Purpose: To determine whether -CONH-(CH 2 ) 6 -NH 3 + Cl À functionalized single-walled carbon nanotubes (SWNT) carrying complexed small interfering RNA (siRNA) can enter into tumor cells, wherein they release the siRNA to silence the targeted gene. Experimental Design: -CONH-(CH 2 ) 6 -NH 3 + Cl À was used to mediate the conjugation of telomerase reverse transcriptase (TERT) siRNA to SWNTs. The ability of TERT siRNA delivered via SWNTcomplexes to silence the expression of TERT was assessed by their effects on the proliferation and growth of tumor cells both in vitro and in mouse models. Results: The functionalized SWNTs -CONH-(CH 2 ) 6 -NH 3 + Cl À could facilitate the coupling of siRNAs that specifically target murine TERT expression to form the mTERT siRNA:SWNT+ complex. These functionalized SWNTs rapidly entered three cultured murine tumor cell lines, suppressed mTERT expression, and produced growth arrest. Injection of mTERT siRNA:SWNT+ complexes into s.c. Lewis lung tumors reduced tumor growth. Furthermore, human TERT siRNA:SWNT+ complexes also suppressed the growth of human HeLa cells both in vitro and when injected into tumors in nude mice. Conclusions: -CONH-(CH 2 ) 6 -NH 3 + Cl À functionalized SWNTs carry complexed siRNA into tumor cells, wherein they release the siRNA from the nanotube sidewalls to silence the targeted gene.The -CONH-(CH 2 ) 6 -NH 3 + Cl À functionalized SWNTs may represent a new class of molecular transporters applicable for siRNA therapeutics.

No abstract

Background: Bone morphogenetic protein-6 (BMP-6) is critically involved in many developmental processes. Recent studies indicate that BMP-6 is closely related to tumor differentiation and metastasis.

Endochondral bone formation, including chondrocyte proliferation, maturation, terminal differentiation and apoptosis, is one major type of bone formation in the vertebrate skeleton. Numerous signaling molecules, cell cycle regulatory proteins, and transcription factors precisely control the balance between chondrocyte proliferation and differentiation.Runx2 is a critical transcription factor that promotes chondrocyte maturation. In Runx2-knockout (Runx2 -/-) mice, the formation of hypertrophic chondrocytes is severely impaired in some skeletal elements including the femur and the humerus (Inada et al., 1999). Targeted expression of Runx2 in non-hypertrophic Col2a1-expressing chondrocytes accelerates chondrocyte differentiation and rescues the chondrocyte phenotype in Runx2 -/-mice (Takeda et al., 2001;Ueta et al., 2001). By contrast, over expression of a dominantnegative Runx2 in Col2a1-expressing chondrocytes inhibits chondrocyte maturation (Takeda et al., 2001;Ueta et al., 2001). These results indicate that Runx2 plays an important role in chondrocyte maturation and also suggests that Runx2 acts not only in hypertrophic chondrocytes but also in Col2a1-expressing proliferating chondrocytes.Runx3, which also belongs to the Runt-domain family of transcription factors, is crucial for gastric epithelial cell growth, neurogenesis of the dorsal root ganglia and CD8-lineage T cell differentiation (Li et al., 2002;Levanon et al., 2002;Taniuchi et al., 2002;Woolf et al., 2003). In addition, Runx3 also plays an In chondrocytes, PTHrP maintains them in a proliferative state and prevents premature hypertrophy. The mechanism by which PTHrP does this is not fully understood. Both Runx2 and Runx3 are required for chondrocyte maturation. We recently demonstrated that cyclin D1 induces Runx2 protein phosphorylation and degradation. In the present studies, we tested the hypothesis that PTHrP regulates both Runx2 and Runx3 protein stability through cyclin D1. We analyzed the effects of cyclin D1 on Runx3 protein stability and function using COS cells, osteoprogenitor C3H10T1/2 cells and chondrogenic RCJ3.1C5.18 cells. We found that cyclin D1 induced Runx3 degradation in a dose-dependent manner and that both Myctagged Runx3 and endogenous Runx3 interact directly with CDK4 in COS and RCJ3.1C5.18 cells. A conserved CDK recognition site was identified in the C-terminal region of Runx3 by sequence analysis (residues 356-359). Pulse-chase experiments showed that the mutation of Runx3 at Ser356 to alanine (SA-Runx3) increased the half-life of Runx3. By contrast, the mutation at the same serine residue to glutamic acid (SE-Runx3) accelerated Runx3 degradation. In addition, SA-Runx3 was resistant to cyclin D1-induced degradation. GSTRunx3 was strongly phosphorylated by CDK4 in vitro. By contrast, CDK4 had no effect on the phosphorylation of SARunx3. Although both wild-type and SE-Runx3 were ubiquitylated, this was not the case for SA-Runx3. Runx3 degradation by cyclin D1 was completely blocked by the proteasome inhibitor PS1. In C3H10T1/2...

MicroRNAs (miRNAs), which are small noncoding RNA molecules, play important roles in the post-transcriptional regulation process. The microRNA-21 gene (miR-21) has been reported to be highly expressed in various solid tumors, including breast cancer. Bone morphogenetic protein-6 (BMP-6) has been identified as an inhibitor of breast cancer epithelial-mesenchymal transition (EMT) through rescuing E-cadherin expression. We initiated experiments to identify the relationships between miR-21 and BMP-6 in breast cancer progression. Real-time PCR analysis showed that miR-21 expression was very high in MDA-MB-231 cells that expressed little BMP-6. A reverse correlation between BMP-6 and miR-21 was also determined in breast cancer tissue samples. Moreover, BMP-6 inhibited miR-21 transcription in MDA-MB-231 cells. In order to investigate how BMP-6 inhibited the miR-21 promoter (miPPR-21), we constructed a series of miPPR-21 reporters. Luciferase assay results indicated that BMP-6 inhibited miPPR-21 activity through the E2-box and AP-1-binding sites. We also demonstrated that both δEF1 and TPA induced miR-21 expression. Using site-directed mutation and CHIP assay, we found that δEF1 induced miPPR-21 activity by binding to the E2-box on miPPR-21. Moreover, TPA triggered miPPR-21 activity through the AP-1 binding sites. BMP-6 treatment significantly reduced the binding of these factors to miPPR-21 by decreasing the expression of δEF1 and c-Fos/c-Jun. We also demonstrated that BMP-6-induced downregulation of miR-21 modified the activity of PDCD4 3′UTR and inhibited MDA-MB-231 cell invasion. δEF1 overexpression and TPA induction blocked this inhibitory effect of BMP-6. In conclusion, BMP-6-induced inhibition of miR-21 suggests that BMP-6 may function as an anti-metastasis factor by a mechanism involving transcriptional repression of miR-21 in breast cancer.