Programmed cell death 4 mechanism of action: The model to be updated?

Programmed cell death 4 (Pdcd4) is frequently suppressed in tumors of various origins and its suppression correlates with tumor progression. Pdcd4 inhibits cap-dependent translation from mRNAs with highly structured 5'-regions through interaction with the eukaryotic translation initiation factor 4A (eIF4A) helicase and a target transcript. Decrease in Pdcd4 protein is believed to provide a relief of otherwise suppressed eIF4A-dependent translation of proteins facilitating tumor progression. However, it remains unknown if lowered Pdcd4 levels in cells suffices to cause a relief in translation inhibition through appearance of the Pdcd4-free translation-competent eIF4A protein, or more complex and selective mechanisms are involved. Here we showed that eIF4A1, the eIF4A isoform involved in translation, significantly over-represents Pdcd4 both in cancerous and normal cells. This observation excludes the possibility that cytoplasmic Pdcd4 can efficiently exert its translation suppression function owing to excess of eIF4A, with Pdcd4-free eIF4A being in excess over Pdcd4-bound translation-incompetent eIF4A, thus leaving translation from Pdcd4 mRNA targets unaffected. This contradiction is resumed in the proposed model, which supposes initial complexing between Pdcd4 and its target mRNAs in the nucleus, with subsequent transport of translation-incompetent, Pdcd4-bound target mRNAs into the cytoplasm. Noteworthy, loss of nuclear Pdcd4 in cancer cells was reported to correlate with tumor progression, which supports the proposed model of Pdcd4 functioning.

A gel-less isolation of untagged plasmid DNA insert from vector backbone in homogeneous format.

Agarose gel electrophoresis with subsequent DNA extraction from gel is routinely used for DNA fragment isolation after plasmid DNA digestion. We describe a gel-less method for DNA fragment isolation after plasmid DNA digestion which is based on in-solution negative selection through depletion of vector backbone bearing LoxP sites by sorption on solid phase-immobilized mutated Cre recombinase. The method might be especially useful in preparation of DNA fragments for transgenic animal generation where residual agarose presence is a concern, and DNA fragments are frequently large in size and thus might be mechanically damaged during purification with conventional affinity-based gel extraction methods.

hTERT and BIRC5 gene promoters for cancer gene therapy: A comparative study.

Human telomerase reverse transcriptase (hTERT) and survivin (BIRC5) gene promoters are frequently used for transcriptional targeting of tumor cells, yet there is no comprehensive comparative analysis allowing rational choice of a promoter for a particular therapy. In the current study, the transcriptional activity of hTERT, human BIRC5 and mouse Birc5 promoters and their modifications were compared in 10 human cancer cell lines using the luciferase reporter gene activity assay. The results revealed that BIRC5- and hTERT-based promoters had strikingly different cell specificities with comparable activities in only 40% of cell lines. Importantly, relative hTERT and BIRC5 transcript abundance cannot be used to predict the most potent promoter. Among the hTERT-based promoters that were assessed, modification with the minimal cytomegalovirus promoter generally resulted in the most potent activity. Mouse Birc5 and modified human BIRC5 promoters were superior to the unmodified human survivin promoter; however, their tumor specificities must be investigated further. In summary, the present results emphasize the desirability for construction of more universal tumor-specific promoters to efficiently target a wide spectrum of tumor cells.

A pilot comparative study of topical latanoprost and tacrolimus in combination with narrow-band ultraviolet B phototherapy and microneedling for the treatment of nonsegmental vitiligo.

Prostaglandins and their analogues are beneficial as topical agents in vitiligo treatment, yet neither of the previous study addressed their comparative efficiency with conventional topical agents used in vitiligo treatment. In this pilot (24 patients) left-right comparative study we addressed efficiency of prostaglandin F2α analogue latanoprost versus tacrolimus when combined with narrow-band ultraviolet B and microneedling in repigmentation of nonsegmental vitiligo lesions. Our results confirm potency of prostaglandins, in particular, that of latanoprost, in inducing repigmentation, with the efficiency being at least comparable to that of tacrolimus, while contribution of microneedling remains unclear. In summary, results of our study provide further evidences for justified use of prostaglandins, in particular, latanoprost, in vitiligo treatment. In turn, this warrants future studies on the topic aiming to conclusively introduce prostaglandin-based formulations as conventional agents for vitiligo management.

