Mitogenic Pathways Research Articles

Alpha‐2 adrenergic stimulation triggers Achilles tenocyte hypercellularity: Comparison between two model systems

The histopathology of tendons with painful tendinopathy is often tendinosis, a fibrosis-like condition of unclear pathogenesis characterized by tissue changes including hypercellularity. The primary tendon cells (tenocytes) have been shown to express adrenoreceptors (mainly alpha-2A) as well as markers of catecholamine production, particularly in tendinosis. It is known that adrenergic stimulation can induce proliferation in other cells. The present study investigated the effects of an exogenously administered alpha-2 adrenergic agonist in an established in vivo Achilles tendinosis model (rabbit) and also in an in vitro human tendon cell culture model. The catecholamine producing enzyme tyrosine hydroxylase and the alpha-2A-adrenoreceptor (α2A AR) were expressed by tenocytes, and alpha-2 adrenergic stimulation had a proliferative effect on these cells, in both models. The proliferation was inhibited by administration of an α2A AR antagonist, and the in vitro model further showed that the proliferative alpha-2A effect was mediated via a mitogenic cell signaling pathway involving phosphorylation of extracellular-signal-regulated kinases 1 and 2. The results indicate that catecholamines produced by tenocytes in tendinosis might contribute to the proliferative nature of the pathology through stimulation of the α2A AR, pointing to a novel target for future therapies. The study furthermore shows that animal models are not necessarily required for all aspects of this research.

Estrogen Receptor-Alpha 36 Mediates Mitogenic Antiestrogen Signaling in ER-Negative Breast Cancer Cells

It is prevailingly thought that the antiestrogens tamoxifen and ICI 182, 780 are competitive antagonists of the estrogen-binding site of the estrogen receptor-alpha (ER-α). However, a plethora of evidence demonstrated both antiestrogens exhibit agonist activities in different systems such as activation of the membrane-initiated signaling pathways. The mechanisms by which antiestrogens mediate estrogen-like activities have not been fully established. Previously, a variant of ER-α, EP–α36, has been cloned and showed to mediate membrane-initiated estrogen and antiestrogen signaling in cells only expressing ER-α36. Here, we investigated the molecular mechanisms underlying the antiestrogen signaling in ER-negative breast cancer MDA-MB-231 and MDA-MB-436 cells that express high levels of endogenous ER-α36. We found that the effects of both 4-hydoxytamoxifen (4-OHT) and ICI 182, 780 (ICI) exhibited a non-monotonic, or biphasic dose response curve; antiestrogens at low concentrations, elicited a mitogenic signaling pathway to stimulate cell proliferation while at high concentrations, antiestrogens inhibited cell growth. Antiestrogens at l nM induced the phosphorylation of the Src-Y416 residue, an event to activate Src, while at 5 µM induced Src-Y527 phosphorylation that inactivates Src. Antiestrogens at 1 nM also induced phosphorylation of the MAPK/ERK and activated the Cyclin D1 promoter activity through the Src/EGFR/STAT5 pathways but not at 5 µM. Knock-down of ER-α36 abrogated the biphasic antiestrogen signaling in these cells. Our results thus indicated that ER-α36 mediates biphasic antiestrogen signaling in the ER-negative breast cancer cells and Src functions as a switch of antiestrogen signaling dependent on concentrations of antiestrogens through the EGFR/STAT5 pathway.

Integrated analysis of mRNA and microRNA expression in mature neurons, neural progenitor cells and neuroblastoma cells

Mature neurons (MNs), neural progenitor cells (NPCs) and neuroblastoma cells (NBCs) are all neural-derived cells. However, MNs are unable to divide once differentiated; NPCs are able to divide a limited number of times and differentiate to normal brain cell types; whereas NBCs can divide an unlimited number of times but rarely differentiate. Here, we perform whole transcriptome (mRNA, miRNA) profiling of these cell types and compare expression levels of each cell type to the others. Integrated mRNA–miRNA functional analyses reveal that: 1) several very highly expressed genes (e.g., Robo1, Nrp1, Epha3, Unc5c, Dcc, Pak3, Limk4) and a few under-expressed miRNAs (e.g., miR-152, miR-146b, miR-339-5p) in MNs are associated with one important cellular process—axon guidance; 2) some very highly expressed mitogenic pathway genes (e.g., Map2k1, Igf1r, Rara, Runx1) and under-expressed miRNAs (e.g., miR-370, miR-9, miR-672) in NBCs are associated with cancer pathways. These results provide a library of negative mRNAmiRNA networks that are likely involved in the cellular processes of differentiation and division.

