Search

This article presents a new method for understanding and visualizing convolutional neural networks (CNNs). Most existing approaches to this problem focus on a global score and evaluate the pixelwise contribution of inputs to the score. The analysis of CNNs for multilabeled outputs or regression has not yet been considered in the literature, despite their success on image classification tasks with well-defined global scores. To address this problem, we propose a new inverse-based approach that computes the inverse of a feedforward pass to identify activations of interest in lower layers. We developed a layerwise inverse procedure based on two observations: 1) inverse results should have consistent internal activations to the original forward pass and 2) a small amount of activation in inverse results is desirable for human interpretability. Experimental results show that the proposed method allows us to analyze CNNs for classification and regression in the same framework. We demonstrated that our method successfully finds attributions in the inputs for image classification with comparable performance to state-of-the-art methods. To visualize the tradeoff between various methods, we developed a novel plot that shows the tradeoff between the amount of activations and the rate of class reidentification. In the case of regression, our method showed that conventional CNNs for single image super-resolution overlook a portion of frequency bands that may result in performance degradation.

This paper presents a new variational inference framework for image restoration and a convolutional neural network (CNN) structure that can solve the restoration problems described by the proposed framework. Earlier CNN-based image restoration methods primarily focused on network architecture design or training strategy with non-blind scenarios where the degradation models are known or assumed. For a step closer to real-world applications, CNNs are also blindly trained with the whole dataset, including diverse degradations. However, the conditional distribution of a high-quality image given a diversely degraded one is too complicated to be learned by a single CNN. Therefore, there have also been some methods that provide additional prior information to train a CNN. Unlike previous approaches, we focus more on the objective of restoration based on the Bayesian perspective and how to reformulate the objective. Specifically, our method relaxes the original posterior inference problem to better manageable sub-problems and thus behaves like a divide-and-conquer scheme. As a result, the proposed framework boosts the performance of several restoration problems compared to the previous ones. Specifically, our method delivers state-of-the-art performance on Gaussian denoising, real-world noise reduction, blind image super-resolution, and JPEG compression artifacts reduction. Our code and more details are available on our project page, https://github.com/JWSoh/VDIR.

Transmembrane Bax Inhibitor Motif-containing 6 (TMBIM6) is upregulated in several cancer types and involved in the metastasis. Specific downregulation of TMBIM6 results in cancer cell death. However, the TMBIM6 gene transcriptional regulation in normal and cancer cells is least studied. Here, we identified the core promoter region (-133/+30 bp) sufficient for promoter activity of TMBIM6 gene. Reporter gene expression with mutations at transcription factor binding sites, EMSA, supershift, and ChIP assays demonstrated that Sp1 is an essential transcription factor for basal promoter activity of TMBIM6. The TMBIM6 mRNA expression was increased with Sp1 levels in a concentration dependent manner. Ablation of Sp1 through siRNA or inhibition with mithramycin-A reduced the TMBIM6 mRNA expression. We also found that the protein kinase-C activation stimulates promoter activity and endogenous TMBIM6 mRNA by 2- to 2.5-fold. Additionally, overexpression of active mutants of PKCι, PKCε, and PKCδ increased TMBIM6 expression by enhancing nuclear translocation of Sp1. Immunohistochemistry analyses confirmed that the expression levels of PKCι, Sp1, and TMBIM6 were correlated with one another in samples from human breast, prostate, and liver cancer patients. Altogether, this study suggests the involvement of Sp1 in basal transcription and PKC in the enhanced expression of TMBIM6 in cancer.

Rationale: Degenerative spondylolisthesis is defined as forward slippage of a vertebra with respect to the underlying vertebra and is associated with the induction of lumbar canal stenosis. The use of anterior column support for degenerative lumbar conditions has been well documented. Direct lateral interbody fusion (DLIF) gains access via a lateral approach through the retroperitoneal fat and psoas muscle. It avoids many of the access-related complications yet comes with its own risks and limitations. The location of the iliac wing precludes exposure of the L5-S1 disc space and may make L4-5 surgery difficult. Therefore, accurate preoperative patient positioning is essential., Patient Concerns: A 71-year-old female with a body mass index (BMI) of 39.2 kg/m (height 155.9 cm, weight 79.5 kg) presented with lumbar pain radiating to the left lower limb. She complained of neurologic claudication with more than 100 m ambulation., Diagnosis: Plain standing view on spine radiography revealed L4-5 spondylolisthesis and disc-space narrowing. Magnetic resonance imaging (MRI) revealed severe L4-5 bilateral foraminal stenosis. After the first surgery, simple rib cage radiography was performed to examine the source of her right-sided flank pain and it revealed acute fracture of the right ninth and tenth ribs., Interventions: The patient was laterally positioned on the table in an extreme bending position. The intervertebral cage was inserted in the L4-5 disc space and disc height was restored. With respect to the rib fracture, treatment was conservative., Outcomes: The patient's radiating pain was immediately relieved and her lower back pain disappeared at 3 months after surgery. The patient reported right-sided flank pain after the first surgery. Simple rib cage radiography was performed and revealed fracture of the right ninth and tenth ribs. Follow-up assessments conducted 2 months later revealed complete bony union., Lessons: DLIF avoids many access-related complications. However, it is associated with other intraoperative complications, including injury to the lumbar nerve root and plexus. In addition, there are preoperative complications associated with improper patient placement on the table. In this case, the patient's obesity and strict positional requirements resulted in rib fracture. We suggest that surgeons consider this complication and exercise care in preoperative positioning.

Purpose: The purpose of this study is to present the effective design of N-plasty of the iliotibial band and surgical results of its use as a treatment for refractory external snapping hip., Materials and Methods: We evaluated 17 patients (24 cases) with external snapping hip who underwent N-plasty between October 2013 and May 2016 and who were followed up for at least 12 months. All patients were male and the mean age was 20.8 years. The mean duration of symptoms prior to surgical intervention was 28.5 months with an average follow up of 24.5 months. Surgery was defined as being successful when patients could carry out their daily activities and exercise without a clicking sensation or pain 6 months after surgery until their last follow-up. Failure was defined when either a clicking sensation or pain was present. The visual analog scale (VAS) and modified Harris hip score (mHHS) were measured and compared preoperatively and at last follow-up., Results: All patients had complete resolution of pain and snapping. The VAS decreased from 6.77 preoperatively to 0.09 postoperatively and mHHS improved from 69.5 to 97.8 after surgery., Conclusion: Modified designed N-plasty is considered to be an excellent treatment method facilitating operation reproducibility with maximum elongation effect of the iliotibial band., Competing Interests: CONFLICT OF INTEREST: The authors declare that there is no potential conflict of interest relevant to this article.

KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR) signaling may cause dysregulation of microRNA (miRNA) and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRAS(G12V) into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR)-4689, one of the significantly down-regulated miRNAs in KRAS(G12V) overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS) and v-akt murine thymoma viral oncogene homolog 1(AKT1), key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC.