Hunk/Mak-v is a negative regulator of intestinal cell proliferation.

BACKGROUND: Conditional deletion of the tumour suppressor gene Apc within the murine intestine results in acute Wnt signalling activation. The associated over-expression of a myriad of Wnt signalling target genes yields phenotypic alterations that encompass many of the hallmarks of neoplasia. Previous transcriptomic analysis aimed at identifying genes that potentially play an important role in this process, inferred the Hormonally upregulated Neu-associated kinase (HUNK/Mak-v/Bstk1) gene as a possible candidate. Hunk is a SNF1 (sucrose non fermenting 1)-related serine/threonine kinase with a proposed association with many different tumour types, including colorectal cancer. METHODS: Here we describe the generation of a novel Hunk kinase deficient mouse which has been used to investigate the involvement of Hunk-kinase activity in intestinal homeostasis and tumourigenesis. RESULTS: We show that in the morphologically normal intestine, Hunk-kinase negatively regulates epithelial cell proliferation. However, the increase in cell proliferation observed in the Hunk kinase deficient intestine is counteracted by increased cell migration, thereby maintaining intestinal homeostasis. Using qRT-PCR, we further demonstrate that Hunk is significantly over-expressed in Apc deficient / Wnt-signalling activated intestinal tissue. Using the classical intestinal tumourigenesis Apc (Min) mouse model we show that loss of Hunk-kinase activity significantly reduced tumour initiation rates in the small intestine. However, an accompanying increase in the size of the tumours counteracts the impact this has on overall tumour burden or subsequently survival. CONCLUSIONS: In the intestinal setting we demonstrate that Hunk has a role in normal intestinal proliferation and homeostasis and, although it does not alter overall survival rates, activity of this kinase does impact on tumour initiation rates during the early stages in tumourigenesis in the small intestine.

Expression of Pdcd4 tumor suppressor in human melanoma cells.

BACKGROUND: Programmed cell death 4 (Pdcd4) is a tumor suppressor frequently lost in tumors of various origins thus contributing to tumor progression. Expression of Pdcd4 in melanoma, however, has not been extensively studied to date. MATERIALS AND METHODS: Pdcd4 protein levels were assessed in 23 human melanoma cell lines and in normal melanocytes by western blot analysis. Also, effects of LY294002, rapamycin and PD098059 on Pdcd4 protein levels were analyzed. RESULTS: Pdcd4 is suppressed in ~25% of human cell lines established from advanced melanoma lesions. Pdcd4 protein levels in melanoma cells were up-regulated by treatment with inhibitors of Akt signaling, one of the key pathways leading to Pdcd4 suppression, and to a lesser extent by inhibiting MEK/ERK pathway. CONCLUSION: Pdcd4 loss is not a common event in melanoma progression yet suppression of Pdcd4 defines a subset of melanoma cells and can be used for molecular typing of melanoma. Our results help determine the significance of Pdcd4 loss in melanoma as well as its up-regulation by Akt pathway inhibitors, which are promising tools in melanoma treatment.

Acridone acetic acid, sodium salt, as an agent to stop vitiligo progression: a pilot study.