Global Gene Expression Analysis of Very Small Embryonic-Like Stem Cells Reveals that the Ezh2-Dependent Bivalent Domain Mechanism Contributes to Their Pluripotent State

Recently, we identified a population of Oct4(+)Sca-1(+)Lin(-)CD45(-) very small embryonic-like stem cells (VSELs) in murine and human adult tissues. VSELs can differentiate in vitro into cells from all 3 germ layers and in vivo tissue-committed stem cells. Open chromatin structure of core pluripotency transcription factors (TFs) supports the pluripotent state of VSELs. However, it has been difficult to determine how primitive VSELs maintain pluripotency, owing to their limited number in adult tissues. Here, we demonstrate by genome-wide gene-expression analysis with a small number of highly purified murine bone marrow-derived VSELs that Oct4(+) VSELs (i) express a similar, yet nonidentical, transcriptome as embryonic stem cells (ESCs), (ii) highly express cell cycle checkpoint genes, (iii) express at a low level genes involved in protein turnover and mitogenic pathways, and (iv) highly express enhancer of zeste drosophila homolog 2 (Ezh2), a polycomb group protein. Furthermore, as a result of high expression of Ezh2, VSELs, like ESCs, exhibit bivalently modified nucleosomes (trimethylated H3K27 and H3K4) at promoters of important homeodomain-containing developmental TFs, thus preventing premature activation of the lineage-committing factors. Notably, spontaneous or RNA interference-enforced downregulation of Ezh2 during VSEL differentiation removes the bivalent domain (BD) structure, which leads to de-repression of several BD-regulated genes. Therefore, we suggest that Oct4(+) VSELs, like other pluripotent stem cells, maintain their pluripotent state through an Ezh2-dependent BD-mediated epigenetic mechanism. Furthermore, our global survey of VSEL gene expression signature would not only advance our understanding of biological process for their pluripotency, differentiation, and quiescence but should also help to develop better protocols for ex vivo expansion of VSELs.

Witnessing a New Era?—The Emerging Role of Targeted Drugs in the Medical Treatment of Advanced Medullary and Anaplastic Thyroid Cancer

The treatment of advanced thyroid cancer is currently entering a new era due to the introduction of targeted therapy into modern cancer treatment. The growing insight into the molecular biology of thyroid cancer and on the development of numerous mainly multitargeted agents provide the basis for new treatment strategies. In particular, activation of mitogenic and angiogenic signaling pathways are suitable targets as preclinical and clinical data suggest. Several Phase II and a few Phase III studies were launched in thyroid cancer which included medullary thyroid cancer (MTC) and anaplastic thyroid cancer (ATC) but only a few focused specifically on theses subtypes. A number of smaller Phase II trials reported promising response rates and progression-free survival. Results from a randomized Phase III trial in MTC with vandetanib, a combined vascular endothelial growth factor receptor 2 + 3 (VEGF-R2+3) and RET multi tyrosine kinase inhibitor demonstrated significant clinical activity and resulted in the first approval of a kinase inhibitor for the treatment of MTC in 2011. Unlike in MTC, in ATC the prognosis is dismal due to the aggressive nature of the disease. Some mainly vascular targeting agents alone or in combination with chemotherapy have shown interesting activity in this disease and have raised new hope. Particularly the combination of fosbretabulin with a chemotherapy backbone of paclitaxel and carboplatin tripled the one-year survival rate in a recent Phase II trial which included 80 patients with ATC. In this review, we provide a brief overview of the general treatment concept of MTC and ATC and summarize the compiled evidence published on targeted agents in these rare thyroid cancer subtypes.

