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Airway smooth muscle thickening, a key characteristic of chronic asthma, is largely attributed to increased smooth muscle cell proliferation and reduced smooth muscle apoptosis. Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that participates in the pathogenesis of airway smooth muscle remodeling. Although the role of Plk1 in cell proliferation and migration is recognized, its function in smooth muscle apoptosis has not been previously investigated. Caspase-9 (Casp9) is a key enzyme that participates in the execution of apoptosis. Casp9 phosphorylation at Ser-196 and Thr-125 is implicated in regulating its activity in cancer cells and epithelial cells. Here, exposure of human airway smooth muscle (HASM) cells to platelet-derived growth factorfor 24 hours enhanced the expression of Plk1 and Casp9 phosphorylation at Ser-196, but not Thr-125. Overexpression of Plk1 in HASM cells increased Casp9 phosphorylation at Ser-196. Moreover, the expression of Plk1 increased the levels of pro-Casp9 and pro-Casp3 and inhibited apoptosis, demonstrating a role of Plk1 in inhibiting apoptosis. Knockdown of Plk1 reduced Casp9 phosphorylation at Ser-196, reduced pro-Casp9/3 expression, and increased apoptosis. Furthermore, Casp9 phosphorylation at Ser-196 was upregulated in asthmatic HASM cells, which was associated with increased Plk1 expression. Knockdown of Plk1 in asthmatic HASM cells decreased Casp9 phosphorylation at Ser-196 and enhanced apoptosis. Together, these studies disclose a previously unknown mechanism that the Plk1-Casp9/3 pathway participates in the controlling of smooth muscle apoptosis.

Smooth muscle cell migration is essential for many diverse biological processes such as pulmonary/cardiovascular development and homeostasis. Abi1 (Abelson interactor 1) is an adapter protein that has been implicated in nonmuscle cell migration. However, the role and mechanism of Abi1 in smooth muscle migration are largely unknown. Here, Abi1 knockdown by shRNA reduced human airway smooth muscle cell migration, which was restored by Abi1 rescue. Abi1 localized at the tip of lamellipodia and its protrusion coordinated with F-actin at the leading cell edge of live cells. In addition, we identified profilin-1 (Pfn-1), a G-actin transporter, as a new partner for Abi1. Abi1 knockdown reduced the recruitment of Pfn-1 to the leading cell edge. Moreover, Abi1 knockdown reduced the localization of the actin-regulatory proteins c-Abl (Abelson tyrosine kinase) and N-WASP (neuronal Wiskott–Aldrich Syndrome Protein) at the cell edge without affecting other migration-related proteins including pVASP (phosphorylated vasodilator stimulated phosphoprotein), cortactin and vinculin. Furthermore, we found that c-Abl and integrin β1 regulated the positioning of Abi1 at the leading edge. Taken together, the results suggest that Abi1 regulates cell migration by affecting Pfn-1 and N-WASP, but not pVASP, cortactin and focal adhesions. Integrin β1 and c-Abl are important for the recruitment of Abi1 to the leading edge.

Summary: Asthma is a complex pulmonary disorder with multiple pathological mechanisms. A key pathological feature of chronic asthma is airway remodeling, which is largely attributed to airway smooth muscle (ASM) hyperplasia that contributes to thickening of the airway wall and further drives asthma pathology. The cellular processes that mediate ASM cell proliferation are not completely elucidated. Using multiple approaches, we demonstrate that the adapter protein Abi1 (Abelson interactor 1) is upregulated in ∼50% of ASM cell cultures derived from patients with asthma. Loss-of-function studies demonstrate that Abi1 regulates the activation of Jak2 (Janus kinase 2) and STAT3 (signal transducers and activators of transcription 3) as well as the proliferation of both nonasthmatic and asthmatic human ASM cell cultures. These findings identify Abi1 as a molecular switch that activates Jak2 kinase and STAT3 in ASM cells and demonstrate that a dysfunctional Abi1-associated pathway contributes to the progression of asthma.

