[CANCER RESEARCH 59, 4793–4797, October 1, 1999]

Advances in Brief

The Expression of a TruncatedHMGI-C Gene Induces GigantismAssociated with Lipomatosis

Sabrina Battista, Vincenzo Fidanza, Monica Fedele, Andres J. P. Klein-Szanto, Eric Outwater, Han Brunner,Massimo Santoro, Carlo M. Croce, and Alfredo Fusco1

Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107 [S. B., V. F., M. F., C. M. C.]; Dipartimento di Biologia e Patologia Cellulare e Molecolare,c/o Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Facolta di Medicina e Chirurgia, Universita degli Studi di Napoli “FedericoII,” 80131 Naples, Italy [S. B., M. F., M S.]; Experimental Histopathology, Fox-Chase Cancer Center, Philadelphia, Pennsylvania 19111 [A. J. P. K-S.]; Image Center, JeffersonMedical College, Philadelphia, Pennsylvania 19107 [E. O.]; Department of Clinical Genetics, University Hospital Nijmegen, 417 Nijmegen, the Netherlands [H. B.]; andDipartimento di Medicina Sperimentale e Clinica, Facolta di Medicina e Chirurgia di Catanzaro, Universita di Catanzaro, 88100 Catanzaro, Italy [A. F.]

Abstract

Rearrangements of theHMGI-C gene have frequently been detected inhuman benign tumors of mesenchymal origin, including lipomas. TheHMGI-C protein has three AT-hook domains and an acidic COOH-terminal tail. The HMGI-C modifications consist in the loss of the C-tailand the fusion with ectopic sequences. Recent results show that the loss ofthe COOH-terminal region, rather than the acquisition of new sequences,is sufficient to confer to HMGI-C the ability to transform NIH3T3 cells.Therefore, transgenic mice carrying a HMGI-C construct (HMGI-C/T),containing only the three AT-hook domains, were generated. The HMGI-C/T mice showed a giant phenotype, together with a predominantlyabdominal/pelvic lipomatosis, suggesting a pivotal role of theHMGI-Ctruncation in the generation of human lipomas.

Introduction

HMGI-C belongs to the HMGI protein family (1), also includingthe HMG-I and HMGI-Y proteins. HMGI proteins bind to the minorgroove of AT-rich DNA sequences, thereby inducing a bend withinthe DNA (2, 3). They are not able to stimulate initiation of transcrip-tion, but they can enhance promoter binding of transcription factors(3–5).

The HMGI-C protein has three separate DNA binding domainsreferred to as “AT-hook” motifs. The COOH-terminal region con-tains a highly acidic tail (1). Rearrangements of theHMGI-C genehave been frequently detected in human benign tumors of mesen-chymal origin, including lipomas, lung hamartomas, uterineleiomyomas, endometrial polyps, fibroadenomas, and adenolipo-mas of the breast (6 –9). Chromosomal translocations involving theregion 12q13–15, where theHMGI-C gene is located, account forthese rearrangements. In most of the human benign tumors, breaksoccur within the third intron of the gene, resulting in chimerictranscripts containing exons 1–3 ofHMGI-C (encoding the AT-hook domains) and ectopic sequences from other genes (6, 7). Insome cases, only a few amino acids are fused to the HMGI-C DNAbinding domains (8, 10). We have recently demonstrated that atruncated HMGI-C, deprived of the acidic tail, is able to neoplas-tically transform the murine fibroblasts NIH3T3 and that theacquisition of ectopic sequences does not increase the transformingability of the truncated form of HMGI-C (11), indicating that thetruncation ofHMGI-C, rather than its fusion with other genes, isresponsible for cell transformation.

Transgenic mice provide a powerful experimental approach todefine the role of oncogenes in neoplastic processesin vivo (12).Therefore, the aim of our work has been to generate transgenic micecarrying a truncatedHMGI-C gene.

In this report, we show that transgenic mice carrying a truncatedHMGI-C gene develop a giant phenotype, together with a drasticexpansion of the retroperitoneal and s.c. white adipose tissue.

Materials and Methods

Generation of Transgenic Mice. The construct carrying the cDNA encod-ing the truncated form of murineHMGI-C gene (pRc/CMV2-HMGI-C/T) hasbeen described previously (11). pRc/CMV-HMGI-C/T construct was electro-porated into ES AB2.2 cells (13), and G418-resistant clones were selected andanalyzed by Southern blot hybridization with a CMV promoter probe (data notshown). Ten positive clones were expanded, and the expression of HMGI-C/Twas evaluated by a semiquantitative RT-PCR-based assay, using a construct-specific primer set, described in the following paragraph. The highest twoHMGI-C/T expressing ES cell clones were microinjected into C57BL6/Jmouse blastocysts and then transferred to pseudopregnant foster mothers(Laboratory Animal Facility, Thomas Jefferson University, Philadelphia, PA).Chimeric mice were crossed to wild-type C57BL6/J mice (Taconic Farm), andgerm-line transmission of the transgene was checked by Southern blot analysisof tail DNA from agouti coat-colored F1 offspring.