Activin induces cortical interneuron identity and differentiation in embryonic stem cell-derived telencephalic neural precursors

Understanding the mechanisms underlying neural progenitor differentiation and neuronal fate specification is critical for the use of embryonic stem cells (ESCs) for regenerative medicine. Cortical interneurons are of particular interest for cell transplantation; however, only a limited subset of these neurons can be generated from ESCs. Here we uncover a pivotal role for Activin in regulating the differentiation and identity of telencephalic neural precursors derived from mouse and human ESCs. We show that Activin directly inhibits the mitogenic sonic hedgehog pathway in a Gli3-dependent manner while enhancing retinoic acid signalling, the pro-neurogenic pathway. In addition, we demonstrate that Activin provides telencephalic neural precursors with positional cues that specifically promote the acquisition of a calretinin interneuron fate by controlling the expression of genes that regulate cortical interneuron identity. This work demonstrates a novel means for regulating neuronal differentiation and specification of subtype identity.

Cell cycle inhibition without disruption of neurogenesis is a strategy for treatment of aberrant cell cycle diseases: an update.

Since publishing our earlier report describing a strategy for the treatment of central nervous system (CNS) diseases by inhibiting the cell cycle and without disrupting neurogenesis (Liu et al. 2010), we now update and extend this strategy to applications in the treatment of cancers as well. Here, we put forth the concept of “aberrant cell cycle diseases” to include both cancer and CNS diseases, the two unrelated disease types on the surface, by focusing on a common mechanism in each aberrant cell cycle reentry. In this paper, we also summarize the pharmacological approaches that interfere with classical cell cycle molecules and mitogenic pathways to block the cell cycle of tumor cells (in treatment of cancer) as well as to block the cell cycle of neurons (in treatment of CNS diseases). Since cell cycle inhibition can also block proliferation of neural progenitor cells (NPCs) and thus impair brain neurogenesis leading to cognitive deficits, we propose that future strategies aimed at cell cycle inhibition in treatment of aberrant cell cycle diseases (i.e., cancers or CNS diseases) should be designed with consideration of the important side effects on normal neurogenesis and cognition.

AMPK in BCR-ABL expressing leukemias. Regulatory effects and therapeutic implications

The abnormal BCR-ABL oncoprotein is a constitutively active tyrosine kinase driving aberrant proliferation of transformed hematopoietic cells. BCR-ABL regulates activation of many mitogenic and pro-survival pathways, including the PI 3'K/AKT/mTOR pathway that controls various effectors and regulates initiation of mRNA translation in mammalian cells. Although tyrosine kinase inhibitors (TKIs) that target the ABL kinase domain have remarkable clinical activity and have dramatically changed the natural history of Ph+ leukemias, resistance to these agents also develops via a wide range of mechanisms. Efforts to target the PI3'K/AKT/mTOR signaling pathway using kinase inhibitors have been the focus of extensive ongoing investigations by several research groups. Here we review the effects of activation of the AMPK kinase, which regulates downstream targeting and inhibition of mTOR. The potential for future clinical-translational applications of AMPK activators such as AICAR, metformin and resveratrol for the treatment of chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) are discussed.

Cellular and molecular mechanisms of silibinin induces cell‐cycle arrest and apoptosis on HeLa cells

Silibinin, an effective anti-cancer and chemopreventive agent in various epithelial cancer models, has been reported to inhibit cancer cell growth through mitogenic signalling pathways including cervical cancer. However, the underlying mechanisms are still not well elucidated. Here, we assessed the effect of silibinin on human cervical carcinoma cell cycle modulation, apoptosis induction and associated molecular alterations by employing HeLa cell line. Silibinin treatment of HeLa cells resulted in a G2 arrest and induced a decrease in cyclin-dependent kinases involved in both G1 and G2 progression. In addition, silibinin showed a dose-dependent and a time-dependent apoptotic death in HeLa cells in both the mitochondrial pathway and the death receptor-mediated pathway, providing a strong rationale for future studies evaluating preventive and/or intervention strategies for silibinin in cervical cancer pre-clinical models.

