two factors in the processes of chromatin condensation–de-condensation which are in turn related to the phosphorylation of these proteins, both well known substrates for cyclin-dependent kinases p34-Cdc2 and Cdk2 (20,21,51).

HMGA1a protein could occupy several distinct subnuclear positions roughly grouped into two categories: specific micro-regions, in which HMGA1a acts as `architectural transcription factor and is involved in both positive and negative gene regulation, and macro-regions, in which HMGA1a is a structural special component of the chromatin of cancer cells as suggested (48), being possible that micro-regions are included in macro-regions. The understanding, by ultrastructural techniques, of the identity of the nuclear domains in which HMGA1a protein localizes and the identification by immunoprecipitation of its proteic partners could constitute an advancement of the simple use of the expression of HMGA1a protein as immunohistochemical marker of neoplastic transformation.

Expression of the HMGA proteins in normal and neoplastic tissues
The HMGA2 gene is not expressed in any of the several adult mouse (3) and human tissues tested (52). A very low expression has been also observed in CD34 positive hematopoietic stem cells (53), and recently in mouse pre-adipocytic proliferating cells (54). The HMGA1 gene is expressed at low levels in adult murine and human tissues: a higher expression was observed in testis, skeletal muscle and thymus. Conversely, both the genes are widely expressed during embryogenesis (55,56).

Hmga1 and Hmga2 over-expression was first described in rat thyroid transformed cells and in experimental thyroid tumors (5759). Over-expression of the HMGA proteins was then found to be a common feature of experimental and human malignant neoplasias, including thyroid (6062), prostate (63), uterus (64), breast (Chiappetta et al., manuscript in preparation), colorectum (6567), ovary (Masciullo,V., personal communication) and pancreas (68) carcinomas. Moreover, the expression level of the HMGA proteins is significantly correlated with parameters of a poor prognosis in patients with colorectal cancer (67).

Over-expression of the HMGA proteins is a necessary event in in vivo cell transformation. This was demonstrated by experiments in which Hmga2 expression was blocked by transfecting rat thyroid cells with an antisense Hmga2 cDNA construct. When these cells were infected by the myeloproliferative sarcoma virus (MPSV) and the Kirsten murine sarcoma virus (KiMSV) carrying the v-mos and v-ras-Ki oncogenes, respectively, they did not acquire the typical markers of neoplastic transformation (ability to grow in soft agar and induce tumors after injection into athymic mice). Conversely, these markers were shown by the untransfected rat thyroid cells infected with the same murine retroviruses. Interestingly, the cells carrying the Hmga2 antisense construct did not express HMGA1a/b proteins suggesting the possibility that the block of cell transformation may be dependent on the inhibition of the Hmga1 expression (69). Further investigations showed that over-expression of HMGA1 proteins is also essential in the development of cancer in humans so that it is possible to suggest a cause and effect relationship. In fact, an adenovirus carrying the HMGA1 gene in an antisense orientation induces programmed cell death in carcinoma cell lines derived from human thyroid, lung, colon and breast cancers (70). Moreover, it has been reported that the over-expression of HMGA1a or HMGA2 leads to neoplastic transformation of both Rat-1a fibloblasts and CB33 cells, whereas the decrease of HMGA1a/b expression abrogates transformation in Burkitt's lymphoma cells (71,72).

The lack of induction of AP-1 transcriptional activity by suppression of HMGA1 and HMGA2 synthesis may account for the inhibition of the neoplastic cell transformation in absence of the HMGA proteins. In fact, thyroid neoplastic transformation is associated with a drastic increase of AP-1 activity, which is prevented by the suppression of the HMGA protein synthesis. The absence of AP-1 transcriptional activity induction, directly or indirectly regulated by the HMGA proteins, would inhibit the expression of AP-1-dependent genes, such as VEGF, collagenase I and stromelysin, which are required for cell neoplastic transformation (73).


    HMGA genes in human lipomas
 Top
 Abstract
 Introduction
 HMGA genes in human...
 Conclusions
 References
 
Rearrangements of HMGA2 and HMGA1 genes in human benign tumors
Several cytogenetic studies have demonstrated a non-random association between rearrangements of bands 12q13-15 or 6p21 and a variety of benign tumors mainly of mesenchymal origin: uterine leiomyomas, lipomas, pulmonary chondroid hamartomas, aggressive angiomyxomas, pleomorphic adenomas of the salivary glands, and fibroadenomas and adenolipomas of the breast (7491). The translocation occurred with different chromosome partners. In lipomas, for example, translocations have been identified between 12q13-15 and chromosomes 1 through 7, 10, 11, 13, 15, 17, 21 and X, even though translocations involving 12q13-15 with chromosomal region 3q27-28 and the long arm of chromosome 13 are known as the most frequent.

These data suggested that on the chromosomal region 12q13-15 should be located a gene playing a critical role in these tumors. This gene has been identified as the HMGA2 gene, which is affected by translocation breakpoints mapping to the chromosome region 12q13-14 (92,93).

In one study, the HMGA2 gene was identified by positional cloning, that led to the identification of a 175-kb region in which breakpoints were identified in eight benign tumors with 12q13-15 aberrations (92). Then, by using 3' terminal exon trapping, sequences identical to HMGA2 gene have been identified. Another study (94) started from earlier work that suggested an important role of the HMGA2 gene in adipocytic cell growth and development. In fact, it has been shown that mice carrying a disrupted HMGA2 presented a pygmy phenotype with a drastic reduction of the adult body weight, mainly affecting fat tissue (55). Moreover, this group located the HMGA2 gene in a region close to the translocation breakpoint. Then fluorescent in situ hybridization (FISH) analysis, using the cloned human HMGA2 gene, demonstrated that it was disrupted in all three lipomas analyzed.

The chromosome breakpoints occurring at 12q13-15 are preferentially clustered in the large (>160 kb) third intron of HMGA2, which separates the third DNA-binding domains from the acidic domain. It is noteworthy that intron 3 of HMGA2 has been basically conserved for at least 30 million years (95). As a consequence of translocation, the HMGA2 gene is disrupted and the AT-hooks fused to ectopic sequences. However, in some of these mesenchymal tumors, only a few amino acids are fused to the HMGA2 DNA-binding domains (96,97).

HMGA2 gene modifications in human lipomas
The lipoma preferred partner (LPP) gene, located on chromosome 3q27-28, is the most likely fusion partner gene of HMGA2 (9899). The LPP gene product is a proline-rich protein that shares 41% of sequence identity with the focal adhesion protein zyxin (100). LPP protein localizes in focal adhesions as well as in cell-to-cell contacts; it binds VASP, a protein implicated in the control of actin organization. In addition, LPP protein accumulates in the nucleus of cells upon treatment with leptomycin B, an inhibitor of the export factor CRM1. The nuclear export of LPP depends on an N-terminally located leucine-rich sequence that shares sequence homology with well-defined nuclear export signals. The LPP protein contains a leucine-zipper motif in its N-terminal region and three LIM domains in its C-terminal region (100,101). The fusion with the HMGA2 gene generally involves the three LIM domains of the LPP protein (98), and results in HMGA2/LPP and LPP/HMGA2 fusion transcripts in lipoma cells carrying a t(3;12)(q27;q14-q15) translocation (98,99). The LIM domains are cysteine-rich, zinc-binding sequences that are present in a variety of proteins involved in cell fate regulation and differentiation (100102). They dimerize with other nuclear proteins, many of which are transcription factors (103,104). Therefore, the LIM domains of the LPP protein may serve as a scaffold upon which distinct protein complexes are assembled in both cytoplasm and nucleus.

