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Huang S-L, Shyu R-Y, Yeh M-Y, Jiang S-YThe retinoid-inducible gene I: effect on apoptosis and mitogen-activated kinase signal pathway. Anticancer Res 22: 799-804
Abstract
The retinoid-inducible gene I (RIG1), belonging to the family of type II tumor suppressor genes, was isolated from human gastric cancer cells treated with all-trans retinoic acid. The activity of the RIG1 gene was investigated in this study.
HtTA cervical and TSGH9201 gastric cancer cells were transiently transfected with expression vectors that synthesized RIG1-myc or RIG1-EGFP fusion protein. Cell growth was analyzed by measuring the incorporation of bromodeoxyuridine. Apoptosis was evaluated by the formation of in situ DNA breakage. The activities of mitogen-activated kinase signal pathways were analyzed using signal pathway trans-reporting systems.
Expression of the RIG1-myc fusion protein resulted in decreased cell growth. Both RIG1-EGFP and RIG1-myc fusion proteins induced cellular apoptosis that was characterized by the presence of apoptotic bodies and in situ DNA breakage. The transactivation activities of Elk1, c-Jun and CHOP proteins were suppressed by 80, 50 and 88%, respectively, in HtTA cells expressing the RIG1-myc fusion protein for two days. Similarly, the transactivation activities of the CHOP protein was suppressed in TSGH9201 and HtTA cells transiently expressing RIG1-myc and RIG1-EGFP, respectively.
The RIG1 fusion proteins exhibited growth suppressive and apoptosis-inducing activity. The protein negatively-regulated signal pathways of extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 mitogen-activated kinase.
... To further understand the effect of ATRA on GC, studies have been conducted and three main anti-cancerous effect of this molecule have been identified: (i) inhibition of cell cycle and induction of cell differentiation [88][89][90][91][92]; (ii) pro-apoptotic action [88,[93][94][95], and (iii) inhibition of CSCs properties [88,96,97] (Table 2). ...
... ATRA can inhibit AP1, which is a transcription factor implicated in inflammation and cell proliferation [91] through RARα and RARβ in GC cell lines [90]. ATRA has shown a decreasing effect on GC cell line growth by downregulating the ERK/MAPK pathway, which is implicated in cellular proliferation, survival, differentiation, migration, and angiogenesis [92]. Moreover, according to Nguyen et al., ATRA induces the expression of GCSCs differentiation markers such as cytokeratin 7 (KRT7), osteopontin (SSP1), cytokeratins (PanCK), mucin 6 (MUC6), and trefoil factor 3 (TTF3) in tumorspheres from two GC cell lines and in mice with subcutaneous tumor xenografts of two GC cell lines and two patient derived xenografts (PDX) [19]. ...
... Inhibition of cell proliferation by the inhibition of AP1 transcription factor [90,91]. Downregulation of ERK/MAPK pathway [92]. Induction of the expression of GCSCs differentiation markers [19]. ...
Gastric cancer (GC) is the third leading cause of cancer-related death worldwide with a five-year survival rate of around 25%, and 4% when diagnosed at a metastatic stage. Cancer stem cells (CSC) have recently been characterized as being responsible for resistance to radio/chemotherapies and metastasis formation, opening up perspectives for new targeted therapies. Those CSCs express biomarkers such as cluster of differentiation 44 (CD44) and display high aldehyde dehydrogenase activity that converts vitamin A-derived retinal into retinoic acids. All-trans retinoic acid (ATRA), which has pro-differentiating properties, has revolutionized the prognosis of acute promyelotic leukemia by increasing its remission rate from 15% to 85%. Recent studies have started to show that ATRA also has an anti-tumoral role on solid cancers such as GC. The purpose of this review is therefore to summarize the work that evaluated the effects of ATRA in GC and to evaluate whether its anti-cancerous action involves gastric CSCs targeting. It has been demonstrated that ATRA can block the cell cycle, enhance apoptosis, and decrease gastric CSCs properties in GC cell lines, tumorspheres, and patient-derived xenograft mice models. Therefore, retinoids and new synthetic retinoids seem to be a promising step forward in targeted therapy of gastric CSC in combination with existing chemotherapies. Future studies should probably focus on these points.
... In this family, the protein contains an NC domain at the N-terminus and a hydrophobic membrane-anchoring domain at the C-terminus [11,12]. The proteins in this family exhibit activities that regulate cellular growth, differentiation, and apoptosis [13][14][15][16][17][18][19][20]. ...
... Murine H-rev107, human H-REV107, and human HRASLS were also shown to inhibit RAS-mediated transformation of fibroblasts, EC cells, and tumor cell lines [4,9,21]. Similar inhibition of RAS signaling pathways has been observed in HRASLS2expressing [7] or RIG1-expressing cervical and gastric cancer cells [13,19,20]. In keratinocytes, RIG1 has been shown to stimulate cellular differentiation that is mediated by activating type I tissue transglutaminase or regulating tubulin to drive the formation of the peripheral microtubule ring [15,17,18,22]. ...
... HtTA cervical cancer cells [20] were maintained in RPMI-1640 medium supplemented with 25 mM HEPES, 26 mM NaHCO 3 , 2 mM L-glutamine, penicillin (100 units/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum (FBS) at 37°C in an atmosphere of 5% CO 2 in air. For DNA transfection, cells plated in culture dishes were transfected with the expression vectors using the method of liposome-mediated transfection. ...
Background
H-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family. Previous studies showed that H-rev107 exhibits phospholipase A/acyltransferase (PLA/AT) activity and downregulates H-RAS expression. However, the mode of action and the site of inhibition of H-RAS by H-rev107 are still unknown.
Results
Our results indicate that H-rev107 was co-precipitated with H-RAS and downregulated the levels of activated RAS (RAS-GTP) and ELK1-mediated transactivation in epidermal growth factor-stimulated and H-RAS-cotransfected HtTA cervical cancer cells. Furthermore, an acyl-biotin exchange assay demonstrated that H-rev107 reduced H-RAS palmitoylation. H-rev107 has been shown to be a PLA/AT that is involved in phospholipid metabolism. Treating cells with the PLA/AT inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) or methyl arachidonyl fluorophosphate (MAFP) alleviated H-rev107-induced downregulation of the levels of acylated H-RAS. AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor. In contrast, treating cells with the acyl-protein thioesterase inhibitor palmostatin B enhanced H-rev107-mediated downregulation of acylated H-RAS in H-rev107-expressing cells. Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation. These results suggest that H-rev107 decreases H-RAS activity through its PLA/AT activity to modulate H-RAS acylation.
Conclusions
We made the novel observation that H-rev107 decrease in the steady state levels of H-RAS palmitoylation through the phospholipase A/acyltransferase activity. H-rev107 is likely to suppress activation of the RAS signaling pathway by reducing the levels of palmitoylated H-RAS, which decreases the levels of GTP-bound H-RAS and also the activation of downstream molecules. Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.
... Titres of the antiserum were determined using an enzyme immunoassay. The specificity of the antiserum was determined by Western blotting of cytosol extracts prepared from cells expressing the RARRES3- fusion protein (Huang et al, 2002). ...
... Moderately and poorly differentiated colorectal tumours had progressive loss of the RARRES3 expression. In addition, in vitro studies investigating the transient expression of RARRES3 protein or fusion proteins also detected the growth suppressive and proapoptotic activity of these proteins in several cancer cells (DiSepio et al, 1998; Deucher et al, 2000; Huang et al, 2002). Therefore, RARRES3 functions as a negative growth regulator. ...
... Mutation at KRAS is frequently observed in adenoma tissues (Vogelstein et al, 1988). Our previous studies showed that RARRES3 negatively regulated signal pathways of extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 mitogen-activated kinase, downstream kinases following activation of the Ras protein (Huang et al, 2002). It therefore is likely that high levels of RARRES3 expression in adenoma tissues as observed in this study may prevent malignant transformation in cells harbouring mutations of the Ras family genes through negative regulation of downstream signal pathways of Ras. ...
RARRES3 is a retinoid-inducible class II tumour-suppressor gene. This study analysed the expression of RARRES3 protein in normal, adenoma and carcinoma tissues of the colorectum and its correlation with tumour differentiation. The expression of RARRES3 protein in 151 paraffin-embedded colorectal tissues (11 distal normal mucosa, 20 adenoma and 120 colorectal adenocarcinoma) was determined by immunohistochemistry. RARRES3 protein was expressed in all 11 distal normal, 120 adjacent normal and 20 adenoma tissues. In distal normal tissues, RARRES3 protein was expressed at the highest levels in differentiated mucosal epithelial cells. Among 120 carcinoma tissues, RARRES3 protein was detected in 97.6% (40 out of 41), 79.4% (54 out of 68) and 17.3% (three out of 11) of well-, moderately and poorly differentiated tumours, respectively. The expression of RARRES3 protein was positively correlated to tumour differentiation (test for trend, P<0.0001). Also, levels of RARRES3 protein were found to be higher in the normal tissues adjacent to 14.6% (six out of 41), 51.5% (35 out of 68), and 90.1% (10 out of 11) of well-, moderately and poorly differentiated tumours, respectively. The decreases in tumour differentiation and RARRES3 expression were significantly correlated compared to the adjacent normal tissues (test for trend, P<0.0001). The prognostic implication of RARRES3 protein expression was studied in 107 tumour, and no statistical difference in survival was observed. The expression of RARRES3 protein was positively correlated to cellular differentiation of normal and adenocarcinoma tissues of the colorectum, which supports the role of RARRES3 in normal and malignant epithelial differentiation of colorectum. RARRES3 expression was decreased only in carcinoma tissue, which suggests that altered RARRES3 expression occurs late in colorectal carcinogenesis.
... 14,15 The TIG3 gene is downregulated in basal and squamous cell carcinomas of the skin and its expression can be reconstituted upon administration of tazarotene, a retinoic acid analogue. 16,17 TIG3 is able to suppress proliferation and colony formation, to impair activation of several mitogen activated protein kinases 15,18 and to induce differentiation in human foreskin keratinocytes. 19 In the current study we sought to identify those human tumor types where TIG3 might act as a growth suppressor and further define the signaling pathways that are involved in the downregulation of the gene. ...
