HMGI-C gene expression is not required for in vivo thyroid cell transformation

Stefania Scala, Giuseppe Portella, Donatella Vitagliano, Catherine Ledent1, Gennaro Chiappetta2, Vincenzo Giancotti3, Jacques Dumont1 and Alfredo Fusco4,5

Dipartimento di Biologia e Patologia Cellulare e Molecolare c/o Centro di Endocrinologia ed Oncologia Sperimentale del CNR, Facoltà di Medicina e Chirurgia di Napoli, Università degli Studi di Napoli `Federico II', via Pansini, 5, 80131 Naples, Italy,
1 Université Libre de Bruxelles, Facultè de Medecine, Institute de Recherches Interdisciplinaires Campus Erasme, Bruxelles, Belgium,
2 Istituto Nazionale dei Tumori di Napoli, Fondazione Senatore Pascale, via M. Semmola, 80131 Naples, Italy,
3 Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universita' degli Studi di Trieste, 34127 Trieste, Italy and
4 Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia di Catanzaro, Università di Catanzaro, via Tommaso Campanella, 88100 Catanzaro, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have previously demonstrated that HMGI proteins are required for the transformation of rat thyroid cells by v-mos and v-ras-Ki oncogenes. To determine whether HMGI proteins are also required for in vivo thyroid carcinogenesis, mice carrying a disrupted HMGI-C gene (pygmy mice) were either treated with radioactive iodine or crossed with transgenic mice carrying the E7 papilloma virus oncogene under the transcriptional control of thyroglobulin gene promoter. The pygmy mice developed thyroid carcinomas with the same frequency as occurred in wild-type mice without significant macroscopic and microscopic differences. Therefore, these results indicate that HMGI-C gene expression is not required in in vivo thyroid cell malignant transformation.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The HMGI protein family comprises three members: HMGI-C, HMGI and HMGY. The last two proteins are products of alternative splicing of the same gene, named HMGI(Y) (1), the HMGI-C protein is coded for by a related gene (2). The HMGI proteins bind DNA and participate in the assembly of protein complexes on the promoters of several inducible genes, thus they are defined as architectural transcription factors (35).

The HMGI genes are abundantly and, almost ubiquitously, expressed during embryogenesis (6,7), and are absent or expressed at low levels in adult mouse and human tissues (2,7). HMGI gene overexpression was first described in rat thyroid transformed cells and in experimental thyroid tumors (810). Further studies assessed HMGI overexpression as a common feature of experimental and human malignant neoplasias (1117).

To determine whether overexpression of HMGI-C was important for tumorigenesis or a result of cell transformation, HMGI-C expression was blocked by introducing an antisense HMGI-C cDNA expression construct into rat thyroid cells. When these cells were subsequently infected by Myeloproliferative sarcoma virus (MPSV) and the Kirsten murine sarcoma virus (KiMSV) carrying the v-mos and v-ras-Ki oncogenes, respectively, they were unable to grow in soft agar or to form tumors when injected into athymic mice. Conversely, untransfected rat thyroid cells after retroviral infection formed colonies in soft agar and were highly tumorigenic after injection into athymic mice. Interestingly, in these experiments expression of HMGI(Y) was totally abolished at the RNA level, raising the possibility that blocking transformation also secondarily inhibited expression of these related family members (18). These results suggested that the induction of HMGI-C, and/or HMGI(Y), expression is required in the process of thyroid cell transformation.

Therefore, we aimed to verify whether the HMGI-C gene has a pivotal role in in vivo thyroid neoplastic transformation, evaluating the susceptibility of HMGI-C null mice for developing thyroid malignancies. In fact, transgenic insertional mice with the pygmy phenotype were generated (19). Analysis of the integration locus revealed that it occurred in the HMGI-C locus. Then, mice carrying a disrupted HMGI-C gene were produced (6). These mice also showed a pygmy phenotype with craniofacial defects and decrease in fat tissue with no alteration in the growth hormone–insulin-like growth factor endocrine pathway. The phenotype shown by the HMGI-C–/– and mutant insertional mice was very similar to spontaneous dwarf mice (pg), described previously (20), in which both the HMGI-C alleles are deleted (6).