Actin cytoskeletal reorganization plays an important role in regulating smooth muscle contraction, which is essential for the modulation of various physiological functions including airway tone. The adapter protein Abi1 (Abelson interactor 1) participates in the control of smooth muscle contraction. The mechanisms by which Abi1 coordinates smooth muscle function are not fully understood. Here, we found that contractile stimulation elicited Abi1 acetylation in human airway smooth muscle (HASM) cells. Mutagenesis analysis identified lysine-416 (K416) as a major acetylation site. Replacement of K416 with Q (glutamine) enhanced the interaction of Abi1 with N-WASP (neuronal Wiskott - Aldrich syndrome Protein), an important actin-regulatory protein. Moreover, the expression of K416Q Abi1 promoted actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19 and vimentin phosphorylation at Ser-56. Furthermore, p300 is a lysine acetyltransferase that catalyzes acetylation of histone and non-histone proteins in various cell types. Here, we discovered that a portion of p300 was localized in the cytoplasm of HASM cells. Knockdown of p300 reduced the agonist-induced Abi1 acetylation in HASM cells and in mouse airway smooth muscle tissues. Smooth muscle conditional knockout of p300 inhibited actin polymerization and the contraction of airway smooth muscle tissues without affecting myosin light chain phosphorylation and vimentin phosphorylation. Together, our results suggest that contractile stimulation induces Abi1 acetylation via p300 in smooth muscle. Acetylation at K416 promotes the coupling of Abi1 with N-WASP, which facilitates actin polymerization and smooth muscle contraction. This is a novel acetylation-dependent regulation of the actin cytoskeleton in smooth muscle.

Airway smooth muscle cells require coordinated protrusion and focal adhesion dynamics to migrate properly. However, the signaling cascades that connect these two processes remain incompletely understood. Glia maturation factor (GMF)-γ has been implicated in inducing actin debranching and inhibiting nucleation. In this study, we discovered that GMFγ phosphorylation at Y104 regulates human airway smooth muscle cell migration. Using high-resolution microscopy coupled with three-dimensional object-based quantitative image analysis software, Imaris 9.2.0, phosphomimetic mutant, Y104D-GMFγ, was enriched at nascent adhesions along the leading edge where it recruited activated neural Wiskott-Aldrich syndrome protein (N-WASP; pY256) to promote actin-branch formation, which enhanced lamellipodial dynamics and limited the growth of focal adhesions. Unexpectedly, we found that nonphosphorylated mutant, Y104F-GMFγ, was enriched in growing adhesions where it promoted a linear branch organization and focal adhesion clustering, and recruited zyxin to increase maturation, thus inhibiting lamellipodial dynamics and cell migration. The localization of GMFγ between the leading edge and focal adhesions was dependent upon myosin activity. Furthermore, c-Abl tyrosine kinase regulated the GMFγ phosphorylation-dependent processes. Together, these results unveil the importance of GMFγ phosphorylation in coordinating lamellipodial and focal adhesion dynamics to regulate cell migration.

Allergic asthma is characterized by airway smooth muscle layer thickening, which is largely attributed to cell division that requires the formation of centrosomes. Centrosomes play a pivotal role in regulating bipolar spindle formation and cell division. Before mitosis, centrosomes undergo maturation characterized by expansion of pericentriolar material proteins, which facilitates spindle formation and mitotic efficiency of many cell types. Although polo-like kinase 1 (Plk1) has been implicated in centrosome maturation, the mechanisms by which Plk1 regulates the cellular process are incompletely elucidated. Here, we identified paxillin as a new Plk1-interacting protein in human airway smooth muscle cells. We unexpectedly found that phosphorylated paxillin (Ser-272) was localized in centrosomes of human smooth muscle cells, which regulated centrosome maturation and spindle assembly. Plk1 knockdown inhibited paxillin Ser-272 phosphorylation, centrosome maturation, and cell division. Furthermore, exposure to allergens enhanced airway smooth muscle layer and paxillin phosphorylation at this residue in mice, which was reduced by smooth muscle conditional knockout of Plk1. These findings suggest that Plk1 regulates centrosome maturation and cell division in part by modulating paxillin phosphorylation on Ser-272. Furthermore, Plk1 contributes to the pathogenesis of allergen-induced thickening of the airway smooth muscle layer by affecting paxillin phosphorylation at this position.