... The 2 closely related genes TIG3 and H-REV107-1 display largely overlapping expression patterns and both can induce apoptosis upon expression in carcinoma cell lines. 8, 18 We have described upregulation of H-REV107-1 upon administration of interferon gamma (IFNg) in human ovarian carcinoma cell lines A27/80 and OVCAR-3 and induction of apoptosis in the H-REV107-1 positive cells. Analysis of the TIG3 regulatory sequence 5 0 upstream of the putative transcriptional start site showed the presence of a well conserved IRSE (interferon response element) in close proximity to the translational start site in the first exon. ...
... This finding is of particular interest because TIG3 has been shown to exert a negative influence on the activation of the p38 MAP kinase, the JNK and the MEK-ERK signaling pathways in cells transiently transfected with a TIG3 expression construct. 18 In addition, loss of TIG3 protein was reported in human colon carcinomas 28 that show a high frequency of KRAS mutations suspected to direct activation of the mitogenic MEK-ERK signaling pathways. 29 In ovarian carcinomas activation of MEK-ERK signaling may occur mainly as a result of increased tyrosine kinase receptor expression and activation. ...
The TIG3 gene is a retinoic acid inducible class II tumor suppressor gene downregulated in several human tumors and malignant cell lines. Diminished TIG3 expression correlates with decreased differentiation whereas forced expression of TIG3 suppresses oncogenic signaling pathways and subsequently induces differentiation or apoptosis in tumor cells. Analysis of TIG3 mRNA expression in a large set of cDNA pools derived from matched tumor and normal human tissues showed a significant downregulation of TIG3 in 29% of the cDNA samples obtained from ovarian carcinomas. Using in situ hybridization, we demonstrated expression of TIG3 in the epithelial lining of 7 normal ovaries but loss of TIG3 expression in 15/19 of human ovarian carcinoma tissues. In SKOV-3, CAOV-3 and ES-2 ovarian carcinoma cell lines, downregulation of TIG3 mRNA was reversible and dependent on an activated MEK-ERK signaling pathway. Re-expression of TIG3 mRNA in these cells upon specific interference with the MEK-pathway was correlated with growth inhibition of the cells. In OVCAR-3 and A27/80 ovarian carcinoma cells, TIG3 suppression is MEK-ERK independent, but expression could be reconstituted upon interferon gamma (IFNgamma) induction. Overexpression of TIG3 in A27/80 ovarian carcinoma cells significantly impaired cell growth and despite increased mRNA levels, TIG3 protein was hardly detectable. These results suggest that TIG3 is negatively regulated by an activated MEK-ERK signaling pathway. Further mechanisms must interfere with TIG3 expression that are independent of MEK and partially include interferon-responsive components.
... The genes are expressed in normal tissues in a tissue-specific manner, and are downregulated in various cancer tissues (Duvic et al., 2000;Siegrist et al., 2001;Shyu et al., 2003;Lotz et al., 2005). The proteins exhibit growth suppressive activities when ectopically expressed in various types of cancer cells and RAS transformed fibroblasts (Hajnal et al., 1994;Sers et al., 1997;Akiyama et al., 1999;Huang et al., 2002;Higuchi et al., 2003;Sturniolo et al., 2003;Lotz et al., 2005;Tsai et al., 2007). In addition, HRASLS3 and RARRES3 induce cellular apoptosis that is mediated through caspase-dependent and independent pathways (Sturniolo et al., 2003;Lotz et al., 2005;Tsai et al., 2007). ...
... The plasmid encoding the constitutively activated RAS G12V was purchased from UMR cDNA Resource Center (Rolla, MO). The pRIG1-myc that encoded the RARRES3 fusion protein with myc and His epitopes has been described previously (Huang et al., 2002). The open reading frame of HRASLS2 cDNA, corresponding to nucleotides 57-545 of the sequence deposited in the NCBI database under the accession number NM_017878.1 was amplified from SW480 cells using 5 0 (5 0 -AAGACATCATCTTGAA GGAAGG-3 0 ) and 3 0 (5 0 -TGGATTTATTGCCTTTCCCG-3 0 ) primers. ...
... Three members of the HREV107 protein family (i.e., RARRES3, HRASLS3, and HRASLS) exhibit growth suppressive activities (Hajnal et al., 1994;Sers et al., 1997;Akiyama et al., 1999;Huang et al., 2002;Higuchi et al., 2003;Sturniolo et al., 2003;Lotz et al., 2005;Tsai et al., 2007). Therefore, we investigated the involvement of myctagged wild-type and truncated HRASLS2 fusion proteins in the growth and death of cancer cells. ...
The HRAS-like suppressor 2 (HRASLS2) gene belongs to the H-REV107 gene family involved in the regulation of cell growth and differentiation. HRASLS2 is expressed at high levels in normal tissues of the small intestine, kidney, and trachea. We cloned HRASLS2 cDNA from human SW480 colon cancer cells. Most wild-type, and some N- and C-terminal truncated HRASLS2 (HRASLS2DeltaNDeltaC) were expressed as a granular pattern located at perinuclear region in HtTA cervical cancer cells, while truncation at the C-terminus only (HRASLS2DeltaC) resulted in a diffuse pattern. Wild-type HRASLS2 significantly suppressed colony formation of HeLa and HCT116 cells. HRASLS2DeltaNDeltaC significantly inhibited colony formation of HCT116 cells, but HRASLS2DeltaC did not affect cell growth. HRASLS2 suppressed the RAS-GTP levels and total RAS protein by 44% and 25%, respectively in HtTA cells; however, the suppression was not observed in truncated HRASLS2 variants. In conclusion, the HRASLS2 protein suppressed growth and RAS activities of cancer cells, and the C-terminal hydrophobic domain appeared to be indispensable for both activities.
... Phospholipase A and acyltransferase 4 (PLAAT4) is a member of the HREV107 protein family [1,2]. HREV107 family proteins have been considered tumor suppressors because the expression of HREV107 family proteins induces keratinocyte differentiation, suppresses cell proliferation, and promotes cell death [3][4][5][6][7][8][9][10][11]. All HREV107 members have an N-terminal H-box motif, an NC domain, and a hydrophobic membrane anchor domain at the C-terminus. ...
... HREV107 was first identified in RAS-reverted cell clones, and many members of this family exert similar effects to those of the negative regulation of the RAS signaling pathway [6,11,27,28]. Subsequently, it was found that each member in the HREV107 protein family had PLA/ AT activity that influenced the lipidation of target proteins. A PLA/AT inhibitor alleviated HREV107 family proteinmediated RAS suppression and increased the level of acylated RAS, indicating that HREV107 family proteins are capable of suppressing RAS via its PLA/AT enzymatic activity [23]. ...
Phospholipase A and acyltransferase 4 (PLAAT4) is a member of the HREV107 tumor suppressor gene family. The expression of PLAAT4 has been shown to induce cell death; however, the underlying mechanism remains unknown. Here, we found that RPLP0, a ribosomal protein, can interact with PLAAT4, as determined by yeast two-hybrid screening, coimmunoprecipitation, and colocalization. The level of RPLP0 was suppressed in HtTA cervical cancer cells expressing PLAAT4. In PLAAT4-expressing or RPLP0-silenced cells, decreased cell viability and cell proliferation combined with increased cell death were observed. Furthermore, the levels of cell cycle-associated proteins and anti-apoptotic proteins decreased in PLAAT4-expressing or RPLP0-silenced cells. Similar patterns of cell viability and expression levels of cell-cycle-associated proteins and apoptosis-related proteins were observed in PLAAT4-expressing and RPLP0-knockdown cells, indicating that RPLP0 deficiency might be involved in PLAAT4-mediated growth inhibition and cellular apoptosis.
... The proteins in this family contain a proline-rich motif located at the N-terminus followed by a conserved H-box, an NC domain and a hydrophobic membrane-anchoring domain at the C-terminus [9,10]. HREV107 family proteins play important roles in the regulation of cellular growth, differentiation, and apoptosis [11][12][13][14][15][16][17]. ...
... Each member of the HREV107 H-rev107 Inhibits POR Activity family acts as a phospholipid-related enzyme catalyzing the release of fatty acid from glycerophospholipid [3,7,18,19]. In addition, they have been shown to negatively regulate RAS [1,6,11,17]. PLA 2 activity plays an important role in H-rev107-mediated RAS suppression [36]. ...
H-rev107 is a member of the HREV107 type II tumor suppressor gene family and acts as a phospholipase to catalyze the release of fatty acids from glycerophospholipid. H-rev107 has been shown to play an important role in fat metabolism in adipocytes through the PGE2/cAMP pathway, but the detailed molecular mechanism underlying H-rev107-mediated lipid degradation has not been studied. In this study, the interaction between H-rev107 and cytochrome P450 reductase (POR), which is involved in hepatic lipid content regulation, was determined by yeast two-hybrid screen and confirmed by using in vitro pull down assays and immunofluorescent staining. The expression of POR in H-rev107-expressing cells enhanced the H-rev107-mediated release of arachidonic acid. However, H-rev107 inhibited POR activity and relieved POR-mediated decreased triglyceride content in HtTA and HeLa cervical cells. The inhibitory effect of H-rev107 will be abolished when POR-expressing cells transfected with PLA2-lacking pH-rev107 or treated with PLA2 inhibitor. Silencing of H-rev107 using siRNA resulted in increased glycerol production and reversion of free fatty acid-mediated growth suppression in Huh7 hepatic cells. In summary, our results revealed that H-rev107 is also involved in lipid accumulation in liver cells through the POR pathway via its PLA2 activity.
... Murine H-rev 107 was first isolated from revertants of HRAS-transformed fibroblasts [6]. Subsequently, RIG1, HREV107, and HRASLS were shown to inhibit RAS-mediated signaling of fibroblasts or cancer cell lines [8,10,11,18,19]. Our study further demonstrated a down-regulation caused by RIG1 of activated RAS and total RAS protein, via a posttranslational mechanism [10,11]. ...
... RIG1 belongs to the HREV107 type II tumor suppressor family, which regulates cellular apoptosis, differentiation, and growth suppression. Previous studies have focused on the effects of the HREV107 protein family in suppressing RAS-mediated transformation in cancer cells [6,8,10,11,19,27]. Although the anti-RAS effect induced by HREV107 protein family has been verified, the direct interaction of RAS and RIG1 in cancer cells has not been observed [11]. ...