Vitiligo progression is attributed to immune system malfunctioning, thus immunomodulating compounds might be beneficial in stopping vitiligo progression which is a prerequisite for successful repigmentation. The goal of this study was to assess efficacy of acridone acetic acid, sodium salt (Na-AAA), an immunomodulating compound with favorable safety profile, in stabilizing active vitiligo, and to reveal prognostic factors of treatment outcome. Sixty consecutive patients with progressing nonsegmental vitiligo were treated with 10 i.m. injections of Na-AAA every other day. Disease stability was assessed in 1, 3, 6, and 12 months post-treatment. Statistical analysis was applied to correlate treatment outcome and available clinical parameters. Of the 60 patients treated, vitiligo stopped progression in 44 patients (73.3%). Older age (p = 0.0219), age of 35 and older (p = 0.0189, odds ratio (OR) = 5.2, 95% confidence interval (CI) 1.30-20.84) or age of 40 and older (p = 0.0039, OR = 6.48, 95% CI 1.86-22.61), longer disease duration (p = 0.0234), pre-treatment interleukin-6 level over 2 pg/mL (p = 0.0005, OR = 13.7, 95% CI 2.97-63), and over the reference threshold value 5.9 pg/mL (p = 0.0009, OR = 25.8, 95% CI 2.8-239) as well as presence of other autoimmune diseases (p = 0.038, OR = 7.0, 95% CI 1.14-42.97) were negative prognostic factors of treatment success. In conclusion, acridone acetic acid, sodium salt, emerges as an efficient option for stopping vitiligo progression.

LY294002 enhances expression of proteins encoded by recombinant replication-defective adenoviruses via mTOR- and non-mTOR-dependent mechanisms.

Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replication-defective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirus-delivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery.

The RHOV gene is overexpressed in human non-small cell lung cancer.

Rho family GTPases act as molecular switches to regulate numerous cellular processes, including malignant transformation. Commonly, overexpression of Rho GTPases contributes to tumorigenesis. Elevated expression of several Rho GTPases has been reported in lung cancer and is associated with poor prognosis. The RHOV gene encodes the atypical Rho family GTPase Chp/RhoV, which is capable of transforming fibroblasts, although other functions of Chp remain largely elusive. RHOV is expressed in normal lung tissue in rats, but not in humans. RHOV expression was found in several human cancer cell lines, including non-small-cell lung cancer (NSCLC) cell line A549, but expression of RHOV in NSCLC tumors has never been investigated. Here we studied the expression of the RHOV gene in lung cancer cell lines and in 29 matched pairs of NSCLC tumors and adjacent nontumorous tissues. We found that RHOV is expressed in lung cancer cell lines and is upregulated in the majority of studied lung tumors. Analysis of the Oncomine database revealed correlation between elevated RHOV level and poor patient survival. We propose that the RHOV gene could be validated as a diagnostic or prognostic marker for NSCLC, and that observed overexpression of RHOV might contribute to tumorigenesis.

Possible contribution of GSTP1 and other xenobiotic metabolizing genes to vitiligo susceptibility.

Vitiligo is an acquired pigmentary disorder with several proposed pathogenesis mechanisms and complex multifactorial genetic predisposition. We analyzed 65 polymorphisms in genes potentially relevant to vitiligo pathogenesis mechanism to reveal novel and confirm reported genetic risk factors in general Russian population. We found that polymorphism rs1138272 (TC + CC) in GSTP1 gene encoding enzyme involved in xenobiotic metabolism is associated with vitiligo (Bonferroni adjusted P value 0.0015) with extraordinary high odds ratio 13.03, and haplotype analysis confirmed association of GSTP1 gene with vitiligo risk. Moreover, analysis of variations in several genes encoding enzymes of xenobiotic metabolism showed that higher risk of vitiligo is associated with higher number of risk alleles. This finding reveals possible contribution of genetic background to observed imbalance of oxidative stress control in vitiligo through cumulative effect of multiple genetic variations in xenobiotic metabolizing genes, supporting the concept of multigenic nature of vitiligo with multiple low-risk alleles cumulatively contributing to vitiligo risk.

Multidisciplinary approach to R&D in vitiligo, a neglected skin disease.

A global interest in therapies for neglected diseases is rising, but traditional biopharma research and development (R&D) process is prohibitively expensive to justify cost of their development. Vitiligo is a multifactorial orphan disease that affects at minimum 35 million people worldwide, yet no therapeutic solutions exist. The present authors describe a budget-minded pursuit of the new therapy development for vitiligo, which includes a multidiscipline collaboration and effective bridging between academic research, biobanking, and bioinformatics. The present authors anticipate that the present authors' "theoretically induced and empirically guided" discovery process will enable development of more leads, with a much greater probability of success and under tighter budgets compared with those of the biopharma company. Ultimately, the multidisciplinary approach described below facilitates the collaborative development of personalized treatments for different patient subpopulations in vitiligo and other neglected diseases.

