Overexpression of ADP-ribosylation factor 1 in human gastric carcinoma and its clinicopathological significance.

Gastric cancer is the sixth leading cause of cancer-related death in Taiwan, and the identification of related factors is essential to increase patient survival. ADP-ribosylation factor 1 (ARF1) was initially identified using 2-D electrophoresis combined with MALDI-time-of-flight mass spectrometry. ADP-ribosylation factor 1 belongs to the Ras superfamily or GTP-binding protein family and has been shown to enhance cell proliferation. In the current study, we evaluated the potential of ARF1 as a biomarker for gastric cancer detection. ADP-ribosylation factor 1 mRNA was upregulated in tumor tissues (compared with adjacent non-tumor tissues, n = 55) in approximately 67.2% of gastric cancer patients. Expression of ARF1 protein was additionally observed using Western blot and immunohistochemistry (IHC) analyses. The clinicopathological correlations of ARF1 were further evaluated. Elevated ARF1 expression was strongly correlated with lymph node metastasis (P = 0.008), serosal invasion (P = 0.046), lymphatic invasion (P = 0.035), and pathological staging (P = 0.010). Moreover, the 5-year survival rate for the lower ARF1 expression group (n = 50; IHC score < 90) was higher than that of the higher expression group (n = 60; IHC score ≥ 90) (P = 0.0228, log-rank test). To establish the specific function of ARF1 in human gastric cancer, isogenic ARF1-overexpressing cell lines were prepared. Our results showed that ARF1-overexpressing clones display enhanced cell proliferation, migration, and invasion. Furthermore, ARF1-overexpression might contribute to poor prognosis of patients. These findings collectively support the utility of ARF1 as a novel prognostic marker for gastric cancer and its role in cell invasion.

Regulation of dendritic development by the ARF exchange factor ARNO.

Here we analyzed the role of ARF6, a member of the ADP-ribosylation factor (ARF) family of small GTPases, in dendritic arbor development in rat hippocampal neurons in culture. Overexpression of the inactive form of the GTP exchange factor ARNO (ARF nucleotide binding site opener) or inactive ARF6 enhanced dendritic branching, whereas coexpression of either Rac1 (a member of the Rho family of small GTPases known to control dendritic dynamics and growth) or active ARF6 with inactive ARNO eliminated the enhanced branching effect. These results indicate that the ARF family of small GTPases contributes to the regulation of dendritic branching, and that ARF6 activation turns on two independent pathways that suppress dendritic branching in vivo: one through Rac1 and the other through ARF6.

Ink4a/Arf expression is a biomarker of aging.

The Ink4a/Arf locus encodes 2 tumor suppressor molecules, p16INK4a and Arf, which are principal mediators of cellular senescence. To study the links between senescence and aging in vivo, we examined Ink4a/Arf expression in rodent models of aging. We show that expression of p16INK4a and Arf markedly increases in almost all rodent tissues with advancing age, while there is little or no change in the expression of other related cell cycle inhibitors. The increase in expression is restricted to well-defined compartments within each organ studied and occurs in both epithelial and stromal cells of diverse lineages. The age-associated increase in expression of p16INK4a and Arf is attenuated in the kidney, ovary, and heart by caloric restriction, and this decrease correlates with diminished expression of an in vivo marker of senescence, as well as decreased pathology of those organs. Last, the age-related increase in Ink4a/Arf expression can be independently attributed to the expression of Ets-1, a known p16INK4a transcriptional activator, as well as unknown Ink4a/Arf coregulatory molecules. These data suggest that expression of the Ink4a/Arf tumor suppressor locus is a robust biomarker, and possible effector, of mammalian aging.

ADP-ribosylation factor 1 expression regulates epithelial-mesenchymal transition and predicts poor clinical outcome in triple-negative breast cancer.

Metastatic capacities are fundamental features of tumor malignancy. ADP-ribosylation factor (ARF) 1 has emerged as a key regulator of invasion in breast cancer cells. However, the importance of this GTPase, in vivo, remains to be demonstrated. We report that ARF1 is highly expressed in breast tumors of the most aggressive and advanced subtypes. Furthermore, we show that lowered expression of ARF1 impairs growth of primary tumors and inhibits lung metastasis in a murine xenograft model. To understand how ARF1 contributes to invasiveness, we used a poorly invasive breast cancer cell line, MCF7 (ER+), and examined the effects of overexpressing ARF1 to levels similar to that found in invasive cell lines. We demonstrate that ARF1 overexpression leads to the epithelial-mesenchymal transition (EMT). Mechanistically, ARF1 controls cell-cell adhesion through ß-catenin and E-cadherin, oncogenic Ras activation and expression of EMT inducers. We further show that ARF1 overexpression enhances invasion, proliferation and resistance to a chemotherapeutic agent. In vivo, ARF1 overexpressing MCF7 cells are able to form more metastases to the lung. Overall, our findings demonstrate that ARF1 is a molecular switch for cancer progression and thus suggest that limiting the expression/activation of this GTPase could help improve outcome for breast cancer patients.