Although the HMGA2–LPP fusion has been frequently found in human lipomas, it does not seem exclusive of this kind of tumor, as the FISH analysis of five pulmonary chondroid hamartomas all showing a t(3;12)(q27;q14-q15) revealed that both HMGA2 and LPP are disrupted by this translocation, which indicates that this fusion is not specific for lipomas (105).

In one case of lipoma, the fusion occurs with DNA sequences located on chromosome 15. These sequences encode an acidic peptide rich in serine and threonine residues. These features have been observed in a number of transcriptional activation domains, including the C-terminal domains of homeobox proteins and NF-{kappa}B. So the acquisition of a trans-activation domain by the DNA-binding domains of HMGA2, which normally possesses a transcriptional inactive acidic domain, can easily be reconciled with aberrant regulation of the HMGA2 target genes (96).

Recently, a novel human gene, LHFP (lipoma HMGA2 fusion partner), that acts as a translocation partner of HMGA2 in a lipoma with t(12;13), has been isolated (106). The LHFP gene was mapped to the long arm of chromosome 13, a region recurrently targeted by chromosomal aberrations in lipomas. By northern blot analysis, a transcript of 2.4 kb was detected in a variety of human tissues. Nucleotide sequence analysis of the composite LHFP cDNA revealed an open reading frame encoding a protein of 200 amino acids. BLAST searches showed that the LHFP protein belongs to a new protein family consisting of at least four or five members. In the lipoma studied, the expressed HMGA2–LHFP fusion transcript encodes the three DNA-binding domains of HMGA2 followed by 69 amino acids encoded by frameshifted LHFP sequences.

Some studies suggest that, besides intragenic HMGA2 rearrangements, transcriptional activation of the gene can also initiate tumor growth. In fact, three pulmonary chondroid hamartomas (PCH) showing a rare variant type of the translocation t(12;14)(q14-15;q24) with presence of two normal chromosomes 12 and a der(14), but missing the der(12), showed that the breakpoint is located 5' to HMGA2 (107). However, so far no extragenic rearrangements have been described in lipomas. It cannot be excluded that this kind of alteration could occur also in lipomas. Lipomatosis induced by HMGA2 wild-type over-expression in transgenic mice (Fedele et al., submitted for publication) and the ability of HMGA2 to transform rat fibroblasts in culture (72) seem to confirm a potential role of HMGA2 over-expression in the process of lipomagenesis.

HMGA2 amplification in atypical lipomatous tumors
Atypical lipomatous tumors (ALTs) are a distinctive subset of mesenchymal neoplasms, characterized by a mature adipocytic differentiation and a tendency to arise in the somatic soft tissue of the limbs and in the retroperitoneum. These tumors often tend to recidive. The cytogenetic hallmark of these lesions is the presence of ring and/or long marker chromosomes derived from the chromosomal region 12q13-15. Eighty-three percent of ALTs showed HMGA1a immunopositivity associated with HMGA2 amplification. It is likely that higher expression of HMGA2 in ALTs compared with benign lipomas may be responsible for the greater aggressiveness of ALTs (108110).

What is the role of the HMGA2 rearrangement in the generation of human lipomas?
The findings summarized above raised several important questions.

(i) Do rearrangements of the HMGA2 gene have a causal role in the development of human lipomas or are they only casually associated with them?
(ii) Does the translocation partner have a specific role in tumorigenesis or does it merely deregulate expression of the DNA-binding region of HMGA2?
(iii) Is the lack of the C-terminal tail sufficient to confer cell growth advantage? Indeed, even though some published data seem to suggest that the fusion partner has a specific role in tumor formation, many more data indicate that the simple over-expression of the first three exons of HMGA2 is sufficient to cause transformation (52). In fact, in some of these mesenchymal tumors, only few amino acids are fused to the HMGA2 DNA-binding domains, suggesting that the truncation of the HMGA2 gene, rather than its fusion with other genes, is the event responsible for cell transformation.
(iv) Does the loss of the C-terminal tail in HMGA2 result in a gain-of-function or in a loss-of-function? The answer to this question determines whether the rearranged HMGA2 should be viewed as the generation of a new oncogene or as the disruption of a gene able to inhibit cell growth and consequently considered as a suppressor gene.

A rearranged HMGA2 gene is able to transform NIH 3T3 cells
In order to answer the above-mentioned questions, the murine fibroblasts NIH 3T3 were transfected with cDNAs coding for (i) a truncated form of the HMGA2 protein constituted by only the three DNA-binding domains (HMGA2/T); (ii) a fusion protein constituted by the three DNA-binding domains of HMGA2 and the LIM domains of the LPP protein (HMGA2/C); (iii) the wild-type HMGA2 protein. HMGA2/T and HMGA2/C caused malignant transformation of NIH3T3 cells. Conversely, the wild-type HMGA2 cDNA did not exert transforming activity. Moreover, the acquisition of ectopic sequences did not increase the transforming ability of the HMGA2 truncated form. The number of foci was significantly lower (~30-fold) than that obtained transfecting Ret/MEN2A or activated ras genes. Moreover, these foci appeared with a longer latency period (3–4 versus 1–2 weeks for the appearance of Ret/MEN2A foci). Finally, the colony-forming efficiency in soft-agar of the HMGA2/T and HMGA2/C transfectants was lower (30–35%) compared with that of Ret/MEN2A transfectants (80%) (111).

These results led to the following conclusions:

(i) the rearranged forms of HMGA2 must be considered oncogenes;
(ii) the loss of the two last exons of HMGA2 is sufficient to confer transformation ability;
(iii) the fusion with ectopic sequences does not increase the transforming ability of the rearranged HMGA2 gene;
(iv) the transforming ability of the rearranged forms of the HMGA2 gene is quite weak. This is consistent with the benign and non-aggressive behaviour of tumors associated with rearranged HMGA2 forms.

Transgenic mice carrying a truncated HMGA2 gene develop abdominal/pelvic lipomatosis
Subsequent to these findings, two independent groups generated transgenic mice carrying a truncated HMGA2 construct. In fact, transgenic mice provide a powerful experimental approach to define the role of oncogenes in neoplastic processes in vivo.

In one study (112), the transgenic mice were obtained with an innovative ES mediated strategy. The truncated HMGA2 cDNA (HMGA2/T), deprived of the C-terminal tail, under the transcriptional control of the cytomegalovirus promoter (CMV), was transfected into the ES cells AB2.2, and G418-resistant clones were selected. Two transfected cell clones expressing the highest levels of HMGA2/T mRNA were microinjected into C57BL6/J mouse blastocysts, which were then transferred to pseudopregnant foster mothers. Several chimeric mice were obtained and crossed with wild-type C57BL6/J mice to generate HMGA2/T mouse strains. This technique resulted in a very high expression of the transgene. Indeed, the authors showed that the expression of the transgene in these mice was higher than that detected in mice generated using the classical approach of microinjecting the HMGA2/T construct into fertilized mouse eggs (112). The second group generated the transgenic mice microinjecting the HMGA2/T construct under the transcriptional control of H-2Kb, a well characterized class I major histocompatibility complex promoter/enhancer, into fertilized mouse eggs (113). In both the studies, the transgene was expressed in all the tissues, and transgenic mice showed an increased body weight with a drastic expansion of the retroperitoneal and subcutaneous white adipose tissue (Figure 2Go). Moreover transgenic mice had an overabundance of fat at many anatomic sites. The most dramatic examples were found at the hilus of the kidney and at the base of the heart. In both the studies, the overall hypertrophy of the adipose tissue in certain sectors of the anatomy of these mice is consistent with a diagnosis of abdominal/pelvic lipomatosis. Analysis of the lipomatous tissue for the expression of several adipocytic terminal differentiation markers demonstrated that the differentiation state of the adipose tissue was not affected by the expression of the HMGA2/T gene. However, in the first study only one classical lipoma was detected in 10 mice; conversely seven lipomas among 33 mice analyzed were found in the second study. In the first study there was an average increase of 15% in body length (naso-anal) in 12-month-old animals, compared with wild-type littermates. Moreover, the animals generated in the first study showed bladder enlargement (Figure 2Go) consequent to compression of the urinary tract by the adipose tissue. The differences shown by animals generated in the two studies may be explained by the different expression of the transgene.