Therefore, spontaneous HMGI-C mouse pygmy mutants (pg) were either treated with radioactive iodine (131I) or crossed with the TgE7 mice, which carried the E7 gene of the papilloma virus, under the transcriptional control of bovine thyroglobulin gene promoter. The TgE7 mice developed a well-differentiated thyroid carcinoma at 9–11 months of age (21).

Here, we show that the frequency of radiation- and E7-induced thyroid carcinomas was the same in pygmy and wild-type mice, indicating that HMGI-C is not required for in vivo thyroid malignant transformation. On the basis of the previously published data (18) and the HMGI(Y) gene overexpression in all of these carcinomas, we suggest that the expression of HMGI(Y) is sufficient for in vivo thyroid carcinogenesis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Transgenic mice carrying the papilloma type 16 E7 transgene under the transcriptional control of bovine thyroglobulin gene promoter (TgE7) have been described previously (21). Spontaneous mutant pygmy mice have also been described previously (20) and were obtained from The Jackson Laboratory (Bar Harbor, ME).

Experimental thyroid carcinogenesis
For the induction of radiation-induced thyroid neoplasias, 5–8-week-old C3H, homozygous (pg–/–) and heterozygous (pg+/–) spontaneous pygmy mutant mice were treated with 131I at the dose of 10 µCi by intraperitoneal injection. Then 0.5% propyl-thiouracil was added to the drinking water until animals were killed after 50–61 weeks.

Generation of TgE7/pg+/– and TgE7/pg–/– mice
TgE7 mice in a C57 genetic background, were bred for five generations in a C3H genetic background to reduce the strain-related genetic effect on tumor development. They were then crossed with C3H pygmy+/– mice to generate the TgE7/pg+/– mice. Since homozygous pg–/– mice were unfertile, the TgE7/pg+/– were crossed with each other to generate the homozygous TgE7/pg–/– mice.

Southern blot analysis
Southern blotting was performed according to a standard procedure (22). DNA was extracted from tail biopsies and the genomic DNA was digested. For the screening of TgE7 mice, DNA was digested with BamHI. The blots were hybridized with a labeled probe corresponding to the 2.0 kb fragment of the bovine thyroglobulin promoter (21).

The screening for the pygmy genotype was performed by digestion of genomic DNA with EcoR1. The blots were hybridized with a labeled probe corresponding to the 1.7 kb cDNA of mouse HMGI-C (2).

RT–PCR analysis of the expression of HMGI(Y) and HMGI-C
Total RNA was extracted by RNAzol (Tel-Test, Inc., Friendswood, TX). Total RNA, digested with DNase, was reverse transcribed using random exonucleotides as primers (100 mM) and 12 U AMV reverse transcriptase (Gibco) and subsequent PCR amplification was performed as reported previously (23). cDNA (200 ng) was amplified in a 25 µl reaction mixture containing Taq DNA in polymerase buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.4 mM of each primer, 1 U Taq DNA polymerase (Perkin Elmer). The PCR amplification was performed for 30 cycles (94°C for 30 s, 55°C for 2 min and 72°C for 2 min). For the detection of the HMGI(Y) and HMGI-C specific sequences, the primers used were: 5'-AGGAGAATGAGCGAGTCG-3' and 5'-CAGTTTCTTGGGTCTGCC-3' [corresponding to nucleotides 197–214 and 421–438, respectively, of HMGI(Y) cDNA]; and 5'-GGTACCGGTAGAGGCAGTGG-3' and 5'-ACCCCGCAGGAAGTAGAAAG-3' (corresponding to nucleotides 47–66 and 507–526, respectively, of the HMGI-C cDNA). Amplification of the constitutively expressed enzyme glyceraldehyde phosphate dehydrogenase (GAPDH) was performed as an internal control for the amount of cDNA tested. The GAPDH specific primers were forward: 5'-ACATGTTCCAATATGATTCC-3' (corresponding to nucleotides 194–214) and reverse, 5'-TGGACTCCACGACGTACTCAG-3' (corresponding to nucleotides 336–356). The products of the reactions were analyzed on a 2% agarose gel, and then transferred by electroblotting to GeneScreen plus nylon membrane (Du Pont, Boston, MA). DNA was fixed to the membranes by air-drying and UV crosslinking, then membranes were hybridized with the specific probes.