Simple in vitro assay of Arf GAPs and preparation of Arf proteins as substrates.

Defining the interaction of Arf GAPs with specific Arfs is important for understanding their functions in the endocytic system. Cell-based approaches have been valuable for identifying Arfs and Arf GAPs active in the endocytic compartment; however, the cell-based assays have some limitations in establishing relationships among the Arfs and ArfGAPs. Here we describe a simple in vitro assay that will provide a means for comparing Arfs as substrates and serve to complement cell-based studies.

Characterization of two novel ADP ribosylation factors from giant freshwater prawn Macrobrachium rosenbergii and their responses to WSSV challenge.

ADP-ribosylation factors (Arfs) are small GTP-binding proteins that have an essential function in intracellular trafficking and organelle structure. To date, little information is available on the Arfs in the economically important giant freshwater prawn Macrobrachium rosenbergii and their relationship to viral infection. Here we identified two Arf genes from M. rosenbergii (MrArf1 and MrArf2) for the first time. Phylogenetic analysis showed that MrArf1, together with MjArf1 from shrimp Marsupenaeus japonicus belonged to Class I Arfs. By contrast, MrArf2 didn't not match any of the Arfs classes of I/II/III, although it could be clustered with an Arf protein from M. japonicas called MjArfn, which may represent an analog of the Arf. MrArf1 was ubiquitously expressed in all the examined tissues, with the highest transcription level in the hepatopancreas, whereas MrArf2 was only highly expressed in the hepatopancreas and exhibited very low levels in the heart, stomach, gills and intestine. The expression level of MrArf1 in the gills was down-regulated post 24 h WSSV challenge, and reached the maximal level at 48 h. MrArf1 in the hepatopancreas went up from 24 to 48 h WSSV challenge. MrArf2 transcript in the gill also went down at 24 h and then was upregulated at 48 h WSSV challenge. MrArf2 increased significantly in the hepatopancreas 24 h after infection and then went down at 48 h WSSV challenge. RNAi results showed that knockdown of MrArf1 or MrArf2 could inhibit the expression of the envelope protein gene vp28 of the WSSV. So, it could be speculated that MrArf1 and MrArf2 might play important roles in the innate immune system against WSSV infection.

Expression characteristics of ARF1 and SAR1 during development and the de-etiolation process.

ARF1 (ADP-ribosylation factor 1) and SAR1 (secretion-associated RAS super family) are involved in the formation and budding of vesicles throughout plant endomembrane systems. The molecular mechanisms of this transport have been studied extensively in mammalian and yeast cells. However, very little is known about the mechanisms of coat protein complex (COP) formation and recruitment of COP-vesicle cargoes in plants. To provide insights into vesicular trafficking in Pisum sativum L., we investigated mRNA and protein expression patterns of ARF1 and SAR1 in roots and shoots at early growth stages and in the de-etiolation process. We showed that ARF1 was concentrated mostly in the crude Golgi fractions, and SAR1 was concentrated predominantly in the crude ER fractions of de-etiolated shoots. ARF1 and SAR1 proteins were several times more abundant in shoots relative to roots. In total protein homogenates, the expression level of SAR1 and ARF1 was higher in shoots of dark-grown pea plants than light-grown plants. In contrast, ARF1 was higher in roots of light-grown pea relative to roots of dark-grown pea. With ageing, the ARF1 mRNA in roots was reduced, while SAR1 expression increased. Unlike ARF1 transcripts, ARF1 protein levels did not fluctuate significantly in root and shoot tissue during early development. The relative abundance of SAR1 protein in root tissues may suggest a high level of vesicular transport from the ER to the Golgi. Experimental results suggested that white light probably affects the regulation of ARF1 and SAR1 protein levels. On the other hand, short-term white light affects SAR1 but not ARF1.

[Expression and significance of ADP-ribosylation factor 1 (ARF1) in colorectal laterally spreading tumor].

BACKGROUND & OBJECTIVE: Laterally spreading tumor (LST) behaves close to malignant tumor. This study was to search for the genes and functional proteins correlated to growth and development of colorectal LST (CLST), and to reveal the molecular mechanisms of CLST growth pattern and malignant transformation. METHODS: cDNA microarray was used to screen differentially expressed genes among CLST cell line and other 2 colorectal cell lines LoVo and SW480. Different mRNA and protein expressions of the genes were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Compared with LoVo and SW480 cells, 58 genes were up-regulated and 39 genes were down-regulated in CLST cells. mRNA level of ADP-ribosylation factor 1 (ARF1) was significantly higher in CLST cells than in SW480 and LoVo cells (0.93 vs. 0.74 and 0.47, P = 0.04); protein level of ARF1 was significantly higher in CLST cells than in LoVo cells. CONCLUSIONS: CLST has specific gene expression profile. ARF1 is overexpressed in CLST, and may mediate specific biological function of CLST.