Retinoid-inducible gene 1 (RIG1), also called tazarotene-induced gene 3, belongs to the HREV107 gene family, which contains five members in humans. RIG1 is expressed in high levels in well-differentiated tissues, but its expression is decreased in cancer tissues and cancer cell lines. We found RIG1 to be highly expressed in testicular cells. When RIG1 was expressed in NT2/D1 testicular cancer cells, neither cell death nor cell viability was affected. However, RIG1 significantly inhibited cell migration and invasion in NT2/D1 cells. We found that prostaglandin D2 synthase (PTGDS) interacted with RIG1 using yeast two-hybrid screens. Further, we found PTGDS to be co-localized with RIG1 in NT2/D1 testis cells. In RIG1-expressing cells, elevated levels of prostaglandin D2 (PGD2), cAMP, and SRY-related high-mobility group box 9 (SOX9) were observed. This indicated that RIG1 can enhance PTGDS activity. Silencing of PTGDS expression significantly decreased RIG1-mediated cAMP and PGD2 production. Furthermore, silencing of PTGDS or SOX9 alleviated RIG1-mediated suppression of migration and invasion. These results suggest that RIG1 will suppress cell migration/invasion through the PGD2 signaling pathway. In conclusion, RIG1 can interact with PTGDS to enhance its function and to further suppress NT2/D1 cell migration and invasion. Our study suggests that RIG1-PGD2 signaling might play an important role in cancer cell suppression in the testis.
... Moreover, phospholipase A1/2 activity does not require the c-terminal hydrophobic domain [40]. TIG3 has been shown to reduce cell survival in a number of cell types [6,10,11,34,41,42], but the mechanism responsible for the suppression is not well understood. In some cell types, TIG3 may act via regulation of MAPK and PI3K/Akt signaling [1, 3,41]. ...
... TIG3 has been shown to reduce cell survival in a number of cell types [6,10,11,34,41,42], but the mechanism responsible for the suppression is not well understood. In some cell types, TIG3 may act via regulation of MAPK and PI3K/Akt signaling [1, 3,41]. ...
TIG3 is a tumor suppressor protein that limits keratinocyte survival during normal differentiation. It is also important in cancer, as TIG3 level is reduced in tumors and in skin cancer cell lines, suggesting that loss of expression may be required for cancer cell survival. An important goal is identifying how TIG3 limits cell survival. In the present study we show that TIG3 expression in epidermal squamous cell carcinoma SCC-13 cells reduces cell proliferation and promotes morphological and biochemical apoptosis. To identify the mechanism that drives these changes, we demonstrate that TIG3 localizes near the centrosome and that pericentrosomal accumulation of TIG3 alters microtubule and microfilament organization and organelle distribution. Organelle accumulation at the centrosome is a hallmark of apoptosis and we demonstrate that TIG3 promotes pericentrosomal organelle accumulation. These changes are associated with reduced cyclin D1, cyclin E and cyclin A, and increased p21 level. In addition, Bax level is increased and Bcl-XL level is reduced, and cleavage of procaspase 3, procaspase 9 and PARP is enhanced. We propose that pericentrosomal localization of TIG3 is a key event that results in microtubule and microfilament redistribution and pericentrosomal organelle clustering and that leads to cancer cell apoptosis.
... The closely related RARRES3 (also known as TIG3) gene is suppressed in basal and squamous cell carcinomas of the skin, and in breast and gastric cancer cell lines (DiSepio et al., 1998; Huang et al., 2000). RARRES3 impairs the function of several mitogen-activated and extracellular signal-regulated protein kinases in human cervical cancer cells (Huang et al., 2002) and was recently shown to suppress RAS activation (Tsai et al., 2006). Furthermore, the protein induces differentiation in human foreskin keratinocytes (Sturniolo et al., 2003). ...
... It can activate type I transglutaminase by an unknown mechanism thereby inducing terminal differentiation (Sturniolo et al., 2003). In cervical cancer cells, RARRES3 suppresses the activation of RAS proteins (Tsai et al., 2006) and negatively regulates the activity of Jun and p38 MAPKs (Huang et al., 2002). Interestingly, both HRASL3 and RARRES3 were identified as downstream targets of IFN (Sers et al., 2002) and are suggested to regulate ovarian carcinoma cell growth by a concerted action (Lotz et al., 2005). ...
HRSL3 (also known as H-REV107-1) belongs to a class II tumor suppressor gene family and is downregulated in several human tumors including ovarian carcinomas. To unravel the mechanism of HRSL3 tumor suppressor action, we performed a yeast two-hybrid screen and identified the alpha-isoform of the regulatory subunit A of protein phosphatase 2A (PR65alpha) as a new interaction partner of HRSL3. Interaction between HRSL3 and PR65alpha was confirmed in vitro and by co-immunoprecipitation in mammalian cells. We demonstrate that HRSL3 binds to the endogenous PR65alpha, thereby partially sequestering the catalytic subunit PR36 from the PR65 protein complex, and inhibiting PP2A catalytic activity. Furthermore, binding of HRSL3 to PR65 induces apoptosis in ovarian carcinoma cells in a caspase-dependent manner. Using several mutant HRSL3 constructs, we identified the N-terminal proline-rich region within the HRSL3 protein as the domain that is relevant for both binding of PR65alpha and induction of programmed cell death. This suggests that the negative impact of HRSL3 onto PP2A activity is important for the HRSL3 pro-apoptotic function and indicates a role of PP2A in survival of human ovarian carcinomas. The analysis of distinct PP2A target molecules revealed PKCzeta as being involved in HRSL3 action. These data implicate HRSL3 as a signaling regulatory molecule, which is functionally involved in the oncogenic network mediating growth and survival of ovarian cancer cells.
... Overexpression of RIG1 suppresses colony formation in T47D breast cancer cells and HaCaT keratinocytes. Ectopic RIG1 expression leads to cellular apoptosis and suppression of growth in cancer cells and human keratinocytes (3,(8)(9)(10). RIG1 also facilitates the terminal stages in keratinocyte differentiation by activating type I transglutaminase (11). ...
... RIG1 also facilitates the terminal stages in keratinocyte differentiation by activating type I transglutaminase (11). Previous studies have demonstrated that RIG1 decreases protein levels and activities of Ras, one of the HER2 downstream proteins, while interacting with Ras protein in the cytoplasmic compartment in HtTA cervical cancer cells (9,12). However, the molecular mechanisms responsible for the effects of RIG1 remain unclear. ...
Interferon-gamma (IFN-gamma) is known to downregulate HER2 oncoprotein (p185(HER2) or briefly p185) in prostate cancer cells. We demonstrate that the IFN-gamma-induced retinoid-inducible gene 1 (RIG1) acts as a transrepressor of p185. Furthermore, we exhibit that RIG1 downregulates the activated (phosphorylated) form of p185 and phosphoinositide-3 kinase (PI3K)/serine/threonine-specific protein kinase (Akt) and the mammalian target of rapamycin (mTOR), downstream substrates of HER2. We also elucidate that heregulin (HRG) specifically restores the activation of p185 and Akt after their activities are reduced by RIG1. Additionally, expression of vascular endothelial growth factor (VEGF) increases through the HER2- and Akt/mTOR-signaling pathways, indicating that VEGF is downregulated by RIG1 within the cell. These findings suggest that RIG1 plays a role in IFN-gamma-mediated therapy by downregulating p185 and its downstream PI3K/Akt/mTOR/VEGF-signaling pathway. These results may provide a new therapeutic mechanism for the clinical use of IFN-gamma and RIG1.
... In this study, we have evaluated the treatment effects of the combination of sorafenib and Platycladus orientalis L. leaf extract on the expression levels of proliferation marker, PCNA, tumor suppressor, TIG3, and apoptosis suppressor, Bcl-2. The synergetic effect of sorafenib and Platycladus orientalis L. on cervical cancer treatment efficiency might have been resulted by targeting TIG3, and an enhanced TIG3 mRNA has been proven in suppressing the RAS protein which is activated by Ras/MAPK and other signalling molecules in cervical cancer cells (Huang et al. 2002;Tsai et al. 2006), thereby decreasing the growth of cells. Besides, The Bcl-2 family which consists of pro-apoptotic proteins that are activated by the intrinsic apoptotic pathway by oligomerization, mediates the release of ROS and Cyt-C from the mitochondrial intermembrane space. ...
The extracts of Platycladus orientalis (L.) Franco leaves have shown promising anti-cancer, anti-oxidant and anti-inflammatory potency with the traditional knowledge of healing HPV associated warts. The purpose of this research is to assess the synergistic activity of sorafenib and Platycladus orientalis (L) leaf extraction on cervical cancer cells. The cytotoxicity efficiency of different concentrations of Sorafenib and ethanol extract of Platycladus orientalis (L.) leaves were tested on HeLa cells by MTT and Trypan blue assays. The synergistic effect of the IC50 concentrations of Sorafenib and Platycladus orientalis (L.) on HeLa cell by MTT assay, and mRNA expression levels of tumor suppressor tazarotene-induced gene 3 (TIG3), proliferating cell nuclear antigen (PCNA) gene and apoptosis modulator (Bcl-2) gene by RT-PCR were evaluated with individual treatments. Combination treatment showed a relatively more expression of TIG3 and less expression of Bcl-2 and PCNA was observed. Growth factor-induced MAPKP activation was arrested by compound combination treatment, which and suppression of proliferation-induced apoptosis of cervical cancer cells. Based on the our results, the combination of sorafenib and crude leaf extract from Platycladus orientalis (L.) can effectively suppress cervical cancer cell growth, thereby providing an interesting rationale for further clinical trials and in-vivo studies.
... [12][13][14] Previous studies have shown that TIG3 played a major role in cell proliferation, differentiation, and apoptosis and loss of TIG3 function would contribute to malignant transformation. [15][16][17][18] Consistently, decreased expression of TIG3 was detected in many types of cancers, such as breast cancer, epidermal squamous cell carcinoma, colorectal cancer, B-cell lymphocytic leukemia, hepatocellular carcinoma, and cholangiocarcinoma. [19][20][21][22][23] Furthermore, restoration of TIG3 expression could significantly suppress hepatocellular carcinoma growth via ERK1/2 inhibition by promoting apoptosis and inhibiting proliferation and migration, antagonize breast cancer cell growth and stemness properties through regulation of WNT proteins acylation and signaling activities, and inhibit metastasis of colorectal cancer via suppression of MTDH to regulate epithelial-mesenchymal transition. ...