Vimentin intermediate filaments (IFs) link to desmosomes (intercellular junctions) on the membrane and dense bodies in the cytoplasm, which provides a structural base for intercellular and intracellular force transmission in smooth muscle. There is evidence to suggest that the vimentin framework plays an important role in mediating smooth muscle mechanical properties such as tension and contractile responses. Contractile activation induces vimentin phosphorylation at Ser-56 and vimentin network reorientation, facilitating contractile force transmission among and within smooth muscle cells. p21-activated kinase 1 and polo-like kinase 1 catalyze vimentin phosphorylation at Ser-56, whereas type 1 protein phosphatase dephosphorylates vimentin at this residue. Vimentin filaments are also involved in other cell functions including migration and nuclear positioning. This review recapitulates our current knowledge how the vimentin network modulates mechanical and biological properties of smooth muscle.

Polo-like kinase 1 (Plk1) has been implicated in mitosis, cytokinesis, and proliferation. The mechanisms that regulate Plk1 expression remain to be elucidated. It is reported that miR-100 targets Plk1 in certain cancer cells. Here, treatment with miR-100 did not affect Plk1 protein expression in human airway smooth muscle cells. In contrast, treatment with miR-509 inhibited the expression of Plk1 in airway smooth muscle cells. Exposure to miR-509 inhibitor enhanced Plk1 expression in cells. Introduction of miR-509 reduced luciferase activity of a Plk1 3′UTR reporter. Mutation of miR-509 targeting sequence in Plk1 3′UTR resisted the reduction of the luciferase activity. Furthermore, miR-509 inhibited the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, and cell proliferation without affecting the expression of c-Abl, a tyrosine kinase implicated in cell proliferation. Moreover, we unexpectedly found that vimentin filaments contacted paxillin-positive focal adhesions. miR-509 exposure inhibited vimentin phosphorylation at Ser-56, vimentin network reorganization, focal adhesion formation, and cell migration. The effects of miR-509 on ERK1/2 and vimentin were diminished in RNAi-resistant Plk1 expressing cells treated with miR-509. Taken together, these findings unveil previously unknown mechanisms that miR-509 regulates ERK1/2 and proliferation by targeting Plk1. miR-509 controls vimentin cytoskeleton reorganization, focal adhesion assembly, and cell migration through Plk1.

During cell migration a critical interdependence between protrusion and focal adhesion dynamics is established and tightly regulated through signaling cascades. Here we demonstrate that c-Abl, a non-receptor tyrosine kinase, can control these migratory structures through the regulation of two actin-associated proteins, glia maturation factor-γ (GMFγ) and Neural Wiskott-Aldrich syndrome protein (N-WASP). Phosphorylation of GMFγ at tyrosine-104 by c-Abl directs activated N-WASP (pY256) to the leading edge, where it can promote protrusion extension. Non-phosphorylated GMFγ guides N-WASP (pY256) to maturing focal adhesions to enhance further growth. Antagonizing this signaling pathway through knockdown or mutation of tyrosine-104 to its non-phosphorylated form attenuates migration, whereas the phospho-mimic mutant GMFγ enhances migration, thus demonstrating c-Abl, GMFγ, and activated N-WASP (pY256) as a critical signaling cascade for regulating migration in a primary human cell line.