RT-PCR Analyses. Tissues from transgenic animals were rapidly dis-sected, frozen on dry ice, and stored at280°C. Total RNA was extracted usingTRI-reagent solution (Molecular Research Center, Cincinnati, OH) accordingto the manufacturer’s protocol and treated with DNase I (GenHunter Corpo-ration, Nashville, TN). Onemg of RNA was reverse transcribed using randomexonucleotides as primers (100 mM) and MuLV reverse transcriptase (Perkin-Elmer). Fiveml of cDNA was amplified in a 25-ml reaction mixture containing1 unit of Taq DNA polymerase (Roche Molecular Biochemicals), 0.4 mM

dNTPs, 2.0 mM MgCl2, 0.2 mM of each primer. The PCR amplification wasperformed for 25 cycles (94°C for 300, 55°C for 300, and 72°C for 300), usingthe Protocol thermal cycler (AMS Biotechnology). Primers designed to spe-cifically amplify the transcripts of the transfected constructs (forward primer,59-ATATAAGCTTGGTACCGGTAGAGGCAGTGG-39; reverse primer, 59-AGTCGAGGCTGATCAGCGAG-39) overlapped the 59end of the clonedgene and the vector pRc/CMV downstream from the cloned gene but upstreamfrom the poly(A) signal site. For detection of the endogenousHMGI-C geneexpression, primers specific for exon 1 and 5 (forward, 59-ATATAAGCTT-GGTACCGGTAGAGGCAGTGG-39; reverse, 59-ATATAAGCTTACCCCG-CAGGAAGTAGAAAG-39) coding sequence were used. In addition, a set ofprimers specific for glyceraldehyde-3-phosphate dehydrogenase (forward, 59-ACATGTTCCAATATGATTCC-39; reverse, 59-TGGACTCCACGACG-TACTCAG-39) was added to each reaction to serve as internal control for theamount of cDNA tested. Amplification of RNA that was not reverse tran-scribed before PCR amplification was performed as well (data not shown). ThePCR products were separated on 2% agarose gel, blotted, and hybridized with

Received 5/24/99; accepted 8/18/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Dipartimento di Biologia ePatologia Cellulare e Molecolare, Facolta di Medicina e Chirurgia di Napoli, via Pan-sini 5, 80131 Naples, Italy. Phone: 39-081-7463056; Fax: 39-081-7463037; E-mail:afusco@synapsis.it.

2 CMV, cytomegalovirus; ES, embryonic stem; RT, reverse transcription; MRI, mag-netic resonance imaging.

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specific probes labeled with [a-32P]dATP using an oligolabeling method(Megaprime, Amersham Pharmacia Biotech) to specific activity equal to orhigher than 73 108 cpm/mg. Hybridization and detection were performed asdescribed previously (14).

Fig. 1. Generation of HMGI-C/T transgenic mice through an ES cell-mediated strategy.

Fig. 2. Detection of HMGI-C and HMGI-C/T expression in transfected ES cells and transgenic mouse tissues by RT-PCR.a, expression of the HMGI-C/T gene in transfected EScells. Construct-specific primer sets, overlapping the 59of the cloned gene and a pRc/CMV sequence, downstream from the cloned gene were used. RNA from NIH3T3 and NIH3T3transfected with the HMGI-C/T construct (11) was used as negative and positive control, respectively.WT,wild type;GAPDH,glyceraldehyde-3-phosphate dehydrogenase.b, transgeneexpression in adult transgenic mouse tissues.c, expression of the endogenousHMGI-C gene. As expected, adult tissues did not express detectable levels of HMGI-C. As positivecontrols, RNAs from a cell line derived from a spindle cell cutaneous carcinoma (A5) and from NIH3T3 fibroblasts were used. Primers specific for exons1 and 5 of theHMGI-C genewere used.d, HMGI-C/T expression in “classical” and “ES-mediated” transgenic mice. The expression level of the transgene was lower in classical HMGI-C/T transgenic mice thanin transgenic mice obtained by the ES-mediated strategy. Construct-specific primers (see “Materials and Methods”) were used.

Fig. 3. Increased weight of transgenic mice. Mean and 95% confidence intervals ofweights of 10 wild-type (M) and transgenic (HMGI-C/T;l) male (a) and female (b) miceas a function of age.