TIG3 (tazarotene-induced gene 3) has been reported to suppress the progression of several malignancies, where this gene is universally downregulated. However, the expression of TIG3 in primary glioblastoma and its relevance to patient’s prognosis have not been elaborated. Thus, this study was aimed to evaluate TIG3 expression level in primary glioblastoma and investigate the prognostic value of TIG3 for patients. The Cancer Genome Atlas database was first utilized to analyze the expression and prognostic potential of TIG3 in 528 glioblastoma cases. Compared with control group, glioblastoma showed significantly elevated TIG3 expression (p < 0.001). Log-rank analysis revealed that higher expression of TIG3 was associated with shorter overall survival (358vs 383 days, p = 0.039). Furthermore, TIG3 protein expression detected by immunohistochemistry confirmed positive correlation of TIG3 expression and glioma grade and upregulation of TIG3 in our cohort of 101 primary glioblastoma patients compared to 16 normal brains. Finally, Kaplan–Meier analysis and Cox regression analysis identified high TIG3 expression as an independent risk factor for overall survival of primary glioblastoma patients (overall survival, 10 vs 13 months, p = 0.033; hazard ratio = 1.542, p = 0.046). Together, this study indicated that increased expression of TIG3 in primary glioblastoma is a novel biomarker for predicting poor outcome of patients. We then hypothesize that TIG3 may function in a different pattern in glioblastoma.
... Like other HRASLS subfamily members, HRASLS4 also inhibits H-RAS-mediated signalling [5,39,40]. The anti-cancer activity of HRASLS4 has been localized specifically to its action within the Golgi. ...
The H-RAS-like suppressor (HRASLS) subfamily consists of five enzymes (1–5) in humans and three (1, 3, and 5) in mice and rats that share sequence homology with lecithin:retinol acyltransferase (LRAT). All HRASLS family members possess in vitro phospholipid metabolizing abilities including phospholipase A 1/2 (PLA 1/2) activities and O-acyltransferase activities for the remodeling of glycerophospholipid acyl chains, as well as N-acyltransferase activities for the production of N-acylphosphatidylethanolamines. The in vivo biological activities of the HRASLS enzymes have not yet been fully investigated. Research to date indicates involvement of this subfamily in a wide array of biological processes and, as a consequence, these five enzymes have undergone extensive rediscovery and renaming within different fields of research. This review briefly describes the discovery of each of the HRASLS enzymes and their role in cancer, and discusses the biochemical function of each enzyme, as well as the biological role, if known. Gaps in current understanding are highlighted and suggestions for future research directions are discussed.
... Others reported that expression of RARRES3 reduced proliferation, promoted cellular apoptosis through redistribution of microfilament [25], and Golgi apparatus [31]. As for the variation of signal pathway, Huang et al observed that RARRES3 negatively regulated extracellular signal-regulated kinase (EKR), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated kinase both in cervical and gastric cancer cell lines [32], which was partly consistent with and supported by Tsai et al [31] that RARRES3 suppressed Ras activation. On the other hand, Ou et al reported that down-regulation of HER2 by RARRES3 was mediated by the PI3K/AKT pathway in ovarian cancer cells [28]. ...
It has been reported that Retinoic acid receptor responder 3 (RARRES3) could suppress the metastasis of colorectal cancer (CRC). However, the underlying mechanism by which RARRES3 suppresses metastasis remains unknown. To investigate the functional involvement of RARRES3 in CRC, we first analyzed the expression of this protein between human CRC clinical samples and their corresponding normal controls and tested its correlation with clinicopathology as well as prognosis of CRC. We also examined the endogenous expression of RARRES3 by western-blot in a panel of CRC cell lines with different metastatic capacity. Cell proliferation, migration and invation of the CRC cell lines with either knockdown or reexpression of RARRES3 were examined by MTT, transwell and wound healing assays, respectively. The intrecellular signaling pathways affected by manipulations of RARRES3 in CRC cells were determined by western blot. Immunoprecipitation (IP) was employed to assess the interactionbetween proteins. To investigate the metastatic ability in vivo, CRC cell lines with manipulations of RARRES3 expression were inoculated in nude mice through tail vein injection. We confirmed that RARRES3 was significantly down-regulated in CRC tissues compared with normal controls. RARRES3 expression was not correlated with prognosis but significantly associated with CRC differentiation and lymphnodes metastases. We also found that RARRES3 was able to significantly suppress the metastasis of CRC cells both in vitro and in vivo through the regulation of epithelial-mesenchymal transition (EMT) process during which RARRES3 interactedwith MTDH in an opposite way. Taken together, we for the first time found that RARRES3 was able to suppress the metastasis of CRC both in vitro and in vivo via suppression of MTDH so as to regulate EMT.
... Die Wachstumshemmung durch IFN-γ und ATRA bei diesen Zelllinien ging mit zunehmender Differenzierung entlang der granulozytären bzw. monozytären Reihe einher ((Lanotte et al., 1991) (Dimberg et al., 2000)) Dabei war der Anteil der Zellen in der S-Phase erniedrigt und der apoptotischer Zellen erhöht (Huang et al., 2002). Diese relativ geringe Wachstumshemmung ist eventuell durch die komplexen genetischen Veränderungen einer von einem malignen soliden ...
Das Gen H-rev107 wurde mit einer subtraktiven cDNS-Bibliothek zwischen H-ras-transformierten und H-ras-resistenten Fibroblasten isoliert. Es wurde als ein Klasse II Tumorsuppressorgen bezeichnet, dessen Expression die H-ras-induzierte maligne Transformation unterdrückt. Weitere Untersuchungen zeigten die Existenz einer Genfamilie mit dem Gen H-rev107-1 auf Chromosom 11q11-12 und H-rev107-2/TIG3/RARRES3 auf 11q23. Die Induzierbarkeit in epithelialen Zellen durch Interferon-gamma (H-rev107-1) und Retinoide (H-rev107-2) legten eine Beteiligung der Expression beider Gene bei der Regulation der Hämatopoese nahe. In der vorliegenden Arbeit wurde mittels RT-PCR in 8/15 Patientenproben (10 AML, 5 ALL) eine Expression von H-rev107-2 gefunden. H-rev107-2 wurde durch IFN-gamma in 14/14 Proben und durch all-trans Retinsäure (ATRA) in 5/15 Proben induziert. In Zelllinien maligner hämatopoetischer Erkrankungen konnte mit RT-PCR und Northern-Blot-Analysen bei CEM eine deutliche, bei Raji und HL-60 eine schwache Expression und bei NB-4, U937, K562 und Jurkat keine Expression von H-rev107-2 nachgewiesen werden. In allen 7 Zelllinien konnte H-rev107-2 durch IFN-gamma induziert werden. Die Induzierbarkeit durch ATRA und IFN-alpha war Zelllinien abhängig. Die stärkste Induktion durch ATRA erfolgte bei der akuten Promyelozytenleukämie-Zelllinie NB-4. Eine Induktionskinetik erbrachte eine H-rev107-2-Expression frühestens nach 4 h Inkubation mit IFN-gamma oder ATRA. Die Inhibition des MAPK-Signalwegs durch den MEK-Inhibitor PD098059 und des Signaltransduktors JAK2 durch AG490 hatte bei den myeloischen Zelllinien weder Einfluss auf die H-rev107-2-Expression noch auf die H-rev107-2-Induktion durch IFN-gamma oder ATRA. Die Expression von H-rev107-1 konnte bei U937, CEM und K562 gezeigt werden, wogegen die RT-PCR-Analysen bei NB-4, HL-60, Raji und Jurkat sowie bei 8 Patientenproben (6 AML, 2 ALL) keine H-rev107-2-Transkripte nachwiesen. Eine schwache Induktion von H-rev107-1 konnte nur bei den Zelllinien NB-4 durch ATRA und IFN-gamma und bei K562 durch IFN-gamma und IFN-alpha erzielt werden. Zusammenfassend legen die Ergebnisse dieser Arbeit eine Beteiligung von H-rev107-2/TIG3/RARRES3 bei der Regulation der Hämatopoese als ein mögliches Interferon- und ATRA-Target nahe.
... Overexpression of either TIG3 or H-REV107 can inhibit proliferation and induce apoptosis in tumor cell lines [3,11,[20][21][22][23]. It was proposed that the pro-apoptotic activity of TIG3 and H-REV107 is dependent on the NC motifs (residues 111-123) located on the N-terminal domain, and NC-motifs of both proteins can induce cell death independently [24,25]. ...
H-REV107-like family proteins TIG3 and H-REV107 are class II tumor suppressors. Here we report that the C-terminal domains (CTDs) of TIG3 and H-REV107 can induce HeLa cell death independently. The N-terminal domain (NTD) of TIG3 enhances the cell death inducing ability of CTD, while NTD of H-REV107 plays an inhibitory role. The solution structure of TIG3 NTD is very similar to that of H-REV107 in overall fold. However, the CTD binding regions on NTD are different between TIG3 and H-REV107, which may explain their functional difference. As a result, the flexible main loop of H-REV107, but not that of TIG3, is critical for its NTD to modulate its CTD in inducing cell death.
Copyright © 2015. Published by Elsevier B.V.
... Loss of TIG3 expression leads to hyperproliferative diseases including psoriasis and cancer. Restoration of TIG3 expression to cancer cell lines decreases cell cycle progression and induces apoptosis causing an overall decrease in viable cells (DiSepio et al., 1998;Huang et al., 2002;Higuchi et al., 2003;Tsai et al., 2009;Scharadin et al., 2011). Localization of TIG3 to the centrosome is believed to be responsible for this decrease in cell survival, which leads to an increase in p21 level, a G1/S phase block, an activation of the caspase cascade, and a reorganization of the microtubule network (Scharadin et al., 2011). ...
... Also, H-REV107 and HRASLS were shown to inhibit the RAS-mediated transformation of fibroblasts [8,20]. Similar inhibition of the RAS signal pathways has been observed in HRASLS2-expressing [6] or RIG1-expressing cervical and gastric cancer cells [21]. The results of our studies further demonstrated a downregulation of activated RAS and total RAS by RIG1 through the post-translational mechanism [11,24]. ...
Background
H-rev107 is a member of the HREV107 type II tumor suppressor gene family which includes H-REV107, RIG1, and HRASLS. H-REV107 has been shown to express at high levels in differentiated tissues of post-meiotic testicular germ cells. Prostaglandin D2 (PGD2) is conjectured to induce SRY-related high-mobility group box 9 (SOX9) expression and subsequent Sertoli cell differentiation. To date, the function of H-rev107 in differentiated testicular cells has not been well defined.