Histological and immunohistochemical procedures
For light microscopy, tissues were fixed by immersion for 24 h in Bouin's solution and embedded in paraffin using standard procedures. Sections (5 µm) were stained with hematoxylin and eosin or hematoxylin and periodic acid–Schiff (PAS) reagent. Frozen sections (4–8 µm) of normal and pathological tissues were cut in a frozen microtome and allowed to dry for 1 h at room temperature, before fixing in acetone for 10 min. The slides were air dried for 2 h at room temperature and then placed in a buffer bath (phosphate buffered saline, PBS) for 5 min before the immunoperoxidase staining procedure.

For the immunohistochemical studies of paraffin-embedded samples, 3–4 µm paraffin sections were deparaffined and then placed in a solution of absolute methanol and 0.3% hydrogen peroxide for 30 min and then washed in PBS before immunoperoxidase staining.

The slides were then incubated overnight at 4°C in a humidified chamber with the antibodies diluted 1:100 in PBS. The slides were subsequently incubated with biotinylated goat anti-rabbit IgG for 20 min (Vectostain ABC kits, Vector Laboratories) and then with premixed reagent ABC (Vector) for 20 min. The immunostaining was performed by incubating the slides in diaminobenzidine (DAB-DAKO) solution containing 0.06 mM DAB and 2 mM hydrogen peroxide in 0.05% PBS (pH 7.6) for 5 min. After chromogen development, the slides were washed, dehydrated with alcohol and xylene, and mounted with coverslips using a permanent-mounting medium (Permount). Micrographs were taken on Kodak Ektachrome film with a photo Zeiss system.

The antibodies used in this study were raised against the synthetic peptide SSSKQQPLASKQ specific for the HMGI(Y) proteins (14). For the HMGI-C immunoreactivity, the antibodies used in this study were raised against the recombinant HMGI-C protein (18).

The specificity of the reaction was validated by the absence of staining when carcinoma samples were stained with antibodies pre-incubated with the peptide against which the antibodies were raised (data not shown). Similarly, no positivity was observed when tumor samples were stained with a pre-immune serum (see Figure 3BGo).



View larger version (147K):
[in this window]
[in a new window]
 
Fig. 3. Immunohistochemical detection of HMGI(Y) in thyroid carcinomas. Paraffin sections from normal thyroid and carcinomas were analyzed by immunohistochemistry using antibodies raised against a specific HMGI(Y) peptide. (A) Immunostaining of a normal thyroid (20x) using antibodies raised versus HMGI(Y). No immunoreactivity was observed. (B) A thyroid papillary carcinoma of a pg+/+TgE7 mouse has been incubated with a pre-immune serum. No nuclear staining was observed. (C and D) Immuno- staining of thyroid papillary carcinomas from pg–/– (C) and pg+/+ (D) treated with 131I. Nuclear staining was observed. (E and F) Immunostaining of thyroid papillary carcinomas from pg–/–Tg E7 ((E) and pg+/+ TgE7 (F). Nuclear staining was observed.

 

    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pygmy (pg–/–) and wild-type (pg+/+) mice showed the same susceptibility to radiation-induced thyroid carcinomas
Eleven pg–/–, 20 pg+/– and 18 C3H mice were treated with radioactive iodine and then a goitrogenic agent as described in Materials and methods. The animals were killed within 40–60 weeks of treatment and their thyroids were examined. The results of the histological examination of the thyroid nodules that developed in these animals indicate that no differences, in terms of carcinoma frequency and histological features, were observed among the different groups (Table IGo and Figure 1A and BGo). These results suggest that the HMGI-C gene is not required for in vivo radioiodine-induced thyroid malignant cell transformation.


View this table:
[in this window]
[in a new window]
 
Table I. Thyroid lesions detected in C3H and pygmy mice after treatment with radioactive iodine
 


View larger version (152K):
[in this window]
[in a new window]
 
Fig. 1. Thyroid carcinoma in pigmy (pg–/–) and wild-type (pg+/+) mice. (A and B) Radiation-induced thyroid carcinomas in pygmy (pg–/–) and wild-type (pg+/+) mice. (C and D) 20x Thyroid carcinomas in mice expressing the HPV E7 oncogene in pygmy (pg–/–) and wild-type (pg+/+) mice, respectively. (E and F) As above, at a higher magnification.