Background: There have been few randomized control trials comparing the incidence of stent fracture and primary patency among different self-expanding nitinol stents to date. The SMART™ CONTROL stent (Cordis Corp, Miami Lakes, Florida, United States) has a peak-to-valley bridge and inline interconnection, whereas the COMPLETE™-SE stent (Medtronic Vascular, Santa Rosa, California, United States) crowns have been configured to minimize crown-to-crown interaction, increasing the stent's flexibility without compromising radial strength. Further, the 2011 ESC (European society of cardiology) guidelines recommend that dual antiplatelet therapy with aspirin and a thienopyridine such as clopidogrel should be administered for at least one month after infrainguinal bare metal stent implantation. Cilostazol has been reported to reduce intimal hyperplasia and subsequent repeat revascularization. To date, there has been no randomized study comparing the safety and efficacy of two different antiplatelet regimens, clopidogrel and cilostazol, following successful femoropopliteal stenting., Methods/design: The primary purpose of our study is to examine the incidence of stent fracture and primary patency between two different major representative self-expanding nitinol stents (SMART™ CONTROL versus COMPLETE™-SE) in stenotic or occlusive femoropopliteal arterial lesion. The secondary purpose is to examine whether there is any difference in efficacy and safety between aspirin plus clopidogrel versus aspirin plus cilostazol for one month following stent implantation in femoropopliteal lesions. This is a prospective, randomized, multicenter trial to assess the efficacy of the COMPLETE™-SE versus SMART™ CONTROL stent for provisional stenting after balloon angioplasty in femoropopliteal arterial lesions. The study design is a 2x2 randomization design and a total of 346 patients will be enrolled. The primary endpoint of this study is the rate of binary restenosis in the treated segment at 12 months after intervention as determined by catheter angiography or duplex ultrasound., Discussion: This trial will provide powerful insight into whether the design of the COMPLETE™-SE stent is more fracture-resistant or effective in preventing restenosis compared with the SMART™ CONTROL stent. Also, it will determine the efficacy and safety of aspirin plus clopidogrel versus aspirin plus cilostazol in patients undergoing stent implantation in femoropopliteal lesions., Trial Registration: Registered on 2 April 2012 with the National Institutes of Health Clinical Trials Registry (ClinicalTrials.gov identifier# NCT01570803).

Sclerotium rot was found on Cymbidium orchids at Seosan-si, Chungcheongnam-do, Korea, in July, 2010. Symptoms occurred on low leaves, which turned yellowish, after which the entire plant wilted. Severely infected plants were blighted and eventually died. White mycelial mats and sclerotia appeared on pseudobulbs. Based on the mycological characteristics and pathogenicity, the causal fungus was identified as Sclerotium rolfsii. This is the first report of new Sclerotium rot on Cymbidium spp. caused by S. rolfsii in Korea.

Objectives: This study compared rate of cell proliferation, viability, cell size, expression patterns of genes related to pluripotency and epigenetic modification between canine foetal fibroblasts (cFF) and canine adipose tissue-derived mesenchymal stem cells (cAd-MSC)., Materials and Methods: Proliferation pattern, cell viability as well as cell size at each passage of cFF and cAd-MSC were measured when cultures reached confluence. In addition, real-time PCR was performed to investigate expression of Dnmt1, HDAC1, OCT4, SOX2, BAX, BCL2 genes with reference to β-actin gene expression as an endogenous control in both cell lines., Results: cFF and cAd-MSC differed in number of generations, but not in doubling times, at all passages. Mean cell size of cAd-MSC was significantly smaller than that of cFF. Cell viability was significantly lower in cFFs and apoptotic level was significantly lower in cAd-MSC compared to passage-matched cFF. In the expression of genes related to pluripotency and epigenetic modification, level of HDAC1 in cAd-MSC was significantly higher than in cFF, but expression of Dnmt1 did not differ between the two groups. OCT4 and SOX2 were significantly more highly expressed in cAd-MSC compared to cFF., Conclusions: cAd-MSC have higher stem-cell potential than cFF in terms of proliferation patterns, epigenetic modification and pluripotency, thus cAd-MSC could be more appropriate than cFF as donors of nuclei in somatic cell nuclear transfer for transgenesis., (© 2012 Blackwell Publishing Ltd.)

Protein kinase C (PKC) is a multigene family of serine/threonine kinases. PKC is involved in regulating adrenal and gonadal steroidogenesis; however, the functional relevance of the different PKC isoenzymes remains obscure. In this study, we demonstrate that MA-10 mouse Leydig tumor cells express several PKC isoforms to varying levels and that the activation of PKC signaling, by phorbol 12-myristate 13-acetate (PMA) elevated the expression and phosphorylation of PKCα, -δ, -ε, and -μ/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKCα, δ, and ε and PKD, using small interfering RNAs, resulted in deceases in basal and PMA-mediated StAR and steroid levels and demonstrated the importance of PKD in steroidogenesis. PKD was capable of controlling PMA and cAMP/PKA-mediated synergism involved in the steroidogenic response. Further studies pointed out that the regulatory events effected by PKD are associated with cAMP response element-binding protein (CREB) and c-Jun/c-Fos-mediated transcription of the StAR gene. Chromatin immunoprecipitation studies revealed that the activation of phosphorylated CREB, c-Jun, and c-Fos by PMA was correlated with in vivo protein-DNA interactions and the recruitment of CREB-binding protein, whereas knockdown of PKD suppressed the association of these factors with the StAR promoter. Ectopic expression of CREB-binding protein enhanced the trans-activation potential of CREB and c-Jun/c-Fos in StAR gene expression. Using EMSA, a -83/-67-bp region of the StAR promoter was shown to bind PKD-transfected MA-10 nuclear extract in a PMA-responsive manner, targeting CREB and c-Jun/c-Fos proteins. These findings provide evidence for the presence of multiple PKC isoforms and demonstrate the molecular events by which selective isozymes, especially PKD, influence PMA/PKC signaling involved in the regulation of the steroidogenic machinery in mouse Leydig cells.

Nitric oxide (NO) production in endothelial cells (EC) is regulated by multisite phosphorylation of specific serine and threonine residues in endothelial NO synthase (eNOS). Among these, eNOS-Ser116 is phosphorylated in the basal state, and its phosphorylation contributes to basal NO production. Here, we investigated the mechanism by which eNOS-Ser116 is phosphorylated during the basal state using bovine aortic EC. Although a previous study suggested that protein kinase C was involved in eNOS-Ser116 phosphorylation, overexpression of various protein kinase C isoforms did not affect eNOS-Ser116 phosphorylation. An in silico analysis using a motif scan revealed that the eNOS-Ser116 residue might be a substrate for proline-directed protein kinases. Roscovitine, a specific inhibitor of cyclin-dependent kinase (CDK), 1, 2, and 5, but not an inhibitor of mitogen-activated protein kinase kinase or glycogen synthase kinase 3beta, inhibited eNOS-Ser116 phosphorylation dose dependently. Furthermore, purified CDK1, 2, or 5 directly phosphorylated eNOS-Ser116 in vitro. Ectopic expression of the dominant-negative CDK5 but not dominant-negative CDK1 or dominant-negative CDK2 repressed eNOS-Ser116 phosphorylation and increased NO production. In addition, CDK5 activity was detected in bovine aortic EC, and coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and CDK5. Cotransfection of CDK5 and p25, the specific CDK5 activator, increased eNOS-Ser116 phosphorylation and decreased NO production, but its parent molecule, p35, and p39, another activator, were not detected in bovine aortic EC, which suggests the existence of a novel CDK5 activator. Overall, this is the first study to find that CDK5 is a physiological kinase responsible for eNOS-Ser116 phosphorylation and regulation of NO production.

