Dianqi Xin

Peroxisome proliferator-activated receptor γ ligands induce cell cycle arrest and apoptosis in human renal carcinoma cell lines

AbstractAim:To study the effect of peroxisome proliferator-actived receptor γ (PPARγ) ligands on cell proliferation and apoptosis in human renal carcinoma cell lines.Methods:The expression of PPARγ was investigated by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. The effect of thiazolidinedione (TZD) PPARγ ligands on growth of renal cell carcinoma (RCC) cells was measured by MTT assay and flow cytometric analysis. Cell death ELISA, Hoechst 33342 fluorescent staining and DNA ladder assay were used to observe the effects of PPARγ ligands on apoptosis. Regulatory proteins of cell cycle and apoptosis were detected by Western blot analysis.Results:PPARγ was expressed at much higher levels in renal tumors than in the normal kidney (2.16±0.85 vs 0.90±0.73; P<0.01). TZD PPARγ ligands inhibited RCC cell growth in a dose-dependent manner with IC50 values of 7.08 μmol/L and 11.32 μmol/L for pioglitazone, and 5.71 μmol/L and 8.38 μmol/L for troglitazone in 786-O and A498 cells, respectively. Cell cycle analysis showed a G0/G1 arrest in human RCC cells following 24-h exposure to TZD. Analysis of cell cycle regulatory proteins revealed that TZD decreased the protein levels of proliferating cell nuclear antigen, pRb, cyclin D1, and Cdk4 but increased the levels of p21 and p27 in a time-dependent manner. Furthermore, high doses of TZD induced massive apoptosis in renal cancer cells, with increased Bax expression and decreased Bcl-2 expression.Conclusion:TZD PPARγ ligands showed potent inhibitory effect on proliferation, and could induce apoptosis in RCC cells. These results suggest that ligands for PPARγ have potential antitumor effects on renal carcinoma cells.

The leader sequence triggers and enhances several functions of clusterin and is instrumental in the progression of human prostate cancer in vivo and in vitro

To investigate the role of the leader sequence (which during clusterin biosynthesis facilitates its proper post‐translational processing and secretion) in the functional activities of clusterin, a ubiquitous secretory glycoprotein with many biological functions, reported to be pro‐apoptotic and anti‐apoptotic in target cells, but for which the dual mechanism remains unclear.

ELL is an HIF-1α partner that regulates and responds to hypoxia response in PC3 cells

Eleven–nineteen lysine‐rich leukemia (ELL) plays an important role in tumorigenesis and animal development. HIF‐1 is a transcriptional factor that functions as a master regulator of O2 homeostasis. Our previous studies showed that a binding partner of ELL, U19/Eaf2, can modulate HIF‐1α activity and hypoxia response, suggesting that ELL may also influence HIF‐1α pathway and hypoxia response.

Identification of over-expressed genes in human renal cell carcinoma by combining suppression subtractive hybridization and cDNA library array

To isolate the over-expressed genes in human renal cell carcinoma (RCC) and analyze its molecular basis of carcinogenesis, we used the mRNA from human RCC tissues as tester and that from the matched normal kidney tissues as driver to construct the suppression subtractive hybridization library. 379 of the subtracted clones were arrayed onto a nylon membrane and the over-expressed genes were then screened by hybridizing the filter with radioactively labeled cDNA from RCC and matched normal kidney tissues. 67 clones over-expressed in RCC by a factor of 6 or more were sequenced and its identities were analyzed in GenBank database. 4 clones were previously unknown fragments and 2 clones represent KIAA genes. The rest clones were the known genes and some of them were RCC-related, including vascular endothelial growth factor, vimentin and tissue factor. Most of the known genes were the RCC-related genes previously unknown, including zinc ribbon domain-containing 1 protein (ZNRD1), pituitary tumor transforming gene1 (PTTG1). Northern blot and semi-quantitative RT-PCR confirmed that the mRNA levels of the 3 novel fragments and 1 KIAA and 3 known genes were significantly higher in RCC than in the matched normal kidney tissues. Immunohistochemical and Western blot analysis for PTTG1 and ZNRD1 revealed increased protein level in RCC. The over-expressed genes in RCC are the potential molecular targets for diagnosis and therapy and it is very important to understand the molecular mechanism of RCC through the profile of over-expressed genes.