Chronological lifespan in stationary culture: from yeast to human cells

A decade ago, Mikhail Blagosklonny predicted that cellular senescence is driven by mitogenic pathways, when the cell cycle is blocked and actual growth is impossible [1]. In particular, the mitogen- and nutrient-sensing mTOR (Target of Rapamycin) pathway drives either cell mass growth or aging [2]. Rapamycin prevents conversion of reversible cell cycle arrest to senescence [3-11]. When the cell cycle is blocked, but mTOR is still active, then cells become senescent. This process was named gerogenic conversion or simply geroconversion [12]. Rapamycin did not by-pass arrest but suppress geroconversion. Cells remained resting but not senescent. The discovery of mTOR-dependent geroconversion allowed Blagosklonny to connect cellular aging to age-related diseases and organismal aging [13, 14]. Furthermore, this predicted that rapamycin is a gerosuppressant, which could be used to prevent age-related diseases by slowing down the aging process [14]. Independently, it was discovered that rapamycin suppresses chronological aging of yeast cells [15]. Chronological lifespan of yeast cells in stationary culture is the most fruitful model in aging research and dozens of papers have been published in Nature, Science, Cell and Cell Cycle. Although yeast model was so useful to identify genes involved in mammalian aging, the reason remained unclear. Yeast only loosely resemble post-mitotic cells in human tissues. Unfortunately, there was no model of mammalian chronological aging in cell culture. In one model developed by Fabrizio and Valter Longo [16] as well as by Matt Kaeberlein and Brian K. Kennedy [17], yeast chronological senescence (CS) is caused by acidosis due to overproduction of acetic acid. Obviously, neither replicative nor accelerated senescence of human cells resembles yeast CS in the stationary culture. Surprisingly, the exact replica was so well known and so trivial that it was overlooked by decades. Here Leontieva and Blagosklonny describe the mammalian cellular model: a neglected flask with overgrown cancer cells that turn medium “yellow” (due to lactate accumulation). Such flasks left and forgotten over weekend could be found in any CO2-incubator. The paper is simultaneously startling and obvious. It is obvious from an everyday experience that highly glycollytic cells can destroy cell culture. But like it was known to most researches (90 years ago) that fungi can destroy bacterial culture, it took a special insight to recognize the potential of this seemingly useless phenomenon. There is an intriguing parallel between penicillin and rapamycin. As described in this issue of Aging, mTOR pathway is involved in glycolytic phenotype, causing self-poisoning due to over-production of lactic acid. By decreasing lactate production, rapamycin prevents chronological senescence (CS). CS can be manipulated genetically and pharmacologically. Most importantly, the same agents that suppress geroconversion, organismal aging and cancer also suppress CS. This study does not break any dogma because there was no dogma as the field did not exist. This paper opens a new filed in both aging and cancer research. Blagosklonny and collaborators are currently preparing follow-up papers with special emphasis to cancer research: the ability of rapamycin to decrease lactate production independently of respiration, selection of highly glycolytic cancer cell clones, tumor progression and resistance to therapy. Many questions will be answered. But this first paper defines the field, providing description of the phenomenon, its mechanism and the ways of pharmacological manipulation. It illuminates the place of yeast CS in aging research and its indirect (via common signaling pathways) relevance to cancer and organismal aging. It also rules out altruistic (programmed) aging of yeast because no one would suspect altruistic nature of cancer cells. I invite the readers to enjoy this first paper in the filed of mammalian cell chronological senescence.

STAT3 Is Required for Notch-Induced Leukemogenesis, and Functions As a Critical Survival Effector in T-Cell Leukemia