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Histological Analysis. For light microscopy, tissues were fixed by immer-sion in 10% formalin and embedded in paraffin by standard procedures. Fivemm sections were stained with H&E or hematoxylin and periodic acid/Schiffreagent. Frozen sections, 4–8mm thick, of wild-type and transgenic tissueswere cut in a frozen microtome and allowed to dry for 1 h at room temperaturebefore being fixed in acetone for 10 min. The slides were air dried for 2 h atroom temperature and then placed in PBS for 5 min before the immunoper-oxidase staining procedure.

MRI. Imaging was performed on a 1.5-T magnet system (GE medicalSystems) using local receiver coils and an 8-cm field of view. Coronal slices3 mm thick were obtained with T1-weighting (TR/TE5 400/11 ms) with andwithout fat saturation to unequivocally identify lipids in fat.

Results

Generation of Transgenic Mice Carrying a Truncated HMGI-CConstruct. An ES cell-mediated strategy has been used to generatetransgenic mice. It is summarized in Fig. 1. A truncated HMGI-C

cDNA (HMGI-C/T), deprived of the COOH-terminal tail, under thetranscriptional control of the CMV promoter, was transfected into theES cells AB2.2. G418-resistant clones were selected and analyzed bySouthern blot hybridization with a CMV promoter probe (data notshown). Ten positive clones were expanded, and the expression ofHMGI-C/T was evaluated by a semiquantitative RT-PCR-based assay(Fig. 2a). Two transfected cell clones expressing the highest levels ofHMGI-C/T mRNA were microinjected into C57BL6/J mouse blasto-cysts, which were then transferred to pseudopregnant foster mothers.Several chimeric mice, identified by the mixed black/agouti coatcolor, were obtained and crossed with wild-type C57BL6/J mice. Twoindependent HMGI-C/T strains, identified by Southern blot hybrid-ization, were generated and examined. Both the mouse lines showedthe same phenotype. We used as wild-type controls the offspring micefrom the chimera that did not inherit the transgene.

The HMGI-C/T expression was detected by RT-PCR in all of the

Fig. 4. MRI analysis. MRI of wild-type, HMGI-C/T chimeric, and HMGI-C/T transgenic mice.a,frontal sections of 1-year-old mice showing testes(t), bladder (bl), and s.c. (sf) and retroperitoneal(rpf) fat. b, frontal sections of 1-year-old miceshowing kidney (k) and epididimal fat (ef).

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analyzed tissues of transgenic animals (Fig. 2b), without any signif-icant difference among them. Conversely, no detection of the endog-enous gene was observed both in transgenic and wild-type mice (Fig.2c). Immunohistochemical and Western blot analyses, confirmed theexpression of the HMGI-C/T protein in transgenic mice (data notshown).

HMGI-C/T Transgenic Mice Exhibit a Giant Phenotype Asso-ciated with Abdominal/Pelvic Lipomatosis. All of the HMGI-C/Ttransgenic mice (deriving from both the lines) exhibited a giant andobese phenotype (data not shown). At 12 months of age, they showedan average 15% increase in body length (naso-anal), compared towild-type littermates. Moreover, male and female transgenic micerevealed, at the same age, a drastic weight gain (;36%), startingaround the third month (Fig. 3).

MRI of the transgenic and wild-type mice was performed to eval-uate the extent of fat deposition and the presence of other abnormal-ities. Transgenic and chimeric mice showed a drastic expansion of theretroperitoneal and s.c. white adipose tissue (Fig. 4a). Abundant fatstores were also observed in the perirenal and epididimal areas (Fig.4b). Moreover, bladder enlargement was observed (Fig. 4a). MRIanalysis has also shown that the accumulation of fat pads becomesapparent at 3 months of age (not shown).

Autopsy examination of the HMGI-C/T transgenic mice confirmedthe presence of a very abundant abdominal fat mass. Moreover, largeadipose pads were found in other body sites, such as around the

kidneys, in the mediastinum, and at the base of the heart (data notshown).

Histological analysis of the s.c. tissues revealed a prominent s.c.adipose tissue, which was not different from the normal adipose tissue(Fig. 5a). In addition to the increase in fat tissue, chimeric andtransgenic mice showed varying degrees of urogenital abnormalities,including hydrosalpinx (Fig. 5,b and c), clitoral gland hyperplasia(Fig. 5e) and epidermoid cysts (Fig. 5d), dilated bladder, mild hydro-nephrosis (Fig. 5g), cystitis, urinary infections, chronic balanitis (Fig.5f), and testis hypotrophy with hypozoospermia (Fig. 5h). Compres-sion of the urinary tract by the adipose tissue may account for someof the genitourinary pathology. BecauseHMGI-C rearrangementshave been found in several benign mesenchymal tumors, a detailedhistological analysis of all of the tissues was performed to findlipomas or other related tumors. One transgenic female out of 10animals analyzed showed a well circumscribed lipoma (Fig. 5b).However, the overall hypertrophy of the adipose tissue in certainsectors of the anatomy of these mice is consistent with a diagnosis ofabdominal/pelvic lipomatosis.