Transfer of melanin-containing melanosomes from melanocytes to neighboring keratinocytes results in skin pigmentation. To provide a more practical method of visualizing melanosomes in melanocytes as well as in keratinocytes, we attempted to use murine cell lines instead of human primary cells. We generated various fluorescent fusion proteins of tyrosinase, a melanin synthesis enzyme located in the melanosome, by using green fluorescent protein and red fluorescent protein. The intracellular localization of tyrosinase was then examined by fluorescence and confocal microscopy. Co-culture of murine melanocytes and keratinocytes was optimized and melanosome transfer was either stimulated with alphaMSH or partially inhibited by niacinamide. To the best of our knowledge, this is the first study showing that a murine co-culture model, in addition to human primary cell co-culture, can be a good tool for depigmenting agent screening by monitoring melanosome transfer.

TPA is known to cooperate with an activated Ras oncogene in the transformation of rodent fibroblasts, but the biochemical mechanisms responsible for this effect have not been established. In the present study we used c-fos promoter-luciferase constructs as reporters, in transient transfection assays, in NIH3T3 cells to assess the mechanism of this cooperation. We found a marked synergistic interaction between TPA and a transfected v-Ha-ras oncogene in the activation of c-fos promoter and SRE. SRE has binding sites for TCF and SRF. A dominant-negative Ras (ras-N17) inhibited the TPA-Ras synergy by blocking the PKC-MAPK-TCF pathway. Dominant-negative RhoA and Rac1 (but not Cdc42Hs) inhibited the TPA-Ras synergy by blocking the Ras-Rho-SRF signaling pathway. Constitutively active PKCalpha and PKCepsilon showed synergy with v-Ras. These results suggest that the activation of two distinct pathways such as Ras-Raf-ERK-TCF pathway and Rho-SRF pathway are responsible for the induction of c-fos by TPA and Ras in mitogenic signaling pathways.

It has been reported previously that cyclin G1 enables cells to overcome radiation-induced G(2) arrest and increased cell death and that these effects are mediated by transcriptional activation of cyclin B1. In this study, we further investigated the mechanism by which cyclin G1 transcriptionally activates cyclin B1. Deletion or point mutations within the cyclin B1 promoter region revealed that the c-Myc binding site (E-box) is necessary for cyclin G1-mediated transcriptional activation of cyclin B1 to occur. In addition, the kinase activity of Cdk5 was increased by cyclin G1 overexpression, and Cdk5 directly phosphorylated c-Myc on Ser-62. Furthermore, cyclin G1 mediated increased radiosensitivity, and radiation-induced M phase arrest was attenuated when RNA interference of Cdk5 was treated. Taken together, the results of this study indicate that Cdk5 activation in cells that overexpress cyclin G1 leads to c-Myc phosphorylation on Ser-62, which is responsible for cyclin G1-mediated transcriptional activation of cyclin B1.

Eukaryotic protein kinases, containing a conserved catalytic domain, represent one of the largest superfamilies of the eukaryotic proteins and play distinct roles in cell signaling and diseases. Near completion of rat genome sequencing project enables the evaluation of a near complete set of rat protein kinases. Publicly accessible genetic sequence databases were searched for rat protein kinases, and 515 eukaryotic protein kinases, 40 atypical protein kinases and 45 kinase pseudogenes were identified. The rat has 509 putative protein kinases orthologous to human kinases. Unlike microtubule affinity-regulating kinases, the rat has a few more kinases, in addition to the orthologous pairs of mouse kinases. The comparison of 11 different eukaryotic species revealed the evolutionary conservation of this diverse family of proteins. The evolutionary rate studies of human disease and non-disease associated kinases suggested that relatively uniform selective pressures have been applied to these kinase classes. This bioinformatic study of the rat protein kinases provides a suitable framework for further characterization of the functional and structural properties of these protein kinases.

Heat shock protein 27 (HSP27), which is highly expressed in human lung and breast cancer tissues, induced resistance to cell death against various stimuli. Treatment of NCI-H1299 cells, which express a high level of HSP27, with small interference RNA specifically targeting HSP27 resulted in inhibition of their resistance to radiation or cisplatin, suggesting that HSP27 contributed to cellular resistance in these lung cancer cells. Furthermore, because HSP27 interacts directly with the COOH terminus of the protein kinase C delta (PKC delta)-V5 region with ensuing inhibition of PKC delta activity and PKC delta-mediated cell death, we wished to determine amino acid residues in the V5 region that mediate its interaction with HSP27. Investigation with various deletion mutants of the region revealed that amino acid residues 668 to 674 of the V5 region mediate its interaction with HSP27. When NCI-H1299 cells were treated with biotin or with FITC-tagged heptapeptide of the residues 668 to 674 (E-F-Q-F-L-D-I), the cells exhibited dramatically increased cisplatin or radiation-induced cell death with the heptapeptide having efficient interaction with HSP27, which in turn restored the PKC delta activity that had been inhibited by HSP27. In vivo nude mice grafting data also suggested that NCI-H1299 cells were sensitized by this heptapeptide. The above data strongly show that the heptapeptide of the PKC delta-V5 region sensitized human cancer cells through its interaction with HSP27, thereby sequestering HSP27. The heptapeptide may provide a novel strategy for selective neutralization of HSP27.

GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although the cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LbetaT2 gonadotrope cells (maximal increase, 2.5-fold; EC(50), 0.30 nm), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca(2+) independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, indicating that the GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with Gö6976 did not prevent GnRH-induced cAMP production, whereas down-regulation of novel PKCdelta, -epsilon, and -theta by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKCdelta or -epsilon but not PKCtheta impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKCdelta and -epsilon are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LbetaT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms, i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP, suggesting that in vivo, the cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.

Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by beta-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCepsilon (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, beta-phorbol myristate acetate (PMA), but not 4alpha-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events.

Although cyclin G1 has been implicated in certain p53-related biological phenomena, other aspects of its function remain unclear. Here we report hitherto unknown mechanism by which cyclin G1 increases radiation sensitivity by regulating the level of cyclin B1. Overexpression of cyclin G1 was observable in lung carcinoma tissues. Irradiation of human lung cells with cyclin G1 overexpression resulted in increased cell death and gamma-H2AX foci suggesting that cyclin G1 rendered the cells more susceptible to DNA damage. Enhanced radiosensitivity by cyclin G1 was correlated with increased cyclin B1, CDC2/cyclin B1 complex, and MPM2. Cell cycle synchronization clearly showed coexpression of cyclin G1 and cyclin B1 in G2/M phase. Depletion of cyclin G1 by interference RNA revealed that cyclin G1 regulated transcription of cyclin B1 in a p53-independent manner, and confirmed that the increased mitotic cells and cell death by cyclin G1 were dependent upon cyclin B1. Therefore, our data suggest that cyclin G1 enhanced radiation sensitivity by overriding radiation-induced G2 arrest through transcriptional upregulation of cyclin B1.