Results
In the study, we found that H-rev107 was co-localized with prostaglandin D2 synthase (PTGDS) and enhanced the activity of PTGDS, resulting in increase of PGD2 production in testis cells. Furthermore, when H-rev107 was expressed in human NT2/D1 testicular cancer cells, cell migration and invasion were inhibited. Also, silencing of PTGDS would reduce H-rev107-mediated increase in PGD2, cAMP, and SOX9. Silencing of PTGDS or SOX9 also alleviated H-rev107-mediated suppression of cell migration and invasion.
Conclusions
These results revealed that H-rev107, through PTGDS, suppressed cell migration and invasion. Our data suggest that the PGD2-cAMP-SOX9 signal pathway might play an important role in H-rev107-mediated cancer cell invasion in testes.
... While being almost ubiquitously expressed in normal tissues, down-regulation or complete loss of these genes in tumours and tumour cell lines have been reported. Expression can be reconstituted by different anti-proliferative signals such as interferons and retinoids, as well as by the inhibition of oncogenic pathways and interference with DNA methylation (Alessi et al., 1994;Husmann et al., 1998;Akiyama et al., 1999;Siegrist et al., 2001;Ito et al., 2001;Roder et al., 2002;Huang et al., 2002;Higuchi et al., 2003;Duvic et al., 2003). Re-activation of the H-REV107-1-related proteins and overexpression of the genes induce apoptosis or differentiation of tumour cells. ...
... However, nuclear localization has been reported for the HREV107 proteins [11,14]. RIG1 exhibits growth suppressive and proapoptotic activities in normal keratinocytes [13] and cancer cells of various origins [10,12,15,16]. The proapoptotic activities of RIG1 are mediated through caspase-dependent [12] or independent pathways [13], and are initiated only by the Golgi-, but not the ER-targeted RIG1 [12]. ...
Retinoid-inducible gene 1 (RIG1), also known as tazarotene-induced gene 3 or retinoic-acid receptor responder 3, is a growth regulator, which induces apoptosis and differentiation. RIG1 is classified into the NC protein family. This study investigated functional domains and critical amino acids associated with RIG1-mediated cell death and apoptosis.
Using enhanced green fluorescence protein (EGFP)-tagged RIG1 variants, RIG1 proteins with deletion at the NC domain significantly decreased cell death induced by RIG1, and fusion variants containing only the NC domain significantly induced apoptosis of HtTA cervical cancer cells. The EGFP-RIG1-induced apoptosis was significantly decreased in cells expressing N(112)C(113) motif double- (NC-->FG) or triple- (NCR-->FGE) mutated RIG1 variants. Using dodecapeptides, nuclear localization and profound cell death was observed in HtTA cells expressing wild type RIG1(111-123) or Leu121-mutated RIG1(111-123):L--> C peptide, but peptides double- or triple-mutated at the NC motif alone, RIG1(111-123):NC-->FG or RIG1(111-123):NCR-->FGE, were cytoplasmically localized and did not induce apoptosis. The RIG1(111-123) also induced apoptosis of A2058 melanoma cells but not normal human fibroblasts.
The NC domain, especially the NC motif, plays the major role in RIG1-mediated pro-apoptotic activity. The RIG1(111-123) dodecapeptide exhibited strong pro-apoptotic activity and has potential as an anticancer drug.
... For example, RARRES3 (retinoic acid receptor responder 3) is a class II tumor suppressor gene (i.e., downregulated in tumorigenesis rather than mutated) with growth suppressive and apoptosis-inducing activity. 32 It has previously been found to be downregulated in a manner correlated with progression of B-CLL 33 and cellular de-differentiation in colorectal adenocarcinoma, 34 consistent with our observation that this gene is downregulated in G2 and G3 HCCs. The majority of oncogenes and tumor suppressors identified here demonstrate expression patterns that systematically change from dysplasia to carcinoma, and in some cases, with alterations in expression already detectable in the pre-neoplastic state. ...
Progression of hepatocellular carcinoma (HCC) is a stepwise process that proceeds from pre-neoplastic lesions--including low-grade dysplastic nodules (LGDNs) and high-grade dysplastic nodules (HGDNs)--to advanced HCC. The molecular changes associated with this progression are unclear, however, and the morphological cues thought to distinguish pre-neoplastic lesions from well-differentiated HCC are not universally accepted. To understand the multistep process of hepato-carcinogenesis at the molecular level, we used oligo-nucleotide microarrays to investigate the transcription profiles of 50 hepatocellular nodular lesions ranging from LGDNs to primary HCC (Edmondson grades 1-3). We demonstrated that gene expression profiles can discriminate not only between dysplastic nodules and overt carcinoma but also between different histological grades of HCC via unsupervised hierarchical clustering with 10,376 genes. We identified 3,084 grade-associated genes, correlated with tumor progression, using one-way ANOVA and a one-versus-all unpooled t test. Functional assignment of these genes revealed discrete expression clusters representing grade-dependent biological properties of HCC. Using both diagonal linear discriminant analysis and support vector machines, we identified 240 genes that could accurately classify tumors according to histological grade, especially when attempting to discriminate LGDNs, HGDNs, and grade 1 HCC. In conclusion, a clear molecular demarcation between dysplastic nodules and overt HCC exists. The progression from grade 1 through grade 3 HCC is associated with changes in gene expression consistent with plausible functional consequences.
Mutated genes of the RAS family encoding small GTP-binding proteins drive numerous cancers, including pancreatic, colon and lung tumors. Besides the numerous effects of mutant RAS gene expression on aberrant proliferation, transformed phenotypes, metabolism, and therapy resistance, the most striking consequences of chronic RAS activation are changes of the genetic program. By performing systematic gene expression studies in cellular models that allow comparisons of pre-neoplastic with RAS-transformed cells, we and others have estimated that 7 percent or more of all transcripts are altered in conjunction with the expression of the oncogene. In this context, the number of up-regulated transcripts approximates that of down-regulated transcripts. While up-regulated transcription factors such as MYC, FOSL1, and HMGA2 have been identified and characterized as RAS-responsive drivers of the altered transcriptome, the suppressed factors have been less well studied as potential regulators of the genetic program and transformed phenotype in the breadth of their occurrence. We therefore have collected information on downregulated RAS-responsive factors and discuss their potential role as tumor suppressors that are likely to antagonize active cancer drivers. To better understand the active mechanisms that entail anti-RAS function and those that lead to loss of tumor suppressor activity, we focus on the tumor suppressor HREV107 (alias PLAAT3 [Phospholipase A and acyltransferase 3], PLA2G16 [Phospholipase A2, group XVI] and HRASLS3 [HRAS-like suppressor 3]). Inactivating HREV107 mutations in tumors are extremely rare, hence epigenetic causes modulated by the RAS pathway are likely to lead to down-regulation and loss of function.
Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences.
Bioactive N-acylethanolamines (NAEs) are ethanolamides of long-chain fatty acids, including palmitoylethanolamide, oleoylethanolamide and anandamide. In animal tissues, NAEs are biosynthesized from membrane phospholipids. The classical "transacylation-phosphodiesterase" pathway proceeds via N-acyl-phosphatidylethanolamine (NAPE), which involves the actions of two enzymes, NAPE-generating Ca(2+)-dependent N-acyltransferase (Ca-NAT) and NAPE-hydrolyzing phospholipase D (NAPE-PLD). Recent identification of Ca-NAT as Ɛ isoform of cytosolic phospholipase A2 enabled the further molecular biological approaches toward this enzyme. In addition, Ca(2+)-independent NAPE formation was shown to occur by N-acyltransferase activity of a group of proteins named phospholipase A/acyltransferases (PLAAT)-1-5. The analysis of NAPE-PLD-deficient mice confirmed that NAEs can be produced through multi-step pathways bypassing NAPE-PLD. The NAPE-PLD-independent pathways involved three members of the glycerophosphodiesterase (GDE) family (GDE1, GDE4, and GDE7) as well as α/β-hydrolase domain-containing protein (ABHD)4. In this review article, we will focus on recent progress made and latest insights in the enzymes involved in NAE synthesis and their further characterization.
Tazarotene-induced gene 3 (TIG3) is a tumor suppressor protein. In normal human epidermis, TIG3 is present in the differentiated, suprabasal layers, and it regulates terminal differentiation. TIG3 level is reduced in hyperproliferative diseases, including psoriasis and skin cancer, suggesting that loss of TIG3 is associated with enhanced cell proliferation. Moreover, transient expression of TIG3 leads to terminal differentiation in normal keratinocytes and apoptosis in skin cancer cells. In both cell types, TIG3 distributes to the cell membrane and to the centrosome. At the cell membrane, TIG3 interacts with and activates type I transglutaminase to enhance keratinocyte terminal differentiation. TIG3 at the centrosome acts to inhibit centrosome separation during mitosis and to alter microtubule function. These findings argue that TIG3 is involved in the control of keratinocyte differentiation and that loss of TIG3 in transformed cells contributes to the malignant phenotype.Journal of Investigative Dermatology advance online publication, 6 March 2014; doi:10.1038/jid.2014.79.
Human TIG3 protein is a member of H-REV107 protein family which belongs to the type II tumor suppressor family. TIG3 can induce apoptosis in cancer cells, and it also possesses Ca2+-independent phospholipase A1/2 activity. The NMR assignments of the N-terminal domain of TIG3 are essential for its solution structure determination.
Background:
Gastric cancer is frequently lethal despite aggressive multimodal therapies, and new treatment approaches are therefore needed. Retinoids are potential candidate drugs: they prevent cell differentiation, proliferation and malignant transformation in gastric cancer cell lines. They interact with nuclear retinoid receptors (the retinoic acid receptors [RARs] and retinoid X receptors [RXRs]), which function as transcription factors, each with three subclasses, α, β and γ. At present, little is known about retinoid expression and influence on prognosis in gastric cancers.
Patients and methods:
We retrospectively analyzed the expression of the subtypes RARα, RARβ, RARγ, RXRα, RXRβ, RXRγ by immunohistochemistry in 147 gastric cancers and 51 normal gastric epithelium tissues for whom clinical follow-up data were available and correlated the results with clinical characteristics. In addition, we quantified the expression of retinoid receptor mRNA using real- time PCR (RT-PCR) in another 6 gastric adenocarcinoma and 3 normal gastric tissues. From 2008 to 2010, 80 patients with gastric cancers were enrolled onto therapy with all-trans-retinoic acid (ATRA).