Analysis of the lipomatous tissue for the expression of severaladipocytic terminal differentiation markers, such asadipocyte aP2gene (aP2),peroxisome-proliferator-activated receptorg (PPARg),lipoprotein lipase(LPL), leptin,andphosphoenolpyruvate carboxyki-nase(PEPCK), by an RT-PCR assay (data not shown) demonstrated

Fig. 5. Histological analysis of transgenic mouse tissues.a, pelvic adipose tissue exhibiting normal cytological and histological features.b, circumscribed and encapsulated adiposetissue nodule (lipoma) in close proximity with a dilated and cystic Fallopian tube (hydrosalpinx).c, another view of the same dilated Fallopian tube showing marked dilation and atrophyof the epithelium.d,epidermoid cyst in clitoral gland.e,clitoral gland hyperplasia showing cystic duct dilation.f, chronic balanitis and ulceration of epithelium.g,hydronephrotic kidneyexhibiting dilated renal calyces.h, decreased cell population in seminiferous tubules (hypozoospermia).

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that the differentiation state of the adipose tissue was not affected bythe expression of the HMGI-C/T gene.

Transgenic mice have been also generated by the classical approachmicroinjecting the same HMGI-C/T construct directly into fertilizedmouse eggs. The expression levels of the transgene were much lowerthan those detected in tissues of the mice generated through theES-mediated strategy (Fig. 2d). Accordingly, only the mice carryingmultiple copies of the transgene showed the giant phenotype associ-ated with lipomatosis (data not shown), indicating that the transgeneexerts its effects in a dose-dependent manner.

Discussion

Here, we report the generation of transgenic mice carrying a trun-cated HMGI-C construct deprived of its COOH-terminal tail by usinga new ES cell-mediated strategy that allows us to generate miceshowing high transgene expression levels. These transgenic miceshowed a giant phenotype, together with a drastic expansion of theretroperitoneal and s.c. white adipose tissue. The great expansion ofthe adipose tissue observed in HMGI-C/T mice suggests a pivotal roleof the HMGI-C rearrangements in generation of human lipomas.Moreover, the giant phenotype shown by the HMGI-C/T mice is themirror image of that of theHMGI-C null mice. In fact, these mice arecharacterized by a pigmy phenotype with a reduction of the adult bodyweight, mainly affecting the fat tissue (15). Based on the combineddata, we suggest that the truncation of theHMGI-C gene leads to anincreased activity of the HMGI-C protein, which in turn stimulatesadipocyte cell growth.

Recently, we have shown that the block of the synthesis of therelated HMGI(Y) protein, by the expression of an antisense construct,induced a drastic increase in the growth rate of the 3T3-L1 cells.3

Moreover, single- and double-HMGI(Y)knockout chimeric mice havean enormous increase in fat tissue.4 These results would indicate thatHMGI(Y), unlikeHMGI-C, exerts a negative role on adipocytic cellgrowth and that the regulation of adipocytic cell proliferation mayresult from the balance of HMGI(Y) and HMGI-C protein functions:a gain in HMGI-C protein activity induces adipocyte cell hyperpro-liferation, whereas the dominance of HMGI(Y) has an opposite effect.

The phenotype shown by the HMGI-C/T transgenic mice closelyresembles a rare benign human disease, known as pelvic lipomatosis,characterized by the proliferation of normal fatty tissue limited to theperirectal and perivisceral spaces of the pelvis; it usually affects menin the fourth decade of life or older (16, 17). Major complications ofthis syndrome are infection and obstruction of the urinary tract andvarious degrees of bladder deformity. Moreover, several other casesof familial lipomatosis have also been described (18). Finally, the

Proteussyndrome, mainly characterized by partial gigantism, lipoma-tosis, and vascular tumors (19), shares several features of the HMGI-C/T mouse phenotype.

Therefore, the HMGI-C/T mice may represent an experimentalmodel for these syndromes, and germ-line HMGI-C rearrangements/mutations may be responsible for some of these lipomatosis diseases.

References

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3 Melillo et al., submitted for publication.4 Manuscript in preparation.

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1999;59:4793-4797. Cancer Res   Sabrina Battista, Vincenzo Fidanza, Monica Fedele, et al.   Gigantism Associated with Lipomatosis

Gene InducesHMGI-CThe Expression of a Truncated

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