View larger version (71K):
[in this window]
[in a new window]
 
Fig. 2. Magnetic resonance imaging analysis. Frontal sections of 1-year-old wild-type and HMGA2/T transgenic mice (112) showing testes (te), bladder (bl) and subcutaneous and retroperitoneal (srf) fat.

 
The dramatic expansion of the adipose tissue observed in HMGA2/T mice suggests that HMGA2 rearrangements play a pivotal role in the generation of human lipomas. Moreover, the giant phenotype shown by the HMGA2/T mice is the mirror image of that of the HMGA2 null mice. In fact, these mice are characterized by a pigmy phenotype with a reduction of the adult body weight, mainly as a result of a decrease in fat tissue (55). Based on these data, we suggest that the truncation of the HMGA2 gene leads to an increased activity of the HMGA2 protein, which in turn stimulates adipocyte cell growth.

HMGA1 alterations in human lipomas
Recent studies have demonstrated that also the HMGA1 gene is rearranged in the benign tumors characterized with 6p21 chromosome aberrations (114117).

So far two cases have been described in human lipomas. In both instances there were deletions in the HMGA1 gene. In the first case, 10 bp of exon 6, 51 bp of exon 7 and 961 of exon 8, for a total of 1022 nucleotides, were deleted. The predicted protein includes the first two DNA-binding domains fused in-frame to an uninterrupted open reading frame (ORF) encoding 108 amino acids with a high content in proline which is indicative of an unusually huge content of domains potentially regulators of transcription. In the second case, 923 nucleotides of exon 8 were deleted, and this does not affect the HMGA1 ORF (118).

Role of the HMGA2 and HMGA1 in process of adipogenesis
The first evidence implicating Hmga2 in adipogenesis came from the observation that Hmga2–/– mice express a pygmy phenotype together with a drastic reduction (87%) of the adipose tissue (55). Conversely mice carrying a truncated Hmga2 gene showed an obese phenotype (112).

More recently it was found that Hmga2 gene was not expressed in wild-type adipose tissue, whereas it was expressed in fat deposits of both wild-type and genetically obese mice (Lepob/Lepob and Leprdb/Leprdb) after 1 week of high fat diet. Moreover, disruption of Hmga2 gene prevents both diet- and gene-induced obesity: in fact, Lepob/Lepob, Hmga2–/– double-homozygous mice (54) do not show an obese phenotype. HMGA2 appears epistatic to Lep in the fat tissue. However, as other phenotypic features of Lepob/Lepob mice are retained in Hmga2–/–, Lepob/Lepob mice, HMGA2 and Lep seem to function via independent genetic pathways. It has been proposed that HMGA2 regulates the proliferation of undifferentiated pre-adipocytes: the inability of these cells to proliferate in absence of Hmga2 could explain the reduced adipose tissue mass in Hmga2–/– mice. This conclusion is also supported by data showing that the suppression of Hmga2 expression by antisense technology suppresses the growth of 3T3-L1 pre-adipocytic murine cells (Battista et al., manuscript in preparation). The model-system of mouse 3T3-L1 fibroblasts, which rapidly differentiate to adipocytes upon treatment with several agents (119), has been used to better define HMGA1 involvement in adipocytic growth and differentiation (120). HMGA1 gene expression was induced soon after differentiation of the pre-adipocytic 3T3-L1 cell line. Suppression of HMGA1 expression by antisense technology dramatically increased growth rate and impaired adipocytic differentiation in these cells. Moreover, the authors show that HMGA1 proteins physically interact C/EBPß and C/EBP{alpha} (transcription factors involved in adipocytic cell differentiation) and bind to their specific binding sites on DNA, suggesting a functional cooperation between HMGA1 proteins and these proteins. Moreover, HMGA1 proteins enhance the transactivation of a c/EBPß responsive promoter, such as the leptin promoter, an adipocytic specific gene. Taken together, these results indicate that HMGA1 proteins exert a critical role in adipocytic cell growth and differentiation (120).

From these data, the role of HMGA1 seems to be multiform, depending on the cellular context. Indeed, HMGA1 proteins have been often associated with cell proliferation and transformation: in fact HMGA1a/b have been found over-expressed in several experimental and human malignant tumors, and HMGIA1a over-expression causes transformation of Burkitt's lymphoma cells. The hypothesis that the different cellular context may account for the different effects of the HMGA1 gene over-expression, is supported by recent data showing that HMGA1 over-expression impairs the growth of normal PC Cl 3 rat thyroid cells by inducing apoptosis. In our opinion, this hypothesis seems to be likely as several genes involved in the control of cell proliferation can induce different biological effects, such as cell growth, differentiation and apoptosis, depending on the cellular context.


    Conclusions
 Top
 Abstract
 Introduction
 HMGA genes in human...
 Conclusions
 References
 
The block of the synthesis of the HMGA1a/b proteins, by the expression of an antisense construct, induced a drastic increase in the growth rate of the 3T3-L1 cells. Moreover, preliminary data indicate an enormous increase in fat tissue in heterozygous-Hmga1 knockout mice [the Hmga1/ status is lethal at the embryonic level (Fedele et al., manuscript in preparation)]. These results would indicate that Hmga1a/b proteins exert a negative role on adipocytic cell growth. Conversely Hmga2 expression seems to be necessary for physiological proliferation of adipocytes. Indeed, Hmga2 knockout mice display a pygmy phenotype with a remarkable reduction of the adipose tissue (55) and disruption of Hmga2 gene prevents both diet- and gene-induced obesity (54). Moreover, the suppression of HMGA2 synthesis blocks the proliferation of the 3T3-L1 pre-adipocytic cells (Baista et al., in preparation).

These data would suggest that the regulation of adipocytic cell proliferation could result from the balance of HMGA1a/b and HMGA2 protein functions: a gain in HMGA2 protein activity induces adipocyte cell hyperproliferation, whereas the dominance of HMGA1a/b has an opposite effect. Therefore, even though the mechanisms underlying the counteracting activity of HMGA1a/b and HMGA2 proteins needs to be defined, it is reasonable to think that any modification of this balance, by impairing the HMGA1a/b activity or by increasing the HMGA2 function, may affect adipocytic cell growth resulting in the generation of lipomas.


