Search

Aging impairs the IGF-I signaling of bone marrow mesenchymal stem cells (bmMSCs), but the mechanism is unclear. Here, we found that the ability to auto-phosphorylate IGF-I receptor (IGF-IR) in response to IGF-I was decreased in the bmMSCs of aged donors. Conversely, data showed that decorin (DCN) expression was prominently increased in aged bmMSCs, and that under IGF-I treatment, DCN knockdown in serum-starved aged bmMSCs potentiated their mitogenic activity and IGF-IR auto-phosphorylation, whereas DCN overexpression in serum-starved adult bmMSCs decreased both activities. Co-immunoprecipitation assays suggested that IGF-I and DCN bound to IGF-IR in a competitive manner. Online MethPrimer predicted 4 CpG islands (CGIs) in the introns of DCN gene. RT-qPCR and bisulfite sequencing showed that dimethyloxalylglycine, an inhibitor of DNA demethylation, increased DCN mRNA expression and CGI-I methylation in adult bmMSCs, whereas 5-aza-2’-deoxycytidine, a DNA methylation inhibitor, decreased DCN mRNA expression and CGI-I methylation in aged bmMSCs, and ultimately enhanced the proliferation of serum-starved aged bmMSCs under IGF-I stimulation. Thus, IGF-IR could be the prime target of aging in down-regulating the IGF-I signaling of bmMSCs, where DCN could be a critical mediator.

Previous, we found that the small molecules capable of inhibiting the expression and the pro-adipogenic activity of ZNF521 might improve the osteogenic performance of aging human bone marrow MSCs (bmMSCs), and that fatty acid synthase (FASN) was a critical effector of ZNF521's pro-adipogenic activity. Here, by characterizing the netoglitazone (MCC-555), one of the thiazolidinediones known as adipogenic enhancers, as an inhibitor of ZNF521 expression, we found that MCC-555 indeed also harbored pro-osteoblastic effect. Investigation revealed that MCC-555 might function as a GSK3β inhibitor to promote osteoblastogenesis and bone formation. Importantly, combination of MCC-555 with FASN knockdown, but not with GW9662 (a PPARγ2 antagonist), blocked the pro-adipogenic but retained the pro-osteoblastic effect of MCC-555. Using a 3-dimentional culture system, we showed that MCC-555 facilitated the FASN-knockdown of aging human bmMSCs to form cell clusters in scaffolds, and to promote osteoblastic differentiation and biomineralization in cell clusters. These data indicated that MCC-555 promoted bmMSCs to produce bone-like tissues. Our data narrate a thiazolidinedione-based novel strategy to improve the osteogenic performance of aging bmMSCs to support the application of autologous aging bmMSCs in cell therapy and in producing bone-like tissues for repairing bone injury in the elderly.

POU-homeodomain transcription factor POU3F2 is a critical transcription factor that participates in neuronal differentiation. However, little is known about its downstream mediators. Here genome-wide analyses of a human neuronal differentiation cell model, NT2D1, suggested neurotrophin-3 (NTF3), a key mediator of neuronal development during the early neurogenic period, as a putative regulatory target of POU3F2. Western blot, cDNA microarray, and real-time quantitative PCR analyses showed that POU3F2 and NTF3 were upregulated during neuronal differentiation. Next-generation-sequence-based POU3F2 chromatin immunoprecipitation-sequencing and genome-wide in silico prediction demonstrated that POU3F2 binds to the NTF3 promoter during neuronal differentiation. Furthermore, unidirectional deletion or mutation of the binding site of POU3F2 in the NTF3 promoter decreased promoter-driven luciferase activity, indicating that POU3F2 is a positive regulator of NTF3 promoter activity. While NTF3 knockdown resulted in decreased viability and differentiation of NT2D1 cells, and POU3F2 knockdown downregulated NTF3 expression, recombinant NTF3 significantly rescued viable neuronal cells from NTF3- or POU3F2-knockdown cell cultures. Moreover, immunostaining showed colocalization of POU3F2 and NTF3 in developing mouse neurons. Thus, our data suggest that NTF3 is a novel target gene of POU3F2 and that the POU3F2/NTF3 pathway plays a role in the process of neuronal differentiation. Electronic supplementary material The online version of this article (10.1007/s12035-018-0995-y) contains supplementary material, which is available to authorized users.