Intracellular signaling molecules and apoptotic factors seem to play an important role in determining the radiation response of tumor cells. However, the basis for the link between signaling pathway and apoptotic cell death machinery after ionizing irradiation remains still largely unclear. In this study, we showed that c-Abl-PKCdelta-Rac1-p38 MAPK signaling is required for the conformational changes of Bak and Bax during ionizing radiation-induced apoptotic cell death in human non-small cell lung cancer cells. Ionizing radiation induced conformational changes and subsequent oligomerizations of Bak and Bax, dissipation of mitochondrial membrane potential, and cytochrome c release from mitochondria. Small interference (siRNA) targeting of Bak and Bax effectively protected cells from radiation-induced mitochondrial membrane potential loss and apoptotic cell death. p38 MAPK was found to be selectively activated in response to radiation treatment. Inhibition of p38 MAPK completely suppressed radiation-induced Bak and Bax activations, dissipation of mitochondrial membrane potential, and cell death. Moreover, expression of a dominant negative form of protein kinase Cdelta (PKCdelta) or siRNA targeting of PKCdelta attenuated p38 MAPK activation and conformational changes of Bak and Bax. In addition, ectopic expression of RacN17, a dominant negative form of Rac1, markedly inhibited p38 MAPK activation but did not affect PKCdelta activation. Upon stimulation of cells with radiation, PKCdelta was phosphorylated dramatically on tyrosine. c-Abl-PKCdelta complex formation was also increased in response to radiation. Moreover, siRNA targeting of c-Abl attenuated radiation-induced PKCdelta and p38 MAPK activations, and Bak and Bax modulations. These data support a notion that activation of the c-Abl-PKCdelta-Rac1-p38 MAPK pathway in response to ionizing radiation signals conformational changes of Bak and Bax, resulting in mitochondrial activation-mediated apoptotic cell death in human non-small cell lung cancer cells.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived mature B cells with the distinctive phenotype CD19(hi) CD5+ CD23+ IgM(lo), which are refractory to apoptosis. An increased level of apoptosis has been observed on treatment of human B-CLL cells with protein kinase C (PKC) inhibitors, suggesting that this family of protein kinases mediate survival signals within B-CLL cells. Therefore, to investigate the ability of individual PKC isoforms to transform developing B cells, we stably expressed plasmids encoding PKC mutants in fetal liver-derived hematopoietic progenitor cells (HPC) from wild-type mice and then cultured them in B-cell generation systems in vitro and in vivo. Surprisingly, we noted that expression of a plasmid-encoding dominant-negative PKC alpha (PKC alpha-KR) in HPCs and subsequent culture both in vitro and in vivo resulted in the generation of a population of cells that displayed an enhanced proliferative capacity over untransfected cells and phenotypically resemble human B-CLL cells. In the absence of growth factors and stroma, these B-CLL-like cells undergo cell cycle arrest and, consistent with their ability to escape growth factor withdrawal-induced apoptosis, exhibited elevated levels of Bcl-2 expression. These studies therefore identify a unique oncogenic trigger for the development of a B-CLL-like disease resulting from the subversion of PKC alpha signaling. Our findings uncover novel avenues not only for the study of the induction of leukemic B cells but also for the development of therapeutic drugs to combat PKC alpha-regulated transformation events.

Since radiation-induced caspase-dependent apoptosis and ROS generation were partially prevented by HSP25 overexpression, similar to the treatment of control cells with antioxidant agents such as DPI and tiron, questions arise whether radiation-mediated ROS generation contributes to the apoptotic cell death, and also whether HSP25 overexpression can reduce ROS mediated apoptotic cell death. In the present study, radiation-induced cytochrome c release from mitochondria and activation of caspases accompanied by a decrease of mitochondrial membrane potential in Jurkat T cells were shown to be inhibited by mitochondrial complex I inhibitor rotenone, suggesting that mitochondrial ROS might be important in radiation-induced caspase-dependent apoptosis. When HSP25 was overexpressed, effects similar to the treatment of cells with the antioxidants were obtained, indicating that HSP25 suppressed radiation-induced mitochondrial alteration that resulted in apoptosis. Furthermore, activation of p38 MAP kinase by radiation was associated with radiation-induced cell death and ROS production and PKCdelta was an upstream molecule for p38 MAP kinase activation, ROS generation and subsequent caspase-dependent apoptotic events. However, in the HSP25 overexpressed cells, the above-described effects were blocked. In fact, radiation-induced membrane translocation of PKCdelta and tyrosine phosphorylation were inhibited by HSP25. Based on the above data, we suggest that HSP25 downregulates PKCdelta, which is a key molecule for radiation-induced ROS generation and mitochondrial-mediated caspase-dependent apoptotic events.

Heat shock protein 25 (HSP25) interferes negatively with apoptosis through several pathways that involve its direct interaction with cytochrome c or Akt. Here we show that HSP25 inhibits protein kinase C (PKC) delta-mediated cell death through direct interaction. HSP25 binds to kinase-active PKCdelta to inhibit its kinase activity and translocation to the membrane, which results in reduced cell death. Deletion constructs of HSP25 and PKCdelta identified amino acids 90-103 of HSP25 and the C-terminal V5 region of PKCdelta as binding sites. In addition, the interaction between HSP25 and PKCdelta induced HSP25 phosphorylation at Ser-15 and Ser-86, and these phosphorylations permitted HSP25 release from PKCdelta. Based on these observations, we propose that after PKCdelta activation, HSP25 binds to the exposed V5 region of PKCdelta. This novel function of HSP25 accounts for its cytoprotective properties via the inhibition of PKCdelta and the enhancement of HSP25 phosphorylation.

We previously demonstrated that protein kinase C-eta (PKC-eta) mediates a phorbol 12-myristate-13-acetate (PMA)-induced proliferative response in human glioblastoma (GBM) cells. In this report, we show that PMA-stimulated activation of PKC-eta in U-251 GBM cells resulted in activation of both Akt and the mammalian target of rapamycin (mTOR) signaling pathways and an increase in cell proliferation. Expression of a kinase dead PKC-eta (PKC-etaKR) construct reduced the basal and PMA-evoked proliferation of PKC-eta-expressing U-251 GBM cells, as well as abrogated the PMA-induced activation of Akt, mTOR, and the mTOR targets 4E-BP1 and STAT-3. Treatment of cells with the PI-3 kinase inhibitor LY294002 (10 muM) or the mTOR inhibitor rapamycin (10 nM) also reduced PMA-induced proliferation and cell-cycle progression. Expression of a constitutively active PKC-eta (PKC-etaDeltaNPS) construct in a GBM cell line with no endogenous PKC-eta (U-1242) also provided evidence that PKC-eta targets the Akt and mTOR signaling pathways. Moreover, activation of 4E-BP1 and STAT-3 in both PMA-treated U-251 and PKC-etaDeltaNPS-expressing U-1242 GBM cells was inhibited by rapamycin. However, activation of Akt, but not mTOR was inhibited by the PI-3 kinase inhibitor LY294002. This study identifies Akt and mTOR as downstream targets of PKC-eta that are involved in GBM cell proliferation.

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKCa isoform. When PKCalpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKCalpha kinase active mutant (PKCalpha- CAT) in the undifferentiated keratinocyte, but not PKCbeta-CAT, also increased differentiation marker expressions. On the other hand, PKCalpha dominant negative mutant (PKCbeta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKCbeta-KR did not, suggesting that PKCalpha is responsible for keratinocyte differentiation. When downstream pathway of PKCalpha in Ca2+ -mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+ -mediated differentiation, and that only ERK1/2 pathway is specific for PKCalpha-mediated differentiation in mouse keratinocytes.