    Notes
 
5 To whom correspondence should be addressedEmail: afusco{at}napoli.com Back

* The nomenclature of the high mobility group (HMG) proteins has been recently revised; see Chromosomal Proteins Nomenclature Home Page: http://www.informatics.jax.org/mgihome/nomen/genefamilies/hmgfamily.shtml. In this review we use the new nomenclature, but we report the old one in parenthesis. HMGA1a, high mobility group A1a (HMGI); HMGA1b, high mobility group A1b (HMGY); HMGA1c, high mobility group A1c (HMG-I/R); HMGA2, high mobility group A2 (HMGI-C). Back


    References
 Top
 Abstract
 Introduction
 HMGA genes in human...
 Conclusions
 References
 

  1. Johnson,K.R., Lehn,D.A., Elton,T.S., Barr,P.J. and Reeves,R. (1988) Complete murine cDNA sequence, genomic structure and tissue expression of the high mobility group protein HMG-I (Y). J. Biol. Chem., 263, 18338–183342.[Abstract/Free Full Text]
  2. Johnson,K.R., Lehn,D.A. and Reeves,R. (1989) Alternative processing of mRNAs encoding mammalian chromosomal high- mobility-group proteins HMG-I and HMG-Y. Mol. Cell. Biol., 9, 2114–2123.[ISI][Medline]
  3. Manfioletti,G., Giancotti,V., Bandiera,A., Buratti,E., Sautiere,P., Cary,P., Crane-Robinson,C., Coles,B. and Goodwin,G.H. (1991) cDNA cloning of the HMGI-C phosphoprotein, a nuclear protein associated with neoplastic and undifferentiated phenotypes. Nucleic Acids Res., 19, 6793–6797.[Abstract]
  4. Patel,U.A., Bandiera,A., Manfioletti,G., Giancotti,V., Chau,K.Y. and Crane-Robinson,C. (1994) Expression and cDNA cloning of human HMGI-C phosphoprotein. Biochem. Biophys. Res. Commun., 201, 63–70.[ISI][Medline]
  5. Friedmann,M., Holth,L.T., Zoghbi,H.Y. and Reeves,R. (1993) Organization, inducible-expression and chromosome localization of the human HMG-I (Y) non-histone protein gene. Nucleic Acids Res., 21, 4259–4267.[Abstract]
  6. Manfioletti,G., Rustighi,A., Mantovani,F., Goodwin,G.H. and Giancotti,V. (1995) Isolation and characterization of the gene coding for murine high- mobility-group protein HMGI-C. Gene, 167, 249–253.[ISI][Medline]
  7. Chau,K.Y., Patel,U.A., Lee,K.L., Lam,H.Y. and Crane-Robinson,C. (1995) The gene for the human architectural transcription factor HMGI-C consists of five exons each coding for a distinct functional element. Nucleic Acids Res., 23, 4262–4266.[Abstract]
  8. Claus,P., Schulze,E. and Wisniewski,J.R. (1994) Insect proteins homologous to mammalian high mobility group proteins I/Y (HMG I/Y). Characterization and binding to linear and four-way junction DNA. J. Biol. Chem., 269, 33042–33048.[Abstract/Free Full Text]
  9. Zhou,X., Benson,K.F., Przybysz,K., Liu,J., Hou,Y., Cherath,L. and Chada,K. (1996) Genomic structure and expression of the murine Hmgi-c gene. Nucleic Acids Res., 24, 4071–7.[Abstract/Free Full Text]
  10. Ghidelli,S., Claus,P., Thies,G. and Wisniewski,J.R. (1997) High mobility group proteins cHMG1a, cHMG1b and cHMGI are distinctly distributed in chromosomes and differentially expressed during ecdysone dependent cell differentiation. Chromosoma, 105, 369–79.[ISI][Medline]
  11. Nagpal,S., Ghosn,C., DiSepio,D., Molina,Y., Sutter,M., Klein,E.S. and Chandraratna,R.A. (1999) Retinoid-dependent recruitment of a histone H1 displacement activity by retinoic acid receptor. J. Biol. Chem., 274, 22563–22568.[Abstract/Free Full Text]
  12. Elton,T.S., Nissen,M.S. and Reeves,R. (1987) Specific A. T DNA sequence binding of RP-HPLC purified HMG-I. Biochem. Biophys. Res. Commun., 143, 260–265.[ISI][Medline]
  13. Reeves,R. and Nissen,M.S. (1990) The ATDNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure. J. Biol. Chem., 265, 8573–8582.[Abstract/Free Full Text]
  14. Geierstanger,B.H., Volkman,B.F., Kremer,W. and Wemmer,D.E. (1994) Short peptide fragments derived from HMG-I/Y proteins bind specifically to the minor groove of DNA. Biochemistry, 33, 5347–5355.[ISI][Medline]
  15. Ashar,H.R., Cherath,L., Przybysz,K.M. and Chada,K. (1996) Genomic characterization of human HMGIC, a member of the accessory transcription factor family found at translocation breakpoints in lipomas. Genomics, 31, 207–14.[ISI][Medline]
  16. Liu,J., Schiltz,J.F., Shah,P.C., Benson,K.F. and Chada,K.K. (2000) Genomic structure and expression of the murine Hmgi (y) gene. Gene, 246, 197–207.[ISI][Medline]
  17. Elton,T.S. and Reeves,R. (1986) Purification and postsynthetic modifications of Friend erythroleukemic cell high mobility group protein HMG-I. Anal. Biochem., 157, 53–62.[ISI][Medline]
  18. Banks,G.C., Li,Y. and Reeves,R. (2000) Differential in vivo modifications of the HMGI (Y) non-histone chromatin proteins modulate nucleosome and DNA interactions. Biochemistry, 39, 8333–8346.[ISI][Medline]
  19. Xiao,D.M., Pak,J.H., Wang,X., Sato,T., Huang,F.L., Chen,H.C. and Huang,K.P. (2000) Phosphorylation of HMG-I by protein kinase C attenuates its binding affinity to the promoter regions of protein kinase C gamma and neurogranin/RC3 genes. J. Neurochem., 74, 392–399.[ISI][Medline]
  20. Reeves,R., Langan,T.A. and Nissen,M.S. (1991) Phosphorylation of the DNA-binding domain of non-histone high-mobility group I protein by cdc2 kinase: reduction of binding affinity. Proc. Natl Acad. Sci. USA, 88, 1671–1675.[Abstract]
  21. Nissen,M.S., Langan,T.A. and Reeves,R. (1991) Phosphorylation by cdc2 kinase modulates DNA binding activity of high mobility group I nonhistone chromatin protein. J. Biol. Chem., 266, 19945–19952.[Abstract/Free Full Text]
  22. Ferranti,P., Malorni,A., Marino,G., Pucci,P., Goodwin,G.H., Manfioletti,G. and Giancotti,V. (1992) Mass spectrometric analysis of the HMGY protein from Lewis lung carcinoma. Identification of phosphorylation sites. J. Biol. Chem., 267, 22486–22489.[Abstract/Free Full Text]
  23. Diana,F., Sgarra,R., Manfioletti,G., Rustighi,A., Poletto,D., Sciortino,M.T., Mastino,A. and Giancotti,V. (2001) A link between apoptosis and degree of phosphorylation of high mobility group A1a protein in leukaemic cells. J. Biol. Chem., 276, 11354–11361.[Abstract/Free Full Text]