AXL is expressed in many types of cancer and promotes cancer cell survival, metastasis and drug resistance. Here, we focus on identifying modulators that regulate AXL at the mRNA level. We have previously observed that the AXL promoter activity is inversely correlated with the AXL expression levels, suggesting that post-transcriptional mechanisms exist that down-regulate the expression of AXL mRNA. Here we show that the RNA binding protein PTBP1 (polypyrimidine tract-binding protein) directly targets the 5′-UTR of AXL mRNA in vitro and in vivo. Moreover, we also demonstrate that PTBP1, but not PTBP2, inhibits the expression of AXL mRNA and the RNA recognition motif 1 (RRM1) of PTBP1 is crucial for this interaction. To clarify how PTBP1 regulates AXL expression at the mRNA level, we found that, while the transcription rate of AXL was not significantly different, PTBP1 decreased the stability of AXL mRNA. In addition, over-expression of AXL may counteract the PTBP1-mediated apoptosis. Knock-down of PTBP1 expression could enhance tumor growth in animal models. Finally, PTBP1 was found to be negatively correlated with AXL expression in lung tumor tissues in Oncomine datasets and in tissue micro-array (TMA) analysis. In conclusion, we have identified a molecular mechanism of AXL expression regulation by PTBP1 through controlling the AXL mRNA stability. These findings may represent new thoughts alternative to current approaches that directly inhibit AXL signaling and may eventually help to develop novel therapeutics to avoid cancer metastasis and drug resistance.

Previously, we found that ZNF521 expression was up-regulated with advancing age in human bone marrow mesenchymal stem cells (bmMSCs). Here, we investigated the regulatory role of ZNF521 in the differentiation of mouse C3H10T1/2 cells and human bmMSCs. Our data show that ZNF521 overexpression repressed osteoblastic differentiation of C3H10T1/2 cells, accompanied by a decrease in Runx2 expression and an increase in PPARγ2 expression. In contrast, ZNF521 overexpression enhanced adipogenic differentiation of C3H10T1/2 cells, concomitant with increased expression of PPARγ2, aP2, adiponectin and C/EBPδ. Chromatin immunoprecipitation followed by quantitative PCR analyses and luciferase reporter assays suggested that ZNF521 overexpression enhances PPARγ2 expression at the transcriptional level. The enhancing effect of ZNF521 overexpression on the adipogenic differentiation of C3H10T1/2 cells was also observed ex vivo. Finally, similar to those noted in C3H10T1/2 cells, ZNF521 overexpression in human bmMSCs was found to promote adipogenic differentiation in vitro and ex vivo, but repressed osteoblastic differentiation in vitro. ZNF521 knockdown significantly repressed adipogenic differentiation in vitro and ex vivo, but promoted osteoblastic differentiation in vitro. We propose that ZNF521 can function as a repressor of osteoblastic differentiation of bmMSCs while promoting adipogenesis, and that elevated ZNF521 expression might play a role in the age-related bone loss.

Many studies have indicated that loss of the osteoblastogenic potential in bone marrow mesenchymal stem cells (bmMSCs) is the major component in the etiology of the aging-related bone deficit. But how the bmMSCs lose osteogenic capability in aging is unclear. Using 2-dimentional cultures, we examined the dose response of human bmMSCs, isolated from adult and aged donors, to exogenous insulin-like growth factor 1 (IGF-1), a growth factor regulating bone formation. The data showed that the mitogenic activity and the osteoblastogenic potential of bmMSCs in response to IGF-1 were impaired with aging, whereas higher doses of IGF-1 increased the proliferation rate and osteogenic potential of aging bmMSCs. Subsequently, we seeded IGF-1-overexpressing aging bmMSCs into calcium-alginate scaffolds and incubated in a bioreactor with constant perfusion for varying time periods to examine the effect of IGF-1 overexpression to the bone-forming capability of aging bmMSCs. We found that IGF-1 overexpression in aging bmMSCs facilitated the formation of cell clusters in scaffolds, increased the cell survival inside the cell clusters, induced the expression of osteoblast markers, and enhanced the biomineralization of cell clusters. These results indicated that IGF-1 overexpression enhanced cells' osteogenic capability. Thus, our data suggest that the aging-related loss of osteogenic potential in bmMSCs can be attributed in part to the impairment in bmMSCs' IGF-1 signaling, and support possible application of IGF-1-overexpressing autologous bmMSCs in repairing bone defect of the elderly and in producing bone graft materials for repairing large scale bone injury in the elderly.