Results:
RARα, RARβ, RARγ and RXRγ positively correlated with each other (p<0.001) and demonstrated significantly lower levels in the carcinoma tissue sections (p<0.01), with lower RARβ, RARγ and RXRα expression significantly related to advanced stages (p<=0.01). Tumors with poor histopathologic grade had lower levels of RARα and RARβ in different histological types of gastric carcinoma (p<0.01). Patients whose tumors exhibited low levels of RARa expression had significantly lower overall survival compared with patients who had higher expression levels of this receptor (p<0.001, HR=0.42, 95.0% CI 0.24-0.73), and patients undergoing ATRA treatment had significantly longer median survival times (p=0.007, HR=0.41, 95.0% CI 0.21-0.80).
Conclusions:
Retinoic acid receptors are frequently expressed in epithelial gastric cancer with a decreased tendency of expression and RARa may be an indicator of a positive prognosis. This study provides a molecular basis for the therapeutic use of retinoids against gastric cancer.
Genes and molecular pathways involved in familial clustering of gastric cancer have not yet been identified. The purpose of the present study was to investigate gene expression changes in response to a cellular stress, and its link with a positive family history for this neoplasia. To this aim leukocytes of healthy first-degree relatives of gastric cancer patients and controls were challenged in vitro with ionizing radiation and gene expression evaluated 4 h later on microarrays with 1,800 cancer-related genes. Eight genes, mainly involved in signal transduction and cell cycle regulation, were differentially expressed in healthy relatives of gastric cancer cases. Functional class scoring by Gene Ontology classification highlighted two G-protein related pathways, implicated in the proliferation of neoplastic tissue, which were differentially expressed in healthy subjects with positive family history of gastric cancer. The relative expression of 84 genes related to these pathways was examined using the SYBR green-based quantitative real-time PCR. The results confirmed the indication of an involvement of G-protein coupled receptor pathways in GC familiarity provided by microarray analysis. This study indicates a possible association between familiarity for gastric cancer and altered transcriptional response to ionizing radiation.
Tarzarotene-induced gene 3 (TIG3) and HRAS-like suppressor (HRASLS3) are members of the HREV107 family of class II tumor suppressors, which are down-regulated in various cancer cells. TIG3 and HRASLS3 also exhibit phospholipase activities. Both proteins share a common domain architecture with hydrophilic N-terminal and hydrophobic C-terminal regions. The hydrophobic C-terminal region is important for tumor suppression. However, the function of the hydrophilic N-terminal region remains elusive. To facilitate biochemical characterizations of TIG3 and HRASLS3, we expressed and purified the N-terminal regions of TIG3 and HRASLS3, designated TIG3 (1-134) and HRASLS3 (1-133), in a bacterial system. We found that the N-terminal regions of TIG3 and HRASLS3 have calcium-independent phospholipase A(2) activities. Limited proteolysis revealed that TIG3 (1-132) is a structural domain in the N-terminal region of TIG3. Our data suggest that the hydrophobic C-terminal regions might be crucial for cellular localization, while the hydrophilic N-terminal regions are sufficient for the enzymatic activity of both TIG3 and HRASLS3.
Berlin, Humboldt-Univ., Diss., 2003 (Nicht für den Austausch).
Tazarotene-induced protein 3 (TIG3) and HRAS-like suppressor family 2 (HRASLS2) exhibit tumor-suppressing activities and belong to the lecithin retinol acyltransferase (LRAT) protein family. Since Ca(2+)-independent N-acyltransferase and H-rev107 (another tumor suppressor), both of which are members of the LRAT family, have been recently reported to possess catalytic activities related to phospholipid metabolism, we examined possible enzyme activities of human TIG3 and HRASLS2 together with human H-rev107. The purified recombinant proteins of TIG3, HRASLS2, and H-rev107 functioned as phospholipase (PL) A(1/2) in a Ca(2+)-independent manner with maximal activities of 0.53, 0.67, and 2.57 micromol/min/mg of protein, respectively. The proteins were active with various phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs), and for most of substrates the PLA(1) activity was much higher than the PLA(2) activity. In addition, HRASLS2 catalyzed N-acylation of PE to form N-acyl-PE and O-acylation of lyso PC to form PC. TIG3 and H-rev107 catalyzed the N-acylation and O-acylation at relatively low rates. Moreover, these three proteins showed different expression profiles in human tissues. These results suggest that the tumor suppressors TIG3, HRASLS2 and H-rev107 are involved in the phospholipid metabolism with different physiological roles.
Retinoids can regulate the proliferation and differentiation of various tumor cells. It is thought that nuclear retinoid receptors mediate these effects by regulating gene transcription. The identity of specific retinoid target genes is only beginning to be unraveled. One candidate for mediating retinoid-induced growth suppression is the novel class II tumor suppressor gene tazarotene-induced gene 3 (TIG3). We examined the constitutive and all-trans retinoic acid (ATRA)-inducible expression of TIG3 mRNA in five head and neck squamous cell carcinoma (HNSCC) and five nonsmall cell lung carcinoma (NSCLC) cell lines to determine whether it is associated with their responsiveness to ATRA. The expression patterns of retinoic acid receptor beta (RARbeta), another putative retinoid-inducible tumor suppressor gene, were also examined. The constitutive TIG3 expression was high in one HNSCC cell line and two NSCLC cell lines, and moderate to very low in the other cells. Some RARbeta-expressing cells had either low or undetectable TIG3 levels and vice versa. ATRA (1 microM; 48 h) increased TIG3 mRNA in 4/5 HNSCCs and 3/5 NSCLCs and RARbeta mRNA in some of the same cell lines, but also in cells that did not show TIG3 induction. TIG3 mRNA was induced by ATRA between 6 and 12 h in most of the responsive cells. ATRA concentrations required for TIG3 induction ranged from 1 to 500 nM depending on the cell line. The pan-RAR antagonists AGN193109 and the RARalpha antagonist Ro 41-5253 blocked TIG3 induction by ATRA. ATRA suppressed anchorage-independent colony formation in most cells that had a high or moderate constitutive or induced TIG3 expression level. In contrast, RARbeta mRNA expression pattern was not correlated with sensitivity to ATRA. These results suggest that TIG3 is regulated by ATRA via retinoid receptors in certain aerodigestive tract cancer cells, and its induction by ATRA is associated with the suppression of anchorage-independent growth.
To analyze the expression of retinoic acid receptor responder 3 (RARRES3) protein in paraffin-embedded tissues of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), and the correlation of RARRES3 production with tumor differentiation.
Expression of RARRES3 in tissues from 21 CC (10 well-, 7 moderately- and 4 poorly-differentiated) and 32 HCC was determined by immunohistochemistry.
Among 21 CC tissues, RARRES3 was detected in 8 (80%) of 10 well-differentiated tumors. Only 2 (18.2%) out of 11 tumors with moderate or poor differentiation showed positive RARRES3 expression. RARRES3 expression in well-differentiated CC was significantly higher than that in tumors with moderate or poor differentiation (Fisher exact test, P<0.01). Expression of RARRES3 was not different between early (I and II) and late (III and IV) stages of CC. Among 30 HCC tissues, 17 (56.7%) weakly expressed RARRES3 in HCC cells, and 25 (83.3%) normal tissues adjacent to HCC expressed the protein. RARRES3 expression was significantly decreased in HCC tissues compared to that in adjacent normal tissues (logistic regression analysis, OR = 0.27, 95% CI (0.11-0.62), P<0.01).
Expression of RARRES3 is positively correlated to well-differentiated CC, which supports the role of RARRES3 in malignant epithelial differentiation of the tumor. The decrease in RARRES3 expression in tissues of HCC and CC with moderate and poor differentiation suggests that altered RARRES3 expression may play a role in the carcinogenesis of the liver and biliary tract.
The retinoid-inducible gene 1 (RIG1) protein is a retinoid-inducible growth regulator. Previous studies have shown that the RIG1 protein inhibits the signaling pathways of Ras/mitogen-activated protein kinases. However, neither the mode of action nor the site of inhibition of RIG1 is known. This study investigated the effects of RIG1, and the mechanisms responsible for these effects, on the activation of Ras proteins in HtTA cervical cancer cells. RIG1 reduced the levels of activated Ras (Ras-GTP) and total Ras protein in cells transfected with mutated H-, N-, or K-Ras(G12V), or in cells transfected with the wild type H- or N-Ras followed by stimulation with epidermal growth factor. The half-life of Ras protein decreased from more than 36 h in control cells to 18 h in RIG1-transfected cells. RIG1 immunoprecipitated with the Ras protein in co-transfected cellular lysates. In contrast to the predominant plasma membrane localization in control cells, the H-Ras fusion protein EGFP-H-Ras was localized within a discrete cytoplasmic compartment where it co-localized with RIG1. RIG1 inhibited more than 93% of the Elk- and CHOP-mediated transactivation induced by H- or K-Ras(G12V). However, RIG1 did not inhibit the transactivation induced by MEK1 or MEK3, and failed to suppress the phosphorylation of extracellular signal-regulated kinases 1 and 2 induced by the constitutively activated B-Raf(V599E). The RIG1 with carboxyl terminal truncation (RIG1DeltaC) did not immunoprecipitate with Ras and had no effect on Ras activation or transactivation of the downstream signal pathways. These data indicate that RIG1 exerts its inhibitory effect at the level of Ras activation, which is independent of Ras subtype but dependent on the membrane localization of the RIG1 protein. This inhibition of Ras activation may be mediated through downregulation of Ras levels and alteration of Ras subcellular distribution.
Retinoid-inducible gene I (RIG1) is a growth regulator protein that exhibits activities to suppress cellular growth and induce cellular differentiation and apoptosis. This study analyzed the expression and regulation of RIG1 in breast cancer cells in vitro and in vivo.
Expression of RIG1 RNA in breast cancer tissues was analyzed using RNA in situ hybridization. Regulation of RIG1 expression by 17beta-estradiol (E2) was analyzed by semi-quantitative reverse transcription polymerase chain reaction.