  24. Munshi,N., Merika,M., Yie,J., Senger,K., Chen,G. and Thanos,D. (1998) Acetylation of HMG I (Y) by CBP turns off IFN beta expression by disrupting the enhanceosome. Mol. Cell, 2, 457–467.[ISI][Medline]
  25. Merika,M., Williams,A.J., Chen,G., Collins,T. and Thanos,D. (1998) Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription. Mol. Cell, 1, 277–87.[ISI][Medline]
  26. Giancotti,V., Bandiera,A., Sindici,C., Perissin,L. and Crane-Robinson,C. (1996) Calcium-dependent ADP-ribosylation of high-mobility-group I (HMGI) proteins. Biochem. J., 317, 865–870.[ISI][Medline]
  27. Lovell-Badge,R. (1995) Developmental genetics. Living with bad architecture [news; comment]. Nature, 376, 725–726.[Medline]
  28. Chuvpilo,S., Schomberg,C., Gerwig,R., Heinfling,A., Reeves,R., Grummt,F. and Serfling,E. (1993) Multiple closely-linked NFAT/octamer and HMG I (Y) binding sites are part of the interleukin-4 promoter. Nucleic Acids Res., 21, 5694–5704.[Abstract]
  29. Wood,L.D., Farmer,A.A. and Richmond,A. (1995) HMGI (Y) and Sp1 in addition to NF-kappa B regulate transcription of the MGSA/GRO alpha gene. Nucleic Acids Res., 23, 4210–4219.[Abstract]
  30. Powell,D.R., Allander,S.V., Scheimann,A.O., Wasserman,R.M., Durham,S.K. and Suwanichkul,A. (1995) Multiple proteins bind the insulin response element in the human IGFBP-1 promoter. Prog. Growth Factor Res., 6, 93–101.[Medline]
  31. Abdulkadir,S.A., Casolaro,V., Tai,A.K., Thanos,D. and Ono,S.J. (1998) High mobility group I/Y protein functions as a specific cofactor for Oct-2A: mapping of interaction domains. J. Leukoc. Biol., 64, 681–691.[Abstract]
  32. Mantovani,F., Covaceuszach,S., Rustighi,A., Sgarra,R., Heath,C., Goodwin,G.H. and Manfioletti,G. (1998) NF-kappa B mediated transcriptional activation is enhanced by the architectural factor HMGI-C. Nucleic Acids Res., 26, 1433–1439.[Abstract/Free Full Text]
  33. John,S., Reeves,R.B., Lin,J.X., Child,R., Leiden,J.M., Thompson,C.B. and Leonard,W.J. (1995) Regulation of cell-type-specific interleukin-2 receptor alpha-chain gene expression: potential role of physical interactions between Elf-1, HMG-I (Y) and NF-kappa B family proteins. Mol. Cell. Biol., 15, 1786–1796.[Abstract]
  34. Leger,H., Sock,E., Renner,K., Grummt,F. and Wegner,M. (1995) Functional interaction between the POU domain protein Tst-1/Oct-6 and the high-mobility-group protein HMG-I/Y. Mol. Cell. Biol., 15, 3738–3747.[Abstract]
  35. Thanos,D., Du,W. and Maniatis,T. (1993) The high mobility group protein HMG I (Y) is an essential structural component of a virus-inducible enhancer complex. Cold Spring Harb. Symp. Quant. Biol., 58, 73–81.[ISI][Medline]
  36. Thanos,D. and Maniatis.T. (1992) The high mobility group protein HMG I (Y) is required for NF-kappa B-dependent virus induction of the human IFN-beta gene. Cell, 71, 777–789.[ISI][Medline]
  37. Thanos,D. and Maniatis,T. (1995) Virus induction of human IFN beta gene expression requires the assembly of an enhanceosome. Cell, 83, 1091–1100.[ISI][Medline]
  38. Yie,J., Liang,S., Merika,M. and Thanos,D. (1997) Intra- and intermolecular cooperative binding of high-mobility-group protein I (Y) to the beta-interferon promoter. Mol. Cell. Biol., 17, 3649–3662.[Abstract]
  39. Falvo,J.V., Thanos,D. and Maniatis,T. (1995) Reversal of intrinsic DNA bends in the IFN beta gene enhancer by transcription factors and the architectural protein HMG I (Y). Cell, 83, 1101–1111.[ISI][Medline]
  40. Kim,T.K. and Maniatis,T. (1997) The mechanism of transcriptional synergy of an in vitro assembled interferon-beta enhanceosome. Mol. Cell, 1, 119–129.[ISI][Medline]
  41. Reeves,R., Leonard,W.J. and Nissen,M.S. (2000) Binding of HMG-I (Y) imparts architectural specificity to a positioned nucleosome on the promoter of the human interleukin-2 receptor alpha gene. Mol. Cell. Biol., 20, 4666–4679.[Abstract/Free Full Text]
  42. Bustin,M. and Reeves,R. (1996) High-mobility-group chromosomal proteins: architectural components that facilitate chromatin function. Prog. Nucleic Acids Res. Mol. Biol., 54, 35–100.[ISI][Medline]
  43. Bustin,M. (1999) Regulation of DNA-dependent activities by the functional motifs of the high-mobility-group chromosomal proteins. Mol. Cell. Biol., 19, 5237–5246.[Free Full Text]
  44. Käs,E. and Laemmli,U.K. (1992) In vivo topoisomerase II cleavage of the Drosophila histone and satellite III repeats: DNA sequence and structural characteristics. EMBO J., 11, 705–716.[Abstract]
  45. Romig,H., Fackelmayer,F.O., Renz,A., Ramsperger,U. and Richter,A. (1992) Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear. EMBO J., 11, 3431–3440.[Abstract]
  46. Luderus,M.E., de Graaf,A., Mattia,E., den Blaauwen,J.L., Grande,M.A., de Jong,L. and van Driel,R. (1992) Binding of matrix attachment regions to lamin B1. Cell, 70, 949–59.[ISI][Medline]
  47. Zhao,K., Käs,E., Gonzales,E. and Laemmli,U.K. (1993) SAR-dependent mobilization of histone H1 by HMG-I/Y in vitro: HMG-I/Y is enriched in H1-depleted chromatin. EMBO J., 12, 3237–3247.[Abstract]
  48. Liu,W.-M., Guerra-Vladusic,F.K., Kurakata,S., Lupu,R. and Kohwi-Shigematsu,T.K. (1999) HMG-I (Y) recognizes base-unpairing regions of matrix attachment sequences and its increased expression is directly linked to metastatic breast cancer phenotype. Cancer Res., 59, 5695–5703.[Abstract/Free Full Text]
  49. Hill,D.A., Pedulla,M.L. and Reeves,R. (1999) Directional binding of HMG-I (Y) on four-way junction DNA and the molecular basis for competitive binding with HMG-1 and histone H1. Nucleic Acids Res., 27, 2135–2144.[Abstract/Free Full Text]
  50. Martelli,A.M., Riccio,M., Bareggi,R., Manfioletti,G., Tabellini,G., Baldini,G., Narducci,P. and Giancotti,V. (1998) Intranuclear distribution of HMGI/Y proteins. An immunocytochemical study. J. Histochem. Cytochem., 46, 863–864.[Abstract/Free Full Text]
