High Mobility Group Protein AT-hook Research Articles

High mobility group protein AT‐hook 1 (HMGA1) is associated with the development of androgen independence in prostate cancer cells

We previously reported that the level of high mobility group protein AT-hook 1 (HMGA1) is low in androgen-dependent prostate cancer (PCa) cells (LNCaP), but is high in androgen-independent PCa cells (DU145 and PC-3) and that HMGA1 is a strong candidate gene playing a potential role in the progression of PCa. These findings have prompted us to evaluate the effect of HMGA1 on developing androgen independency, which is associated with the progression of PCa. Expression of HMGA1 in PCa cells and mouse tissues was examined by Western blot. In order to examine the effect of HMGA1 on cell growth under androgen-deprived condition, we transfected HMGA1 into LNCaP cells, and siRNA into both DU145 and PC-3 cells, respectively. Androgen-deprivation induced an increase in the level of HMGA1 in LNCaP cells in vitro and in vivo, but did not in normal prostate tissue. Overexpression of HMGA1 maintained the cell growth of LNCaP under androgen-deprived condition. Furthermore, knockdown of HMGA1 suppressed the cell growth of DU145 and PC-3. These data suggest that elevated expression of HMGA1 is associated with the transition of PCa cells from androgen-sensitive to androgen-independent growth and plays a role in the cell growth of androgen-independent PCa cells.

Abstract 606: Expression and role of high mobility group protein AT-hook 1 (HMGA1) in human renal cell carcinoma

Abstract Even though molecular targeted therapy has improved the clinical outcome of metastatic renal cell carcinoma (mRCC) patients, complete response is rarely observed. Identification of molecules possessing significant molecular functions in mRCC is mandatory for developing new therapeutic modalities for mRCC. High mobility group protein AT-hook 1 (HMGA1) is overexpressed in many types of cancer cells, but is not or at very low levels expressed in normal adult tissues. Expression of HMGA1 has been reported to be associated with metastatic potential and progression of cancer. We have reported that transfection of HMGA1 into prostate cancer cell lines induces chromosomal rearrangement (Cancer Res, 2002) and expression of matrix metalloproteinase 2 (Prostate, 2004) suggesting that HMGA1 may play an important role in the progression of prostate cancer. In this study, we examined expression and function of HMGA1 in human renal cell carcinoma (RCC). Expression of HMGA1 in six human RCC cell lines, Caki-1, ACHN, NC 65, RCC-4, 786-O, and A498, was examined by immunoblot and immunohistochemistry. The expression level of HMGA1 detected by immunoblot was Caki-1=ACHN>NC65>786-O, while RCC-4 and A498 barely expressed HMGA1. Immunohistochemistry showed nuclear localization of HMGA1 in these cell lines. Expression of HMGA1 in renal tumor tissue and normal kidney tissue from the same patients who underwent radical nephrectomy was examined by immunoblot. Immunoblot of surgical samples of 41 cases of RCC revealed that HMGA1 is not expressed in normal kidney tissues from all the cases. On the other hand, immunoblot showed that expression of HMGA1 was observed in 1 out of 23 (4%) non-metastatic cases (M0) compared to 6 out of 18 (33%) metastatic cases (M1) (P=0.031), and 3 out of 29 (10%) G1-2 cases compared to 4 out of 9 (44%) G3 cases (P=0.041). Colony formation assay and flowcytometry were performed using Caki-1 after transfecting with siRNA to knock down HMGA1. Knock-down of HMGA1 expression in Caki-1 cells by siRNA remarkably suppressed colony formation (control siRNA 86.0±5.3 vs HMGA1 siRNA 7.3±1.5, P<0.001)and also induced apoptosis associated with increased subG1 fraction (control siRNA 1.9% vs HMGA1 siRNA 34.6%). These findings suggest that HMGA1 might be correlated with histological grade and metastatic potential of RCC associated with enhanced anti-apoptotic mechanism. No expression in normal kidney tissues, preferential expression in renal tumor tissues of metastatic cases, and remarkable suppression of colony formation by siRNA suggest that HMGA1 might be a potential target molecule for novel therapeutic modalities of mRCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 606. doi:10.1158/1538-7445.AM2011-606