 
Thyroid targeted expression of HPV E7 oncogene induces thyroid carcinomas in pygmy mice (pg–/–) as well as in wild-type (pg+/+) mice
It has been reported previously that transgenic mice expressing the E7 papilloma virus oncogene under the transcriptional control of thyroglobulin develop differentiated and functionally regulated thyroid goiters. Old mice display secondary tumor nodules that mimic the various histological aspects of the human differentiated thyroid cancer (21).

pg–/– mice expressing the E7 transgene under the transcriptional control of the thyroglobulin bovine promoter (TgE7-pg–/–) were generated. Sixteen pg–/–TgE7, 16 pg–/+ TgE7 and 15 TgE7 mice were bred. Two mice from each group were killed at 6 and 9 months, and the thyroids examined. At 12 months, all of the remaining mice were killed. The results of the histological diagnoses of the removed thyroids are summarized in Table IIGo.


View this table:
[in this window]
[in a new window]
 
Table II. Thyroid lesions detected in C3H and pygmy mice carrying the TgE7 transgene
 
At 6 months, nodules characterized by high mitotic rates were observed. Within 9 months we observed the appearance of nodules characterized by cytological and architectural disorders such as micropapillary architecture developed within a macrofollicular area. At 12 months, a large majority of the animals carrying the E7 gene developed a generalized thyroid hyperplasia and hypertrophy with the presence of focal papillary lesions, characterized by cytologic aspects found in the papillary subtype of human thyroid carcinomas, i.e. nuclear polymorphism with nuclear grooves and ground glass cells. However, no differences were observed in the three different groups in tumor development (Table IGo) or histological aspects (Figure 1C–FGo).

Expression of HMGI(Y) and HMGI-C genes in the thyroid carcinomas induced in the pygmy mice
HMGI genes are induced in thyroid cell transformation in vitro and in vivo (810,14), and the expression of the HMGI-C gene is required for in vitro thyroid cell malignant transformation (18). As such, we analyzed the expression of the HMGI genes in the neoplastic thyroid samples originating from wild-type and pygmy mice that had either been treated with radioactive iodine or crossed with the TgE7 mice.

Immunohistochemical analysis of the thyroid carcinoma samples did not show expression of HMGI-C in thyroid carcinomas of pygmy mice (Figure 2B and DGo), while HMGI-C induction was observed in all of the carcinomas from the wild-type C3H (Figure 2C and EGo) and the heterozygous mice (data not shown). As far as the HMGI(Y) gene expression was concerned, the induction was observed in all of the carcinoma samples derived from C3H (Figure 3CGo), homozygous (pg–/–) (Figure 3DGo), heterozygous (pg+/–) (data not shown), TgE7/pg–/– (Figure 3EGo) and TgE7 mice (Figure 3FGo). No staining for HMGI-C and HMGI(Y) was detected in normal thyroid of C3H mice (Figure 2A and 3AGoGo, respectively). The specificity of the reaction was validated by the absence of staining when a neoplastic section was incubated with a pre-immune serum (Figure 3BGo) or with antibodies pre-incubated with the peptide against which antibodies were raised (data not shown).



View larger version (128K):
[in this window]
[in a new window]
 
Fig. 2. Immunohistochemical detection of HMGI-C in thyroid carcinomas. Paraffin sections from normal thyroid and carcinomas were analyzed by immunohistochemistry using antibodies raised against the recombinant HMGI-C protein. (A) Immunostaining of a normal thyroid (20x) using antibodies raised versus HMGI-C. No immunoreactivity was observed. (B and D) Immunostaining of a thyroid papillary carcinoma in pg–/– treated with 131I (B) or in pg–/–TgE7 (D). No nuclear staining was observed. (C and E) Immunostaining of a thyroid papillary carcinoma in pg+/+ treated with 131I (C) or in pg+/+TgE7 (E). Nuclear staining was observed.

 
The same carcinoma samples were analyzed by RT–PCR. The results are shown in Figure 4Go. They parallel those obtained by immunohistochemistry. HMGI-C and HMGI(Y) overexpression was observed in all the carcinoma samples, apart from the absence of the HMGI-C expression in the mice carrying a disrupted HMGI-C genes in both the alleles.