Protein kinase C (PKC) family, which functions through serine/threonine kinase activity, is involved in signal transduction pathways necessary for cell proliferation, differentiation, and apoptosis. Its critical role in neoplastic transformation and tumor invasion renders PKC a potential target for anticancer therapy. In this study, we investigated the effect of targeting individual PKCs on gastric carcinogenesis. We established gastric cancer cell lines stably expressing antisense PKCalpha, PKCbeta1, and PKCbeta2 cDNA. These stable transfectants were characterized by cell morphology, cell growth, apoptosis, and tumorigenicity in vitro and in vivo. PKCalpha-AS and PKCbeta1-AS transfectants showed a different morphology with flattened, long processes and decreased nuclear:cytoplasmic ratio compared with the control cells. Cell growth was markedly inhibited in PKCalpha-AS and PKCbeta1-AS transfectants. PKCalpha-AS and PKCbeta1-AS cells were more responsive to mitomycin C- or 5-fluorouracil-induced apoptosis. However, antisense targeting of PKCbeta2 did not have any significant effect on cell morphology, cell growth, or apoptosis. Furthermore, antisense inhibition of PKCalpha and PKCbeta1 markedly suppressed colony-forming efficiency in soft agar and in nude mice xenografts. Inhibition of PKCalpha or PKCbeta1 significantly suppressed transcriptional and DNA binding activity of activator protein in gastric cancer cells, suggesting that PKCalpha or PKCbeta1 exerts their effects on cell growth through regulation of activator protein activity. These data provide evidence that targeting PKCalpha and PKCbeta1 by antisense method is a promising therapy for gastric cancer.

Epidemiological and experimental carcinogenesis studies provide evidence that components of garlic (Allium sativum) have anticancer activity. We recently reported that the garlic derivative S-allylmercaptocysteine (SAMC) inhibits growth, arrests cells in G(2)-M, and induces apoptosis in human colon cancer cells (Shirin et al., Cancer Res., 61: 725-731, 2001). Because a fraction of the SAMC-treated cells are specifically arrested in mitosis, we examined the mechanism of this effect in the present study. Immunofluorescent microscopy revealed that the treatment of SW480 cells or NIH3T3 fibroblasts with 150 micro M SAMC (the IC(50) concentration) caused rapid microtubule (MT) depolymerization, MT cytoskeleton disruption, centrosome fragmentation and Golgi dispersion in interphase cells. It also induced the formation of monopolar and multipolar spindles in mitotic cells. In vitro turbidity assays indicated that SAMC acted directly on tubulin to cause MT depolymerization, apparently because it interacts with -SH groups on tubulin. To investigate the signaling pathways involved in SAMC-induced apoptosis, we assayed c-Jun NH(2)-terminal kinase (JNK) activity and found that treatment with SAMC caused a rapid and sustained induction of JNK activity. The selective JNK inhibitor SP600125 inhibited the early phase (24 h) but not the late phase (48 h and later) of apoptosis induced by SAMC. Expression of a dominant-negative mutant of JNK1 in SW480 cells inhibited apoptosis induced by SAMC at 24 h but had no protective effect at 48 h. JNK1(-/-) mouse embryonic fibroblasts were resistant to SAMC-induced apoptosis at 24 h but not at 48 h. On the other hand, the inhibition or abrogation of JNK1 activity did not inhibit the G(2)-M arrest induced by SAMC. SAMC also activated caspase-3. The general caspase inhibitor z-VAD-fmk inhibited both early and late phases of apoptosis induced by SAMC. We conclude that the garlic-derived compound SAMC exerts antiproliferative effects by binding directly to tubulin and disrupting the MT assembly, thus arresting cells in mitosis and triggering JNK1 and caspase-3 signaling pathways that lead to apoptosis.

Although protein kinase C (PKC) has been implicated in cell cycle progression, cell proliferation, and tumor promotion, the precise roles of specific isoforms in these processes is not clear. Therefore, we constructed and analyzed a series of expression vectors that encode hemagglutinin-tagged wild type (WT), constitutively active mutants (Delta NPS and CAT), and dominant negative mutants of PKCs alpha, beta 1, beta 2, gamma, delta, epsilon, eta, zeta, and iota. Cyclin D1 promoter reporter assays done in serum-starved NIH3T3 cells indicated that the constitutively active mutants of PKC-alpha and PKC-epsilon were the most potent activators of this reporter, whereas the constitutively active mutant of PKC-delta inhibited its activity. Transient transfection studies with a series of 5'-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-alpha. Deletion of the AP-1 enhancer element located at position -954 upstream from the initiation site abolished PKC-alpha-dependent activation of cyclin D1 expression. Deletion of the SP1 or CRE enhancer elements did not have any effect. A dominant negative mutant of c-Jun inhibited activation of the cyclin D1 promoter in a concentration-dependent manner, providing further evidence that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-alpha and PKC-epsilon. The constitutively active mutants of PKC-alpha and PKC-epsilon also activated c-fos, c-jun, and cyclin E promoter activity. Furthermore, NIH3T3 cells that stably express the constitutively active mutants of PKC-alpha or PKC-epsilon displayed increased expression of endogenous cyclins D1 and E and faster growth rates. These results provide evidence that the activation of PKC-alpha or PKC-epsilon in mouse fibroblasts can play an important role in enhancing cell cycle progression and cell proliferation.

The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), utilizes multiple signaling pathways to activate extracellularly regulated mitogen-activated protein kinases (ERK1/2) in normal and immortalized pituitary gonadotrophs and transfected cells expressing the GnRH receptor. In immortalized hypothalamic GnRH neurons (GT1-7 cells), which also express GnRH receptors, GnRH, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) caused marked phosphorylation of ERK1/2. This action of GnRH and PMA, but not that of EGF, was primarily dependent on activation of protein kinase C (PKC), and the ERK1/2 responses to all three agents were abolished by the selective EGF receptor kinase inhibitor, AG1478. Consistent with this, both GnRH and EGF increased tyrosine phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase, Pyk2, at Tyr(402). This was reduced by Ca(2+) chelation and inhibition of PKC, but not by AG1478. GnRH stimulation caused translocation of PKC alpha and -epsilon to the cell membrane and enhanced the association of Src with PKC alpha and PKC epsilon, Pyk2, and the EGF receptor. The Src inhibitor, PP2, the C-terminal Src kinase (Csk), and dominant-negative Pyk2 attenuated ERK1/2 activation by GnRH and PMA but not by EGF. These findings indicate that Src and Pyk2 act upstream of the EGF receptor to mediate its transactivation, which is essential for GnRH-induced ERK1/2 phosphorylation in hypothalamic GnRH neurons.

Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of gastrointestinal cancers. Recently, a similar protective effect has been demonstrated by the specific cyclo-oxygenase-2 (COX-2) inhibitors. However, the exact mechanism that accounts for the anti-proliferative effect of specific COX-2 inhibitors is still not fully understood, and it is still controversial whether these protective effects are predominantly mediated through the inhibition of COX-2 activity and prostaglandin synthesis. Identification of molecular targets regulated by COX-2 inhibitors could lead to a better understanding of their pro-apoptotic and anti-neoplastic activities. In the present study, we investigated the effect and the possible molecular target of a COX-2-specific inhibitor SC-236 on gastric cancer. We showed that SC-236 induced apoptosis in gastric cancer cells. However, this effect was not dependent on COX-2 inhibition. SC-236 down-regulated the protein expression and kinase activity of PKC-beta(1), increased the expression of PKCdelta and PKCeta, but did not alter the expression of other PKC isoforms in AGS cells. Moreover, exogenous prostaglandins or PGE(2) receptor antagonists could not reverse the inhibition effect on PKCbeta(1) by SC-236, which suggested that this effect occurred through a mechanism independent of cyclo-oxygenase activity and prostaglandin synthesis. Overexpression of PKCbeta(1) attenuated the apoptotic response of AGS cells to SC-236 and was associated with overexpression of p21(waf1/cip1). Inhibition of PKCbeta(1)-mediated overexpression of p21(waf1/cip1) partially reduced the anti-apoptotic effect of PKCbeta(1). The down-regulation of PKCbeta(1) provides an explanation for COX-independent apoptotic effects of specific COX-2 inhibitor in cultured gastric cancer cells. We also suggest that PKCbeta(1) act as survival mediator in gastric cancer, and its down-regulation by COX-2 inhibitor SC-236 may provide new target for future treatment of gastric cancer.