The tyrosine kinase c-Abl participates in the regulation of various cellular functions including cell proliferation, adhesion, migration, smooth muscle contraction and cancer progression. However, knowledge regarding transcriptional regulation of c-Abl is surprisingly limited. Sp1 is a founding member of the Sp1 transcription factor family that has been implicated in housekeeping gene expression, tumor cell proliferation and differentiation. Here, we show that knockdown and rescue of Sp1 affected growth factor-mediated c-Abl expression in cells. c-Abl promoter activity was also affected by Sp1 knockdown. This is the first evidence to suggest that Sp1 is an important transcription factor to regulate c-Abl expression. In addition, Sp1 phosphorylation at Thr-453 and Thr-739 has been proposed to regulate its activity in Drosophila cells. We unexpectedly found that growth factors did not induce Sp1 phosphorylation at these two residues. In contrast, growth factor stimulation upregulated Sp1 expression. Intriguingly, inhibition of ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) reduced expression of Sp1 and c-Abl. Furthermore, c-Abl knockdown diminished ERK1/2 phosphorylation and Sp1 expression. Taken together, these studies suggest that Sp1 can modulate c-Abl expression at transcription level. Conversely, c-Abl affects ERK1/2 activation and Sp1 expression in cells.

Cell migration plays an important role in many normal and pathological functions such as development, wound healing, immune defense, and tumor metastasis. Polarized migrating cells exhibit asymmetric distribution of many cytoskeletal proteins, which is believed to be critical for establishing and maintaining cell polarity and directional cell migration. To target these proteins to the site of function, cells use a variety of mechanisms such as protein transport and messenger RNA (mRNA) localization-mediated local protein synthesis. In contrast to the former which is intensively investigated and relatively well understood, the latter has been understudied and relatively poorly understood. However, recent advances in the study of mRNA localization and local translation have demonstrated that mRNA localization and local translation are specific and effective ways for protein localization and are crucial for embryo development, neuronal function, and many other cellular processes. There are excellent reviews on mRNA localization, transport, and translation during development and other cellular processes. This review will focus on mRNA localization-mediated local protein biogenesis and its impact on somatic cell migration.

Polo-like kinase 1 (Plk1) is a serine/threonine-protein kinase that has been implicated in mitosis, cytokinesis, and smooth muscle cell proliferation. The role of Plk1 in smooth muscle contraction has not been investigated. Here, stimulation with acetylcholine induced Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in smooth muscle. Contractile stimulation also activated Plk1 in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer signal of a Plk1 sensor. Moreover, knockdown of Plk1 in smooth muscle attenuated force development. Smooth muscle conditional knock-out of Plk1 also diminished contraction of mouse tracheal rings. Plk1 knockdown inhibited acetylcholine-induced vimentin phosphorylation at Ser-56 without affecting myosin light chain phosphorylation. Expression of T210A Plk1 inhibited the agonist-induced vimentin phosphorylation at Ser-56 and contraction in smooth muscle. However, myosin light chain phosphorylation was not affected by T210A Plk1. Ste20-like kinase (SLK) is a serine/threonine-protein kinase that has been implicated in spindle orientation and microtubule organization during mitosis. In this study knockdown of SLK inhibited Plk1 phosphorylation at Thr-210 and activation. Finally, asthma is characterized by airway hyperresponsiveness, which largely stems from airway smooth muscle hyperreactivity. Here, smooth muscle conditional knock-out of Plk1 attenuated airway resistance and airway smooth muscle hyperreactivity in a murine model of asthma. Taken together, these findings suggest that Plk1 regulates smooth muscle contraction by modulating vimentin phosphorylation at Ser-56. Plk1 activation is regulated by SLK during contractile activation. Plk1 contributes to the pathogenesis of asthma.