Abstract 920The disruption of main signaling mediators of critical leukemia cell functions represents the most promising approach for the development of more effective targeted therapies for acute lymphoblastic leukemia (ALL). Increasing evidence suggests an important combinatorial role for cell autonomous, epigenetic and extrinsic factors in tumorigenesis and tumor progression. However, little is known on how oncogenic and microenvironmental signals are integrated within leukemia T-cells and how they influence T-cell leukemogenesis. Our studies in patients’ leukemia T-cells showed that oncogenic Notch enhances their fitness by regulating the transcription of IL-7Rα, thus increasing the sensitivity of leukemia cells to IL-7, which triggers survival and mitogenic pathways. Interestingly, analysis of the Notch1/CBF-1 complex regulating IL7-Rα transcription show that it contains STAT3, but not STAT5, the main STAT molecule implicated in lymphoid cell signaling. Further analyses showed that STAT3 signaling is active in patients’ T-ALL specimens and in leukemia cells from mice with Notch-induced leukemia. We observed that IL-7 signaling triggers STAT3 phosphorylation and transcriptional activity, regulating the expression of the STAT3 target genes Bcl-xL, survivin, Cyclin D3 and miR-21.We then performed studies using a genetic model of conditional STAT3 deletion in the hematopoietic system. We observed that STAT3 loss-of-function markedly decreased the response of T-lineage cells (including thymocytes) to IL-7 stimulation, even if these cells exhibit functional STAT5 signaling. Importantly, in comparison to wild-type animals, STAT3-null mice did not exhibit defects in common lymphoid progenitors (CLP) in the bone marrow (BM) or in T-cell development, namely on DN thymocyte subsets. Also, no changes were observed in STAT3-null mice in the surface expression or transcriptional levels of IL-7Rα in CLP or thymocytes. To investigate the putative role of STAT3 in leukemia development, we performed the Notch-induced tumor model using BM primitive hematopoietic cells from STAT3-null mice, which could be effectively transduced with the constitutively-active Notch (ICN) construct. We observed that STAT3 deletion significantly inhibited the development of T-cell leukemia, with animals surviving for long periods without evidence of leukemia. Importantly, this was not due to defects in homing or in early engraftment of transplanted cells, which was further documented using intravital microscopy.Finally, pre-clinical studies were performed to evaluate the therapeutic potential of STAT3 pharmacological targeting on leukemia T-cells. STAT3 blockade by the selective inhibitors STATTIC or S3I-201 or the JAK2 inhibitor WP-1066 abrogated STAT3 phosphorylation, inhibiting the transcription of Bcl-xL and Survivin. This inhibition resulted in marked inhibition of leukemia T-cells, which was observed in primary T-ALL cells, in a IL-7-dependent T-ALL line and in leukemia cells from mice carrying ICN-induced leukemia. Mechanistically, we observed that STAT3 blockade results in and massive leukemia cell apoptosis, which is associated with activation of the pro-apoptotic p53(Ser46), and PARP cleavage. Also, STAT3 silencing by shRNA prevented leukemia cell expansion. Strong anti-leukemia synergism was observed between low doses of STATTIC and the frontline therapeutic drug Dexamethasone, or selective PI3K or mTOR inhibitors.In summary, our studies indicate that STAT3 signaling plays an important role in the integration of IL-7 and Notch signals in T-cell leukemia, and unveiled an unexpected role for STAT3 in T-cell leukemogenesis. Finally, our work suggests that the targeted disruption of STAT3 represents a valid therapeutic approach in this cancer. Disclosures:No relevant conflicts of interest to declare.

Single Cell Level Genome-Wide Gene Expression Analysis of Bone Marrow-Derived Oct-4+ very Small Embryonic-Like Stem Cells (VSELs) Revealed That a Polycomb Group Protein Ezh2 Regulates VSELs Pluripotency by Maintaining Bivalent Domains At Promoters of Important Homeodomain-Containing Developmental Transcription Factors