238 EXPRESSION AND ROLE OF HIGH MOBILITY GROUP PROTEIN AT-HOOK 1 (HMGA1) IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011238 EXPRESSION AND ROLE OF HIGH MOBILITY GROUP PROTEIN AT-HOOK 1 (HMGA1) IN RENAL CELL CARCINOMA Natsuki Takaha, Ichiro Takeuchi, Yoshihiro Sowa, Yasunori Kimura, Terukazu Nakamura, Fumiya Hongo, Kazuya Mikami, Akihiro Kawauchi, and Tsuneharu Miki Natsuki TakahaNatsuki Takaha Kyoto, Japan More articles by this author , Ichiro TakeuchiIchiro Takeuchi Kyoto, Japan More articles by this author , Yoshihiro SowaYoshihiro Sowa Kyoto, Japan More articles by this author , Yasunori KimuraYasunori Kimura Kyoto, Japan More articles by this author , Terukazu NakamuraTerukazu Nakamura Kyoto, Japan More articles by this author , Fumiya HongoFumiya Hongo Kyoto, Japan More articles by this author , Kazuya MikamiKazuya Mikami Kyoto, Japan More articles by this author , Akihiro KawauchiAkihiro Kawauchi Kyoto, Japan More articles by this author , and Tsuneharu MikiTsuneharu Miki Kyoto, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.307AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Even though molecular targeted therapy has improved the clinical outcome of metastatic renal cell carcinoma (mRCC) patients, complete response is rarely observed. Identification of molecules possessing significant molecular functions in mRCC is mandatory for developing novel therapeutic modalities to improve clinical outcome. High mobility group protein AT-hook 1 (HMGA1) is overexpressed in many types of cancer cells, but is not or at very low levels expressed in normal adult tissues. Expression of HMGA1 has been reported to be associated with metastatic potential and progression of cancer. In this study, we examined expression and function of HMGA1 in human renal cell carcinoma (RCC). METHODS Expression of HMGA1 in six human RCC cell lines, Caki-1, ACHN, NC 65, RCC-4, 786-O, and A498 was examined by immunoblot and immunohistochemistry. Expression of HMGA1 in renal tumor and normal kidney tissue from the same patients who underwent radical nephrectomy was examined by immunoblot. Colony formation assay and flowcytometry were performed using Caki-1 after transfecting with siRNA to knock down HMGA1. RESULTS The expression level of HMGA1 detected by immunoblot was Caki-1=ACHN>NC65>786-O, while RCC-4 and A498 barely expressed HMGA1. Immunohistochemistry showed nuclear localization of HMGA1 in these cell lines. Immunoblot of surgical samples of 41 cases of RCC revealed that HMGA1 is not expressed in normal kidney tissues of all the cases. On the other hand, immunoblot showed that expression of HMGA1 was observed in 1 out of 23 (4%) non-metastatic cases (M0) compared to 6 out of 18 (33%) metastatic cases (M1) (P=0.031), and 3 out of 29 (10%) G1–2 cases compared to 4 out of 9 (44%) G3 cases (P=0.041). Knock-down of HMGA1 expression in Caki-1 cells by siRNA remarkably suppressed colony formation (control siRNA 86.0±5.3 vs HMGA1 siRNA 7.3±1.5, P<0.001) and also induced apoptosis associated with increased subG1 fraction (control siRNA 1.9% vs HMGA1 siRNA 34.6%). CONCLUSIONS These findings suggest that HMGA1 might be correlated with histological grade and metastatic potential of RCC associated with enhanced anti-apoptotic mechanism. No expression in normal kidney tissues, preferential expression in renal tumor tissues of metastatic cases, and remarkable suppression of colony formation by siRNA suggest that HMGA1 might be a potential target molecule for novel therapeutic modalities of mRCC. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e97 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Natsuki Takaha Kyoto, Japan More articles by this author Ichiro Takeuchi Kyoto, Japan More articles by this author Yoshihiro Sowa Kyoto, Japan More articles by this author Yasunori Kimura Kyoto, Japan More articles by this author Terukazu Nakamura Kyoto, Japan More articles by this author Fumiya Hongo Kyoto, Japan More articles by this author Kazuya Mikami Kyoto, Japan More articles by this author Akihiro Kawauchi Kyoto, Japan More articles by this author Tsuneharu Miki Kyoto, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