In order to screen the genes differentially expressed in two human prostate cancer cells with different metastasis potentials, suppression subtractive hybridization (SSH) was done twice on human prostate cancer cell line with high potential of metastasis PC3M-1E8 and its synogenetic cell line PC3M-2B4 with low metastasis potential. In the first subtraction PC3M-2B4 was used as tester and PC3M-1E8 as driver and the forward subtractive library was constructed. In the second on the tester and driver were interchanged and the reverse subtractive library was constructed. The screened clones of both libraries were sequenced and Gene Bank homology search was performed. Some clones were confirmed by quantitative real-time PCR. The results showed that two subtractive libraries containing 238 positive clones were constructed. Analysis of 16 sequenced clones randomly picked from two libraries showed that 4 differentially expressed gene fragments were identified as new EST with unknown functions. It was concluded that two subtractive libraries of human prostate cancer cell lines with different metastasis potentials were constructed successfully.

Many peroxidase inhibitors have been used in horseradish peroxidase (HRP) mediated immunostaining and in situ hybridization to quench background peroxidase activity. However, the efficacy of these inhibitors has been controversial, partially due to the lack of a quantitative study. Tyramide signal amplification (TSA) is much more sensitive than other HRP-mediated methods but its super-sensitivity also demands effective inhibition of background peroxidase activity. In searching for an effective peroxidase inhibitor, we have systematically evaluated the efficacy of several peroxidase inhibitors by quantifying the fluorescence intensity in cultured fibroblasts and tissue sections treated with the inhibitors. For cultured cells, 0.05 mM of phenylhydrazine and 1 unit/ml of glucose oxidase gave only moderate inhibition of HRP activity while 1 mM of sodium azide (NaN(3)), 3% of hydrogen peroxide (H(2)O(2)), NaN(3)/H(2)O(2) combined and 0.02 N hydrochloric acid (HCl) provided more complete inhibition. However, the inhibitory effect of NaN(3)/H(2)O(2) is reversible upon removal of the inhibitors and followed by incubation and wash to mimic antibody interactions. Similar results were obtained from rat skin wound tissues that have strong endogenous peroxidase activity. Our results recommend the use of HCl and caution the use of phenylhydrazine, glucose oxidase, NaN(3) and H(2)O(2) as potent peroxidase inhibitors.

The relationship of connexin43 (Cx43) and bystander effect in ovarian tumor cells in herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in vitro was explored and the effect of all-trans retinoic acid (RA) on the expression of Cx43 and bystander effect investigated. The Cx43 expression was detected by flowcytometry, Western blot, and immunofluorescence in two ovarian tumor cell lines OVCAR3, CaOV3 before and after RA treatment. Bystander effect was determined by the cells growth inhibitory rate with methyl thiazolyl tetrazolium. Following exposure to ganciclovir, there was much greater bystander killing in OVCAR3 than that in CaOV3 (P

Messenger RNA (mRNA) localization plays an important role in various cellular functions. To date, two general mechanisms have been identified for intracellular mRNA localization. The first one was identified by Blobel and colleagues more than three decades ago, by which mRNAs encoding for membrane and secreted proteins are targeted to the endoplasmic reticulum (ER) in a signal peptide dependent manner.1 The second mechanism is for the intracellular targeting of mRNAs encoding cytosolic proteins, which is dependent on specific sequence on the mRNA called zipcode.2 Recently, we have identified a new mechanism which targets Dia1 mRNA to the perinuclear ER in a zipcode-independent manner, even though the mRNA encodes a cytosolic protein.3 Here, we provide an updated discussion on how the Dia1 mRNA is targeted and what might be its physiological significance.

Cell migration plays an important role in many normal and pathological functions such as development, wound healing, immune defense, and tumor metastasis. Polarized migrating cells exhibit asymmetric distribution of many cytoskeletal proteins, which is believed to be critical for establishing and maintaining cell polarity and directional cell migration. To target these proteins to the site of function, cells use a variety of mechanisms such as protein transport and messenger RNA (mRNA) localization-mediated local protein synthesis. In contrast to the former which is intensively investigated and relatively well understood, the latter has been understudied and relatively poorly understood. However, recent advances in the study of mRNA localization and local translation have demonstrated that mRNA localization and local translation are specific and effective ways for protein localization and are crucial for embryo development, neuronal function, and many other cellular processes. There are excellent reviews on mRNA localization, transport, and translation during development and other cellular processes. This review will focus on mRNA localization-mediated local protein biogenesis and its impact on somatic cell migration.