Abstract 2345Recently, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult bone marrow (BM) (Leukemia 2006:20;857). These Oct4+CXCR4+SSEA-1+Sca-1+CD45−Lin− VSELs are capable of differentiation in vitro into cells from all three germ lineages and in in vivo animal models they can be specified into mesenchymal stem cells (MSCs) (Stem Cells Dev 2010:19;1557), cardiomyocytes (Stem Cell 2008:26;1646), and long-term engrafting hematopoietic stem cells (HSCs) (Exp Hematol 2011:39;225). Be employing gene-expression and epigenetic profiling studies we reported that VSELs in BM have germ-line stem cell like epigenetic features including i) open/active chromatin structure in Oct4 promoter, ii) parent-of-origin specific reprogramming of genomic imprinting (Leukemia 2009, 23, 2042–2051), and iii) that they share several markers with epiblast-derived primordial germ cells (PGCs), in particular with migratory PGCs (Leukemia 2010, 24, 1450–1461). However, it was not clear how VSELs maintain pluripotent state. To address this issue we recently employed single cell-based genome-wide gene expression analysis and found that, Oct4+ VSELs i) express a similar, yet nonidentical, transcriptome as embryonic stem-cells (ESCs), ii) up-regulate cell-cycle checkpoint genes, and iii) down-regulate genes involved in protein turnover and mitogenic pathways. Interestingly, our single cell library studies also revelaed that Ezh2, a polycomb group protein, is highly expressed in VSELs. This protein is well known to be involved in maintaining a bivalent domains (BDs) at promoters of important homeodomain-containing developmental transcription factors. Of note a presence of BDs is characteristic for pluripotent stem cells (e.g., ESCs) and as result of Ezh2 overexpression, VSELs, like ESCs, exhibit BDs - bivalently modified nucleosomes (trimethylated H3K27 and H3K4) at promoters of important homeodomain-containing developmental transcription factors (Sox21 Nkx2.2 Dlx1 Zfpm2 Irx2 Lbx1h Hlxb9 Pax5 HoxA3). Of note, spontaneous (as seen during differentiation) or RNA interference-enforced down-regulation of Ezh2 removes BDs what, results in lose of their plurioptentiality and de-repression of several BD-regulated genes that control their tissue commitment. In conclusion, Our results show for first time that in addition to the expression of pluripotency core transcription factor Oct-4, VSELs, like other pluripotent stem-cells, maintain their pluripotent state through an Ezh2-dependent BD-mediated epigenetic mechanism. Based on this our genome-wide gene expression study not only advances our understanding of biological processes that govern VSELs pluripotency, differentiation, and quiescence but will also help to develop better protocols for ex vivo expansion of these promising cells for potential application in regenerative medicine. Disclosures:Ratajczak:Neostem Inc: Consultancy, Research Funding.

Insulin resistance in breast cancer: relevance and clinical implications

Breast cancer risk is increased in women who have attributes of the insulin resistance syndrome, such as obesity (postmenopausal), central obesity (premenopausal and postmenopausal), high endogenous insulin levels, clinical diabetes and physical inactivity. There is a large body of evidence that obesity is associated with a 25 to 50% relative increase in risk of breast cancer recurrence or death, with adverse effects that appear to be independent of hormone receptor status. Obesity, particularly when it is central, is strongly associated with insulin resistance in healthy individuals and breast cancer patients. Several studies have shown that higher insulin and/or C-peptide levels (a marker of insulin secretion), both of which are linked to insulin resistance, are associated with an increased risk of recurrence and death in women with early stage breast cancer, even in the absence of diabetes. Risk is increased twofold to threefold in those with insulin levels in the highest (versus lowest) quartile. Data from our group suggest that these prognostic associations of insulin are most marked in the first 5 years post diagnosis. A role of insulin in breast cancer outcomes is biologically plausible given overexpression of insulin receptors (IR), most frequently the fetal form of the receptor (IR-A), by the majority of human breast cancers. IR-A often hybridizes with insulin-like growth factor 1 receptors to stimulate mitogenic signaling pathways; hybrid receptor activation has been associated with poor clinical outcomes. The current observational and preclinical evidence linking insulin to breast cancer is sufficiently compelling that neoadjuvant and adjuvant intervention studies have been initiated to evaluate clinical anti-cancer effects of metformin, an agent that lowers insulin levels and has other potential non-insulin-mediated anti-cancer effects (mainly through activation of adenosine monophosphate-activated protein kinase). Early results from window of opportunity neoadjuvant studies suggest short-term, single-agent metformin (2 to 3 weeks) lowers insulin levels, reduces proliferation and increases apoptosis. NCIC MA32, an ongoing randomized, multicenter, placebo-controlled, adjuvant trial involving 3,582 women with early stage breast cancer, will provide more definitive evidence regarding potential anti-cancer effects. Additional studies of metformin in the metastatic setting are underway and/or planned. Because other factors (such as inflammation, adipocytokines (leptin, adiponectin), higher estrogen levels) may also mediate prognostic effects of obesity and/or insulin resistance in breast cancer, additional research targeting these mediators as well as obesity per se is also needed.

Substance P Is a Mechanoresponsive, Autocrine Regulator of Human Tenocyte Proliferation

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell© technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10−7 M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.