Aging is associated with bone loss and degenerative joint diseases, in which the aging of bone marrow-derived mesenchymal stem cell (bmMSC)[1] may play an important role. In this study, we analyzed the gene expression profiles of bmMSC from 14 donors between 36 and 74 years old, and obtained age-associated genes (in the background of osteoarthritis) and osteoarthritis-associated genes (in the background of old age). Pathway analysis of these genes suggests that alterations in glycobiology might play an important role in the aging of human bmMSC. On the other hand, antigen presentation and signaling of immune cells were the top pathways enriched by osteoarthritis-associated genes, suggesting that alteration in immunology of bmMSC might be involved in the pathogenesis of osteoarthritis. Most intriguingly, we found significant age-associated differential expression of HEXA, HEXB, CTSK, SULF1, ADAMTS5, SPP1, COL8A2, GPNMB, TNFAIP6, and RPL29; those genes have been implicated in the bone loss and the pathology of osteoporosis and osteoarthritis in aging. Collectively, our results suggest a pathological role of bmMSC in aging-related skeletal diseases, and suggest the possibility that alteration in the immunology of bmMSC might also play an important role in the etiology of adult-onset osteoarthritis.

Previously, we showed that CL1-5 cells express more hypoxia-inducible factor-1alpha (HIF-1alpha) than the parental CL1 cells, which bestows CL1-5 cells a stronger invasive activity. Here, we investigated the mechanisms underlying the differential expression of HIF-1alpha mRNA in CL1 and CL1-5 cells. Data showed that the transcription rate of HIF-1alpha gene in CL1 cells was slightly higher than that of CL1-5 cells, suggesting that the expression of HIF-1alpha mRNA in CL1 cells was repressed by post-transcriptional mechanisms. RNA electrophoretic mobility shift assays revealed a 61-base segment (designated as D5) within the 5'-untranslated repeat of HIF-1alpha mRNA, with which the CL1 cell lysates formed more prominent complexes (including complex I) than did CL1-5 cell lysates. Insertion of D5 into a reporter construct reduced the half-life of the chimeric transcripts in transfected CL1 but not CL1-5 cells; conversely, overexpression of D5-containing reporter construct in CL1 cells increased HIF-1alpha mRNA. We also identified the polypyrimidine tract-binding protein (PTB) as a required component of complex I. Deletion of the RNA recognition motif 1 (RRM1) or RRM3 of PTB abolished the formation of complex I. Our data showed that CL1 cells expressed more PTB than CL1-5 cells. Inhibition of PTB expression in CL1 cells decreased the formation of complex I, whereas overexpression of PTB in CL1-5 cells increased the levels of complex I, decreased the stability of HIF-1alpha and D5-containing chimeric mRNAs, and decreased cell invasiveness. In sum, we have identified in lung adenocarcinoma cells a mechanism that regulates HIF-1alpha expression by modulating HIF-1alpha mRNA stability.

We examined the mRNA levels of hypoxia-inducible factor-1alpha (HIF-1alpha) in bone marrow mesenchymal stem cells (bmMSCs) of eight osteoarthritis patients. BmMSC-1, expressing higher HIF-1alpha mRNA and protein than bmMSC-5, elicited higher matrix metalloproteinase-1 (MMP1) activity and stronger invasive capacity. In vitro invasion assays and quantitative PCR analyses showed that targeted inhibition of HIF-1alpha in bmMSC-1 decreased its invasion and expressions of MMP1 and MMP3, whereas overexpression of HIF-1alpha in bmMSC-5 increased its invasion and expressions of MMP1 and MMP3. Therefore, HIF-1alpha can regulate MMP1 and MMP3 expressions in human bmMSCs, which might suggest a pathophysiological role of bmMSC expressing high HIF-1alpha in bone diseases.