DNA Bending by the Mammalian High-Mobility Group Protein AT Hook 2

The mammalian high-mobility group protein AT hook 2 (HMGA2) is a DNA binding protein that specifically recognizes the minor groove of AT-rich DNA sequences. Disruption of its expression pattern is directly linked to oncogenesis and obesity. In this paper, we constructed a new plasmid pBendAT to study HMGA2-induced DNA bending. pBendAT carries a 230 bp DNA segment containing five pairs of restriction enzyme sites, which can be used to produce a set of DNA fragments of identical length to study protein-induced DNA bending. The DNA fragments of identical length can also be generated using PCR amplification. Since pBendAT does not contain more than three consecutive AT base pairs, it is suitable for the assessment of DNA bending induced by proteins recognizing AT-rich DNA sequences. Indeed, using pBendAT, we demonstrated that HMGA2 is a DNA bending protein and bends all three tested DNA binding sequences of HMGA2, SELEX1, SELEX2, and PRDII. The DNA bending angles were estimated to be 34.2 degrees , 33.5 degrees , and 35.4 degrees , respectively.

Binding the Mammalian High Mobility Group Protein AT-hook 2 to AT-Rich Deoxyoligonucleotides: Enthalpy-Entropy Compensation

HMGA2 is a DNA minor-groove binding protein. We previously demonstrated that HMGA2 binds to AT-rich DNA with very high binding affinity where the binding of HMGA2 to poly(dA-dT)2 is enthalpy-driven and to poly(dA)poly(dT) is entropy-driven. This is a typical example of enthalpy-entropy compensation. To further study enthalpy-entropy compensation of HMGA2, we used isothermal-titration-calorimetry to examine the interactions of HMGA2 with two AT-rich DNA hairpins: 5′-CCAAAAAAAAAAAAAAAGCCCCCGCTTTTTTTTTTTTTTTGG-3′ (FL-AT-1) and 5′-CCATATATATATATATAGCCCCCGCTATATATATATATATGG-3′ (FL-AT-2). Surprisingly, we observed an atypical isothermal-titration-calorimetry-binding curve at low-salt aqueous solutions whereby the apparent binding-enthalpy decreased dramatically as the titration approached the end. This unusual behavior can be attributed to the DNA-annealing coupled to the ligand DNA-binding and is eliminated by increasing the salt concentration to ∼200 mM. At this condition, HMGA2 binding to FL-AT-1 is entropy-driven and to FL-AT-2 is enthalpy-driven. Interestingly, the DNA-binding free energies for HMGA2 binding to both hairpins are almost temperature independent; however, the enthalpy-entropy changes are dependent on temperature, which is another aspect of enthalpy-entropy compensation. The heat capacity change for HMGA2 binding to FL-AT-1 and FL-AT-2 are almost identical, indicating that the solvent displacement and charge-charge interaction in the coupled folding/binding processes for both binding reactions are similar.

Specific Recognition of AT-Rich DNA Sequences by the Mammalian High Mobility Group Protein AT-hook 2: A SELEX Study

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a transcriptional factor involved in cell differentiation and transformation. Disruption of its normal expression pattern is directly linked to oncogenesis and obesity. HMGA2 contains three "AT-hook" DNA binding domains, which specifically bind to the minor groove of AT-rich sequences. Using a PCR-based systematic evolution of ligands by exponential enrichment (SELEX) procedure, we have identified two consensus sequences for HMGA2, 5'-ATATTCGCGAWWATT-3' and 5'-ATATTGCGCAWWATT-3', where W represents A or T. These two consensus sequences have a unique and interesting feature: the first five base pairs are AT-rich, the middle four base pairs are GC-rich, and the last six base pairs are AT-rich. Our results showed that all three of these segments are critical for high-affinity binding of HMGA2 to DNA. For example, if one of the AT-rich sequences is mutated to a non-AT-rich sequence, the DNA binding affinity of HMGA2 is reduced at least 100-fold. Intriguingly, if the GC-segment is replaced by an AT-rich segment, the binding affinity of HMGA2 is reduced approximately 5-fold. Identification of the consensus sequences for HMGA2 represents an important step toward finding its binding sites within the genome.