  51. Choi,K.S., Eom,Y.W., Kang,Y., Ha,M.J., Rhee,H., Yoon,J.W. and Kim,S.J. (1999) Cdc2 and Cdk2 kinase activated by transforming growth factor-beta 1 trigger apoptosis through the phosphorylation of retinoblastoma protein in FaO hepatoma cells. J. Biol. Chem., 274, 31775–31783.[Abstract/Free Full Text]
  52. Rogalla,P., Drechsler,K., Frey,G., Hennig,Y., Helmke,B., Bonk,U. and Bullerdiek,J. (1996) HMGI-C expression patterns in human tissues. Implications for the genesis of frequent mesenchymal tumors. Am. J. Pathol., 149, 775–779.[Abstract]
  53. Rommel,B., Rogalla,P., Jox,A., Kalle,C.V., Kazmierczak,B., Wolf,J. and Bullerdiek,J. (1997) HMGI-C, a member of the high mobility group family of proteins, is expressed in hematopoietic stem cells and in leukemic cells. Leuk. Lymphoma, 26, 603–607.[ISI][Medline]
  54. Anand,A. and Chada,K. (2000) In vivo modulation of Hmgic reduces obesity. Nature Genet., 24, 377–380.[ISI][Medline]
  55. Zhou,X., Benson,K.F., Ashar,H.R. and Chada,K. (1995) Mutation responsible for the mouse pygmy phenotype in the developmentally regulated factor HMGI-C. Nature, 376, 771–774.[ISI][Medline]
  56. Chiappetta,G., Avantaggiato,V., Visconti,R. et al. (1996) High level expression of the HMGI (Y) gene during embryonic development. Oncogene, 13, 2439–2446.[ISI][Medline]
  57. Giancotti,V., Pani,B., D'Andrea,P. et al. (1987) Elevated levels of a specific class of nuclear phosphoproteins in cells transformed with v-ras and v-mos oncogenes and by cotransfection with c-myc and polyoma middle T genes. EMBO J., 6, 1981–1987.[Abstract]
  58. Giancotti,V., Berlingieri,M.T., Di Fiore,P.P., Fusco,A., Vecchio,G. and Crane-Robinson,C. (1985) Changes in nuclear proteins following transformation of rat thyroid epithelial cells by a murine sarcoma retrovirus. Cancer Res., 45, 6051–6057.[Abstract]
  59. Giancotti,V., Buratti,E., Perissin,L., Zorzet,S., Balmain,A., Portella,G., Fusco,A. and Goodwin,G.H. (1989) Analysis of the HMGI nuclear proteins in mouse neoplastic cells induced by different procedures. Exp. Cell. Res., 184, 538–545.[ISI][Medline]
  60. Chiappetta,G., Bandiera,A., Berlingieri,M.T., Visconti,R., Manfioletti,G., Battista,S., Martinez-Tello F.J., Santoro,M., Giancotti,V. and Fusco,A. (1995) The expression of the high mobility group HMGI (Y) proteins correlates with the malignant phenotype of human thyroid neoplasias. Oncogene, 10, 1307–1314.[ISI][Medline]
  61. Chiappetta,G., Tallini,G., DeBiasio,M.C. et al. (1998) Detection of high mobility group I HMGI (Y) protein in the diagnosis of thyroid tumors: HMGI (Y) expression represents a potential diagnostic indicator of carcinoma. Cancer Res., 58, 4193–4198.[Abstract]
  62. Kim,S.J., Ryu,J.W. and Choi,D.S. (2000) The expression of the high mobility group I (Y) mRNA in thyroid cancers: useful tool of differential diagnosis of thyroid nodules. Korean J. Intern. Med., 15, 71–75.[Medline]
  63. Tamimi,Y., van der Poel,H.G., Denyn,M.M., Umbas,R., Karthaus,H.F.M., Debruyne,F.M.J. and Shalken,J.A. (1993). Increased expression of high mobility group protein I (Y) in high grade prostate cancer detemined by in situ hybridization. Cancer Res., 53, 5512–5516.[Abstract]
  64. Bandiera,A., Bonifacio,D., Manfioletti,G., Mantovani,F., Rustighi,A., Zanconati,F., Fusco,A., Di Bonito,L. and Giancotti,V. (1998). Expression of high mobility group I (HMGI) proteins in squamous intraepithelial lesions (SILs) of uterine cervix. Cancer Res., 58, 426–431.[Abstract]
  65. Fedele,M., Bandiera,A., Chiappetta,G., Battista,S., Viglietto,G., Manfioletti,G., Casamassimi,A., Santoro,M., Giancotti,V. and Fusco,A. (1996) Human colorectal carcinomas express high levels of high mobility group HMGI (Y) proteins. Cancer Res., 56, 1896–1901.[Abstract]
  66. Abe,N., Watanabe,T., Sugiyama,M., Uchimura,H., Chiappetta,G., Fusco,A. and Atomi,Y. (1999) Determination of high mobility group I (Y) expression level in colorectal neoplasias: a potential diagnostic marker. Cancer Res., 59, 1169–1174.[Abstract/Free Full Text]
  67. Chiappetta,G., Manfioletti,G., Pentimalli,F. et al. (2001) High mobility group HMGI (Y) protein expression in human colorectal hyperplastic and neoplastic diseases. Int. J. Cancer, 91, 147–151.[ISI][Medline]
  68. Abe,N., Watanabe,T., Masaki,T., Mori,T., Sugiyama,M., Uchimura,H., Fujioka,Y., Chiappetta,G., Fusco,A. and Atomi,Y. (2000) Pancreatic duct cell carcinomas express high levels of high mobility group I (Y) proteins. Cancer Res., 60, 3117–3122.[Abstract/Free Full Text]
  69. Berlingieri,M.T., Manfioletti,G., Santoro,M., Bandiera,A., Visconti,R., Giancotti,V. and Fusco,A. (1995) Inhibition of HMGI-C protein synthesis suppresses retrovirally induced neoplastic transformation of rat thyroid cells. Mol. Cell. Biol., 15, 1545–1553.[Abstract]
  70. Scala,S., Portella,G., Fedele,M., Chiappetta,G. and Fusco,A. (2000) Adenovirus-mediated suppression of HMGI (Y) protein synthesis as potential therapy of human malignant neoplasias. Proc. Natl Acad. Sci. USA, 97, 4256–4261.[Abstract/Free Full Text]
  71. Wood,L.J., Murkherjee,M., Dolde,C.E., Xu,Y., Maher,J., Bunton,T.E., Willams,J.B. and Resar,L.M.S. (2000) HMG-I/Y, a new c-Myc target gene and potential oncogene. Mol. Cell. Biol., 20, 5490–5502.[Abstract/Free Full Text]
  72. Wood,L.J., Maher,J.F., Bunton,T.E. and Resar,L.M. (2000) The oncogenic properties of the HMG-I gene family. Cancer Res., 60, 4256–4261.[Abstract/Free Full Text]
  73. Vallone,D., Battista,S., Pierantoni,G.M., Fedele,M., Casalino,L., Santoro,M., Viglietto,G., Fusco,A. and Verde,P. (1997) Neoplastic transformation of rat thyroid cells requires the junB and fra-1 gene induction which is dependent on the HMGI-C gene product. EMBO J., 16, 5310–5321.[Abstract/Free Full Text]
  74. Sait,S.N., Dal,Cin P., Ovanessoff,S. and Sandberg,A.A. (1989) A uterine leiomyoma showing both t (12;14) and del (7) abnormalities. Cancer Genet. Cytogenet., 37, 157–161.[ISI][Medline]
  75. Heim S., Nilbert,M., Vanni,R., Floderus,U.M., Mandahl,N., Liedgren,S., Lecca,U. and Mitelman,F. (1988) A specific translocation, t (12;14) (q14-15;q23-24), characterizes a subgroup of uterine leiomyomas. Cancer Genet. Cytogenet., 32, 13–17.[ISI][Medline]
