Vincenzo Giancotti

Inhibition of HMGI-C protein synthesis suppresses retrovirally induced neoplastic transformation of rat thyroid cells.

Elevated expression of the three high-mobility group I (HMGI) proteins (HMGI, HMGY, and HMGI-C) has previously been correlated with the presence of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells and in experimental thyroid, lung, mammary, and skin carcinomas. Northern (RNA) blot and run-on analyses demonstrated that the induction of HMGI genes in transformed thyroid cells occurs at the transcriptional level. An antisense methodology to block HMGI-C protein synthesis was then used to analyze the role of this protein in the process of thyroid cell transformation. Transfection of an antisense construct for the HMGI-C cDNA into normal thyroid cells, followed by infection with transforming myeloproliferative sarcoma virus or Kirsten murine sarcoma virus, generated cell lines that expressed significant levels of the retroviral transforming oncogenes v-mos or v-ras-Ki and removed the dependency on thyroid-stimulating hormones. However, in contrast with untransfected cells or cells transfected with the sense construct, those containing the antisense construct did not demonstrate the appearance of any malignant phenotypic markers (growth in soft agar and tumorigenicity in athymic mice). A great reduction of the HMGI-C protein levels and the absence of the HMGI(Y) proteins was observed in the HMGI-C antisense-transfected, virally infected cells. Therefore, the HMGI-C protein seems to play a key role in the transformation of these thyroid cells.

Nuclear phosphoproteins HMGA and their relationship with chromatin structure and cancer

The structural characteristics of the three nuclear phosphoproteins of the high mobility group A family are outlined and related to their participation in chromatin structure alteration in many biological processes such as gene expression, neoplastic transformation, differentiation, and apoptosis. The elevated expression of these proteins in tumor cells and their post‐translational modifications, such as phosphorylation, acetylation and methylation, are discussed and suggested as suitable targets for cancer chemotherapy.

Studies on the Role and Mode of Operation of the Very‐Lysine‐Rich Histone H1 (F1) in Eukaryote Chromatin

The nuclear magnetic resonance (NMR) spectrum of chromatin at ionic strengths below about 0.5 M may be attributed solely to its histone H1 component. The effect of various ions and urea on the complex has been investigated using NMR and confirm that the contraction of the complex on increase of ionic strength is largely due to electrostatic interactions. A detailed study of the H1 - DNA complex has also been undertaken. The behaviour of H1 in the two cases is virtually identical, implying that in chromatin the H1 is complexed with the DNA rather than with the other histones. Microcalorimetric measurements reveal that the binding of H1 to DNA is athermic or involves a heat of reaction which is very small indeed.

Analysis of the HMGI nuclear proteins in mouse neoplastic cells induced by different procedures

Four malignant tumors induced in mouse by different experimental procedures were compared as regards their high-mobility-group (HMG) proteins. All tumors showed the complete set of three HMG proteins which we call HMGI-C, I-D, and I-E. The presence of the three HMGI proteins is a characteristic of the transformed phenotype regardless of whether the tumor was chemically, virally, or spontaneously derived. However, the level of expression of the HMGI proteins is not constant in the four tumors. Using reverse-phase HPLC, the individual HMGI proteins were isolated from the spontaneously derived tumor (Lewis lung carcinoma) and shown by amino acid analysis to be similar to those previously obtained from a tumor grown in nude mice by inoculation of in vitro-transformed cells.

Transcriptional Activation of the Cyclin A Gene by the Architectural Transcription Factor HMGA2

ABSTRACT The HMGA2 protein belongs to the HMGA family of architectural transcription factors, which play an important role in chromatin organization. HMGA proteins are overexpressed in several experimental and human tumors and have been implicated in the process of neoplastic transformation. Hmga2 knockout results in the pygmy phenotype in mice and in a decreased growth rate of embryonic fibroblasts, thus indicating a role for HMGA2 in cell proliferation. Here we show that HMGA2 associates with the E1A-regulated transcriptional repressor p120E4F, interfering with p120E4F binding to the cyclin A promoter. Ectopic expression of HMGA2 results in the activation of the cyclin A promoter and induction of the endogenous cyclin A gene. In addition, chromatin immunoprecipitation experiments show that HMGA2 associates with the cyclin A promoter only when the gene is transcriptionally activated. These data identify the cyclin A gene as a cellular target for HMGA2 and, for the first time, suggest a mechanism for HMGA2-dependent cell cycle regulation.