The precise mechanism by which protein kinase C-delta (PKCdelta) inhibits cell cycle progression is not known. We investigated the regulation of cyclin D1 transcription by PKCdelta in primary bovine airway smooth muscle cells. Overexpression of the active catalytic subunit of PKCdelta attenuated platelet-derived growth factor (PDGF)-mediated transcription from the cyclin D1 promoter, whereas overexpression of a dominant-negative PKCdelta increased promoter activity. A PKCdelta-specific pseudosubstrate increased cyclin D1 protein abundance. To determine the transcriptional mechanism by which PKCdelta negatively regulates cyclin D1 expression, we transiently transfected cells with cDNAs encoding cyclin D1 promoter 5' deletions and site mutations in the context of a -66 promoter fragment. We found that the -57 to -52 CRE/ATF2 site functions as a basal level and PDGF enhancer, whereas the -39 to -30 nuclear factor-kappaB site functions as a basal level suppressor. Further, PDGF and PKCdelta responsiveness of the cyclin D1 promoter was maintained following 5' deletion to the Ets-containing -22 minimal promoter. Finally, using electrophoretic mobility gel shift and reporter assays, we determined that PKCdelta inhibits CRE/ATF2 binding and transactivation, activates nuclear factor-kappaB binding and transactivation, and attenuates Ets transactivation. These data suggest that PKCdelta attenuates cyclin D1 promoter activity via the regulation of three distinct cis-acting regulatory elements.

Recent studies provide evidence that exisulind and two potent derivatives, CP461 and CP248, induce apoptosis in colon cancer cells by inhibiting cyclic GMP (cGMP)-specific phosphodiesterases (phosphodiesterases 2 and 5). This causes an increase in intracellular levels of cGMP, thus activating the cGMP-dependent protein kinase G (PKG), which then activates pathways that lead to apoptosis. To further examine this mechanism and to provide a potential in vivo biomarker for activation of this pathway, we examined phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), a ubiquitously expressed endogenous substrate for PKG. We found that VASP was phosphorylated after treating SW480 colon cancer cells with exisulind, CP461, or CP248. CP248-induced VASP phosphorylation was inhibited by a specific PKG inhibitor but not by a protein kinase A inhibitor. The drug 3-(5'-hydroxymethyl-2'-furyl)-benzylindazole and nitric oxide donors that activate cellular guanylyl cyclase and thus increase cellular levels of cGMP also caused VASP phosphorylation. With all of these agents, the phosphorylation of VASP was associated with increased intracellular levels of cGMP and the induction of apoptosis. We also demonstrated direct in vivo phosphorylation of VASP with constitutively activated mutants of PKG. These results suggest that VASP phosphorylation can provide a useful endogenous cellular biomarker for anticancer agents that cause cGMP-mediated apoptosis.

Peroxiredoxin I (Prx I) is an oxidative stress-inducible antioxidant protein with thioredoxin peroxidase activity. Here we report that the levels of Prx I mRNA and protein are dramatically increased in a murine osteoblast cell line, MC3T3-E1, by treatment with sodium arsenate. We further studied the signaling pathways that control the induction of Prx I expression. The treatment of osteoblasts with arsenate activated ERK1/2, JNK, and p38 MAPK. Pre-treating cells with inhibitors of p38 MAPK abolished the induction of Prx I protein but had minimal effect on the induction of Prx I mRNA, suggesting that p38 MAPK activity was required for post-transcriptional regulation. The inhibition of ERK1 and ERK2 had no effect on the induction of Prx I expression. Furthermore, rottlerin, an inhibitor of protein kinase Cdelta (PKCdelta) and calmodulin kinase III, abrogated the up-regulation at both protein and mRNA levels. Staurosporine and Go6983, inhibitors for PKC, also inhibited the induction of Prx I, suggesting that protein kinase Cdelta is required for the induction by arsenate. PKCdelta was activated by arsenate treatment by in vitro kinase assays. The inhibition of PKCdelta by rottlerin did not affect the activation of p38 MAPK by arsenate. These results suggest that there are two separate signaling pathways involved in the up-regulation of Prx I protein in response to arsenate, PKCdelta required for transcriptional activation and p38 MAPK required for post-transcriptional regulation.

We have studied the role of phosphorylation in the activation of metal-regulatory transcription factor-1 (MTF-1) and metallothionein (MT) gene expression. We showed that MTF-1 is phosphorylated in vivo and that zinc stimulates MTF-1 phosphorylation 2-4-fold. Several kinase inhibitors were used to examine the possible involvement of kinase cascades in the activation of MTF-1. Metal-induced MT gene expression was abrogated by protein kinase C (PKC), c-Jun N-terminal kinase (JNK), phosphoinositide 3-kinase, and tyrosine-specific protein kinases inhibitors, as assayed by Northern analysis and by cotransfection experiments using a metal regulatory element-luciferase reporter plasmid. The extracellular signal-activated protein kinase and the p38 kinase cascades did not appear to be essential for the activation of MT gene transcription by metals. By using dominant-negative mutants of PKC, JNK, mitogen-activated kinase kinase 4 (MKK4), and MKK7, we provide further evidence supporting a role for PKC and JNK in the activation of MTF-1 in response to metals. Notably, increased MTF-1 DNA binding in response to zinc and MTF-1 nuclear localization was not inhibited in cells preincubated with the different kinase inhibitors despite strong inhibition of MTF-1-mediated gene expression. This suggests that phosphorylation is essential for MTF-1 transactivation function. We hypothesize that metal-induced phosphorylation of MTF-1 is one of the primary events leading to increased MTF-1 activity. Thus, metal ions such as cadmium could activate MTF-1 and induce MT gene expression by stimulating one or several kinases in the MTF-1 signal transduction pathway.

Expression of the COOH-terminal residues 179-330 of the LSP1 protein in the LSP1(+) B-cell line W10 increases anti-IgM- or ionomycin-induced apoptosis, suggesting that expression of this LSP1 truncate (B-LSP1) interferes with a Ca(2+)-dependent step in anti-IgM signaling. Here we show that inhibition of Ca(2+)-dependent conventional protein kinase C (cPKC) isoforms with Gö6976 increases anti-IgM-induced apoptosis of W10 cells and that expression of B-LSP1 inhibits translocation of PKCbetaI but not of PKCbetaII or PKCalpha to the plasma membrane. The increased anti-IgM-induced apoptosis is partially reversed by overexpression of PKCbetaI. This shows that the B-LSP1-mediated inhibition of PKCbetaI leads to increased anti-IgM-induced apoptosis. Expression of constitutively active PKCbetaI protein in W10 cells activates the mitogen-activated protein kinase ERK2, whereas expression of B-LSP1 inhibits anti-IgM-induced activation of ERK2, suggesting that anti-IgM-activated PKCbetaI is involved in the activation of ERK2 and that inhibition of ERK2 activation contributes to the increased anti-IgM-induced apoptosis. Pull-down assays show that LSP1 interacts with PKCbetaI but not with PKCbetaII or PKCalpha in W10 cell lysates, while in vitro LSP1 and B-LSP1 bind directly to PKCbetaI. Thus, B-LSP1 is a unique reagent that binds PKCbetaI and inhibits anti-IgM-induced PKCbetaI translocation, leading to inhibition of ERK2 activation and increased apoptosis.