Review of current clinical studies of vitiligo treatments.

There is a limited number of options in vitiligo treatment, with the disease frequently refractory to all existing treatment modalities. This warrants development of novel and improving existing vitiligo treatments as well as finding predicting factors to improve treatment outcome through appropriate selection and the most efficient application of a treatment. These issues are addressed in clinical studies aiming to evaluate safety and efficiency of novel treatments, improvements and modifications introduced to existing treatments, and to define predictors of treatment efficiency and their limitations. Here, results of recent (since year 2009) clinical studies in vitiligo field are overviewed, with the emphasis on their contribution to improved vitiligo management.

Identification of Nedd4 E3 ubiquitin ligase as a binding partner and regulator of MAK-V protein kinase.

MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function.

Rho family GTPase Chp/RhoV induces PC12 apoptotic cell death via JNK activation.

Rho GTPases regulate numerous cellular processes including apoptosis. Chp/RhoV is an atypical Rho GTPase which functions are poorly understood. Here we investigated the role of Chp in regulation of cell viability using PC12 cells with inducible expression of Chp as a model. We found that expression of Chp results in apoptosis in PC12 cells. Chp-induced apoptosis was accompanied by activation of JNK signaling and both death receptor-mediated and mitochondrial apoptotic pathways as justified by caspase-8 and caspase-9 activation, respectively. Moreover, inhibition of JNK by SP600125 rescued PC12 cells from Chp-triggered cell death and attenuated activation of caspases-9 and -3/7 suggesting that activation of JNK mediates pro-apoptotic effect of Chp. Expression of Chp resulted in increased phosphorylation of c-Jun in PC12 cells, and Chp expression in HE K293 cells upregulated AP-1-dependent transcription in a JNK-dependent manner. Together results of our study reveal the role of Chp GTPase as a putative regulator of JNK-dependent apoptotic death in PC12 cells, similarly to previously described pro-apoptotic activity of the related Cdc42 and Rac1 GTPases.

Pdcd4 protein and mRNA level alterations do not correlate in human lung tumors.

Pdcd4 (programmed cell death 4) gene is tumor suppressor which expression is frequently down-regulated in tumors, which is considered as a diagnostic and prognostic marker as well as promising target for anti-cancer therapy. Pdcd4 protein is a target for post-translational regulation by phosphorylation marking Pdcd4 for degradation. We questioned if Pdcd4 mRNA decline in human lung tumors is accompanied by proportional depletion of Pdcd4 protein. We found that Pdcd4 protein-to-mRNA ratio varies greatly in human lung cancer cell lines. In squamous cell carcinoma samples where Pdcd4 mRNA suppression was found to be a typical event, Pdcd4 protein level frequently remained unchanged or even up-regulated. Our studies demonstrate that at least in squamous cell carcinoma, alterations in Pdcd4 mRNA and protein levels are not directly linked, and this fact should be taken into consideration when developing Pdcd4-based anti-cancer therapeutic approaches.

Apoptotic cleavage of rabaptin-5-like proteins and a model for rabaptin-5 inactivation in apoptosis.

Intracellular membrane transport from the plasma membrane is one of the processes affected in apoptotic cells. Apoptotic inhibition of endosomal transport occurs due to cleavage of Rabaptin-5, an effector of small GTPase Rab5, which results in inhibition of early endosome fusion. Recently several novel Rabaptin-5-like proteins were identified. We investigated whether Rabaptin-5-like proteins, Rabaptin-5gamma and Rabaptin-5delta, are also cleaved in apoptosis and found that both proteins are cleaved in apoptotic cell extracts by caspase-3-related proteases. This suggests that functional inactivation of these proteins is necessary for apoptotic cell death. We also mapped a novel, N-terminal, putative Rab5 binding site in Rabaptin-5-like proteins, which becomes physically separated from the previously known C-terminal Rab5 binding site after apoptotic cleavage of these proteins. Presence of the second Rab5 binding site provides a new insight into Rabaptin-5 function in early endosome fusion and a mechanistic model for functional inactivation of Rabaptin-5 in apoptosis.