We studied the role of hypoxia-inducible factor-1alpha (HIF-1alpha) in human lung adenocarcinoma cell invasion using a metastatic cell model composed of low invasive CL1 and highly invasive CL1-5 cells. We showed that HIF-1alpha was expressed in CL1-5 but not in CL1 cells under normoxic condition, and that inhibition of HIF-1alpha expression by a small interfering RNA decreased invasiveness of CL1-5 cells. Complementary, overexpression of HIF-1alpha increased the invasiveness of CL1 and gastric cancer SC-M1 cells. Subsequently, we showed that urokinase-type plasminogen activator receptor (uPAR), and matrix metalloproteinases (MMPs) 1 and 2 were critical in HIF-1alpha-induced invasion. Mechanistic studies revealed that HIF-1alpha overexpression could increase the expression of uPAR and MMP1, but not MMP2. However, ELISA assays on the conditioned media generated from control CL1 and CL1 cells overexpressing HIF-1alpha showed that overexpression of HIF-1alpha increased the levels of endogenous free active MMP2 and total free MMP2, and the former was blocked by inhibition of MMP1 expression. We conclude that (i) HIF-1alpha overexpression enhances lung cancer cell invasion at least through up-regulating the expression and activities of uPAR, MMP1, and MMP2; and (ii) induction of MMP1 participates in cell invasion and also plays an important role in HIF-1alpha-induced activation of MMP2.

Mechanical forces have profound effects on endothelial cells. This study was undertaken to examine the hypothesis that tumor necrosis factor-alpha (TNF-alpha) is a potential mediator of stretch-induced effects on matrix metalloproteinase (MMP).Human umbilical vein endothelial cells (HUVECs) grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. We used the TNF-alpha monoclonal antibody and c-Jun N-terminal kinase (JNK) inhibitor, SP600125, to investigate the cyclical stretch-induced expression of MMP-14 and -2 in cultured HUVECs.Cyclical mechanical stretch significantly increased protein synthesis and mRNA expression for MMP-14 and -2 from 2 to 24 h. The increased MMP-14 and-2 proteins after stretch were completely blocked after the addition of TNF-alpha monoclonal antibody (5 microg/ml) or SP600125 (20 microM) 30 min before stretch. By zymography, MMP-2 expression was induced by cyclical stretch and was attenuated by TNF-alpha monoclonal antibody and SP600125. Cyclical stretch increased the immunohistochemical labeling of MMP-14 and -2 and significantly increased release of TNF-alpha into the culture media from 120+/-2 to 331+/-2 pg/ml (P0.001) after stretch for 12 h. Cyclical stretch increased and SP600125 decreased the phosphorylated JNK. Gel-shifting assay showed that DNA-protein binding activity of AP-1 increased after cyclical stretch and TNF-alpha monoclonal antibody and SP600125 abolished the binding activity induced by cyclical stretch.These findings indicate that cyclical stretch augments TNF-alpha production and MMP genes expression in HUVECs. TNF-alpha mediates the stretch-induced MMP genes expression, at least in part, through the JNK pathway.

We have studied GL331's anti-cancer mechanisms by studying their effect on the tumor-induced angiogenesis. Human lung adenocarcinoma CL1-5 cells were treated with GL331 and then maintained in serum-reduced, GL331-free medium for the preparation of condition mediums. These condition mediums were tested for their capability to induce in vitro angiogenesis, i.e., HUVEC tube formation and migration. We found that mediums generated from GL331-treated CL1-5 cells presented reduced ability of inducing in vitro angiogenesis. Western blot analyses showed that both VEGF and HIF-1alpha were down-regulated in GL331-treated CL1-5 cells. Northern blot and EMSA analyses showed that GL331 down-regulated HIF-1alpha expression without decreasing the stability of HIF-1alpha mRNA, and that GL331 decreased the binding of CL1-5-derived nuclear components to the promoter of HIF-1alpha gene. Therefore, our data showed that GL331 is a potent inhibitor of tumor-induced angiogenesis. The underlying mechanisms might involve at least the inhibition of HIF-1alpha expression, probably through transcriptional repression.

