A tumor suppressor function has been attributed to RUNX3, a member of the RUNX family of transcription factors. Here, we examined alterations in the expression of three members, RUNX1, RUNX2, and RUNX3, and their interacting partner, CBFb, in breast cancer. Among them, RUNX3 was consistently underexpressed in breast cancer cell lines and primary tumors. Fifty percent of the breast cancer cell lines (n = 19) showed hypermethylation at the promoter region and displayed significantly lower levels of RUNX3 mRNA expression (P < 0.0001) and protein (P < 0.001). In primary Singaporean breast cancers, 9 of 44 specimens showed undetectable levels of RUNX3 by immunohistochemistry. In 35 of 44 tumors, however, low levels of RUNX3 protein were present. Remarkably, in each case, protein was mislocalized to the cytoplasm. In primary tumors, hypermethylation of RUNX3 was observed in 23 of 44 cases (52%) and was undetectable in matched adjacent normal breast epithelium. Mislocalization of the protein, with or without methylation, seems to account for RUNX3 inactivation in the vast majority of the tumors. In in vitro and in vivo assays, RUNX3 behaved as a growth suppressor in breast cancer cells. Stable expression of RUNX3 in MDA-MB-231 breast cancer cells led to a more cuboidal phenotype, significantly reduced invasiveness in Matrigel invasion assays, and suppressed tumor formation in immunodeficient mice. This study provides biological and mechanistic insights into RUNX3 as the key member of the family that plays a role in breast cancer. Frequent protein mislocalization and methylation could render RUNX3 a valuable marker for early detection and risk assessment.

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(2010) Proteomic analysis revealed association of aberrant ROS signaling with suberoylanilide hydroxamic acid-induced autophagy in Jurkat T-leukemia cells, Autophagy,6:6,[711][712][713][714][715][716][717][718][719][720][721][722][723][724]

Hepatitis B virus (HBV) is one of the major etiological factors responsible for acute and chronic liver disease and for the development of hepatocellular carcinoma (HCC). To determine the effects of HBV replication on host cell-protein expression, we utilized 2-DE and MS/MS analysis to compare and identify differentially expressed proteins between an HBV-producing cell line HepG2.2.15 and its parental cell line HepG2. Of the 66 spots identified as differentially expressed (+/- over twofold, p <0.05) between the two cell lines, 62 spots (corresponding to 61 unique proteins) were positively identified by MS/MS analysis. These proteins could be clearly divided into three major groups by cluster and metabolic/signaling pathway analysis: proteins involved in retinol metabolism pathway, calcium ion-binding proteins, and proteins associated with protein degradation pathways. Other proteins identified include those that function in diverse biological processes such as signal transduction, immune regulation, molecular chaperone, electron transport/redox regulation, cell proliferation/differentiation, and mRNA splicing. In summary, we profiled proteome alterations between HepG2.2.15 and HepG2 cells. The proteins identified in this study would be useful in revealing the mechanisms underlying HBV-host cell interactions and the development of HCC. This study can also provide some useful clues for antiviral research.

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BackgroundThe chemokine CXCL14 has been reported to play an important role in the progression of many malignancies such as breast cancer and papillary thyroid carcinoma, but the role of CXCL14 in colorectal carcinoma (CRC) remains to be established. The purpose of this study was to investigate the expression pattern and significance of CXCL14 in CRC progression.Method265 colorectal carcinoma specimens and 129 matched adjacent normal colorectal mucosa specimens were collected. Expression of CXCL14 in clinical samples was examined by immunostaining. The effect of CXCL14 on colorectal carcinoma cell proliferation was measured by MTT assay, BrdU incorporation assay and colony formation assay. The impact of CXCL14 on migration and invasion of colorectal carcinoma cells was determined by transwell assay and Matrigel invasion assay, respectively.ResultsCXCL14 expression was significantly up-regulated in tumor tissues compared with adjacent nontumorous mucosa tissues (P < 0.001). Tumoral CXCL14 expression levels were significantly correlated with TNM (Tumor-node-metastasis) stage, histodifferentiation, and tumor size. In multivariate Cox regression analysis, high CXCL14 expression in tumor specimens (n = 91) from stage I/II patients was associated with increased risk for disease recurrence (risk ratio, 2.92; 95% CI, 1.15-7.40; P = 0.024). Elevated CXCL14 expression in tumor specimens (n = 135) from stage III/IV patients correlated with worse overall survival (risk ratio, 3.087; 95% CI, 1.866-5.107; P < 0.001). Functional studies demonstrated that enforced expression of CXCL14 in SW620 colorectal carcinoma cells resulted in more aggressive phenotypes. In contrast, knockdown of CXCL14 expression could mitigate the proliferative, migratory and invasive potential of HCT116 colorectal carcinoma cells.ConclusionTaken together, CXCL14 might be a potential novel prognostic factor to predict the disease recurrence and overall survival and could be a potential target of postoperative adjuvant therapy in CRC patients.