To investigate the reliability and feasibility of human papillomavirus (HPV) DNA test in cervical scraping smears with polymerase chain reaction (PCR), 131 cases of cervical scraping specimens were collected, and the positive rates and accuracy of HPV infection were determined in normal subjects and cervical cancer patients. GP5+/GP6+ and E7 primer pairs designed for detecting HPV L1 and HPV type 16 E7 were tested in this study. Our results showed that positive rates of HPV DNA in normal population and cervical cancer patients were 32.99% and 73.53% respectively and there was significant difference between them (P0.001). In normal subjects, detection rates of HPV DNA with GP5+/GP6+ and E7 primer pairs were 27.84% and 16.49% respectively, with statistically significant difference between them (P0.05). However the detection rates in cervical cancer patients were 38.24% and 67.65% for the two markers, with a significant difference found between them (P0.05). It is concluded that HPV DNA test with PCR for cervical scraping smears was feasible. GP5+/GP6+ primer pairs may be a useful probe to screen HPV infection in normal population, but they are not sensitive enough in cervical cancer patients. It is suggested that high risk type HPV DNA test was very useful in population with high risk of cervical cancer.

This paper presents a dynamically scheduled parallel DSP architecture for general purpose DSP computations. The architecture consists of multiple DSP processors and of one or more scheduling units. DSP applications are first captured by stream flow graphs, and then stream flow graphs are statically mapped onto a parallel architecture. The ordering and starting time of DSP tasks are determined by the scheduling unit(s) using a dynamic scheduling algorithm. The main contributions of this paper are summarized as follows: • A scalable parallel DSP architecture : The parallel DSP architecture proposed in this paper is scalable to meet signal processing requirements. For parallel DSP architectures with large configurations, the scheduling unit may become a performance bottleneck. A distributed scheduling mechanism is proposed to address this problem. • A mapping algorithm : An algorithm is proposed to systematically map a stream flow graph onto a parallel DSP architecture. • A dynamic scheduling algorithm : We propose a dynamic scheduling algorithm that will only schedule a node for execution when both input data and output storage space are available. Such scheduling algorithm will allow buffer sizes to be determined at compile time. • A simulation study : Our simulation study reveals the relationships among the grain-size, the processor utilization, and the scheduling capability. We believe these relationships have significant impact on parallel computer architecture design involving dynamic scheduling.

Signal-peptide-mediated ER localization of mRNAs encoding for membrane and secreted proteins, and RNA-zipcode-mediated intracellular targeting of mRNAs encoding for cytosolic proteins are two well-known mechanisms for mRNA localization. Here, we report a previously unidentified mechanism by which mRNA encoding for Dia1, a cytosolic protein without the signal peptide, is localized to the perinuclear ER in an RNA-zipcode-independent manner in fibroblasts. Dia1 mRNA localization is also independent of the actin and microtubule cytoskeleton but requires translation and the association of Dia1 nascent peptide with the ribosome–mRNA complex. Sequence mapping suggests that interactions of the GTPase binding domain of Dia1 peptide with active Rho are important for Dia1 mRNA localization. This mechanism can override the β-actin RNA zipcode and redirect β-actin mRNA to the perinuclear region, providing a new way to manipulate intracellular mRNA localization.