RIG1 expression in 47 breast cancer tissues was detected mostly in the cytoplasm and in some nuclei. Levels of both cytoplasmic and nuclear RIG1 mRNA were significantly lower in 20 estrogen receptor-positive (ER+) than in 27 ER-negative (ER-) tissues (p < 0.05), in 20 progesterone receptor-positive (PR+) than in 27 PR-negative (PR-) tissues (p < 0.01), and in 14 ER+/PR+ than in 21 ER-/PR-tissues (p < 0.05). Basal levels of RIG1 and ER mRNA were inversely related between ER+ (MCF-7 WS8 and ZR75-1) and ER- (ZR-75-30) breast cancer cells. E2 (1 nM) treatment for two days suppressed RIG1 mRNA levels in MCF-7 WS8 and ZR-75-1 cells, but not in the ER- ZR-75-30 cells. The E2-mediated down-regulation of RIG1 expression was time- and concentration-dependent in ZR-75-1 cells.
The negative association between RIG1 and ER expression in breast cancer tissues and down-regulation of RIG1 by E2 in breast cancer cells in vitro suggest that RIG1 expression is negatively regulated by E2 through activation of the ER in ER+ breast cancer cells.
Retinoic acid receptor responder 1 (RARRES1) is a retinoid regulated gene. Its expression is frequently down-regulated through DNA hypermethylation in several types of malignant tissues. This study investigated the clinical significance of RARRES1 protein and its association with RARRES3 protein expression in 161 (26 adenoma, 13 distal normal mucosa and 122 primary colorectal adenocarcinoma) paraffin-embedded colorectal tissues by immunohistochemistry. RARRES1 protein was detected at the highest levels in terminally differentiated cells of normal mucosal tissues and all 26 adenoma tissues. Among 122 colorectal adenocarcinomas, the poorly differentiated adenocarcinomas and Dukes' stage D tumours showed a significant decrease in RARRES1 expression (P < 0.001 and P < 0.01, respectively). RARRES1 expression was significantly (P < 0.001) correlated with RARRES3 expression, which was positively associated with tumour differentiation (P < 0.001). Difference in expression of RARRES1 among 119 patients had no apparent effect on patient survival. Our results suggest the role of RARRES1 in colorectal epithelial differentiation, and the down-regulation of RARRES1 is related to stage D progression.
H-REV107-1, a known member of the class II tumor suppressor gene family, is involved in the regulation of differentiation and survival. We analyzed H-REV107-1 in non-small cell lung carcinomas, in normal lung, and in immortalized and tumor-derived cell lines. Sixty-eight percent of lung tumors revealed positive H-REV107-1-specific staining. Furthermore, survival analysis demonstrated a significant association of cytoplasmic H-REV107-1 with decreased patient survival. This suggested that H-REV107-1, known as a tumor suppressor, plays a different role in non-small cell lung carcinomas. Knock-down of H-REV107-1 expression in lung carcinoma cells inhibited anchorage-dependent and anchorage-independent growth whereas overexpression of H-REV107-1 induced tumor cell proliferation. Consistent with results of the survival analysis, cytoplasmic localization of the protein was essential for this growth-inducing function. Analysis of signaling pathways potentially involved in this process demonstrated that overexpression of H-REV107-1 stimulated RAS-GTPase activity, ERK1,2 phosphorylation, and caveolin-1 expression in the cell lines analyzed. These results indicate that H-REV107-1 is deficient in its function as a tumor suppressor in non-small cell lung carcinomas and is required for proliferation and anchorage-independent growth in cells expressing high levels of the protein, thus contributing to tumor progression in a subset of non-small cell lung carcinomas.
Retinoid-inducible gene 1 encodes RIG1 is a growth regulator, which inhibits the pathways of the RAS/mitogen-activated protein kinases by suppressing the activation of RAS. Confocal microscopic analysis demonstrated that RIG1 is localized in the endoplasmic reticulum (ER) and Golgi apparatus in HtTA cervical cancer cells. Carboxyterminal-deleted RIG1 targeted to the Golgi or ER was constructed and validated. The activation of HRAS was inhibited by 25.1% or 81.4% in cells cotransfected with wild-type or Golgi-targeted RIG1, respectively. Expression of wild-type or Golgi-targeted RIG1 for 24 h induced cellular apoptosis in HtTA cells, as assessed by MTT assay, the release of lactate dehydrogenase, and chromatin condensation. In contrast, ER-targeted RIG1 and carboxyterminal-deleted RIG1 (RIG1DeltaC) exhibited no activity. Caspase-2, -3, and -9 were activated following the expression of wild-type and Golgi-targeted RIG1. Although the caspase-3 inhibitor Z-DEVD-FMK partially or completely reversed the cell death induced by wild-type or Golgi-targeted RIG1, it did not prevent the anti-RAS effect of RIG1. In conclusion, the proapoptotic and anti-RAS activities of RIG1 are primarily associated with the Golgi localization of the protein. The proapoptotic activities of RIG1 are mediated through the activation of caspase-2 and -3 and are independent of its effect on RAS.
Dual speci®city kinases that phosphorylate the Thr-and Tyr-residues within the TXY motif of MAP-kinases of play a central role in the regulation of various processes of cell growth. These dual speci®city kinases also known as MAP kinase kinases are constituents of the sequential kinase signaling modules. Seven distinct mammalian MAP kinases kinases have been identi®ed. Some of the unique signaling properties of these kinases are discussed here.
Retinoids, synthetic and natural analogs of retinoic acid, exhibit potent growth inhibitory and cell differentiation activities
that account for their beneficial effects in treating hyperproliferative diseases such as psoriasis, actinic keratosis, and
certain neoplasias. Tazarotene is a synthetic retinoid that is used in the clinic for the treatment of psoriasis. To better
understand the mechanism of retinoid action in the treatment of hyperproliferative diseases, we used a long-range differential
display–PCR to isolate retinoid-responsive genes from primary human keratinocytes. We have identified a cDNA, tazarotene-induced gene 3 (TIG3; Retinoic Acid Receptor Responder 3) showing significant homology to the class II tumor suppressor gene, H-rev 107. Tazarotene treatment increases TIG3 expression in primary human keratinocytes and in vivo in psoriatic lesions. Increased TIG3 expression is correlated with decreased proliferation. TIG3 is expressed in a number
of tissues, and expression is reduced in cancer cell lines and some primary tumors. In breast cancer cell lines, retinoid-dependent
TIG3 induction is observed in lines that are growth suppressed by retinoids but not in nonresponsive lines. Transient over-expression
of TIG3 in T47D or Chinese hamster ovary cells inhibits colony expansion. Finally, studies in 293 cells expressing TIG3 linked
to an inducible promoter demonstrated decreased proliferation with increased TIG3 levels. These studies suggest that TIG3
may be a growth regulator that mediates some of the growth suppressive effects of retinoids.
Members of a family of intracellular molecular switches, the small GTPases, sense modifications of the extracellular environment and transduce them into a variety of homeostatic signals. Among small GTPases, Ras and the Rho family of proteins hierarchically and/or coordinately regulate signaling pathways leading to phenotypes as important as proliferation, differentiation and apoptosis. Ras and Rho-GTPases are organized in a complex network of functional interactions, whose molecular mechanisms are being elucidated. Starting from the simple concept of linear cascades of events (GTPaseactivator GTPase), the work of several laboratories is uncovering an increasingly complex scenario in which upstream regulators of GTPases also function as downstream effectors and influence the precise biological outcome. Furthermore, small GTPases assemble into macromolecular machineries that include upstream activators, downstream effectors, regulators and perhaps even final biochemical targets. We are starting to understand how these macromolecular complexes work and how they are regulated and targeted to their proper subcellular localization. Ultimately, the acquisition of a cogent picture of the various levels of integration and regulation in small GTPase-mediated signaling should define the physiology of early signal transduction events and the pathological implication of its subversion.
Insulin treatment (Kact, 5 X 10(-9) M) of serum-starved 3T3-L1 adipocytes stimulates a soluble serine/threonine kinase that catalyzes phosphorylation of microtubule-associated protein 2 (MAP-2) in vitro. Maximal activation of MAP-2 kinase activity by 80 nM insulin was observed after 10 min of hormonal stimulation, prior to maximal stimulation of S6 kinase activity (20 min). The insulin-stimulatable MAP-2 kinase activity is not adsorbed to phosphocellulose, whereas the principal S6 kinase activity is retained and elutes at approximately 0.5 M NaCl. The insulin-stimulatable MAP-2 kinase is less stable during incubation at 30 degrees C than S6 kinase activity. Inclusion of phosphatase inhibitors decreases the rate at which the stimulated MAP-2 kinase activity is lost from extract supernatants incubated at 30 degrees C. p-Nitrophenyl phosphate is more effective than DL-phosphotyrosine, whereas DL-phosphoserine is without effect at the concentration used (40 mM). The difference in MAP-2 kinase activity in extract supernatants from control and insulin-treated cells is also preserved after rapid chromatography on Sephadex G-25. These results show that a soluble serine/threonine kinase is rapidly activated by insulin, possibly by phosphorylation of either the kinase itself or an interacting modulator.
The intracellular signalling field is dominated by the mitogen-activated protein kinase (MAPK) cascade and its control, which involves the small GTPase Ras and sequential kinase activation. Until recently, ERK1 and ERK2 were the only cloned and well-characterized mammalian MAPKs; diverse ligand-stimulated, proline-directed protein phosphorylation events were attributed to these kinases. The recent discovery of two other MAPK subtypes, the JNK/SAPK subfamily and p38/RK (mammalian equivalents of HOG1 in yeast), reveals extreme complexity within the family and, most intriguingly, the existence in mammalian cells of parallel MAPK cascades that can be activated simultaneously.
The H-rev107 gene is a new class II tumor suppressor, as defined by its reversible downregulation and growth-inhibiting capacity in HRAS transformed cell lines. Overexpression of the H-rev107 cDNA in HRAS-transformed ANR4 hepatoma cells or in FE-8 fibroblasts resulted in 75% reduction of colony formation. Cell populations of H-rev107 transfectants showed an attenuated tumor formation in nude mice. Cells explanted from tumors or maintained in cell culture for an extended period of time no longer exhibited detectable levels of the H-rev107 protein, suggesting strong selection against H-rev107 expression in vitro and in vivo. Expression of the truncated form of H-rev107 lacking the COOH-terminal membrane associated domain of 25 amino acids, had a weaker inhibitory effect on proliferation in vitro and was unable to attenuate tumor growth in nude mice. The H-rev107 mRNA is expressed in most adult rat tissues, and immunohistochemical analysis showed expression of the protein in differentiated epithelial cells of stomach, of colon and small intestine, in kidney, bladder, esophagus, and in tracheal and bronchial epithelium. H-rev107 gene transcription is downregulated in rat cell lines derived from liver, kidney, and pancreatic tumors and also in experimental mammary tumors expressing a RAS transgene. In colon carcinoma cell lines only minute amounts of protein were detectable. Thus, downregulation of H-rev107 expression may occur at the level of mRNA or protein.