Signal transduction pathways involving the c-Raf protein kinase are frequently activated in tumor cells. We have addressed the relevance of this activation by a loss-offunction approach. An anti-sense phosphorothioate oligonucleotide (ODN) speci®cally targeted against craf mRNA (Monia et al., 1996a) was used to block cRaf protein expression in four di erent cell lines derived from lung, cervical, prostate and colon carcinomas. Concomitant with the abrogation of c-Raf expression we observed the occurrence of classical apoptotic markers, including chromatin condensation, inter-nucleosomal DNA cleavage, annexin V binding and cleavage of PARP, which was followed by cell death, a ecting most of the cell population. This induction of apoptosis occurred independent of the p53 status of the cell. These ®ndings demonstrate that c-Raf can protect tumor cells from undergoing programmed cell death, and suggest that the interference with c-Raf expression or function by ODNs or speci®c drugs could represent a powerful means for improving the e cacy of anti-cancer therapy.Keywords: apoptosis; programmed cell death; Raf; antisense; phosphorothiate ODN The occurrence of pro-apoptotic lesions, including the deregulation of Myc or the loss of pRB, is a hallmark of oncogenesis (Evan et al., 1992). The onset of programmed cell death (PCD) is prevented, however, by the concomitant activation of anti-apoptotic mechanisms, such as the deregulation of speci®c signal transduction cascades, the loss of p53 (Kinzler and Vogelstein, 1994) or the activation of protective Bcl-2 family members (Korsmeyer, 1995;Reed, 1996; Kroemer, 1997). Any manipulation that would shift this intricate balance between pro-and anti-apoptotic pathways could have profound e ects on the fate of a tumor cell, either on its own or in combination with conventional anti-cancer drugs. Since the Raf-Ras pathway is activated in many human tumors, we have been interested in studying its role in controlling PCD in tumor cells. This interest was fostered by two recent reports (Monia et al., 1996a, b) which showed that an anti-sense phosphorothioate oligonucleotide (ODN) speci®cally targeted against c-raf mRNA (ISIS5132) had profound e ects on the growth of the human lung adenocarcinoma cell line A549 both in culture and on xenografts in nude mice. The underlying mechanisms remained, however, unclear.In a ®rst set of experiments, we investigated whether the anti-sense ODN ISIS5132 (Monia et al., 1996a) (subsequently referred to as AS) would also reduce the expression of c-Raf protein in, and a ect the growth of, other tumor cell lines. As shown by the immunoblot in Figure 1, treatment of the lung tumor line A549, the cervical carcinoma cell line HeLa, the prostate carcinoma cell line LNCaP or the colon tumor cell line SW620 with AS led to a dramatic reduction of cRaf protein to levels below the detection limit of the assay. In contrast, a mismatched version of this ODN (Monia et al., 1996a) (termed MM in the present study) had no detectable e ect relative to untreated cont...

The hepatitis B virus-encoded X (HBx) protein coactivates transcription of a variety of viral and cellular genes and it is believed to play essential roles in viral replication and hepatocarcinogenesis. To examine the pleiotropic effects of HBx protein on host cell protein expression, we utilized 2-DE and MS analysis to compare and identify differentially expressed proteins between a stable HBx-transfected cell line (HepG2-HBx), constitutively expressing HBx, and vector control cells. Of the 60 spots identified as differentially expressed (+/- over 2-fold, p < 0.05) between the two cell lines, 54 spots were positively identified by MS/MS analysis. Several recent studies suggested that HBx was involved in regional hypermethylation of tumor suppressor genes and global hypomethylation of satellite 2 repeats during hepatocarcinogenesis; however, no specific gene has been reported as hypomethylated by HBx. Promoter methylation analysis was examined for those protein spots showing significant alterations, and our results revealed that specific genes, such as aldehyde dehydrogenase 1 (ALDH1), can be hypomethylated by HBx, and two calcium ion-binding proteins, S100A6 and S100A4, were hypermethylated by HBx and could be re-expressed by AZA (DNA methylase inhibitor) treatment. Moreover, via cluster and pathway analysis, we proposed a hypothetical model for the HBx regulatory circuit involving aberrant methylation of retinol metabolism-related genes and calcium homeostasis-related genes. In summary, we profiled proteome alterations between HepG2-HBx and control cells, and found that HBx not only induces regional hypermethylation but also specific hypomethylation of host cell genes.