We recently obtained evidence that treatment of human colon cancer cells with exisulind (sulindac sulfone) and related compounds induces apoptosis by activation of protein kinase G (PKG) and c-Jun kinase (JNK1). The present study further explores this mechanism. We demonstrate that in NIH3T3 cells a constitutively active mutant of PKG causes a dose-dependent activation of JNK1 and thereby transactivates c-Jun and stimulates transcription from the AP-1 enhancer element. The activation of JNK1 and the transactivation of c-Jun by this mutant of PKG were inhibited by a dominant negative MEKK1. In vitro assays showed that a purified PKG directly phosphorylated the N-terminal domain of MEKK1. PKG also directly phosphorylated a full-length MEKK1, and this was associated with enhanced MEKK1 phosphorylation. Thus, it appears that PKG activates JNK1 through a novel PKG-MEKK1-SEK1-JNK1 pathway, by directly phosphorylating and activating MEKK1.

Pre-T cell receptor (preTCR)-derived signals mediate the transition of thymocytes from the CD4(-) CD8(-) double-negative (DN) to CD4(+) CD8(+) double-positive stage of T lymphocyte development. This progression, termed beta-selection, is limited to thymocytes that have generated a functional TCR-beta chain able to associate with pTalpha to form the preTCR complex. Formation of the preTCR complex not only induces differentiation, survival, and proliferation of DN thymocytes; it also inhibits further TCR-beta gene rearrangement through an ill-defined process known as allelic exclusion. The signaling pathways controlling this critical developmental checkpoint have not been characterized. Here we demonstrate that formation of the preTCR complex leads to the activation of protein kinase C (PKC), and that activation of PKC is necessary for the differentiation and expansion of DN thymocytes. Importantly, we also show that allelic exclusion at the TCR-beta gene loci is enforced by PKC-mediated signals. These results define PKC as a central mediator of both differentiation and allelic exclusion during thymocyte development.

Epidemiological studies link increased garlic (Allium sativum) consumption with a reduced incidence of colon cancer in various human populations. Experimental carcinogenesis studies in animal models and in cell culture systems indicate that several allium-derived compounds exhibit inhibitory effects and that the underlying mechanisms may involve both the initiation and promotion phases of carcinogenesis. To provide a better understanding of the effects of allium derivatives on the prevention of colon cancer, we examined two water-soluble derivatives of garlic, S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC), for their effects on proliferation and cell cycle progression in two human colon cancer cell lines, SW-480 and HT-29. For comparison, we included the compound sulindac sulfide (SS), because sulindac compounds are well-established colon cancer chemopreventive agents. We found that SAMC, but not SAC, inhibited the growth of both cell lines at doses similar to that of SS. SAMC also induced apoptosis, and this was associated with an increase in caspase3-like activity. These affects of SAMC were accompanied by induction of jun kinase activity and a marked increase in endogenous levels of reduced glutathione. Although SS caused inhibition of cell cycle progression from G1 to S, SAMC inhibited progression at G2-M, and a fraction of the SW-480 and HT-29 cells were specifically arrested in mitosis. Coadministration of SS with SAMC enhanced the growth inhibitory and apoptotic effects of SS. These findings suggest that SAMC may be useful in colon cancer prevention when used alone or in combination with SS or other chemopreventive agents.

Multiple studies have shown that intracellular signal transduction by the protein kinase C (PKC) family participates in the initiation of megakaryocyte differentiation. In this study, multiple approaches addressed the functional contributions by specific PKC isozymes to megakaryocytic lineage commitment of two independent cell lines, K562 and human erythroleukemia (HEL). Pharmacologic profiles of induction and inhibition of megakaryocytic differentiation in both cell lines suggested a role for the calcium-independent novel PKCs, in particular PKC-epsilon. In transfection studies, the isolated variable domain of PKC-epsilon selectively blocked exogenous activation of the megakaryocyte-specific alpha IIb promoter. Constitutively active mutants of PKC-epsilon, but not of other PKC isozymes, cooperated with the transcription factor GATA-1 in the activation of the alpha IIb promoter. The functional cooperation between GATA-1 and PKC-epsilon displayed dependence on cellular milieu, as well as on the promoter context of GATA binding sites. In aggregate, the data suggest that PKC-epsilon specifically participates in megakaryocytic lineage commitment through functional cooperation with GATA-1 in the activation of megakaryocytic promoters.

Sulindac sulfone (Exisulind) induces apoptosis and exhibits cancer chemopreventive activity, but in contrast to sulindac, it does not inhibit cyclooxygenases 1 or 2. We found that sulindac sulfone and two potent derivatives, CP248 and CP461, inhibited the cyclic GMP (cGMP) phosphodiesterases (PDE) 2 and 5 in human colon cells, and these compounds caused rapid and sustained activation of the c-Jun NH2-terminal kinase 1 (JNK1). Rapid activation of stress-activated protein/ERK kinase 1 (SEK1) and mitogen-activated protein kinase kinase kinase (MEKK1), which are upstream of JNK1, was also observed. Other compounds that increase cellular levels of cGMP also activated JNK1, and an inhibitor of protein kinase G (PKG), Rp-8-pCPT-cGMPS, inhibited JNK1 activation by the sulindac sulfone derivatives. Expression of a dominant-negative JNK1 protein inhibited CP248-induced cleavage of poly(ADP-ribose) polymerase, a marker of apoptosis. Thus, it appears that sulindac sulfone and related compounds induce apoptosis, at least in part, through activation of PKG, which then activates the MEKK1-SEK1-JNK1 cascade. These studies also indicate a role for cGMP and PKG in the JNK pathway.

Although mitogenic and differentiating factors often activate a number of common signaling pathways, the mechanisms leading to their distinct cellular outcomes have not been elucidated. In a previous report, we demonstrated that mitogen-activated protein (MAP) kinase (ERK) activation by the neurogenic agents fibroblast growth factor (FGF) and nerve growth factor is dependent on protein kinase Cdelta (PKCdelta), whereas MAP kinase activation in response to the mitogen epidermal growth factor (EGF) is independent of PKCdelta in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells. We now show that EGF activates MAP kinase through a PKCzeta-dependent pathway involving phosphatidylinositol 3-kinase and PDK1 in H19-7 cells. PKCzeta, like PKCdelta, acts upstream of MEK, and PKCzeta can potentiate Raf-1 activation by EGF. Inhibition of PKCzeta also blocks EGF-induced DNA synthesis as monitored by bromodeoxyuridine incorporation in H19-7 cells. Finally, in embryonic rat brain hippocampal cell cultures, inhibitors of PKCzeta or PKCdelta suppress MAP kinase activation by EGF or FGF, respectively, indicating that these factors activate distinct signaling pathways in primary as well as immortalized neural cells. Taken together, these results implicate different PKC isoforms as determinants of growth factor signaling specificity within the same cell. Furthermore, these data provide a mechanism whereby different growth factors can differentially activate a common signaling intermediate and thereby generate biological diversity.