Aberrant activity of Wnt/Frizzled signaling pathway in renal cancer cell lines

The expression ofWnt, Wntreceptor-Frizzled, and several other key components in Wnt pathway in renal cancer cell lines was studied. The result of semi-quantitative RT-PCR has shown that the expression level ofWnt5A andhFz5 mRNA were higher in renal cancer cell line (GRC-1) than in normal renal cell line (HK-2). This result has been confirmed byin situ hybridization. The expression of β-catenin protein was obviously higher in GRC-1 than in HK-2 (P< 0.01), but there were no different expressions of its mRNA between 3 lines. The reasons of the overexpression of β-catenin has been investigated by means of immunocytochemistry, SSCP and so on, no mutation ofβ-catenin gene and APC were found. That means that the overexpression ofWnt5A/hFz5 might be the reason of overexpression of β-catenin. It was concluded that the aberrant activity of Wnt pathway might play an important role in renal cell carcinoma.

Changes in the androgen levels in the ventral prostate of spontaneously hypertensive rats after castration.

To characterize the changes in androgen levels in the prostate after castration, as androgens are critical in the progression of prostate cancer after castration, but the time at which the androgen remaining in the prostatic cancer tissue after castration exerts its effects is poorly understood.

Use of suppression subtractive hybridization strategy for cloning and identifying specifically expressed genes of renal cell carcinoma

Using suppression subtractive hybridization, a renal cell carcinoma (RCC) cDNA subtractive library which only contains differently expressed cDNAs between human RCC and normal kidney has been constructed. 200 clones were picked out randomly to perform enzyme digest analysis, a part of them underwent sequence analysis and Northern blot to identify RCC specially expressed genes. Results showed that 190 clones contain 50–400 bp inserts respectively. Sequence analysis was performed in 10 clones. All the 10 sequences were unknown before and derived from 6 unique novel genes among which the cDNA insert RCC18 has five copies. Northern blot analysis showed that RCC18 cDNA expressed highly in RCC, but there was no signal detected in normal kidney, and the full length of RCC18 was about 2.5 kb. The constructed cDNA subtractive library of human RCC is a highly efficient one and lays the solid foundation for large-scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specially expressed genes provided an important clue for researching the mechanism of the occurrence and development of RCC.

Functional interaction of TCF4 with ATF5 to regulate the Wnt signaling pathway

Wnt signaling directs cell-fate choices during embryonic development and tissue tumorigenesis. T cell factor 4 (TCF4) plays a pivotal role in the Wnt signaling pathway. We demonstrate that a specific protein-protein interaction occurs between TCF4 and ATF5 (activating transcription factor 5) —a new member of cAMP response element binding protein (CREB) with the yeast two-hybrid system. The N-terminal and DNA binding domain of TCF4 (TCF4ND, 1–495 aa) and the C-terminal spanning bZIP domain of ATF5 (162–282 aa) were found to be responsible for the interaction, and the C-terminal of ATF5 (ATF5/C) showed a much stronger interaction with TCF4ND than the full-length of ATF5 by detecting the β-gal activity. Furthermore, overexpression of ATF5/C enhanced transcriptional activation by TCF4 proteins in luciferase assay by transient transfection. Taken together, these data suggest that ATF5 may function as a co-activator to potentiate the ability of TCF4 to activate transcription.

Androgen receptor coregulator ARA267-a interacts with death receptor-6 revealed by the yeast two-hybrid

ARA267-α is a newly identified androgen receptor coactivator. In order to further elucidate its precise role in cells, using the ARA267-α fragment containing four PHD and one SET conserved domains as bait we revealed an ARA267-α-PHD-SET-interacting protein, death receptor-6 (DR6), in the yeast two-hybrid screening. DR6 is the member of TNF receptor family and has a death domain in its intracellular cytoplasmic portion (DR6cp) to mediate the cell apoptosis. The interaction between ARA267-α-PHD-SET and DR6cp was confirmedin vitro andin vivo. Our finding implied that androgen signaling pathway might cross talk with apoptosis signaling pathway through the interaction between ARA267-α and DR6.

Interaction of hTCF4 by yeast two-hybrid system

To investigate the interaction of hTCF4, the yeast two-hybrid system has been used for testing the interaction of mutants of hTCF4 with themselves. Mutants of hTCF4 (hTCF4 1 ) and hTCF4 II) have been obtained by polymerase chain reaction (PCR). Bait (hTCF4 I -pDBLeu and hTCF4 II -pDBLeu) and prey (hTCF4 I -pPC86 and hTCF4 II -pPC86) have been constructed by DNA recombination for yeast two-hybrid. Interaction of hTCF4 II with itself is found in the reverse yeast two-hybrid system (GIBCOBRL Co.). However, no interactions are found in hTCF4 II with hTCF4 1 and in hTCF4 1 with hTCF4 1. These results suggest that hTCF4 could interact with itself to form homodimer or homocopolymer and perform the transcriptional activating function through LZ or HLH motifs in nucleus of renal cell carcinoma.