  76. Walter,T.A., Fan,S.X., Medchill,M.T., Berger,C.S., Decker,H.J. and Sandberg,A.A. (1989) Inv (12) (p11.2q13) in an endometrial polyp. Cancer Genet. Cytogenet., 41, 99–103.[ISI][Medline]
  77. Turc-Carel,C., Limon,J., Dal Cin,P., Rao,U., Karakousis,C. and Sandberg,A.A. (1986) Cytogenetic studies of adipose tissue tumors. II. Recurrent reciprocal translocation t (12;16) (q13;p11) in myxoid liposarcomas. Cancer Genet. Cytogenet., 23, 291–299.[ISI][Medline]
  78. Sreekantaiah,C., Leong,S.P., Karakousis,C.P. et al. (1991) Cytogenetic profile of 109 lipomas. Cancer Res., 51, 422–433.[Abstract]
  79. Rohen,C., Caselitz,J., Stern,C., Wanschura,S., Schoenmakers,E.F., Van de Ven,W.J., Bartnitzke,S. and Bullerdiek,J. (1995) A hamartoma of the breast with an aberration of 12q mapped to the MAR region by fluorescence in situ hybridization. Cancer Genet. Cytogenet., 84, 82–84.[ISI][Medline]
  80. Fletcher C.D., Akerman,M., Dal Cin,P. et al. (1996) Correlation between clinicopathological features and karyotype in lipomatous tumors. A report of 178 cases from the Chromosomes and Morphology (CHAMP) Collaborative Study Group. Am. J. Pathol., 148, 623–630.[Abstract]
  81. Pandis,N., Teixeira,M.R., Gerdes,A.M., Limon,J., Bardi,G. Andersen,J.A., Idvall,I., Mandahl,N., Mitelman,F. and Heim,S. (1995) Chromosome abnormalities in bilateral breast carcinomas. Cytogenetic evaluation of the clonal origin of multiple primary tumors. Cancer, 76, 250–258.[ISI][Medline]
  82. Mandahl,N., Hoglund,M., Mertens,F., Rydholm,A., Willen,H., Brosjo O. and Mitelman,F. (1994) Cytogenetic aberrations in 188 benign and borderline adipose tissue tumors. Genes Chromosomes Cancer, 9, 207–215.[ISI][Medline]
  83. Heim,S., Nilbert,M., Vanni,R., Floderus,U.M., Mandahl,N., Liedgren,S., Lecca,U. and Mitelman,F. (1988) A specific translocation, t (12;14) (q14-15;q23-24), characterizes a subgroup of uterine leiomyomas. Cancer Genet. Cytogenet., 32, 13–17.[ISI][Medline]
  84. Vanni,R., Dal Cin,P., Marras,S., Moerman,P. Andria,M., Valdes,E., Deprest,J. and Van den Berghe,H. (1993) Endometrial polyp: another benign tumor characterized by 12q13-q15 changes. Cancer Genet. Cytogenet., 68, 32–33.[ISI][Medline]
  85. Dal Cin,P., Vanni,R., Marras,S., Moerman,P., Kools,P. Andria,M., Valdes,E., Deprest,J., Van de Ven,W. and Van den Berghe,H. (1995) Four cytogenetic subgroups can be identified in endometrial polyps. Cancer Res., 55, 1565–1568.[Abstract]
  86. Mandahl,N. (1996) Cytogenetics and molecular genetics of bone and soft tissue tumors. Adv. Cancer Res., 69, 63–99.[ISI][Medline]
  87. Mandahl,N., Orndal,C., Heim,S., Willen,H., Rydholm,A., Bauer,H.C. and Mitelman,F. (1993) Aberrations of chromosome segment 12q13-15 characterize a subgroup of hemangiopericytomas. Cancer, 71, 3009–3013.[ISI][Medline]
  88. Wanschura,S., Kazmierczak,B., Schoenmakers,E., Meyen,E., Bartnitzke,S., Van de Ven,W., Bullerdiek,J. and Schloot,W. (1995) Regional fine mapping of the multiple-aberration region involved in uterine leiomyoma, lipoma and pleomorphic adenoma of the salivary gland to 12q15. Genes Chromosomes Cancer, 14, 68–70.[ISI][Medline]
  89. Kazmierczak,B., Wanschura,S., Meyer-Bolte,K., Caselitz,J., Meister,P., Bartnitzke,S., Van de Ven,W. and Bullerdiek,J. (1995) Cytogenic and molecular analysis of an aggressive angiomyxoma. Am. J. Pathol., 147, 580–585.[Abstract]
  90. Kools,P.F., Wanschura,S., Schoenmakers,E.F., Geurts,J.M., Mols,R., Kazmierczak,B., Bullerdiek,J., Van den Berghe,H. and Van de Ven,W.J. (1995) Identification of the chromosome 12 translocation breakpoint region of a pleomorphic salivary gland adenoma with t (1;12) (p22;q15) as the sole cytogenetic abnormality. Cancer Genet. Cytogenet., 79, 1–7.[ISI][Medline]
  91. Schoenmakers,E.F., Kools,P.F., Mols,R., Kazmierczak,B., Bartnitzke,S., Bullerdiek,J., Dal Cin,P., De Jong,P.J., Van den Berghe,H. and Van de Ven,W.J. (1994) Physical mapping of chromosome 12q breakpoints in lipoma, pleomorphic salivary gland adenoma, uterine leiomyoma and myxoid liposarcoma. Genomics, 20, 210–222.[ISI][Medline]
  92. Schoenmakers,E.F., Wanschura,S., Mols,R., Bullerdiek,J., Van den Berghe,H. and Van de Ven,W.J. (1995) Recurrent rearrangements in the high mobility group protein gene, HMGI-C, in benign mesenchymal tumours. Nature Genet., 10, 436–444.[ISI][Medline]
  93. Kazmierczak,B., Hennig,Y., Wanschura,S., Rogalla,P., Bartnitzke,S., Van de Ven,W. and Bullerdiek,J. (1995) Description of a novel fusion transcript between HMGI-C, a gene encoding for a member of the high mobility group proteins and the mitochondrial aldehyde dehydrogenase gene. Cancer Res., 55, 6038–6039.[Abstract]
  94. Ashar,H.R., Fejzo,M.S., Tkachenko,A., Zhou,X., Fletcher,J.A., Weremowicz,S., Morton,C.C. and Chada,K. (1995) Disruption of the architectural factor HMGI-C: DNA-binding AT hook motifs fused in lipomas to distinct transcriptional regulatory domains. Cell, 82, 57–65.[ISI][Medline]
  95. Kazmierczak,B., Bullerdiek,J., Pham,K.H., Bartnitzke,S. and Wiesner,H. (1998) Intron 3 of HMGIC is the most frequent target of chromosomal aberrations in human tumors and has been conserved basically for at least 30 million years [letter]. Cancer Genet. Cytogenet., 103, 175–177.[ISI][Medline]
  96. Kazmierczak,B., Wanschura,S., Rosikeit,J., Meyer-Bolte,K., Uschinsky,K., Hampt,R., Schoenmakers,E.F.P.M, Bartnitzke,S., Van de Ven,M. and Bullerdiek,J. (1995) Molecular characterization of 12q14-15 rearrangements in three pulmonary chondroid hamartomas. Cancer Res., 55, 2497–2499.[Abstract]
  97. Kools,P.F. and Van de Ven,W.J. (1996) Amplification of a rearranged form of the high-mobility group protein gene HMGI-C in OsA-CI osteosarcoma cells. Cancer Genet. Cytogenet., 91, 1–7.[ISI][Medline]
  98. Petit,M.M.R., Mols,R., Schoenmakers,E.F., Mandahl,N. and Van de Ven,W.J. (1996) LPP, the preferred fusion partner gene of HMGIC in lipomas, is a novel member of the LIM protein gene family. Genomics, 36, 118–129.[ISI][Medline]