Differential Regulation of N-Myc and c-Myc Synthesis, Degradation, and Transcriptional Activity by the Ras/Mitogen-activated Protein Kinase Pathway

Myc transcription factors are important regulators of proliferation and can promote oncogenesis when deregulated. Deregulated Myc expression in cancers can result from MYC gene amplification and translocation but also from alterations in mitogenic signaling pathways that affect Myc levels through both transcriptional and post-transcription mechanisms. For example, mutations in Ras family GTPase proteins that cause their constitutive activation can increase cellular levels of c-Myc by interfering with its rapid proteasomal degradation. Although enhanced protein stability is generally thought to be applicable to other Myc family members, here we show that c-Myc and its paralog N-Myc respond to oncogenic H-Ras (H-Ras(G12V)) in very different ways. H-Ras(G12V) promotes accumulation of both c-Myc and N-Myc, but although c-Myc accumulation is achieved by enhanced protein stability, N-Myc accumulation is associated with an accelerated rate of translation that overcomes a surprising H-Ras(G12V)-mediated destabilization of N-Myc. We show that H-Ras(G12V)-mediated degradation of N-Myc functions independently of key phosphorylation sites in the highly conserved Myc homology box I region that controls c-Myc protein stability by oncogenic Ras. Finally, we found that N-Myc and c-Myc transcriptional activity is associated with their proteasomal degradation but that N-Myc may be uniquely dependent on Ras-stimulated proteolysis for target gene expression. Taken together, these studies provide mechanistic insight into how oncogenic Ras augments N-Myc levels in cells and suggest that enhanced N-Myc translation and degradation-coupled transactivation may contribute to oncogenesis.

Ginsenoside Rp1 Inhibits Proliferation and Migration of Human Lung Cancer Cells

Ginsenoside Rp1 (G-Rp1) is a novel ginseng saponin derivative with anti-tumor activity. However, the biochemical and molecular mechanisms of G-Rp1 on anti-tumor activity are not fully understood. In the present study, we report that G-Rp1 inhibits lung cancer cell proliferation, migration and adhesion in p53 wild-type A549 and p53-defi cient H1299 cells. Anti-proliferative activity of G-Rp1 in lung cancer cells is mediated by enhanced nuclear localization of cyclin-dependent kinase inhibitors including <TEX>$p27^{Kip1}$</TEX> and <TEX>$p21^{WAF1/Cip1}$</TEX>, and subsequent inhibition of pRb phosphorylation. We also show that these anti-tumor activities of G-Rp1 in both A549 and H1299 cells appear to be mediated by suppression of mitogenic signaling pathways such as ERK, Akt and <TEX>$p70^{S6K}$</TEX>. Taken together, these findings suggest further development and evaluation of G-Rp1 for the treatment of lung cancers with mutated p53 as well as wild-type p53.

The cell cycle and cancer

Deregulation of the cell cycle underlies the aberrant cell proliferation that characterizes cancer and loss of cell cycle checkpoint control promotes genetic instability. During the past two decades, cancer genetics has shown that hyperactivating mutations in growth signalling networks, coupled to loss of function of tumour suppressor proteins, drives oncogenic proliferation. Gene expression profiling of these complex and redundant mitogenic pathways to identify prognostic and predictive signatures and their therapeutic targeting has, however, proved challenging. The cell cycle machinery, which acts as an integration point for information transduced through upstream signalling networks, represents an alternative target for diagnostic and therapeutic interventions. Analysis of the DNA replication initiation machinery and mitotic engine proteins in human tissues is now leading to the identification of novel biomarkers for cancer detection and prognostication, and is providing target validation for cell cycle-directed therapies.