One of the biological effects of hyperbaric oxygen (HBO) therapy in enhancing ischemia-related wound healing is the induction of angiogenesis. To elucidate the mechanism(s) underlying the HBO-induced angiogenesis, we studied the expression of several angiogenesis-related genes in human umbilical vein endothelial cells exposed to HBO. Western blot analyses showed that HBO enhanced the expression of angiopoietin-2 (Ang2) with no effect on the expression of Tie2, angiopoietin-1, and VEGF. The induction of Ang2 was further confirmed by immunohistochemistry, quantitative PCR, and Northern blot analyses. Inhibition of endothelial nitric oxide synthase blocked the HBO-induced Ang2 expression, but failed to block hypoxia-induced Ang2 expression. These data indicated that HBO-induced Ang2 expression may be through transcriptional stimulation, and requires the nitric oxide signaling pathway, which may play an important role in HBO-induced angiogenesis.

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of both tissue- and urokinase-type plasminogen activators (t-PA, u-PA). PAI-1 also regulates the attachment of cells to the adhesive glycoprotein vitronectin (VN). PAI-1 gene expression has been observed in various cell types, and many regulatory factors have been identified to play a role in PAI-1 gene transcription. The complete picture of how the PAI-1 gene is expressed when cells adhere to a culture plate has not been fully elucidated. We found that in anchorage-dependent cells, PAI-1 gene was up-regulated when cells were beginning to attach to a culture dish and was down-regulated when cells had attached completely. The PAI-1 gene expression was induced only in adhered cells but not in non-adhered cells. The regulation of PAI-1 protein was also found in both culture medium and cell lysate when cells were attached to a culture dish. Our experiment indicates that vitronectin and fibronectin, as components of ECM, may be the factors involved in the regulation of PAI-1 gene expression. PAI-1, as an inhibitor of the interaction between vitronectin and integrin alphavbeta3, may also be a regulator of its own expression.

Type-I plasminogen activator inhibitor (PAI-1) is the primary inhibitor of both tissue- and urokinase-type plasminogen activators (t-PA, u-PA) and is thus a primary regulator of plasminogen activation and possibly of extracellular proteolysis. In anchorage-dependent cells, the PAI-1 gene was regulated by cell adhesion. PAI-1 gene expression was induced more evidently in cells adhered to the culture plate than in nonadherent cells. In this study, we investigated the signal pathway of the PAI-1 gene expression regulated by cell adhesion. We found the induction of both PAI-1 mRNA and protein, when cells adhered to culture dish, was inhibited by the PI-3 kinase specific inhibitors (Ly294002 and wortmannin). The cells seeded on collagen-1 coated plate with low serum further demonstrated that the PAI-1 gene expression was prolonged by the cell adhesion. The above-mentioned PI-3 kinase specific inhibitors also blocked the PAI-1 maintenance when cell adhered to collagen-1 coated plate. In addition, we found that both PI-3 kinase and its downstream molecule, Akt, were activated more evidently in adherent cells than in nonadherent cells. Furthermore, we transfected antisense oligodeoxynucleotides of Akt (AS-ODN-Akt) into cells to block the expression of Akt and found that the induction of PAI-1 mRNA was also inhibited. Hence, we conclude that the induction of PAI-1 gene expression is cell adhesion dependent and is through PI-3 kinase and Akt activation.

Evodiamine, the major bioactive compound isolated from Chinese herbal drug named Wu-Chu-Yu, has been reported to exhibit anti-tumor growth and metastasis. However, the effect of evodiamine on angiogenesis remains to be investigated. We used the fresh medium containing evodiamine or human lung adenocarcinoma cell (CL1 cells) derived conditioned media free of evodiamine to test their capability to induce in vitro angiogenesis, i.e., human umbilical vein endothelial cells (HUVECs) tube formation and invasion. We demonstrated that evodiamine could directly inhibit in vitro HUVECs tube formation and invasion. Locally administered evodiamine also inhibited the in vivo angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay. The gene expression of vascular endothelial growth factor (VEGF) and the p44/p42 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells angiogenesis were inhibited by evodiamine. We found that the evodiamine-treated CL1 cells derived conditioned medium showed decreased VEGF release and reduced ability of inducing in vitro tube formation. After the collection of conditioned media, the VEGF expression of remaining CL1 cells were determined by Western analyses and revealed that evodiamine decreased VEGF expression. Moreover, administration of recombinant human VEGF(165) (rhVEGF(165)) induced tube formation and ERK phosphorylation by HUVECs, and partially attenuated inhibitory effect of evodiamine. From these results, we suggested that evodiamine is a potent inhibitor of angiogenesis. The mechanism might involve at least the inhibition of VEGF expression, probably through repression of ERK phosphorylation.