Multiple domains of Ruk/CIN85/SETA/CD2BP3 are involved in interaction with p85alpha regulatory subunit of PI 3-kinase.

Ruk/CIN85/SETA/CD2BP3 and CD2AP/CMS/METS-1 comprise a new family of proteins involved in such fundamental processes as clustering of receptors and rearrangement of the cytoskeleton in regions of specialised cell-cell contacts, ligand-activated internalisation and targeting to lysosome degradation pathway of receptor tyrosine kinases, and apoptotic cell death. As typical adapter proteins they execute these functions by interacting with other signalling molecules via multiple protein-protein interaction interfaces: SH3 domains, Pro-rich region and coiled-coil domain. It has been previously demonstrated that Ruk is able to interact with the p85alpha regulatory subunit of PI 3-kinase and that the SH3 domain of p85alpha is required for this interaction. However, later observations hinted at a more complex mechanism than simple one-way SH3-Pro-rich interaction. Because interaction with p85alpha was suggested to be important for pro-apoptotic activity of the long isoform of Ruk, Ruk(l)/CIN85, we carried out detailed studies of the mechanism of this interaction and demonstrated that multiple domains are involved; SH3 domains of Ruk are required and sufficient for efficient interaction with full-length p85alpha but the SH3 domain of p85alpha is vital for their "activation" by ousting them from intramolecular interaction with the Pro-rich region of Ruk. Our data also suggest that homodimerisation via C-terminal coiled-coil domain affects both intra- and intermolecular interactions of Ruk proteins.

Subcellular localization of MAK-V/Hunk protein kinase expressed in COS-1 cells.

MAK-V/Hunk is a MARK/Par-1-related protein kinase, whose function is unknown. We studied the subcellular localization of MAK-V/Hunk in COS-1 cells by immunofluorescence. It has a nucleocytoplasmic distribution and is localized to the centrosome, as indicated by co-localization with gamma-tubulin. A putative kinase-deficient mutant, with a mutation in the invariant lysine residue in the catalytic domain, was not targeted to the nucleus or centrosome. These results suggest that the nuclear and centrosomal targeting of MAK-V/Hunk is specific, and is likely to be coupled to its catalytic activity.

Cloning and developmental expression of MARK/Par-1/MELK-related protein kinase xMAK-V in Xenopus laevis.

MAK-V/Hunk is a recently identified MARK/Par-1-related mammalian protein kinase. Although the precise function of this protein kinase is yet to be established, available data suggest its involvement in animals' development and in the physiology of the nervous system. Here we report characterization of a cDNA encoding Xenopus laevis orthologue of MAK-V/Hunk protein kinase, xMAK-V. The in silico analysis also revealed MAK-V/Hunk orthologues in the fish Fugu rubripes and primitive chordate Ciona intestinalis but not in invertebrate species such as Drosophila melanogaster and Caenorhabditis elegans, suggesting that MAK-V/Hunk is a chordate-specific protein kinase. The expression of xmak-v in X. laevis embryos was analyzed using whole-mount in situ hybridization. Expression of xmak-v has been detected in all developmental stages studied including maternal expression in unfertilized eggs. The xmak-v mRNA has a predominant occurrence on the animal hemisphere of the egg, and this pattern of expression is sustained throughout cleavage and blastula stages. At the gastrula stage xmak-v expression is restricted to the ectoderm. In the later stage embryos xmak-v is expressed over the entire embryonic surface including the open neural plate at stage 15 and also in neural tube at stage 22. At tadpole stage xmak-v expression is strong in embryonic epidermis, nervous system and sensory organs, and is also obvious in perisomitic mesoderm and brachial arches.