Clozapine induction of ERK1/2 cell signalling via the EGF receptor in mouse prefrontal cortex and striatum is distinct from other antipsychotic drugs

Treatment resistance remains a major obstacle in schizophrenia, with antipsychotic drugs (APDs) being ineffective in about one third of cases. Poor response to standard therapy leaves the APD clozapine as the only effective treatment for many patients. The reason for the superior efficacy of clozapine is unknown, but as we have proposed previously it may involve modulation of neuroplasticity and connectivity through induction of interconnected mitogenic signalling pathways. These include the mitogen-activated protein kinase-extracellular signal regulated kinase (MAPK-ERK) cascade and epidermal growth factor (EGF)/ErbB systems. Clozapine, distinct from other APDs, induced initial inhibition and subsequent activation of the ERK response in prefrontal cortical (PFC) neurons in vitro and in vivo, an action mediated by the EGF receptor (ErbB1). Here we examine additionally the striatum of C57Bl/6 mice to determine if clozapine, olanzapine, and haloperidol differentially regulate the ERK1/2 pathway in a region or time-specific manner conditional on the EGF receptor. Following acute treatment, only clozapine caused delayed striatal ERK phosphorylation through EGF receptor phosphorylation (tyrosine 1068 site) and MEK that paralleled cortical ERK phosphorylation. Olanzapine induced initial pERK1-specific blockade and an elevation 24-h later in PFC but had no effect in the striatum. By contrast, haloperidol significantly stimulated pERK1 in striatum for up to 8 h, but exerted limited effect in PFC. Clozapine but not olanzapine or haloperidol recruited the EGF receptor to signal to ERK. These in-vivo data reinforce our previous findings that clozapine's action may be uniquely linked to the EGF signalling system, potentially contributing to its distinctive clinical profile.

CMyc Is a Principal Upstream Driver of β-Cell Proliferation in Rat Insulinoma Cell Lines and Is an Effective Mediator of Human β-Cell Replication

Adult human β-cells replicate slowly. Also, despite the abundance of rodent β-cell lines, there are no human β-cell lines for diabetes research or therapy. Prior studies in four commonly studied rodent β-cell lines revealed that all four lines displayed an unusual, but strongly reproducible, cell cycle signature: an increase in seven G(1)/S molecules, i.e. cyclins A, D3, and E, and cdk1, -2, -4, and -6. Here, we explore the upstream mechanism(s) that drive these cell cycle changes. Using biochemical, pharmacological and molecular approaches, we surveyed potential upstream mitogenic signaling pathways in Ins 1 and RIN cells. We used both underexpression and overexpression to assess effects on rat and human β-cell proliferation, survival and cell cycle control. Our results indicate that cMyc is: 1) uniquely up-regulated among other candidates; 2) principally responsible for the increase in the seven G(1)/S molecules; and, 3) largely responsible for proliferation in rat β-cell lines. Importantly, cMyc expression in β-cell lines, although some 5- to 7-fold higher than normal rat β-cells, is far below the levels (75- to 150-fold) previously associated with β-cell death and dedifferentiation. Notably, modest overexpression of cMyc is able to drive proliferation without cell death in normal rat and human β-cells. We conclude that cMyc is an important driver of replication in the two most commonly employed rat β-cell lines. These studies reverse the current paradigm in which cMyc overexpression is inevitably associated with β-cell death and dedifferentiation. The cMyc pathway provides potential approaches, targets, and tools for driving and sustaining human β-cell replication.

Multikinase inhibitors: a new option for the treatment of thyroid cancer

Thyroid cancer typically has a good outcome following standard treatments, which include surgery, radioactive iodine ablation and treatment with TSH-suppressive levothyroxine. Thyroid cancers that persist or recur following these therapies have a poorer prognosis. Activation of mitogenic and angiogenic signaling pathways occurs in these cancers, and preclinical models have shown that inhibition of key kinase steps in these pathways can have antitumoral effects. Several of these kinase inhibitors have now been tested in phase II and phase III trials, with modestly encouraging results. Some promising data exist for the use of vandetanib (also known as ZD6474), motesanib, axitinib, cabozantinib (also known as XL184), sorafenib, sunitinib, pazopanib and lenvatinib (also known as E7080) in progressive thyroid cancer of medullary, papillary and follicular subtypes. These drugs are generally well-tolerated, although dose-limiting toxicities are common, and a few (probable) treatment-related deaths have been reported. Additional phase III trials will be needed to conclusively show that treatment benefit exceeds risk. Drug resistance can occur via activation of alternate mitogenic signals (pathway switching), as has been reported for the use of kinase inhibitors in other malignancies, such as melanoma. The hypothesis that combinations of kinase inhibitors targeting different pathways might produce better results is currently being tested in several clinical trials.