Sesamin has been proved to be antihypertensive. Nitric oxide (NO) is the most important vascular relaxing factor that is regulated in endothelium. Endothelin-1 (ET-1) is characterized as a potent vasoconstrictor and is also regulated in endothelium. Alterations in the endothelial production of NO and ET-1 are known to correlate with hypertension. This study investigated the effect of sesamin on NO and ET-1 in the human umbilical vein endothelial cells (HUVECs).The concentrations of NO and ET-1 in the medium of HUVECs treated by sesamin were measured. The mRNA and protein expressions of nitric oxide synthase (NOS), endothelin converting enzyme-1 (ECE-1), and endothelin-1 (ET-1) were also investigated. Other than the mRNA and protein expression, NOS activity and cyclic GMP (cGMP) were detected.The NO concentration was detected by colorimetric assay. The cGMP and ET-1 were analyzed by EIA. The eNOS, ECE-1, and ET-1 mRNA expressions were assayed by Northern blot. The eNOS and ECE-1 protein expressions were analyzed by Western blot. The NOS activity was assayed by detecting the level of [H]-1-citrullin transformed from [H]-1-arginine.Sesamin not only increased the NO concentration in the medium of HUVECs in a dose-dependent manner after 24 h, but also induced eNOS mRNA and protein expressions. NOS activity in the HUVECs was also induced by sesamin. The content of cGMP was induced by sesamin through NO signaling. On the other hand, the ET-1 concentration in the medium of HUVECs treated by sesamin was suppressed in a dose-dependent manner after 24 h. The ECE-1 protein and mRNA expressions were also inhibited by sesamin. However, the mRNA expression of prepro ET-1 was not influenced by sesamin.From the above results, it is suggested that sesamin may improve hypertension by its ability to induce NO and inhibit ET-1 production from endothelial cells. The increase of NO by sesamin is through the induction of eNOS gene expression. The decrease of ET-1 by sesamin is through the inhibition of ECE gene expression, but is not through the inhibition of prepro ET-1 gene expression.

We have studied the antiangiogenic property of berberine. We showed that berberine could directly inhibit in vitro human umbilical vein endothelial cell (HUVEC) tube formation and migration. In addition, to determine whether berberine could influence the cross-talk between the gastric adenocarcinoma cell line SC-M1 and vascular endothelial cells, we performed modified confrontation culture experiments and showed that berberine (7.5 microM, 16 h) could inhibit the capacity of hypoxic SC-M1 cells to stimulate HUVEC migration. These results demonstrated berberine's antiangiogenic property and its clinical potential as an inhibitor of tumor angiogenesis. Parallel Western blot analyses revealed that berberine prevented hypoxic SC-M1 cultures from expressing vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1alpha, two key factors in mediating tumor angiogenesis. However, overexpression of HIF-1alpha in SC-M1 cells dramatically reversed the inhibitory effect of berberine on SC-M1-induced in vitro HUVEC migration. These data indicated that HIF-1alpha repression is a critical step in the inhibitory effect of berberine on tumor-induced angiogenesis. Northern blot analyses plus pulse-chase assays revealed that berberine did not down-regulate HIF-1alpha mRNA but destabilized HIF-1alpha protein. We found that berberine-induced HIF-1alpha degradation was blocked by a 26S proteasome inhibitor. Moreover, immunoprecipitation and Western blot analyses showed that berberine increased the lysine-acetylated HIF-1alpha in hypoxic SC-M1 cultures. These data indicated that a proteasomal proteolytic pathway and lysine acetylation were involved in berberine-triggered HIF-1alpha degradation. In conclusion, our data provided molecular evidence to support berberine as a potent antiangiogenic agent in cancer therapy.