Arp2/3 complex is an actin polymerization nucleator and localized in the leading protrusions of migrating cells. It has been unclear how this complex is targeted to the protrusions and whether its localization is functionally important. We previously demonstrated that mRNAs encoding for the subunits of the complex were localized in the protrusions of fibroblasts, suggesting a mechanism to target the complex to the protrusions. We here present data demonstrating the importance of Arp2/3 complex mRNA localization in directional cell migration. Using a novel mechanism by which Dia1 mRNA is targeted to the perinuclear endoplasmic reticulum, we re-directed the mRNA encoding Arp2, a subunit of the Arp2/3 complex, to the perinuclear region in fibroblasts. Knockdown of Arp2 alone caused dramatic reduction of the complex and resulted in narrow protrusions, increased random cell migration speed and loss of directionality. Rescue with a protrusion-localizing Arp2 mRNA restored normal cell migration behavior, whereas rescue with a mis-localizing Arp2 mRNA failed to restore speed and directionality. These results demonstrate that localization of Arp2/3 complex mRNAs in the leading protrusions is functionally important for directional cell migration.

We previously demonstrated that mRNAs for the subunits of the Arp2/3 complex localize to protrusions in fibroblasts (Mingle et al. in J Cell Sci 118:2425-2433, 2005). However, the signaling pathway that regulates Arp2/3 complex mRNA localization remains unknown. In this study we have identified lysophosphatidic acid (LPA) as a potent inducer of Arp2 mRNA localization to protrusions in fibroblasts via the RhoA-ROCK pathway. As RhoA is known to be activated locally in the cells, we sought to understand how spatial activation of Rho affects Arp2 mRNA localization. By sequentially performing fluorescence resonance energy transfer (FRET) and fluorescence in situ hybridization (FISH), we have visualized active RhoA and Arp2 mRNA in the same cells. Upon LPA stimulation, approximately two times more cells than those in the serum-free medium showed mutually exclusive localization of active RhoA and Arp2 mRNA. These results demonstrate the importance of localized activation of Rho in Arp2 mRNA localization and provide new insights as to how Rho regulates Arp2/3 complex mRNA localization. To our best knowledge, this is the first report in which FRET and FISH are combined to detect localized protein activity and mRNA in the same cells. This method should be easily adopted for the detection of other fluorescence protein based biosensors and DNA/RNA in the same cells.

Background PC cell-derived growth factor (PCDGF), also called epithelin/granulin precursor (GEP), is an 88-kDa secreted glycoprotein with the ability to stimulate cell proliferation in an autocrine fashion. In addition, some studies indicated that PCDGF participated in invasion, metastasis and survival of cancer cells by regulating cell migration, adhesion and proliferation. Yet the effects of PCDGF on proliferation and invasion of ovarian cancer cells in vitro and the mechanisms by which PCDGF mediates biological behaviors of ovarian cancer have rarely been reported. In the present study we investigated whether and how PCDGF/GEP mediated cell proliferation and invasion in ovarian cancer. Methods PCDGF/GEP expression level in three human ovarian cancer cell lines of different invasion potential were detected by RT-PCR and western blot. Effects of inhibition of PCDGF expression on cell proliferation and invasion capability were determined by MTT assay and Boyden chamber assay. Expression levels of cyclin D1 and CDK4 and MMP-2 activity were evaluated in a pilot study. Results PCDGF mRNA and protein were expressed at a high level in SW626 and A2780 and at a low level in SKOV3. PCDGF expression level correlated well with malignant phenotype including proliferation and invasion in ovarian cancer cell lines. In addition, the proliferation rate and invasion index decreased after inhibition of PCDGF expression by antisense PCDGF cDNA transfection in SW626 and A2780. Furthermore expression of CyclinD1 and CDK4 were downregulated and MMP-2 was inactivated after PCDGF inhibition in the pilot study. Conclusion PCDGF played an important role in stimulating proliferation and promoting invasion in ovarian cancer. Inhibition of PCDGF decreased proliferation and invasion capability through downregulation of cyclin D1 and CDK4 and inactivation of MMP-2. PCDGF could serve as a potential therapeutic target in ovarian cancer.