  99. Petit M.M., Swarts,S., Bridge,J.A. and Van de Ven,W.J. (1998) Expression of reciprocal fusion transcripts of the HMGIC and LPP genes in parosteal lipoma. Cancer Genet. Cytogenet., 106, 18–23.[ISI][Medline]
  100. Freyd G., Kim,S.K. and Horvitz,H.R. (1990) Novel cysteine-rich motif and homeodomain in the product of the Caenorhabditis elegans cell lineage gene lin-11. Nature, 344, 876–879.[ISI][Medline]
  101. Valge-Archer,V.E., Osada,H., Warren,A.J., Forster,A., Li,J., Baer,R. and Rabbitts,T.H. (1994) The LIM protein RBTN2 and the basic helix–loop–helix protein TAL1 are present in a complex in erythroid cells. Proc. Natl Acad. Sci. USA, 91, 8617–8621.[Abstract]
  102. Cohen,B., McGuffin,M.E., Pfeifle,C., Segal,D. and Cohen,S.M. (1992) apterous, a gene required for imaginal disc development in Drosophila encodes a member of the LIM family of developmental regulatory proteins. Genes Dev., 6 (5), 715–729.[Abstract]
  103. German,M.S., Wang,J., Chadwick,R.B. and Rutter,W.J. (1992) Synergistic activation of the insulin gene by a LIM-homeo domain protein and a basic helix–loop–helix protein: building a functional insulin minienhancer complex. Genes Dev., 6 (11), 2165–2176.[Abstract]
  104. Archer,V.E., Breton,J., Sanchez-Garcia,I., Osada,H., Forster,A., Thomson,A.J. and Rabbitts,T.H. (1994) Cysteine-rich LIM domains of LIM-homeodomain and LIM-only proteins contain zinc but not iron. Proc. Natl Acad. Sci. USA, 91 (1), 316–120.[Abstract]
  105. Rogalla,P., Kazmierczak,B., Meyer-Bolte,K., Tran,K.H. and Bullerdiek,J. (1998) The t (3;12) (q27;q14-q15) with underlying HMGIC–LPP fusion is not determining an adipocytic phenotype. Genes Chromo. Cancer, 22, 100–104.[ISI]
  106. Petit,M.M., Schoenmakers,E.F., Huysmans,C., Geurts,J.M., Mandahl,N. and Van de Ven,W.J. (1999) LHFP, a novel translocation partner gene of HMGIC in a lipoma, is a member of a new family of LHFP-like genes. Genomics, 57, 438–441.[ISI][Medline]
  107. Wanschura,S., Kazmierczak,B., Pohnke,Y., Meyer-Bolte,K., Bartnitzke,S., Van de Ven,W.J. and Bullerdiek,J. (1996) Transcriptional activation of HMGI-C in three pulmonary hamartomas each with a der (14)t (12;14) as the sole cytogenetic abnormality. Cancer Lett., 102 (1–2), 17–21.[ISI][Medline]
  108. Mandahl,N., Akerman,M., Aman,P. et al. (1996) Duplication of chromosome segment 12q15-24 is associated with atypical lipomatous tumors: a report of the CHAMP collaborative study group. Chromosomes and Morphology. Int. J. Cancer, 67, 632–635.[ISI][Medline]
  109. Tallini,G., Dal Cin,P., Rhoden,K.J., Chiapetta,G., Manfioletti,G., Giancotti,V., Fusco,A., Van den Berghe,H. and Sciot,R. (1997) Expression of HMGI-C and HMGI (Y) in ordinary lipoma and atypical lipomatous tumors: immunohistochemical reactivity correlates with karyotypic alterations. Am. J. Pathol., 151, 37–43.[Abstract]
  110. Del Tos,A.P., Doglioni,C., Piccinin,S., Sciot,R., Furlanetto,A., Boiocchi,M., Maestro,R., Fletcher,C.D. and Tallini,G. (2000) Coordinated expression and amplification of the MDM2, CDK4 and HMGI-C genes in atypical lipomatous tumours. J. Pathol., 190, 531–536.[ISI][Medline]
  111. Fedele,M., Berlingieri,M.T., Scala,S., Chiariotti,L., Viglietto,G., Rippel,V., Bullerdiek,J., Santoro,M. and Fusco,A. (1998) Truncated and chimeric HMGI-C genes induce neoplastic transformation of NIH3T3 murine fibroblasts. Oncogene, 17, 413–418.[ISI][Medline]
  112. Battista,S., Fidanza,V., Fedele,M., Klein-Szanto,A.J., Outwater,E., Brunner,H., Santoro,M., Croce,C.M. and Fusco,A. (1999) The expression of a truncated HMGI-C gene induces gigantism associated with lipomatosis. Cancer Res., 59, 4793–4797.[Abstract/Free Full Text]
  113. Arlotta,P., Tai,A.K., Manfioletti,G., Clifford,C., Jay,G. and Ono,S.J. (2000) Transgenic mice expressing a truncated form of the high mobility group I-C protein develop adiposity and an abnormally high prevalence of lipomas. J. Biol. Chem., 275, 14394–14400.[Abstract/Free Full Text]
  114. Fletcher,J.A., Pinkus,G.S., Donovan,K., Naeem,R., Sugarbaker,D.J., Mentzer,S., Pinkus,J.L. and Longtine,J. (1992) Clonal rearrangement of chromosome band 6p21 in the mesenchymal component of pulmonary chondroid hamartoma. Cancer Res., 52, 6224–6228.[Abstract]
  115. Kazmierczak,B., Dal Cin,P., Wanschura,S., Borrmann,L., Fusco,A., Van den Berghe,H. and Bullerdiek,J. (1998) HMGIY is the target of 6p21.3 rearrangements in various benign mesenchymal tumors. Genes Chromo. Cancer, 23, 279–285.[ISI]
  116. Xiao,S., Lux,M.L., Reeves,R., Hudson,T.J. and Fletcher,J.A. (1997) HMGI (Y) activation by chromosome 6p21 rearrangements in multilineage mesenchymal cells from pulmonary hamartoma. Am. J. Pathol., 150, 901–910.[Abstract]
  117. Williams,A.J., Powell,W.L., Collins,T. and Morton,C.C. (1997) HMGI (Y) expression in human uterine leiomyomata. Involvement of another high-mobility group architectural factor in a benign neoplasm. Am. J. Pathol., 150, 911–918.[Abstract]
  118. Tkachenko,A., Ashar,H.R., Meloni,A.M., Sandberg,A.A. and Chada,K.K. (1997) Misexpression of disrupted HMGI architectural factors activates alternative pathways of tumorigenesis. Cancer Res., 57, 2276–2280.[Abstract]
  119. Student,A.K., Hsu,R.Y. and Lane,M.D. (1980) Induction of fatty acid synthetase synthesis in differentiating 3T3-L1 preadipocytes. J. Biol. Chem., 255, 4745–4750.[Abstract/Free Full Text]
  120. Melillo,R.M., Pierantoni,G., Scala,S. et al. (2001) Critical role of the HMGI (Y) proteins in adipocytic cell growth and differentiation. Mol. Cell. Biol., 21, 2485–2495.[Abstract/Free Full Text]
  121. Fedele,M., Piezantoni,G.M., Bezlingiezi,M.T., Baista,S., Balolassazze, G., Munshi,N., Dentice,M., Thamos,D., Santoro,M., Viglietto,G. and Fusco,A. (2001) Overexpression of proteins HMGA1 induces cell cycles deregulation and apoptosis in normal rat thyroid cells. Cancer Res., 61, 4583–4590.[Abstract/Free Full Text]
Received February 20, 2001; revised May 15, 2001; accepted May 22, 2001.