Matrix metalloproteinase-9 (MMP-9) associates with cancer cell invasion and metastasis. CL1-5 cells, a human lung adenocarcinoma cell line, expressed an elevated level of MMP-9 and exhibited a highly invasive and metastatic ability. By Matrigel assay and gelatinase zymography, the topoisomerase II poison GL331 was found to dose-dependently inhibit the invasiveness and the level of secreted MMP-9 of CL1-5 cells. Northern blot analysis indicated that cellular MMP-9 mRNA level was decreased after GL331 treatment. Furthermore, GL331-induced down-regulation of mmp-9 gene promoter was demonstrated by using a luciferase reporter gene driven by the -216 to -13 region of the mmp-9 gene promoter cloned from CL1-5 cells. By PCR amplification and gel electrophoresis, we found that GL331 caused shortening of the -216 to -13 region of the mmp-9 promoter. Direct sequencing analysis revealed that the number of d(CA) was reduced from 24 to 18 at the microsatellite d(CA) repeat region of the mmp-9 promoter. The CL1-5 cells transfected with the luciferase reporter containing 18 d(CA)s expressed only 53% of those when the reporter contained 24 d(CA)s. The promoter region of mmp-9 gene contains other positive regulatory elements, such as TRE and kappaB. We found that GL331 did not significantly influence the luciferase activity driven by TRE or kappaB. Taken together, these data suggested that GL331 inhibited MMP-9 mRNA expression at least partly through the selective induction of shortening of microsatellite d(CA) repeats. This is the first report that an anti-cancer agent can inhibit mmp-9 gene expression by inducing microsatellite DNA shortening.

Prostaglandin A(2) (PGA(2)), an experimental chemotherapeutic agent, causes growth arrest associated with decreased cyclin D1 expression in several cancer cell lines. Here, using human non-small-cell lung carcinoma H1299 cells, we investigated the mechanisms whereby PGA(2) down-regulates cyclin D1 expression. Transcription rates of the cyclin D1 gene, studied using a cyclin D1 promoter-luciferase construct and nuclear run-on assays, were not affected by PGA(2) treatment. Instead, the cyclin D1 mRNA was rendered unstable after exposure to PGA(2). Since the stability of labile mRNA is modulated through binding of proteins to specific mRNA sequences, we sought to identify protein(s) recognizing the cyclin D1 mRNA. In electrophoretic mobility-shift assays using radiolabeled RNA probes derived from different regions of cyclin D1 mRNA, we observed that (i) lysates prepared from PGA(2)-treated cells exhibited enhanced protein-cyclin D1 RNA complex formation; (ii) the kinetics of complex formation correlated closely with that of cyclin D1 mRNA loss; and (iii) binding occurred within a 390-base cyclin D1 3' untranslated region (UTR) (K12). This binding activity could be cross-linked, revealing proteins ranging from 30 to 47 kDa. The RNA-binding protein AUF1, previously associated with the degradation of target mRNAs, bound cyclin D1 mRNA, because anti-AUF1 antibodies were capable of supershifting or immunoprecipitating cyclin D1 mRNA-protein complexes. Finally, insertion of K12 in the 3'UTR of reporter genes markedly reduced the expression and half-life of the resulting chimeric mRNAs in transfected, PGA(2)-treated cells. Our data demonstrate that PGA(2) down-regulates cyclin D1 expression by decreasing cyclin D1 mRNA stability and implicates a 390-base element in the 3'UTR in this regulation.