We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic
fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 consists of 167 amino acids and shows
46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated
ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic
MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1
protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate
that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.
Since 1982, Ras has been the subject of intense research scrutiny, focused on determining the role of aberrant Ras function in human cancers and defining the mechanism by which Ras mediates its actions in normal and neoplastic cells. The long-term goal has been to develop antagonists of Ras as novel approaches for cancer treatment. Although impressive strides have been made in these endeavours, and our knowledge of Ras is quite extensive, it appears that we are at the beginning, rather than at the end, of fully understanding Ras function. This review highlights new issues that have further complicated our efforts to understand Ras.
We have shown that stimulation of beta-adrenergic receptors (beta-AR) by norepinephrine (NE) increases apoptosis in adult rat ventricular myocytes (ARVMs) via a cAMP-dependent mechanism that is antagonized by activation of G(i) protein. The family of mitogen-activated protein kinases (MAPKs) is involved in the regulation of cardiac myocyte growth and apoptosis. Here we show that beta-AR stimulation activates p38 kinase, c-jun N-terminal kinases (JNKs), and extracellular signal-regulated kinase (ERK1/2) in ARVMs. Inhibition of p38 kinase with SB-202190 (10 micrometer) potentiated beta-AR-stimulated apoptosis as measured by flow cytometry and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. SB-202190 at this concentration specifically blocked beta-AR-stimulated activation of p38 kinase and its downstream substrate MAPK-activated protein kinase-2 (MAPKAPK2). Pertussis toxin, an inhibitor of G(i)/G(o) proteins, blocked the activation of p38 kinase and potentiated beta-AR-stimulated apoptosis. Activation of G(i) protein with the muscarinic receptor agonist carbachol protected against beta-AR-stimulated apoptosis. Carbachol also activated p38 kinase, and the protective effect of carbachol was abolished by SB-202190. PD-98059 (10 micrometer), an inhibitor of ERK1/2 pathway, blocked beta-AR-stimulated activation of ERK1/2 but had no effect on apoptosis. These data suggest that 1) beta-AR stimulation activates p38 kinase, JNKs, and ERK1/2; 2) activation of p38 kinase plays a protective role in beta-AR-stimulated apoptosis in cardiac myocytes; and 3) the protective effects of G(i) are mediated via the activation of p38 kinase.
A human signet ring gastric carcinoma cell line TSGH9201 was established in vitro. The cells grew in vitro as a monolayer with polygonal morphology and had a population doubling time of 34 hours. The cells secreted tumor markers CEA and CA 125. They were, however, not tumorigenic in athymic nude mice. Karyotypic analysis demonstrated a near tetraploidy with a modal chromosome number of 98. Northern blotting and immunocytochemical analysis revealed the expression of both transforming growth factor alpha and high levels of epidermal growth factor receptor. Cell growth was inhibited by the epidermal growth factor in vitro. The cell line may be a useful tool to study autocrine growth regulation through the epidermal growth factor receptor.
Excitatory amino acids induce both acute membrane depolarization and latent cellular toxicity, which often leads to apoptosis in many neurological disorders. Recent studies indicate that glutamate toxicity may involve the c-Jun amino-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases. One member of the JNK family, Jnk3, may be required for stress-induced neuronal apoptosis, as it is selectively expressed in the nervous system. Here we report that disruption of the gene encoding Jnk3 in mice caused the mice to be resistant to the excitotoxic glutamate-receptor agonist kainic acid: they showed a reduction in seizure activity and hippocampal neuron apoptosis was prevented. Although application of kainic acid imposed the same level of noxious stress, the phosphorylation of c-Jun and the transcriptional activity of the AP-1 transcription factor complex were markedly reduced in the mutant mice. These data indicate that the observed neuroprotection is due to the extinction of a Jnk3-mediated signalling pathway, which is an important component in the pathogenesis of glutamate neurotoxicity.
The H-rev107 tumour suppressor was isolated as a gene specifically expressed in rat fibroblasts resistant toward malignant transformation by the activated HRAS gene (Sers et al., 1997; Hajnal et al., 1994). Here we describe the human homologue of the rat H-rev107 gene. The predicted rat and human proteins are highly conserved exhibiting an overall amino acid identity of 83%. The H-REV107-1 gene is ubiquitously expressed with the exception of haematopoetic cells and tissues. In contrast, H-REV107-1 mRNA was found only in eight of 27 cell lines derived from mammary carcinoma, lung carcinoma, gastric carcinoma, kidney carcinoma, melanoma, neuroblastoma and other tumours. The H-REV107-1 protein was not detectable in any of these tumour cells. Loss of H-REV107-1 expression was not restricted to cultured human tumour cell lines, but also found in primary squamous cell carcinomas. Gross structural aberrations of the H-REV107-1 gene were absent in tumorigenic cell lines. Thus, the block to H-REV107-1 expression is achieved both at the level of transcription and translation. By fluorescence in situ hybridisation the human H-REV107-1 gene was localised to chromosome 11q11-12.
6-[3-(1-Adamantyl)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) is a novel retinoid which induces apoptosis in the retinoic acid-resistant HL-60R human leukemia cell line. CD437-mediated poly(ADP-ribose) polymerase (PARP) cleavage and apoptosis of HL-60R cells does not require gene transcription or protein synthesis since it occurs in the presence or absence of either actinomycin D or cycloheximide. Marked activation of both the p38 and the JNK/SAPK serine and threonine kinases occurs at 1 h of exposure to CD437 with subsequent PARP cleavage at 2 h and apoptosis noted at 4 to 6 h. CD437 concentrations as little as 10 nM result in p38 activation and apoptosis of HL-60R cells. However, inhibition of p38 activation utilizing the specific inhibitor SB203580 does not block CD437-mediated PARP cleavage or apoptosis. In addition, p38 activation is dependent upon the activation of the caspase system since p38 activation is blocked by the pan ICE inhibitor Z-VAD fmk, which also inhibits CD437-mediated apoptosis and PARP cleavage in these cells. CD437-mediated activation of JNK/SAPK is not inhibited by Z-VAD fmk, suggesting that it lies upstream of CD437 activation of caspase activity and subsequent apoptosis. The role of JNK/SAPK activation in CD437-mediated apoptosis remains to be defined.
Retinoids exert wide-spectrum anti-tumor activities, which are mediated via the induction of growth arrest, differentiation or apoptosis. To determine whether the effects of retinoids are mediated by specific gene activation or repression, SC-M1 CL23 gastric cancer cells, pretreated with either vehicle alone or all-trans retinoic acid (10 microM) for 1 day, were analyzed using the technique of differential display. A novel retinoid-inducible gene 1 (RIG1) was isolated. The full-length RIG1 cDNA contained 768 base pairs and encoded a protein of 164 amino acids with a molecular weight of 18 kDa. The RIG1 gene was ubiquitously expressed in normal tissue, and its expression was positively associated with cellular density. Nucleotide sequence analysis demonstrated that the RIG1 gene was similar to a recently-isolated TIG3 gene, and displayed 54% nucleotide sequence homology with a type II tumor suppressor gene H-REV-107-1. RIG1 cDNA, however, contained an extra 32 base pairs located at its 5' end and revealed three base pair differences for the remaining sequences leading to two amino acids substitution between the two encoded proteins. All-trans retinoic acid increased the level of RIG1 mRNA in a time- and concentration-dependent manner in SC-M1 CL23 gastric cancer cells. This was not observed for the H-REV-107-1 gene. The RIG1 regulation was related to cellular retinoid sensitivity. Both retinoic acid receptor alpha- and retinoic acid receptor gamma-selective agonists increased RIG1 mRNA level, and the retinoid x receptor-selective agonist potentiated this regulation. In conclusion, the cDNA of a novel retinoid-inducible gene RIG1 has been cloned. This gene is regulated by retinoic acid through the heterodimer of retinoic acid receptor and retinoid x receptor.
In this study, the downstream signaling of Bcr-Abl tyrosine kinase responsible for apoptosis resistance was investigated. DNA fragmentation, a hallmark of apoptosis, was observed after 2 days of herbimycin A treatment with a peak on 3 day. During the apoptosis induced by the treatment of herbimycin A, stress-activated protein kinase (SAPK) and p38 kinase were activated time- and dose-dependently, while extracellular signal-regulated kinase (ERK) was inhibited. However, apoptosis was induced by the treatment of PD98059, a specific inhibitor of MEK (MAPK or ERK kinase), not by the treatment of sorbitol, a strong activator of SAPK and p38 kinase. Although K562 cells were very resistant to sorbitol-induced apoptosis, DNA fragmentation was induced rapidly in Jurkat, HL-60 and U937 cells after exposure to sorbitol, despite that these apoptosis-sensitive cells have similar or lower activities of JNK/SAPK and p38 kinase compared with K562 cells after treatment of sorbitol. K562 cells had a much higher basal activity of ERK/MAPK than other apoptosis-sensitive cell lines, which were very susceptible to apoptosis induced by low dose of PD98059 compared with K562 cells. In HL-60 cells, sorbitol-induced apoptosis was prevented by the treatment of phorbol myristate 13-acetate (PMA), which activates the ERK/MAPK pathway, and this was blocked by PD98059. From these results, it could be suggested that the inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in K562 cells.
Ras and Rho GTPases are among the best studied signaling molecules in molecular biology. Essential cellular processes, such as cell growth, lipid metabolism, cytoarchitecture, membrane trafficking, transcriptional regulation, apoptosis, and response to genotoxic agents, are directly modulated by different members of this superfamily of proteins. Not until recently have we begun to understand the physiological implications of Ras and Rho GTPases, linking them to processes such as embryonic development, tissue remodeling, tumorigenesis and metastasis. In this sense, uncontrolled activation, due to overexpression of different members of the Rho family in a variety of tissues, leads to uncontrolled proliferation and invasiveness of human tumors. In this review, an attempt to briefly integrate recent findings in transcriptional regulation by Rho GTPases in the context of carcinogenesis and metastasis as well as apoptosis is made.