View larger version (50K):
[in this window]
[in a new window]
 
Fig. 4. Expression of HMGI(Y) and HMGI-C gene in the thyroid carcinomas induced in the pygmy mice. Total RNA was extracted from thyroid nodules and amplified by RT–PCR using HMGI(Y) and HMGI-C specific primers. The product of the reaction was analyzed on a 2% agarose gel, transferred by electroblotting to GeneScreen plus nylon membrane and hybridized with the probes corresponding to the the HMGI(Y) probe and HMGI-C genes. The cDNA was co-amplified with GAPDH gene as an internal control. (A) RT–PCR assay for the HMGI-C expression in 3T3-L1 cells (used as positive control), and in thyroid carcinoma derived from pg–/+, pg+/+ and pg–/–. (B) RT–PCR assay for the HMGI(Y) in 3T3-L1, mouse pre-adipocytic cells cells (used as positive control), and in thyroid carcinoma derived from pg–/–, pg+/– and pg+/+.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
HMGI proteins are overexpressed in all of the malignant neoplasias so far analyzed (1117). We previously demonstrated that HMGI protein overexpression has a causal role in thyroid cell transformation, since blocking the HMGI-C synthesis by an antisense methodology prevents the acquisition of the malignant phenotype induced in thyroid cells by the MPSV and KiMSV virus. The lack of induction of AP-1 transcriptional activity by suppression of HMGI synthesis may account for inhibition of the neoplastic cell transformation in absence of HMGI proteins. In fact, thyroid neoplastic transformation was associated with a drastic increase in AP-1 activity, which was blocked by the suppression of the HMGI protein synthesis. The absence of AP-1 transcriptional activity induction, directly or indirectly regulated by the HMGI proteins, would inhibit the expression of AP-1-dependent genes, such as VEGF, collagenase I and stromelisin, which are required to achieve cell neoplastic transformation (24).

To assess the role of the HMGI-C gene in in vivo thyroid cell transformation, spontaneous pygmy mice, in which both the HMGI-C alleles have been deleted, were treated with radioactive iodine. It is worth noting that previous experiments have shown a high frequency of Ki-ras activation in radiation-induced thyroid tumors (25). The animals treated developed thyroid papillary carcinomas with the same efficiency and without any significant morphological differences either in the presence or absence of the HMGI-C protein. The same results were obtained when the pygmy mice were crossed with the TgE7 mice: the hybrid mice continued to develop papillary carcinomas.

The tumors induced in pygmy and wild-type mice were analyzed for the expression of the HMGI genes. Papillary carcinomas from the pg–/– mice were obviously negative for the expression of HMGI-C showing the induction of the HMGI(Y) gene. Conversely, in the pg+/+ mice both the HMGI genes were expressed at high levels.

In conclusion, the results presented here indicate that the HMGI-C is not necessary for thyroid carcinogenesis in vivo. Several hypotheses can be envisaged to explain this discrepancy with our published data showing that HMGI-C is required for v-mos- and v-ras-Ki-induced cell transformation in vitro (18). The models used do not correspond perfectly, and we could hypothesize that radiation-induced transformation might follow a different pathway, which is HMGI-C-independent, and that E7, a nuclear oncogene, may work downstream of the HMGI proteins, even though induction of the HMGI(Y) proteins has been found in all the carcinomas induced by iodine or by the E7 gene. However, we should also consider the hypothesis that HMGI(Y) proteins, rather than HMGI-C, may be required for thyroid cell transformation. This hypothesis is supported by evidence of the blocking of transformed thyroid cell growth by an adenovirus carrying HMGI(Y) antisense sequences which is able to block HMGI(Y) proteins synthesis (26). Alternatively, the expression of only one of the HMGI genes may be sufficient to lead thyroid cells to the malignant phenotype. The absence of HMGI(Y) gene induction in the HMGI-C antisense virally-infected thyroid cells (18) would be consistent with this hypothesis. Obviously, we cannot exclude completely the possibility that HMGI proteins are not required at all in these murine models of thyroid carcinogenesis.

The generation of HMGI(Y) knock-out mice, currently in progress, and analysis of their susceptibility for developing thyroid malignancies should help in the definition of the role of the single HMGI genes in the process of in vivo thyroid carcinogenesis.