Colorectal carcinoma RKO cells expressing reduced levels of the RNA-binding protein HuR (ASHuR) displayed markedly reduced growth. In synchronous RKO populations, HuR was almost exclusively nuclear during early G(1), increasing in the cytoplasm during late G(1), S and G(2). The expression and half-life of mRNAs encoding cyclins A and B1 similarly increased during S and G(2), then declined, indicating that mRNA stabilization contributed to their cell cycle-regulated expression. In gel-shift assays using radiolabeled cyclin RNA transcripts and RKO protein extracts, only those transcripts corresponding to the 3'-untranslated regions of cyclins A and B1 formed RNA-protein complexes in a cell cycle-dependent fashion. HuR directly bound mRNAs encoding cyclins A and B1, as anti-HuR antibodies supershifted such RNA-protein complexes. Importantly, the expression and half-life of mRNAs encoding cyclins A and B1 were reduced in ASHuR RKO cells. Our results indicate that HuR may play a critical role in cell proliferation, at least in part by mediating cell cycle-dependent stabilization of mRNAs encoding cyclins A and B1.

In this report we describe, for the first time, the purification and characterization of a replication-competent multiprotein form of DNA polymerase (designated the DNA synthesome) from the human leukemia cell line (HL-60) using a series of centrifugation, ion-exchange chromatography and velocity sedimentation steps. The proteins and enzymatic activities thus far identified to co-purify with the leukemia cell DNA synthesome include the DNA polymerases alpha and delta, DNA primase, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), replication protein A (RP-A), and DNA topoisomerases I and II. We have demonstrated that the DNA synthesome is fully competent to replicate simian virus 40 (SV40) replication origin containing DNA in vitro in the presence of the viral large T-antigen. This result implies that all of the cellular activities required for large T-antigen-dependent in vitro SV40 DNA synthesis are present in the isolated human leukemia cell DNA synthesome. Since SV40 is extensively dependent on the host cell's DNA synthetic machinery for its own DNA replication, our results indicate that the isolated leukemia cell DNA synthesome may play a role not only in viral DNA synthesis but also in human leukemia cell DNA replication. We recently proposed a model to represent the DNA synthesome that was isolated from HeLa and murine cells. Our data indicate that the organization of the DNA synthesome from HL-60 cells also fits this proposed model. The purified DNA synthesome will not only allow the further study of the molecular mechanisms required to carry out human leukemia cell DNA replication, but may also provide a tool for eventually dissecting some of the regulatory controls of the cell's DNA synthetic machinery.

The expression of polypyrimidine tract-binding protein (PTB) is up-regulated in many types of cancer. Here, we studied the role of PTB in the growth of non small cell lung cancer cells. Data showed that PTB overexpression inhibited the growth of H1299 cells at least by inhibiting DNA synthesis. Quantitative real-time PCR and Western blot analyses showed that PTB overexpression in H1299 cells specifically induced the expression of p19(Ink4d), an inhibitor of cyclin-dependent kinase 4. Repression of p19(Ink4d) expression partially rescued PTB-caused proliferation inhibition. PTB overexpression also inhibited the growth and induced the expression of p19(Ink4d) mRNA in A549 cells. However, Western blot analyses failed to detect the presence of p19(Ink4d) protein in A549 cells. To address how PTB induced p19(Ink4d) in H1299 cells, we showed that PTB might up-regulate the activity of p19(Ink4d) gene (CDKN2D) promoter. Besides, PTB lacking the RNA recognition motif 3 (RRM3) was less effective in growth inhibition and p19(Ink4d) induction, suggesting that RNA-binding activity of PTB plays an important role in p19(Ink4d) induction. However, immunoprecipitation of ribonuclearprotein complexes plus quantitative real-time PCR analyses showed that PTB might not bind p19(Ink4d) mRNA, suggesting that PTB overexpression might trigger the other RNA-binding protein(s) to bind p19(Ink4d) mRNA. Subsequently, RNA electrophoretic mobility-shift assays revealed a 300-base segment (designated as B2) within the 3'UTR of p19(Ink4d) mRNA, with which the cytoplasmic lysates of PTB-overexpressing cells formed more prominent complexes than did control cell lysates. Insertion of B2 into a reporter construct increased the expression of the chimeric luciferase transcripts in transfected PTB-overexpressing cells but not in control cells; conversely, overexpression of B2-containing reporter construct in PTB-overexpressing cells abolished the induction of p19(Ink4d) mRNA. In sum, we have shown that PTB plays as a negative regulator in H1299 cell proliferation at least by inducing p19(Ink4d) expression at transcriptional and post-transcriptional levels.