Thyroid cell transformation requires the expression of the HMGA1 proteins

Elevated expression of HMGA1 and HMGA2 proteins is correlated with a highly malignant phenotype in several human tumors. We previously demonstrated that the block of HMGA2 protein synthesis prevented rat thyroid cell transformation by murine retroviruses. Suppression of HMGA2 synthesis was associated with lack of induction of HMGA1 proteins suggesting that both HMGA1 and HMGA2 play a role in the process of neoplastic transformation. To determine the role of the HMGA1 gene in thyroid cell transformation, we blocked HMGA1 protein synthesis by an antisense methodology. Here we report that transfection of an HMGA1 cDNA antisense construct into a normal rat thyroid cell line (FRTL-5 Cl2), followed by infection with Kirsten murine sarcoma virus (KiMSV), generated a transformed cell line that expresses high levels of the v-ras-Ki oncogene and that does not require thyroid-stimulating hormones for growth. However, this cell line does not show the malignant phenotype, i.e., it neither grows in soft agar nor induces tumors after injection in athymic mice. Moreover, the lack of the neoplastic phenotype in the virus-infected thyroid cells carrying the HMGA1 antisense construct correlates with the absence of induction of AP-1 transcriptional activity.

The AT-hook of the Chromatin Architectural Transcription Factor High Mobility Group A1a Is Arginine-methylated by Protein Arginine Methyltransferase 6

The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg57 and Arg59, two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization.

HMGA1 inhibits the function of p53 family members in thyroid cancer cells

HMGA1 is an architectural transcription factor expressed at high levels in transformed cells and tumors. Several lines of evidence indicate that HMGA1 up-regulation is involved in the malignant transformation of thyroid epithelial cells. However, the mechanisms underlying the effect of HMGA1 on thyroid cancer cell phenotype are not fully understood. We now show that in thyroid cancer cells, HMGA1 down-regulation by small interfering RNA and antisense techniques results in enhanced transcriptional activity of p53, TAp63alpha, TAp73alpha, and, consequently, increased apoptosis. Coimmunoprecipitation and pull-down experiments with deletion mutants showed that the COOH-terminal oligomerization domain of p53 family members is required for direct interaction with HMGA1. Moreover, inhibition of HMGA1 expression in thyroid cancer cells resulted in increased p53 oligomerization in response to the DNA-damaging agent doxorubicin. Finally, electrophoretic mobility shift assay experiments showed that the p53-HMGA1 interaction results in reduced DNA-binding activity. These results indicate a new function of HMGA1 in the regulation of p53 family members, thus providing new mechanistic insights in tumor progression.

High mobility group HMGI(Y) protein expression in human colorectal hyperplastic and neoplastic diseases.

HMGI(Y) proteins are overexpressed in experimental and human malignancies, including colon, prostate and thyroid carcinomas. To determine at which step of the carcinogenic process HMGI(Y) induction occurs, we analysed the expression of the HMGI(Y) proteins in hyperplastic, preneoplastic and neoplastic tissues of colorectal origin by immunohistochemistry. All the colorectal carcinomas were HMGI(Y)‐positive, whereas no expression was detected in normal colon mucosa tissue. HMGI(Y) expression in adenomas was closely correlated with the degree of cellular atypia. Only 2 of the 18 non‐neoplastic polyps tested were HMGI(Y)‐positive. These data indicate that HMGI(Y) protein induction is associated with the early stages of neoplastic transformation of colon cells and only rarely with colon cell hyperproliferation.

HMGA molecular network: From transcriptional regulation to chromatin remodeling.

Nuclear functions rely on the activity of a plethora of factors which mostly work in highly coordinated molecular networks. The HMGA proteins are chromatin architectural factors which constitute critical hubs in these networks. HMGA are referred to as oncofetal proteins since they are highly expressed and play essential functions both during embryonic development and neoplastic transformation. A particular feature of HMGA is their intrinsically disordered status, which confers on them an unusual plasticity in contacting molecular partners. Indeed these proteins are able to bind to DNA at the level of AT-rich DNA stretches and to interact with several nuclear factors. In the post-genomic era, and with the advent of proteomic tools for the identification of protein-protein interactions, the number of HMGA molecular partners has increased rapidly. This has led to the extension of our knowledge of the functional involvement of HMGA from the transcriptional regulation field to RNA processing, DNA repair, and chromatin remodeling and dynamics. This review focuses mainly on the protein-protein interaction network of HMGA and its functional outcome. HMGA molecular partners have been functionally classified and all the information collected in a freely available database (http://www.bbcm.units.it/ approximately manfiol/INDEX.HTM).