Background & Aims: We have previously reported that nonsteroidal anti-inflammatory drugs (NSAIDs) could induce apoptosis of gastric epithelial cells both in vivo and in vitro. This study investigated the role of protein kinase C (PKC) isoforms in the regulation of NSAID-induced apoptosis., Methods: Protein levels of 12 PKC isoforms in AGS cells, in the presence or absence of indomethacin, were determined by Western blot. The effect of PKC-beta1 overexpression by transfection with its complementary DNA (cDNA) on indomethacin-induced apoptosis and apoptosis-related genes, including p53, p21(waf1/cip1), and c-myc, was further investigated., Results: Treatment with indomethacin decreased the abundance of PKC-beta1 and increased that of PKC-beta2, eta, and epsilon, but did not alter the expression of PKC alpha, gamma, zeta, delta, iota, and micro. Overexpression of PKC-beta1 attenuated the apoptotic response of AGS cells to indomethacin, associated with overexpression of p21(waf1/cip1) in both messenger RNA and protein levels. Inhibition of PKC-beta1-mediated overexpression of p21(waf1/cip1) by its antisense cDNA partially reduced the antiapoptotic effect of PKC-beta1., Conclusions: Indomethacin-induced apoptosis in gastric cancer cells is partly mediated by differential regulation of PKC isoform expression. Enhanced expression of exogenous PKC-beta1 protects against indomethacin-induced apoptosis through up-regulation of p21(waf1/cip1).

The retinoblastoma (Rb) gene is inactivated in a variety of human cancers, but in colorectal carcinomas there is frequently increased expression of this gene. This is paradoxical in view of the known role of Rb as a tumor suppressor gene. In the present study, we compared the levels of expression of the Rb protein (pRb) in normal human colorectal mucosa, adenomatous polyps, and carcinomas by immunohistochemistry. In vitro studies were also done to examine the phenotypic effects of an antisense oligodeoxynucleotide (AS-Rb) targeted to Rb mRNA in the HCT116 colon carcinoma cell line that expresses a relatively high level of pRb. The incidence of pRb-positive cells was increased during multistage colorectal carcinogenesis. In vitro treatment of HCT116 cells with AS-Rb decreased the level of pRb by about 70% and also decreased the levels of the cyclin D1 protein and cyclin D1-associated kinase activity. AS-Rb inhibited growth of HCT116 cells and induced apoptosis. Reporter assays indicated about a 17-fold increase in E2F activity. These findings suggest that the increased expression of pRb in colorectal carcinoma cells may provide a homeostatic mechanism that protects them from growth inhibition and apoptosis, perhaps by counterbalancing potentially toxic effects of excessive E2F activity.

Protein kinase C (PKC) is a multigene family of enzymes consisting of at least 11 isoforms. It has been implicated in the induction of c-fos and other immediate response genes by various mitogens. The serum response element (SRE) in the c-fos promoter is necessary and sufficient for induction of transcription of c-fos by serum, growth factors, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). It forms a complex with the ternary complex factor (TCF) and with a dimer of the serum response factor (SRF). TCF is the target of several signal transduction pathways and SRF is the target of the rhoA pathway. In this study we generated dominant-negative and constitutively active mutants of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta to determine the roles of individual isoforms of PKC in activation of the SRE. Transient-transfection assays with NIH 3T3 cells, using an SRE-driven luciferase reporter plasmid, indicated that PKC-alpha and PKC-epsilon, but not PKC-delta or PKC-zeta, mediate SRE activation. TPA-induced activation of the SRE was partially inhibited by dominant negative c-Raf, ERK1, or ERK2, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of Elk-1. TPA-induced activation of the SRE was also partially inhibited by a dominant-negative MEKK1. Furthermore, TPA treatment of serum-starved NIH 3T3 cells led to phosphorylation of SEK1, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of c-Jun, a major substrate of JNK. Constitutively active mutants of PKC-alpha and PKC-epsilon could also induce a mutant c-fos promoter which lacks the TCF binding site, and they also induce transactivation activity of the SRF. Furthermore, rhoA-mediated SRE activation was blocked by dominant negative mutants of PKC-alpha or PKC-epsilon. Taken together, these findings indicate that PKC-alpha and PKC-epsilon can enhance the activities of at least three signaling pathways that converge on the SRE: c-Raf-MEK1-ERK-TCF, MEKK1-SEK1-JNK-TCF, and rhoA-SRF. Thus, specific isoforms of PKC may play a role in integrating networks of signal transduction pathways that control gene expression.

Recent studies have shown that decreased expression of p27Kip1 is associated with high grade tumors and an unfavorable prognosis in several types of human cancer. To clarify the role of p27Kip1 in colon cancer, we have overexpressed this protein in the HT29 colon cancer cell line. The derivatives displayed an increase in the p27Kip1 protein in cyclin E/CDK2 immunoprecipitates and a decrease in cyclin E-associated kinase activity when compared to vector control clones, providing evidence that the overexpressed protein was functional. Clones with a high level of p27Kip1 displayed partial growth inhibition in monolayer culture and a decrease in plating efficiency, even though they expressed increased levels of the cyclin D1 protein. Using alkaline phosphatase expression as a marker, we found that the p27Kip1 overexpressor clones displayed a 2-3-fold increase in sensitivity to induction of differentiation by 2 mM sodium butyrate. In contrast to these results, derivatives of HT29 cells that stably overexpressed p21Cip1/Waf1 displayed decreased sensitivity to the induction of differentiation. These findings may explain why decreased levels of p27Kip1 in certain human cancers is associated with high grade (poorly differentiated) tumors, and suggest that strategies that increase the level of p27Kip1 may be useful in cancer therapy.

We previously reported increased expression of p27Kip1 in a series of human breast cancer cell lines, as compared to cell lines established from normal mammary epithelial cells. These data were surprising because this protein exerts a growth-inhibitory function in normal cells, and p27Kip1 has been proposed as a candidate tumor suppressor gene. A possible explanation for the paradoxical increase in p27Kip1 in the breast cancer cell lines is that they had become refractory to the inhibitory effects of this protein. To address this question, here, we transfected the MCF7 breast cancer cell line and the MCF10F nontumorigenic mammary epithelial cell line with a vector containing the p27Kip1 cDNA to obtain derivatives that express increased levels of p27Kip1. The increased expression of p27Kip1 in both of these cell lines was associated with lengthening of the G1 phase, an increase in the doubling time, a decreased saturation density, and a decreased plating efficiency. In the MCF7 cells, anchorage-independent growth and in vivo tumorigenicity were also suppressed. These effects were associated with decreased cyclin E-associated in vitro kinase activity in both cell lines. The increased expression of p27Kip1 was associated with a decreased level of expression of cyclin D1 in the MCF10F cells but an increased level of the cyclin D1 protein in the MCF7 cell line. Both derivatives expressed slightly increased levels of the cyclin E protein. Thus, breast cancer cells are still responsive to p27Kip1-mediated inhibition of cell growth despite the high basal level of this protein. These results suggest that therapeutic strategies that further increase the level of expression of p27Kip1 or mimic its activity might be useful in cancer therapy.