The nonreceptor tyrosine kinase c-Abl has a role in regulating smooth muscle cell proliferation, which contributes to the development of airway remodeling in chronic asthma. MicroRNAs (miRs) are small noncoding RNA molecules that regulate gene expression by binding to complementary sequences in the 3' untranslated regions (3' UTR) of target mRNAs. Previous analysis suggests that miR-203 is able to bind to the 3' UTR of human c-Abl mRNA. In this report, treatment with miR-203 attenuated the expression of c-Abl mRNA and protein in human airway smooth muscle (HASM) cells. Furthermore, transfection with an miR-203 inhibitor enhanced the expression of c-Abl at mRNA and protein levels in HASM cells. Treatment with platelet-derived growth factor (PDGF) induced the proliferation and ERK1/2 phosphorylation in HASM cells. Exposure to miR-203 attenuated the PDGF-stimulated proliferation and ERK1/2 phosphorylation in HASM cells. The expression of c-Abl at protein and mRNA levels was higher in asthmatic HASM cells, whereas the level of miR-203 was reduced in asthmatic HASM cells as compared to control HASM cells. Taken together, our present results suggest that miR-203 is a negative regulator of c-Abl expression in smooth muscle cells. miR-203 regulates smooth muscle cell proliferation by controlling c-Abl expression, which in turn modulates the activation of ERK1/2.

Overexpression of breast cancer resistance protein (BCRP) and mitoxantrone (MX) resistance protein can confer resistance to a variety of cytostatic drugs, such as MX, topotecan (TPT), doxorubicin, and daunorubicin. This study investigates the role of BCRP in resistance of ovarian cancer to TPT treatment. We have developed TPT-resistant human ovarian cancer cell line. Intracellular concentration of fluorescent dye rhodamine 123 (Rh123) was measured by flow cytometry. The expression of several membrane transporter proteins including P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), and BCRP were determined by reverse transcription-polymerase chain reaction and Western blotting. The Rh123 concentration in parental cells was approximately three times of those in TPT-resistant cells. In contrast to undetectable level of P-gp messenger RNA (mRNA) and minimal level of MRP1 expression in TPT-resistant cells, overexpression of both the BCRP mRNA and the protein was detected in these cells. Introduction of antisense-phosphorothioate oligonucleotide derived from BCRP mRNA into TPT-resistant cells resulted in a significant increase in the concentration of intracellular Rh123. These results suggested a novel mechanism in which a reduced intracellular drug concentration may be mediated by BCRP gene products in human ovarian cancer cells.

Objective: To select a target molecule associated with invasive potential in PC-3M cell. Methods: Cell subclones were isolated from PC-3M cell line with the method of limited dilution and their invasive ability characterized by monolayer invasion assay. The expression of u-PAR in the cell subclones at mRNA and protein levels was assayed respectively by non-competitive quantitative RT-PCR and immunohistochemical assay. Results: The expression level of u-PAR in highly invasive cell subclones was higher than that of lower invasive subclones. Conclusion: The higher expression level of u-PAR is associated with the relative strong invasive ability of PC-3M subclones. It is indicated that the u-PAR might be a promising target molecule for inhibiting invasion of highly invasive PC-3M cell subclones.

To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79% and 60%, respectively. Although the antisense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P0.01). The antisense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.

Presents a new and efficient error detection and fault location technique suitable for high performance application specific processing element (PE) arrays. In the proposed scheme, two spare PEs are located within four PEs, which forms a reconfigurable fault-tolerant module (RFTM). Any spare can functionally replace any one of the primary PEs within the RTFM. Since spares are physically close to the PEs that they replace, reconfiguration interconnections are short, thus minimizing the performance degradation. The new fault tolerant structure, RFTM, not only provides error detection mechanism, but also achieves fault tolerance. The authors have developed an error detection and fault location algorithm for the RFTM, which makes use of the information that is only available in a fault tolerant structure, but not available in other local redundancy techniques. Then, a systematic way of fault isolation was presented. A major contribution of this paper is that the authors approached the error detection, fault location and fault isolation in a unified way within the RFTM. The simple error detection and fault location mechanism turned out to result in a lean implementation of RFTM. >