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


    Acknowledgments
 
We would like to thank Anna Maria De Bernardo for editing the manuscript. This work has been supported by the Progetto Finalizzato Biotecnologie of the CNR, by the Progetto Finalizzato Ingegneria Genetica, by the `Associazione Italiana per la Ricerca sul Cancro' and the MURST Project `Terapie antineoplastiche innovative' and `Piani di Potenziamento della rete scientifica e tecnologica'. We are indebted to Jean Gilder for editing the text.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Johnson,K.R., Lehn,D.A. and Reeves,R. (1989) Alternative processing of mRNAs coding for human HMG I and HMG Y proteins. Mol. Cell. Biol., 9, 2114–2123.[ISI][Medline]
  2. Manfioletti,G., Giancotti,V., Bandiera,A., Buratti,E., Sautiewre,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]
  3. Thomas,D. and Maniatis,T. (1992) The high mobility group protein HMGI(Y) is required for IFKB-dependent virus induction of the human IFN beta gene. Cell, 71, 777–789.[ISI][Medline]
  4. 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]
  5. Lovell-Badge,R. (1995) Developmental genetics. Living with bad architecture. Nature, 376, 725–726[Medline]
  6. 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]
  7. 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]
  8. 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]
  9. Giancotti,V., Pani,B., D'Andrea,P. et al. (1987) Elevated levels of a specific class of nuclear phosphoroproteins in cells transformed with ras and v-mos oncogenes and by co-transfection with c-myc and Polyoma Middle T genes. EMBO J., 6, 1981–1987.[Abstract]
  10. 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]
  11. Ram,T.G., Reeves,R. and Hosick,H.L. (1993) Elevated High Mobility Group I (Y) gene expression is associated with progressive transformation of mouse mammary epithelial cells. Cancer Res., 53, 2655–2660.[Abstract]
  12. Bussemakers,M.J.G., Van de Ven,W.J.M., Debruyne,F.M.J. and Schalken,J. (1991) Identification of high mobility group protein I (Y) as potential progression marker for prostate cancer by differential hybridization analysis. Cancer Res., 51, 606–611.[Abstract]
  13. 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]
  14. 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 neoplasms. Oncogene, 10, 1307–1314.[ISI][Medline]
  15. Fedele,M., Battista,S., Manfioletti,G., Chiappetta,G., Viglietto,G., Casamassimi,A., Bandiera,A., Santoro,M., Giancotti,V. and Fusco,A. (1996) High mobility group protein I (Y) as potential tumor marker in human colorectal carcinomas. Cancer Res., 56, 1896–1901.[Abstract]
  16. 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]
  17. Hess J.L. (1998) Chromosomal translocations in benign tumors. The HMGI proteins. Am. J. Clin. Pathol., 109, 251–261.[ISI][Medline]
  18. 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]
  19. Xiang,X., Benson,F.B. and Chada,K. (1990) Mini-mouse: disruption of the pygmy locus in a transgenic insertional mutant. Science, 247, 967–969.[ISI][Medline]
  20. Green,M.C. (1989) In Lyon,M. and Searie,A. (eds) Genetic Variants and Strains of the Laboratory Mouse. Oxford University Press, pp. 12–403.
  21. Ledent,C., Marcotte,A., Dumont,J.E., Vassart,G. and Parmentier,M. (1995) Differentiated carcinomas develop as a consequence of the thyroid specific expression of a thyroglobulin-human papillomavirus type 16 E7 transgene. Oncogene, 10, 1789–1797.[ISI][Medline]
  22. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  23. Santoro,M., Carlomagno,F., Hay,I.D. et al. (1992) RET oncogene activation in human thyroid neoplasms is restricted to the papillary carcinoma subtype. J. Clin. Invest., 89, 1517–1522.[ISI][Medline]
  24. 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 Fra1 gene induction which is dependent on the HMGI-C gene products. EMBO J., 17, 5310–5321.
  25. Lemoine,N.R., Mayall,E.S., Williams,E.D., Thurston,V. and Wynford-Thomas,D. (1988) Agent-specific, ras oncogene activation in rat thyroid tumors. Oncogene, 3, 541–544.[ISI][Medline]
  26. Scala,S., Portella,G., Fedele,M., Chiappetta,G. and Fusco,A. (2000) Adenovirus-mediated suppression of the HMGI(Y) protein synthesis as a potential therapy of human malignant neoplasias. Proc. Natl Acad. Sci. USA, 97, 4256–4261.[Abstract/Free Full Text]
Received June 2, 2000; revised November 14, 2000; accepted November 15, 2000.